Article

Optimization of Liquid Fructose Sugar Production from Cassava Peel Waste

using the Isomerization Process

Ratu Mayoreta Debora^1*, Dicky Chandra Hermawan², Isni Utami³, Fachrul Nurcholis⁴

^1,2,3Department of Chemical Engineering, Univeristy of Pembangunan Nasional Veteran Jawa Timur, Surabaya

60294, Indonesia

⁴Department of Chemical Engineering, National Taipei University of Technology, Taipei City 10608, Taiwan

Abstract

Cassava is a type of tuber that is commonly consumed in Indonesia. The peel is one of the parts of

cassava that is occasionally used. Cassava peel has a carbohydrate content of 74.73%. Cassava peel has

the potential to be processed into liquid fructose sugar through an isomerization process. This research

aims to determine optimal results based on results and equations through Response Surface

Methodology (RSM). The research was carried out in the stages of making cassava peel flour and

saccharification, then through a hydrolysis process to break down the starch with the help of 3.4 ml of

α-amylase enzyme and 3.4 ml of glucoamylase enzyme. The final stage is isomerization, which converts

glucose into fructose by adding the enzyme glucose isomerase. Variations in adding the glucose

isomerase enzyme were 250, 300, 350, 400, and 450 mg at 60⁰C for 40, 44, 48, 52, 56, 64, and 72 hours.

The best research results were under an isomerization time of 72 hours and a glucose isomerase enzyme

weight of 450 mg with a fructose content of 26.05%. Meanwhile, the optimization results using the RSM

method were under conditions of an isomerization time of 66.5 hours and a glucose isomerase enzyme

weight of 450 mg with a fructose content of 26.8%

Keywords: Cassava peel, Fructose, Isomerization, RSM

Graphical Abstract

*

Corresponding author

Email addresses: ratumayoretadebora25@gmail.com

DOI: https://doi.org/10.22437/chp.v8i1.33117

Received May 11^th2024; Accepted June 26^th2024; Available online June 30^th2024

(https://creativecommons.org/licenses/by/4.0 )

11

Chempublish Journal, 8(1) 2024, 11 - 18

Introduction

times sweeter than glucose. Fructose as a

sweetener can be produced from cassava peel^[9].

Hydrolysis of starch into glucose syrup involves

the stages of gelatinization, breakdown of starch

molecules, and saccharification^[10]. Gelatinization

is the expansion of starch granules caused by

heat, by disrupting the hydrogen bonds in starch

The cassava plant is a perennial woody shrub

that can grow up to three meters in tropical

regions. Cassava has a big role in meeting

people's food needs compared to other types of

tuber plants. Usually, the edible parts of the

cassava plant are the tubers and leaves.

Meanwhile, cassava peel is generally considered

a useless by-product, so it is not used and is often

wasted. This waste can cause severe problems if

not treated. Every 1 kg of cassava produces 0.2

glycosidic

molecules

gelatinization

bonds.

without

Breaking

going

down

starch

the

through

process

will

take

longer^[11]

.

Enzymatic starch hydrolysis refers to the

breakdown of starch molecules (polysaccharides)

kg of skin^[1,2]

.

into

smaller

elements,

including

dextrin,

maltotriose, maltose and glucose. The speed of

the process of breaking down starch molecules is

determined by the enzymatic activity of α-

amylase. The rate of enzymatic activity can be

increased by utilizing substrates that have

Cassava peel has quite large economic potential,

the carbohydrate content in cassava peel is

higher than the flesh itself, namely around

74.73% (cassava peel) compared to 36.8%

(flesh)^[3]. Cassava peel is something that can be

useful, especially when used for the production

of liquid fructose sugar. By using the method of

starch hydrolysis and glucose isomerization,

cassava peel waste can be converted into liquid

fructose sugar^[4]. According to research that has

been carried out, the carbohydrate content in

corn can be used as high fructose corn syrup

using a catalyst^[5].

previously

undergone

gelatinization^[12]

.

