JOURNAL OF BIOSCIENCE AND BIOENQINEERING

Vol. 90, No. 4, 442-446. 2000

Antifungal Activity of Plant Extracts against Arthrinium sacchari

and Chaetomium funicola
JUN SATO,‘* KEIICHI GOT0,2 FUMIO NANJ0,2 SHIGEYUKI KAWAI,3 AND KOUSAKU MURATA3
Quality Assurance, Coca-Cola (Japan) Company, Limited, 6-3 Shibuya 4-chome, Shibuya-ku, Tokyo 150-0002,1 Food Research
Laboratories, Mitsui Norin Co. Ltd., 223-l Miyabara, Fujieda, Shizuoka 426-0133,2 and Research Institute for
Food Science, Kyoto University, Uji, Kyoto 611-0011,3 Japan
Received30 May 20OO/Accepted
21 July 2000

Various plant extracts were examined for antifungal activity with the objective of improving the commercial
sterility of aseptically filled tea beverage products in PET bottles. When the hot water extract and the methanol
extract of 29 samples were measured for their antifungal activity against Arthrinium sacchari MOO1 and
Chaetomium f&cola MOO2strains, five samples, Acer nikoense, Glycyrrhiza glabra, Lagerstroemia speciosa,
Psidium guajava and Thea sinensis, showed high activity. Of these, the extracts from A. nikoense, G. glabra and
T. sinensiswere fractionated by extraction with CHQ, and the CHQ-soluble fractions from G. glabra showed
antifungal activity with minimum inhibitory concentrations WCs) between 62.5 and 125 B/ml against the
above-mentioned two fungi. When the EtOAc-soluble fraction of A. nikoense was used, the MIC against A.
sacchari MOO1was 62.5 ,f.&ml. However, none of the fractions from A. nikoense or T. sine&s showed high
activity against C. funicola MOO2 and their MICs were greater than 500 ~/ml. A licorice preparation made
from the commercially available oil-based extract of G. glabra showed a low MIC of 25 ,&ml against five tested
strains of iilamentous fungi, but not against Aspergillus fumigatus MOOS,in a blended tea. Consequently, the
possibility of adding a licorice preparation made from the oil-based extract of G. glabra to tea beverages
(aseptically filled into PET bottles) was suggested.
[Key words: antifungal activity, Arthrinium sacchari, Chaetomium funicola, licorice, plant extract]

Arthrinium sacchari strain MOO1 and Chaetomium
funicola strain MOO2 are filamentous fungi isolated from
the production environment of tea beverages. Assuming
them to be indicator microorganisms in the PET bottle
aseptic filling method (hereafter abbreviated as PET
AFM), we investigated their microbiological characteris-
tics, basic heat resistance and elimination with a peracet-
ic acid solution (1, 2). The PET AFM is a beverage
manufacturing technique wherein an UHT (ultra-high
temperature) sterilized liquid (i.e. beverage) is placed in
chemically sterilized containers under an aseptic at-
mosphere. In the PET AFM, the killing of indicator
microorganisms is often achieved by presetting F at 6D (a
sterilization effect that decreases the microbial count to
l/106 of the initial number). This value is equal to or
even higher than those for other aseptically filled prod-
ucts including “long-life milk” products, but stochastical-
ly it does not mean complete eradication of defective
products. Recently, small-sized PET-bottle beverages are
markedly gaining more popularity in the soft drink mar-
ket in Japan. At the same time, there have been con-
cerns regarding filamentous fungi contamination in
small-sized PET-bottle beverages which was apparently
caused by contamination after opening by the consumer
(secondary contamination). This problem cannot be
prevented by the manufacturer even though product
sterilization has been achieved prior to opening of the
bottle. To deal with this problem, addition of sucrose es-
ters to beverages is common in the Japanese soft drinks
industry. Some food emulsifiers, such as the sucrose es-
ters of fatty acids, have been used in the production of
canned milk coffee products for their antibacterial activ-
ity against mainly thermophilic bacterial spores (Clos-
* Correspondingauthor.
