Faculty of Resource Science and Technology
PRODUCTION OF LACTIC ACID FROM TAPIOCA SUGARS
NURFAZIELA BINTI ONET (24668)
Bachelor of Science with Honours
(Biotechnology Resource)
2012
PRODUCTION OF LACTIC ACID FROM TAPIOCA SUGARS
NURFAZIELA BINTI ONET (24668)
This project is submitted in partial fulfillment of the requirement for the degree of
Bachelor of Science with Honours
(Resource Biotechnology)
ACKNOWLEDGEMENTS
First of all, praise to ALLAH S.W.T, the Almighty One, for His blessing in giving me the
strength, the will, and good health to complete this project.
Most appreciation to my supervisor, Professor Dr. Kopli bin Bujang and co-supervisor,
Assoc. Prof. Dr. Cirilo N. Hipolito for his dedicated supervision, patience and advice
throughout this project. A special appreciation goes to Research Assistant of the
Biochemistry Laboratory, Faculty of Resource Science Technology, Miss Rubena Kamal,
and postgraduates students for giving me excellent guidance and knowledge throughout the
making and conduction of this project. Thank you also for spending a lot of time in order to
make me completely understands the tasks given to us.
Last but not least, my sincere thanks to my beloved family for their encouragement, pray,
love, motivation, and support while this project is on-going. Thank you for always being
there for me in the time of need. Also, special thanks to my coursemates and friends for their
support and assistance along the way to finish project.
Thank you.
ii
TABLE OF CONTENTS
Acknowledgements
ii
Table of Contents
iii
List of Abbreviations
v
List of Tables
vi
List of Figures
vii
Abstract
viii
1
CHAPTER 1: INTRODUCTION
1.1 Background of Study
1
1.2 Problem Statement
3
1.3 Objectives
4
5
CHAPTER 2: LITERATURE REVIEW
2.1 Tapioca (Manihot esculenta)
5
2.2 Enzymatic Hydrolysis of Tapioca Starch
7
2.3 Lactic Acid
8
2.3.1 Historical Background
8
2.3.2 Properties of Lactic Acid
9
2.3.3 Lactic Acid Bacteria
10
2.3.4 Lactic Acid Fermentation
11
2.3.5 Applications of Lactic Acid
12
CHAPTER 3: MATERIALS AND METHODS
14
14
3.1 Materials
3.1.1 Tapioca Starch
14
3.1.2 Enzymes for Hydrolysis
15
3.1.3 Microorganism
15
3.1.4 Medium Composition
15
16
3.2 Methods
3.2.1 Preparation and Extraction of Tapioca Starch for Fresh Tapioca
16
3.2.2 Enzymatic Hydrolysis of Tapioca Starch
17
iii
3.3 Fermentation Medium
19
3.4 Lactic Acid Fermentation
20
3.5 Analytical Techniques
21
3.5.1 Dry Matter and Moisture Content
21
3.5.2 Dried Cell Weight Determination
22
3.5.3 Reducing Sugar Analysis
22
3.5.4 Total Starch Determination
23
3.5.5 Lactic Acid Concentration Determination
23
24
CHAPTER 4: RESULTS
4.1 Characterization of Fresh Tapioca (FT) and Tapioca Flour (TF)
24
4.2 Enzymatic Hydrolysis of FT and TF to Sugars
26
4.3 Lactic Acid Fermentation of Tapioca Sugars
27
4.3.1 Utilisation of TFS at different concentrations
27
4.3.1.1 50g/L
27
4.3.1.2 100g/L
28
4.3.1.3 150g/L
29
4.3.1.4 Short Discussion
30
4.3.2 Utilisation of FTS at different concentrations
31
4.3.2.1 50g/L
31
4.3.2.2 100g/L
32
4.3.2.3 150g/L
33
4.3.2.4 Short Discussion
34
CHAPTER 5: GENERAL DISCUSSION
35
CHAPTER 6: CONCLUSIONS AND RECOMMENDATION
38
CHAPTER 7: REFERENCES
40
CHAPTER 8: APPENDIX
43
iv
LIST OF ABBREVIATIONS
cm
Centimetre
g
Gram
g/L
Gram per litre
kg
Kilogram
L
Litre
Mg
Microgram
mL
Millilitre
mm
Millimetre
nm
Nanometre
V
