Food Microbiology, 1996, 13, 407–415

ORIGINAL ARTICLE

The isolation and identification of

microbes from a fermented tea beverage,
Haipao, and their interactions during
Haipao fermentation

C.-H. Liu, W.-H. Hsu, F.-L. Lee and C.-C. Liao*

Samples of Haipao from three cities of Taiwan were analyzed for their microbial population.
Two species of acetic acid bacteria and three species of yeast were isolated from tea
fungus Haipao using appropriate isolation media. The isolated bacteria were identified as
Acetobacter aceti and Acetobacter pasteurianus, based on their biochemical properties, and
compared with those of the type strains of the genus Acetobacter. The yeasts were
Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis
according to conventional phenotypic characterization combined with the Yeast Identifi-
cation Program. The brew broth analyzed by high−performance liquid chromatography
(HPLC) was shown to contain glycerol, acetic acid and ethanol. The symbiosis phenomenon
between the yeast and Acetobacter was studied. It was found that the autoclaved yeast cells
and ethanol produced by yeasts were helpful for Acetobacter to grow or produce acetic
acid. The acetic acid produced by Acetobacter could stimulate the yeast to produce ethanol.
The ethanol and acetic acid produced by yeasts and Acetobacter might prevent the
competition from other micro-organisms.  1996 Academic Press Limited

Introduction cartilaginous substance is called ‘tea fungus’

Haipao is a sour, slightly sparkling beverage
prepared by the fermentation of a mixture of
black tea and sucrose with tea fungus. After
static cultivation for a few days, a layer of
cellulose pellicle can be seen on the top of the
brew tea, which is suitable for drinking
(Kozaki et al. 1972). Hitokoto et al. (1978)
reported that acetic acid (1000 ppm) occupied
more than 95% of the total acid, and formic
acid and lactic acid were 17 ppm after 7 days
of cultivation in Japan tea fungus drink. This
*Corresponding author.
or ‘nata’ (Lapuz et al. 1967). The drink is
called ‘Kocha Kinoko’ in Japan (Terada and
Nishimura 1976), and ‘Hongo’ in Germany
(Benk 1988). Haipao is said to be beneficial to
human health, but this has not yet been
proved. In Taiwan, Haipao has been quite
popular recently. The symbiosis between
yeasts and bacteria is quite common in
fermented food such as western sugary kefir Received:
(Leroi and Pidoux 1993) and eastern 21 November 1995
Tou-pan-chiang (Hwang et al. 1988). It has Food Industry
been reported that tea fungus is a symbiosis Research and
of acetic acid bacteria (Acetobacter xyli- Development
Institute, P.O. Box
num, Acetobacter xylinoides, or Bacterium 246, Hsinchu,
gluconicum) and yeast (Schizosaccharomyces Taiwan, R.O.C