Generally, the enzymes that are often used are

the α-amylase enzyme and the glucoamylase

enzyme. The α-amylase enzyme plays a role in

breaking down the α-(1,4) glycosidic bonds of

starch

into

glucose

and

maltose.

The

glucoamylase enzyme plays a role in converting

maltose produced by α-amylase into glucose^[13]

.

Optimal conditions for the α-amylase enzyme are

between 90 and 105°C and pH 6. The enzyme will

be disrupted and damaged by temperatures that

are too high, and partial gelatinization of starch

will occur due to temperatures that are too

low^[14].

The conversion of glucose to fructose expands

the range of biomass utilization. Not only is it

widely used as a sweetener in food and drinks,

fructose is more easily changed and converted

into other compounds than glucose itself.

Examples are the compounds 5-HMF and

levulinic acid which are materials used to

produce medicines, polymers and biofuels^[6].

Saccharification is continued hydrolysis after the

process of breaking down starch molecules. The

glycoamylase ecoenzyme will hydrolyze α-1,4

bonds and a small amount of α-1,6 bonds at the

branching point. This enzyme will hydrolyze

glucose from maltose and starch from maltose.

Saccharification takes two hours and can be

carried out at temperatures between 60°C and

pH 4^[12].

Starch is a type of polysaccharide consisting of

glucose linked together via α-glycosidic bonds.

Starch consists of two parts, soluble (amylose)

and insoluble (amylopectin)^[7]. Glucose (C₆H₁₂O₆)

is a monosaccharide that has six carbon chains

with an aldehyde group. Glucose generally

comes from starch and is often used as a food

sweetener^[8]. Glucose and fructose have the

same chemical formula, the difference is the

different arrangement of their atoms. Glucose

has a six-carbon ring structure, while fructose

has a carbon ring. Fructose is also often referred

to as fruit sugar. In general, fructose is marketed

in crystal and syrup form. Fructose is about 1.8

The isomerization process is used to convert

glucose into fructose. The isomerization process

is assisted by the enzyme glucose isomerase and

the process is carried out in an incubator for 48

hours^[15]. The process of glucose isomerase

enzyme activity begins with the opening of the

carbon chain ring, isomerization using a hybrid

12

Chempublish Journal, 8(1) 2024, 11 - 18

shift mechanism, and the closing of the carbon

more ideal results. The optimization process can

be carried out in two ways, namely empirical and

statistical methods. In empirical methods, each

factor is usually tested and optimized once,

resulting in a whole process but it is very time

consuming and ignores the interactions between

factors that influence the research. The statistical

method is not too time consuming and is able to

process interactions between influential factors.

Statistical methods are more reflected in the

Response Surface Methodology (RSM) method

used to evaluate various variables in this

chain ring^[16]

.

Optimum conditions for the productivity of the

glucose isomerase enzyme are influenced by

several factors. Higher operating temperatures

will increase enzyme activity while reducing the

level of enzyme stability. The isomerization

process of glucose to fructose is generally carried

out at a neutral pH solution. Glucose cannot be

completely converted into fructose in one step,

because of its thermodynamic properties, the

equilibrium ratio between glucose and fructose

at a normal temperature of 25⁰C is 54:46.

research ^[22]

.

Meanwhile,

at

higher

temperatures,

the

Optimization is carried out using the response

surface method (RSM) to identify ideal conditions

for building an experiment. RSM analysis uses a

response function parameter estimation process

based on a least squares approach, which is

basically comparable to regression analysis.

Simply put, the optimal point is determined by

combining RSM studies with mathematical

equilibrium shifts towards fructose. Therefore,

most of the temperatures used for the

isomerization process are high temperatures ^[17]

.

According to Mahreni’s research, the right

conditions for the glucose isomerase enzyme to

work optimally are at a temperature of around

60°C and the pH is set at 7.8 to 8.3 ^[15]. In addition,

the contact time was set at 48 hours to minimize

techniques to obtain maximum response ^[23]

.

the formation of by-products such as dyes ^[18]

.

