Animal Feed Science and Technology 120 (2005) 159168

Effect of different physical and chemical treatments on detoxication of ricin in castor cake
S. Anandan , G.K. Anil Kumar, J. Ghosh, K.S. Ramachandra
National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore 560 030, India Received 28 October 2003; received in revised form 16 September 2004; accepted 1 October 2004

Abstract In spite of its high protein content, castor cake is not used as livestock feed due to the presence of toxic factorsricin, ricinine and allergen. Of the three, ricin is the most detrimental to the animals. In order to detoxify the cake, a number of physical and chemical methods were employed. Soaking (3, 6 and 12 h), steaming (30 and 60 min), boiling (30 and 60 min), autoclaving (15 psi, 30 min; 15 psi, 60 min) and heating (100 C 30 min; 120 C 25 min) were the physical methods employed, while the chemical methods consisted of ammonia (7.5, 12.5 ml/kg of castor cake), formaldehyde (5, 10 g/kg), lime (10, 20 and 40 g/kg), sodium chloride (5, 10 and 20 g/kg), tannic acid (5, 10 g/kg) and sodium hydroxide (2.5, 5 and 10 g/kg). The efcacy of the treatments was assessed, based on the qualitative and quantitative changes in ricin content. Of all the methods employed, autoclaving (15 psi., 60 min) and lime treatment (40 g/kg) completely destroyed the toxin. 2004 Published by Elsevier B.V.
Keywords: Ricin; Castor cake; Detoxication; Physical treatment; Chemical treatment

1. Introduction Castor bean, Ricinus communis is grown in temperate and tropical parts of the world for its oil, which is extensively used for medicinal and industrial purposes. India is currently the
Abbreviations: CB-1A, castor bean allergen 1; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis Corresponding author. Tel.: +91 80 5711304; fax: +91 80 5711420. E-mail address: anandsrp@yahoo.co.in (S. Anandan). 0377-8401/$ see front matter 2004 Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2004.10.002

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leading producer of castor with the annual production of 590,000 tonnes in the year 2002 (FAO, 2002). The residue, castor cake that is left after the extraction of the oil represents about one half of the weight of the castor bean (Robb et al., 1974) and it has a protein content of 3436%. With decortication protein content of the cake can further be increased to 60% (Mottola et al., 1968) and thus, it can be a good source of protein for animals. In spite of its availability and high protein content, castor cake has not found a place as protein supplement due to its toxicity and currently is used as organic fertilizer. Ricin is the most lethal of the toxins present in castor cake. Ricin is reported to be present to the extent of 1.5% in the castor cake (Ambekar and Dole, 1957). Ricinine is the poisonous alkaloid that exists at very low levels (0.23%) of the cake (Hinkson et al., 1972) and presents little problem (Horton and Williams, 1989). The castor bean allergen is a nontoxic unusually stable protein that exhibits an extraordinary capacity to sensitize individuals exposed to small concentrations of the dust from castor beans or the castor cake. Alilaire was the rst to describe human hypersensitivity to castor bean (Jones Breese, 1947). Castor bean allergen-1 (CB-1A) is the principal allergen of the castor bean. It is a polysaccharididic protein factor. The allergen contents of decorticated defatted castor beans ranged from 6.1 to 9.0% while the commercial castor cake contained 0.094.2% of the same (Coulson et al., 1960). Of the three toxins listed above, ricin is the most potent and any attempt to detoxify the cake should principally address this problem. The remaining threericinine, ricinus communis agglutinin and allergen CB-1A are of little consequence with regard to the feeding value in animals either due to presence in lower concentration or insignicant toxic effects. Allergen is a matter of concern for the people handling the cake and animals are not affected by the allergen (UNIDO, 1989). The present investigation has been carried out to assess the effect of different detoxication methods on the ricin content. The efcacy has been assessed based on the quantitative and qualitative changes in ricin content.

2. Materials and methods 2.1. Processing of castor cake The solvent extracted and toasted castor cake obtained from oil mills in Gujarat, India, were subjected to different physical and chemical processes for detoxication of the castor cake. Five physical processing methods, i.e., soaking, steaming, boiling, autoclaving and heating at different time intervals and six chemical treatments, i.e., ammonia, formaldehyde, lime, sodium chloride, tannic acid and sodium hydroxide at different concentrations were tried to detoxify the cake. 2.1.1. Physical treatments Soaking: Castor cake (1000 g) was steeped in 10 l water (1:10) at three different time intervals of 3, 6 and 12 h. The cake was ltered using a muslin cloth, air dried and stored at 4 C for further evaluation. Steaming: Powdered castor (1000 g) cake was moistened with water to have moisture content of 150 g/kg in the cake. It was spread on a muslin cloth in a perforated dish and