Working Volume
µl
Microliter
α
Alpha
˚C
Celsius
KNU/g
Kilo Novo Unit per gram
AGU/ml
Antigen Unit per millilitre
KI
Potassium Iodide
OD
Optical Density
LA
Lactic acid
FT
Fresh Tapioca
TF
Tapioca Flour
FTS
Fresh Tapioca Sugars
TFS
Tapioca Flour Sugars
v
LIST OF TABLES
Figure
Title
Page
1
Maximum recorded yield and food energy of some important tropical
food crops. Source : de Vries et al. (1967)
6
2
Physical Properties of Lactic Acid
10
3
Analyses was made by blending 500g FT or 500g TF in 1L distilled
water
25
4
Fermentation of LA on 50g/L TFS
27
5
Fermentation of LA on 100g/L TFS
28
6
Fermentation of LA on 150g/L TFS
29
7
Kinetics parameters of batch fermentation at different TFS feed
concentration at 24 hours
30
8
Fermentation of LA on 50g/L FTS
31
9
Fermentation of LA on 100g/L FTS
32
10
Fermentation of LA on 150g/L FTS
33
11
Kinetics parameters of batch fermentation at different FTS feed
concentration at 24 hours
34
12
Starch content
47
vi
LIST OF FIGURES
Figure
Title
Page
1
Different configuration of lactic acid molecule, L (+) lactic acid and D
(-) lactic acid
9
2
Fresh Tapioca (FT)
14
3
Tapioca Flour (TF)
14
4
The pink skin of fresh tapioca after removing the brown outer layer
16
5
The white flesh of fully deskinned fresh tapioca tubers
16
6
Slices of tapioca tubers, ready for pulverising using a blender
17
7
Pulverising of fresh tapioca in high speed wet blender
17
8
Typical process of enzymatic starch hydrolysis
18
9
Hydrolysis of tapioca starch using a Hotplate and Aluminium pot
18
10
Shake flasks fermentation
20
11
Viscous starch syrup from FT during enzymatic hydrolysis
26
12
Less viscous starch syrup from TF during enzymatic hydrolysis
26
13
Glucose consumption and lactic acid fermentation of 50g/L TFS
27
14
Glucose consumption and lactic acid fermentation of 100g/L TFS
28
15
Glucose consumption and lactic acid fermentation of 150g/L TFS
29
16
Glucose consumption and lactic acid fermentation of 50g/L FTS
31
17
Glucose consumption and lactic acid fermentation of 100g/L FTS
32
18
Glucose consumption and lactic acid fermentation of 150g/L FTS
33
19
Graph of starch content
47
vii
Production of Lactic Acid from Tapioca Sugars
Nurfaziela Binti Onet
Program Resource Biotechnology
Faculty Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Tapioca (Manihot esculenta) also known as ubi kayu among Malaysian is a starchy plant material that can be
hydrolyzed to produced reducing sugars by bacterial enzymes. Subsequently, these reducing sugars can be
fermented to produce lactic acid using Lactococcus lactis IO-1. Exactly 500g of FT was added to 1000ml of
distilled water. Then, the mixture is enzymatically hydrolysed, filtered and autoclaved to produce tapioca sugar
syrup. Enzymatic hydrolysis of FT and TF were carried out in 2 stages namely liquefaction and saccharification
with help of enzymes Termamyl-SC and Dextrozyme under optimum conditions. The tapioca sugar with
different concentration (50g/L, 100g/L and 150g/L) were used in batch fermentation using 10% of inoculum of
Lactococcus lactis IO-1 in production of lactic acid. The glucose recovery from both FT and TF were compared,
and glucose recovery from TF was higher (62.7% to 68.1%) than FT (39.3% to 45.4%). Then lactic acid
production from TFS and FTS were compared under three glucose concentrations (50g/L, 100g/L and 150g/L).
