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Dietary effect of probiotics on innate immune response and disease resistance in aquaculture fish

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Abstract
국내의 어류 양식 산업은 크게 성장하고 있지만, 양식 도중 발생하는 어류질병이 양식 산업 발전에 심각한 악 영향을 미치고 있다. 일반적으로 육상수조 양식의 경우 단위 면적당 생산량을 극대화 시키기 위해 고밀도 사육을 하고 있는데, 이러한 고밀도 사육은 증체율 저하 및 각종 질병 발생의 직접적인 원인을 제공하여 폐사에 따른 경제적 손실을 일으킨다. 양식 산업에서의 어류질병의 발생과 확산은 생산성을 현저하게 감소시킬 수 있으며, 이를 예방하기 위해 어류 양식장에서는 어류질병 관리 전략으로 항생제를 광범위하게 사용하고 있다. 하지만 일부 양식장에서는 어류질병의 치료 및 예방의 목적으로 항생제를 오남용 하고 있어 2차적인 문제를 야기시키며, 그 중 가장 대표적인 것이 항생제 내성균주와 항생제 잔존 어류이다.
지난 20년동안 어류 질병의 예방 및 관리에 있어서 probiotics의 적용은 많은 관심을 받고 있다. Probiotics로 인정받은 대부분의 미생물은 약물이나 식품으로 판매되고 있으며 여러 가지 측면에서 안전성이 확인되었다. 항생제보다 효과적이고 안전한 probiotics는 생물학적 제어로 그 쓰임이 증가하고 있다. 생균제로 가장 일반적으로 사용되는 유산균은 장내 미생물의 균형을 개선하는 probiotics로써 사용되고 있으며, 주로 Bacillus sp., Pediococcus sp., Lactobacillus sp., Bifidobacterium sp., Lactococcus sp. 등이 있으며 현재까지 정장작용, 면역증강작용, 영양학적 가치 증진, 간경화 개선 작용, 항암 작용 등에 대한 연구가 진행 중에 있다.
이에 따라 본 연구에서는 전통 발효 식품에서 분리한 Bacillus amyloliquefaciens JFP-02를 양식 어류에 급이 하였을 때 일반 사료와 비교하여 나타나는 성장도, 비특이적 면역반응, 병 저항성을 확인하여 새로운 사료첨가제로써의 응용 가능성을 확인하고자 하였다.
제주민속오일시장에서 전통 발효식품인 된장, 새우젓, 오징어젓, 전어젓, 꽃멸치젓, 갈치속젓, 자리젓에서 총 8 종의 probiotics 후보 균주를 분리하였다. NCBI 의 BLAST 를 이용하여 분리된 균주의 염기서열을 확인한 결과 pediococcus acidilactici, Bacillus amyloliquefaciens, Lactobacillus parabuchneri, Enterococcus durans, Pediococcus pentosaceus, Lactobacillus sakei, Lactobacillus plantarum, Lactobacillus brevis 로 동정 되었다.
분리된 probiotics 후보균주 중, pH, 온도, 배양조건, 인공 위액, 인공 담즙산, 내염성, 내열성에 대한 내성을 확인한 결과 Bacillus amyloliquefaciens JFP-02 을 공시균주로 선정하였다. B. amyloliquefaciens JFP-02 의 형태학적 특성은 집락의 모양은 고르지 못하고, 집락의 중앙이 볼록 솟아 있으며, 백금이로 취할 때 점성을 띈다. 장방출주사전자현미경을 이용하여 관찰한 결과 단간균으로 길이는 1.575 um, 폭은 0.727 um 으로 나타났다. 생육조건은 saccharose, yeast extract, MgSO_4·7H_2O 를 배지에 첨가하여 pH 9 로 조정한 후 30℃에서 생육 시킬 때 활성이 가장 높았다.
2015 년도와 2016 년도에 넙치 양식장 18 개소에서 총 13 개의 어류질병세균을 확보하였다. 분리된 균주의 염기서열을 확인한 결과 Photobacterium damselae sub sp. 2 종, Edwardsiella tarda 2 종, Streptococcus parauberis 2 종, Streptococcus iniae, Tenacibaculum maritimum, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Staphylococcus caprae 가 동정되었다.
넙치 양식장에서 분리한 어류질병세균을 대상으로 B. amyloliquefaciens JFP-02의 배양 조건에 따른 항균활성을 측정한 결과, pH4~pH9와 10℃~50℃에서 T. maritimum, E. tarda 에서 가장 항균활성이 높았다. 또한 탄소원, 질소원, 무기염의 종류에 상관없이 T. maritimum, E. tarda, V. campbellii S. caprae 에 대하여 높은 항균활성을 보였다.
