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Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98
Journal of Bioscience and Bioengineering VOL. 117 No. 2, 208e214, 2014 www.elsevier.com/locate/jbiosc
Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98 Mohankumar Bavimane Veerabhadrappa, Sharath Belame Shivakumar, and Somashekar Devappa* Fermentation Technology and Bioengineering Department, CSIR-Central Food Technological Research Institute, Mysore 570020, Karnataka, India Received 10 May 2013; accepted 2 July 2013 Available online 16 August 2013
This study focused on the solid-state fermentation of Jatropha seed cake (JSC), a byproduct generated after biodiesel production. Presence of anti-nutritional compounds and toxins restricts its application in livestock feed. The disposal of the JSC is a major environmental problem in the future, due to the generation of huge quantity of JSC after biodiesel extraction. Hence the JSC was assessed for its suitability as substrate for production and optimization of lipase and protease from Aspergillus versicolor CJS-98 by solid-state fermentation (SSF). The present study was also focused on the biodetoxification of anti-nutrients and toxins in JSC. The SSF parameters were optimized for maximum production of lipase and protease. Under the optimized conditions, the JSC supplemented with maltose and peptone (2%), adjusted to pH 7.0, moisture content 40%, inoculated with 1 3 107 spores per 5 g cake and incubated at 25 C, produced maximum lipase, 1288 U/g and protease, 3366 U/g at 96 h. The anti-nutrients like phytic acid (6.08%), tannins (0.37%), trypsin inhibitors (697.5 TIU/g), cyanogenic glucosides (692.5 mg/100 g), and lectins (0.309 mg/ml), were reduced to 1.70%, 0.23%, 12.5 TIU/g, 560.6 mg/100 g and 0.034 mg/ml respectively. The main toxic compound phorbol esters content in the JSC was reduced from 0.083% to 0.015% after SSF. Our results indicate that viability of SSF to utilize the huge amount of seed cake generated after extraction of biodiesel, for production of industrial enzymes and biodetoxification of anti-nutrients, toxins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Jatropha seed cake; Solid-state fermentation; Protease and lipase; Anti-nutrients; Aspergillus versicolor CJS-98]
Jatropha curcas is commonly called as ‘physic nut’, grows in wild and also cultivated in many parts of the world (Central and South America, Africa and Asia), because of the potential application of Jatropha seeds for the production of biodiesel. Jatropha plant can grow readily in arid regions with an annual yield of up to 5 tonnes of seeds per hectare (1). The processing of 5 tonnes seeds would yield approximately 1.4 tonnes of oil and over 1 tonne of protein rich seed meal (2). The seed kernels contain 30e40% oil (3) and seed cake is a byproduct generated from the oil extraction of seed in a biodiesel processing plant. The Jatropha seed cake (JSC) is considerably high in protein content (50e60%) and it also contains a significant amount of anti-nutrients like phorbol esters, phytate, cyanogenic glucosides, phenols, tannins, lectins, trypsin inhibitors and saponins (4). The phorbol esters are the main toxic compounds present in the seed are well known for their tumour promoting properties (5). Toxicity of Jatropha seeds has been studied extensively in different animal models like goats, sheep, mice, rats and fish when fed with phorbol ester containing feeds (6e9). Therefore unlike other edible oil seed cakes, the JSC has a limitation in using it as an animal feed. * Corresponding author. Tel.: þ91 821 2515792; fax: +91 821 2517233. E-mail addresses: [email protected], [email protected] (S. Devappa).
Solid-state fermentation (SSF) is a process by which microorganisms are grown on a solid substrate in the absence of free water; however, substrate must possess enough moisture to support growth and metabolism of microorganisms. SSF is environmental friendly and offers numerous opportunities in processing of agroindustrial residues. SSF has been applied in the production of enzymes and other value added products such as organic compounds (10). SSF has been employed for enzyme production utilizing agroindustrial wastes, for example, nigerseed oil cake (11), wheat bran (12), rice bran (13), JSC (14). The SSF was also used for detoxification of anti-nutrient components of the JSC (15,16). SSF has been used in the detoxification of caffeine and tannin in coffee husk by fungi (17) and phytic acid reduction from rapeseed meal by Aspergillus niger (18). Unlike other edible oil seed cakes employed for SSF, the reports on utilization of JSC and biodetoxification of JSC are very scarce. The international Jatropha Organization has claimed that in 2017, there will be around 330,000 km2 of land cultivated worldwide producing 160 Mt of seeds and a major portion of its production will be concentrated in Asia. The total projected annual Jatropha oil production in Asian countries will be 47 Mt, with India and China together playing a major role (19). This indicates that there is a huge quantity of seed cake generation after biodiesel production in the future and disposal of seed cake is a major
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.07.003
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environmental problem. Hence utilization of JSC is a major challenge for biotechnologists while converting the same to value added products. The seed cake can be used as an animal feed, if the toxins are removed. The objective of this study was to investigate the potential of JSC for production and optimization of the conditions for lipase and protease enzymes by SSF using fungus, Aspergillus versicolor CJS-98. The aim of the present research work was also to study the biological detoxification of anti-nutritional factors and toxins present in the seed cake by SSF. MATERIALS AND METHODS Materials The media components were obtained from Hi Media Laboratories (Mumbai, India). p-Nitrophenyl palmitate (pNPP) substrate for lipase, phorbol-12myristate 13-acetate (PMA), Na-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPA) were obtained from Sigma Chemical Co., USA. Casein (Hammersten) was obtained from Sisco Research Laboratories (Mumbai, India). All other chemicals used were of analytical grade. Culture and inoculum The fungus A. versicolor CJS-98 strain, isolated from soil sample collected from Mysore was used in the present investigation. It was maintained on Czapek-Dox agar media (pH 5.0) at 4 C and subcultured every 15 days. To the 7 day old slant culture, 2 ml sterile saline containing, 0.85% NaCl þ 0.1% Tween-20 was added and the spores on the surface were scraped gently. The spore suspension was used as inoculums for SSF. The spores were counted in haemocytometer (Neubauer, Germany). The spore counts were adjusted to required range by using sterile saline solutions. Solid-state fermentation The Jatropha curcus seeds were procured from local market in Mysore, India. The JSC was prepared by deoiling the seeds in a hydraulic press. Then the seed cake was air dried and finely powdered before use. Deoiled seed cake thus obtained was used for SSF and further analysis. JSC (5 g) was taken in 150 ml Erlenmeyer flask and autoclaved at 121 C for 20 min after moistening with 1 ml of distilled water. The spore suspension (0.5 ml) of A. versicolor CJS98 was inoculated into these Erlenmeyer flasks. The physical and chemical parameters responsible for the organism’s growth and optimum production of lipase and protease enzymes were studied. To optimize the inoculum size, spore suspension containing 103, 104, 105, 106, 107, and 108 spores/ml were prepared in sterile saline and inoculated to the above flasks. Similarly incubation time (24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h), moisture content (20%, 30%, 40%, 50%, 60%, and 70%), temperature (20 C, 25 C, 30 C, and 35 C) and pH (5.0, 6.0, 7.0, and 8.0) were the parameters used for optimization studies. The pH of the SSF was adjusted