Initial evaluation of a field-friendly extraction procedure for the enzymatic assay of cassava cyanogens

A novel ‘field friendly’ extraction procedure has been developed for the enzymatic colorimetric determination of cyanogenic potential (CNP) in fresh cassava root parenchyma. The novel procedure does not require electrical power or vacuum, and employs inexpensive lightweight equipment, making it suitable for remote field sites. Testing of the procedure involved ten fresh roots (24–80 mg kg^?1 total CNP, as HCN, fresh basis). From the parenchyma of each root, one extract was made using the novel procedure, and a ‘control’ extract was made using a traditional laboratory‐based procedure. Total CNP assay of the extracts indicated strong (y = mx) or very strong (y = mx + c) correlation of results obtained using the two procedures, while a very strong correlation (y = mx) was obtained for free HCN. Based on this preliminary evidence, the novel procedure is satisfactory at least for total CNP assay of fresh low‐CNP cassava roots.     

The retail market for fresh cassava root tubers in the European Union (EU): the case of Copenhagen,Denmark — a chemical food safety issue?

BACKGROUND: A number of retail shops in Copenhagen sell fresh cassava roots. Cassava roots contain the toxic cyanogenic glucoside linamarin. A survey was made of the shop characteristics, origin of the roots, buyers, shop owner's knowledge of toxicity levels, and actual toxicity levels. RESULTS: Shops selling fresh cassava were shown mostly to be owned by persons originating in the Middle East or Afghanistan, buyers were found to predominantly be of African origin, and sellers' knowledge concerning the potential toxicity was found to be very restricted. Seventy‐six per cent of the roots purchased had a total cyanogenic potentials (CNp) above the 50 mg HCN equivalents kg^?1 dry weight (d.w.) proposed as acceptable by an EU working group. Two of 25 roots purchased had CNp higher than 340 mg HCN eq. kg^?1 d.w. CONCLUSION: The EU has previously made risk assessments concerning cassava and cyanogenic compounds. In the light of the conclusions drawn, the EU needs to make decisions about how to deal with the regulation and control of fresh cassava roots imported to the European food market. Also cassava root products and cassava leaves should be considered. Copyright © 2009 Society of Chemical Industry     

Cyanogenic potential of randomly sampled fresh and processed cassava on retail sale in Singapore

A random sample of fresh cassava roots and cassava products, purchased at various retail outlets in Singapore during 2016–2017, had total cyanogenic potential (CNp–expressed as mean ± standard deviation, mg kg⁻¹ HCN, fresh weight basis) as follows: fresh roots (n = 66) from three SE Asian countries, 59.0 ± 19.2; peeled chilled root pieces from Malaysia (20 × 1-kg packs), 38.5 ± 16.5; cassava flour from Indonesia (8 × 1-kg packs), 11.7 ± 8.2; dried Indonesian chips for home frying (5 × 250 g packs), 61.6 ± 16.5; one pack (120 g) of Malaysian ready-to-eat (RTE) chips, 17.1 ± 3.2. CNp in all flour and RTE chips was below 20 mg kg⁻¹. The majority of fresh roots (59.1%) and packs of dried chips (80%), and 15% of packs of peeled chilled root parenchyma, exceeded the Codex Alimentarius limit (50 mg kg⁻¹) for ‘sweet’ (boil-and-eat) cassava.     

Cyanogenic potential of fresh and frozen cassava on retail sale in three Irish cities: a snapshot survey

Imported cassava roots can be found on retail sale in several Irish cities and towns. Fresh roots (n = 36 roots) and peeled frozen root pieces (n = 28 packs) were randomly purchased from five retailers in Belfast, Dublin and Limerick and assayed for cyanogenic potential (CNp). Total CNp of fresh root parenchyma varied from 37.5 to 242.9 mg kg^?1 as HCN, dry weight basis – dwb), averaging 104.4 mg kg^?1 HCN (dwb). Total CNp of frozen root parenchyma (n = 28 packs) ranged from 28.5 to 258.6 mg kg^?1 HCN (dwb), averaging 81.7 mg kg^?1 HCN (dwb). Around 78% of fresh roots, and 93% of packs of frozen parenchyma, complied with the Codex Alimentarius definition of ‘sweet’ cassava, but most (86.1% and 64.3%, respectively) exceeded European Union NETTOX recommendations for total CNp. In around one‐third of frozen parenchyma packs, nonglycosidic cyanogens accounted for 83–100% of total CNp. The toxicological implications are briefly discussed.     

Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava products

The simple semiquantitative picrate method for the determination of total cyanogens in cassava flour has been modified by increasing the concentration of the picrate solution used to make up the picrate papers, such that a linear Beer's Law relation between absorbance and cyanogen content is obtained over the range 0–800 mg HCN equivalents kg⁻¹ cassava. The method has been adapted to determine the total cyanogen content of cassava roots and the results compared using the picrate method and the acid hydrolysis method for six different roots from five cultivars. The agreement between the results is satisfactory. The simple method for determination of total cyanogens in cassava roots in the field is available in kit form. The methodology has been modified to allow determination of the three different forms of cyanogens present in cassava flour, viz HCN/CN⁻, acetone cyanohydrin and linamarin. HCN/CN⁻ is determined by the picrate method in which cassava flour is reacted with 0.1 M sulphuric acid for 3 h at room temperature. HCN/CN⁻ plus acetone cyanohydrin is also determined by the picrate method after treating cassava flour with 4.2 M guanidine hydrochloride at pH 8 for 3 h at room temperature. A comparison has been made of the amounts of the three cyanogens present in six cassava flour samples using the semiquantitative picrate and the acid hydrolysis methods. The agreement between the two methods is satisfactory, which shows that the new methodology works well. The picrate method for determination of the three cyanogens in cassava flour is also available as a kit. © 1999 Society of Chemical Industry     

Cyanogenicity of cassava varieties and risk of exposure to cyanide from cassava food in Nigerian communities

BACKGROUND: High‐cyanogenic cassava varieties are cultivated in many tropical communities that are free of neurological syndromes attributed to consumption of cassava foods. This study was done in four geographical areas of Nigeria (northern, southwestern, southeastern and an area endemic for ataxic polyneuropathy) to determine if cyanogenicity of cassava is associated with geographical area, altitude or level of cyanogenic compounds in gari, a popular cassava food in West Africa. RESULTS: Mean levels of cassava cyanogens were 153, 127, 68 and 65 mg HCN equivalents (eq.) kg^?1 dry weight (DW) in the endemic, southeastern, southwestern and northern areas respectively (P < 0.0001), while mean levels of gari cyanogens were 9, 4, 7 and 13 mg HCN eq. kg^?1 DW in the respective areas (P < 0.0001). The mean altitude was 35 m in the endemic area, 55 m in the southeastern area, 220 m in the southwestern area and 273 m in the northern area (P < 0.0001). Altitude was associated with cyanogenicity of cassava in univariate and multivariate models (P < 0.0001). One hundred and twenty‐six (93%) farmers and 255 (77%) processors did not perceive cassava or its food products as toxic. CONCLUSION: The findings indicate that cyanogenicity of cassava is determined by environmental factors rather than by conscious selection of varieties by farmers. Farming high‐cyanogenic cassava is not associated with high levels of residual cyanogens in gari. Cassava is not perceived as toxic by farmers and processors. Copyright © 2008 Society of Chemical Industry     

Simple picrate paper kit for determination of the cyanogenic potential of cassava flour

A simple picrate paper kit method was developed for the semiquantitative determination of the cyanogenic potential for cassava flour. The method involved the addition of linamarase and pH 8 phosphate buffer (absorbed in a filter paper disc) to 100 mg of flour +0·5 ml water placed in a small vial. A rectangle of yellow picrate paper attached to a plastic strip was added and the vial capped and left overnight. The yellow paper changed colour towards brown and its colour was compared with that of a standard colour card with 10 shades of colour which corresponded with cyanogenic potentials of 0–800 mg HCN equivalents kg⁻¹ flour (ppm). To obtain a more accurate measure of cyanogenic potential (±20%) the paper was eluted with water and the absorbance measured at 510 nm. The cyanogenic potential in ppm was determined from a calibration graph. The methodology is available in kit form. The simple method may be used in the field by a relatively unskilled person. The more accurate method requires a spectrophotometer and is suitable for use in simple laboratories in developing countries. © 1998 SCI.     

