Blackwell Publishing AsiaMelbourne, AustraliaFISFisheries Science0919 92682005 Blackwell Science Asia Pty LtdApril 2006722310321Original ArticleEffects of probiotics on Japanese

flounder
Y Taoka
et al.

FISHERIES SCIENCE 2006; 72: 310–321

Growth, stress tolerance and non-specific immune

response of Japanese flounder Paralichthys olivaceus to
probiotics in a closed recirculating system
Yousuke TAOKA,1 Hiroto MAEDA,2a* Jae-Yoon JO,3 Min-Jee JEON,3 Sungchul C. BAI,3
Won-Jae LEE,4 Kazuya YUGE5 AND Shunsuke KOSHIO6

1
The United Graduate School of Agricultural Sciences, Faculty of Fisheries, Kagoshima University, Kagoshima
890-0065, 2Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056,
Japan, 3Department of Aquaculture, Pukyong National University, Busan 599-1, 4Department of
Microbiology, Pukyong National University, Busan 599-1, Korea, 5DSM Nutrition Japan KK,
Tokyo 143-0016, and 6Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries,
Kagoshima University, Kagoshima 890-0056, Japan

ABSTRACT: Effects of probiotics on growth, stress tolerance and non-specific immune response in
Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival
and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet
group), or into the rearing water (the water supply group), were higher compared to the untreated
group (the control group). Water quality parameters, pH, NH4-N, NO2-N and PO4-P showed lower con-
centration in the probiotic diet group compared with the control group and the supply group. Plasma
lysozyme activity in the probiotic diet group and the water supply group was significantly higher
(P < 0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated
groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group.
Pathogen challenge tests with Vibrio anguillarum (2 × 107 c.f.u./mL) resulted in significantly higher
survival in the probiotics-treated groups than the control group. Results indicated that probiotics sup-
plied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific
immune system of Japanese flounder, providing them a higher resistance against stress conditions
and pathogens.

KEY WORDS: closed recirculating system, flounder, lysozyme, non-specific immune

response, probiotics, water quality.

INTRODUCTION Antibiotics had been frequently used to cure

bacterial infection and prevent fish mortality in
In intensive aquaculture, growth and proliferation aquaculture systems. However, the use of antibiot-
of microorganisms in the rearing habitat are accel- ics has become limited because certain pathogens
erated by excessive feeding.1 Successful aquacul- have developed resistance to the drugs. This out-
ture relies on adequate microbial control to come gave rise to the use of probiotics as method
prevent the proliferation of pathogenic bacteria for preventing fish disease and in improving
that often results in fish mortality. Bacteria can aquaculture conditions and fish health. Generally,
accumulate in fish by ingestion or through the probiotics are described as live microbial feed
body surfaces and is transported by the body fluids additives that improve the microbial condition of
into the tissues. This may be one of the causes of the host animal’s gastrointestinal tract.2 Probiotics
indigenous microflora formation and bacterial include components of microbial cells that stimu-
infection in fish. late the immune system against pathogens.3 Such
immunostimulants enhance the defense system of
host organisms against pathogens by enhance-
*Corresponding author: Tel: 81-59-231-9566.
Fax: 81-59-231-9557. Email: maeda@bio.mie-u.ac.jp
ment of phagocytosis, antibody production,
a
Present address: 1577 Kurimamachiya, Tsu, Mie, 514-8507 chemiluminescent response and superoxide anion
JAPAN production.4 Probiotics are inoculated into the
Received 22 November 2004. Accepted 24 October 2005. rearing water to improve culture conditions or
Effects of probiotics on Japanese flounder FISHERIES SCIENCE 311

