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.

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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 b

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: dqtuan@hcmiu.edu.vn

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

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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

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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 108 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

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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 37oC 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

5

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 60oC 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 95oC 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

6

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

7

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) = [ Adiff 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.

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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.

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

PSP BGR

Nutrients Mean

(w.b)

CV

(%)

Dry

basis

(d.b)

Mean CV

(%)

D.b

Moisture (%)

Protein (%)

Lipid (%)

Ash (%)

Fiber (%)

Anthocyanins

(mg Cyd-3-glu-

E/100fw)

64.50

1.38

0.23

0.88

2.78

58.46

0.19

7.17

1.30

2.84

1.04

2.56

-

3.89

0.65

2.47

7.83

164.69

12.01

6.18

1.20

1.37

3.67

79.27

0.83

8.50

0.88

1.03

1.29

2.67

-

7.02

1.16

1.56

4.17

90.08

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

10

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.

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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.

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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

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

fermentation

After

fermentation

Before

fermentation

After

fermentation

Viable cell

count

(CFU/mL)

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

OD 0.06 - 0.10 0.4 - 0.9 1.01 1.12

Cell number 2.01x 103

3.15 x 103

4.31 x 104

4.07 x 108

4.16 x 108

5.40 x 108

Log ( CFU/mL) 3.30 3.50 4.63 8.61 8.62 8.73

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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.

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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

15

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).

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

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).

IC50 (L) IC50 (g/mL)

Mean Mean CV (%)

PSP Before 186.1 30.07 3.13

After 311.9 31.8 3.21

BGR Before 371.9 33.47 1.23

After 967 34.42 2.09

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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).

18

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

19

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.

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APPENDIX 1 : MRS medium (broth and agar)

1. MRS broth

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

Chemical Amount

Glucose

Peptone

Meat extract

Yeast extract

Tween 80

K2HPO4

CH3COONa

Triamonium citrate

MgSO4.7H2O

MnSO4.4H2O

Distilled water

pH

20g

10g

10g

5g

1ml

2g

5g

2g

0.2g

0.2g

1000ml

6.2 0,2