Microbial Population and Degradation on Rice Granules during Fermentation of Bedak sejuk

Abstract

Bedak sejuk is a traditional fermented rice based cosmetic used by women in Malaysia. To date, the production of both homemade and commercial bedak sejuk depends on natural fermentation without using starter cultures. Here, we monitored the microbial population and evaluate the degradation of rice starch in the initial batch fermentation of bedak sejuk. Bedak sejuk sample was prepared by natural fermentation of local rice grains (5% broken). The microbial population changes and degradation of rice starch were analysed for two weeks. Within two weeks, the total microbial, anaerobic bacteria, yeast and mould count increased while coliforms count decreased. The total microbial, anaerobic bacteria, yeast, mould and coliform counts were 11.44±0.54, 6.14±0.27, 2.78±0.22, 1.31±0.19 and 1.24±0.13 log CFU/mL, respectively. The liquefaction activity of α α α α α-amylase was higher on the second day (42.3±0.6 µg/min/g) but after that it declined. Total carbohydrate decreased from 80.8 to 80.1 % while reducing sugar of the rice granules and in fermented supernatant increased from 0.02 to 0.15 % and 0.03 to 0.92 g/L, respectively. While the degree of hydrolysis shows an increase but the increment was low. From the scanning electron microscope pictures, the rice grains still retained their whole figures but the fermented starch lost their surface smoothness. Taken together, our results suggest that the changes of microbial population affected the rice starch during the natural fermentation.

Full text

JOURNAL OF PURE AND APPLIED MICROBIOLOGY, Nov 2015. Vol. 9(Spl. Edn. 2), p. 513-518
* To whom all correspondence should be addressed.
E-mail: alippipi@yahoo.com
Microbial Population and Degradation on
Rice Granules during Fermentation of Bedak sejuk
Mohd Alif Afife Dzulfakar*, Noorhisham Tan Kofli and Siti Masrinda Tasirin
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment,
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
(Received: 14 September 2015; accepted: 15 November 2015)
Bedak sejuk is a traditional fermented rice based cosmetic used by women in
Malaysia. To date, the production of both homemade and commercial bedak sejuk depends
on natural fermentation without using starter cultures. Here, we monitored the microbial
population and evaluate the degradation of rice starch in the initial batch fermentation
of bedak sejuk. Bedak sejuk sample was prepared by natural fermentation of local rice
grains (5% broken). The microbial population changes and degradation of rice starch
were analysed for two weeks. Within two weeks, the total microbial, anaerobic bacteria,
yeast and mould count increased while coliforms count decreased. The total microbial,
anaerobic bacteria, yeast, mould and coliform counts were 11.44±0.54, 6.14±0.27,
2.78±0.22, 1.31±0.19 and 1.24±0.13 log CFU/mL, respectively. The liquefaction activity of
αα
αα
α-amylase was higher on the second day (42.3±0.6 µg/min/g) but after that it declined.
Total carbohydrate decreased from 80.8 to 80.1 % while reducing sugar of the rice granules
and in fermented supernatant increased from 0.02 to 0.15 % and 0.03 to 0.92 g/L,
respectively. While the degree of hydrolysis shows an increase but the increment was low.
From the scanning electron microscope pictures, the rice grains still retained their whole
figures but the fermented starch lost their surface smoothness. Taken together, our results
suggest that the changes of microbial population affected the rice starch during the
natural fermentation.
Key words: Lactic acid bacteria (LAB), Yeast, Natural Fermentation, Rice Starch, Bedak sejuk.
The traditional production of bedak sejuk
is a somewhat unique fermentation process due to
its repeating fermentation process, resulted longer
fermentation time taken to complete the production.
Bedak sejuk is a fermented rice-based cosmetic,
normally in the form of water droplet pastilles.
These pastilles are natural cosmetic product as
they are produced only using rice grains and water.
The rice grains will be soaked in tap water until
they become white rice paste1. Literally, bedak sejuk
means cool powder which implies when the
pastilles are mixed with water and applied on face
skin, it will give a cooling effect. Sawaki et al.2
reported that the cosmetics produced by adding
the LAB fermented rice have a good feeling when
and after used; expansion and smoothness on
applying and wet feeling after applying on skin.
This implies why the older called these pastilles as
cool powder.
The soaking water during the
fermentation of bedak sejuk is changed
intermittently because of its pungent smell. Battock
& Azam-Ali3 reported that during fermentation,
spoilage organisms will utilize protein as an energy

