Substrate compositions used for
Biodiesel production using the seed oil of
The toxicity of Jatropha seed is mainly attributed to a group of diterpene esters called phorbolesters. These esters are present in high concentrations in toxic seed varieties but in lower concentrations in a non-toxic seed variety from Mexico . Phorbol esters activate protein kinase C, a key signal transduction enzyme released in response to various hormones and developmental processes in most cells and tissues [5,6].
In addition to the phorbol esters, there is also a toxic protein called curcin in Jatropha seed cake. This protein has two polypeptide chains and is able to inhibit protein synthesis . Curcin is a ribosome-inactivating protein and promotes mucosal irritation and gastrointestinal hemagglutinating action .
Phytic acid (myo-inositol hexaphosphoric acid) and tannins are considered anti-nutritional factors because they inhibit the absorption of proteins and minerals [9-11]. Phytic acid is a compound formed during seed maturation . The seed of
The use of residue or by-products in animal nutrition can minimize expenditures on the development of food sources, such as soybean, cotton and wheat meals, without causing undesirable effects on the overall production system. However, it is first necessary to know the nutritional value and effects of the by-product’s inclusion in animal diets.
Some studies have used physical and chemical treatments to detoxify Jatropha seed [2,16,17]. These methods have been effective but require the use of chemicals that may result in other the presence of other residues. Conversely, bio-detoxification does not require the application of any chemical compounds. It may also reduce the concentrations of phorbol esters and anti-nutritional factors to non-toxic levels .
2.1. Microorganism, fungal growth conditions and inoculum production (spawn)
The isolate Plo 6 of
2.2. Substrate and inoculation
To select the most suitable substrates for lignocellulolytic enzyme production, we conducted preliminary experiments with Jatropha seed cake and various lignocellulosic residues. We tested
The compositions selected for biological detoxification were based on the results of the above preliminary experiments (Table 1). The substrates were humidified with water to 75% of their retention capacity. Then, 1.5 kg of each substrate was placed in polypropylene bags and autoclaved at 121 °C for 2 h. After cooling, the substrates were inoculated with 75 g of spawn and incubated at 25 °C. Samples from non-inoculated autoclaved bags were kept as controls.
|Jatropha seed cake (Jc)||20||0|
|Jc + 10% eucalypt bark (JcEb10)||18||2|
2.3. Chemical composition of the substrates and enzymatic assays
The phorbol ester contents were analyzed by high performance liquid chromatography (HPLC), as previously described . A standard curve was made using solutions of phorbol-12-myristate 13-acetate (Sigma Chemical, St. Louis, USA) at concentrations from 0.005 to 0.5 mg mL-1.
To determinate the dry mass, 1.5 kg of the substrate was dried at 105 ºC until a constant weight was obtained.
The laccase and manganese peroxidase activities were measured using 2,2'-azino-bis-3-etilbenzotiazol-6-sulfonic acid  and phenol red solution  as substrates, respectively. Xylanase and cellulase activity was calculated by measuring the levels of reducing sugars produced by the enzymatic reactions [28,29]. Phytase activity (myo-inositol hexakisphosphate phosphohydrolase) was determined using the Taussky-Schoor reagent .
The level of reducing sugars was determined by the dinitrosalicylic acid (DNS) method (99.5% dinitrosalicylic acid, 0.4% phenol and 0.14% sodium metabisulfite).A standard curve was made with D-glucose, with concentrations from 0.5 to 1.5 g L-1 .
2.4. Digestibility of Jatropha seed cake and ammonium production in rumen liquid measured
To analyze the suitability of the chosen substrates (Table 1) in animal feed, we determined their levels of dry matter (DM), organic matter (OM), crude protein (CP), mineral matter (MM), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), non-fiber carbohydrates(NFC), hemicellulose (HEM), cellulose (CEL) and lignin according to previously described methodology [32,33].
