- Email: [email protected]
Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation
Journal of Bioscience and Bioengineering VOL. 115 No. 2, 168e172, 2013 www.elsevier.com/locate/jbiosc
Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation Thanyarat Phengnuam and Worapot Suntornsuk* Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand Received 11 April 2012; accepted 23 August 2012 Available online 24 September 2012
Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Jatropha curcas seed cake; Phorbol esters; Anti-nutritional factors; Fermentation; Detoxification]
Jatropha curcas is a short-lived tree in the Euphorbiaceae family, which can be grown in Central and South America, South-East Asia, India and Africa (1). It is a multipurpose tree because of its industrial and medicinal uses (2). J. curcas is widely planted as a hedge to protect fields since it could not be browsed by cattle or other animals. It adapts well to arid and semi-arid conditions and is often used for the prevention of soil erosion (1). The fruits of J. curcas have 2e4 seeds containing about 30%e35% (w/w) oil (2). The seeds are the most utilized part of the plant because their oils contain fatty acids similar to cooking oils used for human consumption and can be commercially used as bio-diesel. After oil extraction, its seed cake still contains a high amount of protein e approximately 19%e27% (w/w) (3). Seed proteins contain all essential amino acids, except for lysine, which are higher than the FAO/WHO reference protein for 5-year-old children (1). However, the seed cake contains not only proteins but also toxins such as phorbol esters and anti-nutritional factors: trypsin inhibitor, lectin, saponin and phytate (3). The toxin and anti-nutritional factors negatively affect human and animal health (4). Phorbol esters cause inflammation and tumor promotion in cells (4,5). Trypsin inhibitor prevents intestinal protein digestion and protein
* Corresponding author. Tel.: þ66 2 470 8890; fax: þ66 2 470 8891. E-mail address: [email protected] (W. Suntornsuk).
consumption (6). Phytic acid has a chelating activity of various metals and can bind with proteins. It diminishes the bioavailability of protein and important minerals (7). Saponin is a plant steroid causing hemolytic activity (8). Lectin or agglutinin is a carbohydrate-binding protein which causes the agglutination of the red blood cells (9). Removal of toxin and anti-nutritional factors in J. curcas seed cake is important for its application as either a food ingredient or a feed ingredient. Three techniques have been used to detoxify the toxic compounds in the seed cake. Physical methods were used by heat treatment and ionizing radiation (4,10,11), chemical treatments by solvent extraction, bleaching agents, degumming agents, deodorization agents and alkali treatments (4,12), and biological treatments by using Pseudomonas aeruginosa and fungal fermentation (13,14). However, among three detoxification techniques, for environmental awareness with safety and energy concerns, the biological method would be more advantageous than the others. Joshi et al. (13) reported that P. aeruginosa completely detoxified phorbol esters in deoiled J. curcas seed cake in 9 days under solidstate fermentation. Belewu and Sam (14) also demonstrated that Aspergillus niger removed phorbol esters by nearly 100% and remarkably reduced anti-nutritional factors in J. curcas kernel cake. In addition, phorbol esters, phytic acid, trypsin inhibitor, lectin activity and saponin in defatted ground kernel of J. curcas meal were effectively diminished after the meal was hydrolyzed by
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.017
VOL. 115, 2013
BIOLOGICAL DETOXIFICATION OF J. CURCAS SEED CAKE
cellulase with pectinase and then followed by washing with 65% ethanol (15). To the best of our knowledge, application of Bacillus subtilis and Bacillus licheniformis to remove toxins and anti-nutritional factors in J. curcas seed cake has never been investigated. The strains are generally recognized as safe (GRAS), grow well on various substrates and were reported as an esterase and protease producer (16). Both enzymes would be able to degrade phorbol esters and anti-nutritional factors in the seed cake. Therefore, the objectives of this work were to degrade phorbol esters and anti-nutritional factors in J. curcas seed cake by solid-state and submerged fermentations with Bacillus spp. MATERIALS AND METHODS Raw materials, cultures and chemicals Defatted J. curcas seed cake, obtained by a screw press, was kindly provided from Ladda Company Limited (Bangkok, Thailand). It was stored in polyester plastic containers at 20 C before use. Two Bacillus cultures; B. subtilis MIC 001B and B. licheniformis FK1, kept in a culture collection at the department of Microbiology, the faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand, were used throughout study. The cultures were maintained on nutrient agar (NA) slants (0.5% (w/v) peptone, 0.3% (w/v) beef extract and 1.5% (w/v) agar) at 4 C. Chemicals for analysis of phorbol esters and anti-nutritional factors and enzymes were purchased from SigmaeAldrich (Steinheim, Germany) and Fischer Scientific (Loughborough, UK). Other chemicals were analytical grades. Preparation and chemical composition of J. curcas seed cake J. curcas seed cake was ground and dried by a vacuum oven at 55 C until its weight was constant. It was analyzed for moisture, protein, fat, ash and fiber by the standard methods of the Association of Official Analytical Chemists (17). Its carbohydrate content was calculated by difference (100 (protein þ fat þ fiber þ ash) on dry basis). It was also determined for phorbol esters and anti-nutritional factors; phytic acid, trypsin inhibitor and lectin, according to procedures previously described by Saetae et al. (18).
169
appropriated). The fermented seed cake samples were also dried by a vacuum oven at 55 C for 24 h. The dried sample was obtained for the determination of phorbol esters and anti-nutritional factors. For submerged fermentation, fermented seed cake suspensions were readily determined for bacterial growth by a plate count method (19) on the nutrient agar medium. The suspension was also centrifuged at 8400g at 4 C for 15 min. The supernatant was determined for pH and enzyme activities (if appropriated). Precipitated seed cake was collected and dried by a vacuum oven at 55 C for 24 h. The dried sample was obtained for determination of phorbol esters and antinutritional factors. Phorbol esters Phorbol esters were extracted from the seed cake by the previous method described by Saetae and Suntornsuk (12). The dry extract was dissolved by absolute methanol for phorbol ester determination by HPLC (Shimadzu, Kyoto, Japan). The analysis was carried out using a reversed phase chromatography column (Inertsil ODS-3, 4.6 250 mm) with a mixture of acetonitrile and deionized water (80:20, v:v) as an eluent at a flow rate of 1.3 ml/ min. A photo diode array (PDA) detector (Shimadzu, Kyoto, Japan) was set at the wavelength of 280 nm. Phorbol-12-myristate-13-acetate (PMA) dissolved in absolute methanol was used as a standard. Anti-nutritional factors Phytic acid, trypsin inhibitor and lectin were determined according to the procedure previously described by Saetae et al. (18). Enzyme assays Protease, phytase and esterase activities were determined by the methods of Li et al. (20), Angelis et al. (21) and Torres et al. (22), respectively. Nutritional values of fermented J. curcas seed cake J. curcas seed cake fermented by the selected strain under the selected cultivation method was determined for moisture, protein, fat, fiber and ash by the AOAC methods (17). Its carbohydrate content was also calculated by difference. Statistical analysis Triplicate experiments were performed and the means of data were reported. The analysis of variance (ANOVA) and the least statistical difference (LSD) tests were performed according to Montgomery using the SPSS software version 11.5 (SPSS Inc., Chicago, IL, USA) in order to determine significant differences between the treatments at P < 0.05.
RESULTS AND DISCUSSION
Preparation of bacterial starter A loopful of a 24-h-old slant cultures; B. licheniformis and B. subtilis, was inoculated into 250-ml Erlenmeyer flasks containing 100 ml nutrient broth (NB; 0.5% (w/v) peptone and 0.3% (w/v) beef extract). The flasks were incubated at 30 C for B. subtilis and at 37 C for B. licheniformis on a rotary shaker operated at 150 rev/min for 24 h. After incubation, bacterial cells were harvested by centrifugation at 8500 g at 4 C for 15 min, aseptically washed three times with sterile normal saline, and re-suspended in sterile normal saline to reach a cell concentration of 1.0 107 CFU/ml for solid-state fermentation and 1.0 109 CFU/ml for submerged fermentation.
