Development Of Solid-State-Fermented Jatropha Curcas L. Cake As A Protein Feed And An Omics-Based Evaluation Of Its Effect On Growing Pigs

Jatropha curcas L.is widely cultivated in China as a bioenergy plant.Its seed is rich in protein,but the cake’s application as animal feed is limited because of the existence of a variety of toxins.So far,there have been few studies on the application in animal husbandry of Jatropha curcas L.cake（JCC）obtained by solid-state fermentation,and the mechanism of its effects on animal health is not clear.This study improved the toxicity evaluation criteria of JCC,then the microbial strains with high detoxifying efficiency were screened out from the soil to develop a toxin-free product.The effects of fermented Jatropha curcas L.cake（FJCC）on the growth performance and health status of growing pigs were investigated through analysis of intestinal microbiome and metabolomics.The results of this research not only can promote the development of JCC as a feedstuff to alleviate the shortage of protein feed but also help us to understand further the respective feed safety and value of toxic feed resources and solid-state-fermented feed,to facilitate the development of new feed resources and the scientific use of fermented feed.Experiment 1.Effects of JCC on growth performance and health status of pigsThis study was conducted to investigate the effect of dietary JCC on growth performance,organ morphology,apparent nutrient digestibility,redox state,immune stress parameters,cytokines,colonic microflora and metabolites of pigs.Thirty-two DLY growing pigs with similar age and body weight of 25.21±1.34 kg were divided into two treatments for a 28 d trial.The results showed that:1)The 5%JCC diet group developed severe lesions in the intestine,hypothalamus,liver and kidney.Specifically,in the hypothalamic neurons,nuclear pyknosis were observed,and inflammatory cell infiltration in the surrounding area.The intestinal villi atrophied and displayed progressive necrosis accompanied by cell vacuolation.The hepatocytes were enlarged,with loose cytoplasm and pink eosinophilic particles in the cells.Crescent-shapes or ring-shapes were observed around the capillary plexus in the glomerulus.Red blood cells,neutrophils and fibrinous exudate were found between the proliferated renal epithelial cells.2)Dietary JCC decreased the final body weight,ADG and ADFI of pigs（P<0.05）.The apparent digestibility of dry matter,gross energy,crude protein,ash and ether extract of pigs fed with JCC decreased（P<0.05）.The activity of aspartate aminotransferase and content of urea nitrogen,total protein and albumin in the plasma increased significantly（P>0.05）.The concentrations of glucose and immunoglobulins Ig G and Ig M decreased（P<0.05）,and the content of cortisol increased substantially（P<0.05）.Dietary JCC decreased the levels of antioxidant enzymes（GSH-Px,T-SOD and CAT）and total antioxidant capacity（T-AOC）in the hypothalamus,plasma and intestines,and increased the content of lipid peroxide MDA（P<0.05）.The levels of inflammatory cytokines（TNF-α,IFN-γ,IL-1 and IL-6）in the hypothalamus,plasma and intestines also increased（P<0.05）.4)Dietary JCC decreased the relative abundance of Faecalibacterium,Dialister and Acidaminococcus in the colon,and increased the relative abundance of p-75-a5,Desulfovibrio,Selenomonas,Oxalobacter,L7A＿E11,rc4-4,Schwartzia and Coprobacillus（P<0.05）.The metabonomics analysis of colon digesta indicated that there were 114different metabolites between the JCC and control group.The reduced metabolites mainly include secondary bile acid,vitamins（VB₁,VB₆and VB₇）,L-carnitine,acetylcarnitine,betaine,melatonin,4-aminobutyric acid,etc.The increased metabolites mainly include amino acids and amines（such as tyrosine dipeptides and histamines）,primary bile acids（cholic acids）,as well as carbohydrates and sugars（D-mannitose,DL-lactic acid）.The