Journal of Cereal Science 85 (2019) 15–19

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Journal of Cereal Science

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Physicochemical properties of dry- and semi-wet-milled rice flours after T

fermentation by Lactobacillus amylovorus
So-Hyun Choa, Byung-Hoo Leeb, Jong-Bang Euna,∗
a
Department of Food Science and Technology, Chonnam National University, Gwangju, South Korea
b
Department of Food Science & Biotechnology, College of BioNano Technology, Gachon University, Sungnam, South Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Rice milling is one of the important factors that determines the physicochemical properties of rice flour. Dry
Fermentation milling is a simple and quick process, though it produces high levels of damaged starch that is not desirable in
Rice flour some application. Since fermentation can change the structure of starch as well as its physicochemical prop-
Dry-milling erties, the effect of fermentation on dry-milled rice flour was evaluated with the aim of improving flour quality
Semi-wet-milling
for use as food ingredient in food industry. After fermentation at 37 °C for 24 h by Lactobacillus amylovorus, the
Lactobacillus amylovorus
dry-milled rice flour showed similar proximate compositions, physicochemical properties, and surface structures
as semi-wet-milled rice products. Thus, the results suggest that it can be applied to produce desirable food
materials for preparation of rice-based products.

1. Introduction
Rice is one of the world's major staple crops along with corn and
wheat, and it is cultivated in many countries, especially in Asia (Bao,
2012; Mir and Bosco, 2014); it is the world's most consumed cereal after
wheat (Chang, 2003). The quality of foods containing rice flour de-
pends on the rice milling process used and the characteristics of the rice
starch, which is the major component of the rice kernel. To improve the
physicochemical properties of rice flour, a process for increasing the
moisture absorption ability of rice has been previously employed,
which provides the proper physical characteristics before the milling
process (Chiang and Yeh, 2002). There are two major milling methods
for preparing rice flours: dry- and wet-milling (Chen et al., 1999; Kadan
et al., 2008; Lumdubwong and Seib, 2000). Among these processes, the
dry-milling process has the advantages of being simple and requiring a
short processing time, although it produces a high amount of damaged
starch (Morrison et al., 1994; Tester and Morrison, 1994). When rice
kernels are dry-milled into rice flour, some starch granules are damaged
by the mechanical work during the dry-milling process, and this da-
maged starch has many undesirable features for use in processed rice
foods (Ngamnikom and Songsermpong, 2011).
Currently, the use of starter cultures in food fermentation is being
widely studied and is being introduced into commercial practice to
increase processing rates and product consistency (Shin et al., 2006). In
particular, Lactobacillus amylovorus is the most representative species
amongst the amylolytic lactic acid bacteria (ALAB) isolated from cattle
manure corn-enrichments (Nakamura, 1981). This strain produces an
extracellular α-amylase that hydrolyzes the starch molecules to form
small-sized maltooligosaccharides, and the hydrolyzates are then fer-
mented to lactic acid. This Lactobacillus strain is able to simultaneously
hydrolyze and ferment starch to lactic acid (John et al., 2009; Xiaodong
et al., 1997); therefore, ALAB can be used for the preparation of various
starch-containing food materials (Pompeyo et al., 1993). The use of
ALAB as culture starters has been shown to improve the nutritional
quality of complementary foods by combining amylolysis with the re-
cognized beneficial properties of lactic acid bacteria (LAB) (Agati et al.,
1998; Haydersah et al., 2012). However, further fermentation studies
on the physical properties of starch and the rheological characteristics
of starch gel are required to determine the applications of zymotic
starch modifications (Lu et al., 2005). Therefore, the objective of this
study was to evaluate the effect of fermentation on improving the
physicochemical properties of dry-milled rice flour. The results ob-
tained can help produce high-valued and functional rice flours which
have improved physicochemical properties and increased LAB amount
on the kernel surface by fermentation for various food applications in
the cereal and/or starch industry.