Chemical compositions of seed cake Sterilized J. curcas seed cake contained approximately 15% (w/w) protein, 3% (w/w) fat, 6% (w/w) ash, 9% (w/w) fiber and 68% (w/w) carbohydrate on dry basis. In this study, protein and fat in the seed cake was much lower than those in the seed cake previously reported by Saetae and Suntornsuk (12), Belewu and Sam (14), and Belewu et al. (23) possibly due to the differences in seed variety, seed part and oil extraction method.
Detoxification study of J. curcas seed cake by bacterial fermentation Two fermentation methods; solid-state and submerged fermentations, were employed. For solid-state fermentation, 1 ml of freshly prepared bacterial cells were transferred to each of 250-ml Erlenmeyer flasks containing 10 g sterile seed cake with an addition of sterile distilled water (pH 7.0 0.2) to adjust the initial moisture content of the seed cake to 70%. The flasks were then manually shaken well and incubated at 30 C for B. subtilis and 37 C for B. licheniformis for 7 days. During incubation, the flasks were shaken periodically. At an initial day and at the end of fermentation, the flasks were taken for determination of pH, bacterial growth, phorbol esters and antinutritional factors. For submerged fermentation, 1 ml of freshly prepared bacterial cells was transferred to each of 250-ml sterilized Erlenmeyer flasks containing 10 g seed cake in 100 ml distilled water adjusted to a pH of 7.0 0.2. The flasks were then cultivated on a rotary shaker operated at 150 rev/min at 30 C for B. subtilis and 37 C for B. licheniformis for 5 days. At an initial day and at the end of fermentation, the flasks were taken for determination of pH, bacterial growth, phorbol esters and antinutritional factors. Flasks containing seed cake without bacterial inoculation and incubated under the same conditions of both methods were also carried out as control experiments.
Growth of Bacillus on J. curcas seed cake and pH of fermented seed cake J. curcas seed cake is a rich source of protein and other nutrients as described earlier. Therefore, it is a good substrate for microbial cultivation. It has been used to grow P. aeruginosa (13), A. niger, Penicillium chrysogenum, Rhizopus oligosporous, Rhizopus nigricans, Trichoderma longibrachitum (14) and Rhizopus oryzae (24). In this study, B. subtilis and B. licheniformis grew well on J. curcas seed cake in both solid-state and submerged fermentation as shown in Fig. 1A. The growth of B. subtilis was higher than that of B. licheniformis in both solid-state and submerged fermentation. pH of fermented seed cake increased to 8.3e8.8 during cultivation by both strains (Fig. 1B). This was possibly due to ammonium formation by protein digestion and amino acid metabolism by Bacillus sp.
Fermentation study of J. curcas seed cake by Bacillus sp. The most effective strain and method to remove toxins and anti-nutritional factors in J. curcas seed cake were selected for fermentation study. The fermentation was carried out according to the procedure previously described. Samples were taken periodically during fermentation and were analyzed for pH, bacterial growth, phorbol esters and anti-nutritional factors and the activities of enzymes; protease, phytase and esterase. The control experiments were the seed cake without bacterial inoculation. At the end of fermentation, the fermented seed cake was also determined for its chemical compositions. Sample preparation and analysis For solid-state fermentation, fermented seed cake samples were aseptically suspended in 80 ml of sterile normal saline. The suspension was shaken at 30 C and 150 rev/min for 15 min and then determined for bacterial growth by a plate count method (19) on the nutrient agar medium. The suspension was also centrifuged at 8400g at 4 C for 15 min and its supernatant was taken for pH measurement by a pH meter and enzyme activity (if
Detoxification of J. curcas seed cake by Bacillus fermentation J. curcas seed cake contained phorbol esters and anti-nutritional factors as shown in Table 1. The compounds in this study were lower than those in the report of Saetae and Suntornsuk (12) and Saetae et al. (18) possibly due to the differences in seed age and storage. After seed cake sterilization, levels of phorbol esters and phytic acid were retained since they are heat stable compounds (1). However, trypsin inhibitor and lectin toxicity were remarkably decreased by sterilization since they are heat sensitive proteins (4,25). Bacillus fermentation lowered phorbol esters and anti-nutritional factors in J. curcas seed cake as illustrated in Figs. 2e4. It was
170
PHENGNUAM AND SUNTORNSUK
J. BIOSCI. BIOENG.,
FIG. 2. Phorbol esters in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
FIG. 1. Growth of B. subtilis and B. licheniformis (A) and pH (B) in J. curcas seed cake before and after submerged fermentation on day 5 (log CFU/ml) and solid-state fermentation on day 7 (log CFU/g).
also found that their reduction in the control experiments was very minute. Phorbol esters could be degraded by both strains as shown in Fig. 2. With submerged fermentation, they were maximally reduced by nearly 60% with B. licheniformis after 5 days of incubation, while reduced by approximately 40% only with B. subtilis. Submerged fermentation was more effective than solid-state fermentation for phorbol ester degradation by both strains as directly related to bacterial growth. It is observed that B. licheniformis was able to reduce phorbol esters in the seed cake comparable to P. aeruginosa which degraded phorbol esters in the seed cake by 60e73% after 6 days under solid-state fermentation (13) and to A. niger which obviously degraded phorbol esters in the seed cake by 77% under solid-state fermentation after 7 days (14). In addition, Barros et al. (26) found significant reduction of phorbol esters in J. curcas seed cake inoculated with white-rot fungi such as Bjerkandera adusta and Phelebia rufa. Phytic acid was degraded by both strains as shown in Fig. 3. B. licheniformis with solid-state fermentation showed the most effective method to degrade phytic acid. Under solid-state
fermentation, phytic acid was considerably reduced by nearly 50% with B. licheniformis after 7 days of incubation, while by nearly 20% with B. subtilis. Under submerged fermentation, phytic acid was diminished by approximately 40% with both strains. Bacillus isolates eliminated phytic acid significantly in soybean (27). Likewise, Bifidobacterium infantis ATCC 15697 completely reduced phytic acid in a culture medium cultivated at 37 C under an anaerobic condition (28). Phytic acid in plant tubers (Icacina mannii) was potentially removed by Saccharomyces cerevisiae fermentation (29). In addition, phytic acid in soybean, cowpea and groundbean was effectively removed by R. oligosporous (30). Antony and Chandra (31) and Ijarotimi and Esho (32) demonstrated that phytic acid in finger millet flour and bambara groundnut seeds decreased significantly by traditional fermentation. Trypsin inhibitor was considerably reduced by approximately 75% with B. licheniformis under submerged fermentation as shown in Fig. 4. However, it is observed that trypsin inhibitor could not be removed by B. subtilis under submerged fermentation and by both strains under solid-state fermentation (Fig. 4). This is possibly because a small amount of proteases was produced by each strain or the serine protease produced was inhibited by trypsin inhibitor in the seed cake under those conditions. In contrast, solid-state fermentation by fungi; A. niger, R. oligosporous, P. chrysogenum, T. longibrachitum, R. nigricans and R. oryzae, effectively reduced trypsin inhibitor in J. curcas seed cake probably because of a large amount of fungal protease which was an acid protease produced under this condition (14,24). Moreover, solid-state fermentation of soybean meal by B. subtilis obviously reduced the trypsin inhibitor by approximately 96% (33). Rattansi and Dikshit (34) found that
TABLE 1. Toxic compound and anti-nutritional factors of J. curcas seed cake and seed cake fermented by B. licheniformis under submerged fermentation. Chemicals Phorbol esters (mg/g)b Phytic acid (mg/g) Trypsin inhibitor (TIU/g) Lectin (HU/mg)
Initial seed cakea 119.9c 16.1c 23.3e 3.1d
17.9 1.2 2.2 0.6
Sterilized seed cakea 103.5c 15.5c 1.2c 25.0c
1.2 0.3 0.3 0.0
Fermented seed cakea 39.4d 9.2d 0.3d 25.0c
0.6 0.4 0.0 0.0
TIU, trypsin inhibitor units; HU, heamagglutinating units. a Mean SD (on dry basis). b Equivalent to phorbol 12-myristate-13-acetate. c,d,e Different letters in the same rows indicate the significant differences (p < 0.05).
