Dietary Supplementation of Phytosterol for Olive Flounder Paralichthys olivaceus

191

Copyright © 2020 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

서 론

Phytochemical

,

,

,

steroids, alkaloids, flavonoids, terpenoids

.

phytochemi- cal

(Cyprinus carpio) (Yuan et al., 2007),

(Oreochromis mossambicus) (Divyagnaneswari et al., 2007),

(Oncorhynchus mykiss) (Citarasu, 2010),

(Pagrus major) (Ji et al., 2007a)

.

Aeromonas hy- drophila (Pachanawan et al., 2008), Vibrio harveyi (Punitha et

al., 2008), Ichthyophthirius multifiliis (Ekanem et al., 2004), viral haemorrhagic septicaemia virus (VHSV) (Micol et al.,

2005)

었다

.

phytosterol

phytochemical

sterol

, phytosterol

(Moreau et al., 2002; Mackay and Jones, 2011). Phytosterol

,

cholesterol

(Ostlund, 2002). Phytosterol

,

cholesterol

,

넙치(Paralichthys olivaceus) 사료 내 phytosterol의 이용 가능성 평가

신재형·이초롱·김유정·신재범·임현운·윤관식

·이경준

*

제주대학교 해양생명과학과, ¹제주대학교 해양과학연구소, ²(주)시너젠

Dietary Supplementation of Phytosterol for Olive Flounder Paralichthys olivaceus

Jaehyeong Shin, Chorong Lee, Youjeong Kim, Jaebeom Shin, Hyunwoon Lim, Kawn-Sik Yun² and Kyeong-Jun Lee¹*

Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province 63243, Korea

1Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Korea

2SynergenInc., Bucheon 14574, Korea

We evaluated the effects of dietary supplementation of phytosterol on the growth, feed utilization, immunity, digest- ibility, wound healing ability and disease resistance of olive flounder Paralichthys olivaceus . We conducted two con- secutive feeding trials at different growth stages of the fish: EXP-1 (68.9 g) and EXP-2 (16.5 g). The experimental diets were supplemented with graded levels of phytosterol (1% ecdysteroid extracted from Achyranthis radix ) at 0, 0.025, 0.05, 0.1, 0.2 and 0.4% (designated as Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4, respec- tively). Dietary phytosterol did not significantly affect growth and cholesterol concentration. Feed utilization was higher in fish fed phytosterol-supplemented diets than in fish fed the control diet. Dietary phytosterol increased innate immunity and digestibility of protein and dry matter. Wound healing ability was also increased by the phytosterol supplementation. The survival against Edwardsiella tarda challenge was higher in fish fed low-phytosterol diets than in fish fed the control diet. The optimum dietary level of phytosterol seems to be approximately 0.05%. The results in this study indicate that the phytosterol could be used as a functional supplement in diets to improve feed utilization, immunity, digestibility and wound healing ability of olive flounder.

Keywords: Phytosterol, Phytochemical, Immune-stimulant, Olive flounder, Feeds

*Corresponding author: Tel: +82. 64. 754. 3423 Fax: +82. 64. 756. 3493 E-mail address: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 2 March 2020; Revised 7 April 2020; Accepted 14 April 2020

저자 직위: 신재형(대학원생), 이초롱(대학원생), 김유정(대학원생), 신재범(대 학원생), 임현운(대학원생), 윤관식(대표), 이경준(교수)

https://doi.org/10.5657/KFAS.2020.0191

Korean J Fish Aquat Sci 53(2), 191-202, April 2020

증식을 억제하는 효과가 있다고 보고되었다

(Ostlund, 2002;

Ostlund, 2004; Kritchevsky and Chen, 2005).

phytosterol

European sea bass (Dicen- trarchus labrax) (Couto et al., 2015a; b), gilthead sea bream (Sparus aurata) (Couto et al., 2014a; b),

(Salmo sa- lar L.) (Liland et al., 2013; Sissener et al., 2017)

.

수생생물의 피부는외부의병원체가 체내로유입되는 것을 막기때문에면역체계에있어매우중요한역할을한다

(Wahli et al., 2003).