To analyze the production of ammonia, the samples were incubated under the same conditions as described for the IVDMD process. These samples were placed into two flasks containing buffer, rumen fluid and the substrates samples (Table 1) harvested before and after fungal colonization. Ammonia quantification was determined using ammonium chloride as an indicator and absorbance was measured in a spectrophotometer (Spectronic 20D) at 630 nm .
2.5. Animal assay
The experiment was conducted in theGoat Experimental Section from the Department of Animal Science at the Federal University of Viçosa - MG, BRAZIL. Twenty-four healthy female Alpine goats weighing 20 ±1.5 kg, with a mean age of five months, were used. This experiment was performed after the Jatropha seed cake had been bio-detoxified by
2.5.1. Experimental design
The experimental trial lasted 72 days. During the first12 days, animals were allowed to adapt to the experimental diet. The data were collected during the following 60days.
The animals were kept in individual confinement stables (1.5x2.0m) equipped with food and water systems. The stables had fully slatted floors adapted for the total collection of feces and urine. Water was provided
The diets were formulated to meet the nutritional requirements of goats with a starting ody weight of 20 kg and a daily weight gain of 100g . The feed contained an average of 12% crude protein.
The treatments consisted ofthe detoxified substrates at four levels: 0, 7, 14 and 20% (based on total dry matter) inforagehayTifton-85 (
|Forage hay Tifton-85||33.18||31.26||31.26||31.25|
|Jatropha seed cake bio-detoxified||0.00||6.97||13.93||19.90|
|Dry mass (DM)||84.65||84.75||84.80||84.81|
|Crude protein (CP)||12.84||12.05||11.73||11.89|
|Ether extract (EE)||3.38||3.25||3.06||2.87|
|Neutral detergent fiber (NDF)||41.00||42.46||44.42||45.94|
|Net energy (NE, Mcal/kg)||1.87||1.72||1.63||1.54|
The feed was supplied twice a day as a complete mixture to allow intake of approximately 10% of the offered amount. The amount was based on the intake of the previous day.
The fecal metabolic nitrogen level (Nmet.fecal) was calculated according to previously established methods . The amount of undigested nitrogen (Nund) was calculated as the difference between fecal nitrogen (Nfecal) and Nmet.fecal. To determine the fraction of urinary nitrogen of endogenous origin (Nuend), a previously published equation was used . From the difference between the urinary nitrogen and endogenous urinary nitrogen (Nuend) levels, we calculated the exogenous urinary nitrogen (Nuexo). Nitrogen balance (NB) was estimated with the following equation: NB = N ingested - [Nund + Nuexo]. The biological value of protein was calculated according to previous methods .
The chemical compositions of feeds, orts and feces as a percentage of DM, MM, OM, CP, EE, NDF, ADL and NFC were determined according to previous methodology [32, 33, 40]. The total protein level in the bio-detoxified Jatropha seed cake was calculated from the total nitrogen content by applying the correction factor 4.38. The net energy (NE) was obtained by a previously reported equation .
The blood samples were collected in the morning, before supplying the feed, by jugular puncture and vacuum tubes. Blood was stored with and without the anticoagulant EDTA. After this procedure, the tubes were refrigerated and sent to a laboratory for blood biochemical analysis to determine the hemogram compounds. This analysis included the numbers of erythrocytes, hemoglobin, hematocrit and leukocytes. In the Blood serum was analyzed creatinine, alkaline phosphatase, urea and total protein.
2.6. Statistical analyses
The experiments on phorbol ester degradation, anti-nutritional factors,
The experiments on animals were distributed in a completely randomized design with six replicates per diet condition. The resulting data were subjected to analysis of variance (ANOVA) and regression analysis (p < 0.05). Regression models (linear, quadratic or cubic) were fitted to the observed significance (5% level of probability) using the REG procedure (SAS 9.0).