Chemical assay of cyanide levels of short-time-fermented cassava products in the Abraka area of Delta State,Nigeria

Five cassava products, commonly consumed in and around the Abraka area of Delta State, Nigeria were analysed for their cyanide levels. This study, to determine safe levels of cyanide, became necessary as the fermentation time in the processing of these products has been observed to be drastically shortened to as low as 6 h for quick economic returns. In all the products, the cyanide levels were reduced from 30.0–58.0 mg HCN equivalent kg⁻¹ of product. These levels fall below the reported 30.0 mg HCN equivalent kg⁻¹ safe (fresh weight) of raw cassava to 5.4–24.0 mg HCN equivalent kg⁻¹ level for cassava products. Nevertheless, some of the products containing up to 20.0–26.5 g HCN equivalent kg⁻¹ of products are dangerous to health as these levels are higher than the 20.0 mg HCN equivalent kg⁻¹ safe level recommended by the Standard Organization of Nigeria. The current practice of short-time fermentation should therefore be discouraged.     

An evaluation of enzymic and autolytic assays for cyanide in cassava (Manihot esculenta,crantz)

The total cyanide contents of cassava parenchymal tissue (peeled roots), cassava cortex (peel) and cassava leaves were evaluated by autolytic and enzymic assays. Autolysis of parenchymal tissue was studied under different conditions of _pH, time and temperature and addition of exogenous enzyme and antibiotic. Optimal conditions were determined to be 24 h at 37°C in acetate buffer (0.1M ; _pH 5.5) with 0.1 mg ml^?1 chloramphenicol. Total cyanide contents similar (about 90%) to those obtained by enzymic assay could be achieved only by the use of small sample sizes: < 1 g of parenchymal tissue, < 0.3 g of cortex and < 0.1 g of leaf tissue. This caused sampling problems because of the presence of cyanide gradients in cassava tissues, which could only be resolved by tissue homogenisation prior to analysis. A study using the enzymic assay of cyanide stability in such homogenates, at different _pH values and temperatures, has indicated that subsampling must be done within 15 min so as to prevent appreciable losses in the measured cyanide contents. The rate of loss of total cyanide was found to depend not only on the proportion of total cyanide which is non-glucosidic (free), but also on the proportion of the non-glucosidic cyanide present as cyanohydrins. The implications with regard to residual cyanide contents on cassava processing and the advantages of the enzymic assay over autolytic methods are discussed.     

Processing factors affecting the level of residual cyanohydrins in gari

Intake of cyanogens in gari, a food processed from cassava roots, is implicated in the causation of tropical ataxic neuropathy (TAN). This neurological syndrome is endemic in some communities in south‐western Nigeria. Studies have shown that methods of processing cassava roots determine the quantity of cyanogens in gari. This study was conducted to investigate the effects of the method of dewatering and the duration of fermentation on cyanogens in gari. Cassava roots (400 kg) were peeled, washed, grated and divided into 14 woven polyethylene sacks. The mash in seven of the sacks was dewatered continuously during fermentation, while the mash in the remaining seven sacks was fermented without dewatering, but dewatered at the end of fermentation. Cassava mash from each treatment was roasted into gari at 24 h intervals up to 168 h. Mean cyanohydrin content in gari roasted from cassava mash dewatered continuously during fermentation was 10.8 mg HCN eq kg^?1 dw (CI 9.7–11.9), while mean cyanohydrin content in gari roasted from cassava mash dewatered after fermentation was 6.3 mg HCN eq kg^?1 dw (CI 5.3–7.4). Mean linamarin content was 4.0 mg HCN eq kg^?1 dw (CI 3.1–4.9) and mean HCN content was 1.6 mg kg^?1 dw (CI 1.3–1.9) in gari roasted from cassava mash dewatered continuously, while mean linamarin content was 3.2 mg HCN eq kg^?1 dw (CI 2.3–4.0) and mean HCN content was 1.2 mg kg^?1 dw (CI 0.9–1.5) in gari roasted from cassava mash dewatered after fermentation. The method of dewatering cassava mash and the duration of fermentation were significantly associated with the level of cyanohydrin in gari (p < 0.001). This study shows that dewatering of cassava mash continuously during fermentation contributes to the dietary cyanide load in TAN‐affected communities. © 2002 Society of Chemical Industry     

An enzymatic assay for the total cyanide content of cassava (Manihot esculenta Crantz).

