閉鎖式循環水槽で飼育されたヒラメにおける飼育中Lactobacillus plantarumの添加効果

誌名誌名 水産増殖 = The aquiculture

ISSNISSN 03714217

巻/号巻/号 562

掲載ページ掲載ページ p. 193-202

発行年月発行年月 2008年6月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Aquaculture Sci. 56 (2) ， 193 -202 (2008)

The Efficien句Tof Lactobαcillus plantarum in Diet for

Juvenile Japanese Flounder Paralichthys olivaceus Reared

in a Closed Recirculating System

YousukeTAOKA¥ Kazuya YUGE2， Hiroto MAEDA3， * and Shunsuke KOSHl04

Absむ・act:A study was conducted to exar凶nethe effect of probiotics on the growth performance

and survival of juvenile J apanese flounder， water quality， bacterial number and tolerance to stress.

In the present study， Lactobacillus plantarum仏P)was orally gave to juvenile Japanese flounder，

Paralichthys olivaceus by feeding at two levels， 0.1% and 1.0% in the diet (the 0.1% LP group and

1.0% Lp group， respectively)，組dfish were reared in a closed recirculating system for 50 days.

The surviva1 rate and grow由 performanceof flounder， and water quality were not affected by oral

administration of LP. By feeding a diet wi出 Lp， the number of Lactobacillus sp. in the rearing water and the intestine increased， particularly in出e1.0% LP group. In fish出atreceived diets contain-

ing Lp， the better tolerance to high temperature was observed significantly as compared to those

receiving LP仕eediet.百lIsstudy suggested that LP orally administered could survive in出eintes司

tine of flounder and might be effective加 enhancestress tolerance of P. olivaceus.

Key words: Paralichthys olivaceus; Probiotics; Stress; Closed recirculating system

Microbes have been used empirically to main-

tain the hea1th of humans and livestock and have

been applied as food preservatives (Bengmark

1998; Gatescope 1999). Tannock (1997) found

出atliving microbia1 cells have the potentia1 to

improve the hea1th of host anima1s， although

this applied to humans but not aquatic animals.

Probiotics have been applied to humans and

terrestria1 anima1s (Irianto and Austin 2002).

In aquaculture， the app1ication of probiotics is

re1ative1y recent; however， Yasuda and Taga

a1ready anticipated in 1980出atbacteria might be

useab1e as food and as bio10gical con仕01agents

against fish disease (Yasuda and Taga 1980).

In J apan， the Fisheries Agency (http:/ /

www.jfa.maff.go.jp/bouekianzen.htm) has per・

mitted some kinds of antibiotics to be used in

aquaculture for disease prevention. Recent1y， the

Received October 17， 2007: Accepted February 25，2008.

usage of antibiotics has become limited because

severa1 pathogens have deve10ped resistance to

them (Miranda 2002).τbis gave rise to the use

of probiotics as an alternative strategy for the

prevention of infectious disease. In genera1， prか

biotics have been de:fined as a live microbial feed

additive出atbenefits the host animal by improv-

ing the microbial condition of its gas仕ointestinal

tract σuller 1989). Probiotic effects have been

shown to increase disease resistance through

the stimu1ation of immune responses， competi-

tion wi出 pathogenicbacteria for limiting nu仕i-

ents or adhesion points to the mucous， and the

production of inhibitory substances (Va'zquez et

al. 2003). Recent1y， probiotics have included dead

cells， which show no viability and do not contain

metabolites， which function as immunostimu-

1ants and modify enzyme activity or microfl.ora in

lFaculty of Agriculture， Miyazaki University， Miy出品U889-2192， Japan. 2DSM Nutrition Japan K K.， Tokyo 105-0014， Japan. 3Laboratory of Marine Microbiology， Department of Life Sciences， Faculty of Bioresources， Mie University， Tsu， Mie 519-8507， Japan. 4Laboratory of Aquatic Animal Nu仕ition，Faculty of Fisheries， Kagoshima University， Kagoshima 890-0056， Japan. * Corresponding author: E-mail: maeda@bio.mie-u.ac.jp

194 Y. Taoka， K. Yuge， H. Maeda and S. Koshio

出egastrointestina1仕act.