Materials and Methods

Based on similar research that has been carried

out, the process of making fructose sugar from

cassava peel can be varied in various operating

conditions, such as type of enzyme, amount of

enzyme, pH, temperature and operating time.

The results of the fructose sugar content were a

yield value of 9.298% or a fructose concentration

Chemical and Equipments

Some of the materials used in this research

include cassava peel obtained from waste from

the Cap Lumba Lumba cassava chips industry in

Turen, Malang Regency. The analysis results

published by the Nutrition Laboratory, Airlangga

University stated that the starch content in

cassava peel was 69.73%. The next ingredients

are the α-amylase enzyme and the glucoamylase

enzyme as a bond-breaking agent in starch into

glucose, both enzymes were purchased from the

Nanobio Laboratory. Glucose isomerase enzyme

purchased online via the Tokopedia application

in the Agrotekno store. The glucose isomerase

enzyme functions to convert glucose into

fructose and distilled water as a solvent. The rest

of the materials are purchased at Ngagel Jaya

Kimia Store, Surabaya, such as 0.06 M citric acid

and 0.2 M calcium hydroxide as a pH regulator,

of 229.3 g/L conditioned on

a

substrate

concentration of 35% with a solution volume of

300 ml and a pH of 5.5 [19]. Making fructose

using 1.5 mL of Bacillus licheniformis microbes

yielded a yield of 6.07% ^[20]. Meanwhile, research

using a raw material of onggok flour solution with

a

concentration of 12% at pH

7

and

a

temperature of 120℃ obtained a yield value of

36.19% ^[21]

.

The existing data encourages the development of

a method for making liquid fructose sugar from

cassava peel waste. This is done with the

consideration that cassava peel has a higher

starch content so it will be used as a more useful

ingredient, namely fructose liquid sugar. Apart

from that, to support research, it is necessary to

optimize the results so that they are more

consistent and the research carried out gets

and

activated

carbon

to

bind

impurity

compounds.

The tools used in this research are as shown in

Figure 1, with the numbers (1) beaker glass, (2)

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Chempublish Journal, 8(1) 2024, 11 - 18

magnetic bar, (3) magnetic stirrer hotplate, (4)

incubator, (5) erlenmeyer.

in this research. The reaction is as follows in

Equation 1. In Table 1, it can be seen that the

percentage of fructose levels formed increases

along with the amount of glucose isomerase

enzyme used. Under the conditions of using 250

mg of the glucose isomerase enzyme with

variations in isomerization time from 40 hours to

72 hours, fructose levels were obtained of

16.52% to 19.07% with a difference in the

percentage interval of fructose levels of 2.55%.

Meanwhile, under the conditions of using 450 mg

of the glucose isomerase enzyme with variations

in isomerization time from 40 hours to 72 hours,

fructose levels were obtained of 18.34% to

26.05% with a difference in the percentage

interval of fructose levels of 7.71%. Based on the

results obtained, it is known that the use of the

glucose isomerase enzyme weighing 450 mg can

produce greater fructose than the use of the

glucose isomerase enzyme weighing 250 mg.

These results are similar to previous researchers

who showed that the greater the enzyme added,

the greater the fructose levels produced [15].

Figure 1. Equipment Schematic Process

Hydrolysis and Saccharification

Hydrolysis

and

Saccharification

Process.

Preparation of raw materials is done by

preparing 15 grams of cassava peel flour and

dissolving it in distilled water to a volume of 100

mL. The suspension solution formed is then

heated and stirred using a magnetic stirrer at a

temperature of 60℃ for the gelatinization

process until the solution thickens. Followed by

the liquefaction process by adding 3.4 mL of α-

amylase enzyme at a temperature of 90℃ and pH

6. Then the saccharification process by adding

3.4 mL of glucoamylase enzyme at a temperature

of 90℃ and pH 4.5 for 2 hours [24].