0740-0020/96/060407+09 $25.00/0  1996 Academic Press Limited

408 C.-H. Liu et al.
pombe, or Saccharomyces ludwigii) (Reiss
1994). Few studies on tea fungus have been
reported in Taiwan, except for a study by
Kozaki et al. (1972) that isolated Candida
obtusa and Kloeckera apiculata from For-
mosa Haipao samples. In this paper, samples
of tea fungus in Taiwan were analyzed, and
the interaction between the yeasts and bac-
teria investigated.
Materials and Methods
Isolation and enumeration
Haipao samples were collected from three
household tea fungi originated in Taipei,
Hsinchu, and Chiayi. Ten grams of cellulose
pellicle and broth were mixed with 90 ml of
sterile water, and then disrupted in a blender
(Osterizer, Imperial Co., USA) for 3 min. A
decimal dilution series was prepared with
sterile water, and 0.1 ml of the appropriately
diluted suspension was spread on several iso-
lation media, including tryptic soy agar (TSA,
Difco Laboratories), nutrient agar (NA, Difco
Laboratories), MRS (Difco Laboratories), YM
agar (Difco Laboratories), and mannitol agar
(MA, Asai et al. 1964). Single and well-iso-
lated colonies were picked from the isolation
media. A total of 64 isolates were included in
the examination. Isolates were purified by
repeatedly streaking on appropriate media,
and preserved in 200 g l−1 glycerol in a −80°C
freezer (Kelvinator Commercial Products
Inc., Manitowoc, WI, USA).
Strains and cultivation conditions
The reference strains used in this study were
Acetobacter aceti CCRC 11688 (ATCC 15973),
Acetobacter liquefaciens CCRC 12950 (ATCC
14835), Acetobacter pasteurianus CCRC
14145 (ATCC 33445), Acetobacter hansenii
CCRC 14157 (ATCC 35959), Acetobacter xyli-
num CCRC 12952 (ATCC 23767), and Glu-
conobacter oxydans CCRC 14103 (ATCC
19357). These strains were grown and main-
tained at 30°C on GYC agar for identification
experiments (Nanba and Kato 1985). Acetob-
acter isolates were cultivated in Hestrin and
Schramm medium (1954) which contained 20
g l−1 glucose, 5 g l−1 yeast extract, 5 g l−1 pep-
tone, 2·7 g l −1 sodium phosphate, and 1·2 g l−1
citrate. Yeast isolates were maintained in
YM agar and grown in YM broth for 18 h as
inocula. The lab−made traditional Haipao
was initially prepared by inoculating the
sweetened black tea broth (100 g l−1 raw
sugar and 3·3 g l−1 tea extract; Liu et al. 1995)
with pellicle and broth of the household tea
fungus of Hsinchu. For latter preparation,
the sweetened black tea was inoculated with
pellicle and broth of the former Haipao
brewed in the laboratory.
Identification of bacteria
The experiments were performed at 30°C
unless otherwise stated. Overoxidation of
acetate or lactate was determined in a test
tube with a medium consisting of 2 g l−1 yeast
extract, 3 g l−1 polypeptone, 0·02 g l−1 bromo-
thymol blue, and 2 g l−1 sodium acetate or
sodium lactate. Carbon source requirement
was tested in a medium containing 10 g l−1
carbon source, 5 g l −1 yeast extract, and 0·2 g
l −1 bromocresol purple at pH 6·8 after 7 days
of cultivation (Asai et al. 1963). Water-sol-
uble brown pigment formation on GYC agar
was observed after 10 days of cultivation
(Nanba and Kato 1985). Ketogenesis toward
glycerol, mannitol, or sorbitol was measured
with the following components: 5 g l−1 yeast
extract and 10 g l −1 polypeptone (De Ley et al.
1980). The ability to reduce FeCl3 was tested
after the culture was grown for 14 days in
media composed of 5 g l−1 yeast extract, 5 g l−1
polypeptone, 5 g l−1 CaCO3, and 50 g l−1 glu-
cose or fructose (Nanba and Kato 1985).
Nitrate reduction, catalase activity, growth
on mannitol agar, and Gram stain were also
tested.
Identification of yeasts
Yeast isolates were observed for cell mor-
phology at 25°C on malt extract agar (MEA,
Difco Laboratories) and GYP media (Kreger
van Rij 1984). Formation of asci and ascosp-
ores was checked by growing the cultures at
25°C on corn meal agar (CMA, Difco
Laboratories), YM agar, and MEA for 4
weeks with microscopical observation at