Some studies have suggested that the poten-

tial benefits of probiotics in aquaculture include

the recovery of water and sediment quality

through， for example， the decomposition of

organic matter and the reduction of nutrients in

culture ponds (Quetroz and Boyd 1998; Boyd

and Massaut 1999; Prab et al. 1999).τberefore，

probiotics have been expected to be used as

agents for water and sediment quality remedia-

tion in aquaculture.τbe fact that the microbial

flora of the rearing water influence the gastroin-

testinal flora of the cultured organisms is widely

known (Sugita et al. 1984; Olafsen et al. 2001).

As probiotics， lactic acid bacteria have been

widely used to improve the health of humans

and livestock (Irianto and Austin 2002). In fish， it has been confirmed血atprobiotics can enhance

growth and improve survival (Gatescope et al.

1994; Byun et al. 1997; Riquelme et al. 1997;

Rengpipat et al. 1998; Robertson et a1. 2000). The

]apanese flounder， Paralichthys olivaceus， is one

of the highly valued species of cultured fish.τbis

species is cultured on a large scale， and the prか

duction of it is increasing. However， the flounder

market has been damaged by bacterial disease，

such as vibriosis and Edwardsiellosis (Kanai et

al. 1988; Yamanoi et al. 1998). Pathogenic bac-

teria are in some cases opportunistic， and fish

can be easily infected when they訂 ein stressful

conditions. In aquaculture， fish were intensively

cultured and accepted access feeding， which

results in deterioration of the rearing environ-

ment. These culture conditions are stressful for

fish， and increase白epossibility of disease and

infection (Snieszko 1974; Maule et al. 1989; Davis

et al. 2002).百四refore，to achieve sustainable

aquaculture， it is very important that fish have

stress tolerance as well as disease resistance

(immune response). It has been well established

出atstress affects the survival and grow出 offish

(Vijayan and Moon 1992).

The environmental law enacted by the

]apanese Environmenta1 Agency in 1993 requires

more environmentally friendly aquaculture， pa子

ticularly because un-eaten feeds and feces in cage cultures located in coasta1 areas are discharged

凶tothe environment (Maruyama et al. 1998). To

con仕01the effluent from aquaculture farms and

thus promote environmentally friendly aquacul-

卸re，the use of a closed system was recently rec-

ommended (Maruyama and Suzuki 1998).百le

closed recirculating system has some advantages

over the raceway and cage culture systems in血at

(i)出eculture conditions are easy to con仕01，and (u) environmenta1 events， such as typhoons and

red tide blooms， have li仕leeffect on出eculture.

Furthermore，百 probioticsare used in a recircu-

la出19aquaculture system， those introduced into 出erearing system do not flow out of the system

because the system is closed. Therefore， a closed

recirculating system seems to be well suited for

血eapplication of probiotics泊aquaculture.

Until now， there have been few studies of the application of probiotics to flounder culture，

a1though Byun et a1. (1997) reported血atthe ora1 administration of lactic acid bacteria enhanced

出egrowth of adu1t flounder. Therefore，血issttidy

aims to investigate the effect of the oral admin-

istration of probiotic bacterium Lactobacillus plantarum on the growth and survival of the juvenile ]apanese flounder， and on resistance ω stress.百letota1 viable count and the number of

viable Lactobacillus sp. were monitored during a feeding trial to confirm the kinetics of L.

ρlantarum出血erearing water and the intestina1

trac臼 ofthe fish. Water quality in a closed recircu-

lating system was monitored to con宣rm血eeffi-

cacy of L. plantarum as a bioremediation agent.

Materials and Methods

Experimentalβsh Four thousand juvenile ]apanese flounders of

1.30:t 0.10 g (mean:t s同nd訂 ddeviation， n = 20)

泊weightwere purchased企oma commercia1 fish farm (Matsumoto Fisheries CO. LTD.， Miy沼心a，

] apan) and transferred to出erearing facility in

our department faculty. The fish were conditioned

in a running water system for 1 week. One week

before出estart of the experiment， the fish were

transferred恒toa closed recirculating system at a

density of 15 fish per aquarium (30 l). Previously，

natura1 seawater was日tratedby a sand filter and

used as the rearing water. During血econdition-

ing period， the fish were fed the control diet

Efficiency of Lactobacillus plantarum on Flounder 195

(probiotics~企ee) twice dai1y at 0.5 -1.切6of body

weight.τbe recircu1ating system was equipped

wi出 fouraquaria and a宣1terta凶<:(100 l) consist-

ing of a sand filter and a bio10gica1 filter wi由 p1as・

tic media. The water flow rate was 2.0 1 per min.

