ISBB

Development of Processing Technology of High-Protein

Cassava Flour Made from High-Cyanide Cassava
Syamsu Akmal1,*, Beni Hidayat1, and M. Muslihudin1
1Agricultural Technology Department, Politeknik Negeri Lampung, 10 Soekarno-Hatta Street, Bandar Lampung
35144, Indonesia
2Forest Tree Seed Technology Research and Development Center, Jl. Pakuan Ciheuleut PO BOX 105 Bogor, Jawa

Barat, 16001, Indonesia

3Nusa Tenggara Barat University, Jl. Tawak-tawak Karang Sukun, Mataram, Indonesia
4Gadjah Mada University, Jl. Teknika Selatan, Sekip Utara, Yogyakarta, Indonesia
*Correspondence: syamsuakmal@polinela.ac.id

Abstract: The utilization of high-cyanide cassava as food is limited and mostly used as raw material for tapioca
industry. This research aimed to apply the crushing + pressing method and the fermentation process using
Saccharomyces cerevisiae in the powder form to decrease the hydrocyanic acid content and increase the protein
content of high-cyanide cassava. The research was conducted in a Completely Randomized Block Design with
two factors. The first factor was the ratio of water addition on pressing process with three levels that were 10
times, 15 times, and 20 times: the second factor was the concentration of Saccharomyces cerevisiae with four
levels, i.e. 1.0%, 1.5%, 2%, and 2.5%. The research results showed that the ratio of water addition 20 times on
pressing process and the concentration of Saccharomyces cerevisiae 2.5% was the best treatment that would
produce cassava flour with optimal characteristic as food, which has hydrocyanic acid content of 8.54 mg/kg,
protein content of 7.85%, starch content of 48.44%, dietary fiber content of 9.48%, and white degree of 86.3%.
Reduction of cyanide acid content and increased protein content through application of crushing+ pressing
method and fermentation process will increase the potential use of high-cyanide cassava as food.

Abstract: cassava flour; crushing; pressing; fermentation; high-cyanide; high-protein

________________________________________________________________________________________

1. Introduction

Indonesia is one of the world's main producers of cassava after Nigeria and Thailand [1], with 2015
production of 21,790,956 tons [2]. The use of cassava in Indonesia as food is relatively limited and mostly used
as raw material for tapioca industry. The cultivated cassava in Indonesia is mostly cassava with high
hydrocyanic acid (cyanide) content. Cassava has two main types; they are sweet cassava that contains
hydrocyanic acid content which is less than 40 mg/kg and bitter cassava with more than 40 mg/kg of cyanide
content of root cassava. The high content of cyanide is present in Adira 4, Malang 4, Malang 6, UJ-3, and UJ-
5 [1], and Kasetsart [3].
In addition to having a high content of cyanide, the use of bitter cassava as food also has limitations because
it has a low protein content. Another limitation is that cassava root has short shelf life [4]. Therefore, efforts to
increase the potential use of cassava as food is done in the form of high-protein and low-cyanide cassava flour.
The hydrocyanic acid (cyanide) compound in cassava is formed by the hydrolysis of cyanogenic glycosides
by the linamarase enzyme when the plant tissue is damaged during harvesting, processing or other mechanical
processes [5]. Food and Agriculture Organization/FAO [6], recommends a safe limit of cyanide intake from
food ingredients of 10 mg cyanide/kg (Codex standard 176-1989) which is much lower than the acceptable limit
in Indonesia which is 40 mg cyanide/kg [7].
Efforts to reduce the cyanide content of cassava flour are mainly done by the fermentation process [8]. In
addition to lowering the content of cyanide, the fermentation process will also increase the protein content, by
submerged fermentation [9] or by semi-solid fermentation [10][11]. Gunawan et al. [9] using low-cyanide
cassava (17.5 mg/kg), while Oboh and Eluysian [10] and Oboh and Akindahunsi [11] used low-cyanide and
medium cyanide cassava. According to Oboh and Akindahunsi [11], semi-solid fermentation using
Saccharomyces cerevisiae will produce cassava flour with high protein content (10.9%) and low cyanide
content (9.5 mg/kg). In semi-solid fermentation, Saccharomyces cerevisiae is the most efficient microbial to
increase the nutrient content of cassava [10] and decreases the cyanide content [12]. Further research by semi-
solid fermentation using Saccharomyces cerevisiae is required by using high-cyanide cassava.
Referring to the garri processing, the processing of cassava flour is done through a crushing and pressing
method [13]. According to Etsuyankpa et al. [14], the processing of cassava in the form of a gari involving the
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process of crushing, dewatering (pressing), fermentation and roasting would lead to a significant reduction of
total cyanide. The crushing process will allow more intensive hydrolysis of cyanogenic glycosides to produce
hydrocyanic acid [12]. The hydrocyanic acid formed then separated by immersion continued by pressing. This
research aimed to apply the crushing + pressing method and the fermentation process using Saccharomyces
cerevisiae in the powder form to decrease the cyanide acid content and increase the protein content of high-
cyanide cassava.