양식어류인 넙치, 돌돔, 강도다리를 대상으로 B. amyloliquefaciens JFP-02 를 급이하였을 때 나타나는 혈액학적 특성과 비특이적 면역반응, 어류질병에 대한 병 저항성 및 면역 유전자 발현량을 확인하였다.
넙치의 경우 8주동안 B. amyloliquefaciens JFP-02를 급이 시킨 후 혈액학적 분석 결과 hematocrit, total protein, glucose에서 컨트롤 그룹에 비해 유의적으로 증가하는 것을 확인한 반면, triglyceride, total cholesterol, AST, ALT는 컨트롤그룹에 비해 감소 하는 것으로 확인되었다. 비특이적 면역반응인 라이소자임 활성과 대식세포 활성에서는 실험 후반부터 컨트롤그룹에 비해 유의적으로 증가하였으며, S. iniae를 이용한 병 저항성 실험 결과 컨트롤그룹에서 85%의 폐사율을 보인 반면, B. amyloliquefaciens JFP-02의 그룹에선 폐사율이 45%로 약 절반의 폐사율을 보였다.
돌돔의 경우 10주동안 B. amyloliquefaciens JFP-02를 급이 시킨 후 hematocrit, total protein, glucose의 결과 컨트롤그룹에 비해 B. amyloliquefaciens JFP-02의 그룹에서 유의적으로 활성이 증가하였다. triglyceride, total cholesterol의 결과 컨트롤 그룹에 비해 유의적으로 감소 하였으며, AST와 ALT는 실험 초기부터 꾸준히 감소 하였다. 라이소자임과 대식세포의 결과 컨트롤 그룹에 비해 유의적으로 증가 하였으며, S. iniae를 이용한 병 저항성 실험 결과 컨트롤 그룹이 20일째 폐사율이 90%인 반면, B. amyloliquefaciens JFP-02의 그룹에서 35%의 폐사율을 나타냈다.
강도다리를 이용한 실험 결과 성장률에서는 4주동안 B. amyloliquefaciens JFP-02를 급이 하였을 때 혈액분석 결과 hematocrit, glucose, total protein, calcium은 컨트롤 그룹에 비해 유의적으로 증가하였으며, AST와 ALT는 컨트롤 그룹에 비해 감소하는 것을 확인하였다. 라이소자임 활성, 대식세포 활성은 컨트롤 그룹에 비해 유의적으로 증가하였으며, S. parauberis를 이용한 공격실험에선 컨트롤 그룹에선 20일째 75%의 폐사율을 보인 반면 B. amyloliquefaciens JFP-02그룹에선 45%의 폐사율을 보였다.
Real-time PCR을 이용하여 넙치의 TNFR-1과 IL-1b의 유전자 발현량을 확인한 결과 TNFR-1의 경우 신장을 제외한 장, 비장, 간에서 컨트롤 그룹보다 발현량이 적게 나왔으며, IL-1b의 경우 장과 간에선 컨트롤 그룹에 비해 적은 발현량을 보이고, 비장과 신장에서는 컨트롤 그룹에 비해 높은 발현량을 보였다. 돌돔의 경우 FAS의 경우 간에서 가장 많은 발현량을 보이는 반면, 신장에서 가장 적은 발현량을 보였다. Caspase 3의 경우 모든 장기에서 컨트롤에 비해 많은 발현량을 보였다. 강도다리의 경우 TNF와 IL-6의 유전자 발현량은 장에서 가장 많은 발현량을 보였으며, 신장에서 비교적 적은 발현량을 보였다.
본 연구에서는 제주도 전통발효 식품에서 분리한 B.amyloliquefaciens JFP-02가 probiotics가 가져야 할 특성을 지닌 것으로 사료되며, 배양조건에 따른 다양한 어류질병세균에 대한 항균활성을 확인할 수 있었다. 이러한 일련의 실험결과를 토대로 B. amyloliquefaciens JFP-02를 양식 어류에 적용하였을 비특이적 면역반응과 어류질병세균에 대하여 병 저항성을 가지는 것을 확인 하였으며, 향후 어류 양식산업에 도움이 될 것으로 사료된다.
The domestic fish aquaculture industry is growing greatly, but fish diseases that occur during the aquaculture have serious adverse effect on the development of the aquaculture industry. Generally high density breeding is carried out in order to maximize the production amount per unit area, but these high density breeding is accompanied by a decrease in the gain increase rate, occurrence of various disease to cause a direct economic loss The incidence and spreads of fish diseases in the fish aquaculture industry can significantly reduce productivity, and to prevent antibiotics was widely used in fish disease management strategies in fish farms (Smith et al., 1994). However, some fish farms abuse antibiotics for the purpose of treatment and prevention of fish diseases, causing secondary problems, the most representative of which is antibiotic-resistant strains and antibiotic-remaining fish (Karunasagar et al., 1994; Witte et al., 1999).