Experimental groups and diets

As the probiotic， 1yophilized L.ρlantarum

(LP) cells were obtained from Morinaga Milk

Industry CO.， LTD.， Kanagawa， Japan. Three

treatments with two simu1taneous replications

were prepared: a) a contro1 group fed the con-

tro1 diet (LP-free); b) a 0.1% LP group fed a diet

containing 0.1% LP; and c) a 1.0% LP group fed a

diet containing 1.0% LP. Each group was raised

in a separate recircu1ating system to avoid inter-

treatment contamination. The composition of

the three test diets is shown in Tab1e 1.

In the prep訂 ationof the diet containing Lp， an

adequate procedure based on preliminary experi-

ment was emp1oyed. Al1 powders were mixed

well for 10 min. The sources of lipids， except

Table 1. Composition of test diets for growth trial (g/100 g)

Test diets Ingredients

Control O.l%LP 1.0% LP LBp1 0.1 1.0

Fish meal 41.6 41.6 41.6 Soybean meal 30.0 30.0 30.0 α-starch 3.4 3.4 3.4

Pollack liver oil 5.2 5.2 5.2 Soybean lecithin 3.2 3.2 3.2

Palm oil 2.1 2.1 2.1 b-3HUFA 1.0 1.0 1.0

Vitamin mix2 6.0 6.0 6.0 Mineral mix3 4.3 4.3 4.3 Taurine 0.6 0.6 0.6 IMP 0.1 0.1 0.1 Betain 0.6 0.6 0.6 Alanine 0.4 0.4 0.4

α-cellulose 1.0 0.9 Berda 0.5 0.5 0.5

Total 100 100 100 lLactobacillus plantarum lyophilized powder (Morinaga Milk lndus甘yCO.， LTD. Kanagawa， Japan) 2B-carotene; 17.92， Vitamin D3; 1.80， Menadione-NaHS03; 8.54， A-tocophenol; 71.73， Thiamin-HN03; 10.75， Ribof1avin; 35.77， Pyridoxin守HCl;8.54， Cyanocobalamine; 0.01， Cellulose; 358.08， Biotin; 1.08， lnositol; 716.35， Nicotinic acid; 143.27，

Ca-panto白enate;50.16， Folic acid; 2.69， Choline chloride; 1464.50，

P-aminobenzoic acid; 71.35， APM; 37.50 (mg/l00 g-premix). 3MgS04; 512.64， Na2HP04; 326.35， K2HP04; 897.44， Ferric acid; 18.89， Ca1cium lactate; 1223.79， AL(OH3); 0.24， ZnS04; 12.95， CUS04; 0.38， MnS04; 2.99， Ca(I03h; 0.56， COS04; 3.74 (mg/l∞g-premix).

the palm oil， were mixed wi出 vitarr血s，and出is

mixture was added to the powder mixture. The

probiotics were added to the palm oil at 40'C， and

由iswas mixed well with the previous m民側reof

a11 ingredients for 10 min. Water was added at 40% to dry weight of the mixture and mixed well for

10 min to facilitate pelleting by a meat chopper

(diameter; 1.2 -1.5 mm). j凶erpelle出19，the diets

were 1yophilized unti1 the moisture content was

be10w 100A>. For the preparation of the con仕01diet，

the same procedure was emp10yed without the

addition of LP. Test diets were stored at -20'C.

Experimental design for rearing trial

百leexperimenta1 fish were fed twice dai1y at

0.5 -1.00A> per body weight and reared for 50 days

at a water temperature of 24'C with a coo1er. The

water flow rate in the system was 2.0 1 per min.

Every 10 days fish surviva1 was checked， and

fish body weights were measured. At the same

出ne，血erearing water and the fish intestine were

collected for a bacteria1 count to be performed.