weekly intervals. Formation of mycelia on
Dalmau plate was examined after cultivating
the cells at 25°C on CMA for 7 days (Kreger
van Rij 1984).
Fermentations of carbon sources were
determined by the production of carbon diox-
ide using Durham tubes. The abilities to
assimilate carbon and nitrogen sources were
assessed. The carbon source was D-glucose, D-
galactose, sorbose, D-glucosamine, D-ribose, D-
xylose, L-arabinose, D-arabinose, D-rhamnose,
sucrose, maltose, α-trehalose, α-methyl-D-
glucoside, cellobiose, salicin, arbutin, meli-
biose, lactose, raffinose, melezitose, inulin,
starch, glycerol, erythritol, ribitol, xylitol, L-
arabinitol, D-glucitol, D-mannitol, galactitol,
myo-inositol, D-glucono-1,5-lactone, 2-keto-D-
gluconate, D-gluconate, D-glucuronate, D,L-lac-
tate, succinate, citrate, methanol or ethanol.
The nitrogen source was nitrate, nitrite,
ethylamine, L-lysine or cadaverine. The
abilities to grow at 25, 30, 35, 37, 40, 50, and
60°C on YM agar after 5 days of cultivation
were tested. Growths on vitamin-free and
cycloheximide-containing media were also
examined after 4 weeks of cultivation
(Barnett et al. 1990). The results of carbo-
hydrate fermentation, carbon and nitrogen
compound assimilation, growth on vitamin-
free medium, tolerance of antibiotics, and
urea utilization were analyzed using the
Yeast Identification Program (Barnett et al.
1994) in order to determine the species. The
results were confirmed by morphological
observation.
Influence of autoclaved yeast
suspension and other components on
fermentation of isolates
The influence of glycerol, acetic acid, ethanol
or autoclaved yeast suspension on fermen-
tation of Acetobacter and yeast isolates was
carried out in 500 ml Hinton flasks contain-
ing 200 ml sweetened black tea at 30°C and
shaken at 50 r min−1 for 36 h. Each trial was
in duplicate. To prepare autoclaved yeast cell
suspension, the yeast isolate (Saccharomyces
cerevisiae Y1, Brettanomyces bruxellensis Y2,
or Zygosaccharomyces bailii Y6) was culti-
vated in YM broth at 30°C and shaken at 100
r min−1 for 72 h. Cells were centrifuged and
The microbes and their interactions in Haipao 409
washed three times with water. The cell sus-
pension was then autoclaved twice at 121°C
for 15 min.
Analytical methods
The brew tea was centrifuged at 12 000 r
min−1 for 3 min and analyzed by high−
performance liquid chromatography (HPLC)
(Millipore Co., Miford, MA, USA). Organic
acids and ethanol were separated by Poly-
meric Ion-300 column (Interaction Chroma-
tography Inc., San Jose, CA, USA) with 0·005
N H 2SO4 as eluent at 70°C and a flow rate of
0·4 ml min−1 (Togami et al. 1990). Sub-
sequently, these components were detected
by Waters 410 Refractive Index Detector
(Millipore Co.).
Results and Discussion
Twelve bacterial isolates were obtained from
three Haipao samples using the screening
media NA, TSA and MA. Two representative
strains A1 and A2 were all Gram-negative,
able to overoxidize lactate and acetate as
CO2, and produce acetic acid. Therefore, they
belonged to the genus Acetobacter. Six type
strains of the genus Acetobacter were used as
reference to identify the isolates by biochemi-
cal and physiological tests (Table 1). Isolate
A1 was identical to A. pasteurianus CCRC
14145 in all tests except that A1 could pro-
duce acid from inositol but A. pasteurianus
could not. There was a good match between
isolate A2 and A. aceti CCRC 11688, only two
among the tests having different results. Iso-
late A2 could produce acid from sucrose and
fructose, whereas A. aceti could not (Table 1).
Thus, isolate A1 and A2 might belong to A.
pasteurianus and A. aceti, respectively. The
cellulose-synthesizing strains were formerly
classified as A. aceti subsp. xylinum or A.
pasteurianus subsp. estunensis (De Ley and
Frateur 1974), but in the present Bergey’s
Manual they were attributed to A. pasteur-
ianus (De Ley et al. 1980). A. pasteurianus,
found in all Haipao samples might therefore,
be responsible for the production of cellulose
pellicle.
Fifty-two yeast strains were isolated from
410 C.-H. Liu et al.

three samples of Haipao by YM agar and
MRS. Seven representative yeast isolates
were identified based on the results of physio-
logical assimilation abilities of carbon and
nitrogen compounds (Table 2), biochemical
tests (Table 3), and morphological obser-
vation on GYP media (Table 4). Y1, Y4, and
Y5 were identified as S. cerevisiae, Y2, Y3,
and Y7 as B. bruxellensis, and Y6 as Z. bailii
through the analysis of carbon and nitrogen
compounds assimilation results by Yeast
Identification Program. Y1, Y4, and Y5
grown on CMA were observed to develop
pseudohyphae. Ascospores were also found in
cells grown on CMA, MEA, and YM agar.
These observations further confirmed Y1, Y4,
and Y5 as S. cerevisiae. The cultures of Y2,
Y3, and Y7 on CMA Dalmau plates were
found to have pseudomycelia and filamentous
cells. No ascospore was found when Y2, Y3,
and Y7 were grown on CMA, YM agar, or
MEA for 28 days. The results gave a strong
support for the identification of Y2, Y3, and
Y7 as the imperfect state of Dekkera bruxel-
lensis, i.e. B. bruxellensis. The Dalmau plate
of Y6 on YM agar consisted of chains of cylin-
drical cells. Smooth and globose ascospores
were found for Y6 grown on MEA, YM agar,
and CMA. According to the data of mycelia,
ascospores, and physiological tests, Y6 was
identified as Z. bailii. S. cerevisiae is one of
the most common organisms isolated in tra-

Table 1. Characteristics of Gluconobacter, Acetobacter and bacterial isolates of Haipaoa