2. Methodology

2.1. Materials
The raw materials used were cassava var. kasetsart obtained from Farmers in Jati Agung District, Lampung
Selatan Regency, Lampung Province; and Saccharomyces cerevisiae in powder form, "Fermipan" brand
production of S.I.L. France, obtained from a shop in Bandar Lampung. The chemicals used were Starch (The
tools were used include grater, fermentor, cabinet dryer, disk mill, and sieve of 80 mesh.GR, Merck), maltose
(Sigma M5885), dinitrosalicylic acid (DNS, Sigma D-0550), glucose (Sigma G8270), termamyl enzyme (α-
amylase, Sigma A-4862), pepsin enzyme (Sigma P-7000), amyloglucosidase enzyme (Sigma A-9913),
pancreatin enzyme (Sigma P-1750) obtained from PT Elo Karsa, Jakarta. The tools were used include grater,
fermentor, cabinet dryer, disk mill, and the sieve of 80 mesh. The chemical composition of cassava var. kasetsart
were presented in Table 1.

Table 1. Chemical composition of cassava (mean ± SD) var. kasetsart

No Component Wet basis (wb) Dry basis (db)

1 Carbohydrate (%) 36.01 ± 1.25 87.01 ± 3.01
Starch (%) 28.96 ± 2.71 69.97 ± 4.51
Crude fiber (%) 3.35 ± 0.59 8.10 ± 1.41
Dietary fiber (%) 6.84 ± 0.80 16.53 ± 1.92
2 Water (%) 58.61 ± 1.19 141.59 ± 2.89
3 Ash (%) 0.56 ± 0.29 1.35 ± 0.71
4 Fat (%) 0.19 ± 0.07 0.46 ± 0.16
5 Protein (%) 1.28 ± 0.07 3.08 ± 0.16
6 Cyanide (mg/kg) 27.56 ± 1.35 66.59 ± 3.25

2.2. Methods
Sorting of cassava. Cassava used was varieties kasetsart with harvest age, more than nine months sorted to
separate the damaged and injured cassava.
Preparation of cassava mash. Preparation of cassava mash were included peeling of outer skin and pulp,
washing, crushing, immersion for 2 hours (addition of water according to treatment: 10 times, 15 times, and 20
times peeled cassava weight), pressing for 6 hours so that the liquid that comes out was clear and re-addition of
starch precipitation.
The fermentation process of cassava mash. Cassava mash was added of Saccharomyces cerevisiae in
powder form (concentration according to treatment: 1%, 1.5%, 2%, and 2.5% by weight of cassava mash),
stirred evenly, then put in a plastic container jar with a hollow cover. Fermentation was carried out at room
temperature for four days.
Drying of fermented cassava mash. Drying was done using a cabinet dryer at 50oC for 5-6 hours followed
by grinding until 80 mesh cassava flour was obtained.
Analysis of fermented cassava flour. Analysis of fermented cassava flour was done in the form of cyanide
content, protein content, starch content, dietary fiber content, and white degree. Analysis of the cyanide content
was performed by the silver nitrate titration method [15]. Analysis of protein and starch content was performed
according to the method of AOAC [16]. Determination of dietary fiber content was done by the enzymatic
method based on Asp et al. [17].
Experimental design. The research was conducted in a Completely Randomized Block Design with two
factors. The first factor was the ratio of water addition on pressing process with three levels that were 10 times,
15 times, and 20 times: the second factor was the concentration of Saccharomyces cerevisiae with four levels,
i.e. 1.0%, 1.5%, 2%, and 2.5%.
Data analysis. The analysis was carried out in three replicates for all determinations. The data were
analyzed by two-way analysis of variance (ANOVA) and continued with the least significant difference (LSD)
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test. The results were reported as mean ± SD on the dry basis (db). The significance of the differences was
defined as P<0.05.