correlation analysis between microbes,metabolites and growth performance,as well as physiological and biochemical indicators,indicates that the enriched microbes in the JCC dietary group correlate positively with cortisol,inflammatory cytokines（IL-1,IL-6 and IFN-γ）,histamine,primary bile acid,and carbohydrate metabolism intermediates（D-mannose and DL-lactic acid）,but negatively with the levels of ADFI,ADG,vitamin B group,4-aminobutyric acid,melatonin,betaine,L-carnitine,secondary bile acid and immunoglobulins（Ig G and Ig M）.In conclusion,5%JCC has adverse effects on pig’s growth performance and the pigs exhibit lesions on the intestines,liver,kidney and hypothalamus.These changes may be due to the disturbance of intestinal microbes and metabolites arising from JCC intake,thus promoting the levels of toxic metabolites,which indicate changes in important KEGG pathways,such as neuroactive ligand-receptor interaction,central carbon metabolism of cancer,and GABAergic synapses,finally leading to a systemic toxicity.The sensitivity of pigs to JCC highlights the need for its detoxification before feeding.Experiment 2.Establishment of a zebrafish survival model to screen microbial strains,optimization of fermentation parameters and determination of nutritional valueA mortality rate model of JCC toxicity for zebrafish was established to screen out the most effective detoxifying microbial strains.The key parameters of the solid fermentation process were optimized,and changes in proximal nutrients,toxin content and metabolites were measured.The results showed that:1)The LC₉₉value of JCC toxicity to zebrafish was 43.945 mg/L.Three strains were screened out by employing this model,and they were identified as Bacillus megaterium SCYA10,Geotrichum candidum SCYA23 and Mucor circinelloides SCYA25,respectively.An optimal model of toxin degradation was established by applying the single factor and response surface methodology.Under the optimized fermentation conditions（Bacillus megaterium SCYA10:Geotrichum candidum SCYA221.53:Mucor circinelloides SCYA25=2:3:1,total injection amount of 15.25%,feed to water ratio of 1:0.68,fermentation temperature of 30.3℃,and fermentation time of d）,the detoxification rate reached 96%.2)The crude protein content of FJCC increased by 9.92%,while ether extract and crude fiber decreased by 6.19%and 3.96%,respectively.The degradation rates of key toxins such as phorbol esters,trypsin inhibitors,lectins,saponins and total phenols were76.93%,87.25%,73.95%,86.23%and 65.91%,respectively.Scanning electron microscopy showed that the texture of FJCC was loose and porous,which is beneficial,from a nutritional standpoint,for animal digestion and absorption.3)There were 448 differential metabolites between JCC and FJCC.The metabolites that increased in FJCC are as follows（1）carnitine:（5Z,8Z）-tetradecadienoylcarnitine and cis-5-Tetradecenoylcarnitine;（2）GABA and 5-HT receptor modulators:tetrahydrocortisol,tetrahydrodeoxycortisol and 4-hydroxy-3-nitrobenzoamide;（3）flavors and emulsifiers:sorbitan laurate,cinnamate anthracene,3,4-Dihydro-2H-1-benzopyran-2-one;（4）insect repellent:OXIBENDAZOLE;（5）small peptides.The decreased metabolites mainly include（1）carbohydrates:sucrose,D-Maltose,D-Gluconic acid,3-Galactosyllactose,1-[（5-Amino-5-carboxypentyl）amino]-1-deoxyfructose;（2）lipid:glycerophosphocholine,peatidyleetholamine,phosphatidylserine,phosphatidylcholine,stearyl citrate;（3）neurotoxic substance;（4）6’’’-O-Sinapoylsaponarin and others.The KEGG pathway enrichment analysis indicates that there were significant changes in important pathways after fermentation.The representative differential metabolic pathways are as follows:arginine biosynthesis,aminoacyl-t RNA biosynthesis,protein digestion and absorption,taste transduction,galactose metabolism,linoleic acid metabolism,neuroactive ligand-receptor interaction,and GABAergic synapse.The above