442
tridium thermaceticum, Bacillus stearothermophilus),
which could cause “flat sour spoilage” (3-7). Sucrose es-
ters have also been added to tea beverages containing
low concentrations of catechin, such as barley tea, to
suppress the outgrowth of spore-forming bacteria which
derive from the production process and containers. In
this case, however, it is necessary to indicate that the
product contains a synthetic “emulsifier” on the label as
one of the ingredients and this could damage the con-
sumer’s image of the product. In this study, we exam-
ined the antifungal activities of various plant extracts
with the objective of improving the commercial sterility
of PET AFM products (8) by blending into tea bever-
ages natural ingredients that possess antifungal activity.
MATERlALS AND METHODS
Plant materials We examined 29 plants, the ex-
tracts of which are sold as commercial drinks. The plant
materials used are summarized in Table 1.
Test strains and preparation of spore suspension
Two strains of filamentous fungi isolated from the tea
beverage production environment, A. sacchari MOO1 and
C. funicola MOO2 were used. The spore suspension was
prepared as follows (1). The mycelium of the test strain
was suspended in 10 ml of sterile saline containing
O.O5%(w/v) Tween 80 (hereafter referred to as TSS) and
inoculated onto the sporulation medium (50 ml of PDA,
Eiken, Tokyo). After 30d incubation at 25”C, the fun-
gus formed spores which were suspended in 10ml of
TSS. After dispersing the fungal clumps, the suspension
was filtered through glass wool. The filtrate was cen-
trifuged at 3000 x g for 15 min and the residue was
resuspended in TSS. The washing procedure was re-
peated three times before preparation of the sample
VOL. !N,2000 ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS 443

spore suspension.
Water extraction A 100-g sample was extracted
with 1 I of boiling distilled water for 1 h with stirring.
The extract was filtered using gauze, filter paper and a
filter (Advantec, Tokyo). If fine particles were present,
the filtrate was centrifuged at 18,500 Xg for 10 min and
the supernatant was vacuum filtered using a filter plate.
The filtrate was concentrated to around 40 ml in WCUO,
and the residue was freeze dried.
Methanol extraction A 100-g sample was extracted
with 1 I of 80% methanol. After 22 to 24 h of agitation
with stirring, the extract was filtered using gauze, filter
paper and a filter. The subsequent procedure was the
same as that for the hot water extraction.
Measurement of antifungal activity of hot water and
methanol extracts The freeze-dried extract was dis-
solved in a sufficient amount of sterile water before it
was sterilized by filtration. The solution was diluted
with sterile water (hot water extract) or 10% DMSO
(methanol extracts) before it was mixed with PDA con-
taining chloramphenicol (10 mg/l) (hereafter abbreviated
as c-PDA). The final concentrations of extracts in the
medium were fixed at 1 or 10 mg/ml. The spore suspen-
sion was inoculated at the center of the agar plate and
the diameter of the formed colony was measured after
14 d of incubation at 25°C.
Fractionation of extracts from A. nikoense, G. glabra
and T. sinensis The hot water extracts of A. nikoense
(4.5 g), G. glabra (10 g) and T. sinensis (10 g) were dis-
solved separately in 500ml of distilled water. The
methanol extracts (10 g each) of the same plants were
also dissolved separately in 10 ml of 80% methanol
followed by the addition of 500ml of distilled water.
Each solution was extracted with 1 1 of chloroform three
times. The chloroform layer was concentrated and
freeze-dried (MeOH-CHC& and HzO-CHC& fractions).
The aqueous layer was extracted with 500ml of ethyl
acetate three times. The ethyl acetate fraction was con-
centrated and freeze-dried (MeOH-EtOAc and HzO-
EtOAc fractions). The aqueous layer that remained after
ethyl acetate extraction was concentrated in vacua to
about 50ml and then freeze-dried (MeOH-HZ0 and
H20-HZ0 fractions).
Measurement of minimum inhibitory concentration
(MIC!) of the organic solvent fraction The sample
fraction (0.1 g) was dissolved in 10 ml of 10% DMSO
(lOmg/ml). This solution was diluted with 10% DMSO
and was added to c-PDA to final concentrations of 0,
7.8, 15.6, 31.3, 62.5, 125, 250 and 5OO,~g/ml. The spore
suspension of each test strain was inoculated at the cen-
ter of the agar plates which were incubated for 14 d at
25°C. The minimum concentration at which no colony
was formed was defined as the MIC. DMSO did not
affect the growth of the sample strains at the concentra-
tion used in this study (final concentration, 0.5%).