3. Results and Discussion

3.1. Characteristics of cyanide content and protein content of fermented cassava flour
Characteristics of cyanide content and protein content of fermented cassava flour on the various treatment
of water addition ratio and Saccharomyces cerevisiae concentration were presented in Table 2.

3.1.1. Cyanide Content

Treatment of water addition ratio on pressing process and Saccharomyces cerevisiae concentration had a
significant effect (p <0.05) to the cyanide content of fermented cassava flour. The treatment of Saccharomyces
cerevisiae concentration 2.5% and the addition of water 20 times would cause the decrease of cyanide content
as much as 87.18% (66.59 mg/kg to 8.54 mg/kg). The cyanide content of 8.54 mg/kg indicated that cassava
flour from this research qualified as food referring to the Codex standard 176-1989 [6].
The lower of the cyanide content by the higher of Saccharomyces cerevisiae concentration was strongly
associated with the growth of Saccharomyces cerevisiae. Saccharomyces cerevisiae will secrete some
extracellular enzyme such as amylases, linamarase and cellulase during the fermentation process which would
lead to its growth [5]. The higher the growth of Saccharomyces cerevisiae will be the higher decomposition of
the cyanogenic glycosides by linamarase as stated Etsuyankpa et al. [14]. Decomposed cyanogenic glycosides
would more easily break down into hydrocyanic acids which subsequently evaporate during the drying process.

Table 2. Characteristics of cyanide content and protein content of fermented cassava flour on the various
treatment of water addition ratio and Saccharomyces cerevisiae concentration (mean ± SD), % dry
basis.

Treatment Cyanide (mg/kg) Protein (%)

Saccharomyces cerevisiae concentration 1%, 59.19 ± 1.98 a 2.90 ± 0.12 d
water addition ratio 10 times
Saccharomyces cerevisiae concentration 1%, 55.19 ± 0.76 b 2.94 ± 0.02 d
water addition ratio 15 times
Saccharomyces cerevisiae concentration 1%, 49.99 ± 0.43 c 2.87 ± 0.10 d
water addition ratio 20 times
Saccharomyces cerevisiae concentration 1.5%, 51.62 ± 0.33 c 4.53 ± 0.13 c
water addition ratio 10 times
Saccharomyces cerevisiae concentration 1.5%, 45.40 ± 0.86 d 4.42 ± 0.33 c
water addition ratio 15 times
Saccharomyces cerevisiae concentration 1.5%, 35.62 ± 0.79 e 4.44 ± 0.39 c
water addition ratio 20 times
Saccharomyces cerevisiae concentration 2%, 35.00 ± 2.07 e 6.48 ± 0.37 b
water addition ratio 10 times
Saccharomyces cerevisiae concentration 2%, 22.77 ± 2.29 f 6.42 ± 0.39 b
water addition ratio 15 times
Saccharomyces cerevisiae concentration 2%, 13.59 ± 1.05 g 6.39 ± 0.17 b
water addition ratio 20 times
Saccharomyces cerevisiae concentration 2.5%, 20.44 ± 0.68 f 7.47 ± 0.23 a
water addition ratio 10 times
Saccharomyces cerevisiae concentration 2.5%, 11.77 ± 0.36 g 7.64 ± 0.35 a
water addition ratio 15 times
Saccharomyces cerevisiae concentration 2.5%, 8.54 ± 1.18 h 7.85 ± 0.51 a
water addition ratio 20 times
Values with the same letters are not significantly different (P > 0.05)

