Production of Glucose from Cassava using Rice

ABSTRACT

Cassava and rice are abundantly readily available for use as appropriate in form(s) that may
be desired. In this regard, it is necessary to provide information on a novel approach that
would support and boost the production of glucose syrup and other related products through
the use of glucose produced using cassava starch as substrate and glucoamylase as enzyme
sourced from rice. The enzymatic hydrolysis was carried out at optimum conditions of pH 5
and temperature 40 ℃ under normal atmospheric pressure. The use of cassava starch from
TMS 30572 cassava tuber variety in Nigeria revealed that enzyme (glucoamylase) sourced
from rice could be utilized in hydrolysing the cassava starch to produce high yield of glucose
that could possibly support the glucose syrup and other related industries. The production of
glucose was found to be optimal at a specific reaction velocity of
0.00205 ± 0.00005 gDper litre−min per subtrate concentration% w /v . This value was
reported to be close with the values obtained using model Equations 1 and 2.

Keyword:

Cassava starch, Rice amylase, Hydrolysis, Glucose, Reaction velocity, Optimum conditions

1.1 Introduction
Glucose is a form of sugar obtained from processing or consumption of food materials with
carbohydrate content. It is used by the body as its energy source. Its structure has six (6)
carbon and the formula isC 6 H 12 O6. One of the major uses of glucose is in the production of
glucose syrup which finds application in food and pharmaceutical industries, etc. Starch as a
source of glucose for production of syrup is required to be pure even though little or no
protein content starch could also be used (Osuji and Anih, 2011, Onyenekwe,2013, Chopra,
2010, Vander-Veen et al., 2006, Jane, 2006.). Cassava is considered among the primary
sources of pure starch. Cassava is a root tuber and are of two major types namely sweet and
bitter. The bitter type have higher concentration of anti-nutrients. As a good source of
carbohydrate, cassava may be processed and used in various forms as human diets, chips and
pellets.(Voragen, 1998, Adejumo and Ola, 2010, Osipina and Wheatley, 2005, Balagopalan,
2002). In Nigeria there are various varieties of cassava such as TME419, TMS 30572, etc.
Generally, starch may be obtained from various crops. The processed starch may be in
modified or unmodified forms. Starch that are unmodified have several functional properties
as against modified starch. The structure of starch consist of two glycosidic linkages namely
α (1−4) and α (1−6) linkages; that holds polysaccharide molecules to form chains made up
of amylose and amylopectin. These linkages in starch could be broken using acid or enzyme
hydrolysis to obtain basic glucose units from the spiral polysaccharide chain (Odebunmi and
Owalude, 2005,Eric, 2017, Qinyue et al., 2020, Rao, 2009, Bon et al.,2007). The use of
enzyme to carry out hydrolysis may result in having high reaction rate and low viscosity of
the reaction medium. The frequently used enzymes for hydrolysis of starch is α -amylase as
against β -amylase. The amylase are present in all living organism; but its ability to convert
substrate to product may vary depending on its source (Amira et al.,2012, Britannica, 2020).
The common sources of enzyme for starch hydrolysis may be from grains and cereals such as
rice, maize, sorghum and wheat. The extent of enzymatic hydrolysis of starch may depend on
the starch granule size, starch source, enzyme type, enzyme concentration, crystallinity, type
of polymorphism (A, B and C), hydrolytic conditions (temperature, substrate concentration
and pH). The pH range of 5 to 9, temperature up to 70 ℃ and normal atmospheric pressure
are usually employed (Tester et al., 2006,Kolusheva and Marinova, 2007, Veena et al.,2013,
Karolina, 2015, Igor et al., 2019, Franco and Ciacco, 1992).
Usually, in following enzyme actions, the starch (substrate) disappearance or the appearance
of product by using iodine to react with the sample may be used. The use of Benedict solution
gives positive test for monosaccharide and disaccharides within a time range of 2 to 3 and
from 10 minutes upwards respectively. However during enzyme hydrolysis, the period for
which the starch would disappear may depend on the enzyme activity. The rate of this
reaction could be modelled to fit the data obtained during experimental runs (Huifang et
al.,2017, Cepeda et al., 2001, AOAC, 1998).
1.2 Optimum Conditions for Cassava Starch Hydrolysis by Glycoamylase from rice
to yield glucose
The use of normal atmospheric pressure and the determination of optimum pH, temperature
and substrate concentration to enhance glucose production from cassava starch using amylase
sourced from rice are the essentials. Usually in carrying out these tasks the following were
considered in a synchronized approach (Olosunde et al., 2023):
Reagents and equipment, sourcing of cassava and rice, preparation of rice malt, enzyme
(glucoamylase) activation and buffer solutions, preparation of starch, substrate and standard
curve of glucose D concentration, determination of alpha amylase activity (enzyme assay),
and assessment of glucose production from starch using enzyme hydrolysis. The design of the
study involved the use of three (3) factors (pH, temperature and substrate) at six (6) levels
that falls within the ranges of 0.5 to 3.0 % w/ v , 5-9 and 30-80℃ respectively for substrate
concentration, pH and temperature to produce glucose based on time course of the reaction.
The standard glucose calibration curve that was prepared and used in the study is presented in
Figure 1.