Strainb
Type Type Type Type Type Type A1 A2
strain strain strain strain strain strain
1 2 3 4 5 6
Oxidation of ethanolc + + + + + + + +
Acid produced from
Ethanol + + + + + + + +
Sucrose − − − + + + − +
Mannitol − − − + − + − −
Arabinose − − − − + + − −
Inositol − − − − − + + −
Fructose − − − + + + − +
Erythritol + + − − − + − +
Glycerol + + − + − + − +
Maltose − − − + − + − −
Ribose − + − − − + − −
Xylose + + + − + + + +

Pigment synthesis on − + − − − − − −
GYC media

Ketogenesis from
Glycerol + + − − − + − +
Sorbitol − + − − − − − −
Mannitol + + − − − + − +

Ferric chloride reaction

in:
Glucose − + − − − − − −
Fructose + + + + + + + +
Nitrate reduction − − + + − − + −
Gram stain − − − − − − − −
Growth on MA + + + + + + + +
Catalase + + + + + + + +
a
+, positive; −, negative.
b
Type strain 1: A. aceti; 2: A. liquefaciens; 3: A. pasteurianus; 4: A. hansenii; 5: A. xylinum; 6: G. oxydans.
A1 and A2 are bacterial isolates of Haipao.
c
Growth but without gas production.
 The microbes and their interactions in Haipao 411

ditional alcoholic beverages and B. bruxel- special flavors. Z. bailii is present in many
lensis was reported by Kumara (1991) to par- foods and beverages, particularly those with
ticipate in the spontaneous fermentation of low pH and high sugar content. It could also
Belgian lambic beer with the production of produce acetic acid and tolerate 20 g l −1 acetic
acid (Thomas and Davenport 1985). It is,
therefore, reasonable to expect to isolate
Table 2. Assimilation of carbon and nitrogen these yeasts in the Haipao fermentation con-
compounds by the yeast isolates
dition. In Haipao samples of Japan, S. cerevi-
Strain siae, Saccharomyces inoconspicus, Candida
Y1 Y2 Y3 Y4 Y5 Y6 Y7 tropicalis, Candida guilliermondii, Debar-
yomyces hansenii, Torulopsis famata, and
Carbon compound
D-Glucose + + + + + + + Pichia membranefacience are isolated
D-Galactose + + + + − − + (Kozaki et al. 1972, Hitokoto et al. 1978).
Sorbose − − − − − + − These isolates are significantly different from
D-Glucosamine − − + − − − + microbes of Taiwan Haipao samples.
D-Ribose − − − − − − + The composition of traditional fermented
D-Xylose − − − − − − −
L-Arabinose − − − − − − − Haipao produced in the laboratory was ana-
D-Arabinose − − − − − − − lyzed by HPLC. Glycerol, acetic acid, and
D-Rhamnose − − − − − − −
Sucrose − − − − − − −
Maltose − + + + + + + Table 3. Physiological and biochemical
α,α-Trehalose − + + + + − + properties of the yeast isolates
α-Methyl-D-glucoside − + + + − − +
Cellobiose − − + − − − + Strain
Salicin − − + − − − + Y1 Y2 Y3 Y4 Y5 Y6 Y7
Arbutin − − + − − − +
Melibiose − − − + + − − Fermentation test
Lactose − − − − − − − D-Glucose + + + + + + +
Raffinose + − − + + − − D-Galactose + − + + − − −
Melezitose − + + − − − + Maltose + + + + + − +
Inulin − − − − − − − α-Methyl-D-glucoside − + + + − − +
Starch − − − − − − − Sucrose + + + + + − +
Glycerol − − − − − + + α,α-Trehalose − + + − − − +
Erythritol − − − − − − − Melibiose − − − + w − −
Ribitol − − − − − + − Lactose − − − − − − −
Xylitol − − − − − + − Cellobiose − − + − − − +
L-Arabinitol − − − − − − − Melezitose − − + − − − +
D-Glucitol − − − − − + − Raffinose + + − + + − w
D-Mannitol − − − − − + − Inulin − − − − − − −
Galactitol − − − − − − − Starch − − − − − − −
myo-Inositol − − − − − − − D-Xylose − − − − − − −
D-Glucono-1,5-lactone − + + + − + +
2-Keto-D-gluconate − − − − − + − Vitamin-free growth + − − + + − −
D-Gluconate − − − − − − − Growth at elevated
D-Glucuronate − − − − − − − temperature
D,L-Lactate − − − − − − − 25°C + + + + + + +
Succinate − − − − − − − 30°C + + + + + + +
Citrate − − − − − − − 35°C + + + + + + +
Methanol − − − − − − − 37°C + w w + + w +
Ethanol + + + + + + + 42°C − − − w − − −
50°C − − − − − − −
Nitrogen compound 60°C − − − − − − −
Nitrate − + + − − − +
Nitrite − + + − − − + Urea utilization − − − − − − −
Ethylamine − + + − − + + Resistance to antibiotics
L-lysine − + + − − + + 0·01% Cycloheximide − + + − − − +
Cadaverine − + + − − + + 0·1% Cycloheximide − + + − − − +
+, positive; −, negative. +, positive; −, negative; w, weakly positive.
412 C.-H. Liu et al.