Glucose concentration at 50g/L of FT showed the highest fermentation efficiency (29.91%) and lactic acid
production (10.42g/L) than 100g/L and 150g/L of FT at 2.80% and 1.67%, respectively. The high fermentation
efficiency (12.90%) and lactic acid production (6.31g/L) also derived from 50g/L of TFS. Therefore, FTS with
concentration of 50g/L has high possibility in maximizing the lactic acid production (29.91%) and show
economical potential compared to TFS although its glucose recovery (39.3% to 45.4%) was low than TFs
(62.7% to 68.1%).
Key words: tapioca, glucose concentration, enzymatic hydrolysis, lactic acid
ABSTRAK
Manihot esculenta juga dikenali sebagai ubi kayu di kalangan rakyat Malaysia adalah bahan berkanji
tumbuhan yang boleh terhidrolisis dihasilkan mengurangkan gula oleh enzim bakteria. Selepas itu, kandungan
gula penurun ini boleh ditapai untuk menghasilkan asid laktik menggunakan Lactococcus lactis IO-1. Tepat
500g FT telah dimasukkan ke dalam 1000ml air suling. Kemudian, campuran enzim dihidrolisis, ditapis dan
autoklaf untuk menghasilkan ubi sirap gula. Hidrolisis berenzim FT dan TF telah dijalankan dalam 2 peringkat
iaitu pencairan dan pensakarifikasi yang dengan bantuan enzim Termamyl-SC dan Dextrozyme di bawah
keadaan optimum. Gula ubi dengan kepekatan yang berbeza (50g/L, 100g/L dan 150g/L) telah digunakan
dalam Fermentasi menggunakan 10% daripada inokulum Lactococcus lactis IO-1 dalam pengeluaran asid
laktik. Pemulihan glukosa dari kedua-dua FT dan TF telah dibandingkan, dan pemulihan glukosa dari TF
adalah lebih tinggi (62.7% kepada 68.1%) berbanding FT (39.3% kepada 45.4%). Kemudian pengeluaran asid
laktik dari TFS dan FTS berbanding di bawah tiga kepekatan glukosa (50g/L, 100g/L dan 150g/L). Kepekatan
glukosa pada 50g /L menunjukkan penapaian kecekapan tertinggi (29.91%) dan pengeluaran asid laktik
(10.42g/L) daripada 100g/L dan 150g/L pada 2.80% dan 1.67%, masing-masing. Kecekapan penapaian yang
tinggi (12.90%) dan pengeluaran asid laktik (6.31g / L) juga dari 50g/L TFS. Oleh itu, FTS dengan kepekatan
50g/L mempunyai kemungkinan yang tinggi dalam memaksimumkan pengeluaran asid laktik (29.91%) dan
menunjukkan kebolehan menjimatkan berbanding dengan TFS walaupun pemulihan glukosa (39.3% kepada
45.4%) adalah rendah daripada TFS (62.7% kepada 68.1%).
Kata kunci: ubi kayu, kepekatan glukosa, hidrolisis berenzim,asid laktik
viii
CHAPTER 1
INTRODUCTION
1.1 Background of Study
L-lactic acid (CH3-CHOH-COOH) has been produced commercially from many countries
including Thailand, Brazil, America and Indonesia. In early 1780s, lactic acid has been found act
as sour component of milk (Narayanan et al., 2004). Ever since, it is very important in many
applications such as pharmaceutical, cosmetic industry and food industry. It can acts as a natural
preservative that facilitate the inhibition of putrefying bacteria, hence important as ingredient for
the synthesis of cosmetic products (Ãkerberg & Zacchi, 2000). Lactic acid can be produced via
fermentation of simple sugars degraded from starch. There are several varieties products
produced from fermentation, such as pharmaceutical, organic acids, and alcohols (ethanol).
