Assessing the effectiveness of ash soaking in reducing hydrogen cyanide (HCN) levels in cassava peel

Barnabas Gairtua¹, Ristianto Utomo², Chusnul Hanim², Zein Ahmad Baihaqi³ and Wulandari^3*

¹ Faculty of Agriculture, Universitas Pattimura, Jl. Ir. M. Putuhena, Poka, Kecamatan Tlk. Ambon, Kota Ambon, Maluku - 97233, Indonesia
² Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta - 55281, Indonesia
³ Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN), Bogor, Cibinong, Kabupaten Bogor, Jawa Barat - 16915, Indonesia
* wulandari.1@brin.go.id

Abstract

This research aims to investigate the effects of soaking and drying cassava peel with absorbent material (ash) to reduce HCN levels, as well as to determine the concentration of ash and the duration required for HCN reduction. The study was conducted with two factors: the level of ash usage and the duration of soaking and drying. Cassava peel was soaked and dried with ash levels of 0%, 5%, 10%, and 15% dry matter for 12, 24, 36, and 48 hours, respectively. The HCN content of cassava peel was tested using factorial pattern analysis of variance (ANOVA) as the research design. If there were treatment differences, a new Duncan's Multiple Range Test (DMRT) was performed. The results of the study indicate that soaking and drying cassava peel with varying concentrations and durations of ash significantly reduced HCN levels (p<0.05) compared to the control group. The best results in reducing HCN have been achieved by soaking cassava peel with 10% ash for 48 hours (T1) and drying cassava peel with 15% ash for 48 hours (T2). Both underwent in vitro analysis of In Vitro Dry Matter Digestibility (IVDMD) and In Vitro Organic Matter Digestibility (IVOMD), as well as evaluation of rumen fermentation characteristics. Based on the in vitro results, it can be demonstrated that the reduction in HCN also improves feed efficiency, as evidenced by increased levels of IVDMD, microbial protein, and Volatile Fatty Acids (VFA) (p<0.05).

Key words: ash soaking, ash drying, cassava peel, feed additive, HCN reduction, ruminant

Introduction

The livestock industry in Indonesia is currently making progress, but this progress is not balanced with land availability, leading to an impact on feed continuity. Other agricultural products hold significant potential for meeting the feed requirements of ruminants (Prasetyawan et al 2012). However, to use agricultural residues as local feed ingredients, three key aspects must be addressed: quantity, quality, and continuity (Rahmawati et al 2020). Cassava peel is part of agricultural waste products that are abundantly available and have potential as a raw material for feed. The potential abundance of cassava peels presents an opportunity for processing and using it as animal feed.

In Indonesia, cassava peel has been widely used as animal feed for livestock, both for fattening and breeding. Farmers in Indonesia can use cassava peel as an alternative feed or additional feed to meet the needs of their livestock. Cassava peel can be given to livestock directly or can be fermented first (Hersoelistyorini 2012; Agustin et al 2020).

The nutritional value of cassava peel is as follows: dry matter content is 17.45%, protein is 8.11%, crude fiber is 15.20%, crude fat is 1.29%, calcium is 0.63%, and phosphorus is 0.22% (Nurlaili et al 2014). Sandi et al (2013) stated that cassava peel contains 7.2% lignin, 13.8% cellulose, and 109 ppm HCN (Hydrogen Cyanide). The wild yam tubers have a HCN content of 84.26 ppm, and a total cyanide content ranging from 379 to 739 ppm (Kumoro et al 2011; Saleha et al 2018). This allows cassava peel to be used as animal feed, especially for ruminants. However, the acceptable level of HCN that livestock can tolerate is not more than 50 ppm. If the HCN content exceeds this limit, it can have adverse effects on livestock, such as death (Gensa 2019). Therefore, cassava peel can be given to livestock as feed without having a detrimental effect on the animals that consume it. Several ways to process it include soaking and drying using ash material. Due to these considerations, it is essential to research to assess the impact of soaking and drying cassava skins with an absorbent material (ash) for HCN content removal. This research aims to determine the effects of the ash concentration, soaking time, and drying time on HCN content. Additionally, it seeks to investigate digestibility and the characteristics of in vitro rumen fermentation.