Licorice preparation and measurement of MIC

TABLE 1. Antifungal activity of extracts from 29 test plants against A.succhari MOO1 and C. funicolu MOO2
Diameter of A. sacchuriMOO1on PDA (cm) Diameter of C. f&cola MOO2on PDA (cm)
Plant material 80%methanolextract Hot waterextract 80% methanolextract Hot waterextract
lmg/ml lOmg/ml lmg/ml lOmg/ml lmg/ml lOmg/ml lmg/ml lOmg/ml
Acer nikoense Maxim. (megusurinoki, trunk) 0.0 0.0 0.0 0.0 3.1 2.0 3.0 3.1
Aloe arborescens Mill. var. natalensis Bergel. (kidachi-aloe) 8.5< 5.4 8.5< 8.5< 5.4 3.3 5.6 6.7
Artemisia princeps Pamp. (mugwort) 7.0 2.2 8.5< 6.0 5.0 2.1 5.3 5.0
Aspalathus linearis (rooibos) 8.5< 3.0 8.5< 5.7 6.2 2.9 5.8 6.4
Carthamus tinctorius L. (safflower, flower) 7.5 0.0 8.5< 8.5< 5.8 2.1 5.1 5.7
Cassia obtusifolia L. (habu, seed) 3.5 0.0 8.5< 5.7 2.7 0.0 3.8 3.5
Chrysanthemum morifolium Ramat. (chrysanthemum, flower) 4.7 0.0 8.5< 7.8 4.2 0.0 4.9 5.6
Coix lachryma-jobi L. var. mayuen Stapf (Job’s tears, seed) 8.5< 5.6 8.5< 8.5< 5.0 2.4 3.7 4.1
Diospyros kaki Thunb. (Japanese persimmon) 5.5 3.0 7.3 3.7 5.1 1.7 4.8 2.9
Equisetum arvense L. (sugina) 8.5< 4.6 8.5< 8.5< 5.2 3.1 5.5 3.9
Eriobotrya japonica (Thunb.) Lindl. (loquat) 2.1 8.5< 3.8 6.0 3.0 5.7 6.1
Fagopyrum esculentum Moench (buckwheat, seed) it:< 43 8.5< 8.5< 4.9 2.9 5.3 5.3
Glechoma hederacea L. var. grandis Kudo (kakidooshz] 6.4 219 8.5< 8.5< 4.5 2.1 5.2 5.7
GIycyrrhiza glabra L. (licorice, root) 0.0 0.0 5.4 2.1 0.0 0.0 3.8 2.6
Gymnema sylvestre (Retz.) Schult. (gymnema) 1.6 0.0 7.5 0.6 2.5 1.1 4.4 2.0
Houttuynia cordata Thunb. (dokudaml) 6.8 0.0 8.5< 7.3 5.1 1.2 5.2 4.6
Hyssopus oficinalis L. (hyssop) 8.5< 4.3 8.5< 8.5< 5.3 2.6 5.2 5.0
Lagerstroemia speciosa Pers (banaba) 1.9 0.0 3.8 0.0 2.8 0.0 5.4 0.0
Lavandula oJficinalti L. (lavender, flower) 8.5< 2.2 8.5< 7.1 4.7 1.0 5.4 5.4
Lycium chinense Mill. (Chinese matrimony vine) 8.5< 4.2 8.5< 8.5< 5.0 2.4 5.1 3.2
Morus alba L. (mulberry) 6.0 2.3 8.5< 8.5< 5.1 2.7 5.7 6.6
Nelumbo nucifera Gaertn. (lotus) 5.2 1.9 8.5< 8.5< 4.9 2.6 8.5< 5.8
Perillu frutescens Britton var. acuta Kudo (perilla) 6.7 1.2 8.5< 8.5< 4.9 1.5 5.5 4.5
Pinus densiflora Sieb. et Zucc. (pine) 2.7 0.0 8.5< 4.0 3.7 0.6 3.8 3.8
Plantago asiatica L. (plantain) 8.5< 3.8 8.5< 8.5< 5.3 2.7 6.5 8.5<
Psidium guajava L. (guava) 3.2 0.0 3.8 0.0 5.4 0.0 6.1 1.5
Rosmarinus oficinalis L. (rosemary) 2.6 0.0 8.5< 5.4 2.9 0.0 5.5 6.1
Sass veitchii (Carr.) Rehd. (striped bamboo) 8.5< 4.0 8.5< 8.5< 4.8 2.8 5.7 5.7
Thea sinensis L. (green tea) 0.0 0.0 7.2 0.0 3.8 0.0 4.3 1.1
Control 6.8 6.9 8.5< 8.5< 4.1 4.1 4.1 4.1
Incubation conditions: on PDA containing chloramphenicol at 25°C for 14 d.