incorporated in the food of fish through diet.5–7 Table 1 Compositions and proximate analyses of
Probiotics composed of lactic acid bacteria are experimental diets
widely used to improve the health of humans and Diets
livestock.8 In black tiger shrimp, scallop, flounder,
Ingredients (%) Control Probiotic diet
Atlantic salmon, rainbow trout and turbot, the
growth and survival plus water and sediment qual- Fish meal 54.9 54.9
ity of the farm environment were improved, and Probiotics – 1.0
the microbial flora in fish intestine, rearing water Soy bean meal 20.0 20.0
and feces were stabilized by addition of probiot- α-starch 3.4 3.4
ics.8–13 The influence of microbial flora from the Fish oil 5.2 5.2
Soy bean lecithin 3.2 3.2
rearing water on the gastrointestinal flora of the
Corn oil 2.1 2.1
cultured animal is widely recognized.14 Vitamin mix.† 3.0 3.0
Flounder is one of the most highly valued species Mineral mix.‡ 5.0 5.0
in Japanese and Korean aquaculture and its culture Taurine 0.6 0.6
has increased in scale. The fish, however, have IMP 0.1 0.1
been affected by vibriosis caused by Vibrio anguil- Betain 0.6 0.6
larum.15 The bacteria act as an opportunistic Alanine 0.4 0.4
pathogen in various juvenile fishes. However, the α-cellulose 1.0 –
application of probiotics in flounder is still not CMC1 0.5 0.5
enough to produce healthier fish and improve the Total 100 100
immune defense system. Therefore, this study was
conducted to investigate the effects of probiotics Approximate composition
(% wet weight) Pooled SEM2
on growth performance, stress tolerance, non-
specific immune response and the most effective Moisture 11.3 ± 0.05 13.6 ± 0.31 1.35
method of application of probiotics in the culture Protein 50.6 ± 0.09 46.5 ± 0.18 2.37
of Japanese flounder Paralichthys olivaceus. In Fat 12.5 ± 0.90 13.5 ± 0.06 0.50
addition, experiments were conducted to investi- Ash 14.5 ± 0.05 14.1 ± 0.46 0.27
gate if probiotic treatment would enhance toler-
Values are means ± standard error (n = 3).
ance to stress and resistance to pathogens. †
menadione, 3.17; DL-α-tochophenol acetate, 26.67; thiamin-
HCl, 4.0; riboflavin, 13.33; pyridoxine-HC, 3.17; biotin, 0.33;
inositol, 267.0; nicotinic acid, 53.33; Ca panthothenate, 18.67;
folic acid, 1.0; choline chloride, 545.83; vitamin A-palmitate,
MATERIALS AND METHODS 12.83; calcifenol, 0.67; ascorbyl-2-phosphate-Mg, 4.67;
cellulose, 45.33 (g/kg premix).
‡
Experimental animals NaCl, 36.76; MgSO4·7H2O, 137.0; NaHPO4·2H2O, 77.06;
KH2PO4, 239.80; Ca(H2PO4)·2H2O, 6.34; Fe citrate, 29.70; Ca
lactate, 461.80; Al (OH)3, 0.56; ZnSO4·7H2O, 3.58; CuSO4, 0.14;
One thousand Japanese flounder with a mean MnSO4·7H2O, 0.696; Ca(IO3)2, 0.16; CoSO4·7H2O, 2.17; cellulose,
weight of 15 g were obtained from Uljin Marine 4.234 (g/kg premix).
1
Hatchery in Korea and transported to the Depart- CMC; carboxymethyl cellulose.
2
ment of Aquaculture, Pukyong National University. SEM; standard error of mean.
The fish population was conditioned in aerated
1 m3 tanks at 20–22°C for 1 month prior to the
experiment. Two weeks before the start of the
experiment, the fish were transferred into a closed
control diet and reared in water without probiotics;
recirculating system at a density of 20 individuals
(ii) a probiotic diet group fed with a diet containing
per aquarium. During the conditioning period, the
1% probiotics and reared in water without probiot-
fish were fed with the control diet twice daily at 1%
ics; and (iii) a water supply group fed with the con-
of body weight (Table 1).
trol diet and reared in water with probiotics. Each
The recirculating system was equipped with four
group was raised in separate recirculating systems
120-L aquaria and one 450-L filter tank treated
to avoid inter-treatment contamination.
with NH4Cl to accelerate nitrification.
The composition of the experimental diets is
presented in Table 1. The commercial probiotics
(Alchem Poseidon; Alchem- Korea CO., LDT,
Experimental design Wonju, Korea) contained Bacillus subtilis
(>1.6 × 107 c.f.u./g), Lactobacillus acidophilus
Three treatments with three simultaneous replica- (>1.2 × 108 c.f.u./g), Clostridium butyricum (>2.0
tions were used: (i) a control group fed with the 107 c.f.u./g) and Saccharomyces cerevisiae
312 FISHERIES SCIENCE
(>1.6 × 107 c.f.u./g). The probiotics were added in
corn oil at 40°C and mixed with the other ingredi-
ents at a proportion of 1% using a food mixer.
Water was added at 40% to dry weight of the mix-
ture to facilitate pelleting by a meat chopper. After
pelleting, diets were dried with an electric fan at
room temperature. In the water supply group, 10 g
of the probiotics suspended in 100 mL rearing sea
water and aerated vigorously for 12 h was intro-
duced into the rearing system every 10 days.
The experimental fish were fed twice daily at 1%
of body weight and reared for 50 days at water tem-
perature of 17 ± 2°C and salinity of 33–35 psu.
Shortly before feeding, the hands of feeder were
sterilized with 70% ethanol to prevent contamina-
tion of the control diet with the probiotics. Excess
food and feces were removed a few hours after
feeding.
Sample collection
During the 50 days of feeding, the body weight of
fish was collectively measured every 10 days. At
50 days of rearing, moisture attached to body sur-
face of sampled fish was removed in a net and the
fish was quickly placed in a preweighed small ves-
sel with seawater to record the body weight and
the body length was measured. Livers were dis-
sected out from six fish in each group, and
weighed to calculate the hepatosomatic index
(HSI). Growth performance indicators; survival
rate, Fulton’s condition factors, specific weight
gain (SWG) and specific growth rate (SGR) were
calculated. At the end of rearing trial, three fish
were sampled from each recirculating system per
treatment group for approximate analysis of fish
body compositions, and the data from the three
fish were pooled.
Blood samples from four fish in each group were
obtained from the caudal vein by using a heparin-
ized syringe. Each blood sample was centrifuged at
5000 × g for 10 min. The supernatant was then
taken as a plasma sample and stored at −20°C until
analysis.
At the end of experiment, skin mucus of four fish
in each group was sampled according to Aranishi
and Nakane.16 Fish were anesthetized with 0.5%
(w/v) MS-222 (ethyl-3-aminobenzoate methane-
sulforic acid salt) solution for 20 min then
immersed in 10 mM sodium phosphate buffer
(PBS, pH 7.5) containing 115 mM NaCl for 1 min.
Skin mucus was collected by wiping with polyeth-
ylene gloves. The sample was homogenized in four
