J. Biol. Macromol., 18 (1), 3-10 (2018)
ArticleCharacterization of polished rice washing water fermented with milk and its inhibition activity toward
tomato bacterial canker causative agent Clavibacter michiganensis subsp. michiganensis
Tatsuya Nakata1, Kohei Kawakami1, Rafael Prieto2, Hisabumi Takase2, and Jiro Sekiya2*
1

Division of Bioenvironmental Science, Graduate School of Bioenvironmental Science,
Kyoto Gakuen University, Kameoka, Kyoto 621-8555, Japan2

Department of Bioscience, Faculty of Bioenvironmental Science,
Kyoto Gakuen University, Kameoka, Kyoto 621-8555, Japan

Received December 30, 2017; Accepted January 15, 2018
Abbreviations: CMM, Clavibacter michiganensis subsp. michiganensis; PS medium, potato semi-synthetic
medium; RWWM, polished rice washing water fermented with cow’s milk; RWW, polished rice washing water;
PET, polyethylene terephthalate; SD, standard deviation.
Polished rice (seihaku-mai in Japanese) washing water (RWW) exerts a heavy load on gray water treatment.
Some farmers ferment RWW with milk and use it for managing healthy plant growth (Fukushima, 2010).
In this study, we prepared RWW fermented with cow’s milk (RWWM) and found that it generated
flammable gas and protein debris, lowered pH at the early stage of fermentation, and produced lactic acid.
RWWM also inhibited the growth of Clavibacter michiganensis subsp. michiganensis (CMM), a causative
agent of tomato bacterial canker, but not the growth of Bacillus subtilis, Staphylococcus aureus, orEscherichia coli. Thus, we assumed that lactic acid bacteria are involved in the antibacterial activity of
RWWM and that some metabolites in RWWM inhibit the growth of CMM.
Key words: fermented polished rice washing water, Clavibacter michiganensis, characterization, growth inhibition

Introduction
In Japan, polished rice (seihaku-mai in Japanese) is

cow’s milk (RWWM), which was found to protect
plants against southern blight caused by Sclerotium

washed several times with water to remove sticky branrolfsii (4,5).

Fukushima (4) stated that lactic acidbefore cooking. This polished rice washing water

bacteria might be related to the “anti-disease” effect of(RWW) is usually thrown away, thus exerting a heavy

RWWM.

load on gray water treatment (1,2). Farmers often use

contribute to both reducing the load on gray waterhomemade fermented organic materials for thetreatment and managing healthy plant growth.

management of healthy plant growth (3). An

However, RWWM has not been characterized so far.

example is polished rice washing water fermented with

We attempted to characterize RWWM and estimate its

If this were true, the use of RWWM would

antimicrobial activity.
*To whom correspondence should be addressed:

E-mail: sekiyaj@kyotogakuen.ac.jp

We describe herein someproperties of RWWM and its inhibition activity toward
Clavibacter michiganensis subsp. michiganensis(CMM). CMM is known to be a causative agent of

4Characterization of fermented polished-rice washing water
x 108 cell/mL.

tomato bacterial canker (6,7).

RWWM cultured for 14-60 days wasused, unless otherwise noted. RWWM used forMaterials and Methods

measurement of inhibition activity was sterilized byMaterials

passing through a filter (0.2 m, Advantec

The cultivars of rice (Oryza sativa subsp. japonica)

25AS020AS).

used in this study were mostly “Koshihikari” and“Kinuhikari” produced in Kyoto and Shiga Prefecturesand obtained from local markets.

C. michiganensis

The test tube for assay contained 5 mL of PS liquidmedium, 50 L of pre-cultured CMM, and 500 L offilter-sterilized RWWM.

Sterilized water was added

subsp. michiganensis (CMM) strain 05M1-2 (ato the control test tube instead of RWWM. After thegift from Dr. Akira Kawaguchi) was cultured in

test tubes were incubated at 30ºC for 24 hr in the dark

potato semi-synthetic (PS) liquid medium (7).

with reciprocal shaking at 150 rpm, the turbidity at 660

Bacillus subtilis strain 168 (a gift from Dr. Yasutaro

nm was measured. Measurement was routinely

Fujita) was cultured in Luria Bertani (LB) medium.

conducted in triplicate and expressed as growth rate

Escherichia coli AB1157 (ATCC 29055) andrelative to control in %.