C₆H₁₂O₆(glucose) → C₆H₁₂O₆(fructose)......... (1)

Isomerization Process

The use of the glucose isomerase enzyme as a

biocatalyst in this process. So that the use of

more enzymes can effectively increase the levels

of fructose produced. This is also because the

enzymes still get enough nutrition to process

glucose into fructose. The performance of the

glucose isomerase enzyme is still optimal and

has not yet reached the death or inactive phase

[25].

Isomerization Process. The hydrolysis and

saccharification process produces liquid glucose.

The resulting liquid glucose must first be purified

from impurity compounds with the help of

activated carbon. Liquid glucose is heated and

stirred in a magnetic stirrer at 80 ℃ for 15

minutes then 2 grams of activated carbon is

added. Liquid glucose is filtered to separate

impurity compounds. The liquid glucose that has

been filtered is then stirred in a magnetic stirrer

and then the glucose isomerase enzyme is added

with weight variations of 250, 300, 350, 400 and

450 mg. Next, the incubation process continued

at a temperature of 60℃ for 40, 44, 48, 52, 56, 64,

and 72 hours ^[15]. After the isomerization process

was carried out, the fructose syrup produced was

then analyzed for its fructose content using the

luff scroll method at the Nutrition Laboratory,

Airlangga University.

Based on the results of the research that has

been carried out, it is known that the longer the

isomerization time takes, the higher the fructose

content produced and it starts to remain

constant at the isomerization time of 64 hours. In

this study, the isomerization time parameters

were varied in the range of 40 to 72 hours. In

Table 1, it can be seen that the longer the

isomerization time, such as when the glucose

isomerase enzyme weight 450 mg, the lowest

percentage is at an isomerization time of 40

hours, around 18.34%. The increase in the

percentage of fructose content will continue to

increase as the isomerization takes longer until it

reaches 26.05% at an isomerization time of 72

Result and Discussion

The isomerization process of glucose into

fructose from cassava peel has been carried out

14

Chempublish Journal, 8(1) 2024, 11 - 18

hours. These results are similar to previous

researchers who showed that the conversion of

glucose to fructose increased with the length of

isomerization time [17].

Table 1. Yield of Fructose

% Yield of Fructose

Glucose Isomerase Enzyme Weight (mg)

Time

(hour)

40

250

300

350

400

450

16.52

17.27

18.06

18.42

18.70

19.00

19.07

17.09

18.89

19.91

20.06

21.18

22.15

22.21

17.62

20.73

22.04

22.84

23.09

24.56

24.71

18.19

21.08

22.50

23.47

24.15

25.72

25.84

18.34

21.47

22.67

23.76

24.36

25.94

26.05

44

48

52

56

64

72

From the research results, the best fructose level

was 26.05%. Isomerization is a process to convert

glucose from cassava peel sugar hydrolyzate into

fructose syrup. This reaction is a reversible

reaction so that the resulting yield may

experience a decreasing trend depending on

operational conditions such as temperature,

of Yield Optimization. The equation obtained

from optimization is as follows:

H₂O₂has the property of very easily evaporating

into oxygen and water.

2

% Yield = -13.86 + 1.096 X₁- 0.01396 X₂- 0.01065 X₁

0.000708 X₁X₂........................................................................(2)

+

time, enzyme use, and catalyst used^[26]

.

Furthermore, it is necessary to vary the

isomerization time and weight of the glucose

isomerase enzyme over a longer range to

determine the most optimal conditions for this

process.

Based on the results of similar research, the use

of enzymes as biocatalysts to convert glucose

into fructose is also not very effective. The results

of the review stated that the use of enzymes

without a catalyst in the isomerization process

only obtained 42% of the fructose content

Figure 2. Contour Plot Response Surface

Characteristics

produced^[27]

.

The performance of chemical

catalysts is actually lower than enzymatic

conversion, namely 30-40% on average^[28]

.

Optimization was carried out to determine the

optimal results of fructose formation using the

response surface method in Minitab 19 software.