FIG. 3. Phytic acid in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
VOL. 115, 2013
BIOLOGICAL DETOXIFICATION OF J. CURCAS SEED CAKE
171
Suntornsuk (36) which reported the pH of B. licheniformis under submerged fermentation gradually increased during feather fermentation. Protease production during the seed cake fermentation Generally, Bacillus sp. is reported to be a potent extracellular protease producer (37). In this study, protease was produced during J. curcas seed cake fermentation by B. licheniformis and reached the maximum at approximately 0.9 U/ml at the end of fermentation (Fig. 5). Protease activity was parallel to trypsin inhibitor removal. Trypsin inhibitor was reduced from 1.2 TIU/g to 0.3 TIU/g (Fig. 5). It is clearly observed that protease is responsible for the hydrolysis of trypsin inhibitor.
FIG. 4. Trypsin inhibitor in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
trypsin inhibitor in karanja (Pongamia glabra) oil seed residue was reduced by traditional fermentation. Therefore, microbial source, substrate type or chemical compositions in the substrate, and fermentation method affect trypsin inhibitor removal by microorganisms. B. licheniformis and B. subtilis were not able to degrade lectin under both fermentation methods (data not shown). Lectin in J. curcas seed meal, however, can be partly eliminated by sterilization (1). In this study, no such condition could remove phorbol esters and anti-nutritional factors in J. curcas seed cake completely. Combined methods of physical treatment, chemical treatment and biological methods with microbial fermentation or enzymatic hydrolysis could improve the degradation of all toxins and anti-nutritional factors in J. curcas seed cake. However, by overall, B. licheniformis under submerged fermentation was the most effective method to eliminate phorbol ester and anti-nutritional factors. It was selected for further investigation in the fermentation study. Chemical compositions of fermented seed cake by B. licheniformis under submerged fermentation J. curcas seed cake was slightly changed in chemical compositions after bacterial fermentation. Ash and fiber was slightly increased from 6% (w/w) to approximately 7% (w/w) and from 9% (w/w) to 11% (w/w) on dry basis, respectively, while fat slightly decreased from 3% (w/ w) to 2% (w/w) on dry basis. However, protein and carbohydrate contents in the fermented seed cake remained unchanged (15% (w/ w) and 68% (w/w) on dry basis, respectively). In contrast, after fungal fermentation, protein content in J. curcas seed cake slightly increased (14,23,24). In another report, protein, ash, fiber and fat in Jatropha cathatica and J. curcas seeds significantly decreased by the traditional fermentation (35). The reduction of protein, fat and carbohydrate in the seed cake by fermentation was possibly due to transformation and utilization to gain energy for microbial cells. Phorbol esters and anti-nutritional factors in the fermented seed cake were shown in Table 1. By fermentation, phorbol esters were obviously reduced from the initial level of 103.5 mg/g to 39.4 mg/g (62% reduction), while phytic acid was reduced from the initial level of approximately 15.5 mg/g to approximately 9 mg/g (42% reduction). Trypsin inhibitor was almost totally eliminated from 1.2 TIU/g to 0.3 TIU/g (75% reduction). The data agreed with the earlier results. Growth of B. licheniformis and pH of fermented seed cake Under submerged fermentation, the growth of B. licheniformis increased over incubation time and reached its maximum at 3.5 1010 CFU/ml on day 3 of fermentation (data not shown). pH of the seed cake gradually increased during incubation and reached its maximum at the end of fermentation at pH 8.7 (data not shown). This is similar to the fermentation study by Suntornsuk and
Phytase production during the seed cake fermentation Typically, Bacillus sp. was reported to be an effective producer of phytase which hydrolyzes phytic acid to inositol and orthophosphate (38,39). In this study, considerable amounts of phytase was produced during the first 3 days of fermentation and reached a maximum at 12 U/ml as shown in Fig. 5. After that, its production declined at the end of fermentation. In the meantime, phytic acid was obviously reduced from 15.5 mg/g to 9 mg/g (Fig. 5). Its reduction matched the phytase activity. This is similar to the studies of Antony and Chandra (31) which reported that the decrease of phytic acid in the household fermented finger millet flour was well related to phytase activity. Furthermore, endogenous phytase of grain, microbial phytase and commercial phytase were main causes of phytic acid reduction in cereals and legumes (31,40,41). Esterase production during the seed cake fermentation B. licheniformis is the esterase producing strain (22). Esterase hydrolyzes ester bonds which links alcohol moiety and acid moiety (22). In this study, esterase was remarkably produced within 2 days of fermentation and reached a maximum of approximately 300 U/ml at the end of fermentation as shown in Fig. 5. At that time, phorbol esters were reduced from the initial level of 103.5 mg/g to 39.4 mg/g at the end of fermentation (Fig. 5). Their reduction agreed well with esterase activities detected in fermented seed cake. This is similar to Barros et al. (26) which reported that phorbol ester reduction probably related to esterase activities produced by white-rot fungi. In addition, lipase from P. aeruginosa was reported to hydrolyze phorbol esters in J. curcas seed cake under solid-state fermentation (13). It is also reported that Hyles euphorbiae larvae detoxified phorbol ester in their cells (42) and carboxylesterases containing esterases, acylcarnitine hydrolase and diacylglycerol lipase from rat liver homogenate were able to degrade phorbol esters (43).
FIG. 5. Enzyme production and contents of trypsin inhibitor, phytic acid and phorbol esters in J. curcas seed cake during submerged fermentation by B. licheniformis.
172
PHENGNUAM AND SUNTORNSUK
Conclusions It is clearly demonstrated that submerged fermentation of J. curcas seed cake by B. licheniformis could not totally remove toxin and all anti-nutritional factors in the seed cake. However, this method reduced them significantly. The method could be applied after phorbol esters in the seed cake were completely eliminated by ethanol extraction. In addition, a heavy bacterial starter would be used to accelerate bacterial growth and possibly remove phorbol esters, trypsin inhibitor and phytic acid rapidly. The fermented seed cake would then retain high protein content and other nutritional values applicable to the animal feed industry. ACKNOWLEDGMENTS The authors would like to acknowledge the Thailand Research Fund e the Commission of Higher Education for their financial support (RMU5180016) and the Thailand Research Fund for a scholarship and financial support of Ms. Phengnuam under the Royal Golden Jubilee Ph.D. Program e Industry (PHD/0151/2550). We are also grateful to the Ladda Company Limited for kindly providing J. curcas seed cake as the working material. Finally, we appreciate Michael Willing for his English proofreading.