using buffers of 0.1 M citrate buffer (pH 5.0), phosphate (pH 6.0 and 7.0) and TriseHCl (pH 8.0). The supplementation of carbon source (glucose, maltose, starch) and nitrogen source (peptone, ammonium sulphate, ammonium chloride) was done at 2%(w/w). All experiments were done in triplicate and the variation was within 5%. Enzyme extraction The enzyme extraction was carried out by using 25 ml chilled distilled water to the fermented substrate and was kept for shaking in an orbital shaker at 200 rpm for 30 min. The suspension was filtered using coarse filter paper. The filtered crude extract was collected and the residue was re-subjected to extraction as above. The extracts from the above were pooled and centrifuged at 10,000 rpm for 10 min at 4 C (Remi Centrifuge, India) and the supernatant was used for the enzyme assay. Lipase assay To determine the lipase activity, 1.8 ml of 0.1 M TriseHCl buffer (pH 8.0) containing 0.15 M NaCl and 0.5% Triton X-100 was taken and pre-incubated at 37 C with 200 ml of suitable diluted crude enzyme extract. Twenty ml of 50 mM p-nitrophenyl palmitate (pNPP in acetonitrile) was added as a substrate to the reaction mixture and it was incubated at 37 C for 30 min. The amount of liberated p-nitrophenol (p-NP) was measured at 400 nm (14). One unit of activity is defined as the amount of enzyme releasing 1 nmol of pNP/ ml/min under standard assay conditions. Lipase activity was expressed in units/ gram (U/g) of dry substrate used for SSF. Protease assay Protease activity was determined using 3 ml of 0.6% casein substrate prepared in 50 mM phosphate buffer, pH 7.50 was taken and 0.5 ml of suitably diluted enzyme was added. The reaction mixture was incubated at 37 C for 20 min. The reaction was stopped by adding 3 ml of 110 mM trichloroacetic acid and allowed to stand for 30 min to precipitate unhydrolyzed casein (14). The filtrate was collected using Whatman no. 1 paper and tyrosine liberated during casein hydrolysis was estimated by using Lowry’s method (20). A unit of protease activity was defined as the amount of enzyme liberating 1 mg of tyrosine/ml/min under assay conditions. Protease activity was expressed in units/ gram (U/g) of dry substrate used for SSF. Estimation of anti-nutritional components The PE content in unfermented JSC and fermented sample were estimated by extracting with methanol (HPLC grade, Merck, India). 20 ml of methanol was added to a flask containing 5 g sample and kept for shaking in an orbital shaker at 250 rpm for 5 min. The extract was filtered over Whatman filter paper no. 1 and the filtrate was collected. The extraction process was repeated with the residue four times. The pooled methanol
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extract was concentrated by using vacuum flash rotary-evaporator at 65 C (HeildolfRota evaporator, Germany). To determine PE, 20 ml of the sample was injected into HPLC (Shimadzu-LC10AVP) with a reverse phase C-18 column (5 mm, 4.6 250 mm i.d., SGE, Australia). The separation was performed as described by Joshi et al. (15) with slight modifications. The solvents used were acetonitrile (solvent A) and water (solvent B). The gradient used was: Start from 40% (solvent A) and 60% (solvent B); for 15 min, increase solvent A to 75% and decrease solvent B to 25%; for next 20 min, increase solvent A to 100%; for 20 min and then the column was adjusted to the starting conditions-40% (solvent A) and 60% (solvent B); for next 3 min. Separation was performed at 25 C and the flow rate was maintained at 1.3 ml/min. The PE peaks were identified between 35 and 39 min. The peaks were integrated at 280 nm and the PE content expressed as equivalents to standard PMA, which appear at 41.9 min. Phytate content in unfermented and fermented JSC (1 g) was extracted with 50 ml trichloroacetic acid and precipitated as ferric salt. The iron content was determined spectrophotometrically at 480 nm and the phytate phosphorus content calculated from this value by assuming a constant 4Fe:6P molecular ratio in the precipitate (21). Tannins were estimated calorimetrically by vanillineHCl method (22). Tannin content was expressed as catechin equivalents. Lectin activity was determined by haemagglutination (HA) assay by trypsinised human blood erythrocytes. The highest dilution of the meal extract causing visible HA was identified as the titre value. Lectin activity is defined as the minimum amount of meal which is sufficient to cause visible HA per ml of trypsinised blood (23). TIU activity was determined according to the procedure of Kakade et al. (24) using BAPA as substrate and crystalline bovine trypsin. Results were expressed as trypsin inhibitor units (TIU) per gram of sample. The cyanogenic glucosides were extracted from fermented and unfermented seed cake samples as cyanohydrin after treatment of 5 g of samples with 50 ml of orthophosphoric acid. Cyanohydrins rapidly decomposed to cyanide ion in alkali. Excess pH 6.0, 0.2 M phosphate buffer was added, followed by chloramine-T and pyridine/barbituric acid to produce a purple coloured solution which was measured spectrophotometrically at 583 nm. Potassium cyanide was used as standard (25).
RESULTS AND DISCUSSION Due to the depletion of non-renewable energy sources, there is a great demand for exploring renewable energy sources like biodiesel from Jatropha seeds. A large amount of JSC is generated after biodiesel production, which is rich in protein and could not be used as animal feed due to the presence of anti-nutritional factors. The objective of the present work is to make use of the residual seed cake for the production of industrially important enzymes like lipase and protease by SSF. The biotechnological potential of agroindustrial wastes for the production of enzymes has been highlighted by Pandey (10). Unlike other agro-industrial wastes used for the production of various enzymes by SSF, not much work has been done with regard to utilization of JSC for SSF. The fungi A. versicolor CJS-98 was able to utilize JSC as substrate for its growth and was found to secrete both extracellular lipase and protease. The optimization of various parameters for the production of the above enzymes was studied. Effect of process parameters on lipase and protease production The inoculum level had a considerable influence on production of enzyme from A. versicolor CJS-98. The inoculum size has a major influence on enzyme production because lower inoculum level may give insufficient biomass causing reduced product formation and higher inoculum level may produce more biomass leading to poor product formation. The lipase (457.41 U/g) and protease (1553.23 U/g) activity was obtained maximum from inoculum containing 1 107 spores/5 g JSC. The enzyme activity at the various inoculum levels is shown in Fig. 1a. The maximum lipase production reported from Rhizopus homothallicus was with inoculum level 3 107 spores/g (26) and maximum protease production reported from Rhizopus oryzae was at spore density 2 105/g (27). The time course of protease and lipase production from A. versicolor CJS-98 using JSC as substrate is given in Fig. 1b. The fungus was grown for different time periods and the enzyme activity was checked at specific intervals of time. The production of
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a
b
FIG. 1. (a) Effect of inoculum size on lipase (open squares) and protease (closed rhombus) production by A. versicolor CJS-98 grown under SSF condition. (b) Time course of lipase (open squares) and protease (closed rhombus) production in JSC SSF using A. versicolor CJS-98. Incubation was done at 30 C with 40% moisture content. The enzyme activity was determined in aseptically withdrawn samples after 5 days. Error bars show the percent error.
FIG. 2. Effect of moisture levels on (a) lipase and (b) protease production in SSF. Moisture level represents 20% (closed rhombus); 30% (closed squares); 40% (closed triangles); 50% (open squares); 60% (asterisk); 70% (open triangles). Error bars show the percent error.