Water was samp1ed with a po1yethy1ene bo仕le

every 5 days for water quality ana1ysis and was

stored at -20'C. After 50 days of rearing， a11 fish were removed from the recircu1ating system， and

the system continued to circu1ate for 7 days wi出-

out feeding. At 55， 56 and 57 days，出etota1 viab1e

count and the number of Lactobacillus sp. were

enumerated as described below.

時'aterquαlity analyses Urea-ni仕ogenwas determined according to

the method of N ewell et a1. (1967) NH4・N，NOz-N，N03・Nand P04-P were determined using the

method of S凶cklandand Parsons (1972). Tota1

ni仕ogenσ刊 andto凶 phosphorus仔P)were

determined according to the potassium persu1-

fate oxidizing method. COD (chemica1oxygen

demand) was determined according to出epotas-

sium permanganate-iodine titration method

(Japanese Industria1 Standard， 1995). Disso1ved inorganic nitrogen (DIN) was ca1cu1ated as the

sum ofNH4-N， NOz-N and N03-N.

Bacterial count For the enumeration of the tota1 viab1e count

in a water samp1e， 1/2 ZoBell 2216 agar medium

196 Y. Taoka， K Yuge， H. Maeda and S. Koshio

(polypeptone 5.0 g， yeast extract 1.0 g， ferric

phosphate 0.1 g， agar 15.0 g， artificial seawa-

ter [ASW] 1000 ml， pH 7.6) was used. For the

enumeration of the total viable count in the

fish intestine， ZB-n agar medium (polypeptone 5.0 g， yeast ex凶 ct1.0 g， agar 15.0 g， 1/2 ASW

1000 ml， pH 7.6) was used. For the enumeration of viable Lactobacillus sp. (LBS count)， modi-

fied LBS agar medium consisting of LBS agar

medium (BBL， Becton， Dickinson， Sparks， MD)

84.0 g， Lab-Lemco powder (Oxoid， Basingstoke，

England) 8.0 g， sodium acetate 15.0 g， glacial

acetate 3.7 ml， and distilled water 1000 ml was used. The water sample (200 ml) was col-

lected with a sterile glass bo仕le.For the bacte-

rial count in the fish intestine， two fish were

transferred from the rearing tank onto a c1ean

bench. The surface of the fish body was steril-

ized with 70% ethanol.τbe whole fish intestine

(from pyloric caeca to anus) was dissected out

and washed with乱rUTSUOKAbuffer (KH2P04

4.5 g， Na2HP04 6.0 g， L-cystein 0.5 g， tween 80

0.5 g， DW 1000 ml).百leintestine was homog-

enized in the same solution. Ten-fold serial

dilutions were prepared from each sample wi出

MITSUOKA buffer， and the aliquot (100μ1)

was spread on each agar plate. In the case of

出etest diet， the sample was first powdered well

and serially diluted. The agar plate inoculated

with each dilution was incubated for 3 days at

25t and 37t for the total viable count and LBS

count， respectively.

High temperature stress test After a feeding triallasting 50 days， the toler-

ance of fish to exposure to high temperature

was examined. Five fish were starved for 24

hrs and transferred from each tank to fi1tered

natural seawater in a glass aquarium (12 l).

Beforehand， a preliminary experiment was

conducted to confirm the time to death of P.

olivaceus at various temperatures within an hour， and the resu1ts led to the use of a tem-

perature of 34 t for the high-temperature

stress test. The fi1tered natural seawater was

used. The water temperature was maintained

at 34t with a thermostatically controlled glass

heater based on a preliminary experiment. The

pH， salinity and dissolved oxygen in seawater

were 8.15:t 0.03， 34 psu and 6.5:t 0.1 mg/l，

respectively. The time to death of the fish was

recorded， and the time duration for 50% mortal-

ity was determined.τbis test was conducted in

triplicate.

St，αtistical analysis

Data were statistically analyzed using one-

way analysis of variance (ANOVA) followed by

出eTukey-t test.τbese analyses were conducted using SPSS Version. 10.0 (SPSS， Chicago， IL，

USA). In the high-temperature stress test， the

significant difference between groups was

obtained using Student's t-test. Significant dif-

ference was established at Pく0.05.

Results

Change 01 LBS count in a diet with LP stored αt -20

0

C

Initially， the LBS count in the diet contain-

ing 1.0% LP was 1.0 x 108/ g-diet. It was stored

at -20t. One week later， the LBS count in

the diet was 1.2 x 108/ g・diet.One month later，

the number had decreased slight1y to 8.1 x

107/ g-diet.