(Nolan et al., 1999),

(Chakraborty and Hancz,

2011).

.

도가높을경우피부에상처가발생할확률이높아진다

.

. Phytochemical

(Nath and Dutta, 1997; Ghosh and Gaba, 2013).

그러나

,

phytochemical

phytosterol

.

넙치는우리나라의대표적인 양식어종이다

. 2018

37,238

,

46%

(KOSIS, 2019).

. 2018

∙

984

,

25% (242

)

(KFDA,

2019).

,

효율을감소시키고환경적으로도많은문제를일으킨다

(Allen et al., 2010).

,

phytochemical

.

phytosterol

,

,

.

재료 및 방법

실험어와 사육관리

사육실험에사용된넙치는

5

(CJ CheilJedang, Seoul, Korea)

.

2

.

(

1, 68.9 g)

(

2, 16.5 g)

.

1

(

, 68.9 g)

24

acrylonitrile butadiene styrene (ABS)

(300 L)

30

,

4

.

23.1±2.4°C

,

12

.

3 L/min/

tank

,

(aeration)

.

12 light: 12 dark

.

1

2

(08:30, 18:00 h)

.

실험

2

(

, 16.5 g)

20

ABS

(300 L)

28

4

.

18.7±1.5°C

,

8

.

1

.

(

, 2019-0043)

. 실험사료

실험에사용된

phytosterol (ecdysteroid 1%)

(Achy- ranthis radix)

,

(

)

(Bu- cheon, Korea)

.

1

2

(control)

,

53%,

8.8%

(Table 1).

1

phytosterol

0.025, 0.05, 0.1, 0.2, 0.4% (PHY0.025, PHY0.05, PHY0.1, PHY0.2, PHY0.4)

5

.

2

phytosterol

0.025, 0.05, 0.1, 0.2%

(PHY0.025, PHY 0.05, PHY 0.1, PHY 0.2)

4

.

phytosterol

,

.

,

(cod liver oil)

(

15%)

(SP-50, Gumgang ENG, Daegu, Korea)

2-4 mm

.

25°C

8

(-20°C)

.

Sampling과 분석

실험어의증체율

(weight gain, WG)

(survival)

24

.

(feed conversion ra- tio, FCR)

(protein efficiency ratio, PER)

.

,

8

2-phenoxyethanol

(200 mg/L)

,

. 8

4

hematocrit, hemoglobin,

(nitro blue tetrazolium, NBT)

.

(5,000 g, 10 min)

(plasma)

immunoglobulin (Ig)

.

4

30

(5,000 g, 10 min)

(serum)

.

sam-

pling

여

Tris-Buffered Saline (TBS, 50 mM Tris-HCl, pH 8.0, 150

mM NaCl)

.

(4000 rpm, 4°C, 30

min)

alkaline phosphate (ALP)

.

실험어의비특이적면역력을조사하기위해총

6

. NBT

Kumari and Sahoo (2006)

, Ig

Siwicki and Anderson (1993)

, lysozyme

Hultmark (1980)

. SOD (super- oxide dismutase)

SOD kit (19160, Sigma-Aldrich, ST Louis, USA)

, GPx (glutathione peroxidase)

GPx kit (K762-100, Biovision, Milpitas, USA)

.

ALP

Ross et al. (2000)

. Hematocrit

(Micro hemato- crit VS-12000, Vision Scientific, Daejeon, Korea) (10 min)

, hemoglobin, aspartate aminotransfer- ase (AST), total protein, cholesterol

(SLIM, SEAC Inc, Florence, Italy)

.

실험사료와분

(feces)

AOAC (2005)

(125°C, 3 h),

회화법

(550°C, 4 h),

(Kjeltec

2300, FOSS analytical, Hilleroed, Denmark)

,

Folch et al. (1957)

soxhlet

(SOX406 fat analyzer, Jinan Hanon Instruments, Shandong, China)

.

외관상 소화율 측정

평균무게

120 g

사하였다

.

1

(Cr

O

, Daejung, Siheung, Korea)

1%

.

(Guelph system)

.

1

.

cartridge filter

.