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514 DZULFAKAR et al.: RICE GRANULES DURING FERMENTATION OF BEDAK SEJUK
source producing unpleasant odour. This
unpleasant odour normally resulted from a long
fermentation time. As the traditional process of
bedak sejuk production that being used is still a
traditional method which passes from generation
to generation, the changing of the soaking water
is not based on scientific knowledge4. Therefore
the soaking water is frequently changed, leading
to a long overall soaking time to complete the
production. This portrayed that the process is less
effective, timely and costly.
Little information is available about the
microbial composition during the soaking of rice
grains which can show high variability depending
on the rice grain types and fermentation conditions
that are difficult to control even in the laboratory
experiment. The nature of the natural fermentation
with respect to the role played by the
microorganisms and the soaking water conditions
on the physicochemical changes and on the
characteristics of the final product is not known.
Therefore, the aim of this work was to study the
microbial population dynamics during the initial
fermentation and evaluate the degradation of rice
starch.
Preparation of Bedak sejuk
The preparation of bedak sejuk was
followed the traditional process of making bedak
sejuk. The principal ingredients are 5% broken local
polished rice grains (Indica) and tap water. Based
on our previous study, the soaking or fermentation
process will take place for 14 days. The conditions
for the fermentation were; (1) polished rice grains
were not cleaned or washed first, (2) the container
used was not sterilized and (3) polished rice grains
will ferment undisturbed at ambient temperature.
250 g of local polished rice grains will be soaked in
tap water (w/v) and aliquots of the fermented
supernatant were collected every days throughout
the soaking. Sample of bedak sejuk (rice granules)
were collected at the end of soaking process.
Microbial enumeration
The quantities of the dominant microbial
were enumerated on the basis of colony-forming
unis (CFU) in selective media. 1 mL of the fermented
supernatant were homogenized in 9 mL sterile
peptone physiological saline solution (5 g peptone,
8.5 g NaCl, 1000 mL distilled water, pH 7.0 ± 0.2).
Total anaerobic bacteria were enumerated using
MRS agar (Merck) and incubated (in an anaerobic
jar) at 37 oC for 48 h. Yeast and moulds were counted
by surface plating on Dichloran Rose Bengal
Chloramphenicol agar (DRBC) (Oxoid, Unipath,
Basingstoke, UK) and incubated aerobically at 25
oC for 3-6 days. While Coliforms were plated on
Petri-Film (3M, St. Paul, MN, USA) specific for
coliform determinations and incubated at 37 oC.
All samples were measured in triplicate.
The basic statistical method was used to calculate
the means and standard deviation of the triplicate
results of each sample.
Liquefying (αα
αα
α-amylase) activity
The activity of a-amylase was determined
by slightly modifying the method described by
Maity et al.5. Briefly, 0.1 mL of supernatant was
incubated with 0.5 mL of soluble starch (1%, w/v),
and 0.4 mL of buffer (0.1 M phosphate buffer for
pH 7.0) and incubated at 40 oC for 10 min. The
reaction was terminated by the addition of 1 mL of
3,5-dinitrosalicylic acid (DNS) reagent and the
liberated reducing sugars were estimated
colorimetrically according to the method of Miller6.
One unit of amylase activity was defined as the
amount of enzyme releasing 1 µmol of reducing
sugars (glucose equivalents) per minute at pH 7.0
at 40 oC.
Determination total carbohydrate, reducing sugar
and degree of hydrolysis
For the rice granules sample, total
carbohydrate was estimated by an ethanol
extraction by the phenol-sulphuric acid method7.
The ethanol extraction method was altered from
the work done by Anthony & Chandra8; 10 g of
milled rice granules were extracted with 5 ml of hot
80% ethanol twice, then centrifuged at 8000 x g for
10 min and the supernatant was collected. Total
reducing sugar was estimated by the 3,5-
dinitrosalicylic acid method6 with the ethanol
extraction prepared as above for rice granules while
no extraction needed for fermented supernatant.
Reducing sugar in fermented supernatant was
calculated on the basis of total volume of soaking
water. The degree of hydrolysis was evaluated
by monitoring the total carbohydrate of
supernatant which measured by phenol sulphuric
method6. Degree of hydrolysis was calculated
using following equation:
() ()
()
Total carbohydrate of supernatant g
Degree of hydrolysis % 100
Amount of starch g
=×

