VIETNAM NATIONAL UNIVERSITY HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

LACTIC ACID FERMENTATION OF PURPLE SWEET POTATO

(IPOMOEA BATATAS L.) AND BLACK GLUTINOUS RICE (ORYZA
SATIVA L.) BY LACTOBACILLUS ACIDOPHILUS AND ITS EFFECT
ON ANTIOXIDANT CAPACITY AND ANTHOCYANIN CONTENT OF
THE FERMENTED SOLUTION

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology

Student name: Ly Hong Van Nhi ID No. : BTIU08034

Supervisor: Dr. Dang Quoc Tuan

Febuary / 2013

ACKNOWLEDGMENT
Thanks go first and foremost to my supervisor, Dr Dang Quoc Tuan, who
instructed, advised, gave thoughtful comments to me. I have learnt so much
from him.
I am also grateful to all staffs in the laboratories at International University who
provided me with chemicals and equipment needed. Particular thanks go to
laboratory technician Ms Le Tran Hong Ngoc who was always with me at school
until night in these days that I established the growth curve of bacteria. Her
suggestion, support, assistance and courage meant have been of immerse help
me in completing my research.
I would like to acknowledge Ms Nguyen Thi Huong from of Ho Chi Minh City
University of Technology, who gave me bacteria strain as well as shared with me
many experiences. I also thank to Ms Nguyen Thi Tieu Mi, Ms Vu Thanh Nguyen
and her aunt who helped me to find the raw material with best quality, black
glutinous rice and purple sweet potato.
Doing research with my friend at laboratory of International University has been
a wonderful experience. I would like to thank the support and encouragement of
my friends, Ms Le Thi Thanh Thao, Ms Doan Thi Nhu Nguyen, Ms Ngo Thi Thu
Hien, Ms Nguyen Thanh Tram, Ms Nguyen Thi Tieu Mi, Ms Pham Hong Ngoc, Mr
Nghe Van Dat, Mr Huynh Xuan Vu.
Finally, this project would not have been possible without the unfailing support of
our family, my parents Mr Ly Cong Hien; Mrs Nguyen Thi Xuan Hong and my
little sister, Miss Ly Phuong Nhi. Their patience, encouragement, and enthusiasm
have made this endeavor possible.

LACTIC ACID FERMENTATION OF PURPLE SWEET POTATO (IPOMOEA BATATAS

L.) AND BLACK GLUTINOUS RICE (ORYZA SATIVA L.) BY LACTOBACILLUS
ACIDOPHILUS AND ITS EFFECT ON ANTIOXIDANT CAPACITY AND ANTHOCYANIN
CONTENT OF THE FERMENTED SOLUTION
Ly Hong Van Nhi a, Dang Quoc Tuan
a

School of Biotechnology, International University Vietnam National University

in HCMC
b

Dept. of Food Technology, International University Vietnam National

University in HCMC
Corresponding authors email address: [email protected]

ABSTRACT
This study was carried out to find the possibility of fermenting purple sweet
potato (Ipomoea batatas L.) and black glutinous rice (Oryza sativa L.) with
Lactobacillus acidophilus. Two substrates, namely purple sweet potato (PSP) and
black glutinous rice (BGR) were saccharified by the combination of 0.1 % amylase and 0.15% glucoamylase. Saccharified PSP and BGR were subjected to
lactic acid fermentation using 1% starter culture of Lactobacillus acidophilus.
Falcultative anaerobic fermentation was performed for 24 hours at 37oC. The
resulted lactic fermented PSP and BGR contained 6.20 x 108 CFU/mL and 1.12 x
108 CFU/mL viable cell count, respectively. Also, PSP and BGR lactic acid solution
had 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity IC50
values of 31.8g/mL and 34.42 g/mL, respectively. Fermentation had no effect
on the antioxidant activity of fermented solutions. Based on these data, it
suggested that PSP and BGR fermented by L.acidophilus could be used to
develop the healthy food with the supplement of viable cells and antioxidant
activities.
Keywords: lactic acid fermentation, saccharified PSP, BGR, Lactobacillus
acidophilus, anthocyanins, antioxidant activity