ethanol were found in the brew tea (Fig. 1). 9000

The pH of the brew tea dropped rapidly from 8000
6·6 to 4·2 in the first 24 h, and then to 3·5
after 6 days of cultivation. The concentration 7000

Concentration (ppm)
of glycerol was significantly lower than those 6000
of acetate and ethanol. Although glycerol
5000
does not influence the aroma, it does contrib-
ute to the smoothness and viscosity of a drink 4000
(Gardner et al. 1993). The dominant yeast 3000
during the fermentation was Z. bailii, which
was about 1·7×107 cfu ml−1 after 48 h of fer- 2000
mentation. A. pasteurianus was the domi- 1000
nant bacterium and the cells reached about
5·5×106 cfu ml−1 after 2 days of fermentation. 0 20 40 60 80 100 120 140
The cell concentrations of yeast and bac- Time (h)
terium remained stable during the fermen-
tation from 3–6 days. Figure 1. Time course of composition during
The interactions between Saccharomyces static cultivation of Haipao. (s) glycerol; (n) acetic
and lactic acid bacteria during fermentation acid; (h) ethanol.
of sourdough (Gobbetti et al. 1994), sugary
kefir (Leroi and Pidoux 1993), and molasses on the microbes isolated from Haipao was
worts (Essia Ngang et al. 1992) have been evaluated.
investigated. The effect of glycerol, acetic The cell concentration of A. aceti A1
acid, ethanol or autoclaved yeast suspension decreased in the mixture of sucrose and black

Table 4. The morphology of yeast isolates grown on GYPa media for 3 days
Strain Characteristics
Size (µm) Reproduction Shape Occurrence Ascospore shape
Y1 (3·2–7·2)×(3·2–8·8) Multilaterial Cylindrical Single, in pair Round
budding ellipsoidal, globose

Y2 (2·4–4·8)×(4·0–6·4) Multilaterial Cylindrical Single, cluster −b

budding ellipsoidal

Y3 (2·4–4·0)×(4·0–3·6) Multilaterial Cylindrical, Single, in pair −b

budding ellipsoidal,
ovoid, elongate,
filamentous

Y4 (3·2–6·4)×(3·2–9·6) Multilaterial Ellipsoidal, Single, in pair Round

budding ovoid, globose

Y5 (3·2–8·0)×(4–10·4) Multilaterial Cylindrical, Single, in pair Round

budding Ellipsoidal,
globose, ovoid

Y6 (3·2–5·6)×(4·8–9·6) Multilaterial Ellipsoidal, Single, in pair Round

budding ovoid, globose

Y7 (2·4–4·8)×(4·8–8·0) Multilaterial Cylindrical, Single, in pair −b

budding ellipsoidal
ovoid, globose
a
GYP media contained 0·5% glucose, 0·5% peptone, and 0·3% yeast extract.
b
No ascospore.
 The microbes and their interactions in Haipao 413