Nowadays, the most widely known fermentation product is lactic acid since it has many
significant especially in cosmetic industry. Utilizing enzymatic and fermentation technology
lactic acid can be produced from low cost raw material such as agricultural waste, agroindustrial
waste, woody crops, corn, sorghum, potato, tapioca and sago (Tonukari, 2004). Tapioca was
used for this research due to its high availability in the Samarahan areas.
Tapioca (Manihot esculenta) or cassava or ubi kayu in Malaysia belongs to the family
Euphorbiaceae has same potential values or abilities as other starchy materials in producing
sugars, lactic acid and ethanol. Tapioca is one of the examples of starchy materials that grows
and produces best under warm humid tropical conditions and tolerant to stressful environments
1
like drought conditions and soil of low-fertility (Burrell, 2003). Moreover, the carbohydrate
production of tapioca (cassava) is 40% higher than rice and 25% more than maize (Tonukari,
2004). Also, tapioca is the cheapest source of calories for human nutrition and animal feeding
(Tonukari, 2004).
Tapioca starch has characteristics that make it suitable to serve as substrate in
fermentation, since it easy to plant and has lowest price (Tonukari, 2004). According to
International Starch Trading (2009), tapioca starch is easily to be gelatinized under the low
temperature, in range 59˚C to 65˚C. Moreover, its viscosity is higher and makes it easily to be
digested by enzymes, especially during enzymatic hydrolysis (International Starch Trading,
2009). The degree of viscosity of tapioca starch in breaking its starch was decrease due to its
composition of fibrous matter that lowers the fermentation, thus reduce its viscosity of starch
extracted from inoculums (Ho, 2009).
Tapioca starch like others crops has potential to hydrolyse into sugars by either fungal or
bacterial amylase. The examples of fungi used for hydrolyse are Aspergillus niger and Rhizopus
sp., which both of that fungi require minimal nutritional requirements. Tapioca starch also easily
hydrolysed by bacterial amylase, Bacillus amyloliquefaciens, Bacillus acidopullulyticus and
Bacillus licheniformis (Manno & Pekka, 1989).
2
The conversion of starch to simple sugars is called an enzymatic hydrolysis. Fresh
tapioca is washed, peeled, sliced, weighed, and pulverised before undergo enzymatic hydrolysis
process. The fermentable sugars produced then undergo the fermentation process to produce
lactic acid using Lactococcus lactis IO-1, a homolactic (L-lactic only) acid producing organism.
This organism has higher capability in converting the sugars to L-lactic acid product compared to
others microorganisms such as Enterococcus, Streptococcus, or Pediococcus. Lactic acid
fermentation is the biochemical conversion of biomass into chemicals since its production does
not generate carbon dioxide (Bujang, et al., 2000).
1.2 Problem Statement
The high demand for production of lactic acid in the cosmetic industries makes other way to
enhance the lactic acid production by using cheaper substrate since other starchy materials like
sago, corn, wheat and potato are costly. For this study, tapioca will be used since it is less
expensive and easier to obtain than most other starchy materials, which cheapest sources of
starch compared to cereals, tuber, and root crops (Alias, 2009). The utilization of renewable
substrate will indeed provide this process more attractive since the production cost can be
minimized, and substrate can be obtained at all times.
3
1.3 Objectives
The aim of this study is to produce lactic acid from tapioca sugars under variable sugar
concentrations (50g/L, 100g/L and 100g/L). The general objectives of this study are to:
1. Study the process of enzymatic hydrolysis of tapioca in production of fermentable sugars.
2. Maximize the production of lactic acid from tapioca sugars.
3. Observe the best sugar concentrations to produce higher yield of lactic acid.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Tapioca (Manihot esculenta)
Tapioca, also known as cassava is commonly distributed in South America, Africa Southeast
Asia, Brazil, Indonesia and is one of the most important food crops in the tropics (Burrell, 2003),
and contributed to the nutrition and livelihood of many people and traders around the world.