lipase was maximum (971.53 U/g) on 5th day of incubation. There was no increase of lipase activity beyond 5th day of incubation. Mahadik et al. (12) and Mahanta et al. (14) have reported almost similar trend of lipase production by A. niger and Pseudomonas aeruginosa and the maximum production was on the 5th day of incubation. Protease activity (1692.15 U/g) was highest at 24 h period and the activity remained stable after 24 h incubation. Protease production was reported maximum (1818 U/g) on 3rd day by P. aeruginosa (14). Moisture content is an important parameter for SSF which has an influence on the physical properties of the solid substrate. Moisture levels higher than optimum causes decreased porosity, lower oxygen transfer and alteration in substrate’s particle structure. Likewise lower than optimum moisture decreases the solubility of the solid substrate, lowers the degree of swelling and produces a higher water tension (12). To study the effect of moisture on the production of lipase and protease enzymes, the seed cake was adjusted to various moisture levels by using distilled water. The study was carried out for a period of 6 days and the activity was measured at every 24 h. Maximum lipase activity was obtained at 40% (1079.47 U/g) moisture level on the 5th day. The production of protease was highest at 50%, moisture level (1828 U/ g). The results are summarized in Fig. 2a and b. Earlier reports indicated that maximum lipase production from A. niger (12) was at about 71% moisture level and that from P. aeruginosa (14) was at 50% moisture level. Similarly maximum protease activity was reported from P. aeruginosa was at 55% moisture level (14). The incubation temperature is yet another vital parameter that determines the growth and enzyme production. The SSF was
carried out at a temperature range between 20 C and 35 C. It was observed that A. versicolor CJS-98 produced maximum lipase (1229.55 U/g) and protease (2187.25 U/g) activity at 25 C. The variation in lipase and protease production as a function of temperature is shown in Fig. 3a and b. The optimum temperature reported for lipase production from Aspergillus oryzae was at 28 C (28) and that for protease from A. niger was at 28 C (29). pH is an important parameter for any fermentation process, as it may change with the metabolic activities of the organism thus affecting the level of enzyme production. Maximum lipase and protease production was observed at pH e 7.0 (1137.40 U/g) and 6.0 (2715.79 U/g) respectively (Fig. 4a and b). The trend was similar to the reported results of optimum pH for enzyme production by P. aeruginosa. The optimum pH for production of lipase and protease for P. aeruginosa was found to be 7.0 and 6.0 respectively (14). The difference in optimized parameters between lipase and protease were observed with regard to incubation period, pH and moisture content. Since the substrate JSC is rich in protein, maximum protease production was in the first 24 h of growth of the organism. The lipid content is lower in seed cake when compared to protein, hence higher lipase production was observed at a later stage of growth. The difference in pH optimized is due to the production of acidic protease and neutral lipase by the organism. The moisture content in SSF may influence the physical properties of the substrate, which in turn affect enzyme production. Higher moisture content in the cake may help in diffusion of proteins resulting in higher protease production. Whereas lower moisture level in the substrate may stimulate increased production of lipase, as lipids are hydrophobic.
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a
b
FIG. 3. Effect of incubation temperature on (a) lipase and (b) protease production under SSF condition by A. versicolor CJS-98. Temperature range: 20 C (closed rhombus); 25 C (closed squares); 30 C (closed triangles); 35 C (open squares). Error bars show the percent error.
Effect of carbon source supplementation The carbon and nitrogen are essential for robust growth of microorganisms. Different carbon sources like glucose, maltose and starch were used to study their influence on the growth of the organism and also on production of enzymes. All three carbon sources showed a pronounced increase in the enzyme activity when they are supplemented with JSC. Among these, maltose gave maximum lipase (1084.09 U/g) and protease (3260.52 U/g) production. The effect of various carbon sources studied is shown in Fig. 5. This result indicates that supplementing JSC with maltose has a synergistic effect on production of lipase and protease in A. versicolor CJS-98. Earlier reports have shown that increase in lipase and protease production with JSC supplemented with maltose by P. aeruginosa (14). The results are in agreement with Rao et al. (13) who have observed maltose supplementation yielded maximum lipase production by Candida rugosa in SSF. Effect of nitrogen source supplementation Three nitrogen sources (peptone, ammonium sulphate and ammonium chloride) were supplemented to check for increase in lipase and protease production. Out of these, organic nitrogen supplement peptone showed maximum lipase (1234 U/g) and protease (3061 U/g) production. The variation of protease and lipase production with respect to nitrogen sources is shown in Fig. 5. The results are in agreement with the finding of other researchers. Although the seed cake contained sufficient amount of crude protein as per composition, the supplemented nitrogen may be better accessed and utilized leading to better enzyme production (14). Cumulative effect of carbon and nitrogen supplementation The optimized levels of carbon and nitrogen supplementation were used to study the cumulative effect of these
FIG. 4. Influence of initial medium pH on (a) lipase and (b) protease production under SSF condition. Closed rhombus, pH 5.0; closed squares, pH 6.0; open triangles, pH 7.0; open circles, pH 8.0. Error bars show the percent error.
sources on production of lipase and protease. The maximum lipase (1288 U/g) and protease (3366 U/g) production was in supplemented SSF, when compared to control (without supplementation of carbon and nitrogen) where lipase and protease production were found to be 1019 U/g and 2029 U/g respectively. The results also indicate that the organism can grow and produce a significant amount of enzymes with JSC alone as substrate. However, supplementation of carbon and nitrogen source enhances the productivity of lipase and protease by 20% and 39% respectively. The results of enzyme production at different days are shown in Fig. 5a and b. The production of lipase and protease was maximum on the 4th day of incubation after optimization of all the parameters. Detoxification of anti-nutrients by SSF The J. curcus seeds are highly toxic to a number of animal species due to the presence of anti-nutritional components such as phytic acid, trypsin inhibitors, lectins, cyanogenic glucosides, tannins and phorbol esters at high levels (4). PE are considered to be the most toxic compounds and known to be strong tumour promoting agents (5). The PE content was found to vary from 0.87 to 3.32 mg/g in different varieties of Jatropha (30). In the present investigation, biological detoxification of anti-nutrients and toxins were studied after SSF by using A. versicolor CJS-98. The initial PE content in the seed cake was 0.832 mg/g, which after fermentation was reduced to 0.158 mg/g. The HPLC profile of PE before and after JSC SSF is given in Fig. 6. Barros et al. (16) have reported that PE concentration in JSC decreased around 20%, 91%, 97% after SSF with white-rot fungi, Ganoderma resinaceum, Bjerkandera adusta and Phlebia rufa respectively. The results are in agreement with the findings of Belewu and Sam (31) who affirmed that there was a reduction in the anti-nutritional factors including PE of JSC after solid-state fermentation.
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FIG. 5. Effect of carbon and nitrogen sources supplementation on (a) lipase and (b) protease production by A. versicolor CJS-98 under SSF condition. Error bars show the percent error.
The phytate content in JSC was 6.08 g per 100 g. After fermentation the phytate level was decreased to 1.70/100 g, indicating that there was a reduction of phytate level by 72% after solid-state fermentation. The phytate have been implicated in decreasing protein digestibility by forming complexes and also by interacting with enzymes such as trypsin and pepsin. The phytic acid content was decreased from 10% to 6.85% after enzyme and ethanol treatments of JSC (32). The tannin content of JSC was initially 0.373/100 g of seed cake and after solid-state fermentation of JSC reduced to 0.232/100 g of seed cake. The results are in agreement with the report of Oseni and Akindahunsi (33), where the unfermented JSC had 0.92% and after fermentation the tannins were reduced to 0.076%. The lectin is generally considered as another toxic factor in JSC and the lectin activity in JSC was 0.309 mg/ml. The lectin activity
was found to decrease by 88.9% after solid-state fermentation (0.034 mg/ml) of JSC. Trypsin inhibitors are anti-nutritional factors which interfere with the physiological process of digestion through interference with the normal functioning of pancreatic proteolytic enzymes in non-ruminants (34) leading to severe growth depression. Trypsin inhibitor activity in JSC was 697.5 TIU/g sample and after fermentation the TIU was 12.5, with a reduction in 98%. This reduction was probably due to the utilization of TI by the organism for growth. The cyanogenic glucosides were present at concentrations of 692.5 mg per 100 g in unfermented JSC. This was decreased to 560 mg per 100 g after SSF. The anti-nutritional factors reported to be present in JSC (phytate-4.1, tannin-0.76, trypsin inhibitor 11.5%) was reduced to almost fifty percent of original content, when it was fermented using R. oryzae (33). The various anti-nutritional factors in JSC and the reduction of the same after SSF are shown in
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FIG. 6. HPLC chromatogram and absorption spectrum of phorbol esters from (a) unfermented and (b) fermented seed cake. (c) Standard phorbol-12-myristate 13-acetate (PMA). SSF carried out under moisture level 40%, pH 7.0, temperature 25 C condition. The PE was determined after 4th day of fermentation.