1. INTRODUCTION
Fermentation includes various traditional processes which allow fresh food
to be preserved for future uses. People has been fermented food since ancient
times. Nowadays the main purpose of food fermentation is not to preserve but to
produce a wide variety of food fermentation products with specific taste, aroma,
and texture. Being enriched with probiotic bacteria, fermented products have
evolved into one of the most successful class of functional foods. Lactic acid
bacteria (LAB) are principle organisms involved in fermentation for the purpose
of probiotic as well as flavor enhancement and preservation (Anderson, 1988).
Among LAB used in fermentation, Latobacillus acidophilus have been applied
extensively in food fermentation and processing (Lee et al., 2011). Deraz et al.
(2007) reported that L.acidophilus was widely used in fermented dairy products
in oder to reduce the levels of harmful bacteria and yeasts in the small intestine.
L.acidophilus strains have been widely utilized as a dairy starter culture for their
therapeutic activities associated with an intestinal microbial balance, and has
been used in fermented foods, and as a probiotic in dietary supplements
(Sanders & Klaenhammer, 2001). However, L.acidophilus fermentation of
substrates rich in anthocyanins such as purple sweet potato and black glutinous
rice has been very limited. Duangjicharoen et al. (2008) showed that plant and
root beverages are healthy due to their high nutritional value and presence of
bioactive compounds derived from the substrates used and during the
fermenteation process.
Many fruits, vegetables, cereal grains and flowers which have red, purple
and blue colors all contain anthocyanin pigments. Anthocyanins have an electron
deficiency due to their particular chemical structure, which makes them very
reactive toward free radicals present in the body; help them to be powerful
natural antioxidants. In recent years, the interest in anthocyanins pigments in
consumer market has increased due to their possible health benefits as dietary
antioxidants (Bridgers et al., 2010). Moreover, anthocyanins become attractive
sources of natural food colorant and textile industry as an alternative to
synthetic food dyes because of their deep purple-red color (Wegner et al., 2009).
Purple sweet potato (PSP), scientifically known as Ipomoea batatas L., is
easily grown in tropical area. It is rich in vitamin (B1, B2, C, E), minerals
(calcium, magnesium, potassium, zinc), especially anthrocyanins. The purple
sweet potato can be recommended as a superior source for production of foods
with health benefits (Suda et al., 2003). Due to the high level of anthocyanins,
PSP is considered as a healthy food additive and potential source of natural food

colorants. Suda also indicated that PSP belong to a group with highest stability
to heating and intraviolet ray radiation. A study by Kano et al. (2005) showed
that the pigments of PSP anthocyanins have higher levels of radical scavenging
activity than other pigments. Because this reason, acylated anthocyanins from
purple sweet potato can be used as natural colorants due to their high heat and
light stability. Moreover, PSP anthocyanins have useful characteristics for food
manufacturing, remaining stable after heating and ultraviolet irradiation (Kano et
al., 2005). Therefore PSP could be used in food industry as antioxidants to
improve human health.
Like purple sweet potatoes, black glutinous rice (BGR) or Oryza sativa L.,
also possess color substances that belong to the flavonoid. A commonly found
anthocyanin in colored rice is acelylated procyanidins, which is reported to
possess a free radical scavenging activity (Oki et al., 2002). Acoording to
Satharut (2012), black rice contain two main compounds of anthocyanin;
cyanidin 3-glucoside (C3G) and peonidin 3-glucoside (P3G).
Recently, yogurt, drinking-yogurt type beverage have been consumed
widely in Asian countries. Many researchers have used many kinds of raw
materials as a substitution of milk for fermentation. Substrates for cereal-based
lactic fermented products contained corn, sorghum and millet (Nashiru et al.,
1992), and extruded rice (Viet et al., 1992). Lactic acid fermentation of cassava
was also studied in Nigeria (Nashiru et al., 1992). A study by Wongkhalaung
(1995) sucessflully made drinking yogurt-type beverage from sweet potato.
Sweet potato was saccharifized with 2 kinds of enzyme alpha-amylase and
glucoseamylase. Using 1% starter culure Streptococcus thermophilus and
Lactobacillus bulgaricus, fermentation was carried out for 18-21 hours at 37oC.
The product contained about 0.7% acidic as lactic acid and 6.8 x 10 8 CFU/g
viable cell count. Another research from Lee et al., (2011) showed that the
fermented yam with Lactobacillus acidophilus can be served as a functional food
and nutraceutical content, such as allantoin and diosgenin. In a study of Sasaki
and Ohma (2004), purple weet potatoes were added in lactic acid bacteria drink
to develop the anthocyanin content. Nevertheless, researches

on using

substrates rich in anthocyanins for lactic fermentation have still little known in
Viet Nam.
So far, there has been little discussion about the change of antioxidant
activity after fermenting process. A study of Sasaki & Ohba (2004) showed that
lactic acid bacteria drink (LABD) with purple sweet potato had the most
antioxidant activity compared with LABD without PSP and a 10% PSP solution
(which contained the same level of anthocyanins as the PSPLABD prior to

fermentation). The result of antioxidant activity of PSPLAB drink before and after
fermentation did not differ significantly. Another research from Wu et all (2012)
also studied on fermented PSP milk, the authors also used DPPH radical
scavenging assay to measure the antioxidant activity. However the result of
DPPH scavenging activities in their study was not similar to Sasaki & Ohba
(2004).
Thus, further study needed to carry out in an attempt of finding a way to
enhance

ultilization

and

consumption

of

products

rich

in

anthocyanins;

especially, the development in fermentation with different substrate and the

change in some products characteristic after fermenting process. This paper is
designed to investigate the possibility on development of lactic fermentation
from two substrates: purple sweet potato and black glutinous rice. In addition to,
observing the changes in anthocyanin content and antioxidant activity after
fermenting process also the objective of this research.
2. MATERIALS AND METHODS
2.1 Research location
The