After 15 days of inoculation,
3.1. Phorbol ester degradation
Autoclaving the substrates (at 121 °C) reduced the phorbol ester content by an average of 20% (Figure 2). However, these compounds were not degraded at 160 °C for 30 min . Moreover, the addition of sodium hydroxide and sodium hypochlorite combined with heat treatment was able to reduce only 25% of the phorbol concentration .
In this study,
The ability of
After 45 d of substrate incubation with
3.2. Degradation of anti-nutritional factors
Tannin concentrations observed in the seed cake (Figure 4) are similar to those previously reported in the fruit peel of
The thermal treatment of the substrates decreased the tannin concentration by 46% (Figure 4). This result was similar to that observed in vegetables after cooking or autoclaving at 121 °C and 128 °C for different periods of time .
Regardless of the substrate, tannin degradation by
Although phytic acid is considered to be heat-stable , the amount of phytic acid decreased by 20% after sterilization of the substrates, (Figure 5). A degradation of 50% of this anti-nutritional factor was also been observed in legumes subjected to autoclaving at 121 ºC for 90 min .
Phytase activity by
3.3. Digestibility of Jatropha seed cake and ammonium production in rumen liquid
Many agro-industrial residues contain a higher content of fibers, of low digestibility, than proteins, vitamins and minerals. The colonization or fermentation of these by-products by microorganisms, especially lignocellulosic fungi, can efficiently and affordably increase their digestibility and nutritional value . This procedure has been used successfully in cotton waste  by colonization with
Before fungal colonization, we observed higher levels of CP, lignin, ADF and EE in the Jatropha seed cake (Table 3). These data show the importance of adding eucalyptus bark to balance carbon and nitrogen and decrease the fat content, thus resulting in improved fungal growth. Furthermore, these data confirm the potential of using the bio-detoxified seed cake as a source of protein and lipids in ruminant diets . The use of foods rich in these nutrients in animal diets is important because (a) the proteins are the main source of nitrogen and amino acids, and (b) lipids can reduce the production of methane by the rumen . For every 1% increase in the amount of fat added to the diet, there is a 6% reduction in methane emissions by ruminant animals. This reduction in methane production may be due to a negative effect on the lipid protozoa and methanogenic archaea .
In the ruminant diet, proteins and amino acids supply nitrogen for microbial protein production. Proteins synthesized by microorganisms of the rumen have a higher nutritional value than dietary protein. According to Alemawor et al. , the low level of protein in the skin of cocoa limits its use as animal feed. In this context, increasing the CP in Jatropha seed cake by colonization with
|Dry mass (DM)||95.027aB||96.243A||95.028aB||96.076A|
|Organic matter (OM)||93.304aA||91.136B||92.738bA||91.972B|
|Crude protein (CP)||11.438aB||13.158A||11.075bA||11.264A|
|Ether extract (EE)||17.929aA||7.563B||16.214bA||7.097B|
|Non-fiber carbohydrates (NFC)||63.937bB||70.915A||65.259aB||73.800A|
|Neutral detergent fibre (NDF)||49.217aB||53.920A||49.445aB||54.129A|
|Acid detergent fibre (ADF)||37.549aA||35.243B||34.442bB||37.363A|
Ether extract content in substrates also decreased after incubation with
After inoculation with
We observed an increase in carbohydrates after inoculation with
Therefore, colonization of Jatropha seed cake by
3.3.1. Ammonia production by microorganismsin the ruminal liquid
The ruminant’s microorganisms are large and genetically diverse, consisting of bacteria, fungi, protozoa and viruses . These microorganisms contribute to the fermentation of substrates that have low solubility (e.g., plant material rich in fiber) in organic acids, methane, ammonia, acetate, lactate, formate, ethanol, propionate, CO2 and H2 .