volumes of PBS and centrifuged at 15 000 × g for
30 min at 4°C. The supernatant was stored at −20°C
until analysis.
Y Taoka et al.
Analytical methods
Water quality
Rearing water samples (1 L) were collected every
5 days with a polyethylene bottle, and then stored
in a freezer at −20°C until analysis. At the time of
sampling, pH, dissolved oxygen (DO), salinity and
water temperature were checked by using a port-
able DO meter (KDO5151, Kasahara Chemical
Instrument, Saitama, Japan) and a portable pH
meter (Pinpoint pH meter, American Marine INC,
Ridgefield, USA). Urea-nitrogen was analyzed by
the method of Newell et al.17 NH4-N, NO2-N and
PO4-P were analyzed by the method of Strickland
and Parsons.18 NO3-N was analyzed with a DREL
2000 (HACH company, Loveland, Colorado, USA).
Chemical oxygen demand (COD) was analyzed by
potassium permanganate–iodine titration.
Proximate analyses of the feed and carcass
Proximate analyses of the whole body of flounder
and the test diets were carried out. Crude protein,
ash content and moisture were analyzed according
to the method of the Association of Official Analyt-
ical Chemists.19 Crude fat was analyzed by using
the Soxtec system 1046 (Foss-Tecator, Höganäs,
Sweden) after freeze-drying the sample.
Monitoring of non-specific immune responses
Protein concentrations in the plasma and skin
mucus were determined according to Lowly et al.20
Lysozyme activity of fish plasma and skin mucus
were determined by turbidometric assays.21,22
Plasma (100 µL) was added to 900 µL of Micrococ-
cus lysodeikticus (lyophilized cell, Sigma-ALDRICH
Chemical company, St. Louis, Mo, USA) aqueous
solution and incubated at 25°C. The absorbance
was read at 530 nm with a spectrophotometer
(OPRON-3000, Hanson Technology Co., Ltd, Korea)
at 0.5 and 4.5 min after reaction. Fish mucus
(0.5 mL) was added to 2.5 mL of M. lysodeikticus
aqueous solution and incubated at 25°C. The
absorbance was read and recorded at 530 nm with
a spectrophotometer 20 min after reaction. The
M. lysodeikticus aqueous solution was prepared by
adding M. lysodeikticus to 50 mM PBS (for plasma
sample) and 5 mM (for mucus sample), and was
adjusted to the absorbance of 0.7 and 0.6 at
530 nm, respectively. PBS and all apparatus used
for lysozyme activity assay were autoclaved for
15 min at 121°C The unit of lysozyme activity (U)
was defined as the amount of enzyme that caused a
decrease in absorption of 0.001/mg protein.
Effects of probiotics on Japanese flounder FISHERIES SCIENCE
Stress tolerance tests
After feeding for 50 days, flounder in each treat-
ment were subjected to heat shock stress tests. Six
flounder were transferred from each rearing sys-
tem to individual test glass aquaria with a volume
of 30 L. Water temperature was maintained at 30°C
with a thermostatically controlled glass heater,
based on preliminary experiments. The test was
conducted until half of the test population died.
The number of surviving fish was recorded and the
50% lethal time (LT50) was calculated. The blood
from the caudal vein of surviving fish was collected
with a heparinized syringe and plasma sample was
stored at −20°C until analysis. All measurements
were conducted in duplicate.
The tolerance to exposure to air in each treat-
ment was also examined according to the method
of Koshio et al.23 A net with small mesh was spread
out on a plastic container, with a slight sag. Five
flounder from each group were placed in the net
for 105 min. Before exposure to air, moisture on the
body surface of the flounder was removed using
a napkin. After exposure to air, the flounder
were immediately returned to sea water. Survival
and upright curvature or stiffness of fish body
was recorded. The air dive test was conducted in
duplicate.
Pathogen challenge test
The flounder in each treatment were also chal-
lenged with the pathogenic bacteria Vibrio anguil-
larum which had been cultured and maintained
using PPESII medium consisting of proteose pep-
tone (1 g/L), polypeptone (2 g/L), bacto soytone
(1 g/L), bacto yeast extract (1 g/L) and ferric citrate
(0.1 g/L), adjusted to pH 7.8. Seven flounder
were immersed for 60 min in a suspension of
V. anguillarum at 2 × 107 c.f.u./mL according to
Austin et al.24 Before and after immersion, a blood
sample was obtained from the caudal vein by a
heparinized syringe. The flounder were reared for
Table 2 Whole body approximate composition of P. olivaceus after 50 days of culture
Control
Moisture 78.5 ± 0.01 a
Protein 16.8 ± 0.05a
Fat 2.36 ± 0.01c
Ash 4.1 ± 0.25a
Values are means ± standard error (n = 3). The same superscripts indicates not statistically different (P > 0.05, Duncan’s multiple
range test).
313
14 days and fed with the control and the probiotic
diets. In the water supply group, probiotics were
added to the rearing water in the same proportion
as the rearing experiment. Fish survival rate was
monitored every 2 days. After 14 days, all surviving
fish from each aquarium were sampled for plasma
and fish skin mucus analyses. The challenge test
was carried out in triplicate.
Analysis of data
All experimental data were analyzed using one
way-analysis of variance (ANOVA) followed by
Duncan’s multiple range test (SPSS Version. 10.0
software, SPSS, Inc, Chicago, IL). The change of the
lysozyme activity and the plasma protein concen-
tration in heat shock stress test were subjected to a
Student’s t-test to check the differences.
RESULTS
Rearing experiment
The results of approximate analysis of whole body
flounder are presented in Table 2. No significant
differences were observed in the moisture, protein
and ash contents of the fish between treatment
groups (P > 0.05). The fat content, however, was
significantly lower in the probiotic diet group than
the control and the water supply groups (P < 0.05).
The control group had the highest fat content
among the three treatments (P < 0.05).
There were no observable differences in water
quality among groups. Water temperature ranged
14–19°C. During 50 days of culture, pH in the water
supply group was significantly lower than the con-
trol and probiotic diet groups. NH4-N in the control
group was significantly higher compared to the
probiotic diet group. In NO2-N, the probiotic diet
group was significantly lower than the control and
the water-supply groups. PO4-P in the probiotic
diet group was significantly lower than the control
Treatment groups
Probiotic diet Water supply Pooled SEM
79.1 ± 0.61 a
79.1 ± 0.13 a
0.41
16.1 ± 0.79a 16.5 ± 0.01a 0.46
1.16 ± 0.07a 1.83 ± 0.06b 0.54
4.1 ± 0.79a 4.0 ± 0.20a 0.41
314 FISHERIES SCIENCE Y Taoka et al.