For the past decades, the application of probiotics in the prevention and management of fish disease has received more interest (Ganguly et al., 2010). Most probiotic microbes are marketed as drug or foodstuffs and safe application has been confirmed through long-term experience (Lim et al., 2011). Increased use of bacteria as biological controllers is effective and safer than antibiocis (Bansemir et al., 2006). The supplementation of fish diets with probiotics modulate specific functions of the gut and immune system and enhance disease protection (Gullian et al., 2004; Nayak., 2010). The most commonly used probiotics in aquaculture are Bacillus sp., Pediococcus sp., Lactobacillus sp., Bifidobacterium sp., Lactococcus sp (Abdel-Tawwab et al., 2008; Aly et al., 2008; Pal et al., 2007).
In this study, the compared to control feed when fed aquaculture fish with Bacillus amyloliquefaciens JFP-02 isolated traditional fermented foods, also growth performance, innate immune response, disease resistance confirmed the possibility of application as a new feed additive.
Eight species of probiotics candidate strains were isolated from Jeju Island traditional fermented foods: Doenjang, Saeujeot, fermented Squid, salted Damselfish, salted guts of Hairtail, salted Gizzard shad, salted Spratelloides gracilis. As a result of confirmed the nucleotide sequence of the isolated strains used BLAST of NCBI, it was identified by pediococcus acidilactici, Bacillus amyloliquefaciens, Lactobacillus parabuchneri, Enterococcus durans, Pediococcus pentosaceus, Lactobacillus sakei, Lactobacillus plantarum, Lactobacillus brevis (Table 5).
It`s resistance to artificial gastric juice, artificial bile acid, salt tolerance, heat resistance, confirms the characteristics that probiotics should have and confirm the optimum culture conditions Bacillus amyloliquefaciens JFP-02. B. amyloliquefaciens JFP-02 was the shape of colony was uneven, the center of colony was convex (Fig. 10) and loop draws a considerable viscosity when taken. As a result of observation used SEM, it was in the form of single bacillus, the length was 1.575 um, and the width was 0.727 um. The optimal culture conditions were the highest growth activity by added saccharose as a carbon source, yeast extract as a nitrogen source, MgSO_4·7H2_O as a mineral source to MRS medium at pH 9 and 30℃.
In 2015 and 2016 a total 13 species fish disease pathogens were isolated at 18 olive flounder aquaculture farms site. Confirmed the sequence of the isolated strains, Photobacterium damselae sub sp. 2 species, Edwardsiella tarda 2 species, Streptococcus parauberis 2 species, Streptococcus iniae, Tenacibaculum maritimum, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Staphylococcus caprae (Table 11, 14).
As a result of measured the antimicrobial activity according to the culture conditions of B. amyloliquefaciens JFP-02 for fish disease pathogens isolated from the olive flounder farm, T. maritimum, E. tarda at pH 4 to pH 9and 10℃ to 50℃ the antimicrobial activity was the highest. In addition, it showed highest antimicrobial activity against T. maritimum, E. tarda, V. campbellii S. caprae of carbon source, nitrogen source and mineral source.
Confirmed of the hematological characteristics, innate immune response, disease resistance, and immune genes expression level showed when B. amyloliquefaciens JFP-02 was fed for olive flounder, rock bream, and starry flounder of aquaculture fish.
In the case of olive flounder, confirmed that hematocrit, total protein, glucose increased significantly compared to the control group by hematological analysis after fed B. amyloliquefaciens JFP-02 for 8 weeks, it was confirmed that triglyceride, total cholesterol, AST, ALT decreased compared to the control group. Lysozyme activity and macrophage activity which is an innate immune response, increase significantly compared with the control group from the latter part of the experiment and showed mortality rate of 85% in the control group of the disease resistance experiment using S. iniae, than B. amyloliquefaciens JFP-02 group showed mortality rate of 45%.
In the case of rock bream, confirmed that hematocrit, total protein, glucose increased significantly compared to the control group by hematological analysis after fed B. amyloliquefaciens JFP-02 for 10 weeks, it was confirmed that triglyceride, total cholesterol in deceased significantly compared to the control group and AST, ALT decreased. Lysozyme activity and macrophage activity which is an innate immune response, increase significantly compared with the control group and showed mortality rate of 90% in the control group of the disease resistance experiment using S. iniae, than B. amyloliquefaciens JFP-02 group showed mortality rate of 35%.
