• Vol 10, No 3 (2019)
  • Chemical Engineering

Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology

Mohamed Hawashi, Hakun Aparamarta, Tri Widjaja, Setiyo Gunawan

Corresponding email: gunawan@chem-eng.its.ac.id


Cite this article as:
Hawashi, M., Aparamarta, H., Widjaja, T., Gunawan, S., 2019. Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology. International Journal of Technology. Volume 10(3), pp. 624-633
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Mohamed Hawashi -Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS) -Central Scientific Research Laboratory, Sebha University, Sebha
Hakun Aparamarta Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, 60111, Indonesia
Tri Widjaja Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya 60111, Indonesia
Setiyo Gunawan Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya 60111, Indonesia
Email to Corresponding Author

Abstract
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Cassava leaves are a good source of protein. However, their use is limited because of the presence of cyanogenic glucosides. These require a further detoxification process in order to reduce the cyanide to a safe level prior to human consumption. The main objectives of this work are: (i) to demonstrate the effectiveness of solid-state fermentation using Saccharomyces cerevisiae on the cyanide content degradation of cassava leaves; and (ii) to optimize the independent variables for the minimum cyanide content level of cassava leaves by the application of response surface methodology (RSM). The various process parameters investigated for these purposes were sucrose concentration, urea concentration, moisture content, and fermentation time. The degradation of cyanide content was described by the quadratic model, which resulted in an excellent fit of the experimental data (p < 0.01). The statistical tests show that linear terms for sucrose concentration, urea concentration, moisture content and fermentation time had a significant effect on cyanide content (p < 0.01). Moreover, the interaction coefficients between sucrose concentration and fermentation time; urea concentration and moisture content; and nitrogen concentration and fermentation time were significant model terms (p < 0.05). A minimum cyanide content of 0.81 ppm was obtained at 1% (w/w) sucrose concentration, 0.5% (w/w) urea concentration, 60% (v/w) moisture content and with a fermentation time of 78 hours. The optimal level made a significant reduction in cyanide content of 97.96%, which is lower than the toxicity level suggested by the World Health Organization of 10 ppm.

Cassava leaves; Cyanide content; Response surface methodology; Solid state fermentation

Introduction

With the growth in food consumption, the majority of people rely heavily on food crops as their primary food sources. Root crops, such as cassava, are grown in developing countries as a primary source of carbohydrates (Hawashi et al., 2018). This crop represents one of the primary sources of food for Indonesian people, along with other staples such as rice, sago and corn.  Reports indicate a production rate of nearly 20 million tons per year, harvested from 1.93 million hectares (Agustian, 2016). Cultivation of cassava plants can take place even in marginal environmental conditions, due to their high drought tolerance, with an optimal yield of approximately 50% for leaves and 6% for roots upon plant maturity (Tewe & Lutaladio, 2004). Cassava leaves contain valuable protein and nutrients and are consumed in many countries, including Indonesia, Malaysia, the Congo, Madagascar and Nigeria (Latif & Muller, 2015). However, they contain both nutritive and non-nutritive compounds. Among the anti-nutrients, particular concern is paid to cyanide acid (HCN), whose concentration in fresh cassava leaves is much higher than the safe limit recommended by the World Health Organization (WHO) for human consumption (10 ppm). As an effect of consuming a high concentration of cyanide, HCN poses health problems to the human body. Such a condition is known as Konzo disease, an irreversible neurological disorder associated with cyanide consumption (Bradbury, 2006). Other long-term exposure to cyanogenic glycosides from eating cassava includes tropical ataxic neuropathy, neurological effects, and damages to goiter, and thyroid functions (WHO, 2008). In addition, HCN is an inhibitor of the oxidation processes occurring in the mitochondria, which can lead to chronic toxicity. Therefore, it is essential that the content of cyanide be reduced below 10 ppm to allow people to safely consume cassava (WHO, 1995).

Reduction of cyanide levels can also be made in cyanide-rich raw food sources such as cassava.

Worldwide, the most common methods of cassava leaf processing include boiling and soaking in water, steaming, sun drying, and oven drying. These approaches aim to reduce the toxic compounds in the leaves for human consumption (Fasuyi, 2005). Cassava leaf processing is mainly based on the endogenous cassava enzyme (linamarase), which catalyzes the conversion of cyanide-containing compounds (linamarin) into acetone cyanohydrin, which either enzymatically or spontaneously decomposes into HCN and acetone (Montagnac et al., 2009). However, some methods (such as steaming and oven drying) have been proven to be ineffective for lowering the cyanide content in cassava leaves to the safe limit. Studies have shown that the fermentation of cassava leaves is a promising method for reducing cyanide content (Kobawila et al., 2005; Morales et al., 2018). These reports show respective reductions of at least 70% and 94.18% in cyanide content during fermentation. They further validate the preference for the fermentation technique over conventional methods. The SSF technique has several advantages, including high productivity and reduced processing time (Febrianti et al., 2017). However, reports indicating the efficiency of cassava leaf fermentation are quite scarce compared to those which investigate tubers.