Since it has the ability to tolerate stressful environments, it becomes one of the crop for
sustainable agriculture, especially in tropical Africa, Asia, and Latin America (El-Sharkawy,
1993).
Tapioca is quite easy plant to be planted (El-Sharkawy, 2004). The mature stem cuttings
(15 – 30 cm long) can be vegetatively propagated either in a density between 6000 to 20,000
plants per ha which depend on the farmer’s ability (El-Sharkawy, 1993). The tubers can be
harvested after about 7 to 24 months after planting. Since tapioca’s root has high starch content,
the root must be used immediately after processing to avoid from deterioration. Root of tapioca
has high content of starch during the period of lower vegetative growth rates of plants.
5
Moreover, tapioca has higher production yield of 71 tons per ha compared to other
tropical food crops, such as maize, sweet potato, rice and others as in Table 1 (de Vries et al.,
1967 cited by EL-Sharkawy, 1993).
Crop
Annual yield
(t/ha)
Cassava (fresh root)
71
Maize (dry grain)
20
Sweet potato (fresh root)
65
Rice (dry grain)
26
Sorghum (dry grain)
13
Wheat (dry grain)
12
Banana (fruit)
39
Table 1 Maximum recorded yield and food energy of some important tropical food crops.
Source adapted from de Vries et al. (1967) cited by EL-Sharkawy (1993)
6
2.2 Enzymatic Hydrolysis of Starch
The process of degradation of starch into fermentable sugars using water and catalyzed by an
enzymes is known as enzymatic hydrolysis. The tapioca flour is easier to be hydrolyzed
compared to other flours due to the approximately 100% conversion during hydrolysis of tapioca
starch using enzymes (Ho, 2009). Tapioca also has lower gelatinization temperature and higher
swelling power that make it easy to saccharify to fermented sugars with optimum conditions
(temperature, pH). Enzymatic hydrolysis is more preferable method for conversion of starch into
fermentable sugars than other methods, acid hydrolysis (Van der Veen, et al., 2006).
Consequently, enzymatic hydrolysis is the first step that defines the capacity of starch
utilization for lactic acid fermentation (Bujang et al., 2000). There are two steps in this process,
which are Liquefaction and Saccharification (Van der Veen, et al., 2006), which each step needs
a specific enzyme that must be separated and inactivated after each run (Paolucci Jeanjean, et al.,
2000). Before liquefaction, there are gelatinization step to open the starch granule in order to
allow its granule become swell, hence easier for bacterial enzyme accessible. In liquefaction, an
enzyme Termamyl-120L (thermostable α-amylase from Bacillus licheniformis, 120 KNU/g) is
used, and require the incubation at 90˚C and stirred for 2 hours (Bujang et al., 2000). This
enzyme is used for reduce the viscosity and induce partial hydrolysis of starch. Saccharification
is the further step which Dextrozyme (a mixture of glucoamylase form Aspergillus niger and
pullulanase from Bacillus acidopullulyticus, 225 AGU/ml) is added into liquefied suspension,
and the mixture is incubated at 60˚C for another 2 hours (Bujang et al., 2000). This enzyme is act
to remove or breakdown of both a-1, 4- and a-1, 6 glycosidic bond.
7
2.3 Lactic Acid
2.3.1 Historical Background
Lactic acid (C3H6O3) is an organic hydroxyl acid that has been discovered and isolated in 1780s
by Swedish Chemist Carl Wilhem Scheele in sour milk (Narayanan et al., 2004; Datta, 1995).
Scheele isolated it in a rather impure condition as brownish syrup. In the beginning of 1881s,
lactic acid was the first organic acid to be commercially produces by microbial fermentation
(Ruter, 1975; Severson, 1998). According to the Holten (1971), in 1839s area, lactic acid
fermentation was performed by Fremy using of several carbohydrates such as sugar, milk sugar,
mannite, starch and dextrin, and this discovery was established by Gay-Lussac. The first lactic
acid bacteria have been found was Streptococcus lactis that isolated in pure culture of distilleries
(Hilton, 1971).