Staphylococcus aureus (ATCC 31890) were purchased

The agar cup method was used to estimate thefrom the American Type Culture Collection, USA andinhibition activity of RWWM. Hot PS agar medium

cultured in LB medium and Staphylococcus medium

was poured into a sterile Petri dish (9 cm diameter) and

No. 110, respectively. Cow’s milk was obtained fromsolidified. Then, sterilized penicillin cups were put

local markets.

concentrically on the agar medium.

PS agar medium(15 mL) containing CMM was layered onto the
Preparation of RWWM

solidified PS agar medium in the Petri dish, cooled,

RWWM was prepared essentially according to theand solidified. Then, the penicillin cups were

method described by Fukushima (4). Polished rice

removed gently. The filter-sterilized RWWM (150(400 g) was washed four times with 400 mL of tap

L) was pipetted into each well. Water was used aswater.

control. After the Petri dishes were incubated at 30ºC

After washing, the four portions of washingwater were combined and water was added to makefor 48 hr, a clear zone along the well was observed.

1600 mL (RWW). RWW was poured into a 2000 mL
PET bottle together with 400 mL of commerciallyavailable cow’s milk. Then, the bottle was capped

Determination of lactic acids
D-Lactic acid and L-lactic acid were determined

and incubated statically at 25ºC in the dark for aaccording to the instructions attached to the F-kit for

specified period. Due to the generation of gas, thethe determination of D-lactic acid/L-lactic acid (Roche

PET bottle was decapped quickly once a day until the

Diagnostics GmbH).

tenth day after the start of incubation. Preparation of
RWWM on the one-quarter scale was also conductedin a 500 mL PET bottle. We designated the polishedrice washing water fermented with milk as RWWM.

Measurement of whiteness value
Rice whiteness value was measured with a ricewhiteness tester (Model C-300-3, Kett Electric
Laboratory) and expressed as whiteness value in %.

Measurement of inhibition activity of RWWM toward
CMM growth
CMM was pre-cultured in PS liquid medium at

Results and Discussion
Characterization of RWWM

30ºC for 24 hr with reciprocal shaking at 150 rpm.

Whiteness of polished rice and wash-free rice

CMM growth in the 24-hr pre-culture was at the early(musen-mai in Japanese) is approximately 40% andstationary phase and cell density was approximately 2

more than 45%, respectively (8). Therefore, sticky

Nakata et al.

5rice bran is produced while polished rice is refined tothe dark, white milk curd appeared gradually both on

wash-free rice. Whiteness of polished rice used in

top and at the bottom of the PET bottle (Fig. 1). In

this experiment was 42.0 ± 0.4% (mean ± SD, n=4).

addition, the cloudiness of the emulsion was decreased

Thus, it is suggested that RWW contains sticky rice

and its color gradually turned pale yellow.

bran.

later, most of the supernatants of RWWM were clear

RWW was mixed well with milk in the PET bottle

and had a pale yellow color.

One yearDuring the first two

to obtain a milky white homogeneous emulsion.

weeks, PET bottle containing RWWM continued toWhen the emulsion was incubated statically at 25ºC in

expand because of gas generation. We confirmed theFig. 1 Visual changes of RWWM
RWWM in PET bottles was incubated at 25ºC in the dark after RWW was mixed well with milk.

Fig. 2 Changes of pH (A) and lactic acid contents (B) during the incubation of RWWM and RWW
RWWM and RWW in PET bottles were incubated at 25ºC in the dark. pH was measured with a pH meter.
Lactic acid contents were measured with a commercially available kit as described in Materials and Methods.
Data are expressed as means ± SD (n=3). (A): RWWM (○), RWW alone (□). (B): L-Lactic acid (○) and Dlactic acid (●) in RWWM, L-lactic acid (□) and D-lactic acid (■) in RWW.