research

experiments

were

conducted

in

the

laboratory

of

International University, Linh Trung, Thu Duc Dist, HCM city, Vietnam.
2.2 Materials
Purple sweet potato (PSP), scientifically known as Ipomoea batatas L. and
black glutinous rice (BGR), or Oryza sativa L. used in this study were purchased
from the Nguyen Son market, Tan Phu Dist, HCM city and An Giang province,
Vietnam. They were stored at 4oC until used.
Lactobacillus acidophilus was obtained from the Genus collection of Ho
Chi Minh City University of Technology (HCMUT). This strain was propagated in
MRS broth (see Appendix 1) for 24 hours at 37 oC and finally stored at -20oC in
MRS broth containing 20% glycerol, before being subjected to fermentation.
2.3 Chemicals
The -amylase used was Temamyl 120L (Novozymes, produced from
Bacillus licheniformis, stored at 4oC, density 1.26g/mL) with an optimal pH 6
6.5, optimal temperature 85oC and activity of 120 KNU-T/g enzyme. A kilo novo
unit, KNU, is the amount of enzyme necessary for breaking down 5.26g starch
per hour. The glucoamylase used was Amyloglucosidase EC 3.2.1.3 (Sigma,
USA, obtained from Aspergillus niger, stored at 4oC, density 1.2g/mL) with an

optimal pH 3.6 - 4.2, optimal temperature 60oC. Enzyme activity is that 0.1 mL
of this enzyme will digest 1 gram of corn or wheat starch to glucose.
2.4 Experimental design
2.4.1 Saccharification of purple sweet potato and black glutinous
rice
Saccharification of purple sweet potato and black glutinous rice were
carried out by the method described by Wongkhalaung (1995) with a slight
modification. PSP were washed, peeled, sliced and steamed for 15 minutes.
Next, PSP were mashed while hot; the moisture was checked again; and a
mixture was created by mixing the mashed PSP with distilled water to get the
solution of 10% dry matter. Saccharification was carried out at 60 oC in an
incubator for maximum 90 minutes using 0.15% (dry matter) of glucoamylase
and 0.1% (d.m.) -amylase.
BGR were also ground and mixed with distilled water to get the solution of
10% dry matter. Then the mixture was cooked for 25-30 minutes. Stirring was
needed during the cooking process (prevent clotting at the bottom).
Saccharification was carried out in an incubator at 60oC for maximum 90 minutes
using 0.15% (d.m.) of glucoamylase and 0.1% (d.m.) -amylase.
2.4.2 Preparation of lactic fermented PSP and BGR
Lactic fermented PSP and BGR was prepared according to the method of
Lee and others (2011). Saccharified PSP and BGR were heated to 95 oC and held
for 5 minutes to inactivate the enzymes. Then the solutions obtained from the
process were centrifuged to get the clear supernatant and sterilized at 121oC for
15 minutes. L. acidophilus incubated at MRS broth at 370C was collected at the
log phase and centrifuged. The precipitant was washed 3 times with distilled
water and was later used as fermenting microorganism. Lactic acid fermetation
was performed at 37oC in facultative anaerobic condition for 24 hours, using 1%
starter cultures. All steps were executed in a safety cabinet in order to minimize
contamination for medium preparation, culturing bacteria, viable cell counting.

Lactic acid bacteria ( Starter culture )

Stock (-20oC) was primary and secondary increased the number in 20mL

MRS broth and 100mL MRS broth, respectively. The relationship between the
number of colonies and OD value was investigated after each 5 hours from 0 to

20 hours. With each OD value, the number of colonies was calculated and the
growth curve for L.acidophilus was established. From this, the starter culture
could be controlled.
2.4.3 Microbial analysis
MRS plate count agar was used for L.acidophilus counting (Lee et al.,
2011). One mL of sample diluted with 9 mL of sodium chloride solution (0.85%).
Subsequent dilutions of each sample were plated in Petri dishes and incubated at
37oC for 72 hours. Viable cell count of lactic acid bacteria (CFU/mL) was then
enumerated by using the

colony counter, model mrc 570-06. The CFU was

calculated as the following equation:

Mi (CFU/ml) = Ai x Di /V

(1)

Where Ai is an average number of colony of two Petri dish; Di is a dilution factor;

and V is a volume of loading sample in each Petri dish. Average density of colony
number in initial sample is arithmetic mean of Mi at different dilute factor.
2.4.4 Analytical methods
Chemical composition of raw materials (fresh PSP and BGR) were
determined by AOAC and AACC Official Method : moisture (AOAC 1999) and
moisture balance MOC-120H; protein (AACC 46-10); crude fat (AOAC 2003.05);
crude fiber (AOAC 962.09); crude ash (AACC 08-01).
Reducing sugar was determined by using DNS method (Miller., 1959).
pH and titratable acidicity (Lee et al., 2011) of the samples were mesured
at room temperature. After mixing the 9mL samle with the same amount of
distilled water, the acidicity was mesured by titrating with 0.1 N NaOH using a
1% phenolphthalein indicator to an end point of faint pink color. The formula for
calculating percentage of lactic acid follows:
Lactic acid (%) = [ 0.1 N NaOH used (mL) x 0.009x100]/sample (mL)
(2)
2.4.5 Determination of total monomeric anthocyanins
To measure the anthocyanin contents, the experiment was carried out as
described previously ( Bridgers et al., 2010 ; Ohba and Sasaki, 2004) with a