results indicate that the solid state fermentation of JCC by Bacillus megaterium SCYA10,Geotrichum candidum SCYA23 and Mucor circinelloides SCYA25achieved a high detoxification rate.The nutritional value of FJCC was significantly improved,and the levels of carnitine,neurotransmitter receptor modulator,emulsifier,aromatic substances and insecticidal compounds increased during fermentation,while the levels of carbohydrate,lipid,oxidative stress and neurotoxic substances decreased.In conclusion,the fermentation process effectively improves the nutritional value of JCC,which can be applied in animal feed.Experiment 3.Effects of dietary FJCC on growing pigs,pertaining to growth performance,profiles of colon microbiota and metabolites,and regulation of the gut-brain axisA total of 64 male pigs,with an initial body weight of 27.60±1.59 kg,were assigned to 4 treatments for a 28-d trial.The supplemental levels of FJCC in each treatment were0%,2.5%,5%and 7.5%,respectively.The results showed that:1)The 5%FJCC dietary group had the highest ADG among all treatments（P<0.05）.Dietary inclusion of FJCC had a tendency to increase the feed to gain ratio（P=0.071）when compared with the control group.The apparent digestibility of gross energy,crude protein,calcium and phosphorus in the 5%and 7.5%FJCC dietary groups was higher than those in the control group（P<0.05）.The apparent digestibility of ash in the 7.5%FJCC dietary group was significantly higher than other groups（P<0.05）.The villus height of the duodenum and jejunum,and the ratio of villus height to crypt depth in the jejunum were significantly higher in the 5%and 7.5%FJCC dietary groups（P<0.05）.The villus height of the ileum in the 5%FJCC dietary group was significantly higher than in the other groups（P<0.05）.5%and 7.5%FJCC significantly reduced the p H value of colon digesta（P<0.05）.The contents of butyric acid and total SCFAs were significantly increased in the 5%FJCC group（P<0.05）.FJCC had no significant effects on the levels of Ig G,Ig A,Ig M and cortisol in the plasma（P>0.05）.In the hypothalamus,there was no difference in levels of GSH-Px and T-AOC among all groups（P>0.05）,but the highest levels of T-SOD and CAT were observed in the 5%and 7.5%FJCC dietary groups（P<0.05）.Dietary FJCC decreased the MDA level in the hypothalamus（P<0.05）.In the plasma,dietary FJCC had a tendency to decrease the T-AOC level（P=0.062）.In the jejunum,the CAT content in the 5%FJCC dietary group was the highest,while the content of MDA was the lowest（P<0.05）.In the colon,the levels of GSH-Px,T-AOC and T-SOD in the FJCC dietary groups were higher than those in the basal diet group（P<0.05）,while the MDA content was lower（P<0.05）,and there was no significant difference in CAT levels among all groups（P>0.05）.The content of IL-1 in the hypothalamus,as well as the levels of TNF-αand IL-6 in the plasma,were lowest in the 5%FJCC dietary group（P<0.05）.The content of IL-1 and IL-6 in the colon of pigs provided with 5%and 7.5%FJCC significantly decreased（P<0.05）.3)The above results showed that the 5%FJCC dietary group had a better growth performance,apparent nutrient digestibility,intestinal development and intestinal microenvironment than the control diet group.To evaluate whether these above benefits are associated with the changes in intestinal microbiota and their metabolites,comparisons of colonic microflora and metabolite profiles were carried out between these two groups.There was no significant difference in alpha diversity between the two groups（P>0.05）.However,5%dietary FJCC increased the abundance of Prevotella and Streptococcus（P<0.05）,and decreased the abundance of P-75-A5,Anaerovibrio,Desulfovibrio,Methanobrevibacter,Mitsuokella,Schwartzia and Sharpea（P<0.05）.4)The metabolomics analysis found different 47 metabolites between the control group and the 5%FJCC dietary group.Significantly increased metabolites in the 