瓦併ct01 LP on the survivalω2d grow的 ρe幼子

mances 01 flounder

Table 2 shows the survival rate and growth

performance after the 50-day rearing trial in

which fish were fed wi出血econ仕01diet and the

diets containing 0.1% or 1.0% LP. In the survival

rate and all parameters of growth performance，

no significant differences were observed among

three groups after 50 days of rearing. Fぜ句Tdays

after rearing the survival rate was 75 -82%， and

the mean body weight was 8.99 -9.29 g per

individual in the three groups. WG， BWG， feed

intake and FCE ranged from 7.59 -7.89， 541 -

565，9.9 -10.2 and 76.2 -78.1， respectively.

政fects01 LP on wαter quality in a closed recircu-lating system

No obvious differences were observed among

groups in the water quality parameters of the

rearing water throughout the experiment (Fig. 1).

Efficiency of Lactobacillusρlantarum on Flounder 197

Efjセcts01 LP on the bacterial counts in the rear-

ing water and the fish intestine

Fig. 2 shows the results of the bacterial counts.

In the total viable count in the rearing water，

no difference was observed among the groups

during白e50 days of the tria1， a1though the count

泊 the1.0% LP group was higher由加 thosein the

control group and 0.1% LP group at 50 days. In

the tota1 viable count in出eintestine sample， no

differences were observed among the groups.

In the case of the rearing water， the LBS

count was not detected in the control group. In

Table 2. Survival rate and growth performances of P. olivaceus

Treatment Survival rate Mean BW1 (g)

WG2 (g) BWG3 (%) Feed intake

FCE4 (%) (%) Initial Final (g)

Control 80.0:t7.7 1.41土0.03 9.29:t 0.76 7.89:t0.57 565:t38 10.2:t 0.4 77.6:t 4.5 0.1%LP 75.0:t 6.4 1.40:t 0.03 8.99:t 0.58 7.59:t0.76 541:t 56.8 9.9:t 0.3 76.2:t 5.1 1.0%LP 81.7:t 6.4 1.40:t 0.01 9.09:t 0.34 7.69:t 0.33 549:t20 9.9:t 0.4 78.1:t 1.4

lBW; body weight. 2WG; weight gain 3BWG; (WG / initial body weight) x 100. 4FCE; (WG / feed intake) x 100 (n = 4).

0.2 r

Urea-N

LL出Ne4-N

1.5

¥『切立、0.12 1.2

日、、

0.08 ':.Il 0.9 g

0.6

0.04

。o 5 10 15 20 25 30 35 40 45 50

Days

120

100

80 、、、

E ω

40

20

。o 5 10 15 20 25 30 35 40 45 50

Days

弓'

'

n

h

V

F

h

d

4-qa

『¥切

g2

。o 5 10 15 20 25 30 35 40 45 50

Days

0.3

N02-N

0.2 、『、、ー

切E

0.1

。o 5 10 15 20 25 30 35 40 45 50

Days

120

100

80 、目、

E 60 40

20

。o 5 10 15 20 25 30 35 40 45 50

Days

COD 14

12

10 、目 8

8 6

4

2

。o 5 10 15 20 25 30 35 40 45 50

Days

Fig. 1. Change of water quality parameters in a closed recirculating system during 50 days of feeding trial with a control diet

and a diet containing LP at 0.1% and 1.0% concentrations.・;con仕01group，口;0.1% LP group，企;1.0% LP group. Bars repre-

sent mean:t standard error (n = 2).

。o 5 10 15 20 25 30 35 40 45 50

Days

150 T-N

120

AHV

A

HV

A

W

d

F

h

v

『¥切自

o 5 10 15 20 25 30 35 40 45 50

Days

QdQU

司

d

no

T-P

Fhd

N¥凶日

o 5 10 15 20 25 30 35 40 45 50

Days

198 Y. Taoka， K Yuge， H. Maeda and S. Koshio

the 0.1% LP group and the 1.0% LP group， the

LBS count was detected and was observed to

increase throughout the rearing. At 50 days， in

the case of出erearing water， the LBS count in

the 1.0% LP group was higher than出atin the

0.1% LP group. In the case of the intestine， the

LBS count was not detected in仕lecontrol group.