Various process conditions such as moisture content, pH, inoculum size, fermentation time, concentration of nutrient supplementation and temperature can affect the microbial growth, enzyme production, and formation of the product during the fermentation process (Ezekiel & Aworh, 2013). The optimization processes using the “One Variable at One Time (OVAT)” technique (changing one single variable, while keeping others at constant levels) is an inefficient way of determining the interaction between the process variables as it involves high cost and requires various experiments to obtain the optimum levels (Braga et al., 2011; Hadiyat & Wahyudi, 2013). Recently, the application of RSM has attracted the attention of researchers working with fermentation to optimize process conditions and evaluate the correlation between independent variables and their responses (Istianah et al., 2018).

Yeast and lactic acid bacteria (LAB) are the most investigated microorganisms for the production of linamarase during cassava fermentation and the development of flavor. Yeast, such as Saccharomyces cerevisiae, has several advantages, including its availability, low cost, ability to secrete extracellular enzymes, non-pathogenic character, and widespread use in traditional fermentation, particularly in fermented foods (Oboh & Akindahunsi, 2003). Furthermore, Saccharomyces cerevisiae is able to use cyanogenic glucosides and their metabolites during food processing, making it one of the micro-organisms which is most involved in the cassava fermentation process (Lambri et al., 2013). Therefore, the objective of this work is to demonstrate the effectiveness of solid-state fermentation using Saccharomyces cerevisiae in the reduction of cyanide in cassava leaves. Furthermore, the optimization of the independent variables (moisture content, incubation time and nutrient supplementation) to achieve a minimum cyanide content level in cassava leaves by employing response surface methodology (RSM), is also studied in detail.


Conclusion

The research has investigated the effect of solid state fermentation using Saccharomyces cerevisiae on the removal of cyanide content from cassava leaves.  The study has shown that response surface methodology (RSM) was a high-performance technique for optimization of the process conditions for minimizing cyanide content in fermented cassava leaves through solid-state fermentation. The optimal process condition was obtained at 1% (w/w) sucrose concentration, 0.5% (w/w) urea concentration and 60% (v/w) moisture content, with a fermentation time of 78 hours. It was observed that an exponential decrease in cyanide content over time can lead to satisfactory detoxification in cassava leaves, with cyanide concentration falling to levels lower than 10 ppm after 60 hours of fermentation, and thus providing a safe and healthy food source.

Acknowledgement

This work was supported by grant no. 849/PKS/ITS/2018 provided by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia.

Supplementary Material
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References

Agustian, A., 2016. Bioenergy Development in the Agricultural Sector: Potential and Constraints on the Development of Bioenergy Made from Cassava. Agricultural Policy Analysis, Volume 13(1), pp. 19–38

Bradbury, J.H., 2006. Simple Wetting Method to Reduce Cyanogen Content of Cassava Flour. Journal of Food Composition and Analysis, Volume 19(4), pp. 388–393

Braga, F.R., Araújo, J.V., Soares, F.E.F., Araujo, J.M., Geniêr, H.L.A., Silva, A.R., Carvalho, R.O., Queiroz, J.H., Ferreira, S.R., 2011. Optimizing Protease Production from an Isolate of the Nematophagous Fungus Duddingtonia Flagrans using Response Surface Methodology and Its Larvicidal Activity on Horse Cyathostomins. Journal of Helminthology, Volume 85(2), pp. 164–170

Ezekiel, O.O., Aworh, O.C., 2013. Solid State Fermentation of Cassava Peel with Trichoderma Viride (ATCC 36316) for Protein Enrichment. International Journal of Nutrition and Food Engineering, Volume 7(3), pp. 6892–6991

FAO/WHO, 1995. Codex Standard for Edible Cassava Flour. In: Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission (Vol. Codex Standard 176-1989), Rome, Italy

Fasuyi, A.O., 2005. Nutrient Composition and Processing Effects on Cassava Leaf (Manihot esculenta, Crantz) Antinutrients. Pakistan Journal of Nutrition, Volume 4(1), pp. 37–42