Table 1. As per the results, there is a considerable reduction in the anti-nutritional factors after solid-state fermentation by A. versicolor CJS-98. The anti-nutritional compounds and toxins in the seed cake decreased significantly after solid-state fermentation when compared to the unfermented control sample. The decrease in the levels of anti-nutritional compounds and toxins could be due
to production of various enzymes during the growth phases of fungal organism. Thus the biological detoxification method can be used to remove toxin and other anti-nutritional factors in the JSC. The production of biodiesel from non-edible feed stocks is receiving considerable attention recently, as a renewable source of energy, which results in the generation of huge quantity of JSC. The
TABLE 1. Composition of anti-nutrients of unfermented and fermented JSC using A. versicolor CJS-98. Samples
Unfermented JSC Fermented JSC Reduction (%)
Phorbol esters (g/100 g seed cake) 0.0832 0.002 0.0158 0.005 81.1
Phytic acid (g/100 g seed cake) 6.08 0.03 1.70 0.01 72.03
Estimation was done in triplicate samples. Values are mean standard error.
Tannin (g/100 g seed cake)
Cyanogenic glucosides (mg/100 g)
0.373 0.02 0.232 0.02 37.75
692.55 0.36 560.63 0.30 19.04
Trypsin inhibitor activity (TIU/g) 697.5 0.02 12.5 0.03 98.20
Lectin (1/mg meal/ ml assay) 0.309 0.28 0.034 2.20 88.90
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study on the SSF of JSC has indicated that it is an economical source for the production of industrially important enzymes by using fungi. The fungus, A. versicolor CJS-98 was able to utilize the nutrients of JSC by secreting extracellular enzymes. The various antinutrients present in the seed cake were also reduced considerably after SSF, indicating the potential for application of JSC as animal feed. Hence the biological detoxification method can be used to remove toxin and other anti-nutritional factors in the JSC. ACKNOWLEDGMENTS Authors gratefully acknowledge the financial grant provided by Ministry of Environment and Forests, New Delhi, India for carrying out this research work. Authors thank Dr. M.S. Thakur, Dr. S.G. Prapulla, Dr. H.K. Manonmani, Scientists, FTBE Dept, Ms. M. Asha, CIFS Dept and Prof. Ram Rajasekharan, Director, CSIR-CFTRI, Mysore for their encouragement and support. References 1. Heller, J.: Physic nut Jatropha curcas L. promoting the conservation and use of underutilized and neglected crops. Institute of Plant Genetics and Crop Plant Research, Gatersleben. International Plant Genetics Research Institute, Rome (1996). 2. Makkar, H. P. S. and Becker, K.: Plant toxins and detoxification methods to improve feed quality of tropical seeds e review, Asian-Aust. J. Anim. Sci., 12, 467e480 (1999). 3. Makkar, H. P. S., Aderibigbe, A. O., and Becker, K.: Comparative evaluation of nontoxic and toxic varieties of Jatropha curcas for chemical composition, digestibility, protein degradability and toxic factors, Food Chem., 62, 207e215 (1998). 4. Makkar, H. P. S., Francis, G., and Becker, K.: Protein concentrate from Jatropha curcas screw pressed seed cake and toxic and anti-nutritional factors in protein concentrate, J. Sci. Food Agri., 88, 1542e1548 (2008). 5. Vogg, G., Achatz, S., Kettrup, A., and Sandermann, H., Jr.: Fast, sensitive and selective liquid chromatographic tandem mass spectrometric determination of tumor promoting diterpene esters, J. Chromatogr., 853, 563e573 (1999). 6. Adam, S. E.: Toxic effects of Jatropha curcas in mice, Toxicology, 2, 67e76 (1974). 7. Adam, S. E. and Magzomb, M.: Toxic effect of Jatropha in goats, Toxicology, 4, 347e354 (1975). 8. Becker, K. and Makkar, H. P. S.: Toxic effects of phorbol esters in carp (Cyprinus carpio L.), Vet. Human. Toxicol., 40, 82e86 (1998). 9. Makkar, H. P. S. and Becker, K.: Nutritional studies on rats and fish (carp Cyprinus, carpio) fed diet containing unheated and heated Jatropha curcus meal of a non-toxic provenance, Plants Foods Hum. Nutri., 53, 183e192 (1999). 10. Pandey, A.: Solid state fermentation, Biochem. Eng. J., 13, 81e84 (2003). 11. Imandi, S. D., Karanam, S. K., and Garapati, H. R.: Optimization of process parameters for the production of lipase in solid state fermentation by Yarrowia lipolytica from niger seed oil Cake (Guizotia abyssinica), J. Microb. Biochem. Technol., 2, 28e33 (2010). 12. Mahadik, N. D., Puntambekar, U. S., Bastawde, K. B., Khire, J. M., and Gokhale, D. V.: Production of acidic lipase by Aspergillus niger in SSF, Process Biochem., 38, 715e721 (2002). 13. Rao, P. V., Jayaraman, K., and Lakshmanan, C. M.: Production of lipase by Candida rugosa in solid state fermentation. 1: determination of significant process variables, Process Biochem., 28, 385e389 (1993). 14. Mahanta, N., Gupta, A., and Khare, S. K.: Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using
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Jatropha curcas seed cake as substrate, Bioresour. Technol., 99, 1729e1735 (2008). Joshi, C., Mathur, P., and Khare, S. K.: Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid state fermentation of deoiled Jatropha seed cake, Bioresour. Technol., 102, 4815e4819 (2011). Barros, C. R. M. D., Ferreira, L. M. M., Nunes, F. M., Bezerra, R. M. F., Dias, A. A., Guedes, C. V., Cone, J. W., Marques, G. S. M., and Rodrigues, M. A. M.: The potential of white rot fungi to degrade phorbol ester of Jatropha curcas L. seed cake, Eng. Life Sci., 11, 107e110 (2011). Brand, D., Pandey, A., Roussos, S., and Soccol, C. R.: Biological detoxification of coffee husk by filamentous fungi using a solid state fermentation system, Enzyme Microb. Technol., 27, 127e133 (2000). El-Batal, A. I. and Karem, A. H.: Phytase production and phytic acid reduction in rapeseed meal by Aspergillus niger during solid state fermentation, Food Res. Int., 34, 715e720 (2001). Siang, C. C.: Jatropha curcas L: development of a new oil crop for biofuel, http:// eneken.ieej.or.jp/en/data/pdf/467.pdf, The Institute of Energy Economics, Japan (2009). Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randell, R. J.,: Protein measurement by Folin phenol reagent, J. Biol. Chem., 193, 265e275 (1951). Makkar, H. P. S., Siddhuraju, P., and Becker, K.: Plant secondary metabolites, pp. 23e28, in: Methods in molecular biology, vol. 393. Humana Press Inc., Totowa, NJ (2007). Price, M. L., Van Scoyoc, S., and Butler, L. G.: A critical evaluation of the vanillin reaction as an assay for tannins in sorghum grain, J. Agric. Food Chem., 26, 1214e1218 (1978). Lis, H. and Sharon, N.: Soyabean (Glycine max) haemoglutinin in complex carbonates, pp. 360e362, in: Colowick, S. P. and Kaplan, N. O. (Eds.), Methods in enzymology, vol. XXVIIIB. Academic Press, New York (1972). Kakade, M. L., Rackis, J. J., McGhee, J. E., and Puski, G.: Determination of trypsin inhibitor activity of soy products; collaborative analysis of an improved procedure, Cereal Chem., 51, 376e382 (1974). Bradbury, J. H., Egan, S. V., and Lynch, M. J.: Analysis of cyanide in cassava using acid hydrolysis of cyanogenic glucosides, J. Sci. Food Agri., 55, 277e290 (1991). Diaz, M. J. C., Rodríguez, J. A., Roussos, S., Cordova, J., and Abousalham, A.: Lipase from the thermotolerant fungus Rhizopus homothallicus is more thermostable when produced using solid state fermentation than liquid fermentation procedures, Enzyme Microb. Technol., 39, 1042e1050 (2006). Tunga, R., Banerjee, R., and Bhattacharyya, B. C.: Optimizing some factors affecting protease production under SSF, Bioprocess. Eng., 19, 187e190 (1998). Ohnishi, K., Yoshida, Y., and Sekiguchi, J.: Lipase production of Aspergillus oryzae, J. Ferment. Bioeng., 77, 490e495 (1994). Kamath, P., Subrahmanyam, V. M., Venkata Rao, J., and Vasantha Raj, P.: Optimization of cultural conditions for protease production by a fungal species, Ind. J. Pharm. Sci., 72, 161e166 (2010). Makkar, H. P. S., Becker, K., Spores, F., and Wink, M.: Studies on nutritive potential and toxic constituents of different provenances of Jatropha curcas, J. Agric. Food Chem., 45, 3152e3157 (1997). Belewu, M. A. and Sam, R.: Solid state fermentation of Jatropha curcas kernel cake, proximate composition and antinutritional components, J. Yeast Fungal. Res., 1, 44e46 (2010). Xiao, J., Zhang, H., Niu, L., Wang, X., and Lu, X.: Evaluation of detoxification methods on toxic and antinutritional composition and nutritional quality of proteins in Jatropha curcas meal, J. Agric. Food Chem., 59, 4040e4044 (2011). Oseni, O. A. and Akindahunsi, A. A.: Some phytochemical properties and effect of fermentation on the seed of Jatropha curcas L, Am. J. Food Technol., 6, 158e165 (2011). White, C. E., Campbell, D. R., and Combs, G. E.: Effect of moisture and processing temperature on activities of trypsin inhibitor and urease in soybeans fed to swine, pp. 230e234, in: Huisman, J., Van der poel, T. F. B., and Liener, L. E. (Eds.), Recent advances in antinutritional factors in legume seeds. Puduc, Wageningen (1989).
Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98 Mohankumar Bavimane Veerabhadrappa, Sharath Belame Shivakumar, and Somashekar Devappa* Fermentation Technology and Bioengineering Department, CSIR-Central Food Technological Research Institute, Mysore 570020, Karnataka, India Received 10 May 2013; accepted 2 July 2013 Available online 16 August 2013
This study focused on the solid-state fermentation of Jatropha seed cake (JSC), a byproduct generated after biodiesel production. Presence of anti-nutritional compounds and toxins restricts its application in livestock feed. The disposal of the JSC is a major environmental problem in the future, due to the generation of huge quantity of JSC after biodiesel extraction. Hence the JSC was assessed for its suitability as substrate for production and optimization of lipase and protease from Aspergillus versicolor CJS-98 by solid-state fermentation (SSF). The present study was also focused on the biodetoxification of anti-nutrients and toxins in JSC. The SSF parameters were optimized for maximum production of lipase and protease. Under the optimized conditions, the JSC supplemented with maltose and peptone (2%), adjusted to pH 7.0, moisture content 40%, inoculated with 1 3 107 spores per 5 g cake and incubated at 25 C, produced maximum lipase, 1288 U/g and protease, 3366 U/g at 96 h. The anti-nutrients like phytic acid (6.08%), tannins (0.37%), trypsin inhibitors (697.5 TIU/g), cyanogenic glucosides (692.5 mg/100 g), and lectins (0.309 mg/ml), were reduced to 1.70%, 0.23%, 12.5 TIU/g, 560.6 mg/100 g and 0.034 mg/ml respectively. The main toxic compound phorbol esters content in the JSC was reduced from 0.083% to 0.015% after SSF. Our results indicate that viability of SSF to utilize the huge amount of seed cake generated after extraction of biodiesel, for production of industrial enzymes and biodetoxification of anti-nutrients, toxins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Jatropha seed cake; Solid-state fermentation; Protease and lipase; Anti-nutrients; Aspergillus versicolor CJS-98]
Jatropha curcas is commonly called as ‘physic nut’, grows in wild and also cultivated in many parts of the world (Central and South America, Africa and Asia), because of the potential application of Jatropha seeds for the production of biodiesel. Jatropha plant can grow readily in arid regions with an annual yield of up to 5 tonnes of seeds per hectare (1). The processing of 5 tonnes seeds would yield approximately 1.4 tonnes of oil and over 1 tonne of protein rich seed meal (2). The seed kernels contain 30e40% oil (3) and seed cake is a byproduct generated from the oil extraction of seed in a biodiesel processing plant. The Jatropha seed cake (JSC) is considerably high in protein content (50e60%) and it also contains a significant amount of anti-nutrients like phorbol esters, phytate, cyanogenic glucosides, phenols, tannins, lectins, trypsin inhibitors and saponins (4). The phorbol esters are the main toxic compounds present in the seed are well known for their tumour promoting properties (5). Toxicity of Jatropha seeds has been studied extensively in different animal models like goats, sheep, mice, rats and fish when fed with phorbol ester containing feeds (6e9). Therefore unlike other edible oil seed cakes, the JSC has a limitation in using it as an animal feed. * Corresponding author. Tel.: þ91 821 2515792; fax: +91 821 2517233. E-mail addresses: [email protected], [email protected] (S. Devappa).