An enzymatic assay for the cyanide contents of cassava parenchymal tissue (peeled root), cassava peel or cassava leaves is described. The material is homogenised in orthophosphoric acid; filtered through glass-fibre paper and aliquots of the filtrate are neutralised and incubated with exogenous linamarase for 15 min. The cyanogenic glucosides present are hydrolysed to free cyanide which is estimated spectrophotometrically. The acid extraction solution inactivates endogenous linamarase, and assay of aliquots without enzyme treatment gives the free (non-glycosidic) cyanide contents of the extracts. The acid extracts are stable for at least 4 days at 4°C, and the steam-distillation/aspiration of earlier methods is unnecessary. The detection limit is < 0.01 mg (0.1 parts 10^?6) cyanide per 100 g fresh weight and peeled root, and 40-50 samples per day can be handled easily. Analyses of eight cultivars indicated longitudinal and radial cyanide gradients in the roots, and the problem of sampling bulky roots is discussed.     

Bitter taste in cassava roots correlates with cyanogenic glucoside levels

Cassava roots contain cyanogenic glucosides. Malawian farmers classify cultivars into two groups based on the perceived danger of eating raw roots that they associate with bitterness. In the vernacular, cultivars that produce roots with bitter taste are called vyakubaba (bitter), whereas those yielding non‐bitter roots are called vyakuzizra (cool). In the scientific literature they are distinguished as ‘bitter’ or ‘sweet’. Roots from ‘bitter’ cultivars are processed prior to consumption. We studied the ability of farmers to predict the cyanogenic glucoside levels of 492 roots from the 10 most commonly grown cultivars. Twenty‐eight farmers predicted the taste of each of the cultivars that they grew, and scored bitterness on a five‐point scale by tasting the root tip. Thereafter cyanogenic glucosides were determined on half of the root, while a taste panel scored the taste of the other half. The mean cyanogenic glucoside level in 132 roots from ‘cool’ cultivars was 29 mg HCN eq kg^?1 fresh weight (CI 25–33, range 1–123) and in 360 roots from ‘bitter’ cultivars was 153 mg HCN eq kg^?1 fresh weight (CI 143–163, range 22–661). Farmers' distinction of ‘cool’ and ‘bitter’ cultivars predicts glucoside levels. The tasting of the tip of the root improved the farmers' prediction of toxicity. Scoring of bitterness by a trained taste panel showed a stronger correlation with glucoside levels (r² = 0.67). This suggests that cyanogenic glucosides confer the bitter taste, notwithstanding the probability of additional modifying intrinsic factors. Copyright © 2004 Society of Chemical Industry     

Initial evaluation of a field-friendly incubation procedure for the colorimetric assay of cassava linamarase

The Cooke colorimetric assay of cassava linamarase activity is temperature- and time-inflexible, making “real time” monitoring of linamarase activity in remote cassava-processing sites practically impossible. A modified incubation procedure is described, in which the 30 °C linamarase incubation step is terminated through acidification, yielding a stable cyanohydrin solution. Using partially purified linamarase as a “standard extract”, the solution – held for up to 21 days at ambient/refrigeration temperatures before colorimetry – showed reductions of up to 21% compared with the standard Cooke assay. In a separate trial, a strong linear relationship (r² > 0.95) was observed between recorded linamarase activity values and incubation temperature in the 25–40 °C range, indicating that incubation may take place in remote processing sites without a water bath, and resulting data may reliably be adjusted in keeping with the standard 30 °C incubation assay. The novel procedure thus appears to offer a satisfactory “field-friendly” means of assaying linamarase activity.     