5%FJCC dietary group include vitamin B₆,L-carnitine,GABA,5-HT,isocaproic acid,lithocholic acid and stachyose.The significantly reduced metabolites mainly include（1）primary bile acids:cholic acid and chenodeoxycholate;（2）lipids:1-Palmitoylglycerol,16-Hydroxypalmitic acid,linoleic acid,cholesteryl sulfate and N-Oleoylethanolamine;（3）amino acids and their metabolites:indoleacetic acid,indole-2-carboxylic acid cyclohexylamine,linoleoyl ethanolamide,N6-Methyl-L-lysine,Lys-Gly,ketoisocaproic acid,phenylpyruvate,p-Hydroxyphenylacetic acid,betaine,kaempferol,pyrrolidine,sphingosine,sphinganine and creatinine;（4）pyrimidines:thymine,cytosine and thymidine.The KEGG pathway enrichment analysis of the above metabolites indicates that some essential pathways,including GABAergic synapses,vitamin B₆metabolism,synaptic vesicle recycling,arginine and proline metabolism,scabbard lipid metabolism and lipid signalling pathways,retrograde nerve signals,neural activity ligand-receptor interaction,bile secretion,arginine and phenylalanine biosynthesis and metabolism,c AMP signalling,pyrimidine metabolism,cell apoptosis,taste transduction,and biosynthesis of amino acids,as well as alanine,glutamic acid and aspartic acid metabolism,were significantly changed in the 5%FJCC dietary group.5)Correlation analysis between intestinal microbes and physiological and biochemical indexes showed that a higher level of Streptococcus in the 5%FJCC dietary group was negatively correlated with the levels of IL-6 and MDA,and positively correlated with the level of T-SOD.Correlation analysis between microbes and metabolites showed that the Prevotella and Streptococcus enriched in the 5%FJCC group was negatively correlated with the levels of sphingosine,sphinganine,cholesterol sulfate and cholic acid,and positively correlated with the levels of butyric acid,isovaleric acid,vitamin B₆,5-HT,lithocholic acid,L-carnitine and GABA.6)5%FJCC increased the concentrations of 5-HT and GABA in the plasma,with no significant change in DA level（P<0.05）.The levels of 5-HT,GABA and DA in the hypothalamus all increased significantly（P<0.05）.The levels of precursor amino acids corresponding to the above neurotransmitters also significantly increased in the hypothalamus and plasma of the 5%FJCC dietary group（P<0.05）.7)A 5%FJCC diet increased the m RNA levels of Ah R,Nrf2,SOD and GPX in the hypothalamus of pigs（P<0.05）,and decreased the m RNA expression of Keap1（P<0.05）,but had no significant effect on the gene transcription levels of CYP1A1,CAT and NQO1（P>0.05）.The m RNA levels of Ah R,Nrf2,SOD,GPX and NQO1 in the mucosa increased in the 5%FJCC dietary group（P<0.05）,the Keap1 gene transcription level decreased（P<0.05）,but the m RNA levels of CYP1A1 and CAT were not affected（P>0.05）.The above results suggest that 5%FJCC increased the recycling level of aromatic amino acids and thus ensured the synthesis efficiency of neurotransmitters.Furthermore,5%FJCC improved the colon microbiota profile and increased the levels of beneficial metabolites,such as 5-HT,GABA,SCFAs and secondary bile acids.5%FJCC also activated the expression of neuron functional gene Ah R,thereby promoting the expression of its downstream antioxidant genes（Nrf2 etc）.Therefore,5%FJCC may have positive effects on the gut-brain axis of pigs,improving their growth performance and health status.In conclusion,the solid-state fermentation process developed in this study can effectively detoxify JCC,and the nutritional value of mature cake is significantly improved.A 7.5%FJCC diet had no adverse effects on the performance and health status of growing pigs,and a 5%FJCC diet had more beneficial effects than soybean meal on the performance,apparent nutrient digestibility,organ morphology,levels of antioxidant enzyme and intestinal microenvironment of pigs.These beneficial effects may be accredited to the regulations of FJCC on the gut-brain axis.