For plants without a specific designation for the parts to be used, the leaves were used.
Data expressed are results of duplicate experiments for all samples; the margin of error for each datum is -+ 1.O cm.
444 SAT0 ET AL. J. BIOSCI. BIOENG.,

After combining 5 g of the G. glabra oil-based extract TABLE 2. Yields of each organic solventfraction of A. nikoense,
(Maruzen Pharmaceuticals Co. Ltd., Tokyo), 5 g of G. glabra and T. sinensis
emulsifier (L-1695, Mitsubishi Chemical Foods, Tokyo), Yields (%)”
30 ml of propylene glycol and 30 ml of ethanol, distilled Fraction A. nikoense G. glabra T. sinensis
water was added up to 100 ml to make a licorice prepara- (licorice)
(megusurinokr] (green tea)
tion. The G. glabra oil-based extract contains 10% to
20% of a phenolic component the primary component MeOH 1.80 19.20 24.60
MeOH-CHC& 0.66 1.11 1.97
of which is grabridin (9). It was appropriately diluted to MeOH-EtOAc 0.27 2.21 6.89
give a final concentration between 0 and 100 pg/ml in MeOH-Hz0 0.77 14.55 8.88
either c-PDA or blended tea (primary materials: Coix
lacryma-jobi L., green tea, barley and brown rice and Hz0 1.10 13.00 17.20
H20-CHC& 0.10 0.09 1.08
others; 0.4”Bx; pH 6.6; tannin content: 4.6 mg/lOO ml). H1O-EtOAc Ozll 0.95 4.23
HrO-Hz0 0.82 11.00 11.80
RESULTS AND DISCUSSION a The values were calculated per 100 g of each sample.
Data expressed are results of duplicate experiments for all samples.
Antifungal activity of methanol and hot water extracts
The antifungal activity of the 29 plant extracts against
A. sacchari MOO1 and C. funicola MOO2 is shown in From the above results, the plant extracts were sub-
Table 1. The 80% methanol extract of A. nikoense, G. divided into the following antifungal activity categories;
glabra and T. sinensis showed high antifungal activity high activity: A. nikoense, G. glabra, L. speciosa, P.
against A. sacchari MOO1 at a concentration of 1 mg/ml, guajava and T. sinensis; moderate activity: G. sylvestre,
and no colonies developed. T. sinensis contains catechin H. cordata, C. obtusifolia, P. densiflora and R. ofhcina-
compounds (10-12) which may underlie the high anti- lis; low activity: N. nucifera, C. morifolium, A. linearis,
bacterial activity. At a concentration of lOmg/ml, no P. frutescens and L. o@cinalis; no activity: the remain-
colony formation was observed on plates containing ex- ing 14 samples.
tracts from 12 plants including C. obtuszfolia, G. sylves- From a preliminary study on the MICs of five extracts
tre and L. speciosa in addition to the aforementioned in the high activity category, A. nikoense, G. glabra and
three samples. Compared with the 80% methanol ex- T. sinensis were found to be active at a particularly low
tract, the hot water extract generally showed lower activ- concentration (data not shown). Consequently, we per-
ity, but the extract of A. nikoense showed high activity formed an organic solvent fractionation on these three
at 1 mg/ml. At 10 mg/ml, no colony formation was ob- extracts.
served on the plates containing the extracts from L. Antifungal activity of organic solvent fraction of A.
speciosa, P. guajava and T. sinensis in addition to A. nikoense, G. glabra and T. sinensis extracts Table 2
nikoense. shows the yield of each organic solvent fraction of A.