(a) Urea-N (b) NH4-N

0.5 1.4
1.2
0.4
1
0.3

mg/L
mg/L

0.8
0.2 0.6
0.4
0.1
0.2
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Days Days

(c) NO2-N (d) NO3-N

0.5
30
0.4
24
0.3
mg/L

mg/L

18
0.2
12
0.1 6
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Days Days

(e) PO4-N (f) COD

24 8

20
6
16
mg/mL

mg/L

12 4
8
2
4
0 0
0 10 20 40 50 0 10 20 30 40 50

Days Days

(g) pH
8
7.8 Fig. 1 Change of water quality
7.6 parameters in a closed recirculat-
ing system after 50 days of cul-
pH

7.4
ture. (a) urea-N, (b) NH4-N, (c)
7.2 NO2-N, (d) NO3-N, (e) PO4-N, (f)
7 chemical oxygen demand (COD),
6.8
(g) pH. (
) control, (), probiotic
diet, () water supply. Error bars
0 10 20 30 40 50
represent mean ± standard devia-
Days tion (n = 3).

group. There was no significant difference in NO3-
N and COD among groups (Fig. 1, Table 3).
Addition of probiotics in the rearing water pro-
duced the best growth of flounder. Average body
weights and total length in the water supply group
were significantly greater than in the other two
groups (Fig. 2, Table 4). The probiotics-treated
groups showed significantly greater survival rate as
compared to the control group at the end of rearing
experiment (Fig. 2). Although there was no signifi-
cant difference in HSI and condition factor among
groups, HSI in the probiotics-treated groups was
higher than the control group, particularly the pro-
biotic diet group (Table 4). SGR values in the water
Effects of probiotics on Japanese flounder FISHERIES SCIENCE 315

Table 3 Water quality parameters of rearing water during 50 days of culture

Treatment groups
Control Probiotic diet Water supply Pooled SD‡ Pooled SE
pH 7.7 ± 0.2a
7.6 ± 0.2 a
7.4 ± 0.3b
0.24 0.02
NH4-N 0.24 ± 0.22a 0.12 ± 0.10b 0.15 ± 0.25ab 0.21 0.02
NO2-N 0.15 ± 0.08b 0.08 ± 0.08a 0.14 ± 0.16b 0.11 0.01
NO3-N 13.7 ± 6.4a 16.4 ± 7.9a 14.7 ± 8.1a 7.51 0.78
PO4-P 13.0 ± 3.9b 10.2 ± 3.0a 11.7 ± 4.6ab 1.72 0.18
COD† 3.5 ± 1.7a 3.7 ± 1.9a 3.2 ± 1.6a 3.98 0.44

Values are means ± standard deviation (n = 11). Different superscripts indicated statistically different (P < 0.05, Duncan’s multiple
range test).
†
COD, chemical oxygen demand.
‡
SD, standard deviation; SE, standard error.