∗
Corresponding author.
E-mail address: jbeun@jnu.ac.kr (J.-B. Eun).

https://doi.org/10.1016/j.jcs.2018.11.003
Received 7 January 2017; Received in revised form 31 October 2017; Accepted 5 November 2018
Available online 08 November 2018
0733-5210/ © 2018 Elsevier Ltd. All rights reserved.
S.-H. Cho et al.
2. Materials and methods
2.1. Materials
A commercially available non-waxy rice (Calrose, Kokuho rose) was
purchased from a local market in California, USA. L. amylovorus (ATCC
33620), an ALAB species, was obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA) and used to ferment the differ-
ently milled rice flours. All other chemicals in this study were pur-
chased from Sigma-Aldrich Co. (St. Louis, MO, USA).
2.2. Preparation of rice flour samples by the roll-milling method
Rice flours were obtained from both dry and the semi-wet grains.
Dry-milled rice flour was produced by cutting the rice grains with a
Waring blender (Torrington, CT, USA) for 2 min. To prepare semi-wet-
milled rice flours, the rice grains were soaked in distilled water at a
ratio of 1:3 for 8 h at room temperature (25 °C). The soaked rice was
drained for 1 h and air-dried at room temperature (25 °C) for 12 h to
produce semi-wet rice grains. The semi-wet rice grains were milled with
a roll mill (Marga roller; Marcato, Italy) twice and ground for 1 min in a
Waring blender. All dry- and semi-wet-milled rice flours were passed
through a 100-mesh sieve after drying. The moisture content of the final
products was adjusted to 12–14%, and the samples were stored at 4 °C
in a plastic bag. The abbreviations used for the differently milled rice
flour samples in this study are summarized in Table 1.
2.3. Preparation of fermented rice flours
L. amylovorus ATCC 33620 strain was cultured in Lactobacillus MRS
broth medium (Remel, Lenexa, KS) at 37 °C for 48 h, and changes in the
optical density were measured at 600 nm with a Cary 50 Bio spectro-
photometer (Varian INC., Palo Alto, CA, USA). After incubation, the
harvested cell suspension was precipitated by centrifugation (2000×g)
at 4 °C for 15 min and then suspended in 0.85% NaCl solution after
washing twice.
The dried rice flour samples (50 g) were suspended in 150 mL of
sterile 0.85% NaCl solution, and the mixture was then vortexed for
1 min. The strain was inoculated at a concentration of 1% (g/g) for the
differently milled rice flours and fermented at 37 °C for 24 h in a
shaking incubator (120 rpm). The fermented rice flour slurry was dried
using a dry oven (BON-50 Being Instrument Inc. California, USA) at
65 °C for 4 h. The dried fermented rice flour was ground using a Waring
blender for 1 min and then passed through a 100-mesh sieve.
2.4. Analysis of the proximate composition and physicochemical properties
of the differently milled rice flour samples
The crude fat, protein, and ash contents of the differently milled rice
flours were determined by official methods (AOAC, 1998). The color
values of the rice flour samples were assessed in triplicate using a color
difference meter (CR-410 Minolta, Osaka, Japan), and the results were
expressed as lightness (L∗), redness (a∗), and yellowness (b∗) values.
The damaged starch content of the rice flour samples was measured
in triplicate following the method of Boyaci et al. (Boyacı et al., 2004)
Table 1
Abbreviations for the differently milled rice flour samples used in this study.
Abbreviations Sample preparation
DF Rice → milling with cutting blender for 2 min → passing through a 100-mesh sieve
WRF Rice → soaking (water) → draining → drying 12 h → 1st milling with roll mill → 2nd milling with roll mill → passing through a 100-mesh sieve
WBF Rice → soaking (water) → draining → drying 12 h → 1st milling with roll mill → 2nd milling with cutting blender for 1 min → passing through a 100-mesh sieve
FDF DF → fermentation → drying 4 h → milling with cutting blender for 1 min → passing through a 100-mesh sieve
FWRF WRF → fermentation → drying 4 h → milling with cutting blender for 1 min → passing through a 100-mesh sieve
FWBF WBF → fermentation → drying 4 h → milling with cutting blender for 1 min → passing through a 100-mesh sieve
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Journal of Cereal Science 85 (2019) 15–19
based on the standard AACC method (76-30A). A rice flour sample (9 g)
in 45 mL of acetate buffer (100 mM, pH 5.0) was mixed with α-amylase
from Aspergillus oryzae (39.7 mg, 315 sigma units/mg protein) in a
100 mL volumetric flask. The volumetric flask was placed in a shaking
water bath (30 °C and 120 rpm) for 25 min. To this solution, 3 mL of
3.68 N H2SO4 and 2 mL of 12% sodium tungstate were added, and the
solution was filtered through a Whatman No. 4 filter paper. The brix
value of the filtrates was measured using a refractometer (Fisher Sci-
entific Co., Pittsburgh, PA, USA). The percentage of damaged starch
was determined as follows:
Damaged starch (%) = [(brix value of sample filtrate – brix value of
blank filtrate) × volume of slurry (50 mL) × conversion factor (1.64)]/
[sample size (g)]
2.5. Analysis of the morphology and crystal structure of the differently
milled rice flours
The morphology of the rice flour samples was observed using a
scanning electron microscope (SEM; S-2400, Hitachi, Japan) at an ac-
celerating voltage of 25 kV. Micrographs of each sample were taken at
1,000X magnification.
The X-ray diffraction (XRD) patterns of the rice flour samples were
obtained using a D8 advance X-ray diffractometer (Bruker AXS GmbH,
Karlsruhe, Germany). The diffractometer was operated at 40 kV and
40 mA. The samples were scanned over an angular range (2θ) from 0° to
40° at a scan rate of 3 s/step (Srichuwong et al., 2005).
2.6. Thermal properties of the differently milled rice flour samples
The thermal characteristics of the rice flour samples were measured
using a differential scanning calorimeter (DSC; Perkin-Elmer Co.,
Norwalk, CT, USA) calibrated with indium before analysis, and using an
empty pan as a reference. A sample (2 mg, dry basis) was weighed into
a DSC pan, and distilled water (6 mg) was added dropwise. The pan was
sealed and allowed to stand for 1 h at room temperature. The samples
were heated from 30 to 100 °C at the rate of 10 °C/min (Zhou et al.,
2010). The transition temperatures were recorded as the onset tem-
perature (T0), peak temperature (Tp), and conclusion temperature (Tc).
The gelatinization enthalpy (ΔH) was estimated by integrating the area
between the thermogram and a baseline under the peak.
2.7. Statistical analysis
Experimental data were analyzed using analysis of variance
(ANOVA) at a 95% confidence level (p < 0.05). Significant differences
among mean values were determined by Duncan's multiple range tests
using IBM® SPSS® Statistics for Windows (Version 21.0, IBM
Corporation, Armonk, NY, USA). All tests were performed at least in
triplicate, and the results were expressed as the mean ± standard de-
viation.
S.-H. Cho et al. Journal of Cereal Science 85 (2019) 15–19