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steam was allowed to pass through it for 30 and 60 min. A pressure cooker was used for the purpose with out closing the outlet by removing the knob to produce the steam with out any pressure. The treated cake was air dried and stored at 4 C. Boiling: Castor cake (1000 g) steeped in 10 l of water was boiled at 100 C for 30 and 60 min. The water was poured off and the sample ltered through a muslin cloth, air dried and stored at 4 C for further evaluation. Autoclaving: The castor cake samples each weighing 1000 g were autoclaved at 15 psi for 30 and 60 min. The cake was allowed to dry at room temperature before storing. Heating: The castor cake samples of 1000 g each were subjected to dry heat at different time and temperature combination in a hot air oven. The different combinations that were tried were 100 C for 30 min and 120 C for 25 min.

2.1.2. Chemical treatments Ammonia treatment: Ammonia solution (25 ml/l) was added to castor cake samples (each weighing 1000 g) at the rate of 30 and 50 ml to have a concentration of 7.5 and 12.5 ml of ammonia per kg of castor cake sample. The treated samples were kept in airtight plastic containers for 7 days. The samples were then air dried and used for analysis. Formaldehyde treatment: A formaldehyde solution of 400 g/l was added to each of 1000 g of castor cake samples. The samples were subjected to treatment at 5 and 10 g/kg on protein basis by adding 5 and 10 ml of the solution to the respective samples. The treated samples were thoroughly mixed and kept in airtight containers for a period of 7 days. The samples were air dried and used for analysis. Lime treatment: The castor cake samples of 1000 g each were mixed with calcium hydroxide solutions in a ratio of 3 g/ml. Calcium hydroxide concentration were 10, 20 and 40 g/kg. The treated samples were left over night (8 h) and the sun dried lime treated samples were then used for analysis. Sodium chloride treatment: Sodium chloride was used at 0.25, 0.5 and 1.0N strength to treat the castor cake samples. The cake samples of 1000 g each were initially mixed with sodium chloride solution in a ratio of 3 g/ml. The treated samples were left over night and sun dried. Tannic acid treatment: Tannic acid was dissolved in water at the rate of 5 and 10 g/kg. Castor cake samples of 1000 g each were mixed with tannic acid solution in the ratio of 3 g/ml and allowed to react overnight. The treated cake was sun dried and stored for further studies. Sodium hydroxide treatment: Sodium hydroxide at 0.18, 0.38 and 0.75N strength was used to treat the cake. Cake samples of 1000 g each were mixed with sodium hydroxide solution at the rate of 3 g/ml. The treated cake was left overnight and sun dried for further studies.

2.2. Estimation of ricin The untreated and the treated samples were analyzed for the toxin, ricin as per the method of Kabat et al. (1947) with modications as suggested by Waller and Negi (1958).

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2.2.1. Extraction of ricin The solvent extracted castor cake sample of 500 g was extracted with 2.5 l of water acidied with HCl to a pH of 3.8 by shaking the contents in a conical ask for 6 h. The contents were allowed to settle and were then ltered through Whatman lterpaper-1. The residue was treated with 1.5 l of distilled water, shaken for 3 h and ltered through the same paper. A second treatment with water was given to the residue and ltered again through the same lterpaper. This ltrate contains all the ricin and portions of ricinine that are soluble in cold, dilute HCl. It was evaporated to a small volume by vacuum distillation below 40 C. The ltrate was saturated with sodium chloride and centrifuged at 4000 rpm for 20 min to separate the precipitate containing only ricin. 2.2.2. Purication of ricin The precipitate was dissolved in deionised and distilled water. It was reprecipitated at pH 8 by saturation with ammonium sulphate. After reprecipitation twice more with the same salt, they were dissolved in deoinised water and dialyzed at 4 C for a period of 72 h against tris buffer adjusted to pH 6.8 using dialysis membrane-110 (HIMEDIA). The buffer was changed once in 2 h for the rst 12 h and subsequently once in 6 h for the remaining period. After 72 h, the dialysate was centrifuged at 4000 rpm for 10 min to separate the insoluble matter from the clear solution containing ricin. The solution was concentrated under vacuum and the amount of solution was measured. The ricin was estimated as the quantity of protein present in the dialysate as per the method of Lowry et al. (1951). The extract of the castor cake obtained after dialysis were subjected to SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) as per the method of (Laemmli, 1970) for qualitative determination of the ricin. 2.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracted ricin The ricin obtained after dialysis was subjected to denaturing-PAGE analysis using sodium dodecyl sulphate as denaturing agent. An extract from untreated castor cake (control) equivalent to about 50 g protein and protein (ricin extracted and puried) from treated material on an equivalent basis to the amount of untreated cake that yielded 50 g protein were mixed with sample buffer containing 20 ml mercapto-ethanol and 200 ml glycerol per liter of 0.5 M tris HCl (pH 6.8) to make 40 l volume and loaded in each well. Proteins were separated in 12.5% acrylamide gel (10 cm 10 cm 0.1 cm) prepared as per the method of Laemmli (1970) on which 5% stacking gel (4 cm 10 cm 0.1 cm) was layered. Electrophoresis condition was set at 40 V till the dye front reached the separating gel and then xed at 60 V till the samples reached the end. After complete run, protein was xed for 1 h in a solution containing methanol (500 ml) and acetic acid (100 ml) and then stained overnight using a solution containing coomassie brilliant blue-R, dye (2.5 g), methanol (400 ml) and acetic acid (100 ml). The protein bands in the gel were visualized by destaining using a solution containing methanol (250 ml) and acetic acid (70 ml). Molecular weights of the stained bands of extracted ricin in control and treated cake were determined using known molecular weight markers (Sigma, USA).