Febrianti, F., Syamsu, K., Rahayuningsih, M., 2017. Bioethanol Production from Tofu Waste by Simultaneous Saccharification and Fermentation (SSF) using Microbial Consortium. International Journal of Technology, Volume 8(5), pp. 898–908

WHO/FOOD, 2008. Safety Evaluation of Certain Food Additives and Contaminants. Rome, Italy

Grover, A., Maninder, A., Sarao, L.K., 2013. Production of Fungal Amylase and Cellulase Enzymes via Solid State Fermentation using Aspergillus Oryzae and Trichoderma Reesei. International Journal of Advanced Research in Education & Technology, Volume 2(8), pp. 108–124

Gunawan, S., Widjaja, T., Zullaikah, S., Ernawati, L., Istianah, N., Aparamarta, H.W., Prasetyoko, D., 2015. Effect of Fermenting Cassava with Lactobacillus Plantarum, Saccharomyces Cereviseae, and Rhizopus Oryzae on the Chemical Composition of their Flour. International Food Research Journal, Volume 22(3), pp. 1280–1287

Hawashi, M., Ningsih, T.S., Cahyani, S.B.T., Widjaja, K.T., Gunawan, S., 2018. Optimization of the Fermentation Time and Bacteria Cell Concentration in the Starter Culture for Cyanide Acid Removal from Wild Cassava (Manihot glaziovii). In: MATEC Web of Conferences, Article Number 01004, Volume 156

Hadiyat, M.A., Wahyudi, R.D., 2013. Integrating Steepest Ascent for the Taguchi Experiment: A Simulation Study. International Journal of Technology, Volume 4(3), pp. 280–287

Istianah, N., Ernawati, L., Anal, A.K., Gunawan, S., 2018. Application of Modified Sorghum Flour for Improving Bread Properties and Nutritional Values. International Food Research Journal, Volume 25(1), pp. 166–173

Kobawila, S.C., Louembe, D., Keleke, S., Hounhouigan, J., Gamba, C., 2005. Reduction of the Cyanide Content during Fermentation of Cassava Roots and Leaves to Produce Bikedi and Ntoba Mbodi, Two Food Products from Congo. African Journal of Biotechnology, Volume 4(7), pp. 689–696

Lambri, M., Fumi, M.D., Roda, A., De Faveri, D.M., 2013. Improved Processing Methods to Reduce the Total Cyanide Content of Cassava Roots from Burundi. African journal of biotechnology, Volume 12(19), pp. 2685–2691

Latif, S., Müller, J., 2015. Potential of Cassava Leaves in Human Nutrition: A Review. Food Science & Technology, Volume 44(2), pp. 147–158

Mason, R.L., Gunst, R.F., Hess, J.L., 2003. Statistical Design and Analysis of Experiments: With Applications to Engineering and Science. John Wiley & Sons, Hoboken, New Jersey, United States

Montagnac, J.A., Davis, C.R., Tanumihardjo, S.A., 2009. Processing Techniques to Reduce Toxicity and Antinutrients of Cassava for Use as a Staple Food. Food Science and Food Safety, Volume 8(1), pp. 17–27

Morales, E.M., Domingos, R.N., Angelis, D.F., 2018. Improvement of Protein Bioavailability by Solid-State Fermentation of Babassu Mesocarp Flour and Cassava Leaves. Waste and Biomass Valorization, Volume 9(4), pp. 581–590

Nwabueze, T.U., Odunsi, F.O., 2007. Optimization of Process Conditions for Cassava (Manihot esculenta) Lafun Production. African Journal of Biotechnology, Volume 6(5), pp. 603–611

Oboh, G., Akindahunsi, A.A., 2003. Biochemical Changes in Cassava Products (Flour & Gari) Subjected to Saccharomyces Cerevisae Solid Media Fermentation. Food Chemistry, Volume 82(4), pp. 599–602

Rana, B., Awasthi, P., Kumbhar, B.K., 2012. Optimization of Processing Conditions for Cyanide Content Reduction in Fresh Bamboo Shoot during NaCl Treatment by Response Surface Methodology. Journal of Food Science and Technology, Volume 49(1), pp. 103–109

Silvestrini, R.T., Montgomery, D.C., Jones, B., 2013. Comparing Computer Experiments for the Gaussian Process Model using Integrated Prediction Variance. Quality Engineering, Volume 25(2), pp. 164–174

National Standardization Agency of Indonesia (SNI), Standard, 2011. Modified Cassava Flour. SNI 7622, Jakarta, Indonesia

Tewe, O.O., Lutaladio, N., 2004. Cassava for Livestock Feed in Sub-Saharan Africa. FAO/IFAD, Rome, Italy