Table 2 reduced the amount of damaged starch in rice flour.

Proximate compositions (%) of the differently milled rice flours.1)
Samples2) Ash Protein Lipid 3.2. The morphological properties of the differently milled rice flours

DF 0.99 ± 0.03a 8.61 ± 0.10a 0.59 ± 0.07a Scanning electron micrographs (SEMs) revealed the shape of the
WRF 0.43 ± 0.01c 5.92 ± 0.13c 0.33 ± 0.04bc
rice flours prepared by dry- and semi-wet-milling and fermented by L.
WBF 0.43 ± 0.01c 5.34 ± 0.09d 0.32 ± 0.05bc
FDF 0.56 ± 0.02b 7.14 ± 0.10b 0.42 ± 0.08b
amylovorus (Fig. 1). The shape of DF had some lumps of starch granules
FWRF 0.24 ± 0.02d 5.33 ± 0.15d 0.24 ± 0.02bc and large flour particles, whereas WRF and WBF contained individual
FWBF 0.26 ± 0.02d 4.97 ± 0.17e 0.25 ± 0.06bc starch granules and fine particles, which is consistent with a previous
1)
finding (Song and Shin, 2007). The surface of the starch granule was
Values represent means ± standard deviations of replicates. Means with the
surrounded by protein matrix (Chandrashekar and Kirleis, 1988). The
same letter in each row are not significantly different at p < 0.05. All ex-
protein matrix was leached out from the granule surface during the
periments were performed in triplicate.
2) soaking process, and the starch granules were clearly observed on the
The abbreviations refer to those listed in Table 1.
surface of the WRF and WBF particles. The shape of all fermented rice
flours appeared as a smooth surface with clear starch granules. In the
3. Results and discussion
fermented rice flours, the removal of proteins from the rice starch
granules by lactic acid fermentation could be clearly observed. In ad-
3.1. Analysis of the proximate compositions and physicochemical
dition, the inside of fermented rice flour contained some structural
characteristics of rice flour samples
modifications and a reduction in particle size, but the fermented rice
flours retained a regularity of structure. Consequently, following fer-
Table 2 shows the proximate compositions of the rice flours pro-
mentation, the rice flour particles were observed to contain free starch
duced by dry- and semi-wet-milling and fermented by L. amylovorus.
granules and endosperm cells, whereas the untreated rice flour particles
The crude ash and protein contents in the DF group were higher than
contained protein matrix with a smooth surface.
those in the WRF and WBF groups, whereas the fermented rice flour
samples had lower values than those of the non-fermented rice flours. In
3.3. XRD patterns of the differently milled rice flours
addition, the crude lipid content of dry-milled rice flour decreased
following fermentation, which was in agreement with a previous
The XRD patterns of the differently milled rice flours are shown in
finding (Lu et al., 2005). This was because protein and lipid complex
Fig. 2, and they were used to determine the degree and type of crys-
were released out from rice flour, and confirmed the fermentation by L.
tallinity of the starch granules. The crystallinity patterns are divided
amylovorus affected the proximate compositions of the final rice flour
into A, B, and C types depending on the type of the peak obtained
samples.
(Donald, 2004). In general, cereal starches show A-type, whereas tuber
The physicochemical properties of the differently milled rice flours,
starches show B-type patterns. The XRD patterns for all rice flours
including those that were subjected to fermentation are shown in
showed strong peaks at 15.3, 17.1, and 23.5, which is the typical A-type
Table 3. Compared to that in non-fermented rice flours, the lightness L*
crystallinity pattern (2θ = 15.3, 17.1, 23.5°) (Chung et al., 2011; Song
values were slightly lower in both the fermented samples (FWRF and
and Shin, 2007). Rice flour contains proteins and lipids as well as
FWBF). The redness (a*) values of rice flours indicated that all rice flour