6Characterization of fermented polished-rice washing water

generation of flammable gas, probably methane, bythat of RWW, to 4.36. On day 14, pH of RWWM wasburning. The pH change during the first two weeks

3.66 whereas that of RWW was 4.25 (Fig. 2A). Evenof incubation is shown in Fig. 2A. pH of RWWM

after the prolonged incubation period, pH of RWWM

and RWW gradually decreased from the original pH

was maintained around 3.3-3.7, whereas that of RWW

6.8-7.0 during incubation. One week after the start of

did not reach values below 4.0 (Table 1).

incubation, pH of RWWM decreased to 3.75 whereas
Table 1 Effects of RWWM and RWW on CMM growth

Tested material

Relative growth of CMM

Material

Culture period (day)ｐH

Experiment 1
Water alone
RWWM (RWW + milk)
RWWM (RWW + milk)
RWWM (RWW + milk)
RWWM (RWW + milk)

0
10
14
32
55

nm
3.72 ± 0.06
3.66 ± 0.04
3.49 ± 0.01
nm

100 ± 3
2±1
6±1
3±1
1±1

Experiment 2
RWWM (RWW + milk)
RWWM (RWW + milk)

100
200

nm
3.68 ± 0.13

1±1
3±1

Experiment 3
Water alone
RWW + water
RWW + water
Water + milk
RWWM (RWW + milk)

14
14
60
14
14

nm
4.25 ± 0.02
5.85 ± 0.01
5.86 ± 0.02
3.66 ± 0.04

100 ± 286 ± 283 ± 3109 ± 195±1

Experiment 4
Water alone
Autoclaved RWW + milk
RWW + autoclaved milk
RWWM (RWW+ milk)

14
14
14
14

nm
4.97 ± 0.04
4.61 ± 0.18
3.66 ± 0.04

100 ± 3101 ± 13
5±2
5±1

(%)

Tested materials including RWWM were cultured in 2 L PET bottles at 25ºC in the dark for the indicatedperiods, and pH of the tested materials was measured in triplicate at the end of the culture. The assay forinhibition activity toward CMM growth was conducted by using the liquid culture method as described inMaterials and Methods. Data are expressed as means ± SD (n=3). nm, not measured.

It is speculated that lactic acid bacteria are related

resulting in the unchanged lactic acid content (Fig.

to RWWM (4). Thus, we determined the lactic acid

2B).

For RWWM incubated for 180 days, thecontents of RWWM and RWW for 14 days. Total

contents of D-lactic acid and L-lactic acid werelactic acid content in RWWM increased, with the

31.9±3.2 and 26.5±1.6 mmol/L, respectively, and pH

increase in D-lactic acid content contributing to thewas 3.48. Total lactic acid content of RWWM on day

total increase (Fig. 2B). In contrast, neither D-lactic

180 was approximately 300 times higher than that onacid nor L-lactic acid content increased in RWW,

day 14. The increase in D-lactic acid content

Nakata et al.

7

Fig. 3 Effects of RWWM added on CMM growth
CMM cultured in PS liquid medium at 30ºC for 24 hr with reciprocal shaking at 150 rpm was used. One
turbidity unit at 660 nm, attained approximately 24 hr after the start of culture, corresponded to 2 x 108
cells/mL under the culture condition used. The test tube for assay consisted of 5 mL of PS liquid medium,50 L of CMM, and an aliquot volume of RWWM.

In the control test tube, water was added instead of

RWWM. After the test tubes were incubated for 24 hr at 30ºC with reciprocal shaking at 150 rpm, turbidity
at 660 nm was measured. Data are expressed as means ± SD (n=4-10). Different letters attached to thebars in the panel mean significant difference at P < 0.05 by the Student’s t-test.
suggests the involvement of lactic acid bacteria insignificantly compared to no RWWM addition,

RWWM. The isolation and identification of lactic

although no significant difference was observed

acid bacteria in RWWM are in progress.

between the addition of 100 L and 200 L.
Therefore, we employed the reaction mixture