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Treatment of cake was considered effective where no protein bands were visible in the stained gel compared to the two bands obtained in the control sample.

3. Results and discussion The ricin content in the untreated castor sample was 388 mg/kg. Although there are claims (Kim, 2001) that the normal extraction and desolventisation processes for meals are capable of total destruction of ricin, the presence of ricin in the castor cake indicate that the normal processing methods are not capable of destroying the toxin totally and there is a need to detoxify this for further use as livestock feed. The quantity of ricin and the percentage decrease in the differently processed castor cake samples are given in the Table 1. The physical treatments were selected based on the fact that ricin is both heat labile and soluble in water. All the treatments employed were able to decrease ricin content to different extents. Autoclaving at 15 psi for 30 min could decrease the toxin by about 85%. Increasing the time to 60 min completely removed the toxin from castor cake. Autoclaving at various pressures (1020 psi) and duration (1560 min) was earlier tried to detoxify the castor meal (Jaki, 1940; Ambekar and Dole, 1957; Okamato et al., 1965; Mottola et al., 1971). Autoclaving highly toxic castor pomace for periods of 15 min or more had resulted in essentially complete destruction of the toxin with minimal changes in the physical character of the toxin (Kodras Rudolph et al., 1949). Heating the cake (100 C for 30 min and 120 C for 25 min) was least effective of all the methods in decreasing the ricin content. The heating of the cake could decrease the toxin by about 50%. Dry heat does not seem to have much effect on bringing down the toxin levels in the castor cake (Heller, 1932). In their studies Ambekar and Dole (1957) reported the heating of castor bean meal to 150 C for 3 h did not reduce the toxin levels and feeding the heat treated cake resulted in mortality of rats. However, few reports also exist (Tangl, 1938; Okorie et al., 1985) showing the benecial effects of heat treatment at various time and temperatures in removal of the toxin. Boiling in water was more effective than steaming as it takes into account the watersoluble and heat labile nature of the toxin. The earlier attempts to detoxify the toxin by steaming at different temperature and time combinations were not successful (Borchers, 1949; Okorie et al., 1985). The absence of toxic symptoms in rats and chicks fed hot water extracted castor cake indicated that the water treatment was more effective (Ambekar and Dole, 1957; Vilhjalmsdottir and Fisher, 1972). Soaking for 3 h decreased ricin by 65% and when the duration was increased to 6 h, a decrease of 85% was seen. However, no further changes were observed when the duration of soaking was increased to 12 h showing that soaking for 6 h was the optimum one. Among the different chemical treatments that were tried, lime and sodium hydroxide treatments were the most effective. Lime being a weaker alkali at lower levels the efcacy was not optimum and only at higher levels of 40 g/kg optimum efcacy could be achieved. Sodium hydroxide being a stronger alkali it was effective even at smaller levels. About 2.5 g/kg of sodium hydroxide could reduce the toxin by about 82% and when the concentration was increased to 10 g/kg, the reduction was up to 91%. Increasing the concentration of sodium hydroxide could increase the elimination of the toxin showing that the toxin is