Materials and methods

This research was conducted at the Faculty of Animal Science, Universitas Gadjah Mada. The research results were analyzed at the Feed Technology Laboratory and the Agricultural Technology Laboratory at Universitas Gadjah Mada. The cassava peel used in this research was obtained from the bitter cassava variety (known locally as 'singkong pahit') found in Gunungkidul Regency, Yogyakarta, Indonesia. Cassava peel is obtained by peeling cassava to separate the tubers from the peel.

The preparation and the process of soaking and drying cassava peel

Soaking cassava peel with ash uses different levels, namely 5, 10, and 15% dry matter, and the soaking and drying time are 12, 24, 36, and 48 hours. Soaking is carried out with a composition of 2 liters of water, 500 grams of cassava peel, and the addition of ash according to the treatment level. Samples were collected for each soaking treatment and each drying treatment to measure the reduction in HCN content. The HCN was analyzed was conducted according to the official methods of analysis (AOAC 2005).

Digestibility and the characteristics of in vitro rumen fermentation

A 0.5 g sample of cassava peel was weighed to assess its digestibility and fermentability levels. The digestibility values of dry matter and organic matter were determined using the in vitro method (Tilley and Terry 1963). After the in vitro process, rumen fluid was analyzed for ammonia (Chaney and Marbach 1962), microbial protein (Plummer 1987), and volatile fatty acids (Filipek and Dvorak 2009).

Statistical analysis

This research employed a completely randomized design (CRD) with a factorial pattern. Each treatment was replicated five times. The research data were statistically analyzed using analysis of variance (ANOVA). In cases where differences were observed in the data, Duncan's Multiple Range Test (Steel and Torry 1997) was applied. Digestibility data and rumen fermentation parameters were analyzed using the T test. The calculations were performed using SPSS version 25 software.

Results and Discussion

Effect of soaking on HCN concentration

Cyanogen glucoside is a secondary compound in cassava plants, consisting of linamarin and lotaustralin. HCN is produced from the hydrolysis of linamarin by the enzyme linamarase. High HCN content can result in livestock poisoning, so efforts to reduce HCN content, including soaking, are necessary (Wheatley et al 2003; Kennedy et al 2021). The results of the analysis of HCN content in cassava peel after soaking are presented in Table 1.

The data presented in Table 1 show that the treatment of ash levels and soaking time had a significant effect (p<0.05) on reducing the HCN content of cassava peel. The lowest HCN content was obtained in the treatment with an ash level of 10% and a soaking time of 48 hours, resulting in an HCN content of 7.82 ppm. An increase in the addition of 15% ash and 48 hours was observed, but it was not statistically significant and remained within the tolerance limit for livestock, with an HCN content of 13.55 ppm. This phenomenon is influenced by the material's saturation level in absorbing the release of HCN in cassava peel. Soaking with ash can reduce the concentration of HCN because the ash used as an absorbent material contains compounds with basic elements such as Ca and K. The ash solution is alkaline, and polyphenols are soluble in water and alkaline, so the remaining HCN levels decrease after the polyphenols in the dissolved solution are removed through soaking. The husk ash can act as a good absorbent, effectively absorbing and binding polyphenolic compounds (Sembodo 2005; Hawashi et al 2019).

The decrease in HCN levels in cassava peel is attributed to higher ash concentration and longer soaking time. This phenomenon occurs because there is an increasing number of polyphenolic compounds in HCN, which are bound by the calcium (Ca) and potassium (K) elements present in the ash. The longer the cassava peel remains in the ash solution due to prolonged soaking, the more time the ash has to bind HCN, increasing the dissolved HCN content in the cassava peel (Mukhtar et al 2023). Zarnila et al (2021) noted that one of the factors influencing the solubility of a substance is soaking time, where longer contact time between the material and the solvent (water) leads to the dissolution of more compounds from the material.