Changes in scopoletin concentration in cassava chips from four varieties during storage

BACKGROUND: The use of the root crop cassava (Manihot esculenta Crantz) is constrained by its rapid deterioration after harvesting. Chemical and spectroscopic examination earlier revealed the accumulation of the four hydroxycoumarins esculetin, esculin, scopolin and scopoletin derived from the phenylpropanoid pathway, during the time course of postharvest deterioration. In this investigation the scopoletin level in parenchymal samples of four cassava cultivars used in Benin, i.e. Kpaki kpika, Kpaki soan, Logoguesse kotorou and BEN 86052, was investigated by high‐performance liquid chromatography (HPLC). RESULTS: Presence was shown in all four varieties with a mean in fresh roots between 4.1 and 11.1 mg kg^?1 dry weight. A strong increase in the content of scopoletin was noticed after a peeling and drying process (6 days) for chip production, the mean content reaching 242.5 mg kg^?1 dry weight in the cultivar BEN 86052. After 3 months of storage this had decreased to 0.7 mg kg^?1 dry weight. CONCLUSION: Strong accumulation of scopoletin in cassava roots used for chip production in Benin is followed by a decrease in its concentration. Copyright © 2011 Society of Chemical Industry     

Protein Contents,Amino Acid Compositions and Nitrogen-to-Protein Conversion Factors for Cassava Roots

Root protein contents of 15 cassava varieties ( Manihot esculenta Crantz) ranged from 5 to 19 g kg⁻¹ dry matter. Intervarietal differences in amino acid profiles of cassava roots were evident. Differences in the levels of aspartic acid, glutamic acid and arginine were most notable. The nitrogen-to-protein conversion factors ( k _AA ) based on nitrogen recovered from total amino acid analyses including ammonia ranged from 4·75 to 5·87, showing that the traditional conversion factor of 6·25 was not valid for cassava root proteins. Conversion factors ( k _P ) for 15 cassava varieties based on Kjeldahl nitrogen ranged from 2·49 to 3·67. Therefore an average k _P value of 3·24±0·31 may provide a better estimate of the protein content in cassava roots.     

An improved microdiffusion method with solid-phase detection for the determination of total cyanogens in fresh cassava. Comparison to the method of Cooke (1978)

A microdiffusion method with solid-phase detection for the determination of total cyanogens (=cyanogenic potential, CNp) in fresh cassava roots was developed and evaluated against the classical spectrophotometric method of Cooke (J Sci Food Agric 29 (1978) 345–352). Using seven different cassava cultivars, a significant difference (approx 15%) in the determined CNp was observed only for one of these. The new method offers several advantages over the earlier described spectrophotometric methods. Filtration of homogenates is not necessary, neither is cooling nor heating. The developed colour is stable on the reaction sheet, so that levels may be measured immediately or when appropriate, and be filed for documentation purposes. Further the solid-phase reaction may be measured in different ways according to the instrumentation available, ie reflectometry as used in this study, or absorption of transmitted light using a TLC-densitometer or a microplate reader. The amount of enzyme (linamarase) used per analysis is lower than for the spectrophotometric assays, giving economic advantages to the present assay. In addition to the extract, only three solutions are used when performing the assay, ie the buffer, the substrate solution for standards, and the enzyme solution. The assay may be run either according to a ‘slow’ overnight protocol or to a ‘fast’ protocol at 40°C. © 1998 SCI.     

食用木薯块根及其制品中生氰糖苷检测方法的建立与应用

生氰糖苷含量的高低对评估食用木薯块根及其制品的食用安全性及其重要。以木薯块根和木薯粉为试材,本研究建立了亚麻苦苷和百脉根苷2种生氰糖苷（Cyanogenic Glycosides,CNGs）的快速提取和高效液相色谱-蒸发光散射（HPLC-ELSD）检测的方法,分析了不同品种木薯块根、木薯粉及其制品中CNGs含量变化。结果表明,食用木薯块根及木薯粉中CNGs提取最佳的硫酸溶液浓度分别为0.025和0.100 mol/L;HPLC-ELSD检测方法可有效分离亚麻苦苷和百脉根苷,其浓度在4.1～820.0 mg/L和2.5～250.0 mg/L范围内线性相关性较好,相关系数（r）分别达到了0.9996和0.9993,检测限（LOD）分别为2.1和0.5 mg/kg,定量限（LOQ）分别为8.2和2.0 mg/kg;该方法重复性和样品稳定性的标准偏差（RSD）都低于5.0%;木薯块根和木薯粉中2种CNGs的平均加标回收率分别在89.3%～96.3%和107.5%～114.9%之间,重复间RSD小于3.4%。样品验证结果表明:不同的木薯品种CNGs含量差异较大;种植8～9个月的木薯制备木薯粉CNGs含量较低;蒸煮加工方式可有效清除木薯食品中CNGs。本方法操作简单、稳定性较好、准确度高,在食用木薯块根及其制品中CNGs的分析与评价等研究方面具有较强的应用前景。     