The lower of the cyanide content by the higher of water addition ratio was related to the increasing amount
of dissolved hydrocyanic acid and can be separated. The crushing process would cause the cyanogenic
glycosides to react more intensively with the linamarase enzyme and produce water-soluble cyanide [14]. The
decrease of cyanide content was also caused by the pressing process. Lambri et al. [12], reported that the
pressing followed by the fermentation process would decrease the content of cyanide substantially.
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3.1.2. Protein Content

The Saccharomyces cerevisiae concentration treatment had the significant effect (p <0.05) on the protein
content of fermented cassava flour but the water addition ratio on pressing process had no significant effect (p>
0.05) as shown in Table 2. Treatment of Saccharomyces cerevisiae concentration 2.5% and water addition 20
times would cause the increase of protein content 60.7% (3.08% db to 7.86% db).
The higher the protein content with the higher concentration of Saccharomyces cerevisiae concentration
was related to the higher number of the biomass of Saccharomyces cerevisiae cells that formed. Saccharomyces
cerevisiae will secrete some extracellular enzyme such as amylases, linamarase and cellulase during the
fermentation process which would lead to its growth [5]. The increase in protein content during cassava flour
fermentation is also in line with the results of a study conducted by Gunawan et al. [9], Oboh and Elusiyan [10],
Adepoju et al. [18].

3.2. Characteristics of starch content, dietary fiber content, and white degree of Fermented Cassava Flour
Table 3 showed the characteristics of starch content, dietary fiber content, and white degree fermented
cassava flour on the various treatment of Saccharomyces cerevisiae concentration and water addition ratio.

Table 3. Characteristics of starch content, dietary fiber content, and white degree of fermented cassava flour
on the various treatment of Saccharomyces cerevisiae concentration and water addition ratio (mean
± SD), % dry basis.

Dietary fiber White degree

Treatment Starch (%)
(%) (%)
Saccharomyces cerevisiae concentration 1%, 51.39 ± 0.44 d 12.36 ± 0.77 a 77.8 ± 0.9 de
water addition ratio 10 times
Saccharomyces cerevisiae concentration 1%, 55.46 ± 0.04 b 12.25 ± 0.35 a 77.5 ± 0.5 de
water addition ratio 15 times
Saccharomyces cerevisiae concentration 1%, 59.09 ± 0.75 a 11.99 ± 0.19 b 77.3 ± 0.6 e
water addition ratio 20 times
Saccharomyces cerevisiae concentration 45.19 ± 0.99 g 11.19 ± 0.45 c 78.9 ± 1.2 de
1.5%, water addition ratio 10 times
Saccharomyces cerevisiae concentration 50.98 ± 0.57 d 11.07 ± 0.28 c 80.2 ± 1.1 d
1.5%, water addition ratio 15 times
Saccharomyces cerevisiae concentration 55.44 ± 0.60 c 10.99 ± 0.59 c 79.5 ± 0.7 d
1.5%, water addition ratio 20 times
Saccharomyces cerevisiae concentration 2%, 41.89 ± 0.22 i 9.99 ± 0.22 d 80.5 ± 0.6 d
water addition ratio 10 times
Saccharomyces cerevisiae concentration 2%, 46.68 ± 0.32 f 9.99 ± 0.68 d 81.3 ± 2.1 c
water addition ratio 15 times
Saccharomyces cerevisiae concentration 2%, 51.99 ± 0.54 d 10.10 ± 0.54 d 83.1 ± 2.1 bc
water addition ratio 20 times
Saccharomyces cerevisiae concentration 39.90 ± 0.57 j 9.53 ± 0.18 d 83.8 ± 1.1 b
2.5%, water addition ratio 10 times
Saccharomyces cerevisiae concentration 43.53 ± 0.52 h 9.49 ± 0.45 d 84.2 ± 1.5 ab
2.5%, water addition ratio 15 times
Saccharomyces cerevisiae concentration 48.44 ± 0.95 e 9.48 ± 0.37 d 86.3 ± 1.4 a
2.5%, water addition ratio 20 times

