Detoxification Of Latropha Curcas Seed Cake By Solid-state Fermentation And Nutritional Assessment In Rats

Jatropha curcas is a versatile oil plant with its seed oil content of up to 60%.Its remaining seed cake after oil extraction is an important source of feed protein,but it contains a toxic substance-phorbol ester(PE),which limits its use in animal feed.The biological fermentation could degrade phorbol esters content and anti-nutritional facors,and also improve the nutritional value of Jatropha curcas.In this paper,a strain for degrading was isotated at first and the process of fermentation was then studied.The detoxified Jatropha curcas seed cake(JSC)was used to replace different portion of soybean meal in therat diets to assess their nutritional value.1.Isolation of JSC phorbol esters degrading bacteriaA method was established for determenation of phorbol esters in the JSC.Phorbol esters were analyzed by HPLC system using an analytical column(Agilent Extend-C 18,4.6mm×250mm I.D and 5 μm particle size).The mobile phase consisted of 20%water,80%acetonitrile and 0.3%formic acid.Separation was performed at room temperature(25 ℃)with flow rate 1.0 ml/min.The detector wavelength was set at 280 nm.The results were expressed as equivalent to phorbol 12-myristatel 3-acetate(PMA)(Sigma,U.K.)used as an external standard.The linear relationship between phorbol concentration(Y)and peak area(X)was fit in the range of 3～500μg/mL,with the regression equation as Y = 1523X-22.79,r = 0.997.Twenty-eight bacterial strains were isolated from soil sample,which was obtained from the City of Panzhihua,Sichuan where the Jatropha curcas was cultivated,using Jatropha curcas seed oil as the only carbon source.All strains were tested for their efficiency for degradation of PEs or derivatives by the submerged fermentation method.The strain Z11 was found to have the maximum capacity to degrade the PEs in JSC.Based on its morphological characteristics,physiological biochemical characteristics,16S rDNA sequence assessment,and nucleotide alignment of the sequence with the available NCBI public database,the Z11 strain was found to be close to the genus Enterobacter with a maximum homology similarity of 99%(MF102129).Growth activity of the strain was measured by spectrophotometry.The optimum conditions for the growth of the strain was at temperature of 30 ℃,pH 7.8,and the optimum carbon and nitrogen sources were sucrose and peptone,respectively.2.Optimization of parameters for solid-state fermentationWe evaluated the effect of different moisture levels,inoculum size,and incubation time on solid-state fermentation by Enterobacter cloacae Z11 with the aim to identify the optimum conditions for fermentation.The fementation process was optimized by single factor test and response surface method.The optimum condition for Z11 to ferment Jatropha curcas was at 30 ℃,initial pH value of 7.8,inoculation amount of 20%,and initial water content of 50%for 5 days of fermentation.The highest degradation rate of phorbol ester was 50.7%.After fermentation,the contents of crude protein and total essential amino acids in the JSC increased by 29.8 and 16.4%,respectively.The contents of lysine,methionine and threonine in JSC increased by 14.9,50.0 and 38.2%,respectively.After the fermentation,the content of anti-nutritional factors including phytate,tannin,trypsin inhibitor and lectin decreased by 72.0,37.8,98.2 and 88.9%,respectively.3.Effect of replacing soybean meal with the fermentedJSC in SD ratsRats were used to assess the nutritional value of the fermented JSC.The experimental rats were divided into 4 groups and allocated to 4 diets:basal diet(control)and three treatment diets in which soybean meal was replaced by fermented JSC at levels of 5,8 and 10%(as dry matter basis).After 28 days of experiment,feed intake,body weight gain,serum biochemical parameters and organ ratio to body weight rats were determined.With the increasing replacement level,the feed intake and body weight gain of the rats were decreased(P<0.05).The feed effiency in control and 5%groups were significantly higher than in the other two groups(P<0.05).Lower activities of alkaline phosphatase,aspartate transaminase and total protein were observed in 10%groupthan in the other three groups(P<0.05).There was no significant difference(P>0.05)in serum concentration of urea and albumin among four groups.No significant difference(P>0.05)was observed in liver and spleen weight or their ratios over body weight among all treatments.Compared with the control group,the nephrocyte and pneumonocyte in rats fed higher level of fermented JSC showed varying degrees of damage,but no obvious change was observed in spleens and livers of all treatments.In summary,the study was conducted to establish a method for determenation of phorbol esters in the JSC.The Enterobacter cloacae Z11 isolated from the soil could degrade phorbol esters in the JSC.The highest degradation rate of phorbol ester could reach 51.6%under optimized conditions.Replacing soybean meal with different levels of fermented JSC in SD rats diets indicate that it could be used to replace SBM up to 25%with no detrimental impacts on feed effiency and some serum biochemical parameters.