Only the 80% methanol extract of G. glabra showed nikoense, G. glabra and T. sinensis which was calculated
high activity against C. funicola MOO2 at 1 mg/ml. At per 1OOg of each sample. The yield of each fraction of
lOmg/ml, no colony formation was observed for the ex- A. nikoense was generally lower compared with that of
tracts of seven plants, C. obtusifolia, C. morifolium, L. G. glabra and T. sine&s. This means that A. nikoense
speciosa, P. guajava, R. oficinalis and T. sinensis in ad- is not suitable for use in tea beverage production.
dition to G. glabra. None of the hot water extracts ex- Table 3 shows the MIC of each fraction against A. sac-
hibited high activity at 1 mg/ml. The hot water extracts, chari MOO1 and C. funicola MO02. The MIC against A.
except for that of L. speciosa, did not prevent colony sacchari MOO1 was 125 /*g/ml with the MeOH, MeOH-
formation at lOmg/ml. There were several extracts EtOAc and MeOH-Hz0 fractions of A. nikoense, and
which seemed to function as nutrients, judging from the with the MeOH-CHC& fraction of G. glabra; and 62.5
colony diameter. For instance, with the hot water extract pg/ml with the HzO-EtOAc fraction of A. nikoense
of P. asiatica, the colony diameter on the plate contain- and the H20-CHC13 fraction of G. glabra. None of the
ing lOmg/ml of the extract was larger than that on the fractions from T. sinensis showed a MIC less than
plate containing 1 mg/ml. 250 pg/ml.

TABLE 3. MIC of each organic solvent fraction of A. nikoense, G. glabra and T. sinensis against A. sacchari MOO1 and C. funicola MQO2
MIC against A. sacchari MOO1 @g/ml) MIC against C. funicola MOO2 @g/ml)
Fraction A. nikoense G. glabra T. sinensis A. nikoense G. glabra T. sine&s
(megusurinokQ (licorice) (green tea) (megusurinokz] (licorice) (green tea)
MeOH 125 500< 250 500< 500< 500<
MeOH-CHC& 500 125 500 500< 62.5 500<
MeOH-EtOAc 125 500< 500< 500< 500< 500<
MeOH-Hz0 125 500< 250 500< 500< 500<
Hz0 ND” 500< 500< ND 500< 500<
HzO-CHClj 500< 62.5 500< 500< 125 500<
HzO-EtOAc 62.5 500< 500< 500< 500< 500<
HzO-H,O 500< 500< 500< 500< 500-C 500<
a Not done
Incubation conditions: on PDA containing chloramphenicol at 25°C for 14 d.
Data expressed are results of duplicate experiments for all samples. Duplicate experiments produced identical results and no range of error was
found.
VOL. 90, 2000
TABLE 4. MIC of the licorice preparation against various filamen-
tous fungi on c-PDA and in blended tea
MIC @g/ml)
Filamentous fungi c-PDAa Blended teab
Id 14d 7d 14d
Art. sacchariMOO1 46.9 46.9 12.5 25.0
C. funicola MOO2 24.4 24.4 12.5 25.0
Aur. pulhdans MOO6 60.0 ND’ 25.0 50.0
Asp. fumigatus MOO8 lOO< ND lOO< lOO<
Nodulisporium sp. MO10 40.0 ND 25.0 25.0
a PDA containing chloramphenicol.
b Brewed from a mixture of Coix Iacryma-jobi, green tea, barley,
brown rice and others; 0.4” Bx, pH 6.6, tannin content: 4.6
mg/lOO ml.
c Not done.
Incubation temperature: 25°C.
Data expressed are results of duplicate experiments for all samples.
Duplicate experiments produced identical results and no range of error
was found.
The MIC of the G. glabra Me-CHC& fraction against
C. funicola MOO2 was 62.5 ,ug/ml while it was 125 /*g/ml
with the H20-CHC& fraction. None of the fractions
from A. nikoense and T. sinensis showed high activity
and their MICs were greater than 500 pg/ml.