tea after 36 h of cultivation (see control in
Table 5). Leisinger and Muller (1967) have
reported that phosphofructokinase is absent
in Acetobacter, rendering glycolysis by Aceto-
bacter either absent or very weak. Further-
more, A. aceti A1 could not use sucrose to pro-
duce acid (Table 1). It is thus exposed to
starvation and osmotic stress conditions in
the solution of 10% sucrose black tea broth. It
has been reported that starvation and
osmotic stress may decrease the viability and
culturability of Pseudomonas fluorescence
(Jorgensen et al. 1994). Chemielewski and
Frank (1995) reported that formation of
viable but nonculturable Salmonella during
starvation in chemically defined solutions.
These might explain why the cell concen-
tration of A. aceti A1 estimated by plate count
method would decrease after 36 h of culti-
vation in sweetened black tea broth. The
addition of three types of autoclaved yeast
suspensions could maintain the growths of
Acetobacter A1 and A2, even though they
could not induce Acetobacter to produce acetic
acid. The results are different from that of
Czuba (1989) who reported yeast autolysate
could promote the acetate production of Ace-
tobacter. Ethanol could be used by Acetob-
acter to produce acetic acid and grow. As
shown in Table 6, acetic acid could stimulate
the yeast isolates to produce ethanol. The
alcohol production of B. bruxellensis is lower
than those of S. cerevisiae and Z. bailii, but
significant amount of acetate was formed by
B. bruxellensis after 36 h of cultivation.
Because B. bruxellensis did not produce gly-

Table 5. The effects of 2·5% (v/v) autoclaved yeast suspension (AYS) or 1000 ppm ethanol on brew tea
fermented by A. aceti A1 or A. pasteurianus A2 for 36 h
Concentration (ppm)c Cell concentration (cfu ml−1)c
Glycerol Acetate Ethanol
a
A. aceti A1
AYS of S. cerevisiae 0 0 0 1·1×109
Y1 added

AYS of B. bruxellensis 0 0 0 1·5×109

Y2 added

AYS of Z. bailii Y6 0 0 0 1·5×109

added

1000 ppm ethanol 0 521 65 2·5×108

added

Control 0 0 0 7·0×106

A. pasteurianus A2b
AYS of S. cerevisiae 0 0 0 7·5×108
Y1 added

AYS of B. bruxellensis 0 0 0 1·1×109

Y2 added

AYS of Z. bailii Y3 0 0 0 9·0×108

added

1000 ppm ethanol 0 839 409 2·1×109

added

Control 0 74 86 1·3×108
a
The initial cell concentration was 2·0×109 cfu ml−1.
b
The initial cell concentration was 2·4×109 cfu ml−1.
c
Each data was the average from duplicate experiments.
414 C.-H. Liu et al.

Table 6. The effects of 1000 ppm of glycerol, acetate, or ethanol on brew tea fermented by S.
cerevisiae Y1, B. bruxellensis Y2, and Z. bailii Y6 for 36 h
Concentration (ppm) d Cell concentration (cfu ml−1)d
Glycerol Acetate Ethanol
a
S. cerevisiae Y1
Glycerol added 1641 230 2045 2·3×109
Acetate added 244 940 3836 2·0×109
Ethanol added 287 178 2990 2·3×109
Control 290 203 2162 2·3×109

B. bruxellensis Y2b
Glycerol added 1301 512 442 6·6×109
Acetate added 0 1752 641 7·4×109
Ethanol added 0 794 1160 7·0×109
Control 0 563 434 6·5×109

Z. bailii Y6c
Glycerol added 1646 119 3146 2·5×109
Acetate added 405 765 5539 2·8×109
Ethanol added 389 79 4116 2·4×109
Control 427 142 3491 2·0×109
a
The initial cell concentration was 1·8×106 cfu ml−1.
b
The initial cell concentration was 1·5×106 cfu ml−1.
c
The initial cell concentration was 3·6×105 cfu ml−1.
d
Each data was the average from duplicate experiments.

cerol, glycerol in Haipao might be contributed
by S. cerevisiae and Z. bailii. In the fermen-
tation of Haipao, it was found that the auto-
claved yeast cells and ethanol produced by
yeasts were helpful for Acetobacter to grow or
produce acetic acid. The acetic acid produced
by Acetobacter could stimulate the yeast to
produce ethanol. Acetic acid and ethanol
were reported to have antimicrobial activities
(Kurita and Koike 1983, Adams and Hall
1988, Matsuda et al. 1994). The inhibition of
pathogenic bacteria such as Salmonella
typhi, Shigella sonnei, Escherichie coli, and
Staphylococcus aureus when incubated with
Haipao was demonstrated by Hitokoto et al.
(1978). The stable symbiosis between Acetob-
acter and these yeasts could be explained by
these facts and is why Haipao can be so
widely distributed without changing its
microbial population dramatically and the
contamination of other micro-organisms is
very rare.
Acknowledgements
We are grateful to Dr Chu, Wen-Shen for
his critical reading of this manuscript. This
work was supported by grant (FIRDI-133-
p104(84)) from the Ministry of Economic
Affairs of the Republic of China.
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