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Effects of Hypochlorous Acid, Calcium Chloride and Phosphoric Acid in a Highly Saline Solution on Cell Death Rate and Growth Rate of Porphyra yezoensis and Ulva intestinalis

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한수지 51(6), 682-687, 2018

682

Copyright © 2018 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815 Korean J Fish Aquat Sci 51(6),682-687,2018

Original Article

서 론

(Laver Pyropia sp.)

우리나라를포함해서전세계적으로 즐겨먹는기호품으로서탄수화물과단백질을각각

40%

30-40%

정도를함유하고있으며

,

탄수화물의대부분은

다당류에속하는식물성섬유질이어서다이어트식품으로도 광받고있다

(Jimenez-Escrig and Sanchez-Muniz, 2000).

또한 칼슘

,

마그네슘

,

요오드

,

철분

,

아연망간등과같은무기질

10%

함유하고있어국내해조류중에서도가장많이

비되고있다

(KTSPI, 2017).

생산량에있어서도해조류 산량

120

만톤

40

만톤

(

33%)

생산량으로

,

단일 품종으로는해조류양식산업에서번째로부분을차지하

있다

(FAO, 2016).

이에따라관련수출금액은

2007

600

억원

, 2011

1600

억원

, 2016

년에는

3500

억으로매년 출이증대되어전체식품

3

번째로높은수출액을기록하였

(KTSPI, 2017).

김양식은크게지주식

,

부유식으로구분되고

,

부유식은다시 뜸틀식과 침지식으로구분된다

.

전통방식인지주식으로양식 하는경우노출선

5

시간지점에서자연노출에의해파래

,

규조 잡조를탈락시켜최고품질의김을생산하는방법이지만 생산량이많지않다

.

부유식의하나인뜸틀식은생산량은많지 뜸틀을뒤집어서인위적인간출로잡조구제를하게되기 문에일손이많이든다

.

그리고침지식의경우

24

시간해수에 담가져있기때문에생산량은가장많지만파래

,

매생이

고염수 처리제에 차아염소산, 염화칼슘 및 인산 첨가가

방사무늬김( Pyropia yezoensis)과 창자파래(Ulva intestinalis)의 사세포율과 엽체 성장에 미치는 영향

권오남·윤영랑 1 ·신일식 2

강릉원주대학교 동해안생명과학연구소, 1호남남서부김활성처리제사업협동조합, 2강릉원주대학교 해양식품공학과

Effects of Hypochlorous Acid, Calcium Chloride and Phosphoric Acid in a Highly Saline Solution on Cell Death Rate and Growth Rate of

Porphyra yezoensis and Ulva intestinalis

O-Nam Kwon, Yeoung-Rang Yun

1

and Il-Shik Shin

2

East Coastal Life Science Institute, Gangneung-Wonju National University, Gangneung 25457, Korea

1Honam South-Eastern Laver Complex Solution Business Cooperation, Haenam 59031, Korea

2Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung 25457, Korea

We investigated the effects of a highly saline solution (HS) containing hypochlorous acid, calcium chloride (CaCl 2 ), and phosphoric acid (H 3 PO 4 ) on cell death and growth rate of laver Porphyra yezoensis and green laver Ulva intesti- nalis . Cell death rates of laver treated with HS and HS plus hypochlorous acid (HS + HOCl) in the harvesting stage were less than 0.5%, and there were no significant differences between the HS and HS + HOCl treatments. However, cell death of green laver treated with HS + HOCl in the harvesting stage was greater than 81.2%. These results indi- cate that the addition of HOCl is highly effective to eradicate noxious green laver without causing damage to laver.

The addition of HOCl and H 3 PO 4 to HS did not increase the area or weight of laver blades. A combination treatment of CaCl 2 and HS, however, significantly increased the area and weight of laver lades compared to controls (P<0.05).

Key words: laver, green laver, hypochlorous acid, calcium chloride, phosphoric acid

*Corresponding author: Tel: +82. 33-640-2346 Fax: +82. 33-640-2340 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 20 November 2018; Revised 4 December 2018; Accepted 11 December 2018 저자 직위: 권오남(연구교수), 윤영랑(이사장), 신일식(교수)

https://doi.org/10.5657/KFAS.2018.0682

Korean J Fish Aquat Sci 51(6), 682-687, December 2018

(2)

차아염소산, 칼슘 및 인산의 첨가가 김의 성장에 미치는 영향

683

잡조와규조류뻘꼽의부착이많아서김의품질이저하되 단점이있다

(Song et al., 1993).

침지식의단점을보완하 위해서활성처리제처리로잡조를제거하여김의품질을 상시키고있다

.