Because the MeOH-CHC13 and H20-CHC& fractions
of G. glabra showed high activity against both test
strains, it was suggested that the component with anti-
fungal activity in the G. glabra extract was relatively low
in polarity and could be extracted with chloroform.
MIC of licorice preparation against five strains of
filamentous fungi An extract similar to the chloro-
form fraction is commercially available (G. glabra oil-
based extract). This commercial product was solubilized
and the MIC of the resultant licorice preparation was
measured against five filamentous fungi (Table 4). The
five test strains included stock cultures from the Quality
Assurance of Coca-Cola (Japan) Co. Ltd., i.e., Aureo-
basidium pullulans M006, Aspergillus fumigatus MO08
and Nodulisporium sp. MO10 as well as the aforemen-
tioned A. sacchari MOO1 and C. funicola M002. After
7 d of incubation on c-PDA, the licorice preparation
showed the lowest MIC against C. funicola MOO2 at
24.4 pg/ml, whereas it showed the highest MIC against
A. fumigatus MOO8 at greater than lOO,~g/ml. The
licorice preparation also showed a MIC higher than
lOO,~g/ml against A. fumigatus MOOS in blended tea
after 7 or 14d of incubation. As for the other strains,
the MIC (7 d value) of the preparation tended to be low-
er when the strains were incubated in blended tea than
on c-PDA. This was probably because the blended tea
provides less of the nutrients required for the growth of
filamentous fungi than does c-PDA.
From the above results, it was found that the licorice
preparation had a growth inhibiting effect against all of
the five test strains except A. fumigatus MOO8 and its
MIC against the other four strains was 60,0,~g/ml on c-
PDA and 25.0 /*g/ml in blended tea after 7 d of incuba-
tion. The MICs of the preparations against A. sacchari
MOO1 and C. funicola MOO2 were 46.9 and 24.4,~g/ml,
respectively, after 14 d of incubation on c-PDA. Because
the MIC of the MeOH-CHC& or H20-CHC& fraction of
G. glabra against the above two strains was between
62.5 and 125 pg/ml, it appeared that this licorice prepa-
ration contained a greater amount of the component
ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS 445
with antifungal activity. Consequently, if we consider
blending a licorice preparation into beverages, 25 /*g/ml
or higher is generally regarded to be an appropriate con-
centration from a microbiological aspect. At the same
time, the maximum concentration has to be determined
in view of the effect of this preparation on product
flavor.
In addition to its use as a food sweetening agent and
a tobacco flavoring material, the root of G. glabra
(licorice-root) is also an important ingredient in Chinese
herbal medicines (13, 14). Among the chemical compo-
nents of the licorice-root, phenolic components (mainly
flavonoids and saponin (triterpene glycoside)) have been
studied in detail (15). Mitscher et al. (16) studied the anti-
bacterial activity of the G. glabra extract and reported
that it had high activity against Staphylococcus aureus
ATCC13709 and Candida albicans ATCC10231 strains.
Demizu et al. (17) examined a flavonoid in licorice-root
for antibacterial activity and found that its antibacterial
activity was region-specific. Its high activity against gram
positive bacteria (S. aureus and Bacillus subtilis) was
also reported. Murakami (Japan Kokai Tokkyo Koho,
59-46210, 1984, in Japanese) reported that the growth
of S. aureus, Saccharomyces cerevisiae and Penicillium
chrysogenum was suppressed by components which were
extracted using an organic solvent or the combination of
an organic solvent and a lower alcohol from the glycyr-
rhizin extraction residue of G. glabra. It was not clear
whether the components extracted by Murakami were
present in the organic solvent fraction or the oil-based
extract of G. glabra which showed high activity against
A. sacchari MOO1 and C. funicola MOO2 in our study.
In conclusion, it was found that the organic solvent
fraction and the commercial oil-based extract of G.
glabra had high antifungal activity against A. sacchari
MOO1 and C. funicola M002, which are filamentous
fungi isolated from the production environment of tea
beverages. Consequently, the possibility of adding a
licorice preparation made from the oil-based extract of
G. glabra to tea beverages to prevent fungal spoilage
was suggested.
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