References 1. Martinez-Herrera, J., Siddhuraju, P., Francis, G., Dávila-Ortiz, G., and Becker, K.: Chemical composition, toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico, Food Chem., 96, 80e89 (2006). 2. 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 Agric., 88, 542e548 (2008). 3. Makkar, H. P. S., Becker, K., Sporer, F., and Wink, M.: Studies on nutritive potential and toxic constituents of different provenances of Jatropha curcas, J. Agric. Food Chem., 45, 3152e3157 (1997). 4. Goel, G., Makkar, H. P. S., Francis, G., and Becker, K.: Phorbol esters: structure, biological activity, and toxicity in animals, Int. J. Toxicol., 26, 279e288 (2007). 5. Dimitrijevíc, S. M., Humer, U., Shehadeh, M., Ryves, W. J., Hassan, N. M., and Evans, F. J.: Analysis and purification of phorbol ester using normal phase HPLC and photodiode-array detection, J. Pharm. Biomed. Anal., 15, 393e401 (1996). 6. Guillamón, E., Pedrosa, M. M., Burbano, C., Caudrado, C., Sanchez, M. C., and Muzquiz, M.: The trypsin inhibitors present in seed of different grain legume species and cultivar, Food Chem., 107, 68e74 (2008). 7. Park, H. R., Ahn, H. J., Kim, S. H., Lee, C. H., Byun, M. W., and Lee, G. W.: Determination of the phytic acid levels in infant foods using different analytical methods, Food Control., 17, 727e732 (2006). 8. Lin, J. T. and Yang, D. J.: Determination of steroidal saponin in different organs of yam (Dioscorea pseudojaponica Yamamoto), Food Chem., 108, 1068e1074 (2008). 9. Rizzi, C., Galeoto, L., Zoccatelli, G., Vincenzi, S., Chignola, R., and Peruffo, A. D. B.: Active soybean lectin in foods: quantitative determination by ELISA using immobilised asialofetuin, Food Res. Int., 36, 815e821 (2003). 10. 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). 11. Kumar, A. and Sharma, S.: An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): a review, Ind. Crop Prod., 28, 1e10 (2008). 12. Saetae, D. and Suntornsuk, W.: Antifungal activities of ethanolic extraction from Jatropha curcas seed cake, J. Microbiol. Biotechnol., 20, 319e324 (2010). 13. Joshi, C., Mathur, P., and Khare, S. K.: Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake, Bioresour. Technol., 102, 4815e4819 (2011). 14. 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). 15. 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). 16. Torres, S., Baigori, M. D., Swathy, S. L., Pandey, A., and Castro, G. R.: Effect of hydroxylic solvents on cell growth, sporulation, and esterase production of Bacillus licheniformis S-86, Process Biochem., 40, 2333e2338 (2005). 17. Association of Official Analytical Chemists (AOAC): Official methods of analysis of Association of Official Analytical Chemists, Chapter 4, 16th ed., pp. 4e18. AOAC International, Arlington, VA, USA (1995).
J. BIOSCI. BIOENG., 18. Saetae, D., Kleekayai, T., Jayasena, V., and Suntornsuk, W.: Functional properties of protein isolate obtained from physic nut (Jatropha curcas L.) seed cake, Food Sci. Biotechnol., 20, 29e37 (2011). 19. Association of Official Analytical Chemists (AOAC): Official methods of analysis of Association of Official Analytical Chemists, 14th ed., p. 941. AOAC International, Arlington, Washington, DC, USA (1984). 20. Li, C., Yu, H., Liu, S., Xing, R., Guo, Z., and Li, P.: Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye, Bioorg. Med. Chem. Lett., 15, 5370e5374 (2005). 21. Angelis, M. D., Gallo, G., Corbo, M. R., McSweeney, P. L. H., Faccia, M., Giovine, M., and Gobbetti, M.: Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1, Int. J. Food Microbiol., 87, 259e270 (2003). 22. Torres, S., Baigori, M. D., Swathy, S. L., Pandey, A., and Castro, G. R.: Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase, Food Res. Int., 42, 454e460 (2009). 23. Belewu, M. A., Belewu, K. Y., and Ogunsola, F. O.: Nutritive value of dietary fungi treated Jatropha curcas kernel cake: voluntary intake, growth and digestibility coefficient of goat, Agric. Biol. J. North. Am., 1, 135e138 (2010). 24. 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). 25. Li, C. Y., Devappa, R. K., Liu, J. X., Lv, J. M., Makkar, H. P. S., and Becker, K.: Toxicity of Jatropha curcas phorbol esters in mice, Food Chem. Toxicol., 48, 620e625 (2010). 26. Barros, C. R. M., Ferreira, L. M. M., Nunes, F. M., Bezerra, R. M., 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 esters of Jatropha curcas L. seed cake, Eng. Life Sci., 11, 107e110 (2011). 27. Ramesh, A., Sharma, S. K., and Joshi, O. P.: Phytase, phosphatase activity and P-nutrition of soybean as influenced by inoculation of Bacillus, Indian J. Microbiol., 51, 94e99 (2011). 28. Haros, M., Bielecka, M., Honke, J., and Sanz, Y.: Myo-inositol hexaphosphate degradation by Bifidobacterium infantis ATCC 15697, Int. J. Food Microbiol., 117, 76e84 (2007). 29. Antai, S. P. and Nkwelang, G.: Reduction of some toxicants in Icacina mannii by fermentation with Saccharomyces cerevisiae, Plant Food Hum. Nutr., 53, 103e111 (1998). 30. Egounlety, M. and Aworh, O. C.: Effect of soaking, dehulling, cooking and fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soy bean (Glycine max Merr.), cowpea (Vigna unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Harms), J. Food Eng., 56, 249e254 (2003). 31. Antony, U. and Chandra, T. S.: Antinutrients reduction and enhancement in protein, starch, and mineral availability in fermented flour of finger millet (Eleusine coracana), J. Agric. Food Chem., 46, 2578e2582 (1998). 32. Ijarotimi, O. S. and Esho, T. R.: Comparison of nutritional composition and anti-nutrient status of fermented, germinated and roasted Bambara groundnut seeds (Vigna subterranean), Br. Food J., 111, 376e386 (2009). 33. Teng, D., Gao, M., Yang, Y., Liu, B., Tian, Z., and Wang, J.: Bio-modification of soybean meal with Bacillus subtilis or Aspergillus oryzae, Biocatal. Agric. Biotechnol., 1, 32e38 (2012). 34. Rattansi, R. and Dikshit, M.: Protease inhibitor and in vitro digestability of karanja (Pongamia glabra) oil seed residue. A comparative study of various treatments, J. Am. Oil Chem. Soc., 74, 1161e1164 (1997). 35. Peace, O. E. B. and Oshodi, A. A.: Effect of fermentation on some chemical and nutritive properties of berlandier nettle spurge (Jatropha cathartica) and physic nut (Jatropha curcas) seeds, Pak. J. Nutr., 2, 292e296 (2008). 36. Suntornsuk, W. and Suntornsuk, L.: Feather degradation by Bacillus sp. FK46 in submerged cultivation, Bioresour. Technol., 86, 239e243 (2003). 37. Olajuyigbe, F. M. and Ajele, J. O.: Some properties of extracellular protease from Bacillus licheniformis LBBL-11 isolated from “iru”, a traditionally fermented African locust bean condiment, Afr. J. Biochem. Res., 2, 206e220 (2008). 38. Tran, T. T., Hashim, S. O., Gaber, Y., Mamo, G., Mattiasson, B., and HattiKaul, R.: Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability, J. Inorg. Biochem., 105, 1000e1007 (2011). 39. Kim, Y. O., Kim, H. K., Bae, K. S., Yu, J. H., and Oh, T. K.: Purification and properties of a thermostable phytase from Bacillus sp. DS11, Enzyme Microb. Technol., 22, 2e7 (1998). 40. Reddy, N. and Pierson, M. D.: Reduction in antinutritional and toxic components in plant foods by fermentation, Food Chem., 44, 13e17 (1992). 41. Frias, J., Doblado, R., Antezana, J. R., and Vidal-Valverde, C.: Inositol phosphate degradadtion by the action of phytase enzyme in legume seeds, Food Chem., 81, 233e239 (2003). 42. Hundsdoerfer, A. K., Tshibangul, J. N., Wetterauer, B., and Wink, T.: Sequestration of phorbol esters by aposematic larvae of Hyles euphorbiae (Lepidoptera: Sphingidae), Chemoecology, 15, 261e267 (2005). 43. Mentlein, R.: The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase, J. Biol. Chem., 261, 7816e7818 (1986).
Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation Thanyarat Phengnuam and Worapot Suntornsuk* Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand Received 11 April 2012; accepted 23 August 2012 Available online 24 September 2012
Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Jatropha curcas seed cake; Phorbol esters; Anti-nutritional factors; Fermentation; Detoxification]
Jatropha curcas is a short-lived tree in the Euphorbiaceae family, which can be grown in Central and South America, South-East Asia, India and Africa (1). It is a multipurpose tree because of its industrial and medicinal uses (2). J. curcas is widely planted as a hedge to protect fields since it could not be browsed by cattle or other animals. It adapts well to arid and semi-arid conditions and is often used for the prevention of soil erosion (1). The fruits of J. curcas have 2e4 seeds containing about 30%e35% (w/w) oil (2). The seeds are the most utilized part of the plant because their oils contain fatty acids similar to cooking oils used for human consumption and can be commercially used as bio-diesel. After oil extraction, its seed cake still contains a high amount of protein e approximately 19%e27% (w/w) (3). Seed proteins contain all essential amino acids, except for lysine, which are higher than the FAO/WHO reference protein for 5-year-old children (1). However, the seed cake contains not only proteins but also toxins such as phorbol esters and anti-nutritional factors: trypsin inhibitor, lectin, saponin and phytate (3). The toxin and anti-nutritional factors negatively affect human and animal health (4). Phorbol esters cause inflammation and tumor promotion in cells (4,5). Trypsin inhibitor prevents intestinal protein digestion and protein
* Corresponding author. Tel.: þ66 2 470 8890; fax: þ66 2 470 8891. E-mail address: [email protected] (W. Suntornsuk).
consumption (6). Phytic acid has a chelating activity of various metals and can bind with proteins. It diminishes the bioavailability of protein and important minerals (7). Saponin is a plant steroid causing hemolytic activity (8). Lectin or agglutinin is a carbohydrate-binding protein which causes the agglutination of the red blood cells (9). Removal of toxin and anti-nutritional factors in J. curcas seed cake is important for its application as either a food ingredient or a feed ingredient. Three techniques have been used to detoxify the toxic compounds in the seed cake. Physical methods were used by heat treatment and ionizing radiation (4,10,11), chemical treatments by solvent extraction, bleaching agents, degumming agents, deodorization agents and alkali treatments (4,12), and biological treatments by using Pseudomonas aeruginosa and fungal fermentation (13,14). However, among three detoxification techniques, for environmental awareness with safety and energy concerns, the biological method would be more advantageous than the others. Joshi et al. (13) reported that P. aeruginosa completely detoxified phorbol esters in deoiled J. curcas seed cake in 9 days under solidstate fermentation. Belewu and Sam (14) also demonstrated that Aspergillus niger removed phorbol esters by nearly 100% and remarkably reduced anti-nutritional factors in J. curcas kernel cake. In addition, phorbol esters, phytic acid, trypsin inhibitor, lectin activity and saponin in defatted ground kernel of J. curcas meal were effectively diminished after the meal was hydrolyzed by
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.017
VOL. 115, 2013
BIOLOGICAL DETOXIFICATION OF J. CURCAS SEED CAKE
cellulase with pectinase and then followed by washing with 65% ethanol (15). To the best of our knowledge, application of Bacillus subtilis and Bacillus licheniformis to remove toxins and anti-nutritional factors in J. curcas seed cake has never been investigated. The strains are generally recognized as safe (GRAS), grow well on various substrates and were reported as an esterase and protease producer (16). Both enzymes would be able to degrade phorbol esters and anti-nutritional factors in the seed cake. Therefore, the objectives of this work were to degrade phorbol esters and anti-nutritional factors in J. curcas seed cake by solid-state and submerged fermentations with Bacillus spp. MATERIALS AND METHODS Raw materials, cultures and chemicals Defatted J. curcas seed cake, obtained by a screw press, was kindly provided from Ladda Company Limited (Bangkok, Thailand). It was stored in polyester plastic containers at 20 C before use. Two Bacillus cultures; B. subtilis MIC 001B and B. licheniformis FK1, kept in a culture collection at the department of Microbiology, the faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand, were used throughout study. The cultures were maintained on nutrient agar (NA) slants (0.5% (w/v) peptone, 0.3% (w/v) beef extract and 1.5% (w/v) agar) at 4 C. Chemicals for analysis of phorbol esters and anti-nutritional factors and enzymes were purchased from SigmaeAldrich (Steinheim, Germany) and Fischer Scientific (Loughborough, UK). Other chemicals were analytical grades. Preparation and chemical composition of J. curcas seed cake J. curcas seed cake was ground and dried by a vacuum oven at 55 C until its weight was constant. It was analyzed for moisture, protein, fat, ash and fiber by the standard methods of the Association of Official Analytical Chemists (17). Its carbohydrate content was calculated by difference (100 (protein þ fat þ fiber þ ash) on dry basis). It was also determined for phorbol esters and anti-nutritional factors; phytic acid, trypsin inhibitor and lectin, according to procedures previously described by Saetae et al. (18).
169
appropriated). The fermented seed cake samples were also dried by a vacuum oven at 55 C for 24 h. The dried sample was obtained for the determination of phorbol esters and anti-nutritional factors. For submerged fermentation, fermented seed cake suspensions were readily determined for bacterial growth by a plate count method (19) on the nutrient agar medium. The suspension was also centrifuged at 8400g at 4 C for 15 min. The supernatant was determined for pH and enzyme activities (if appropriated). Precipitated seed cake was collected and dried by a vacuum oven at 55 C for 24 h. The dried sample was obtained for determination of phorbol esters and antinutritional factors. Phorbol esters Phorbol esters were extracted from the seed cake by the previous method described by Saetae and Suntornsuk (12). The dry extract was dissolved by absolute methanol for phorbol ester determination by HPLC (Shimadzu, Kyoto, Japan). The analysis was carried out using a reversed phase chromatography column (Inertsil ODS-3, 4.6 250 mm) with a mixture of acetonitrile and deionized water (80:20, v:v) as an eluent at a flow rate of 1.3 ml/ min. A photo diode array (PDA) detector (Shimadzu, Kyoto, Japan) was set at the wavelength of 280 nm. Phorbol-12-myristate-13-acetate (PMA) dissolved in absolute methanol was used as a standard. Anti-nutritional factors Phytic acid, trypsin inhibitor and lectin were determined according to the procedure previously described by Saetae et al. (18). Enzyme assays Protease, phytase and esterase activities were determined by the methods of Li et al. (20), Angelis et al. (21) and Torres et al. (22), respectively. Nutritional values of fermented J. curcas seed cake J. curcas seed cake fermented by the selected strain under the selected cultivation method was determined for moisture, protein, fat, fiber and ash by the AOAC methods (17). Its carbohydrate content was also calculated by difference. Statistical analysis Triplicate experiments were performed and the means of data were reported. The analysis of variance (ANOVA) and the least statistical difference (LSD) tests were performed according to Montgomery using the SPSS software version 11.5 (SPSS Inc., Chicago, IL, USA) in order to determine significant differences between the treatments at P < 0.05.
RESULTS AND DISCUSSION
Preparation of bacterial starter A loopful of a 24-h-old slant cultures; B. licheniformis and B. subtilis, was inoculated into 250-ml Erlenmeyer flasks containing 100 ml nutrient broth (NB; 0.5% (w/v) peptone and 0.3% (w/v) beef extract). The flasks were incubated at 30 C for B. subtilis and at 37 C for B. licheniformis on a rotary shaker operated at 150 rev/min for 24 h. After incubation, bacterial cells were harvested by centrifugation at 8500 g at 4 C for 15 min, aseptically washed three times with sterile normal saline, and re-suspended in sterile normal saline to reach a cell concentration of 1.0 107 CFU/ml for solid-state fermentation and 1.0 109 CFU/ml for submerged fermentation.