Inhibition of CMM growth by RWWM
It is assumed that lactic acid bacteria are present inconsisting of 5 mL of PS liquid medium, 50 L of precultured CMM, and 500 L of filter-sterilized

RWWM, as described above. Lactic acid bacteria

RWWM, as described in Materials and Methods.

usually produce antibacterial substances called

Inhibition of CMM growth was also confirmed by thebacteriocin (9). Many bacteriocins produced by lactic

agar cup method. CMM inhibition zones wereacid bacteria are generally effective against Gram-observed around wells 1 and 3 filled with RWWM, but

positive bacteria (10-12). To evaluate the

not around control wells 2 and 4 filled with water (Fig.

antimicrobial activity of RWWM, CMM was chosen

4).

because it is a Gram-positive phytopathogenic

CMM growth inhibition was first observed

bacterium that causes tomato bacterial canker and its

approximately 10 days after the start of culture of

growth was inhibited by RWWM in a preliminary

RWWM (Experiment 1 in Table 1). The inhibition

experiment.

activity of RWWM was detected even after 200 days

We first tried to establish an assay for the inhibition(Experiment 2 in Table 1). Seventy-six of the 91 lots

activity of RWWM toward CMM growth. As shownof RWWM (culture period, 14 to 60 days) showed

in Fig. 3, the addition of 500 L of RWWM inhibited

more than 50% inhibition of CMM growth under the

CMM growth almost completely.

assay condition described in Materials and Methods.

The addition of

100-200 L of RWWM inhibited CMM growth

Sixty-three lots showed more than 90% inhibition.

8

Characterization of fermented polished-rice washing water

Fig. 4 Inhibition of CMM growth by RWWM
Inhibitory effect of RWWM was estimated by the agar cup method as described in Materials and Methods.
Wells 1 and 3 were filled with RWWM and wells 2 and 4, with water as control. The Petri dish was incubatedat 30ºC for 48 hr in the dark. Growth inhibition was estimated from the inhibition zone around the well.

Table 2 Effect of RWWM on growth of various bacteria

Bacterium species

MediumTested material

Relative growth rate
(%)

CMM

PS
PS

Water
RWWM

100 ± 3
3±1

Bacillus subtilis

LB
LB

Water
RWWM

100 ± 196 ± 5

St. #110
St. #110

Water
RWWM

100 ± 199 ± 2

LB
LB

Water
RWWM

100 ± 296 ± 1

Staphylococcus aureus
Escherichia coli

The assay for the inhibition activity of RWWM was conducted by using the liquid culture method as
described in Materials and Methods with the exception that CMM was substituted by other bacterium. Data
are expressed as means ± SD (n=3).
RWWM consists of RWW and milk. When

autoclaved and non-autoclaved materials revealed that

RWW was cultured with water, only low inhibition

non-autoclaved, RWW was necessary to produce the(less than 20%) was observed (Experiment 3 in Table

inhibition activity (Experiment 4 in Table 1).

1). Culture of milk alone showed no inhibition of

Autoclaved milk was as effective as non-autoclaved

CMM growth. Therefore, both RWW and milk aremilk. These results suggest that RWW is the mainnecessary to produce the inhibition activity towardsource of microorganisms and milk supplies the

CMM growth. Combination experiments ofnutrients required for the growth of microorganisms.

Nakata et al.

9Then, the effect of RWWM on the growth of otherassay tube was inhibited almost completely.

Effect ofbacteria was examined. The growth of B. subtilis andlactic acid addition on CMM growth was also

S. aureus, both Gram-positive bacteria, was not

examined. pH of the assay tube containing PS

inhibited (Table 2). The growth of Escherichia coli, a

medium and CMM was 5.9 when lactic acid was added

Gram-negative bacterium, was not inhibited as well.

to the tube (final concentration: 0.3 mM), but CMM

Thus, the inhibition activity of RWWM was specific togrowth was not inhibited (data not shown). From

CMM among the bacteria examined. Nisin, which isthese results, we conclude that the low pH of the assay

used as an additive for food preservation, is produced

tube was not the cause of the inhibition of CMM

by Lactococcus lactis (9,12). Nisin inhibits thegrowth. We assume that the presence of bacteriocin-

growth of some but not all Gram-positive bacteria.

like compounds is responsible for the inhibition of

These results also suggest that lactic acid bacteria are

CMM growth and are now trying to isolate andinvolved in RWWM.

identify them.