Solid-state fermentation (SSF) is a process by which microorganisms are grown on a solid substrate in the absence of free water; however, substrate must possess enough moisture to support growth and metabolism of microorganisms. SSF is environmental friendly and offers numerous opportunities in processing of agroindustrial residues. SSF has been applied in the production of enzymes and other value added products such as organic compounds (10). SSF has been employed for enzyme production utilizing agroindustrial wastes, for example, nigerseed oil cake (11), wheat bran (12), rice bran (13), JSC (14). The SSF was also used for detoxification of anti-nutrient components of the JSC (15,16). SSF has been used in the detoxification of caffeine and tannin in coffee husk by fungi (17) and phytic acid reduction from rapeseed meal by Aspergillus niger (18). Unlike other edible oil seed cakes employed for SSF, the reports on utilization of JSC and biodetoxification of JSC are very scarce. The international Jatropha Organization has claimed that in 2017, there will be around 330,000 km2 of land cultivated worldwide producing 160 Mt of seeds and a major portion of its production will be concentrated in Asia. The total projected annual Jatropha oil production in Asian countries will be 47 Mt, with India and China together playing a major role (19). This indicates that there is a huge quantity of seed cake generation after biodiesel production in the future and disposal of seed cake is a major
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.07.003
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environmental problem. Hence utilization of JSC is a major challenge for biotechnologists while converting the same to value added products. The seed cake can be used as an animal feed, if the toxins are removed. The objective of this study was to investigate the potential of JSC for production and optimization of the conditions for lipase and protease enzymes by SSF using fungus, Aspergillus versicolor CJS-98. The aim of the present research work was also to study the biological detoxification of anti-nutritional factors and toxins present in the seed cake by SSF. MATERIALS AND METHODS Materials The media components were obtained from Hi Media Laboratories (Mumbai, India). p-Nitrophenyl palmitate (pNPP) substrate for lipase, phorbol-12myristate 13-acetate (PMA), Na-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPA) were obtained from Sigma Chemical Co., USA. Casein (Hammersten) was obtained from Sisco Research Laboratories (Mumbai, India). All other chemicals used were of analytical grade. Culture and inoculum The fungus A. versicolor CJS-98 strain, isolated from soil sample collected from Mysore was used in the present investigation. It was maintained on Czapek-Dox agar media (pH 5.0) at 4 C and subcultured every 15 days. To the 7 day old slant culture, 2 ml sterile saline containing, 0.85% NaCl þ 0.1% Tween-20 was added and the spores on the surface were scraped gently. The spore suspension was used as inoculums for SSF. The spores were counted in haemocytometer (Neubauer, Germany). The spore counts were adjusted to required range by using sterile saline solutions. Solid-state fermentation The Jatropha curcus seeds were procured from local market in Mysore, India. The JSC was prepared by deoiling the seeds in a hydraulic press. Then the seed cake was air dried and finely powdered before use. Deoiled seed cake thus obtained was used for SSF and further analysis. JSC (5 g) was taken in 150 ml Erlenmeyer flask and autoclaved at 121 C for 20 min after moistening with 1 ml of distilled water. The spore suspension (0.5 ml) of A. versicolor CJS98 was inoculated into these Erlenmeyer flasks. The physical and chemical parameters responsible for the organism’s growth and optimum production of lipase and protease enzymes were studied. To optimize the inoculum size, spore suspension containing 103, 104, 105, 106, 107, and 108 spores/ml were prepared in sterile saline and inoculated to the above flasks. Similarly incubation time (24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h), moisture content (20%, 30%, 40%, 50%, 60%, and 70%), temperature (20 C, 25 C, 30 C, and 35 C) and pH (5.0, 6.0, 7.0, and 8.0) were the parameters used for optimization studies. The pH of the SSF was adjusted using buffers of 0.1 M citrate buffer (pH 5.0), phosphate (pH 6.0 and 7.0) and TriseHCl (pH 8.0). The supplementation of carbon source (glucose, maltose, starch) and nitrogen source (peptone, ammonium sulphate, ammonium chloride) was done at 2%(w/w). All experiments were done in triplicate and the variation was within 5%. Enzyme extraction The enzyme extraction was carried out by using 25 ml chilled distilled water to the fermented substrate and was kept for shaking in an orbital shaker at 200 rpm for 30 min. The suspension was filtered using coarse filter paper. The filtered crude extract was collected and the residue was re-subjected to extraction as above. The extracts from the above were pooled and centrifuged at 10,000 rpm for 10 min at 4 C (Remi Centrifuge, India) and the supernatant was used for the enzyme assay. Lipase assay To determine the lipase activity, 1.8 ml of 0.1 M TriseHCl buffer (pH 8.0) containing 0.15 M NaCl and 0.5% Triton X-100 was taken and pre-incubated at 37 C with 200 ml of suitable diluted crude enzyme extract. Twenty ml of 50 mM p-nitrophenyl palmitate (pNPP in acetonitrile) was added as a substrate to the reaction mixture and it was incubated at 37 C for 30 min. The amount of liberated p-nitrophenol (p-NP) was measured at 400 nm (14). One unit of activity is defined as the amount of enzyme releasing 1 nmol of pNP/ ml/min under standard assay conditions. Lipase activity was expressed in units/ gram (U/g) of dry substrate used for SSF. Protease assay Protease activity was determined using 3 ml of 0.6% casein substrate prepared in 50 mM phosphate buffer, pH 7.50 was taken and 0.5 ml of suitably diluted enzyme was added. The reaction mixture was incubated at 37 C for 20 min. The reaction was stopped by adding 3 ml of 110 mM trichloroacetic acid and allowed to stand for 30 min to precipitate unhydrolyzed casein (14). The filtrate was collected using Whatman no. 1 paper and tyrosine liberated during casein hydrolysis was estimated by using Lowry’s method (20). A unit of protease activity was defined as the amount of enzyme liberating 1 mg of tyrosine/ml/min under assay conditions. Protease activity was expressed in units/ gram (U/g) of dry substrate used for SSF. Estimation of anti-nutritional components The PE content in unfermented JSC and fermented sample were estimated by extracting with methanol (HPLC grade, Merck, India). 20 ml of methanol was added to a flask containing 5 g sample and kept for shaking in an orbital shaker at 250 rpm for 5 min. The extract was filtered over Whatman filter paper no. 1 and the filtrate was collected. The extraction process was repeated with the residue four times. The pooled methanol
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extract was concentrated by using vacuum flash rotary-evaporator at 65 C (HeildolfRota evaporator, Germany). To determine PE, 20 ml of the sample was injected into HPLC (Shimadzu-LC10AVP) with a reverse phase C-18 column (5 mm, 4.6 250 mm i.d., SGE, Australia). The separation was performed as described by Joshi et al. (15) with slight modifications. The solvents used were acetonitrile (solvent A) and water (solvent B). The gradient used was: Start from 40% (solvent A) and 60% (solvent B); for 15 min, increase solvent A to 75% and decrease solvent B to 25%; for next 20 min, increase solvent A to 100%; for 20 min and then the column was adjusted to the starting conditions-40% (solvent A) and 60% (solvent B); for next 3 min. Separation was performed at 25 C and the flow rate was maintained at 1.3 ml/min. The PE peaks were identified between 35 and 39 min. The peaks were integrated at 280 nm and the PE content expressed as equivalents to standard PMA, which appear at 41.9 min. Phytate content in unfermented and fermented JSC (1 g) was extracted with 50 ml trichloroacetic acid and precipitated as ferric salt. The iron content was determined spectrophotometrically at 480 nm and the phytate phosphorus content calculated from this value by assuming a constant 4Fe:6P molecular ratio in the precipitate (21). Tannins were estimated calorimetrically by vanillineHCl method (22). Tannin content was expressed as catechin equivalents. Lectin activity was determined by haemagglutination (HA) assay by trypsinised human blood erythrocytes. The highest dilution of the meal extract causing visible HA was identified as the titre value. Lectin activity is defined as the minimum amount of meal which is sufficient to cause visible HA per ml of trypsinised blood (23). TIU activity was determined according to the procedure of Kakade et al. (24) using BAPA as substrate and crystalline bovine trypsin. Results were expressed as trypsin inhibitor units (TIU) per gram of sample. The cyanogenic glucosides were extracted from fermented and unfermented seed cake samples as cyanohydrin after treatment of 5 g of samples with 50 ml of orthophosphoric acid. Cyanohydrins rapidly decomposed to cyanide ion in alkali. Excess pH 6.0, 0.2 M phosphate buffer was added, followed by chloramine-T and pyridine/barbituric acid to produce a purple coloured solution which was measured spectrophotometrically at 583 nm. Potassium cyanide was used as standard (25).
RESULTS AND DISCUSSION Due to the depletion of non-renewable energy sources, there is a great demand for exploring renewable energy sources like biodiesel from Jatropha seeds. A large amount of JSC is generated after biodiesel production, which is rich in protein and could not be used as animal feed due to the presence of anti-nutritional factors. The objective of the present work is to make use of the residual seed cake for the production of industrially important enzymes like lipase and protease by SSF. The biotechnological potential of agroindustrial wastes for the production of enzymes has been highlighted by Pandey (10). Unlike other agro-industrial wastes used for the production of various enzymes by SSF, not much work has been done with regard to utilization of JSC for SSF. The fungi A. versicolor CJS-98 was able to utilize JSC as substrate for its growth and was found to secrete both extracellular lipase and protease. The optimization of various parameters for the production of the above enzymes was studied. Effect of process parameters on lipase and protease production The inoculum level had a considerable influence on production of enzyme from A. versicolor CJS-98. The inoculum size has a major influence on enzyme production because lower inoculum level may give insufficient biomass causing reduced product formation and higher inoculum level may produce more biomass leading to poor product formation. The lipase (457.41 U/g) and protease (1553.23 U/g) activity was obtained maximum from inoculum containing 1 107 spores/5 g JSC. The enzyme activity at the various inoculum levels is shown in Fig. 1a. The maximum lipase production reported from Rhizopus homothallicus was with inoculum level 3 107 spores/g (26) and maximum protease production reported from Rhizopus oryzae was at spore density 2 105/g (27). The time course of protease and lipase production from A. versicolor CJS-98 using JSC as substrate is given in Fig. 1b. The fungus was grown for different time periods and the enzyme activity was checked at specific intervals of time. The production of
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a
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FIG. 1. (a) Effect of inoculum size on lipase (open squares) and protease (closed rhombus) production by A. versicolor CJS-98 grown under SSF condition. (b) Time course of lipase (open squares) and protease (closed rhombus) production in JSC SSF using A. versicolor CJS-98. Incubation was done at 30 C with 40% moisture content. The enzyme activity was determined in aseptically withdrawn samples after 5 days. Error bars show the percent error.