Chemical compositions of seed cake Sterilized J. curcas seed cake contained approximately 15% (w/w) protein, 3% (w/w) fat, 6% (w/w) ash, 9% (w/w) fiber and 68% (w/w) carbohydrate on dry basis. In this study, protein and fat in the seed cake was much lower than those in the seed cake previously reported by Saetae and Suntornsuk (12), Belewu and Sam (14), and Belewu et al. (23) possibly due to the differences in seed variety, seed part and oil extraction method.
Detoxification study of J. curcas seed cake by bacterial fermentation Two fermentation methods; solid-state and submerged fermentations, were employed. For solid-state fermentation, 1 ml of freshly prepared bacterial cells were transferred to each of 250-ml Erlenmeyer flasks containing 10 g sterile seed cake with an addition of sterile distilled water (pH 7.0 0.2) to adjust the initial moisture content of the seed cake to 70%. The flasks were then manually shaken well and incubated at 30 C for B. subtilis and 37 C for B. licheniformis for 7 days. During incubation, the flasks were shaken periodically. At an initial day and at the end of fermentation, the flasks were taken for determination of pH, bacterial growth, phorbol esters and antinutritional factors. For submerged fermentation, 1 ml of freshly prepared bacterial cells was transferred to each of 250-ml sterilized Erlenmeyer flasks containing 10 g seed cake in 100 ml distilled water adjusted to a pH of 7.0 0.2. The flasks were then cultivated on a rotary shaker operated at 150 rev/min at 30 C for B. subtilis and 37 C for B. licheniformis for 5 days. At an initial day and at the end of fermentation, the flasks were taken for determination of pH, bacterial growth, phorbol esters and antinutritional factors. Flasks containing seed cake without bacterial inoculation and incubated under the same conditions of both methods were also carried out as control experiments.
Growth of Bacillus on J. curcas seed cake and pH of fermented seed cake J. curcas seed cake is a rich source of protein and other nutrients as described earlier. Therefore, it is a good substrate for microbial cultivation. It has been used to grow P. aeruginosa (13), A. niger, Penicillium chrysogenum, Rhizopus oligosporous, Rhizopus nigricans, Trichoderma longibrachitum (14) and Rhizopus oryzae (24). In this study, B. subtilis and B. licheniformis grew well on J. curcas seed cake in both solid-state and submerged fermentation as shown in Fig. 1A. The growth of B. subtilis was higher than that of B. licheniformis in both solid-state and submerged fermentation. pH of fermented seed cake increased to 8.3e8.8 during cultivation by both strains (Fig. 1B). This was possibly due to ammonium formation by protein digestion and amino acid metabolism by Bacillus sp.
Fermentation study of J. curcas seed cake by Bacillus sp. The most effective strain and method to remove toxins and anti-nutritional factors in J. curcas seed cake were selected for fermentation study. The fermentation was carried out according to the procedure previously described. Samples were taken periodically during fermentation and were analyzed for pH, bacterial growth, phorbol esters and anti-nutritional factors and the activities of enzymes; protease, phytase and esterase. The control experiments were the seed cake without bacterial inoculation. At the end of fermentation, the fermented seed cake was also determined for its chemical compositions. Sample preparation and analysis For solid-state fermentation, fermented seed cake samples were aseptically suspended in 80 ml of sterile normal saline. The suspension was shaken at 30 C and 150 rev/min for 15 min and then determined for bacterial growth by a plate count method (19) on the nutrient agar medium. The suspension was also centrifuged at 8400g at 4 C for 15 min and its supernatant was taken for pH measurement by a pH meter and enzyme activity (if
Detoxification of J. curcas seed cake by Bacillus fermentation J. curcas seed cake contained phorbol esters and anti-nutritional factors as shown in Table 1. The compounds in this study were lower than those in the report of Saetae and Suntornsuk (12) and Saetae et al. (18) possibly due to the differences in seed age and storage. After seed cake sterilization, levels of phorbol esters and phytic acid were retained since they are heat stable compounds (1). However, trypsin inhibitor and lectin toxicity were remarkably decreased by sterilization since they are heat sensitive proteins (4,25). Bacillus fermentation lowered phorbol esters and anti-nutritional factors in J. curcas seed cake as illustrated in Figs. 2e4. It was
170
PHENGNUAM AND SUNTORNSUK
J. BIOSCI. BIOENG.,
FIG. 2. Phorbol esters in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
FIG. 1. Growth of B. subtilis and B. licheniformis (A) and pH (B) in J. curcas seed cake before and after submerged fermentation on day 5 (log CFU/ml) and solid-state fermentation on day 7 (log CFU/g).
also found that their reduction in the control experiments was very minute. Phorbol esters could be degraded by both strains as shown in Fig. 2. With submerged fermentation, they were maximally reduced by nearly 60% with B. licheniformis after 5 days of incubation, while reduced by approximately 40% only with B. subtilis. Submerged fermentation was more effective than solid-state fermentation for phorbol ester degradation by both strains as directly related to bacterial growth. It is observed that B. licheniformis was able to reduce phorbol esters in the seed cake comparable to P. aeruginosa which degraded phorbol esters in the seed cake by 60e73% after 6 days under solid-state fermentation (13) and to A. niger which obviously degraded phorbol esters in the seed cake by 77% under solid-state fermentation after 7 days (14). In addition, Barros et al. (26) found significant reduction of phorbol esters in J. curcas seed cake inoculated with white-rot fungi such as Bjerkandera adusta and Phelebia rufa. Phytic acid was degraded by both strains as shown in Fig. 3. B. licheniformis with solid-state fermentation showed the most effective method to degrade phytic acid. Under solid-state
fermentation, phytic acid was considerably reduced by nearly 50% with B. licheniformis after 7 days of incubation, while by nearly 20% with B. subtilis. Under submerged fermentation, phytic acid was diminished by approximately 40% with both strains. Bacillus isolates eliminated phytic acid significantly in soybean (27). Likewise, Bifidobacterium infantis ATCC 15697 completely reduced phytic acid in a culture medium cultivated at 37 C under an anaerobic condition (28). Phytic acid in plant tubers (Icacina mannii) was potentially removed by Saccharomyces cerevisiae fermentation (29). In addition, phytic acid in soybean, cowpea and groundbean was effectively removed by R. oligosporous (30). Antony and Chandra (31) and Ijarotimi and Esho (32) demonstrated that phytic acid in finger millet flour and bambara groundnut seeds decreased significantly by traditional fermentation. Trypsin inhibitor was considerably reduced by approximately 75% with B. licheniformis under submerged fermentation as shown in Fig. 4. However, it is observed that trypsin inhibitor could not be removed by B. subtilis under submerged fermentation and by both strains under solid-state fermentation (Fig. 4). This is possibly because a small amount of proteases was produced by each strain or the serine protease produced was inhibited by trypsin inhibitor in the seed cake under those conditions. In contrast, solid-state fermentation by fungi; A. niger, R. oligosporous, P. chrysogenum, T. longibrachitum, R. nigricans and R. oryzae, effectively reduced trypsin inhibitor in J. curcas seed cake probably because of a large amount of fungal protease which was an acid protease produced under this condition (14,24). Moreover, solid-state fermentation of soybean meal by B. subtilis obviously reduced the trypsin inhibitor by approximately 96% (33). Rattansi and Dikshit (34) found that
TABLE 1. Toxic compound and anti-nutritional factors of J. curcas seed cake and seed cake fermented by B. licheniformis under submerged fermentation. Chemicals Phorbol esters (mg/g)b Phytic acid (mg/g) Trypsin inhibitor (TIU/g) Lectin (HU/mg)
Initial seed cakea 119.9c 16.1c 23.3e 3.1d
17.9 1.2 2.2 0.6
Sterilized seed cakea 103.5c 15.5c 1.2c 25.0c
1.2 0.3 0.3 0.0
Fermented seed cakea 39.4d 9.2d 0.3d 25.0c
0.6 0.4 0.0 0.0
TIU, trypsin inhibitor units; HU, heamagglutinating units. a Mean SD (on dry basis). b Equivalent to phorbol 12-myristate-13-acetate. c,d,e Different letters in the same rows indicate the significant differences (p < 0.05).