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Table 1 Quantication of ricin (mg/kg) and percentage decrease in different processed castor cake samples Treatments Untreated sample 1. Soaking (h) 3 6 12 2. Steaming (min) 30 60 3. Boiling (min) 30 60 4. Autoclaving 15 psi, 30 min 15 psi, 60 min 5. Heating 100 C, 30 min 120 C, 25 min 6. Ammonia (ml/kg) 7.5 12.5 7. Formaldehyde (g/kg) 5 10 8. Lime (g/kg) 10 20 40 9. Sodium chloride (g/kg) 5 10 20 10. Tannic acid (g/kg) 5 10 11. Sodium hydroxide (g/kg) 2.5 5 10 Ricin (mg/kg) 388 136 55 62 103 57 38 37 58 187 193 191 158 236 75 129 144 78 85 70 180 282 69 53 36 65 86 84 73 85 90 91 85 100 52 50 51 59 39 81 67 63 100 82 86 91 54 27 82 86 91 %Decrease

highly susceptible to the strong alkalis. Treatment with sodium chloride failed to detoxify the toxin ricin completely as only 80% reduction in the toxin level was recorded. Not much difference was found among the different concentrations of sodium chloride indicating that the maximum reduction is obtained at 5 g/kg levels. Formaldehyde at lower levels (5 g/kg)

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was ineffective while at higher levels (10 g/kg) it was partially effective. The chemical treatments with ammonia and tannic acid were not effective as the percent reduction in the toxin was below 60%. Since the principal toxicants in the castor cake are soluble proteins, tannins could be used to minimize their effects. In studies with rats tannins have been successfully used to neutralize the toxic effect of castor meal extract in rats based on the ability of the tannins to react with proteins to form tannin protein complexes that interfere with digestibility and absorption of the latter (Gandhi and Mulky, 1994). The effect of various treatments on detoxication of ricin by earlier workers is primarily based on oral toxicity studies, neutralization and heamagglutination studies unlike the present study where ricin quantication has been used to assess the efcacy. Oral toxicity and neutralization studies are carried out using experimental animals where the response is inuenced by different factors like species, age, and quantum of the detoxied material and duration of feeding. As far as heamagglutination is concerned it has been identied that a separate protein other than ricin that is closely bound to ricin is responsible for the agglutination property (Ishiguro and Takashishi, 1964; Walkschmidt-Leitz and Keller, 1969). The

Fig. 1. PAGE showing molecular weight of ricin; MM, molecular marker; C, control.

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above factors have to be considered while comparing the present ndings with the earlier ndings. 3.1. PAGE analysis of extracted ricin Ricin, the toxic protein present in the castor cake is a heterodimer consisting of two chains of 32 and 34 kilo dalton (kD) connected with a disulphide bond (Robertus, 1988; Frigerio and Roberts, 1998). Several isoforms of ricin exist (Lord et al., 1994) and the molecular weights of the two chains are reported to be 30 kD in each by Rutenber (1991) but as 32 kD in each by Olsnes and Pihl (1973). Ricin appears as two bands representing two chains on PAGE (poly acryl amid gel electrophoresis). The bands could be identied based on the molecular weights. The ricin extracted from untreated samples (control) showed clear-cut two bands one at approximately 35 kD and another at 29 kD molecular weight range (Fig. 1). The bands in the treated groups varied in their intensity depending on the efcacy of the treatment in the treatment groups. The complete disappearance of the bands suggested that the treatments are most effective in removal of the toxin. Amongst all the treatments tried, Autoclaving at 15 psi for 60 min and lime treatment at 40 g/kg levels were the most effective as evidenced by the total disappearance of the bands (Figs. 2 and 3). These observations were in accordance with the quantication of the toxin (Table 1). In view of the potent nature of the toxin ricin, it is essential that the detoxication process should be capable of destroying the toxin to the maximum extent so that it could be safely fed to any category of livestock irrespective of species and age. The results have revealed that amongst all the methods tried autoclaving at 15 psi for 60 min and 40 g/kg lime treatment gave the best results in terms of reduction in the toxin ricin (100%) and the same was

Fig. 2. PAGE showing the effect of different treatments on ricin; C, control; A1, autoclaving (15 psi, 30 min); A2, autoclaving (15 psi, 60 min); H1, heating (100 C, 30 min); H2, heating (120 C, 25 min); Am1, ammonia treatment (7.5 ml/kg); Am2, ammonia treatment (12.5 ml/kg).

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Fig. 3. PAGE showing the effect of different treatments on ricin; C, control; L1, lime (10 g/kg); L2, lime (20 g/kg); L3, lime (40 g/kg); S1, sodium hydroxide (2.5 g/kg); S2, sodium hydroxide (5 g/kg); S3, sodium hydroxide (10 g/kg).

conrmed by poly acrylamide gel electrophoresis study wherein the bands corresponding to ricin were not visible implying complete destruction.

Acknowledgements The authors are grateful to the Director of the institute for providing the necessary facilities for carrying out the present study. The nancial assistance from NATP is duly acknowledged.

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