From 10 days to 50 days， the LBS count in the

intestine was detected in the 1.0% LP group. It rapidly increased to around 1.0 x 104/g at 20

days and remained at出atlevel until 50 days. In

the 0.1% LP group， the LBS count in出e凶tes出le

was observed at only 50 days，組dthat count was

much lower than that in the 1.0% LP group.

1.0E+06

1.0E+05

1.0E+04

s O 1.0E+03 同u

1.0E+02

1.0E+01

1.0E+00

1.0E+09

1.0E+08

1.0E+07

1.0E+06

妻仁匡J 1 肥+田1.0E+04

1.0E+03

1.0E+02

1.0E+01

1.0E+00

O

O

Water

10 20 30 40 50

Time (days)

Intestine

10 20 30 40 50

Time (days)

Fig. 2. Change of the total bacteria count and LBS count in the rearing water and the intestine of P olivaceus during 50 days of feeding with a control diet and a diet containing LP at 0.1% and 1.0% concentrations. Closed symbol; total viable count， open symbol; LBS count， circular symbol; control group， square symbol; 0.1% LP group， triangle symbol; 1.0% LP group (n = 2).

Change 01 bacteriα1 count in rearing water after the leeding was stopped

Fig. 3 shows the change in the bacteria count

in the rearing water after 50 days of rearing and

after the feeding was stopped. The total viable

count in the rearing water was stable with some

fl.uctuations in the 1.0% LP and 0.1% LP groups.

The LBS count in the 1.0% LP group decreased

slightly. In the case of the 0.1% LP group， the

LBS count dramatically decreased and was not

detected at 57 days.

Ellecお 01LP on the tolerance 01 flounder to high temperature

In the high-temperature stress test， fish

receiving the diet containing LP (the 1.0% LP

group) showed significantly higher tolerance

than the fish in the control group (Fig.4).

Discussion

In this study， the result of the rearing trial

showed that the survival rate and growth rate

of fl.ounder were not affected by the ora1 admin-

istration of LPσ'able 2). This result was in dis-

agreement with the findings of Byun (1997)， who reported that the growth of fl.ounder

Days

Fig. 3. Change of the total viable count and LBS count in the rearing water after the feeding was stopped in the 0.1% LP group and the 1.0% LP group. Closed symbol; total viable count， open symbol; LBS count， circular symbol; 1.0% LP group， square symbol; 0.1% LP group. Arrow means when the feeding was stopped， and all test fish were removed企omthe rearing system (n = 2).

E血ciencyof Lactobacillus plantarum on Flounder 199

60 牢

50

-ロE

30 通常

B も。320

E F4J 10

。Control 1.0% LP

Fig. 4. Time to 50% mortality of P olivaceus in the high temperature stress test (34t). An asterisk shows signifi-cant difference (P < 0.05). Error bars represent means ::t standard deviation (n = 3).

was enhanced by the oral administration of

Lactobacillus sp. One of the reasons for the disagreement of our宣ndingwith that of Byun

(1997) was likely the difference in the bacterial

strain used. Byun (1997) used the Lactobacillus s甘ainisolated from the intestine of the host宣sh，

whi1e we used the Lactobacillus s仕ainfor mam-

malians. In addition， Byun (1997) used adult

f10under in con仕astto juveni1e f10under in our study， meaning白atfish size used for the rearing

凶a1was quite different in each study. Jeong et a1. (2006) used three diets containing probiotic bac-

teria， Bacillus poly，伽menticus，B. licheniformis or B.ρolyfermenticus plus Saccharomyces cerevisiae，

to juveni1e founder as well as a con仕01diet with-

out probiotics and confirmed出at出efeeding of

probiotics did not enhance the fish grow吐1.This

result agreed wi出 ourresult. It seems出at出e

species of probiotic bacteria and the size of the

宣share important factors in whether probiotics

enhance the growth of fish.

Water quality is a very important factor in

sustainable aquaculture. Therefore， sometimes

probiotics are used to recover the water qual-

ity in culture ponds. Probiotic bacteria， genus

Bacillus， have been widely accepted as a biore-

mediation agent in aquaculture (Queiroz and

Boyd 1998; Prabu et al. 1999; Corre et al. 2001).