In the case of starry flounder, confirmed that hematocrit, total protein, glucose, calcium increased significantly compared to the control group by hematological analysis after fed B. amyloliquefaciens JFP-02 for 4 weeks, it was confirmed that AST, ALT in deceased compared to the control group. Lysozyme activity and macrophage activity which is an innate immune response, increase significantly compared with the control group and showed mortality rate of 75% in the control group of the disease resistance experiment using S. parauberis, than B. amyloliquefaciens JFP-02 group showed mortality rate of 45%.
In the case of olive flounder, TNFR-1 the expression level was smaller than that in the intestine, kidney, and liver excluding the kidney, the expression level was lower in the case of IL-1b than in the control group, and in the spleen, kidney, the expression level was higher spleen and kidney than in the control group (Fig. 32). In the case of rock bream, FAS the expression level was highest than that in the liver, whereas the lowest expression level was showed in the kidney. In the case of Caspase 3, it showed high expression than the control in all organs (Fig. 37). In the case of starry flounder TNF and IL-6, the expression amount was showed highest in the intestine, and the expression amount in the kidney was comparatively lower (Fig. 42).
In this study, B.amyloliquefaciens JFP-02isolated from Jeju Island traditional foods had characteristic of probiotic, and it's possible to confirm the antimicrobial activity against various fish disease pathogens according to culture conditions. Series of experimental results, it was confirmed that B. amyloliquefaciens JFP-02 has an innate immune response and disease resistance against fish disease pathogens to aquaculture fish. It seems to be useful for the fish aquaculture industry in the future.
Author(s)
김동휘
Issued Date
2018
Awarded Date
2018. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000008423
Alternative Author(s)
Kim, Dong Hwi
Affiliation
제주대학교 일반대학원
Department
대학원 해양생명과학과
Advisor
허운수
Table Of Contents
국문초록 Ⅵ
LIST OF FIGURES Ⅹ
LIST OF TABLE ⅩⅠⅤ
Part Ⅰ. Characterization analysis of probiotics strains isolated from Jeju Island traditional foods 1
1.1 INTRODUCTION 1
1.2 MATERIALS AND METHODS 4
1.21 Isolated and storage of probiotics candidate strains 4
1.2.2 Sequence analysis of probiotics candidate strains 6
1.2.3 Antibiotics susceptibility test of probiotics candidate strains 7
1.2.4 Optimal culture conditions of probiotics candidate strains 8
1.2.4.1 Optimal culture conditions according to pH 8
1.2.4.2 Optimal culture conditions according to temperature 8
1.2.4.3 Optimal culture conditions according to carbon source 8
1.2.4.4 Optimal culture conditions according to nitrogen source 9
1.2.4.5 Optimal culture conditions according to mineral source 9
1.2.5 Resistance analysis of probiotics candidate strains 10
1.2.5.1 Resistance to artificial gastric juice 10
1.2.5.2 Resistance to artificial bile acids 10
1.2.5.3 Resistance to salt tolerance 10
1.2.5.4 Resistance to heat resistance 11
1.2.6 Probiotic strain selection 11
1.2.7 Isolated of probiotics strain 12
1.2.7.1 Phylogenetic analysis 12
1.2.7.2 Morphological analysis 12
1.2.7.3 Biochemical analysis 13
1.2.8 Isolated and purification of bacteriocin from probiotics strain 13
1.2.9 Tricine SDS-PAGE 14
1.2.10 Statistical analysis 14
1.3 RESULTS 15
1.3.1 Probiotics candidate strains 15
1.3.2 Sequence of probiotics candidate strains 16
1.3.3 Antibiotics susceptibility of probiotics candidate strains 18
1.3.4 Establish of optimal culture conditions for probiotics candidate strains 20
1.3.4.1 Establish of optimal culture conditions according to pH 20
1.3.4.2 Establish of optimal culture conditions according to temperature 21
1.3.4.3 Establish of optimal culture conditions according to carbon source 22
1.3.4.4 Establish of optimal culture conditions according to nitrogen source 24
1.3.4.5 Establish of optimal culture conditions according to mineral source 26
1.3.5 Establish of resistance conditions of probiotics candidate strains 28
1.3.6 Probiotics strain selection 29
1.3.7 Isolated of Bacillus amyloliquefaciens JFP-02 30