The ammonia production observed in this study can be considered low (Figure 6). The production of this compound by rumen ammonia-producing bacteria may vary from 33 to 159% of the dry mass depending on the bacterial species and protein content of the diet . Rumen microorganisms are capable of incorporating a large portion of the produced ammonia by deamination of amino acids and hydrolysis of nitrogen compounds. However, when the rate of deamination exceeds the rate of assimilation, protein catabolism results in the undesirable and inefficient process of high ammonia production and low retention of nitrogen . This undesirable process can be characterized by the loss of protein through the excretion of nitrogen as urea in the animal’s urine . According to previous studies, the rate of dietary protein degradation is directly proportional to the ammonia production and protein nitrogen loss. Therefore, low ammonia production by microorganisms in the rumen fluid demonstrates that the substrates colonized with
The highest ammonia production was observed in substrates colonized by
Finally, it is important to note that
3.4. Animal assay
3.4.1. Food intake, digestibility and nitrogen balance
The intake of dry matter and nutrients was influenced by the different amounts of detoxified Jatropha seed cake in the diet (Table 4). The DM intake (% BW) and NDF showed a quadratic response (P < 0.05), and there was a positive linear effect (P < 0.05) on DM intake (g/kg BW0.75). The DM, OM and CP increased linearly (P < 0.05) and no changes were observed in either the EE and NFC consumption by animals (Table 4).
The increase in DM intake may be attributed to a reduction of the energy values in the experimental diets (Table 2). In this sense, the animals ate more DM to reach their energy requirements. Thus, we can infer that the consumption and palatability of the diets was not restricted by the inclusion of detoxified Jatropha seed cake, although it increased DM intake by the animals. In prior experimental animals, the replacement of soybean meal by Jatropha seed cake resulted in a decrease in DM ingestion, which was attributed to the presence of anti-nutritional factors . In this study, the maximum intake of DM and NDF was 3.68 and 1.67% of BW, respectively. This was not enough to promote the rumen fill effect. In diets with a low energy level, animals tend to exceed the consumption limit of 1.2% of BW, offsetting any food energy deficiency .
The increase in the DM intake resulted in increases in intakes of OM and CP. However, this increase had no effect on the overall consumption of EE and NFC. These results support the theory of compensation in DM intake by animals on diets with a low concentration of energy.
|Dry mass (DM)||701.34||719.90||826.27||891.35||Y = 681.609+10.058x||0.94||13.67|
|DM (%BW)||2.91||2.84||3.19||3.68||Y = 2.901-0.030x+0.003x2||0.99||10.33|
|DM (g/kgBW0.75)||64.41||63.61||71.89||81.65||Y = 61.327+0.883x||0.83||10.42|
|Neutral detergent fiber(%BW)||1.17||1.19||1.38||1.67||Y = 1.170-0.009x+0.001x2||0.99||9.83|
|Organic matter||673.35||696.55||795.80||854.01||Y = 657.092+9.545x||0.95||13.62|
|Crude protein||91.40||87.55||97.29||106.09||Y = 87.469+0.791x||0.72||14.10|
|Dry mass||74.16||68.10||65.40||62.46||Y = 73.328-0.565x||0.97||7.24|
|Organic matter||74.88||69.02||65.90||63.06||Y = 74.136-0.577x||0.97||6.83|
|Crude protein||67.92||54.10||52.14||46.70||Y = 65.305-0.984x||0.89||10.53|
|Neutral detergent fiber||69.32||64.19||60.55||58.05||Y = 68.774-0.560x||0.98||7.44|
|Non-fiber carbohydrates||82.46||78.50||75.75||73.75||Y = 82.050-0.432x||0.98||7.99|
The inclusion of increasing levels of detoxified Jatropha seed cake promoted a linear reduction (P < 0.05) in the digestibility of DM, OM, CP, NDF and NFC diets tested (Table 4). The exception was EE digestibility, which did not show significant variation and had average values of 78.39%. This reduction in dry matter digestibility can be attributed to an increase in passage rate as a function of consumption, resulting in the shorter digestion time of nutrients in the gastrointestinal tract . This effect is associated with the highest possible lignin concentration of the experimental diets.