40

,

16

.

30

,

.

(-40°C)

.

Diva- karan et al. (2002)

.

.

Apparent digestibility coefficient of dry matter (%, ADCd)

= 100-100×(% of Cr

O

in diet/% of Cr

O

in feces) Apparent digestibility coefficient of protein (%, ADCp)

= 100-100×(% of Cr

O

in diet/% of Cr

O

in feces)

×(% of protein in feces/% of protein in diet)

상처치유능력

상처치유능력실험은

Wahli et al. (2003)

.

1

,

6

ABS

(215 L)

16

.

1

2

(08:30, 18:00 h)

.

2-phenoxyethanol

(200 mg/L)

,

(

1 cm,

0.5 cm)

.

6

(0, 1, 3, 7, 9, 16

)

2

.

Bouin’s solution

24

.

tissue processor (TP1020, Leica, Wetzlar, Germany)

,

(par- affin)

harris hematoxylin

0.5% eosin

.

(Olympus CKX41, Tokyo, Japan)

(Image J 1.44 software)

.

공격실험

사육실험

2

, 120 L acryl

11

(

33

)

(3

, 15

). Edwardsiella

basal diet (% of dry matter)

Ingredients g/kg

Fish meal, sardine¹ 500.0

Soybean meal 80.0

Corn gluten meal 80.0

Wheat flour² 230.0

Cod liver oil³ 40.0

Mineral premix⁴ 10.0

Vitamin premix⁵ 10.0

Starch 20.0

Choline chloride (50%) 20.0

Mono calcium phosphate 10.0

Proximate composition (%)

Crude protein 52.7

Crude lipid 8.82

Crude ash 10.5

1Orizon S.A., Corp., Santiago, Chile (crude protein, 69%; crude lipid, 8.6%). ²Deahan Flour Co. Ltd, Incheon, Korea. ³E-wha oil &

fat Industry Corp., Busan, Korea. ⁴Mineral premixcontains(g kg^-

1):MgSO₄∙7H₂O, 80; NaH₂PO₄∙2H₂O, 370; KCl, 130; Ferriccitrate, 40; ZnSO₄∙7H₂O, 20; Ca-lactate, 357; CuCl, 0.2; AlCl₃∙6H₂O, 0.15; Na₂Se₂O₃, 0.01; MnSO₄∙H₂O, 2; CoCl₂∙6H₂O, 1.0. ⁵Vitamin premixcontains (g kg^-1):L-ascorbic acid, 121; DL-α tocopheryl acetate, 19; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridox- ine hydrochloride, 1.8; niacin, 36; Ca-D-pantothenate, 12.7; myo- inositol, 182; D-biotin, 0.27; folic acid, 0.68; p-aminobenzoic acid, 18; menadione, 1.8; retinyl acetate, 0.73; cholecalciferol, 0.003;

cyanocobalamin, 0.003.

tarda

salmonella-shigella (SS, Difco)

(25°C, 48 h)

.

colony

tryptic soy agar (TSA)

(27°C, 24 h)

. Bacteria

Sakai et al. (2007)

kit (Higene genomic DNA prep kit, BIO-FACT, Daegu, Korea)

. E. tarda

(1×10

CFU/mL)

100 µL

.

12

.

통계학적 분석

실험사료의배치는완전확률계획법

(completely randomized design)

,

SPSS (Version 18.0)

One-way ANOVA

.

Duncan’s multiple test (P<0.05)

.

±

(mean±SD)

.

arcsine

.

결 과

사육실험

1

Ig

PHY0.1

PHY0.4

(Table 2). GPx

phytosterol

. NBT, lysozyme, SOD, ALP

.

,

율은모든실험구사이에유의적인차이를보이지않았다

(Table

3).

PHY0.4

phytosterol

.

AST

PHY0.04

phytosterol

(Table 4).

hematocrit, hemo- globin, total protein

.

cholesterol

PHY0.025

구가

PHY0.2

.

PHY0.05, 0.2, 0.4

(Ta- ble 5).

PHY0.025

phytosterol

.

3

,

(epidermis)

.