starch. Although there were interactions between starch and the other
samples had a greenness characteristic (–a* value). In addition, the a*
components, the XRD patterns of rice flour displayed the same trend as
values of FWRF and FWBF were significantly lower than those of non-
for the crystallinity of starch. Thus, this result suggests that fermenta-
fermented semi-wet-milled rice flours. As discussed in previous re-
tion did not affect the XRD patterns of rice flour, and it retained the A-
search (Lu et al., 2005), fermentation can be changed the ratio of pure
type pattern. However, our results contradicted with those described in
starch and ash amount, and lead to change the color of rice flour.
the report previously published by Lu et al. (2005), who stated that the
The damaged starch content in DF was significantly higher than that
amorphous regions of starch granules were hydrolyzed during fer-
in WRF and WBF. Damaged starch is usually formed from broken starch
mentation, which resulted in an increase in the relative crystallinity of
granules during the dry milling process (Barrera et al., 2007), making
the rice flour particles. The contradicting results of our studies might be
the starch molecules, including amylose and amylopectin more exposed
due to differences in the experimental conditions and the fermentation
to water (Tara et al., 1972). During the WRF and WBF treatments, the
process such as different fermentation time and milling processes.
starch molecules or granules are partially soaked by the water soaking
process. Perhaps this mechanism decreases the amount of damaged
3.4. Thermal properties
starch during DF compared to WRF and WBF. Compared to non-fer-
mented rice flours, the damaged starch content was significantly lower
The thermal properties of rice flours were measured by DSC, and the
in fermented rice flours, which suggested that fermentation effectively
onset, peak, and conclusion temperatures and gelatinization enthalpy
(ΔH) of rice flours prepared by dry- and semi-wet-milling and fer-
Table 3 mented by L. amylovorus are compiled in Table 4. The onset tempera-
Color values and damaged starch contents of the differently milled rice flours. tures of the dry-milled samples were significantly higher than those of
Samples1) Color values Damaged starch the semi-wet-milled samples. The gelatinization enthalpy for the order-
(%) disorder transition of DF was higher than that of WRF and WBF, in-
L∗ a∗ b∗ dicating a high crystalline quality within the rice molecules. These re-
DF 70.04 ± 0.20e −0.26 ± 0.01b 5.54 ± 0.09a 4.25 ± 0.38a
sults indicated that the soaking treatment significantly affected the
WRF 74.47 ± 0.49a −0.15 ± 0.01a 2.40 ± 0.08e 3.57 ± 0.34b onset and peak temperatures and the gelatinization enthalpy of the rice
WBF 72.97 ± 0.45b −0.18 ± 0.01a 2.60 ± 0.05d 3.27 ± 0.45b flours. Usually, the particle size of dry-milled rice flours is higher than
FDF 70.62 ± 1.28de −0.31 ± 0.11b 4.19 ± 0.24b 2.48 ± 0.41d that of wet-milled flours, thereby delaying starch gelatinization; hence,
FWRF 72.05 ± 0.45c −0.38 ± 0.09d 2.85 ± 0.04c 3.05 ± 0.17c
dry-milled rice flour has higher onset and peak temperatures than wet-
FWBF 71.33 ± 0.31d −0.30 ± 0.01b 2.64 ± 0.08d 2.03 ± 0.21d
milled flour (Leewatchararongjaroen and Anuntagool, 2016).
1)
The abbreviations refer to those listed in Table 1. The gelatinization enthalpy of WRF was the lowest because of the
a–e
Superscripts within the same column are significantly different (p < 0.05). increased amorphous regions and decreased crystalline regions, or the
Values represent means ± standard deviations of three replicates. ordered structure of the starch due to the grinding process (Song and

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S.-H. Cho et al. Journal of Cereal Science 85 (2019) 15–19

Fig. 1. Scanning electron microphotographs of rice flours (1,000X magnification). The abbreviations refer to those listed in Table 1.