slight modification. Solvent, acidified ethanol (pH ~ 3.5) _ 70% ethanol with 7%
acetic acid, was added to treatment tubes and distilled water was used as
control. With raw material, 5%( DW, w/v) of PSP and BGR were used as solid
loadings. PSP roots were sliced ( 2-3 mm thickness chips) and diced ( 3mm3).
BGR was milled and passed through the 250 m sieve. Diced PSP roots and BGR
flour were measured into 50mL Falcon tubes. All tubes ( except controls) were
shaken (100rpm) and incubated for 1 hour in an incubator at 80oC. After
centrifugation at 6000rpm, 4oC for 15 minutes, the supernatant was taken and
stored at -80oC until anthocyanin analysis. All samples were analyzed within a
week.
With fermented solutions, anthocyanins were extracted by adding 50mL
acidified ethanol into these 10% (DW, w/v) PSP and BGR solutions. The
procedure for measuring anthocyanins from fermented solution was similar to
the one used in raw material solutions.
Total monomeric anthocyanin content of PSP and BGR were determined
by the spectrophotometric pH differential method (Lee et al., 2005) ( AOAC
Official Method 2005.02). To measure the absorbance at pH 1.0 and 4.5, the
samples were diluted in appropriated dilution factor with pH 1.0 potassium
chloride buffer and pH 4.5 sodium acetate buffer, respectively. The absorbance
of each dilution was measured at 520 nm and 700 nm by using a
spectrophotometer (GENESYS 10S UV-Vis, Thermo Fisher Scientific, Madison,
WI, USA). The concentration of anthocyanin pigment was calculated by the
following equation:
Monomeric anthocyanin pigment (mg/L) = [ A diff x MW x DF x 1000] /
(3)
where MW represents molecular weight of cyanidin-3-glucoside (449.2); DF is
dilution factor, is molar absorptivity of cyanidin-3-glucoside (26900 L/mol cm)
and Adiff was calculated from the following equation:
Adiff = (A520nm A700 nm)

pH 1.0

(A520nm A

700nm) pH 4.5

(4)
Note that A700 was measured and subtracted off in order to eliminate the effect
of haze or sediments in the sample.

2.4.6

Assay

of

1,1-Diphenyl-2-picrylhydrazyl

(DPPH)

radical

scavenging activity
To measure antioxidant activity, the DPPH radical scavenging assay was
carried out as described previously (Sasaki & Ohba., 2004 ; Kano et al., 2005)
with a slight modification. The sample solutions were centrifuged at 1300 x g for
10 min. After that 2 mL sample of the supernatant was mixed with 2 ml of 100
M DPPH in ethanol. Ethanol (2mL) with DPPH solution was used as blank. These
solutions were kept in dark for 30 min at room temperature. The absorbance of
the mixture was determined at 517 nm. Three replicates are done. The
antioxidant activity of test compounds is expressed as IC50, which was defined as
the concentration of test compounds required to inhibit DPPH radiacls by 50%.
Percentage of inhibition was calculated using the following formula:
Percent ( %) inhibition of DPPH activity = [ (A-B)/A] x 100

(5)

Where A is the optical density of the blank and B is the optical density of the
sample.
2.5 Data analysis
The experimental results were expressed as average values (means)
standard deviations ( or CV- coefficient of variation). The analysis of variance (
ANOVA) was conducted using SPSS version 16.0 to test the significant different
between groups ( P < 0.05).

3. RESULTS
3.1 Initial analyses of purple sweet potato (PSP) and black glutinous
rice (BGR)
Initial analyses in this study helped to determine compositions of raw
material (PSP and BGR). Moisture content, protein, lipid, ash, crude fiber and the
anthocyanin contents of two above substrates were listed in Table 1. These
results were based on the wet weight of the materials and then they were
converted into dry basis (d.b) for easily compared. The coefficient of variation
(CV) was also included, providing a general evaluation about the performance of
the method.

Table 1. Basic compostions of PSP and BGR ( g/100g Wet basis)

PSP
Nutrients

BGR

Mean

CV

Dry

(w.b)

(%)

basis

Mean

CV

D.b

(%)

(d.b)
Moisture (%)

64.50

0.19

12.01

0.83

Protein (%)

1.38

7.17

3.89

6.18

8.50

7.02

Lipid (%)

0.23

1.30

0.65

1.20

0.88

1.16

Ash (%)

0.88

2.84

2.47

1.37

1.03

1.56

Fiber (%)

2.78

1.04

7.83

3.67

1.29

4.17

58.46

2.56

164.69

79.27

2.67

90.08

Anthocyanins
(mg Cyd-3-gluE/100fw)

Values represent the mean of triplicate with coefficient of variation (CV)

3.2 Reducing sugar of PSP and BGR before saccharification, after
saccharification and after fermentation

Mean values with different letters are significantly different (P<0.05)

Figure

1.