(a) supply group were significantly higher than the

33 other two groups. SWG and WG in the water supply
group were significantly higher than the control
30 and the probiotic diet groups, and the probiotic
diet group also showed a significantly higher value
Average body weight (g/fish)

than the control group.

27
Skin mucus protein concentration in the control
group was significantly higher than in the water
24 supply group (Fig. 3a). Plasma protein at 30 and
50 days increased compared to those at 0 days in
21 all groups. At 30 days, plasma protein in the probi-
otic diet group was significantly higher than that in
18 the control. Finally, the plasma protein concentra-
tion was not significantly different between treat-
15 ment groups (Fig. 3b).
0 10 20 30 40 50 Skin mucus lysozyme activity in the probiotic
Days diet group was significantly higher than the activity
observed in the control group and the water supply
(b) Control Probiotic diet Water supply group (Fig. 3c). Plasma lysozyme activity was sim-
100 ilar among groups (0.5 min incubation). At 4.5 min
incubation, however, the water supply group
showed significantly greater activity than the con-
80
trol group. The probiotics-treated groups in gen-
eral showed a higher trend compared to the control
Survival rate (%)

60 group (Fig. 3d).

40
Tolerance to stress

20 LT50 was not significantly different between

groups, although the probiotics-treated groups
showed slightly higher tolerance than the control
0 group (Fig. 4). Plasma lysozyme activity (0.5 min
0 10 20 30 40 50
incubation) significantly decreased in the control
Days
group and the probiotic diet group. The probiotic
Fig. 2 Experiment holding P. olivaceus for 50 days in a diet group showed significantly higher activity
closed recirculating system. (a) average body weight, (b) compared to the control group, but the water sup-
survival. Error bars represent mean ± standard deviation ply group showed significantly higher activity com-
(n = 3). pared to the probiotic diet group. Plasma lysozyme
316 FISHERIES SCIENCE Y Taoka et al.

Table 4 Growth and feeding performance in P. olivaceus after 50 days of culture

Treatment groups
Control Probiotic diet Water supply ± SEM†
0 day
Initial mean weight (g) 16.6 ± 2 16.6 ± 2 16.6 ± 2
Initial mean length (cm) 13.0 ± 0.6 13.0 ± 0.6 13.0 ± 0.6
Condition factor‡ 0.75 ± 0.03 0.75 ± 0.03 0.75 ± 0.03
HIS (%)§ 1.24 ± 0.19 1.24 ± 0.19 1.24 ± 0.19
50 days
Mean weight (g)§§ 28.1 ± 0.61a 29.2 ± 0.61a 32.1 ± 0.5b 1.97
Mean length (cm)¶¶ 15.1 ± 1.25a 15.2 ± 0.94a 16.1 ± 1.44b 1.97
WG¶ at individual (g) 12.7 ± 0.6a 13.7 ± 0.5b 16.3 ± 0.1c 1.64
SWG (%)†† 80.0 ± 1.3a 87.9 ± 2.1b 107.3 ± 1.9c 12.30
SGR (%)‡‡ 0.52 ± 0.03a 0.55 ± 0.01a 0.63 ± 0.01b 0.05
Condition factor 0.96 ± 0.03a 0.86 ± 0.07a 0.85 ± 0.01a 0.05
HIS (%) 1.96 ± 0.05a 3.39 ± 1.40a 2.36 ± 0.76a 0.94

Values with the different superscript in the same row are significantly different (P < 0.05).
†
Standard error, calculated from the mean square error of the ANOVA.
‡
Condition factor = (body weight/body length3) × 100 (n = 6).
§
HIS (%) = liver weight/body weight (n = 6).
¶
Weight gain, WG (g) = (Final body weight – initial body weight)/n (n = 3).
††
Specific weight gain, SWG = (WG/initial weight) × 100 (n = 3).
‡‡
Specific growth ratio, SGR (%) = [(In final body weight – In initial body weight)/time in days] × 100 (n = 3).
§§
n = 3.
¶¶
n = 10.