이러한김의품질향상을위한활성처리제로서

1980

년대부터

35%

염산인무기산을주로사용하여왔는데

,

기산은인체에대한독성과부식성이강하며

,

해양에배출되면 어패류가폐사하게되고해양생태계를파괴시키는좋지

영향을미친다는연구결과

(MAFF, 1995)

로부터농림수산식

품부

(

해양수산부

)

1998

년부터고시로지정된유기산을 성분으로하는활성처리제를개발하여무기산의이용을규제하 있다

(MIFAFF, 2008).

하지만유기산을주성분으로하는 성처리제는무기산에비해사용량이월등히많아서김양식 업인들에게외면받고있는실정이다

.

이에 새로운 활성처리제에 대한 수요가 급증하고 있어

2017

9

해양수산부에서는소금을주성분으로하는고염수 처리제를고시에적용하여사용하도록권장하고있다

.

고염 수처리제는소금을주성분으로하면서잡조제거율을높이기 위해서차아염소산을첨가하고있으며

,

엽체의성장을촉진

하기위해서칼슘과인산을첨가하고있다

(MOF, 2017).

하지

차아염소산

,

칼슘인산의함유가고염수처리제에서의 할이확실하게밝혀져있지않으며이에대한연구도거의없는 실정이다

.

이에연구에서는고염수처리제

(high salty solu- tion, HS)

차아염소산

(hypochlorous acid, HOCl),

염화칼슘

(calcium chloride, CaCl

2

)

인산

(phosphoric acid, H

3

PO

4

)

가가김의사세포율과성장에미치는영향을규명하기위하여 정제염

10%,

염산

(HCl) 9.0%

함유한고염수처리제에차아 염소산을첨가

,

김과파래엽체의사세포율변화를조사하였으

,

또한차아염소산

,

염화칼슘인산의첨가가엽체의 장에미치는영향을조사하였다

.

재료 및 방법

시료

실험에사용된방사무늬김

(Laver Pyropia yezoensis)

창자 파래

(Green laver Ulva intestinalis)

2016

12

월에전라남도 해남군황산면한자리소재김양식장의김발에서채취하였다

.

유엽기에는엽체를기준으로

1.5 cm

이하의김과파래가 착되어있는김발을채취하였으며

,

수확기

(

성엽기

)

에는

15

cm

이상의엽체와김발에붙은파래를채취하여해남군옥천공

단의대성케미칼공장의연구실로옮겨서실험하였다

. 사세포율 측정

차아염소산첨가에따른김과파래의사세포율은사전실험 에서정해진

10%

정제염과

9.0%

염산

(HCl)

함유하는 고염수처리제

(high salty solution, HS)

HS

유효염소농도

200 ppm

강산성차아염소산수

(strong acidic hypochlorous

water)

첨가하여사전실험에서정해진차아염소산의최종

농도가

4%

되도록조절한처리제

(HS containing HOCl,

HS+HOCl)

제조

(Table 1)

하여측정하였다

.

실험은일정 크기로절단된김과파래엽체

3

이상을

10, 20

40

석된각각의처리제로김양식장에서처리하는방법과동일하

15

초간처리한

,

즉시멸균해수로세척하였다

.

세척한 체들은

0.1% erythrosine

으로염색하여붉은색으로염색된

(

사세포

)

측정하였다

.

염색된사세포율의측정은

100

200

배율의광학현미경

(Leica CME, Wetzlar, Germany)

엽체별

3

이상의사진을촬영

(

스마트폰거치대활용

)

하여

ImageJ (NIH, Bethesda, USA)

프로그램으로전체면적당사세 면적을계산하여사세포율을백분율

(%)

나타내었다

. 엽체 성장률 측정

차아염소산

,

칼슘

,

인산의첨가가김의엽체성장에미치는 향을조사하기위하여

10%

정제염과

9%

염산을함유하는 대조구

(control)

각각다른농도의차아염소산

,

칼슘

,

인산을 첨가하여활성처리제를제조하였다

(Table 2).

김의엽체성장 측정은엽체를

1 cm×1 cm

크기로절단하고

,

절단한엽체를 각각의시료용액에

15

초간침지처리한

,

깨끗한해수로 척하고멸균해수를넣은

250 mL

삼각플라스크에넣어형광등 으로명도를

2,500 lux

조정한

15℃

인큐베이터

(JeioTech, Seoul, Korea)

에서

, 1

3

이상교반하면서배양하였다

.

배양

1

,

위의침지처리를다시

1

실시한같은조건에서

1

주일간배양하였다

.

침지처리는실험시작할때와배양

1

2

실시하였으며

,

엽체꼬임으로측정하기힘들기 전인배양

14

후에엽체의면적과중량을측정하였다

. 통계처리

연구에서 모든실험은반복으로 행하였으며

,

수치 실험값의평균값으로나타내었다

.