Reducing

sugar

(%D.M)

of

PSP

and

BGR

saccharification, after saccharification and after fermentation

solutions

before

The changes in total reducing sugar of two substrates occurred after

saccharification and after fermentation were shown in Figure 1. Determinations
of reducing sugar contents from PSP and BGR before saccharification process
were found to be the same, 2.54% 0.14 and 2.15% 0.11, respectively. After
sachharification, there were high increases in sugar content, 17.85% 0.18 with
PSP substrate and 15.74% 0.28 with BGR substrate. Reducing sugar contents
left after fermentation of PSP and BGR were around 15.45% 0.4 and 14.58%
0.27, respectively.
3.3 Lactic acid fermentation of PSP and BGR
3.3.1 Morphology of Lactobacillus acidophilus and their colonies

Figure 2. Morphology of Lactobacillus acidophilus

After increasing the number of Lactobacillus acidophilus in MRS broth, the
morphology was checked by Gram staining. The result (Figure 2) observed under
100X microscopic objective lens showed that L.acidophilus got the purple colour.
They are bacillus organisms, usually separate from each other or form a short
chain.

10

Figure 3. Colonies of Lactobacillus acidophilus in MRS agar plate

The agar plates were made with the sample on the surface and spread
gradually. One cell can multiply in geometric progression to form one colony.
After incubating at 37oC in 72 hours, the colonies of L. acidophilus had white
round shape, convex surface with a smooth outer edge.

3.3.2 Growth curve of L.acidophilus

Figure 4. Growth curve of Lactobacillus acidophilus

Growth curve of L.acidophilus was established in Figure 4 in which log
(CFU/mL) was recorded every 5 hours over a period of 24 hours.

11

Figure 5. Lactobacillus acidophilus in MRS broth before (left) and after (right)
incubation
By measuring the OD of broth medium (Figure 5) combined with
spreading plates, the number of colony forming unit (CFU) could be measured
(Table 2). The starter culture for lactic acid fermentation was around 107
CFU/mL.
Table 2. The correspond number among OD, cell number and Log (CFU/mL) of
L. acidophilus
OD

0.06 - 0.10

0.4 - 0.9

1.01 1.12

Cell number

2.01x 103

4.31 x 104

4.16 x 108

3.15 x 103

4.07 x 108

5.40 x 108

3.30 3.50

4.63 8.61

8.62 8.73

Log ( CFU/mL)

3.3.3 Lactic fermentation of PSP and BGR

Table 3. Some characteristics of lactic fermented PSP as compared to lactic
fermented BGR
PSP

BGR

Before

After

Before

After

fermentation

fermentation

fermentation

fermentation

1.02 x 107

6.20 x 108

1.02 x 107

1.12 x 108

Acidity (%)

0.1

0.54

0.08

0.35

pH

5.1

3.33

5.8

3.16

Viable cell
count
(CFU/mL)

12

Lactic acid fermentation of saccharified PSP and BGR were carried out and
changes occurred during fermentation period were determined as showed in
Table 3.

Before fermentation

After fermentation

Figure 6. Lactic acid fermentation with PSP substrate

With lactic fermented PSP, pH decreased from 5.1 to 3.33; acidity was increased
from 0.1% to 0.54%; and viable cell count of lactic acid bacteria was raised
about 1 log cycle, from 1.02 x 107 to 6.20 x 108.

Before fermentation

After fermentation

Figure 7. Lactic acid fermentation with BGR substrate

With lactic fermented BGR, pH decreased from 5.8 to 3.16; acidity was increased
from 0.08% to 0.35%; and viable cell count of lactic acid bacteria was also grew
about 1 log cycle, from 1.02 x 107 to 1.12x 108.

13

Figure 8. Colonies of Lactobacillus acidophilus from lactic fermented PSP (left)

and lactic fermented BGR (right)
During fermentation, L. acidophilus could play an important role for fermentation
system which ferments monosaccharide or sugar to alcohols and. The reduction
of pH of fermented PSP and BGR were probably due the formation of acids by
bacteria utilized carbohydrate.

3.4 Anthocyanin concentration of PSP and BGR before and after

fermentation

Mean values with different letters are significantly different (P<0.05)

Figure 9. Anthocyanin concentration of PSP and BGR solutions before and after
fermentation
Figure 9 showed that the anthocyanin contents of PSP and BGR solution
were

changed

after

fermentation.

As

14

raw

material,

the

anthocyanin

concentration of PSP was higher than the one of BGR, 164.69 mg/100g dry basis
(d.b) and 90.08 mg/100g d.b, respectively. After 24 hours of fermentation, PSPs
anthocyanin was decreased from 164.69 mg/100g d.b to 102.07 mg/100g d.b.
BGRs anthocyanin was also reduced from 90.08 mg/100g db to 35.71 mg/100g
d.b.
3.5 DPPH radical scavenging activity of PSP and BGR before and after
fermentation
DPPH radical scavenging activity measures the hydrogen-donating ability of
antioxidants. In this study, the antioxidant activity was evaluated with IC50
value (the concentration at which radical scavenging activity is 50%). Results of
the free radical scavenging activities were presented in Figure 10 and table 4.