(b)
(a)
50
b
15 a
Plasma protein (mg/L)
Mucus protein (mg/L)

40 a
12 ab a a a a
b 30
9

6 20

3 10

0 0
Control Probiotic diet Water Control Probiotic diet Water
supply supply

(d) Fig. 3 Protein concentration

(c)
700 and lysozyme activity in plasma
b ab b and skin mucus. (a) skin mucus
300 600
a protein, (b) plasma protein on
Lysozyme activity

a
(unit/mg-protein)

250 500 day 0 (), day 30 () and day 50

Lysozyme activity

a
(unit/mg-protein)

a a ( ), (c) skin mucus lysozyme

200 400
a activity, (d) plasma lysozyme
150 300 activity at incubation time of
100
0.5 min () and 4.5 min (). Error
200
bars represent mean ± standard
50 100 deviation. Bars denoted by the
same letter are not significantly
0 0
Control Probiotic diet Water Control Probiotic diet Water different (P > 0.05, Duncan’s mul-
supply supply tiple range test) (n = 4).
Effects of probiotics on Japanese flounder FISHERIES SCIENCE 317

Table 5 Lysozyme activity and plasma protein concentration of P. olivaceus after heat shock
Control Probiotic diet Water supply
Responses Before After Before After Before After
Lysozyme activity (units/mg protein)
0.5 min incubation 354 ± 31 117 ± 22*a 413 ± 49 297 ± 12*b 405 ± 37 400 ± 0c
4.5 min incubation 429 ± 67 443 ± 3a 518 ± 84 406 ± 20a 527 ± 59 557 ± 13b
Plasma protein (mg/mL) 28.92 ± 1.67 28.42 ± 0.84a 30.09 ± 1.67 35.12 ± 0.10*b 28.80 ± 1.88 27.65 ± 3.08a

The test was conducted twice and value are mean ± standard deviation (n = 4).
*the value is significantly different compared to that before heat shock (P < 0.05). Values with the same letters are not significantly
different (P > 0.05, Duncan’s multiple range test).

70 a a
a a
60 100
Time of death (min)

50 a
80
40 a

Percentage (%)
60
30 a b
40 b
20
20
10

0 0
Control Probiotic diet Water supply tr
ol
on

et
di
Fig. 4 The 50% lethal time for P. olivaceus submitted to
C

ly
ic

pp
heat shock test (30°C). Bars denotd by the same letter are
t
io

su
ob
not significantly different (P > 0.05, Duncan’s multiple

er
Pr

range test). Error bars represent mean ± standard devia-

at
W
tion (n = 2).
Fig. 5 Responses of P. olivaceus after air dive test. ()
stiffness/curvature, () recovery rate. Bars denoted by
activity (4.5 min incubation) in the water supply the same letter are not significantly different (P > 0.05,
group was significantly higher than the control and Duncan’s multiple range test) (n = 3).
the probiotic diet groups. Following heat shock,
lysozyme activity in the supply group did not sig-
nificantly decrease, although those in the control 100
group and the probiotic diet group significantly
decreased. Plasma protein was significantly higher
in the probiotic diet group after heat shock than in 80
the control and the water supply groups (Table 5).
Survival rate (%)

The control group was greatly affected by the air

60
dive test, evidenced by the greater upright curva-
ture or stiffness of the body compared to the pro-
biotics-treated group. There was no significant
40
difference in recovery rate among groups, although
the water supply group showed 100% recovery rate
(Fig. 5). 20

Tolerance to pathogens 0
0 2 4 6 8 10 12 14
Days
The pathogenic bacteria V. anguillarum affected Control Probiotic diet Water supply
only the fish in the control group whose popula-
tion decreased to 86 on day 4 and 76 on day 6 Fig. 6 Survival of P. olivaceus exposed to Vibrio anguil-
(Fig. 6). However, mortality was not observed larum at 2 × 107 c.f.u./mL (n = 3).
318 FISHERIES SCIENCE Y Taoka et al.

(a) (b)

Lysozyme activity (units/mg-protein)

40 a 500
a a a
Plasma protein (mg/L)

a a
400 a
30 a a
300
20
200
10 100

0 0
Control Probiotic Water Control Probiotic Water
diet supply diet supply

(c) (d) Fig. 7 Lysozyme activity and

Lysozyme activity (units/mg-protein)
protein concentration in plasma
and skin mucus 14 days after
300 b exposure to Vibrio anguillarum at
16 a
2 × 107 c.f.u./mL. (a) plasma pro-
Mucus protein (mg/mL)

14 250
12
tein, (b) plasma lysozyme activity
200 at incubation time of 0.5 min ()
10 a
b b and 4.5 min (), (c) skin mucus
8 150 protein, (d) skin mucus lysozyme
a
6 100 activity. Error bars represent
4 mean ± standard deviation. Bars
50 denoted by the same letter are
2
0 0 not significantly different (P >
Control Probiotic Water Control Probiotic Water 0.05, Duncan’s multiple range
diet supply diet supply test) (n = 4).