FIG. 2. Effect of moisture levels on (a) lipase and (b) protease production in SSF. Moisture level represents 20% (closed rhombus); 30% (closed squares); 40% (closed triangles); 50% (open squares); 60% (asterisk); 70% (open triangles). Error bars show the percent error.
lipase was maximum (971.53 U/g) on 5th day of incubation. There was no increase of lipase activity beyond 5th day of incubation. Mahadik et al. (12) and Mahanta et al. (14) have reported almost similar trend of lipase production by A. niger and Pseudomonas aeruginosa and the maximum production was on the 5th day of incubation. Protease activity (1692.15 U/g) was highest at 24 h period and the activity remained stable after 24 h incubation. Protease production was reported maximum (1818 U/g) on 3rd day by P. aeruginosa (14). Moisture content is an important parameter for SSF which has an influence on the physical properties of the solid substrate. Moisture levels higher than optimum causes decreased porosity, lower oxygen transfer and alteration in substrate’s particle structure. Likewise lower than optimum moisture decreases the solubility of the solid substrate, lowers the degree of swelling and produces a higher water tension (12). To study the effect of moisture on the production of lipase and protease enzymes, the seed cake was adjusted to various moisture levels by using distilled water. The study was carried out for a period of 6 days and the activity was measured at every 24 h. Maximum lipase activity was obtained at 40% (1079.47 U/g) moisture level on the 5th day. The production of protease was highest at 50%, moisture level (1828 U/ g). The results are summarized in Fig. 2a and b. Earlier reports indicated that maximum lipase production from A. niger (12) was at about 71% moisture level and that from P. aeruginosa (14) was at 50% moisture level. Similarly maximum protease activity was reported from P. aeruginosa was at 55% moisture level (14). The incubation temperature is yet another vital parameter that determines the growth and enzyme production. The SSF was
carried out at a temperature range between 20 C and 35 C. It was observed that A. versicolor CJS-98 produced maximum lipase (1229.55 U/g) and protease (2187.25 U/g) activity at 25 C. The variation in lipase and protease production as a function of temperature is shown in Fig. 3a and b. The optimum temperature reported for lipase production from Aspergillus oryzae was at 28 C (28) and that for protease from A. niger was at 28 C (29). pH is an important parameter for any fermentation process, as it may change with the metabolic activities of the organism thus affecting the level of enzyme production. Maximum lipase and protease production was observed at pH e 7.0 (1137.40 U/g) and 6.0 (2715.79 U/g) respectively (Fig. 4a and b). The trend was similar to the reported results of optimum pH for enzyme production by P. aeruginosa. The optimum pH for production of lipase and protease for P. aeruginosa was found to be 7.0 and 6.0 respectively (14). The difference in optimized parameters between lipase and protease were observed with regard to incubation period, pH and moisture content. Since the substrate JSC is rich in protein, maximum protease production was in the first 24 h of growth of the organism. The lipid content is lower in seed cake when compared to protein, hence higher lipase production was observed at a later stage of growth. The difference in pH optimized is due to the production of acidic protease and neutral lipase by the organism. The moisture content in SSF may influence the physical properties of the substrate, which in turn affect enzyme production. Higher moisture content in the cake may help in diffusion of proteins resulting in higher protease production. Whereas lower moisture level in the substrate may stimulate increased production of lipase, as lipids are hydrophobic.
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a
b
FIG. 3. Effect of incubation temperature on (a) lipase and (b) protease production under SSF condition by A. versicolor CJS-98. Temperature range: 20 C (closed rhombus); 25 C (closed squares); 30 C (closed triangles); 35 C (open squares). Error bars show the percent error.
Effect of carbon source supplementation The carbon and nitrogen are essential for robust growth of microorganisms. Different carbon sources like glucose, maltose and starch were used to study their influence on the growth of the organism and also on production of enzymes. All three carbon sources showed a pronounced increase in the enzyme activity when they are supplemented with JSC. Among these, maltose gave maximum lipase (1084.09 U/g) and protease (3260.52 U/g) production. The effect of various carbon sources studied is shown in Fig. 5. This result indicates that supplementing JSC with maltose has a synergistic effect on production of lipase and protease in A. versicolor CJS-98. Earlier reports have shown that increase in lipase and protease production with JSC supplemented with maltose by P. aeruginosa (14). The results are in agreement with Rao et al. (13) who have observed maltose supplementation yielded maximum lipase production by Candida rugosa in SSF. Effect of nitrogen source supplementation Three nitrogen sources (peptone, ammonium sulphate and ammonium chloride) were supplemented to check for increase in lipase and protease production. Out of these, organic nitrogen supplement peptone showed maximum lipase (1234 U/g) and protease (3061 U/g) production. The variation of protease and lipase production with respect to nitrogen sources is shown in Fig. 5. The results are in agreement with the finding of other researchers. Although the seed cake contained sufficient amount of crude protein as per composition, the supplemented nitrogen may be better accessed and utilized leading to better enzyme production (14). Cumulative effect of carbon and nitrogen supplementation The optimized levels of carbon and nitrogen supplementation were used to study the cumulative effect of these
FIG. 4. Influence of initial medium pH on (a) lipase and (b) protease production under SSF condition. Closed rhombus, pH 5.0; closed squares, pH 6.0; open triangles, pH 7.0; open circles, pH 8.0. Error bars show the percent error.
sources on production of lipase and protease. The maximum lipase (1288 U/g) and protease (3366 U/g) production was in supplemented SSF, when compared to control (without supplementation of carbon and nitrogen) where lipase and protease production were found to be 1019 U/g and 2029 U/g respectively. The results also indicate that the organism can grow and produce a significant amount of enzymes with JSC alone as substrate. However, supplementation of carbon and nitrogen source enhances the productivity of lipase and protease by 20% and 39% respectively. The results of enzyme production at different days are shown in Fig. 5a and b. The production of lipase and protease was maximum on the 4th day of incubation after optimization of all the parameters. Detoxification of anti-nutrients by SSF The J. curcus seeds are highly toxic to a number of animal species due to the presence of anti-nutritional components such as phytic acid, trypsin inhibitors, lectins, cyanogenic glucosides, tannins and phorbol esters at high levels (4). PE are considered to be the most toxic compounds and known to be strong tumour promoting agents (5). The PE content was found to vary from 0.87 to 3.32 mg/g in different varieties of Jatropha (30). In the present investigation, biological detoxification of anti-nutrients and toxins were studied after SSF by using A. versicolor CJS-98. The initial PE content in the seed cake was 0.832 mg/g, which after fermentation was reduced to 0.158 mg/g. The HPLC profile of PE before and after JSC SSF is given in Fig. 6. Barros et al. (16) have reported that PE concentration in JSC decreased around 20%, 91%, 97% after SSF with white-rot fungi, Ganoderma resinaceum, Bjerkandera adusta and Phlebia rufa respectively. The results are in agreement with the findings of Belewu and Sam (31) who affirmed that there was a reduction in the anti-nutritional factors including PE of JSC after solid-state fermentation.