Figure 10. DPPH radical scavenging activity of two solutions (PSP and BGR)
before and after fermentation
Figure 10 indicated that the PSP solution before fermentation showed strongest
antioxidant activity (IC50 = 186.1 L).

15

Table 4. IC50 value of PSP and BGR solutions before and after fermentation
IC50 (L)

PSP

BGR

IC50 (g/mL)

Mean

Mean

CV (%)

Before

186.1

30.07

3.13

After

311.9

31.8

3.21

Before

371.9

33.47

1.23

After

967

34.42

2.09

The antioxidant potential is inversely proportional to IC50 value. It means

that the higher the IC50 values were, the lower the antioxidant activities were.
According to table 4, the antioxidant potential of PSP and BGR solutions were not
changed too much after fermentation. IC50 values of PSP solution varied from
30.07g/mL to 31.8g/mL. Meanwhile, IC50 values of BGR solution changed
from 33.47 g/mL to 34.42 g/mL.

4. DISCUSSION
4.1 Initial analyses of purple sweet potato (PSP) and black glutinous
rice (BGR)
In comparison with the USDA nutrient database, the basic compositions
of PSP and BGR including moisture, protein, lipid, ash, fiber (Table 1) in this
study were acceptable.
Total anthocyanin content of PSP was 58.46 mg Cyd-3-glu-E/100 f.w.
(Table 1). This result was higher than those results found in Teow et al. (2007)
24.6-43.0 mg/100g fw and Brown et al. (2005) 15-38 mg/100g f.w. However,
the investigation by Suda et al. (2003) showed anthocyanins in the same data as
this study, 60mg/100g f.w. Extracted anthocyanins from PSP have been reported
in literature ranging from 15mg/100g f.w. to 182 mg/100g f.w. (Brown et al.,
2005).
Meanwhile the total anthocyanin of BGR was 79.27 mg Cyd-3-glu-E/100g
f.w., correspond to 900.08 g/g d.b (dry basis). This result was lower than total
anthocyanin content, 3276 g/g d.b, as reported by Abdel-Aal and Hucl (2003).

16

Other research of Tananuwong (2010) showed lower anthocyanin concentration

from BGR, 288 g/g d.b.
As compared to reported data, the anthocyanin contents gathered in this
study were in middle range. The PSP and BGR samples were used for further
experiments.
4.2 Reducing sugar of PSP and BGR before saccharification, after
saccharification and after fermentation
By using ANOVA analysis, after saccharification, total reducing sugar
content was notably increased. Effects of two enzymes alpha amylase and
glucosamylase on saccharification of PSP were more pronounced than that of
BGR. In the after saccharification phase, with PSP substrate, reducing sugar
content was raised from 2.54% to 17.85% after 90 minutes incubation.
Meanwhile, with BGR substrate, the increase was from 2.15% to 15.74%. Then,
it was a slightly reduction in sugar content of BGR after fermentation from
15.74% to 14.58%. With PSP substrate, reducing sugar content decreased with
a greater amount from 17.85% to 15.45%. As compare with a study of
Wongkhalaung (1995) which researched about lactic acid fermentation of sweet
potato, reducing sugar also reduced approximately 2% after fermenting process.
Generally, the starch molecules after the saccharification are broken
down to monosaccharides by the effect of two enzyme, alpha amylase and
glucoamylase. This leads to the increase of reducing sugar after saccharification.
Alpha amylase randomly cleaves the inner portion of amylase (-1,4 bonds) to
form soluble dextrins. Meanwhile, glucoamylase hydrolyzes -1,4 in addition to
-1,6

glycosidic

linkages

from

the

non-reducing

ends

of

amylase

and

amylopectin. The reducing sugar content decreased after fermentation because

lactic acid bacteria used monosaccharides or sugars and converted them to
acids.
4.3 Lactic acid fermentation of PSP and BGR
4.3.1 Morphology of L.acidophilus and their colonies
L.acidophilus got the purple color because of their thick peptidoglycan.
Therefore, they belong to Gram (+) bacteria group. L.acidophilus can ferment
with or without the presence of oxygen. This bacterium, is homofermentative
and has optimum temperature from 37C - 42C (Todar, 2012).