between day 7 and day 14. All fish in probiotics- to the rearing water, the water quality slightly
treated groups survived throughout the rearing improved as compared to the case without Bacil-
period and the survival rate in the probiotics- lus.5,25 The inoculation of probiotics to the rearing
treated group was significantly higher than that in water in the present experiment did not remark-
the control group. ably influence water quality, but the addition of
The plasma protein concentration was similar probiotics to the diet affected some water quality
among groups, although the water supply group parameters, such as NH4-N, NO2-N, and PO4-P. In
showed a higher level (Fig. 7a). The plasma NH4-N and PO4-P, the differences may be due to the
lysozyme activity of flounder was not significantly nitrogen and phosphorus metabolism because
different among groups (Fig. 7b). Mucus protein nitrogen and phosphorus excretion of fish are
was significantly higher in the control group than affected by components of diet, digestibility and
in the probiotics-treated groups (Fig. 7c). The skin digestive enzyme activity. De Schrijver and
mucus lysozyme activity in the probiotic diet Ollevier26 reported that protein digestion in juve-
group was significantly higher than the water sup- nile turbot was improved by feed supplement of
ply and the control groups (Fig. 7d). probiotic bacteria V. proteolyticus, and that supple-
ment with V. proteolyticus resulted in a signifi-
cantly increased proportion of protein of low
DISCUSSION molecular weight in the gut. Several probiotic bac-
teria excrete extracellular enzymes such as amylase
The decrease of pH in the water supply group was and protease, and these enzymes may affect diges-
distinct compared to the control and the probiotic tion in the gastrointestinal tract.27–29
diet groups. The difference may be due to acid pro- The introduction of probiotics into the rearing
duction by bacteria, because commercial probiot- water appeared to enhance growth and survival
ics used in this study included acid-producing rate of Japanese flounder. Growth trials showed
bacteria. Rengpipat et al.9 reported that probiotics that the introduction of probiotics to the rearing
(Bacillus) added to shrimp diets did not affect water accelerated the growth of flounder and
water quality. However, when Bacillus was added improved the survival significantly. However, a sig-
Effects of probiotics on Japanese flounder FISHERIES SCIENCE
nificant difference in growth rate was not observed
between the control group and the probiotic diet
group. These results are not in agreement with the
findings of Byun,11 who concluded that the growth
rate of flounder was improved by feeding with a
probiotic diet. The survival rate in the probiotic
diet group was significantly higher than that in the
control group. Rengpipat et al.9 showed that the
growth and the survival rates of black tiger shrimp
were improved by feeding with probiotic diet. This
disagreement between the findings of Byun,11
Rengpipat et al.,9 and our findings may be due to
the concentration of probiotics added in diet, the
bacterial strains, and the species of organisms
examined.
Enhancement of lysozyme activity was observed
in the probiotics-treated groups, especially in the
water supply group. Lysozyme has an important
role in non-specific immune defense system and is
contained in the mucus on the fish body surface,
and in plasma and liver. Lysozyme has an anti-
biotic ability and is released by leukocytes. It
can damage bacterial cell walls, especially of
Gram-positive and some Gram-negative bacte-
ria.30,31 Stress conditions induce variation of
lysozyme activity. Some studies suggested that
lysozyme activity was modified by intensity and
duration of the stress.32,33 Caruso and Lazard34
reported that plasma lysozyme activity in sheatfish
Silurus glanis decreased under persistent stress,
random modification of environmental light, and
holding. Yehuda et al.35 also reported that negative
correlation was observed between lysozyme
activity and cortisol. Cortisol is secreted as a
response to stress. Plasma lysozyme activity in tila-
pia Oreochromis nilotocus stressed by social pres-
sure was lower than that of unstressed fish.34
However, there have been few data available to
explain the effect of stress on lysozyme activity.
Demers and Bayne36 reported that simultaneous
increases in cortisol and lysozyme activity were
induced by acute stress in rainbow trout, Onco-
rhynchus mykiss (Wallbaum). In the present study,
lysozyme activity in the control was lower than the
probiotic-treated groups. These results indicated
improvement in antibiotic activity and modifica-
tion of the stress conditions of fish. Probiotic treat-
ment affected the protein concentration and
lysozyme activity in fish skin mucus at the end of
this experiment. As an immune defense system,
skin mucus has an important role and contains
some enzymes to prevent the invasion of patho-
gens, such as lysozyme, lectine, and protease. Pro-
biotic treatment has been shown by this study to
improve lysozyme activity of plasma and skin
mucus, and to increase plasma protein. These
results indicate that the non-specific immune
319
system of fish treated with probiotics is partially
improved. Fish excretes mucus under the stressful
conditions, and protein concentration in the skin
mucus is an indicator for amounts of skin mucus
excreted. In the present study, the concentration of
skin mucus protein in the control group was signif-
icantly higher than that in the water supply group.
In the case of red sea bream Pargus major, oral
administration of lactoferrin as an immunostimu-
lant increased the secretion of skin mucus, lectin