The addition of ash during the soaking process can accelerate the reduction of HCN because the calcium (Ca) and potassium (K) elements contained in the ash can bind cyanide, thus expediting the reduction of cyanide in cassava peel. Ash can draw out cyanide from the material and then move through the pores, being absorbed into the inner walls, resulting in a decrease in cyanide content. The results obtained in this study differ from those reported by Wahyu et al (2020), who stated that soaking cassava with husk ash for 24 hours could reduce HCN levels to 3.07 ppm. Appenteng et al (2021) explained that HCN in food can be reduced by soaking it in water because HCN is a compound that readily dissolves in water.

Soaking with water can break down HCN by breaking the cyanogenic glycoside bonds, allowing much of the HCN to dissolve and be carried away by the water (Sudharmono et al 2016; Kurniawan et al 2019). When immersing in water, diffusion and osmosis processes also occur. Diffusion during soaking happens as the remaining substances in the feed ingredients dissolve. This is characterized by a change in the water's color or foaming, which suggests that one of the soluble substances is HCN due to its easy solubility in water. The osmosis process stops when the levels of the two substances have reached equilibrium. Cyanide exhibits autohydrolysis properties at 25°C. It is believed that HCN will evaporate more readily at higher temperatures; in other words, the higher the temperature, the greater the decrease in HCN levels in feed ingredients (Feng et al 2003). To date, it is known that HCN is highly soluble in water and can be evaporated through heating, as suggested by Das et al (2019).

Effect of drying on HCN concentration

The results of the analysis of HCN content in cassava peel after drying are presented in Table 2. The results of the statistical analysis indicate that the treatment of ash levels has a significant effect (p<0.05) on the reduction of cyanogenic content (HCN) in cassava peels. The lowest HCN content, 75.53 ppm, was obtained with an ash level of 15% and a duration of 48 hours. In contrast, the highest HCN content, 290.32 ppm, was observed in the treatment without ash (0%). The analysis of fresh cassava peel HCN content also showed a value of 291 ppm. Overall, the mean values suggest a decreasing trend in HCN content with longer durations and higher ash levels.

The safe concentration of hydrogen cyanide (HCN) for livestock is 50 ppm. The decrease in cyanide is relatively slow due to the dense structure of bitter cassava (high starch content) and low water content, while water accelerates the activation of glycosides in the cyanide hydrolysis reaction for cyanide release. Cassava containing cyanide up to 100 ppm can still be tolerated in terms of toxicity if the protein supplement ratio (especially sulfur-containing amino acids) and iodine are adequate in the feed mixture because cyanide will bind to sulfur and form thiocyanate, which is excreted through urine, as shown by Tweyongyere and Katongole (2002).

Hydrogen cyanide is a volatile-free compound. The presence of wind and the heat of sunlight at a moderate temperature prevents the deactivation of the enzyme linamarase. Processes such as grating, grinding, and other methods of tissue disruption are employed to effectively decrease cyanide levels by promoting the interaction between cyanogenic compounds in the vacuole and ß glucosidase in the cell wall. This interaction results in the conversion of cyanogenic glucosides into hydrogen cyanide (HCN). The removal of HCN can be easily achieved through heating or solubilization, as highlighted by Ferraro et al (2016). Limited research has been conducted on detoxifying wild yam tubers, with some studied methods including leaching and steaming (Kumoro et al 2011), as well as boiling, roasting, and soaking in flowing water (Ashri et al 2014).

During a process commonly known as cyanogenesis, the stability of certain compounds is compromised when the ß-glycosidic linkage is hydrolyzed by a ß-glycosidase, such as linamarase. This hydrolysis results in the formation of acetonecyanhidrin (Gleadow and Moller 2014). The unstable cyanohydrin can undergo decomposition through the activity of bacterial and/or endogenous ß-glucosidase or spontaneously at temperatures exceeding 30°C (Montagnac et al 2009; Gleadow and Moller 2014). The breakdown of linamarin presented in Figure 1 (adapted from Montagnac et al 2009).