7

, phytos-

terol

로나타났다

(Fig. 1).

16

,

(der-

mis)

.

사육실험

2

Ig

PHY0.05

(Table 2). Lysozyme

PHY0.05

0.2

. NBT, SOD, ALP

.

(Table 3).

PHY0.025

.

PHY0.025

.

hematocrit, hemoglobin, AST, total protein

Table 2. Non-specific immune responses and antioxidant activities of olive flounder Paralichthys olivaceus fed the experimental diets for 12 (Exp-1) and 8 weeks (Exp-2), respectively. The experimental diets were prepared with supplementing phytosterol by 0, 0.025, 0.05, 0.1, 0.2 and 0.4% (Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4)

NBT¹

(absorbance) Ig²

(mg/mL) Lysozyme

(µg/mL) SOD³

(% inhibition) GPx⁴

(mU/mL) ALP⁵

(U/L) Exp-1 (Initial body weight, 68.9g)

Con 1.05±0.09 15.0±1.66^bc 22.3±6.87 79.0±6.91 61.8±1.94^c 199±26.5

PHY0.025 0.96±0.07 17.0±2.42^ab 23.3±2.66 76.3±4.75 95.6±3.93^a 216±38.8

PHY0.05 1.09±0.15 17.7±1.06^ab 22.1±2.16 77.7±8.10 77.3±6.74^b 210±14.7

PHY0.1 0.96±0.14 18.7±2.70^a 25.4±5.86 75.0±7.94 85.4±8.94^ab 212±61.9

PHY0.2 0.91±0.14 18.1±2.16^ab 25.1±4.35 74.6±8.94 91.7±10.2^a 204±24.7

PHY0.4 1.04±0.04 13.6±2.16^c 23.1±3.06 82.2±5.64 93.3±7.93^a 183±30.1

Exp-2 (Initial body weight, 16.5g)

Con 0.98±0.12 24.6±2.49^b 44.5±2.67^c 136±6.40 - 40.1±3.91

PHY0.025 1.04±0.04 33.8±8.19^ab 43.8±8.10^c 139±2.89 - 46.3±4.89

PHY0.05 0.94±0.06 38.4±4.67^a 56.7±4.97^ab 142±8.59 - 56.3±6.35

PHY0.1 0.89±0.05 30.7±4.90^ab 42.5±7.85^c 137±2.28 - 55.3±3.26

PHY0.2 0.96±0.09 33.1±1.56^ab 61.3±2.98^a 140±0.13 - 47.8±5.70

1NBT, Nitro-blue tetrazolium. ²Ig, Immunoglobulin. ³SOD, Superoxide dismutase. ⁴GPx, Glutathione Peroxidase. ⁵Alkaline Phosphatase.

Values are mean of quadruplicates and presented as mean±SD. Values with different superscripts in the same column are significantly dif- ferent (P<0.05). The lack of superscript letter indicates no significant differences among treatments.

EL DL

ML

EL DL 3 days16 days0 day7 days ML

(A) (B)

(C) (D)

(E) (F)

(G) (H)

Fig. 1. Photomicrograph of cross-section of skin incision of olive flounder Paralichthys olivaceus fed the experimental diets. A, initial before incision; B, immediately after incision; C, 3 days after incision of the control fish; D, 3 days after incision of PHY0.025% fish; E, 7 days after incision of the control fish; F, 7 days after incision of PHY0.025% fish; G, 16 days after incision of the control fish; H, 16 days after incision of PHY0.025% fish; EI, epidermis layer; DI, dermis layer; ML, muscle layer; Arrows point to incision site; Staining AB-PAS (pH 2.5). Scale bars=200 µm.

도는모든실험구사이에유의적인차이가없었다

(Table 4). E.

tarda

PHY0.025

(56.8%)

(40.9%)

(Fig. 2).