17

4.3.2 Growth curve of L.acidophilus

According to the growth curve showed in Figure 4, lag phase extended
about 8 hours. At this time, the bacteria had to adapt with the MRS broth
medium. Exponential phase (log phase) lasted for the next 12 hours. After that,
the growth of bacteria turned to stationary phase. This investigate showed that
the best time to collect starter culture was in range of 10-20 hours.
4.3.3 Lactic fermentation of PSP and BGR
According to the table 3, results showed that PSP was the better substrate
for lactic fermentation of L.acidophilus than BGR. With the same amount of
starter culture, 1.02 x 107 (CFU/mL), after 24 hours incubation the viable cell
count of lactic acid bacteria on PSP substrate increased to 6.20 x 108 (CFU/mL)
more than 5.08 x 108 (CFU/mL) as compared with one on BGR substrate (1.12 x
108 CFU/mL).
4.4 Anthocyanin concentration of PSP and BGR before and after
fermentation
According to ANOVA analysis, which was carried out in order to compare
these mean values, the anthocyanin concentrations of two substrates, PSP and
BGR, before and after fermentation were significantly different. In general, the
anthocyanin contents of both PSP and BGR tended to declined.
Decreases in total monomeric anthocyanin content from PSP and BGR
during fermentation were depicted in Figure 9. Generally, cyaniding-3-glucoside
is the most widespread anthocyanin from fruit, vegetables and plants (Kong et
al., 2003).

Acid may cause partial or total hydrolysis of the acyl moieties of

acylated anthocyanins that are present in some plants (Kong et al.,2003). In

addition, degradation of anthocyanins in the presence of weak acids, consists of
direct condensation of acid on the carbon 4 of the anthocyanin molecule, causing
the loss of both (Poei-Langston and Wrolstad, 1981). From the results, the
decrease in total monomeric anthocyanin content resulted from pH lowering
during fermentation (Table 3).
4.5 DPPH radical scavenging activity of PSP and BGR before and after
fermentation
The free radical scavenging activities of the PSP and BGR solution before and
after fermentation were established by examining their abilities to bleach the

18

stable

radical

DPPH.

DPPH

measured

the hydrogen

donating ability

of

antioxidants. Figure 10 shows that the changes in antioxidant activity after

fermentation of two substrates, PSP and BGR, were not too different. The higher
the IC50 values were, the lower the antioxidant activities were. After
fermentation by Lactobacillus acidophilus, IC50 values of PSP and BGR increased
from 30.07g/mL to 31.8g/mL and 33.47 g/mL to 34.42 g/mL, respectively.
Due to this result, PSP solution has more antioxidant activity than PSP solution,
both before and after fermentation. The free radical scavenging activity in the
fermented solution is attributed to the anthocyanin pigment from PSP and BGR.
Therefore, the antioxidant activities of two substrates were reduced a little bit
due to the reductions in anthocyanin contents of both PSP and BGR after
fermentation.

5. CONCLUSION
After saccharifying two substrates, purple sweet potato (PSP) and black
glutinous rice (BGR) with the combination of 0.1 % -amylase and 0.15%
glucoamylase in 90 minutes at 55oC, the reducing sugar content in the solutions
were suitable for the next step, the fermenting step. By using 1% starter culture
of Lactobacillus acidophilus, falcultative anaerobic fermentation of two substrates
were carried out for 24 hours at 37oC. The results indicated that lactic fermented
PSP contained about 10% soluble solid; 15.45% sugar; 0.54% acidity as lactic
acid and 6.20 x 108 CFU/mL viable cell count. Lactic fermented BGR contained
10% soluble solid; 14.58% sugar; 0.35% acidity as lactic acid and 1.12 x 108
CFU/mL viable cell count. The anthocyanin contents after fermentation were
62.62 mg/100g (d.b) with PSP substrate and 55.09 mg/100g (d.b) with BGR
substrate. PSP and BGR lactic acid solution had 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity IC50 values of 31.8g/mL and 34.42 g/mL,
respectively. Statistical

analysis

showed that

the antioxidant

activity

of

fermented PSP and BGR were not significantly different after fermentation. It
means that, fermentation had no effect on antioxidant activities of these
fermented solutions
Based on the viable cell count of these two fermented solutions, this
study showed that PSP and BGR were the appropriate substrates for lactic acid
fermentation of L.acidophilus. Therefore, it can be concluded that there were
virtuous possibilities to develop products of beverage with antioxidant activity
from lactic fermentation of saccharified PSP and BGR.

19

REFERENCES
AOAC., 1999. Official method of analysis (15th ed.). Washington: Association of
Official analytical Chemists
Abdel-Aal, EM., & Hucl, P. (2003). Composition and stability of anthocyanins in
blue-grained wheat. Journal of Agricultural and Food Chemistry, 51, 21742180
Anderson R (1988). Lactic acid bacteria in the production of food. Food
Laboratory Newsletter, 14: 17-21.
Brigers, E.N., Chinn, M. S., & Truong, Van-Den., (2010). Extraction of
anthocyanins from industrial pruple-fleshed sweetpotatoes and enzymatic
hydrolysis of residues for fermentable suger. Industrial Crops and products
32, 613-620
Brown C.R., Culley D., Yang CP., Durst R., & Wrolstad R., (2005). Variation of
anthocyanin and carotenoid contents and associated antioxidant values in
potato breeding lines. Journal of the American Society for Horticulture
Science 130:174-180
Duangjitcharoen, Y., Kantachot, D., Ongsakul, M., Poosaran, N., & Chaiyasut, C.
(2008) Selection of probiotic lactic bacteria isolated from fermented plant
beverages. Pak. J. Biol. Sci 11: 652-5
Deraz, S., Karlsson, N. E., Khalil, A.A., & Mattiasson, B. (2007). Mode of action
of acidocin D20079, a bacteriocin produced by the potential probiotic
strain, Lactobacillus acidophilus DSM 20079. J. Ind. Microbiol. Biotechnol.
34: 373-379.
Lee, J., Durst R.W., & Wrolstad R.E., (2005). Determination of Total
Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages,
Natural Colorants, and Wines by the pH Differential Method: Collaborative
Study. Jounal of AOAC International 88, 5.
Kano, M., Takayanagi, T., & Harada, K. (2005). Antioxidative activity of
anthocyanins from purple sweet potato, Ipomoera batats cultivar
ayamurasaki. Biosci. Biotechnol. Biochem, 69(5), 979-988
Kenneth Todar (2012). Online textbook of Bacteriology. Retrieved September 15
from [email protected]
Kong, J.-M, L.-S. Chia, N. K. Goh, T.-F. Chia, and R. Brouillard. (2003). Analysis
and biological activities of anthocyanins. Phytochemistry. 64: 923-933.