It is also speculated that the low pH of RWWM is a

Fukushima (4) described that RWWM reduced

cause of the inhibition of CMM growth because the pH

damage in green onion caused by southern blightof RWWM is usually around 3.5-3.8. Therefore, thedisease. It is well known that southern blight disease

effect of medium pH on CMM growth was examined.

is caused by Sclerotium rolfsii, which produces many

Instead of RWWM, water was added to PS media

sclerotia under stress conditions. Sclerotia are alive

adjusted to pH 5.0 and 6.8, and this was followed byfor a few years in soil.

the addition of CMM. RWWM and CMM were

RWWM inhibited neither mycelial growth nor

added to the standard PS medium. The pH of thesclerotia germination of S. rolfsii (data not shown).

assay tubes containing CMM in PS medium adjusted

This suggests that RWWM does not act directly on

to pH 5.0 and 6.8 was 5.2 and 7.0, respectively, before

mycelia or sclerotia of S. rolfsii.

the incubation for assay. After the incubation for 24
hr, the pH of the assay tubes was 5.6 and 6.7,

Acknowledgements

respectively (Table 3). The growth rate of CMM in

We thank Dr. Akira Kawaguchi, Research Institutethe medium with pH 5.0 was 82% compared to thefor Agriculture, Okayama Prefectural Technology

100% growth rate in the medium with pH 6.8. pH ofCenter for Agriculture, Forestry and Fishery, for thethe standard assay tube containing CMM and RWWM

kind gift of C. michiganensis subsp. michiganensis andwas 5.4 and 5.6 before and after the assay, respectively

Professor Yasutaro Fujita (presently Emeritus(Table 3). However, CMM growth in the standard

Professor), Faculty of Life Science and Biotechnology,

Table 3 Effect of medium pH on CMM growth

MediumPS medium (pH 6.8)
PS medium (pH 5.0)
PS medium (pH 6.8)

Tested material
Water
Water
RWWM

before assay

pHafter assay

Relative growth of CMM
(%)

7.0
5.2
5.4

6.7
5.6
5.6

100 ± 182 ± 71±1

PS media adjusted to pH 6.8 and 5.0 before autoclaving were prepared. Water (500 L) and CMM (50 L)
were added to each PS medium and pH was measured and designated as “pH before assay”.

After 24 hr

incubation, pH and turbidity of the assay test tubes were measured and designated as “pH after assay” and“relative growth of CMM”. RWWM used in this experiment was a 44-day culture with pH of 3.6. Data forrelative growth of CMM are expressed as means ± SD (n=3).

10Characterization of fermented polished-rice washing water

Fukuyama University, for the kind gift of B. subtilis.

(9) Cotter, P. D., Hill, C. and Ross, R. P. (2005)

We also thank Mr. Masaaki Amano, president ofBacteriocins: developing innate immunity for food.

Hooden Industries, Ltd., for rice whitenessNat. Rev. Microbiol., 3, 777-788.

measurement and Dr. Yukio Takii, former guest(10) Schillinger, U. and Lücke, F. -K. (1989)

researcher of Kyoto Gakuen University, for useful

Antibacterial activity of Lactobacillus sake isolated

advice and discussion. This work was partiallyfrom meat. Appl. Environ. Microbiol., 55, 1901-1906.

supported by Kameoka City Incentive Grant for(11) Rajaram, G., Manivasagan, P., Thilagavathi, B.

Postgraduates Conducting Local Community Researchand Saravanakumar, A. (2010) Purification andto T. Nakata (2113).

characterization of a bacteriocin produced by
Lactobacillus lactis isolated from marine environment.
Adv. J. Food Sci. Technol., 2, 138-144.

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Communicated by Ueno Hiroshi