and lactoferrin, but did not affect the lysozyme
activity of skin mucus.37
In the heat shock and exposure-to-air stress
tests, the stress parameters indicated that stress
tolerance of fish is enhanced by probiotics. Mock
and Peters33 and Weyts et al.38 reported that
lysozyme activity decreased when fish were
stressed due to handling and exposure to polluted
water. In the heat shock stress test, the control
group showed lower lysozyme activity compared to
those of the probiotics-treated groups, especially
the water supply group. In addition, lysozyme
activity in the water supply group did not signifi-
cantly decrease by heat shock, although those in
the control group and the probiotic diet group
decreased significantly. This result indicated that
the water supply group is not stressed as much by
heat shock stress as were the control and probiotic
diet groups, from the viewpoint of lysozyme activ-
ity after heat shock stress.
The present study showed that probiotic treat-
ment enhances disease tolerance in cultured fish.
Increased resistance to the pathogen by probiotics
has been widely reported. Growth inhibition
against pathogens by Carnobacterium was demon-
strated.39 Black tiger shrimp fed with a probiotic
diet had greater tolerance to V. harveyi, and phago-
cytosis and phagocytic activity in hemolymph were
activated.9,40 The tolerance of rainbow trout Onco-
rhynchus mykiss to furunculosis was enhanced
when fed with a diet including the probiotic
L. rhamnosus.41 In Atlantic cod Gadus morhua, tol-
erance to V. anguillarum was measured by feeding
with lactic acid bacteria (Carnobacterium diver-
gens) supplemented in the diet.42 Robertson et al.12
reported that Atlantic salmon Salmo salar and rain-
bow trout O. mykiss, Walbaum fed with Carnobac-
teria spp. supplemented in the diet were more
tolerant to disease. However, adverse effects of pro-
biotics were also observed. The highest mortality
was observed when Atlantic salmon S. salar was fed
a probiotic diet containing lactic acid bacteria.43
Skin mucus lysozyme activity in the probiotics-
treated groups showed significantly better results
than that of the control groups. Takahashi et al.21
suggested that fish mucus included antibacterial
materials and lysozyme or non-specific defense
320 FISHERIES SCIENCE
factors, based on the relationship between bacteria
number attached to the fish body surface and that
of the other parts with stressed fish. Fevolden
et al.44 found a negative relationship between
lysozyme activity and mortality in rainbow trout,
meaning that enhanced lysozyme levels correlated
with significantly higher mortality following ex-
posure to Aeromonas salmonicida. Probiotics
orally administrated colonize the wall of intestine
and several probiotics can produce pathogen-
inhibitory substances (bacteriocin). Invading
pathogens or opportunistic bacteria were sup-
pressed by these substances. Carnobacterium
could stay in the intestine of rainbow trout by con-
tinuous feeding of probiotics.12 In the case of newly
hatched turbot, bacteria introduced to rearing
water colonized and became a major part of the
autochthonous flora of the gut of the larvae.45 From
the enhancement of disease resistance in the
pathogen challenge test, it was supposed that the
immune system is comprehensively improved by
probiotics treatment. In the rearing trial and the
pathogen challenge test, results showed higher
concentration of skin mucus protein in the control
group compared to the probiotics-treated groups.
Stress indicators in the air dive test also supported
that probiotics-treatment may improve tolerance
to stress. Generally, stress can affect the growth of
fish. Skin mucus protein in the water supply group
showed the lowest value. This result may explain
why the growth rate in the water supply group was
significantly higher than the probiotic diet group.
From the viewpoint of non-specific parameters,
mucus secretions, air dive test, and the pathogen
challenge test, it has been indicated that probiotic
treatment may activate fish immune response, and
improve the resistance to disease and tolerance to
stress. In this study, the improvement of flounder
health by probiotics treatment was indicated by
enhanced survival rate and growth rate. Tolerance
to stress in heat shock and air exposure, and resis-
tance to pathogens were enhanced by probiotics
treatment. Moreover, the introduction of probiot-
ics to rearing water seemed to be more effective
than oral administration, from the viewpoint of the
growth rate. This study indicated that stronger and
healthier flounder would be produced if probiotics
were used. However, the mechanisms of probiotic
action on cultured fish are still complicated and
unknown. Thus, more detailed knowledge should
be accumulated and further research is required.
ACKNOWLEDGMENTS
Sincere thanks to Professor T. Sakata and Dr. T.
Yoshikawa, Laboratory of Microbiology, Faculty of
Y Taoka et al.
Fisheries, Kagoshima University, and Professor S.
Teshima, Laboratory of Aquatic Animal Nutrition,
Faculty of Fisheries, Kagoshima University, for
advice. Thanks to Professors IB. Kim and YJ. Chang,
Department of Aquaculture, Pukyong National
University, and Professors MJ. Formacion, EB.
Seraspe and CA. Saclauso, Institute of Aquaculture,
College of Fisheries and Ocean Sciences, Univer-
sity of the Philippines, for their instruction. Thanks
to Dr. XJ. Wang and Mr. S. Choi for advices on test
diets, to Dr. JW. Lee, Dr. L. Peng, Dr. Muslim and
staff of Pukyong National University, Busan, Korea,
and to JH. Kim for support. This study was sup-
ported in part by the exchange student program
from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (MEXT).
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