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515DZULFAKAR et al.: RICE GRANULES DURING FERMENTATION OF BEDAK SEJUK
Scanning electron microscopy (SEM)
The association of microbes onto the rice
grains was examined under scanning electron
microscope. Bedak sejuk was observed using a
scanning electron microscope (High Resolution
Fesem Supra 55VP), under 2-12 KX magnification
and a constant acceleration voltage of 10 kV.
RESULTS AND DISCUSSION
Microbiological analysis
Figure 1 shows the distribution of
microbial in the fermented supernatant during
fermentation. The anaerobic bacteria counts
increased from 3.02 to 6.14 log CFU/mL at the end
of fermentation time. Yeasts count increased from
1.68 to 2.78 log CFU/mL, moulds count also
increased from 0.73 to 1.31 log CFU/mL while
coliforms count decreased from 3.88 to 1.21 log
CFU/mL. A high microbial load was observed in the
fermented supernatant as high initial counts of 9.31
log CFU/mL was recorded. This initial count could
be attributed to the raw material.
The total microbial enumeration showed
almost the same pattern of a growth profile of batch
process. These pattern was observed too during
the previous study4 but it was a growth profile of
cell density of the supernatant. This shows that 14
days of fermentation days are the best fermentation
as the growth cycle of the microbial in the soaking
water is almost completed.
From the microbial count, it can be
suggested that the initial stages of fermentation is
dominated by anaerobic bacteria and coliforms but
in the later stages by anaerobic bacteria and yeasts.
Nche et al.,9 stated that yeasts provide growth
factors like vitamin amino acids for bacteria while
bacteria will create an acidic environment which is
a conducive to the yeast growth. Initially LAB will
dominated the fermentation10 due to their higher
growth rate followed later by yeasts in substrates
that rich in fermentable sugars.
Yeast secreted different hydrolytic
enzymes such as α-amylase, β-amylase,
glucoamylase and proteases11,12 that facilitated a
rapid degradation of available starch of the rice
Table 1. Chemical components of rice grains and fermented supernatants of bedak sejuk
Soaking Polished rice After soaking
Fermentation time (day) 0 14
Rice grains
Total carbohydrate (%) 80.8±0.17 80.1±0.17
Reducing sugar (%) 0.02±0.13 0.15±0.17
Reducing sugar of fermented supernatant (mg/mL) 0.03±0.14 0.92±0.11
Results given as means and standard deviation of triplicate samples
granules. It has been reported that the bacterial
presence during rice fermentation was dominated
by LAB that can create conditions that inhibited
growth of the pathogen13. This is probably why
the coliform count decrease during the soaking
process.
αα
αα
α-Amylase activity
The liquefaction activity of α-amylase
was higher on the second day (42.3±0.6 µg/min/g)
but after that it declined. This hydrolytic enzyme
were produced by yeast and mould during the
fermentation. α-amylase will contribute to the
saccharification and liquefaction of the rice.
Normally, starch was first degrade into limit dextrins
by α-amylase whereas glucose was specifically
produced from dextrins by glucoamylase14.
Variation of total carbohydrates, reducing sugar
during fermentation and degree of hydrolysis
The total carbohydrate decreased while
reducing sugars from the rice granules samples
and reducing sugars in the fermented supernatant
increased throughout the soaking process (Table
1). Total carbohydrate percentage in polished rice
grains slightly decreased after the soaking from
80.8 to 80.1 %. The reducing sugar of the polished
rice grains was increased rapidly after the first
fermentation, from 0.02 to 0.15 %. The reducing
sugar in the fermented supernatant also shows
the same trend as it increased rapidly from 0.03 to
0.92 mg/mL at the end of soaking process.