FIG. 3. Phytic acid in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
VOL. 115, 2013
BIOLOGICAL DETOXIFICATION OF J. CURCAS SEED CAKE
171
Suntornsuk (36) which reported the pH of B. licheniformis under submerged fermentation gradually increased during feather fermentation. Protease production during the seed cake fermentation Generally, Bacillus sp. is reported to be a potent extracellular protease producer (37). In this study, protease was produced during J. curcas seed cake fermentation by B. licheniformis and reached the maximum at approximately 0.9 U/ml at the end of fermentation (Fig. 5). Protease activity was parallel to trypsin inhibitor removal. Trypsin inhibitor was reduced from 1.2 TIU/g to 0.3 TIU/g (Fig. 5). It is clearly observed that protease is responsible for the hydrolysis of trypsin inhibitor.
FIG. 4. Trypsin inhibitor in J. curcas seed cake before and after submerged fermentation on day 5 and solid-state fermentation on day 7 by B. subtilis and B. licheniformis.
trypsin inhibitor in karanja (Pongamia glabra) oil seed residue was reduced by traditional fermentation. Therefore, microbial source, substrate type or chemical compositions in the substrate, and fermentation method affect trypsin inhibitor removal by microorganisms. B. licheniformis and B. subtilis were not able to degrade lectin under both fermentation methods (data not shown). Lectin in J. curcas seed meal, however, can be partly eliminated by sterilization (1). In this study, no such condition could remove phorbol esters and anti-nutritional factors in J. curcas seed cake completely. Combined methods of physical treatment, chemical treatment and biological methods with microbial fermentation or enzymatic hydrolysis could improve the degradation of all toxins and anti-nutritional factors in J. curcas seed cake. However, by overall, B. licheniformis under submerged fermentation was the most effective method to eliminate phorbol ester and anti-nutritional factors. It was selected for further investigation in the fermentation study. Chemical compositions of fermented seed cake by B. licheniformis under submerged fermentation J. curcas seed cake was slightly changed in chemical compositions after bacterial fermentation. Ash and fiber was slightly increased from 6% (w/w) to approximately 7% (w/w) and from 9% (w/w) to 11% (w/w) on dry basis, respectively, while fat slightly decreased from 3% (w/ w) to 2% (w/w) on dry basis. However, protein and carbohydrate contents in the fermented seed cake remained unchanged (15% (w/ w) and 68% (w/w) on dry basis, respectively). In contrast, after fungal fermentation, protein content in J. curcas seed cake slightly increased (14,23,24). In another report, protein, ash, fiber and fat in Jatropha cathatica and J. curcas seeds significantly decreased by the traditional fermentation (35). The reduction of protein, fat and carbohydrate in the seed cake by fermentation was possibly due to transformation and utilization to gain energy for microbial cells. Phorbol esters and anti-nutritional factors in the fermented seed cake were shown in Table 1. By fermentation, phorbol esters were obviously reduced from the initial level of 103.5 mg/g to 39.4 mg/g (62% reduction), while phytic acid was reduced from the initial level of approximately 15.5 mg/g to approximately 9 mg/g (42% reduction). Trypsin inhibitor was almost totally eliminated from 1.2 TIU/g to 0.3 TIU/g (75% reduction). The data agreed with the earlier results. Growth of B. licheniformis and pH of fermented seed cake Under submerged fermentation, the growth of B. licheniformis increased over incubation time and reached its maximum at 3.5 1010 CFU/ml on day 3 of fermentation (data not shown). pH of the seed cake gradually increased during incubation and reached its maximum at the end of fermentation at pH 8.7 (data not shown). This is similar to the fermentation study by Suntornsuk and
Phytase production during the seed cake fermentation Typically, Bacillus sp. was reported to be an effective producer of phytase which hydrolyzes phytic acid to inositol and orthophosphate (38,39). In this study, considerable amounts of phytase was produced during the first 3 days of fermentation and reached a maximum at 12 U/ml as shown in Fig. 5. After that, its production declined at the end of fermentation. In the meantime, phytic acid was obviously reduced from 15.5 mg/g to 9 mg/g (Fig. 5). Its reduction matched the phytase activity. This is similar to the studies of Antony and Chandra (31) which reported that the decrease of phytic acid in the household fermented finger millet flour was well related to phytase activity. Furthermore, endogenous phytase of grain, microbial phytase and commercial phytase were main causes of phytic acid reduction in cereals and legumes (31,40,41). Esterase production during the seed cake fermentation B. licheniformis is the esterase producing strain (22). Esterase hydrolyzes ester bonds which links alcohol moiety and acid moiety (22). In this study, esterase was remarkably produced within 2 days of fermentation and reached a maximum of approximately 300 U/ml at the end of fermentation as shown in Fig. 5. At that time, phorbol esters were reduced from the initial level of 103.5 mg/g to 39.4 mg/g at the end of fermentation (Fig. 5). Their reduction agreed well with esterase activities detected in fermented seed cake. This is similar to Barros et al. (26) which reported that phorbol ester reduction probably related to esterase activities produced by white-rot fungi. In addition, lipase from P. aeruginosa was reported to hydrolyze phorbol esters in J. curcas seed cake under solid-state fermentation (13). It is also reported that Hyles euphorbiae larvae detoxified phorbol ester in their cells (42) and carboxylesterases containing esterases, acylcarnitine hydrolase and diacylglycerol lipase from rat liver homogenate were able to degrade phorbol esters (43).
FIG. 5. Enzyme production and contents of trypsin inhibitor, phytic acid and phorbol esters in J. curcas seed cake during submerged fermentation by B. licheniformis.
172
PHENGNUAM AND SUNTORNSUK
Conclusions It is clearly demonstrated that submerged fermentation of J. curcas seed cake by B. licheniformis could not totally remove toxin and all anti-nutritional factors in the seed cake. However, this method reduced them significantly. The method could be applied after phorbol esters in the seed cake were completely eliminated by ethanol extraction. In addition, a heavy bacterial starter would be used to accelerate bacterial growth and possibly remove phorbol esters, trypsin inhibitor and phytic acid rapidly. The fermented seed cake would then retain high protein content and other nutritional values applicable to the animal feed industry. ACKNOWLEDGMENTS The authors would like to acknowledge the Thailand Research Fund e the Commission of Higher Education for their financial support (RMU5180016) and the Thailand Research Fund for a scholarship and financial support of Ms. Phengnuam under the Royal Golden Jubilee Ph.D. Program e Industry (PHD/0151/2550). We are also grateful to the Ladda Company Limited for kindly providing J. curcas seed cake as the working material. Finally, we appreciate Michael Willing for his English proofreading.