1.3.7.1 Phylogenetic characteristics 30
1.3.7.2 Morphological characteristics 36
1.3.7.3 Biochemical characteristics 38
1.3.8 Confirmation of bacteriocin by tricine SDS-PAGE 41
1.4 DISCUSSION 42
Part Ⅱ. Antimicrobial activity of Bacillus amyloliquefaciens JFP-02 on fish disease pathogens isolated from Jeju Island fish farm 45
2.1 INTRODUCTION 45
2.2 MATERIALS AND METHODS 47
2.2.1 Isolated of fish disease pathogens in 2015 47
2.2.2 Isolated of fish disease pathogens in 2016 51
2.2.3 Sequence analysis of fish disease pathogens 51
2.2.4 Antibiotics susceptibility test of fish disease pathogens 52
2.2.5 Antimicrobial activity of probiotics candidate strains against fish disease pathogens 52
2.2.6 Antimicrobial activity of B. amyloliquefaciens JFP-02 against fish disease pathogens according to optimal culture conditions 53
2.2.6.1 Antimicrobial activity according to pH variation 53
2.2.6.2 Antimicrobial activity according to temperature variation 53
2.2.6.3 Antimicrobial activity according to carbon source variation 54
2.2.6.4 Antimicrobial activity according to nitrogen source variation 54
2.2.6.5 Antimicrobial activity according to mineral source variation 55
2.2.7 Statistical analysis 55
2.3 RESULTS 56
2.3.1 Isolated of fish disease pathogens isolated from olive flounder farm in 2015 56
2.3.2 Sequence of fish disease pathogens isolated from olive flounder farm in 2015 59
2.3.3 Antibiotics susceptibility test of fish disease pathogens in 2015 60
2.3.4 Antimicrobial activity of probiotic candidate strains against fish disease pathogens 62
2.3.5 Isolated of fish disease pathogens isolated from olive flounder fish farm in 2016 64
2.3.6 Sequence of fish disease pathogens isolated from olive flounder farm in 2016 67
2.3.7 Antibiotics susceptibility test of fish disease pathogens in 2016 69
2.3.8 Antimicrobial activity of B. amyloliquefaciens JFP-02 according to optimal culture conditions 71
2.3.8.1 Effect of antimicrobial activity according to pH variation 71
2.3.8.2 Effect of antimicrobial activity according to temperature variation 73
2.3.8.3 Effect of antimicrobial activity according to carbon source variation 75
2.3.8.4 Effect of antimicrobial activity according to nitrogen source variation 77
2.3.8.5 Effect of antimicrobial activity according to mineral source variation 79
2.4 DISCUSSION 81
Part Ⅲ. Dietary effect of B. amyloliquefaciens JFP-02 on innate immune response and disease resistance in aquaculture fish 83
3.1 INTRODUCTION 83
3.2 MATERIALS AND METHODS 85
3.2.1.1 Fish (olive flounder (Paralichthys olivaceus)) 85
3.2.1.2 Experimental diet 85
3.2.2.1 Fish (rock bream (Oplegnathus fasciatus)) 87
3.2.2.2 Experimental diet 87
3.2.3.1 Fish (starry flounder (Platichthys stellatus)) 89
3.2.3.2 Experimental diet 89
3.2.4 Growth survey 91
3.2.5 Hematological analysis 91
3.2.6 Innate immune response of aquaculture fish 92
3.2.6.1 Lysozyme activity 92
3.2.6.2 Macrophage activity 92
3.2.7 Total RNA 93
3.2.8 cDNA 93
3.2.9 Real-time PCR 94
3.2.10 Challenge test 96
3.2.11 Statistical analysis 96
3.3 RESULTS 97
3.3.1.1 Growth of olive flounder 97
3.3.1.2 Hematological analysis of olive flounder 98
3.3.1.3 Innate immune response of olive flounder 101
3.3.1.4 Real-rime PCR expression of immune gene in olive flounder 103
3.3.1.5 Disease resistance of olive flounder 105
3.3.2.1 Growth of rock bream 106
3.3.2.2 Hematological analysis of rock bream 107
3.3.2.3 Innate immune response of rock bream 110
3.3.2.4 Real-rime PCR expression of immune gene in rock bream 112
3.3.2.5 Disease resistance of rock bream 114
3.3.3.1 Growth of starry flounder 115
3.3.3.2 Hematological analysis of starry flounder 116
3.3.3.3 Innate immune response of starry flounder 119
3.3.3.4 Real-rime PCR expression of immune gene in starry flounder 121
3.3.3.5 Disease resistance of starry flounder 123
3.4 DISCUSSION 124
CONCLUSION 127
REFERENCE 131
ACKOWLEDGEMENT 144
Degree
Doctor
Publisher
제주대학교 일반대학원
Citation
김동휘. (2018). Dietary effect of probiotics on innate immune response and disease resistance in aquaculture fish
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General Graduate School > Marine Life Sciences
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