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516 DZULFAKAR et al.: RICE GRANULES DURING FERMENTATION OF BEDAK SEJUK
Fig. 1. The distribution of microbial in the fermented
supernatant during fermentation
Fig. 2. Changes of liquefying activity during bedak sejuk
production
Fig. 3. Degree of hydrolysis of rice during the bedak
sejuk production
The polished rice grains were soaked
without washing in this work. Thus, carbon source
from polishing step is available for the
microorganisms. This supported by Lu et al.15
which reported that rich amount of damaged starch
that occurred during the polishing process of the
rice served as the carbon source for the LAB or
yeast at the first stage. But in this work, the total
carbohydrate was found slightly decreased during
the fermentation which suggests that the damaged
starch was first used as the carbon source instead
of the reducing sugars. This results are not in
agreement to those obtained by other cereals
fermentation16,17.
Fig. 3 shows the degree of hydrolysis.
The degree of hydrolysis shows an increase but
the increment was low. The degree of hydrolysis
at the end of the soaking was only 1.14±1.1%. This
suggested that the degradation rate of rice are slow.
0.92±0.11 mg/mL of reducing sugar was produced
in supernatant after fermentation. Even though the
amount of the reducing sugar was not high but
this suggests that the rice starch was degraded
during the fermentation. But the degradation is
somewhat a partial digestion at a slow degree of
hydrolysis.
From all those results, generally, these
decreases in the starch content with increased
fermentation time are due to the breakdown of
starch molecules into sugars by microorganisms
during the fermentation process. It is suggests that
starch in the rice is hydrolysed into simple sugars
then these sugars are metabolised by
microorganisms to organic acids or other
metabolites. After 14 days of soaking, it is observed
that the rice grains was degrading but they were
still in granules form but a bit smaller than their
original size. This shows that the rice starch
underwent somewhat degradation either because
of the hydrolysis or acidification.
Fig. 4. Scanning electron micrographs of unfermented rice grains (a), natural fermented rice grains (b) and association
of microbes with rice grains at the end of fermentation (c)