Factors in the protein enrichment of cassava by solid state fermentation

The enrichment of cassava protein content using solid substrate fermentation was studied on both laboratory and on-farm scales using Aspergillus niger as a starter. The effects of cassava particle size and of various nitrogen source ratios and mixing methods were investigated. Ammonium sulphate and urea as sources at respective levels of addition (g N kg⁻¹ substrate) of 10:10, 20:10 and 20:20 were used in combination with cassava alone and cassava mixed with rice bran and soya bean each having mixing levels of 50, 100 and 150 g kg⁻¹. The mixture was fermented for 84 h at 35°C and 90–95% RH on the laboratory scale and 29–31°C and 95–99% RH at the on-farm scale. The results indicated that cube sizes ranging from 0.3 to 0.5 cm³ gave good mycelial growth. Pure cassava alone at the 10:10 nitrogen addition level produced the highest protein yield of about 145 g kg⁻¹. The on-farm technique yielded higher protein enrichment compared with laboratory experiments. Cassava alone yielded 230 g kg⁻¹ protein while cassava with rice bran and cassava with soya bean each produced 210 g kg⁻¹ protein.     

Mechanism of cyanogen reduction in cassava roots during cooking

The cyanogen elimination profile during cooking of cassava roots was investigated and correlated with the changes in cyanogenic β-glucosidase (GLase) activity monitored at close intervals in the cook water and root. Cyanogenic β-glucosidase (GLase) activity was detected in the cook water within 5 min of starting cooking. Whilst significant decrease in the GLase activity was noticed in cook water and root within 15 and 10 min, respectively, for varieties M4 and H165, H1687 GLase activity decreased tremendously only at 20 and 15 min in the cook water and root, respectively. There was no GLase activity in cook water after the 30 min pre-boiling phase while GLase activity was present in the roots even after 60 min of cooking. The decrease in GLase activity at 15–20 min pre-boiling phase led to accumulation of cyanogens in cook water. The cyanogen fractions (glucosidic and non-glucosidic) increased significantly with cooking in the cook water in the case of varieties M4 and H165 while their levels were almost static throughout the post-boiling phase for H1687. The amount of HCN escaping through steam did not bear a direct relationship with the initial cyanogen content in cassava roots. Although free cyanogen is highly volatile, all of it does not escape through steam and a certain quantity appears to be stabilised in the cook water. ©1997 SCI     

The effects of time to first shoot removal on leaf vegetable quality in cassava (Manihot esculenta Crantz)

A field trial was established on a sandy soil at IITA. Ibadan. Nigeria to investigate the effects of time to first shoot removal on cyanide content and chemical composition of leaves of two cassava clones: TMS 91934 and TMS 30572. Four different times to first shoot removal were imposed on each clone: 8. 14, 20 and 52 weeks after planting (WAP). Shoot removal was repeated at 8-week intervals until harvesting at 52 WAP. Shoot removal (SR) involved removing the shoot tips just below the most fully expanded leaf. Early shoot removal during the crop growth period (SR at 8 weeks and SR at 14 weeks 14) produced significantly (P < 0.01) more fresh shoot yield than late shoot removal (SR20 and SR52 weeks). Concentrations of crude protein, iron, phosphorus and zinc were higher in the early harvested leaves but declined in concentration with increasing age of the crop (430–280 g kg^?1, 680–85 mg kg^?1, 88–42 g kg^?1, 128–30 mg kg^?1, respectively). Leaf HCN concentration was about three times that in tuberous roots (137 mg kg^?1 fresh weight compared with 43 mg kg^?1 fresh weight). The variation in leaf HCN concentration was not significant for all the treatments over the period.