In relation to nitrogen metabolism, significant effects on Nuendo, nitrogen balance and the biological value of protein from the level of detoxified Jatropha seed cake added were not observed (Table 5). The intake of nitrogen, excretion of Nfecal, Nmet.fecal, Nundig, Nuexo and urinary nitrogen were influenced in a linear manner at the levels studied (Table 5). Losses of nitrogen in the urine and feces were 28.63 and 40.20% of the consumed nitrogen, respectively.
|Consumed nitrogen||14.62||14.01||15.57||16.98||Y = 13.993+0.126x||0.72||14.10|
|Nfecal||4.67||5.22||6.79||7.95||Y = 4.416+0.169x||0.96||20.44|
|Nmet. fecal||0.39||0.37||0.42||0.45||Y = 0.372+0.003x||0.72||14.14|
|Nund||4.28||4.84||6.37||7.50||Y = 4.042+1.166x||0.97||21.64|
|Urinary nitrogen||4.56||3.49||4.29||5.19||Y= 4.495-0.194x+0.011x2||0.70||25.38|
|Nuexo||2.77||1.63||2.39||3.39||Y = 2.703-0.213x+0.012x2||0.75||43.50|
Generally, urea concentration is correlated with ammonia content in ruminants because digestive microorganisms using nitrogen require energy for the synthesis of bacterial proteins. Most likely, there was excess of ruminal ammonia, which increased the excretion of nitrogen in the urine; thus, levels of 12% CP in the diet of growing goats can promote higher levels in waste nitrogen. Valadares et al.  also found an increase in nitrogen excretion in urine when they provided a similar amount of protein to zebu cattle.
Nitrogen balance (NB) and biological value did not differ between the evaluated diets. However, the positive observed values of NB suggest its use in the synthesis of tissue.
3.4.2. Blood parameters
The experimental diets did not significantly alter the blood parameters of the animals (Table 6). The resulting values were similar to those of normal goats . The hemoglobin concentration was similar to that observed in goats fed with Jatropha seed cake .
From the leukocyte values observed in this study, it could be inferred that animals did not experience inflammation after ingesting bio-detoxified Jatropha seed cake (Table 6).
The absence of significant effects in the content of creatinine, alkaline phosphatase and total protein by the different levels of bio-detoxified Jatropha seed cake (Table6) shows that liver function was not altered in animals fed the experimental diets.
|Erythrocytes (x 106 mm3)||4.93||5.16||4.81||5.01||ns||11.88||8 a 18|
|Hemoglobin (g dL-1)||11.61||11.55||11.60||11.82||ns||5.93||8 a 12|
|Hematocrit (%)||31.75||31.83||31.68||32.33||ns||6.18||22 a 38|
|Leukocytes (n µL-1)||12179||12405||10533||11583||ns||14||4000 a 13000|
|Creatinine (mg dL-1)||0.76||0.82||0.76||0.75||ns||7.48||1 a 1.82|
|173.95||154.90||186.62||163.84||ns||18.95||93 a 387|
|Urea (mg dL-1)||20.76||21.53||18.09||20.53||ns||14.47||21.4 a 42.8|
|Total proteins (g dL-1)||6.90||6.66||6.73||6.57||ns||4.71||6.4 a 7|
Urea levels in the blood can increase in response to diets with low energy . However, we did not observe this effect.
Thus, inclusion of Jatropha seed cake bio-detoxified by
The residue of
Thebio-detoxification of Jatropha seed cake promotes the reduction of phorbol ester levels and increases the nutritional value of this residue. The resulting alternative food can be included in amounts up to20% (DM)in the diet of growing goats.
The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support; Biovale Energy for support; and Fuserman Biocombustíveis for kindly donating the Jatropha seed cake. Additionally, the authors thank all friends and collaborators of the Laboratory of Mycorrhizal Association and the Department of Microbiology of the Federal University of Viçosa.