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517DZULFAKAR et al.: RICE GRANULES DURING FERMENTATION OF BEDAK SEJUK
Scanning electron microscopy (SEM)
The granular structure of native and
natural fermentation of rice starches exhibited
significant variations in their shape when viewed
with scanning electron microscopy. Representative
scanning electron micrographs of the residual
starch granules are provided in Figure 4. The rice
grains still retained their whole figures but their
surface changed (Fig. 4a). Typically, the fermented
starch lost their surface smoothness (Fig. 4b). SEM
analysis also revealed a layer of microbes on the
rice grain at the end of fermentation, they formed a
thick biofilm consisting a rod shaped bacteria (Fig.
4c).
CONCLUSION
The results presented in this study have
been intended as a contribution to understanding
of the basis of the natural fermentation process
underlying the production of bedak sejuk, a
traditional Malaysia cosmetic product. It also has
given an insight into the role of microorganisms
within the natural fermentation process. During
the initial stage of fermentation, anaerobic bacteria
provide a favourable environment for the later
stage of fermentations by yeasts. Natural
fermentation on polished rice grains allowing the
growth of anaerobic bacteria, yeasts and moulds
while decreased of coliform. The increasing of
microbial population can alter the structure of rice
starch. From the degree of hydrolysis analysis,
the degradation occurred is slow but the
degradation managed to alter the surface of the
rice grains. However, further studies have to be
performed to understand the microbial population
dynamics and its relation to the final product.
ACKNOWLEDGEMENTS
The authors thank to the Universiti
Kebangsaan Malaysia for Dana Lonjakan
Penerbitan (DLP-2012-022) for financial support.
REFERENCES
1. Dzulfakar, M. A. A., Tasirin, S. M., Tan Kofli,
N. Observation on the soaking water and particle
size changes during “Cool Powder” (Bedak
Sejuk) production. Proceeding of International
Technical Conference (ITC2014), 17-19th Nov
2014, Seoul, Korea., 2014; pp 361-370.
2. Sawaki, S., Sawaki, S., Matsukawa, K., Osawa,
Y., Hirota, A. Use of lactic acid bacteria
fermented rice in cosmetics or as emulsifier.
European Patent Specification, 2008; 1-37.
3. Battock, M., Azam-Ali, S. (eds): Fermented
fruits and vegetables. A global perspectives,
F.A.O Agricultural Services Bulletin No. 134.
Rome: Food and Agriculture Organization of the
United Nations, 1998.
4. Dzulfakar, M. A. A., Tan Kofli, N. The effects
of soaking on rice granules for bedak sejuk
production. Adv. Mat. Res., 2015; 1113:465-470.
5. Maity, C., Samanta, S., Halder, S. K., Das,
Mohapatra, P. K., Patri, B. R., Jana, M., Mondal,
K. C. Isozymes of α-amylase from newly
isolated Bacillus thuringiensis CKB19:
Production from immobilized cells.
Biotechnology and bioprocess Engineering,
2011; 312-319.
6. Miller, G. L. Use of dinitrosalicylic acid reagent
for determination of reducing sugar. Anal. Chem.,
1959; 31:426-428.
7. Dubois, M. K., Gills, K. A., Hamilton, J. K.,
Rebers, P. A., Smith, F. Calorimetric method for
determination of sugars and related substances.
Anal. Chem., 1956; 28:350-356.
8 .Anthony, U., Chandra, T. S. Microbial
population and biochemical changes in
fermenting finger millet (Eleusine coracana).
World J. Microbiol. Biotechnol., 2013; 13: 533-
537.
9. Nche, P. F., Nout, J. R., Rombouts, F. M. Effect
of cowpea supplementation on the quality of
kenkey, a traditional Ghanaian fermented maize
food. J. Cereal Sci., 1994; 19:191-197.
10. Conway, P. L. Selection criteria for probiotics
microorganisms. Asia Pac. J. Clin. Nutr., 1996;
5: 10-14.
11. Wang, H. Y., Gao, Y. B., Fan, Q. W., Xu, Y.
Characterization and comparison of microbial
community of different typical Chinese liquor
Daqus by PCR-DGGE. Letter in Applied
Microbiology, 2011; 53:134-140.
12. Zheng, X.-W., Tabrizi, M. R., Nout, M. J. R.,
Han, B.-Z. Daqu, a traditional Chinese liquor
fermentation starter. Journal of the Institute of
Brewing, 2011; 117:82-90.
13. Jones, R. J., Hussein, H. M., Zagorec, M.,
Brightwell, G., Tagg, J. R. Isolation of lactic acid
bacteria with inhibitory activity against
pathogens and spoilage organisms associated
with fresh meat. Food Microbiology, 2008;
25:228-234.
14. Santoyo, M. C., Loiseau, G., Sanoja, R. R.,

J PURE APPL MICROBIO, 9(SPL. EDN.), NOVEMBER 2015.
518 DZULFAKAR et al.: RICE GRANULES DURING FERMENTATION OF BEDAK SEJUK
Guyot, J. P. Study of starch between
fermentation at low pH by Lactobacillus
fermentum Ogi E1 reveals uncoupling between
growth and á-amylase production at pH 4.0.
International Journal of Gastroenterology, 2003;
126:1620-1633.
15. Lu, Z.-H., Peng, H.-H., Cao, W., Tatsumi, E.,
Li, L. –T. Isolation, characterization and
identification of lactic acid bacteria and yeast
from sour Mifen, a traditional fermented rice
noodle from China. J. Appl. Microbiol., 2008;
105: 893-903.
16. Foma, R. K., Destain, J., Mobinzo, P. K.,
Kayisu, K. Study of physicochemical
parameters and spontaneous fermentation during
traditional production of munkoyo, an indigenous
beverage produced in Democratic Republic of
Congo. Food Contr, 2012; 25: 334-341.
17. Savijoki, K., Ingmer, H., Varmanen, P.
Proteolytic systems of lactic acid bacteria. Appl.
Microbiol. Biotechnol., 2006; 71: 394-406.