To our knowledge， there has been no report on

the improvement of water quality in the culture

ponds by means of lactic acid bacteria such

as genus Lactobacillus. In our study， the oral administration of LP did not affect the water

quality parameters of the rearing water in a

c10sed recirculating system (Fig. 1)， indicating

出atLP has no potential to act as a bioremedia-

tion agent like Bacillus. In the high-temperature stress test， the time

to death of fish fed with LP was longer in com-

parison with that in the control group (Fig. 4)，

which means出atthe oral adminis仕組onofLP is

effective in enhancing the 位 esstolerance of P.

olivaceus. S仕essis well known as a m司orfactor which increases the susceptibi1ity of fish to dis-

ease (Maule et al. 1989). S仕essinhibits antibody

production by circulating lymphocytes in fish

(Ellis 1981; Maule et al. 1987， 1989). Matsuyama

et al. (2000) found血atthe host-defense system

in凶apiawas damaged by the addition of corti-

sol， which is known as a stress hormone， and

cortisol administration inhibited the production

of inter1eukin-like factors (Tripp et a1. 1987).

Maule and Schreck (1991) found that stress

and cortisol altered the affinity of corticoste-

roid receptors in lymphocytes. Yokoyama et al.

(2007) investigated the effect of lactoferrin (LF)，

which is known as an immunostimulant， on high-

temperature stress in J apanese f1ounder.τbey reported出at出es仕esstolerance of宣shin high-

temperature surroundings was improved by the

feeding of a 1000 mg LF /kg diet. In addition， a

significant1y higher level of出eheat shock pro-

tein 70 far凶ly但SP70s)in the skin and livers of

fish fed with LF was observed in comparison to

出atof the control group (0 mg LF)， and this ten-

dency was consistent wi出 theresult of the lethal

stress test in their study. HSP is a molecular

chaperone that binds to and prevents the aggre-gation of proteins， and has been known as a kind

of stress protein (Elicker and Hutson 2007).

This protein is synthesized when organisms are

exposed to various stressors， such as heat ~hock，

cold shock， viral infection， and heavy meta1 expか

sure (Iwama et al. 1998). Therefore HSP level is

thought to ref1ect physicochemical stress.

It has been established that some compo-

nents known as immunostimulants can stimulate

the immune response and the stress response

200 Y. Taoka， K Yuge， H. Maeda and S. Koshio

(Sakai 1999). Yokoyarna et al. (2007) suggested

that oral administration of an immunostimulant

affects not only immune responses but also

the expression of HSP synthesis in fish under

stressful conditions， which implies that the

improvement of stress tolerance relates to immu-

nostimulation in fish. The immunostimulatory

effect of probiotics is well known. Some studies

reported that the mortality of fish artificially

infected with pathogens decreased due to the

use of probiotics. In shrimp， those that received oral adminis仕ationof probiotics showed higher

survival in comparison with the control group

after immersion in pathogens (Rengpipat et al.

1998). The feeding of a probiotic diet containing

L. rhamnosus reduced the mortality of rainbow

仕outby furunculos恒例ikoskelainenet al. 2001).

Atlantic cod Gadus morhua became more toler-

ant to Vibrio anguillarum by means of feeding

with the lactic acid bacteria Carnobacterium

divergens (Gildverg et al. 1998). Jeong et al.

(2006) described白紙 theoral administration of

probiotics to juvenile flounder enhanced the non-

specific immune response of the fish and their

resistance against Edwardsiella tarda. The oral

administration of LP may have potential as an

immunostimulant. The effect of LP on resistance

to disease should be further studied by conduct-

ing a pathogen cha11enge test. τbe LBS count in the water and the intestine

increased as a resu1t of feeding the fish a diet

containing Lp， and the density of LP in the diet

positively correlated with the LBS count in出e

water and the intestineσig. 2). After the feeding

was stopped， the LBS count in the 0.1% LP group

was drarnatically decreased in the rearing water

over the course of 7 days， while血atin the 1.00，6

LP group decreased slightly (Fig. 3). Va'zwuez

et al. (2003) suggested investigating the survival

of lactic acid bacteria as a potential probiotic in

seawater. Based on the action of the LBS count

during the feeding trial and after the feeding was

stopped， continuous feeding and a concentration

of more出an1.0% LP (> 108 CFU/g-feed) would

be required to keep the high occupation of LP to

a total viable count in the water and the intestine.