Early 1960s, lactic acid was produced by chemical process using petroleum by products
in USA (Hilton, 1971). At that time, America became the supplier for lactic acid production.
Ecological Chemical Products (EcoChem), a joint venture of E.I du Pont Nemours & Co., and
Con Agra was contributed in lactic acid production by producing 1 to 2 million pounds of lactic
acid by fermentation of whey permeate (Severson, 1998). The lactic acid production has been
increase by produced 10 to 40 million pound per year at 1990s. According to the Severson
(1998), the demand of lactic acid has been increased by the year until reach about 2000 million
and above per year.
8
2.3.2 Properties of Lactic Acid
Lactic acid is the three carbons organic acid, which one terminal carbon atom is part of an acid
or carboxyl group. The other terminal carbon atom is a part of methyl or hydrocarbon group and
central carbon atom is alcohol carbon group. The molecular structure of lactic acid containing
carbon atom that occurs naturally in two isomer forms (D and L) as shown Figure 1.
COOH
OH
C
COOH
H
H
CH3
C
OH
CH3
L (+) lactic acid
D (-) lactic acid
Figure 1 Different configuration of lactic acid molecule, L (+) lactic acid and D (-) lactic acid
L (+) lactic acid is the normal intermediary product of carbohydrate and amino acid
metabolism in mammals include human. It also has been found in all tissues, body fluids and
excreta. While, D (-) lactic acid has only been detected in excreta (Holten, et al., 1971).
Lactic acid is a soluble in water and water miscible organic solvents but insoluble in
other organic solvents (Narayanan et al., 2004). It is a pure anhydrous with a white crystalline
solid. It has a colorless, sour in taste, odorless and appears generally in form of more or less
concentrated aqueous solution, as syrup liquid (Rashid, 2008). It can be considered as a stable
and combustible substance that compatible with strong oxidizing agents (Narayanan et al.,
9
2004). According to the Rashid (2008), the normally lactic acid is observed as a clear to slightly
yellowish liquid and typically has an 88% to 92% concentration. Thus, it can be appears in
diluted or concentrated aqueous solution. Moreover, lactic acid is a weak acid with a low
volatility (Casida, 1964). Table 1 shows the physical properties of lactic acid (Narayanan, 2004).
Molecular weight
90.08
Melting point
16.8°C
Boiling point
82°C at0.5 mm Hg
122°C at 14 mm Hg
Dissociation constant, Ka at 25°C
1.37 X 10-4
Heat of combustion, ΔHc
1361 KJ/mole
Specific heat, Cp at 20°C
190 J/mole
Table2 Physical properties of lactic acid
Source adapted from Narayanan, et al., 2004
2.3.3 Lactic Acid Bacteria
Lactic acid bacteria are among the best studied microorganism. In early 2000, there were new
developments have been made in the research of lactic acid bacteria in the areas of autolysin,
bacteriocins, multidrug resistance and bacteriophages (Narayanan, et al., 2004). In advance,
there were also been made in the construction of food grade genetically modified of lactic acid
bacteria (Konings et al., 2000).
10
Lactic acid bacteria can be classified into two types based on its shape, rod and coccus.
The taxonomy for rod shaped, mainly belonging to the genus Lactobacillus (Jobli, 2004).
However, the coccus is so sensitive when isolated form its natural source, hence still
unestablished in terms of taxonomy and morphology (Jobli, 2004).
Lactococcus lactis IO-1 species is an important group of lactic acid bacteria that used for
production of lactic acid. It can grew under microphilic conditions (Samaržija et al., 2001).