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FIG. 5. Effect of carbon and nitrogen sources supplementation on (a) lipase and (b) protease production by A. versicolor CJS-98 under SSF condition. Error bars show the percent error.
The phytate content in JSC was 6.08 g per 100 g. After fermentation the phytate level was decreased to 1.70/100 g, indicating that there was a reduction of phytate level by 72% after solid-state fermentation. The phytate have been implicated in decreasing protein digestibility by forming complexes and also by interacting with enzymes such as trypsin and pepsin. The phytic acid content was decreased from 10% to 6.85% after enzyme and ethanol treatments of JSC (32). The tannin content of JSC was initially 0.373/100 g of seed cake and after solid-state fermentation of JSC reduced to 0.232/100 g of seed cake. The results are in agreement with the report of Oseni and Akindahunsi (33), where the unfermented JSC had 0.92% and after fermentation the tannins were reduced to 0.076%. The lectin is generally considered as another toxic factor in JSC and the lectin activity in JSC was 0.309 mg/ml. The lectin activity
was found to decrease by 88.9% after solid-state fermentation (0.034 mg/ml) of JSC. Trypsin inhibitors are anti-nutritional factors which interfere with the physiological process of digestion through interference with the normal functioning of pancreatic proteolytic enzymes in non-ruminants (34) leading to severe growth depression. Trypsin inhibitor activity in JSC was 697.5 TIU/g sample and after fermentation the TIU was 12.5, with a reduction in 98%. This reduction was probably due to the utilization of TI by the organism for growth. The cyanogenic glucosides were present at concentrations of 692.5 mg per 100 g in unfermented JSC. This was decreased to 560 mg per 100 g after SSF. The anti-nutritional factors reported to be present in JSC (phytate-4.1, tannin-0.76, trypsin inhibitor 11.5%) was reduced to almost fifty percent of original content, when it was fermented using R. oryzae (33). The various anti-nutritional factors in JSC and the reduction of the same after SSF are shown in
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FIG. 6. HPLC chromatogram and absorption spectrum of phorbol esters from (a) unfermented and (b) fermented seed cake. (c) Standard phorbol-12-myristate 13-acetate (PMA). SSF carried out under moisture level 40%, pH 7.0, temperature 25 C condition. The PE was determined after 4th day of fermentation.
Table 1. As per the results, there is a considerable reduction in the anti-nutritional factors after solid-state fermentation by A. versicolor CJS-98. The anti-nutritional compounds and toxins in the seed cake decreased significantly after solid-state fermentation when compared to the unfermented control sample. The decrease in the levels of anti-nutritional compounds and toxins could be due
to production of various enzymes during the growth phases of fungal organism. Thus the biological detoxification method can be used to remove toxin and other anti-nutritional factors in the JSC. The production of biodiesel from non-edible feed stocks is receiving considerable attention recently, as a renewable source of energy, which results in the generation of huge quantity of JSC. The
TABLE 1. Composition of anti-nutrients of unfermented and fermented JSC using A. versicolor CJS-98. Samples
Unfermented JSC Fermented JSC Reduction (%)
Phorbol esters (g/100 g seed cake) 0.0832 0.002 0.0158 0.005 81.1
Phytic acid (g/100 g seed cake) 6.08 0.03 1.70 0.01 72.03
Estimation was done in triplicate samples. Values are mean standard error.
Tannin (g/100 g seed cake)
Cyanogenic glucosides (mg/100 g)
0.373 0.02 0.232 0.02 37.75
692.55 0.36 560.63 0.30 19.04
Trypsin inhibitor activity (TIU/g) 697.5 0.02 12.5 0.03 98.20
Lectin (1/mg meal/ ml assay) 0.309 0.28 0.034 2.20 88.90
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study on the SSF of JSC has indicated that it is an economical source for the production of industrially important enzymes by using fungi. The fungus, A. versicolor CJS-98 was able to utilize the nutrients of JSC by secreting extracellular enzymes. The various antinutrients present in the seed cake were also reduced considerably after SSF, indicating the potential for application of JSC as animal feed. Hence the biological detoxification method can be used to remove toxin and other anti-nutritional factors in the JSC. ACKNOWLEDGMENTS Authors gratefully acknowledge the financial grant provided by Ministry of Environment and Forests, New Delhi, India for carrying out this research work. Authors thank Dr. M.S. Thakur, Dr. S.G. Prapulla, Dr. H.K. Manonmani, Scientists, FTBE Dept, Ms. M. Asha, CIFS Dept and Prof. Ram Rajasekharan, Director, CSIR-CFTRI, Mysore for their encouragement and support. References 1. Heller, J.: Physic nut Jatropha curcas L. promoting the conservation and use of underutilized and neglected crops. Institute of Plant Genetics and Crop Plant Research, Gatersleben. International Plant Genetics Research Institute, Rome (1996). 2. Makkar, H. P. S. and Becker, K.: Plant toxins and detoxification methods to improve feed quality of tropical seeds e review, Asian-Aust. J. Anim. Sci., 12, 467e480 (1999). 3. Makkar, H. P. S., Aderibigbe, A. O., and Becker, K.: Comparative evaluation of nontoxic and toxic varieties of Jatropha curcas for chemical composition, digestibility, protein degradability and toxic factors, Food Chem., 62, 207e215 (1998). 4. Makkar, H. P. S., Francis, G., and Becker, K.: Protein concentrate from Jatropha curcas screw pressed seed cake and toxic and anti-nutritional factors in protein concentrate, J. Sci. Food Agri., 88, 1542e1548 (2008). 5. Vogg, G., Achatz, S., Kettrup, A., and Sandermann, H., Jr.: Fast, sensitive and selective liquid chromatographic tandem mass spectrometric determination of tumor promoting diterpene esters, J. Chromatogr., 853, 563e573 (1999). 6. Adam, S. E.: Toxic effects of Jatropha curcas in mice, Toxicology, 2, 67e76 (1974). 7. Adam, S. E. and Magzomb, M.: Toxic effect of Jatropha in goats, Toxicology, 4, 347e354 (1975). 8. Becker, K. and Makkar, H. P. S.: Toxic effects of phorbol esters in carp (Cyprinus carpio L.), Vet. Human. Toxicol., 40, 82e86 (1998). 9. Makkar, H. P. S. and Becker, K.: Nutritional studies on rats and fish (carp Cyprinus, carpio) fed diet containing unheated and heated Jatropha curcus meal of a non-toxic provenance, Plants Foods Hum. Nutri., 53, 183e192 (1999). 10. Pandey, A.: Solid state fermentation, Biochem. Eng. J., 13, 81e84 (2003). 11. Imandi, S. D., Karanam, S. K., and Garapati, H. R.: Optimization of process parameters for the production of lipase in solid state fermentation by Yarrowia lipolytica from niger seed oil Cake (Guizotia abyssinica), J. Microb. Biochem. Technol., 2, 28e33 (2010). 12. Mahadik, N. D., Puntambekar, U. S., Bastawde, K. B., Khire, J. M., and Gokhale, D. V.: Production of acidic lipase by Aspergillus niger in SSF, Process Biochem., 38, 715e721 (2002). 13. Rao, P. V., Jayaraman, K., and Lakshmanan, C. M.: Production of lipase by Candida rugosa in solid state fermentation. 1: determination of significant process variables, Process Biochem., 28, 385e389 (1993). 14. Mahanta, N., Gupta, A., and Khare, S. K.: Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using
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