Figure 1. The breakdown of linamarin

Drying using ash is one of the methods employed to eliminate hydrogen cyanide (HCN) content in cassava peels because ash can inhibit the rate of poison oxidation and neutralize the carcinogenic acid present in it. This has been proven effective in the reduction of HCN content in cassava peels. Soaking Dioscorea hispida Dennst tubers with an ash concentration of 75% for 24 hours can reduce cyanide levels by 38.69% (Harimu et al 2020).

Digestibility and rumen fermentation profile of cassava peel

The digestibility data and in vitro fermentation parameters of cassava peels soaked with the addition of 10% ash level for 48 hours (T1) and cassava peels dried with the addition of 15% ash level for 48 hours (T2) after incubation for 48 hours are presented in Table 3.

The research results indicate that cassava peels soaked with the addition of a 10% ash level (T1) have higher values of IVDMD and IVOMD compared to T2 (p<0.05). This is consistent with the HCN value in the treatment of soaking cassava peel with a 10% ash level for 48 hours, which has a lower HCN value compared to drying cassava peel with a 15% ash level for 48 hours (Tables 1 and 2). The low HCN value may reduce the toxic risk of HCN to rumen microbes, thus optimizing the process of feed degradation by rumen microbes. This decrease in HCN levels is caused by HCN being easily soluble in water. The longer the soaking time, the softer the cassava peel becomes, and the HCN within the cassava peel cells will come out and dissolve in water (Agustin et al 2022).

Digestion of protein by fermentation in the rumen produces a fermentation product, namely ammonia. The low HCN value resulting from the process of soaking cassava peel in ash has a positive effect. This is indicated by the higher IVDMD and IVOMD values at T1 compared to T2. Additionally, it also demonstrates higher NH3 values at T1 (7.53 mg/100 mL) compared to T2 (p<0.05). This suggests that the dry material components, especially the proteins in cassava skin can be degraded by rumen microbes, particularly through proteolysis into ammonia. The NH₃levels in this study met the minimum required for rumen microbial protein synthesis, namely 5 mg/100 mL (Satter and Slyter. 1974). NH ₃ levels are determined not only by the protein content of the feed but also by the availability of energy for microbes to degrade the protein in the feed (Agustin et al. 2022).

The microbial protein concentrations in the two treatments exhibited significant differences (p<0.05). The microbial protein concentration at T2 is lower than that at T1. This decline correlates with the decrease in ammonia and VFA concentrations at T2, as microbial protein production is predominantly influenced by the availability of N-NH ₃ in the rumen. The rumen microbial population increases when the availability of nutrients, in the form of nitrogen and carbon supplies, meets microbial needs, thereby enhancing microbial protein synthesis (Membrive. 2016; Öztürk and Gür. 2021).

The results indicate that the soaking and drying treatments of cassava peel with ash yielded significant differences (p<0.05) in the total VFA and partial VFA concentrations of rumen fluid (Table 3). The total VFA and partial VFA concentrations at T1 were higher than those at T2, which aligns with the higher IVDMD value at T1 compared to T2. The quantity of VFA formed is influenced by the digestibility and quality of the fermented ration. Volatile fatty acids are generated from the degradation process of crude fiber (CF) by microorganisms, thus the CF content in the ration significantly affects the VFA formation (Hapsari et al. 2018). The concentration of VFA in the rumen fluid varies depending on the type of feed provided. The resultant VFA concentration can serve as an energy source and carbon framework for the formation of microbial proteins (McDonald et al. 2011; Agustin et al. 2022). Our assumption regarding the low VFA concentration in this study is attributed to HCN and adjusted according to its concentration. Jayanegara et al. (2019) stated an inverse correlation between active compounds and total VFA. Baihaqi et al. (2023) added that several factors influence VFA concentration, including feed digestibility, the physical form of feed, types of soluble carbohydrates, and the presence of feed additives in the form of active compounds in the feed.

Conclusion

Based on the research results, it was found that soaking treatment with the addition of ash can affect the reduction of HCN levels. Soaking treatment with 10% ash for 48 hours effectively reduces HCN levels to 7.82 ppm. Soaking the ash at higher levels significantly reduced HCN levels and improved rumen fermentation parameters compared with the drying method.

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