References 1. Martinez-Herrera, J., Siddhuraju, P., Francis, G., Dávila-Ortiz, G., and Becker, K.: Chemical composition, toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico, Food Chem., 96, 80e89 (2006). 2. 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 Agric., 88, 542e548 (2008). 3. Makkar, H. P. S., Becker, K., Sporer, F., and Wink, M.: Studies on nutritive potential and toxic constituents of different provenances of Jatropha curcas, J. Agric. Food Chem., 45, 3152e3157 (1997). 4. Goel, G., Makkar, H. P. S., Francis, G., and Becker, K.: Phorbol esters: structure, biological activity, and toxicity in animals, Int. J. Toxicol., 26, 279e288 (2007). 5. Dimitrijevíc, S. M., Humer, U., Shehadeh, M., Ryves, W. J., Hassan, N. M., and Evans, F. J.: Analysis and purification of phorbol ester using normal phase HPLC and photodiode-array detection, J. Pharm. Biomed. Anal., 15, 393e401 (1996). 6. Guillamón, E., Pedrosa, M. M., Burbano, C., Caudrado, C., Sanchez, M. C., and Muzquiz, M.: The trypsin inhibitors present in seed of different grain legume species and cultivar, Food Chem., 107, 68e74 (2008). 7. Park, H. R., Ahn, H. J., Kim, S. H., Lee, C. H., Byun, M. W., and Lee, G. W.: Determination of the phytic acid levels in infant foods using different analytical methods, Food Control., 17, 727e732 (2006). 8. Lin, J. T. and Yang, D. J.: Determination of steroidal saponin in different organs of yam (Dioscorea pseudojaponica Yamamoto), Food Chem., 108, 1068e1074 (2008). 9. Rizzi, C., Galeoto, L., Zoccatelli, G., Vincenzi, S., Chignola, R., and Peruffo, A. D. B.: Active soybean lectin in foods: quantitative determination by ELISA using immobilised asialofetuin, Food Res. Int., 36, 815e821 (2003). 10. 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). 11. Kumar, A. and Sharma, S.: An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): a review, Ind. Crop Prod., 28, 1e10 (2008). 12. Saetae, D. and Suntornsuk, W.: Antifungal activities of ethanolic extraction from Jatropha curcas seed cake, J. Microbiol. Biotechnol., 20, 319e324 (2010). 13. Joshi, C., Mathur, P., and Khare, S. K.: Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake, Bioresour. Technol., 102, 4815e4819 (2011). 14. 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). 15. 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). 16. Torres, S., Baigori, M. D., Swathy, S. L., Pandey, A., and Castro, G. R.: Effect of hydroxylic solvents on cell growth, sporulation, and esterase production of Bacillus licheniformis S-86, Process Biochem., 40, 2333e2338 (2005). 17. Association of Official Analytical Chemists (AOAC): Official methods of analysis of Association of Official Analytical Chemists, Chapter 4, 16th ed., pp. 4e18. AOAC International, Arlington, VA, USA (1995).
J. BIOSCI. BIOENG., 18. Saetae, D., Kleekayai, T., Jayasena, V., and Suntornsuk, W.: Functional properties of protein isolate obtained from physic nut (Jatropha curcas L.) seed cake, Food Sci. Biotechnol., 20, 29e37 (2011). 19. Association of Official Analytical Chemists (AOAC): Official methods of analysis of Association of Official Analytical Chemists, 14th ed., p. 941. AOAC International, Arlington, Washington, DC, USA (1984). 20. Li, C., Yu, H., Liu, S., Xing, R., Guo, Z., and Li, P.: Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye, Bioorg. Med. Chem. Lett., 15, 5370e5374 (2005). 21. Angelis, M. D., Gallo, G., Corbo, M. R., McSweeney, P. L. H., Faccia, M., Giovine, M., and Gobbetti, M.: Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1, Int. J. Food Microbiol., 87, 259e270 (2003). 22. Torres, S., Baigori, M. D., Swathy, S. L., Pandey, A., and Castro, G. R.: Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase, Food Res. Int., 42, 454e460 (2009). 23. Belewu, M. A., Belewu, K. Y., and Ogunsola, F. O.: Nutritive value of dietary fungi treated Jatropha curcas kernel cake: voluntary intake, growth and digestibility coefficient of goat, Agric. Biol. J. North. Am., 1, 135e138 (2010). 24. 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). 25. Li, C. Y., Devappa, R. K., Liu, J. X., Lv, J. M., Makkar, H. P. S., and Becker, K.: Toxicity of Jatropha curcas phorbol esters in mice, Food Chem. Toxicol., 48, 620e625 (2010). 26. Barros, C. R. M., Ferreira, L. M. M., Nunes, F. M., Bezerra, R. M., 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 esters of Jatropha curcas L. seed cake, Eng. Life Sci., 11, 107e110 (2011). 27. Ramesh, A., Sharma, S. K., and Joshi, O. P.: Phytase, phosphatase activity and P-nutrition of soybean as influenced by inoculation of Bacillus, Indian J. Microbiol., 51, 94e99 (2011). 28. Haros, M., Bielecka, M., Honke, J., and Sanz, Y.: Myo-inositol hexaphosphate degradation by Bifidobacterium infantis ATCC 15697, Int. J. Food Microbiol., 117, 76e84 (2007). 29. Antai, S. P. and Nkwelang, G.: Reduction of some toxicants in Icacina mannii by fermentation with Saccharomyces cerevisiae, Plant Food Hum. Nutr., 53, 103e111 (1998). 30. Egounlety, M. and Aworh, O. C.: Effect of soaking, dehulling, cooking and fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soy bean (Glycine max Merr.), cowpea (Vigna unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Harms), J. Food Eng., 56, 249e254 (2003). 31. Antony, U. and Chandra, T. S.: Antinutrients reduction and enhancement in protein, starch, and mineral availability in fermented flour of finger millet (Eleusine coracana), J. Agric. Food Chem., 46, 2578e2582 (1998). 32. Ijarotimi, O. S. and Esho, T. R.: Comparison of nutritional composition and anti-nutrient status of fermented, germinated and roasted Bambara groundnut seeds (Vigna subterranean), Br. Food J., 111, 376e386 (2009). 33. Teng, D., Gao, M., Yang, Y., Liu, B., Tian, Z., and Wang, J.: Bio-modification of soybean meal with Bacillus subtilis or Aspergillus oryzae, Biocatal. Agric. Biotechnol., 1, 32e38 (2012). 34. Rattansi, R. and Dikshit, M.: Protease inhibitor and in vitro digestability of karanja (Pongamia glabra) oil seed residue. A comparative study of various treatments, J. Am. Oil Chem. Soc., 74, 1161e1164 (1997). 35. Peace, O. E. B. and Oshodi, A. A.: Effect of fermentation on some chemical and nutritive properties of berlandier nettle spurge (Jatropha cathartica) and physic nut (Jatropha curcas) seeds, Pak. J. Nutr., 2, 292e296 (2008). 36. Suntornsuk, W. and Suntornsuk, L.: Feather degradation by Bacillus sp. FK46 in submerged cultivation, Bioresour. Technol., 86, 239e243 (2003). 37. Olajuyigbe, F. M. and Ajele, J. O.: Some properties of extracellular protease from Bacillus licheniformis LBBL-11 isolated from “iru”, a traditionally fermented African locust bean condiment, Afr. J. Biochem. Res., 2, 206e220 (2008). 38. Tran, T. T., Hashim, S. O., Gaber, Y., Mamo, G., Mattiasson, B., and HattiKaul, R.: Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability, J. Inorg. Biochem., 105, 1000e1007 (2011). 39. Kim, Y. O., Kim, H. K., Bae, K. S., Yu, J. H., and Oh, T. K.: Purification and properties of a thermostable phytase from Bacillus sp. DS11, Enzyme Microb. Technol., 22, 2e7 (1998). 40. Reddy, N. and Pierson, M. D.: Reduction in antinutritional and toxic components in plant foods by fermentation, Food Chem., 44, 13e17 (1992). 41. Frias, J., Doblado, R., Antezana, J. R., and Vidal-Valverde, C.: Inositol phosphate degradadtion by the action of phytase enzyme in legume seeds, Food Chem., 81, 233e239 (2003). 42. Hundsdoerfer, A. K., Tshibangul, J. N., Wetterauer, B., and Wink, T.: Sequestration of phorbol esters by aposematic larvae of Hyles euphorbiae (Lepidoptera: Sphingidae), Chemoecology, 15, 261e267 (2005). 43. Mentlein, R.: The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase, J. Biol. Chem., 261, 7816e7818 (1986).