고 찰

흥미롭게도본연구에서사료내

phytosterol

Ig, lysozyme, GPx

Table 4. Hematological parameters of olive flounder Paralichthys olivaceus fed the experimental diets for 12 (Exp-1) and 8 weeks (Exp-2), respectively. The experimental diets were prepared with supplementing phytosterol by 0, 0.025, 0.05, 0.1, 0.2 and 0.4% (Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4)

Hematocrit (%) Hemoglobin (g/dL) AST¹ (U/L) Total protein (g/dL) Cholesterol (mg/dL) Exp-1 (Initial body weight, 68.9g)

Con 35.2±3.68 4.44±0.30 40.4±7.99^a 6.08±0.35 210±12.6^ab

PHY0.025 33.4±5.46 4.57±0.36 26.7±10.0^bc 6.33±0.74 240±38.5^a

PHY0.05 33.8±4.92 4.99±0.45 20.5±6.01^c 6.58±0.10 228±7.91^ab

PHY0.1 37.2±3.33 5.11±0.45 24.3±3.10^bc 6.69±0.86 219±26.5^ab

PHY0.2 37.1±4.35 4.55±0.51 27.1±1.41^bc 6.45±0.41 200±19.7^b

PHY0.4 35.0±8.92 4.35±0.83 36.1±12.8^ab 6.38±0.49 223±18.2^ab

Exp-2 (Initial body weight, 16.5g)

Con 21.4±1.79 3.86±0.44 20.0±2.87 4.04±0.34 -

PHY0.025 24.1±2.14 4.04±0.32 18.9±6.40 4.04±0.30 -

PHY0.05 23.1±1.94 3.91±0.36 23.6±4.29 3.91±0.38 -

PHY0.1 24.1±1.04 4.11±1.00 30.0±1.76 4.38±0.56 -

PHY0.2 22.9±0.76 5.15±2.13 24.7±9.01 4.11±0.06 -

1Aspartate aminotransferase. Values are mean of quadruplicates and presented as mean±SD. Values with different superscripts in the same column are significantly different (P<0.05). The lack of superscript letter indicates no significant differences among treatments.

Table 3. Growth performance and feed utilization of olive flounder Paralichthys olivaceus fed the experimental diets for 12 (Exp-1) and 8 weeks (Exp-2), respectively. The experimental diets were prepared with supplementing phytosterol by 0, 0.025, 0.05, 0.1, 0.2 and 0.4%

(Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4)

FBW (g)¹ WG (%)² FCR³ PER⁴ Survival

Exp-1 (Initial body weight, 68.9g)

Con 179±29.7 160±43.4 1.37±0.51 1.65±0.47 82.5±5.00^b

PHY0.025 198±11.8 186±16.8 1.18±0.05 1.73±0.08 91.7±8.39^a

PHY0.05 189±10.1 174±14.2 1.17±0.08 1.78±0.13 95.8±3.19^a

PHY0.1 181±17.6 163±25.4 1.30±0.10 1.60±0.13 92.5±1.67^a

PHY0.2 187±22.7 171±34.4 1.35±0.31 1.59±0.31 95.0±6.38^a

PHY0.4 174±15.4 153±21.5 1.28±0.12 1.62±0.15 88.3±1.92^ab

Exp-2 (Initial body weight, 16.5g)

Con 56.7±1.60 245±12.8 0.91±0.05^a 2.10±0.11^b 99.1±1.79

PHY0.025 59.0±1.51 261±8.23 0.81±0.05^b 2.34±0.15^a 99.1±1.79

PHY0.05 57.7±0.89 253±4.77 0.87±0.03^ab 2.18±0.08^ab 100±0.00

PHY0.1 57.8±1.71 254±8.06 0.89±0.05^ab 2.18±0.13^ab 98.8±2.06

PHY0.2 56.2±1.59 240±12.6 0.92±0.06^a 2.05±0.13^b 97.6±4.12

1Final body weight. ²Weight gain (%)=(final weight-initial weight)×100/initial weight. ³Feed conversion ratio=feed intake/wet weight gain.

4Protein efficiency ratio=wet weight gain/total protein given. Values are mean of quadruplicates and presented as mean±SD. Values with different superscripts in the same column are significantly different (P<0.05). The lack of superscript letter indicates no significant differ- ences among treatments.

났다

. Phytosterol

β-sitosterol, campesterol, stig- masterol, stigmastanol

(Gilman et al., 2003).