3.2.1. Starch content

Treatment of Saccharomyces cerevisiae concentration and water addition ratio on pressing process had a
significant effect (p <0.05) to the starch content of fermented cassava flour. The treatment of Saccharomyces
cerevisiae concentration 2.5% and the addition of water 20 times would cause the decrease of starch content by
30.77% (69.97% db to 48.44% db). The decreased of starch content during the cassava fermentation process
was also reported by Gunawan et al. [9].
The decreased of starch content with higher concentrations of Saccharomyces cerevisiae was in line with
the increase in protein content. The higher of Saccharomyces cerevisiae concentrations would lead to an
increase in its metabolic activity. Saccharomyces cerevisiae would produce amylase [5] which will decompose
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starch into simpler compounds to use as a source of nutrients for its growth. This fact was corresponding to the
opinion of Yuliana et al. [19] that in the fermentation process of high starch products, starch is a major source
of nutrients during the fermentation process.
The hydrolysis of cassava starch into shorter chain polymers during the fermentation process will also
benefit from functional food characteristic. During the fermentation process, the starch will be hydrolyzed into
shorter chain polymers [20], so potentially further modified to resistant starch through the application of
autoclaving-cooling cycles [21].
The decreased of starch content with higher water additions ratio was in line with the decrease in cyanide
content. The higher the water addition ratio would cause the higher the dissolved component (starch and
cyanide). Because the process of settling in all treatments was done at the same time (6 hours), the higher the
ratio of water addition would increase the lower the starch content of the cassava flour.

3.2.2. Dietary fiber content

The Saccharomyces cerevisiae concentration treatment had the significant effect (p <0.05) on the dietary
fiber content of fermented cassava flour but the water addition ratio on pressing process had no significant effect
(p> 0.05) as shown in Table 3. Treatment of Saccharomyces cerevisiae concentration 2.5% and water addition
20 times would cause dietary fiber content to decrease 42.66% (16.53% db to 9.48% db).
The decrease in dietary fiber content during this fermentation process indicated that Saccharomyces
cerevisiae was able to utilize dietary fiber as a source of nutrients. Saccharomyces cerevisiae will secrete
cellulose that will hydrolyze fibers especially cellulose [5]. It also showed that Saccharomyces cerevisiae would
use starch and fiber as its nutritional source.
Olaoye et al. [13], reported an increase in dietary fiber content on the submerged fermentation using lactic
acid bacteria. The opposite phenomena were due to differences in fermentation method. On the submerged
fermentation using lactic acid bacteria, bacteria did not use dietary fiber as a source of nutrients. In contrast to
semi-solid fermentation using Saccharomyces cerevisiae, dietary fiber, especially cellulose was also used by
microorganisms as a source of nutrition.

3.2.3. White degree

Treatment of water addition ratio on pressing process and Saccharomyces cerevisiae concentration had a
significant effect (p <0.05) to the white degree of fermented cassava flour. The treatment of Saccharomyces
cerevisiae concentration 2.5% and the addition of water 20 times would produce cassava flour product with the
white degree of 86.3%, and meets the requirements of Indonesia's national standards [7].
Increased white degree along with increased yeast concentration associated with the intensification of its
metabolic activity to break down anti-nutritional compounds. Tanin was an anti-nutritional compound that also
impacts dull color on cassava product. [22]. According to Oboh and Eluysian 2007 [10], during the fermentation
process, there would be decreased cyanide and antinutritional compounds such as tannins.
The increase of white degree by the higher of water addition ratio was related to the increasing number of
dissolved tannin components and can be separated. This white degree increase was in line with the decrease in
cyanide. The higher the ratio of water addition, the more cyanide and tannin are separated, and the whiter the
color of the flour was produced.

4. Conclusions

Treatment of water addition 20 times on pressing process and concentration of Saccharomyces cerevisiae
2.5% will produce cassava flour with optimal characteristic as food, which has hydrocyanic acid content of 8.54
mg/kg, protein content of 7.85%, starch content of 48.44%, dietary fiber content of 9.48%, and white degree of
86.3%.
Reduction of hydrocyanic acid content to the safe level (≤ 10 mg/kg) and increased protein content through
application of crushing+ pressing method and fermentation process will increase the potential use of high-
cyanide cassava as food.

Acknowledgments: The authors would like to thanks to the Politeknik Negeri Lampung, for funding this
research through the scheme of Applied Research in 2018.
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