In出isstudy， a closed recirculating system was

employed as the rearing method for :fish. Some of

出eprobiotics administered ora11y colonize on出e

intestinal wa11， and others pass through the diges-

tive tract and are expelled with feces. Olafsen

(2001) found that :fish larvae ingest bacteria by

drinking water， resulting in the formation of an

indigenous larval microflota. Therefore， even if

some of the probiotics cannot adhere to出e泊tes-

tinal wall， it is thought that宣shcan repeatedly

ingest白eprobiotic cells expelled by :fish into出e

water by drinking the water in a closed recirculat-

ing system. In出ismanner， a closed recirculating

system increases the opportunities for the colo-

nization of probiotics on the intestinal wall and is

adequate for the application of probiotics in aqua-

culture. 1ρng-term monitoring of the adherence

ofLPto血eintestinal wall should be done.

In general， probiotics administered orally colo-nize the intestinal wall and inhibit the growth of

pathogenic bacteria， which resu1ts in a decrease

in fish mortality due to disease. Previously，

the authors confirmed that the LP used in

this study showed strong antibacterial activity

against a wide spectrum of :fish pathogens， such

as V. anguillarum， V. harveyi， Vibrio spp.， and

Edwardsiella tarda， using the double layer ag訂plate method (data not shown). In our study，

LP with viability was observed in the intestine

of the flounder (Fig. 2). lf pathogenic bacteria

invade the intestines of flounder from the rear-

ing water， there is a possibility that LP with

antibacterial activities can kill or eliminate the

invaders， which would reduce the risk of disease

outbreaks. The interaction of LP and pathogens

in白e:fish intestine should be fur出erstudied.

Some of the orally administered probiotics

are killed and degraded by digestive enzymes

in the gastrointestinal仕actsof the宣sh，and the cell components are absorbed from the intes-

tinal wall. Fish physiological functions， such

as immune responses， might be influenced

without the colonization of probiotics on the

intestinal wall， meaning that the components of probiotic bacteria play the role of an immunos-

timulant. Murosaki et al. (1998) found出atheat-

killed L.ρlant，αrum could induce inter1eukin-12

in mice， which implied that the colonization of

probiotic bacteria is not quite essential for the

stimulation of immune responses. Otherwise，

Efficiency of Lactobacillus plantarum on Flounder 201

some reports have indicated that live bacterial

cells are more potent as probiotics than dead

bacterial cells (De Simone et al. 1986; Panigrahi

et al. 2005). Taoka et al. (2006) found that com-

mercial probiotics consisting of live cells are

superior as immunostimulants for Ni1e ti1apia as compared to those that consist of dead cells.

官官 effectsof LP on the immune response of

f10under should be further researched.

To our knowledge， this study was one of the

first reports on the dietary administration of LP

to juvenile ]apanese f10under in a c10sed recir-culating system. The results showed that LP

could survive in the宣shintestine and that the

oral administration of LP could improve stress

resistance.

Acknowledgements

百leauthors would like to express thanks to

Morinaga Mi1k industry CO.， LTD. that kindly

provided probiotics used in this study.

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閉鎖式循環水槽で飼育されたヒラメにおける飼料中

Lactobacillus plantarumの添加効果

田岡洋介・弓削寿哉・前田広人 ・越塩俊介

ヒラメの生残率，成長，飼育水質，細菌数および高水温ストレス耐性について調べた。

Lactobacillusρlanωrum (LP)を0.1%および1.0%の割合で試験飼料へ添加し，ヒ ラメに経口投与して

50日間閉鎖式循環水槽にて飼育を行った。LP投与によるヒラメの成長，生残および水質への影響は

認められなかった。LPの経口投与に伴い 特に1.0% LP添加区において 飼育水およびヒラメ腸試

料の Lactobacillussp.数が増加した。無添加区と比較してLP添加区のヒラメでは，高温に対するスト

レス耐性が有意に増加した。以上の結果より，経口投与された LPはヒラメ腸内で生残し，ヒラメの

ストレス耐性向上に有効で、あることが示された。