Lee, S.Y., Ganesan, P., Ahn, J., & Kwak ,H.S. (2011). Lactobacillus acidophilus
Fermented Yam (Dioscorea opposita Thunb.) and Its Preventive Effects on
Gastric Lession. Food Sci. Biotechnol, 20(4), 927-932.
Miller., & Gail, L., (19559). Use of dinitrosalicylic acid reagent for determination
of reducing sugar. Anal. Chem 31(3): 426-428
Nashiru, T. O., Chulkyoon-Mok., & Se-Young, L. (1992). Comparative studies on
lactic acid fermentation of different cereals for the production of nonalcoholic yogurt like beverage. UNIDO International Workshop on Lactic
Acid Fermentation of Non- Dairy Food and Beverages Proceeding, pp. 9098
Oki, T., Matsuda, M., Kobayashi, M., Nishiba, Y., Furuta, S., Suda, I., et al
(2002). Polymeric procyanidins as radical-scavenging components in redhulled rice. Journal of Agricultural and Food Chemistry, 50, 7524-759
Poei-Langston, M.S., and R.E.Wrolstad. 1981. Color degradation in an ascorbic
acid-anthocyaninflavonol model system. Journal of Food Science 46: 12811222.
Sanders, M.E., Klaenhammer, T.R. (2001). Invited review: the scientific basis of
Lactobacillus acidophilus NCFM functionality as a probiotic. J. Dairy Sci. 84:
319-331.
Sasaki, Y., & Ohma, R. (2004). Antioxidant activity and optimal manufacturing
conditions of purple sweet potato lactic acid bacteria drink. Food Sci.
Technol. Res., 10(4), 447-452.
Suda, I., Oki, T., Masuda, M., Kobayashi., Nishiba, Y., & Furuta, S. (2003).
Physiological functinality of purple fleshed sweet potatoes containing
anthocyanins and their utilization in food. JARQ, 37(3), 167-173.
Sutharut, J., & Sudarat, J. (2012). Total anthocyanin content and antioxidant
activity of germinated colored rice. International Food Research Journal
19(1): 215-221
Tananuwong, K., & Tewaruth, W., (2010). Extraction and application of
antioxidants from black glutinous rice. Food science and Technology 43,
476-481
Teow C, Truong VD., McFeeters R., Thompson R., Pacota K., Yencho G., (2007).
Antioxidant activities, phenolic and -carotene contents of sweet potato
genotypes with varying flesh colours. Food Chemistry 103:829-838
Viet, V. N., Park, D. J., & Chee, K. M. (1992) Nutritional value of Risogurt
prepared from rice extrudate and soy protein isolate. UNIDO International
Workshop on Lactic Acid Fermentation of Non- Dairy Food and Beverages
Proceeding, pp 81-89

Wegener, C.B., Jansen G., Jillrgens H.U., Schtze, W., (2009). Special quality
traits of coloured potato breeding clones: Anthocyanins, soluble phenols
and antioxidant capacity. Journal Sc. Food Agric. 89: 206215.
Wongkhalaung, C. (1995). Lactic Acid Fermentation of Sweet potato. Kasetsart J.
(Nat. Sci.), 29, 521-526. Thailand: Bangkok
Wu, T-Y., Chiu, C.C., Hwang, Y-T., &Chiu, T.H. (2012). Effect of Antioxidant
Activity and Functional Properties of Chingsheypurple Sweet potato
Fermented milk by Lactobacillus acidophilus, L. debrueckii subsp.lactics,
and L. gasseri strains. Journal of Food Science, 71 (1).

APPENDIX 1 : MRS medium (broth and agar)

1. MRS broth
Chemical

Amount

Glucose

20g

Peptone

10g

Meat extract

10g

Yeast extract

5g

Tween 80

1ml

K2HPO4

2g

CH3COONa

5g

Triamonium citrate

2g

MgSO4.7H2O

0.2g

MnSO4.4H2O

0.2g

Distilled water

1000ml

pH

6.2 0,2

2. MRS agar: Prepared the same as MRS broth and then added 2% agar