Lactococci are homofermentative microaerophilic Gram-positive bacteria which produce L (+)
lactic acid form glucose (Samaržija et al., 2001). The strains of these bacteria can utilize several
of carbohydrate to produce mostly L-lactate with high conversion rate and absence of fatty acid
(Ishizaki et al., 1990; cited by Jobli, 2004). Moreover, it can grow in range of temperature 10°C
to 45°C since the optimal temperature for its production is 37°C. Thus, this strain has been
identified to the Japan Collection of Microorganism as L. lactis IO-1 JCM 7638, and used in this
study.
2.3.4 Lactic acid Fermentation
Lactic acid fermentation is classified into types, which are homolytic and heterolytic
fermentation (Holten et al., 1971). In homolytic fermentation, the pure lactic acid was forms
using homolytic Lactobacteriaceae (Holten, et al., 2004). Streptococci and Lactobacilli arge
generally homolytic lactic acid bacteria that considered to yield 85 to98 per cent of sugars
fermented as lactic acid (Gunsalus & Niven, 1942). However, there has been presence of volatile
acids, acetic acid reported, which suggested as secondary fermentation products or oxidation of
11
lactic acid (Gunsalus & Niven, 1942). While, in heterolyctic fermentation, others products were
formed as well as lactic acid, such as acetic acid, ethanol, carbon dioxide and formic acid. This
type of lactic acid fermentation was done by heterolytic Lactobacteriaceae and others bacteria,
Bacillus, Staphylococcus, Salmonella species and fungi species, Rhizopus.
Thus, the type of homolytic fermentation was used in this study which pure lactic acid
has been produced without carbon dioxide synthesis.
2.3.5 Applications of Lactic Acid
Lactic acid is very important in our industry especially in fermentation (Bujang et al.,
2001) that can be produced by either microbial fermentation or chemical synthesis. However,
fermentation process is more favourable compared to chemical synthesis due to its higher
efficiency together with less wastage (Bujang et al., 2000).
It most useful chemicals, used in the food industry as preservatives, acidulant,
emulsifying agents in baking foods, and flavouring, in the textile and pharmaceutical industries,
and in the chemical industry as a raw materials for the production of lactate ester, propylene
glycol, 2, 3-pentanedione, propanoic acid, acrylic acid, acetaldehyde, and dilactide (8, 9)
(Vickroy, 1991). In addition, lactic acid consumption has increased because of its role as
monomer in the production of biodegradable PLA (well-known as sustainable bioplastic).
12
Recently, L-lactic acid utilization has increased considerably because of its utilization in
pharmaceutical and cosmetic applications and formulations in topical ointments lotion, anti-acne
solutions, humectants, parenteral solutions and dialysis application, for anti carries agent
(Narayanan et al., 2004). Moreover, L-lactic acid also can be used as skins whitening agent that
shows a synergistic effect compared to other skin whitening agent (Narayanan et al., 2004).
13
CHAPTER 3
MATERIALS AND METHODS
3.1 Materials
3.1.1 Tapioca Starch
Fresh tapioca (Figure 2) was purchased from the local wet market in Satok Kuching, Sarawak.
The food TF (Figure 3) purchased from a local market (Unaco brand).
Figure 3: Tapioca Flour (TF)
Figure 2: Fresh Tapioca (FT)
14
3.1.2 Enzymes for Hydrolysis
Two enzymes were used in the hydrolysis. Termamyl-120L, an alpha-amylase from Bacillus
licheniformis (120 KNU/g) was used in the liquefaction step. Detroxzyme, a mixture of
glucoamylase from Aspergillus niger and pullulanase from Bacillus acidopullulyticus, 225
AGU/ml were used in the saccharification step.
3.1.3 Microorganism
The microorganism used in this study was Lactococcus lactis IO-1 (JCM7638). A stock culture
was incubated in Thioglycollate (TGC) liquid medium for 18 hours at 37ºC prior to be as
inoculum.
3.1.4 Medium Composition
The basal medium for fermentation is glucose broth consisting of 5g/L yeast extract (Difco,
USA) and distilled water. Glucose concentrations were set at 50g/L, 100g/L and 150g/L. The
same medium were containing of 10% (volume/volume) inoculum.
15