Shin, 2007). A higher gelatinization enthalpy indicates the need for a regions of rice starch by LAB.
large amount of energy to melt the crystalline structure (Ratnayake and
Jackson, 2006). The gelatinization enthalpy of fermented rice flours
4. Conclusions
was significantly higher than that of non-fermented rice flours. Fer-
mented rice flour can be made into a transparent and clear paste faster
In this study, we investigated the effects of fermentation with L.
than unfermented rice flour because the starch is degraded by micro-
amylovorus on the physicochemical characteristics of dry- and semi-wet-
organisms or modified by acid. The reduction in protein and lipid
milled rice flours. The results clearly indicate that fermentation im-
contents caused the starch granules to absorb water faster and gelati-
proves the physicochemical properties of dry-milled rice flour, making
nize easily with heating. The proportion of crystalline regions in the
it a desirable material for use in rice-based products. Thus, the fer-
rice starch granules increased due to the hydrolysis of the amorphous
mented products can be applied as potential functional rice flours for

Fig. 2. X-ray diffractograms of rice flours. The abbreviations refer to those listed in Table 1.

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S.-H. Cho et al. Journal of Cereal Science 85 (2019) 15–19

Table 4 Chiang, P.Y., Yeh, A.I., 2002. Effect of soaking on wet-milling of rice. J. Cereal. Sci. 35,
Differential scanning calorimetric characteristics of rice flours. 85–94.
Chung, H.-J., Liu, Q., Lee, L., Wei, D., 2011. Relationship between the structure, physi-
Samples1) Temperature (°C) ΔH (J/g)2) cochemical properties and in vitro digestibility of rice starches with different amylose
contents. Food Hydrocolloids 25, 968–975.
Onset Peak Conclusion Donald, A.M., 2004. Understanding starch structure and functionality. In: Eliasson, A.-C.
(Ed.), Starch in Food: Structure, Function and Applications. CRC Press, Boca Raton,
DF 61.89 ± 0.29a
68.77 ± 0.18a
73.08 ± 2.84ns 7.84 ± 1.01b FL, pp. 156–184.
WRF 53.24 ± 3.53de 66.06 ± 0.52b 72.88 ± 0.65 4.73 ± 0.34c Haydersah, J., Chevallier, I., Rochette, I., Mouquet-Rivier, C., Picq, C., Marianne-Pépin,
WBF 58.01 ± 0.54bc 66.09 ± 0.41b 72.61 ± 0.80 5.26 ± 0.36c T., Icard-Vernière, C., Guyot, J.-P., 2012. Fermentation by amylolytic lactic acid
bacteria and consequences for starch digestibility of plantain, breadfruit, and sweet
FDF 58.67 ± 0.75b 66.41 ± 1.35b 72.72 ± 0.25 9.70 ± 0.14a
potato flours. J. Food Sci. 77, M466–M472.
FWRF 52.24 ± 0.44e 65.24 ± 0.53b 71.93 ± 0.41 8.53 ± 0.60b
John, R.P., G.S, A., Nampoothiri, K.M., Pandey, A., 2009. Direct lactic acid fermentation:
FWBF 55.58 ± 0.52cd 65.58 ± 1.43b 71.76 ± 0.66 10.34 ± 0.34a
focus on simultaneous saccharification and lactic acid production. Biotechnol. Adv.
1)
27, 145–152.
The abbreviations refer to those listed in Table 1. Kadan, R.S., Bryant, R.J., Miller, J.A., 2008. Effects of milling on functional properties of
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Gelatinization enthalpy. rice flour. J. Food Sci. 73, E151–E154.
a–e Lu, Z.-H., Li, L.-T., Min, W.-H., Wang, F., Tatsumi, E., 2005. The effects of natural fer-
Superscript within the same column are significantly different (p < 0.05).
ns
Not significant at p < 0.05. mentation on the physical properties of rice flour and the rheological characteristics
of rice noodles. Int. J. Food Sci. Tech. 40, 985–992.
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