This optimization was based on the basic theory

Figure 3. Optimization Result Graph

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Chempublish Journal, 8(1) 2024, 11 - 18

Where X1 is the isomerization time and X2 is the

enzyme weight. The response plan in Minitab

determines the fixed variable (Y) in the form of

the percentage of fructose content formed, while

After applying RSM to the Minitab 19 program,

treatment results can be optimized by analyzing

contour

plots

and

surface

plots.

This

optimization process involves adjusting certain

parameters to obtain the desired results.

Optimization settings are configured to produce

the percentage of fructose levels achieved under

the

independent

variable

consists

of

isomerization time as factor 1 (X1), and the

weight of the glucose isomerase enzyme as

factor 2 (X2). Based on data analysis from Minitab

19 software, a regression equation was obtained

which describes the effect of isomerization time

and the weight of the glucose isomerase enzyme

used on the percentage of fructose content

produced. The optimization results from Minitab

19 show that the entire data is a second order

polynomial equation or quadratic linear model.

The optimization results will show the response

equation function to changed conditions such as

isomerization time and weight of the glucose

isomerase enzyme. This research obtained a

contour plot that illustrates the results of

optimizing the percentage of fructose content

resulting from the isomerization process. The

contour plot graphic can be seen in Figure 2.

the most favorable conditions ^[29]

.

The parameters in the response results include

the term lower for the lowest result and target for

the highest result. The solution for optimization

results using the Surface Response Method has

optimum results at an isomerization time of 66.5

hours and a glucose isomerase enzyme weight of

450 mg with a resulting fructose content of 26.8%

and a D (Desirability) value of 1. Value desirability

which is close to the value of 1 is the most desired

value which can be shown by the model because

it shows the accuracy of the optimization, this

shows the program's ability to produce the

desired product which is close to perfect ^[23]

.

Based on Figure 4, the graph shows the

optimization results. The graph shows the

maximum optimum results. The highest point in

the image above shows the highest point. Where

if the red line is shifted to follow the shape of the

curve it will show the results for each condition.

From this figure it can be concluded that the

optimum value for the percentage of extracted

nickel occurs at an isomerization time of 66.5

hours using 450 mg of the glucose isomerase

enzyme. These results also show that the

resulting D (desirability) value is 1. The optimum

conditions obtained can be used as a reference

[30] for producing fructose sugar from cassava

peel using the isomerization process

The contour plot image shows the differences in

the percentage of fructose levels formed with

different combinations of isomerization time and

weight of the glucose isomerase enzyme. The

variables of isomerization time and weight of the

glucose isomerase enzyme obtained were used

as independent variables in this study. The

darker the blue colour on the contour plot graph,

the lower the fructose levels formed, while the

darker the green colour on the contour plot

graph, the higher the fructose levels formed.

Conclusion

Production of fructose liquid sugar from cassava

peel waste using the isomerization process

produces the best fructose content under the

isomerization time of 72 hours and the weight of

the glucose isomerase enzyme is 450 mg with a

yield of 26.05%. As for the optimization results

using Response Surface Methodology (RSM),

optimum

results

were

obtained

at

an

isomerization time of 66.5 hours and a glucose

isomerase enzyme weight of 450 mg with a yield

Figure 4. Response Optimization

16

Chempublish Journal, 8(1) 2024, 11 - 18

6.

H. Huang, X. G. Meng, W. W. Yu, L. Y. Chen, and

Y. Y. Wu, “High Selective Isomerization of

Glucose to Fructose Catalyzed by Amidoximed

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19860–19866, 2021.

of 26.8%. Variations in the use of isomerization

time and weight of the glucose isomerase

enzyme showed increasing results as these

variables increased. Moreover, research needs to

be

developed

with

variations

in

longer

7.

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8.

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The author expresses his gratitude to the

University of Pembangunan Nasional "Veteran"

Jawa Timur which has supported and facilitated

this research.

10.

11.

12.

Author Contributions

Concept, R.M.D; D.C.H; Methodology, R.M.D;

D.C.H; Validation; F.N, I.U; Analysis, I.U; Review

and Editing, R.M.D, I.U, D.C.M

Conflict of Interest

The authors declare no conflict of interest

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