β-sitosterol

phytosterol

(50-55%)

(Orrego et al., 2010),

(Desai et al., 2009),

(Gupta et al., 1980;

Bouic, 2001; Nirmal et al., 2012),

(Yoshida and Niki, 2003)

,

(Zhao et al., 2009). β-sitosterol

T

(Bouic et al., 1996). Phytosterol

pro-inflammatory cytokine

TNF-α

(Gupta et al., 1980, Bouic, 1996, Bouic and Lamprecht, 1999), anti-inflammatory cyto- kines (IL-10, NF-kB)

(Valerio and Awad, 2011)

.

,

phytosterol

(Kim et al., 2007),

(Yuan et al., 2007),

(Wu et al., 2010)

.

phytosterol

(Hu et al., 2017).

phytos-

terol

키는것으로보아사료내면역증강제로써의이용가능성이매 우높다고판단된다

.

대두사포닌 등의 식물추출물

(extract)

되면오히려생물에부정적인영향을끼칠수있다

(Gathumbi

et al., 2002; Flaoyen et al., 2004; Makkar et al., 2007). Eu- ropean sea bass (27-283 g)

phytosterol

(0.5-1%)

,

(Couto

et al., 2015a, 2015b). Gilthead sea bream (12.5-112 g)

phytosterol

(0.5-1%)

(Couto et al., 2014a, 2014b).

phytosterol

(0.14-0.19%)

치지않는것으로보고되었다

(Liland et al., 2013).

phytosterol (0.025-0.4%, 86

)

(hematocrit, hemoglobin, total protein)

.

phytosterol

(0.025-0.2%)

경우넙치의혈중

AST

났다

(

1). AST

,

노산대사에관여하는효소이다

.

AST

의괴사가일어날경우증가하는것으로알려져있다

(Yousafzai

and Shakoori, 2011). Phytochemical

African catfish Clarias

lazera

로보고되었다

(Mahmoud and AI-Salahy, 2004).

(total serum protein, globulin, albumin)

(Wu et al., 2007; Xie et al., 2008). Ji et al. (2007a)

phytochemical (herb extracts)

.

(Ji et al., 2009).

phytosterol (0.025-0.4%)

,

.

본연구에서사료내

phytosterol

.

Table 5. Apparent digestibility coefficients (ADC) of dry matter and protein in diets for olive flounder (Exp-1) (% of ADC). The experimental diets were prepared with supplementing phytosterol by 0, 0.025, 0.05, 0.1, 0.2 and 0.4% (Con, PHY0.025, PHY0.05, PHY0.1, PHY0.2 and PHY0.4).

ADCd¹ ADCp²

Con 73.9±0.27^d 87.5±0.53^d

PHY0.025 72.9±0.58^d 88.1±0.25^cd

PHY0.05 77.1±0.71^c 89.1±0.38^ab

PHY0.1 71.1±1.15^e 88.4±0.46^c

PHY0.2 80.4±0.11^a 88.7±0.16^bc

PHY0.4 78.8±0.33^b 89.4±0.19^a

1Apparent digestibility coefficient of dry matter (%)=100-100×(%

of Cr₂O₃ in diet/% of Cr₂O₃ in feces). ²Apparent digestibility coef- ficient of protein (%)=100-100×(% of Cr₂O₃ in diet/% of Cr₂O₃ in feces)×(% of protein in feces/% of protein in diet). Mean values of triplicate groups. Values are presented as mean±SD. Values in the same column having different superscript letters are significantly different (P<0.05).

Fig. 2. Survival of olive flounder Paralichthys olivaceus fed the ex- perimental diets for 8 weeks during the challenge with Edwardsi- ella tarda (Exp-2). The fish were injected with E. tarda suspension containing 1×10³ CFU mL^-1. Quadruplicate groups of fish were fed with one of the experimental diets two times a day during the chal- lenge period. The diets were added with graded levels of phytoster- ol by 0, 0.025, 0.05, 0.1 and 0.2% (designated as Con, PHY0.025, PHY0.05, PHY0.1 and PHY0.2, respectively).