Fig.1: Standard glucose calibration curve

The maximum concentration of glucose produced was achieved as 0.253 gDE L−1 using pH
of 5, temperature of 40 ℃ and substrate concentration of 2.5% w/ v or 3 % w/ v at 50minutes
or 40minutes respectively as optimum conditions. This may be presented in Figure 2.

Fig. 2: Glucose concentration produced against reaction time.

1.3 Optimum reaction velocity
The optimum reaction velocity is the velocity at which enzyme will become saturated with
the substrate to give maximum yield of the desired product. Therefore, in determining the
optimum reaction velocity for the production of glucose using Glucoamalyse sourced from
rice with aim to enhance production of glucose syrup and other usages, the following
protocol were carried out (Onumadu et al., 2023):
Assembling of materials/reagents and equipment, preparation of glycoamaylase and buffer
solution; preparation of starch (substrate) and standard curve of glucose D concentration. The
time course of reaction was 60 minutes with glucose production assessed at 10 minutes
intervals. The optimal pH of 5 and temperature of 40℃ was employed.
A model equation to fit the experimental data was given as:

[ ]
p ×t
p= max (1)
t+ t 1
2

Where, p=glucose produced at any given time

−1
pmax =maximum glucose produced .(gDE L ) t
¿ reaction time(minutes)
t 1 =half reaction time(minutes)
2

The Michaelis-Menten model equation given as Equation 2 was also employed to compare
the estimated glucose production from Equation 1.
v max +[s ]
v= (2)
k m +[s ]

Where, v= productionrate of glucose∨reaction velocity at any time[ gDE litre−min−1 ]

v max=maximum velocity of reaction

s=concentration of glucose produced [gDElitre ]

k m=Michaelis Menten Constsnt

The reaction velocity based on the experimental data was determined based on Equation 3 as:

[ Amount of glucose produced (gDE L−1)]

[ ReactionVelocity ] =
Reaction time(minutes)
(3)
In order to standardize the reaction velocity for uniform assessment of the glucose production
irrespective of the substrate concentration, the Equation 4 was employed as:

[ Reaction velocity gDE L−1 . min ] (4)

Reaction velocity per subtrate concentration=
Substrate concentration(% w/v )
The Equation 4 was referred to as specific reaction velocity. It was observed that as the
concentration of substrate is increased, the production of glucose increased up to a
concentration of 0.253 gDE L−1 at 40 and 50 minutes respectively for cassava starch substrate
concentration of 3.0% and 2.5% w/ v . (see Figure 2). It was suggested that at this substrate
concentration, the active’ site for the given concentration of enzyme was optimally saturated
to achieve the optimum reaction velocity range of 0.00506 ¿ 0.00625 gDE per litre−min.
Model Equation 1 proposed was compared with Equation 2 and were validated. The optimum
specific reaction velocity was obtained as
0.00205 ± 0.00005 gDper litre−min per subtrate concentration% w /v .

Conclusion:

The maximum yield of glucose could be achieved at a specific reaction velocity of

0.00206 ± 0.00005 gDE per litre−min per substrate concentration % w /v when temperature of
40 ℃ and pH 5 was employed at normal atmospheric pressure.

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