통계프로그램은

SPSS version 21 software (SPSS Inc., Chicago, IL, USA)

에서

one- way ANOVA

Duncan’s multiple range test (10)

사용하여

Table 1. Composition (%) of high salty complex solution used for measurement of death cell ratio of Laver Pyropia yezoen- sis and Green laver Ulva intestinalis blade

Component

Concentration of component (%) High salty solution

(HS) HS containing HOCl (HS+HOCl)

Purified salt 10.0 10.0

HCl 9.0 9.0

HOCl 0.0 4.0

Water1 81.0 81.0

1

Underground water without hypochlorous acid in Okchun

industrial complex in Haenam-Gun, Jeollanam-do Province.

(3)

권오남

윤영랑

신일식

684

(Duncan, 1955) P<0.05

에서유의성을조사하였다

.

결과 및 고찰

사세포율

고염수처리제

(HS)

차아염소산 첨가에 따른 방사무늬김

(Laver Pyropia yezoensis)

창자파래

(Green laver Ulva in- testinalis)

사세포율을 유엽기와 성엽기

(

수확기

)

구분하

조사한결과는

Fig. 2

같다

. 10

배와

40

배로희석한

HS

처리한유엽기김의사세포율은각각

32.1%, 3.1%

이었으

, 10

배와

40

배로희석한

HS+HOCl

처리구의사세포율은

35.2%, 5.0%

, HS

처리구와유사한경향을나타내었으며 유의적인차이는없었다

(P<0.05).

반면김의유엽기에출현한 파래의경우

, 10

배와

40

배로희석한

HS

처리구의사세포율은 각각

52.4%, 7.2%

이었으나

10

배와

40

배로희석한

HS+HOCl

처리구의사세포율은각각

79%, 11%

HOCl

첨가한처리

Table 2. Composition (%) of high salty complex solutions used for measurement of growth rate of Laver Pyropia yezoensis leaves in this study

Component

Final concentration of component (%) Control Solution 1 Solution

2 Solution

3 Solution

4 Solution

5 Solution

6 Solution

7 Solution

8 Solution

9 MOF’s Official notification1

Purified salt 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0-13.0

HCl 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 8.5-9.5

HOCl -2 1.0 2.0 4.0 - - - - - - 0.25-4.0

H3PO4 - - - - 1.5 2.7 3.8 - - - 0.1-0.6

CaCl2 - - - - - - - 0.1 0.3 0.5 1.5-4.0

Water3 81.0 80.1 81.0 81.0 80.9 80.7 80.5 79.5 78.3 77.2 68.9-79.7

1

Standards of Ministry of Oceans and Fisheries.

2

Not added.

3

Underground water without hypochlorous acid in Okchun industrial complex in Haenam-Gun, Jeollanam-do Province.

Fig. 1. Change of Laver Pyropia yezoensis and Green laver Ulva intestinalis cells by treatment with HS+HOCl on early and harvest stage of Laver. HS, High salty solution.

Laver Treated laver by HS+HOCl

Treated green laver by HS+HOCl Green laver

a 0 20 40 60 80 100

0 20 40 60 80 100

c

10

c

10

a 40

ab

40 c

10

d

10

a

40

ab

40 b

20 HS

b

20 HS

b

20 HS +HOCl

c

20 HS +HOCl Control

(a)

a

Control (c)

a

10

d

10

a 40

b

40 a 10

d

10

a 40

d

40 Control

a

20 HS

c

20 HS

a

20 HS +HOCl

d

20 HS +HOCl a

Control (b)

a (d)

Early stage Harvest stage

Death ratio of green laver (%) Death ratio of laver (%)

(4)

차아염소산, 칼슘 및 인산의 첨가가 김의 성장에 미치는 영향

685

구의사세포율이유의적으로높게나타났다

(P<0.05).

그리고

HS+HOCl

처리구의경우

,

김보다파래의사세포율이

2

높게나타났다

.

성엽기김의경우

HS

처리구와

HS+HOCl

처리구모두

세포율이

0.5%

이하로처리구간의유의적인차이는없었다

.

그러나김의성엽기에출현한파래의경우

, 10

배와

40

배로 석한

HS

처리구의 사세포율은각각

80.2%, 37.9%

이었으나

HS+HOCl

처리구에서는희석배수와관계없이

81.2%

이상의

높은사세포율을나타내어

, HOCl

첨가한처리구의사세포율

HS

처리구보다유의적으로높게나타났으며

(P<0.05),

파래 구제효과도뛰어난것을있었다

.

김은우리나라를포함하여극동아시아

(FAO, 2016)

뿐만아니 뉴질랜드

(Liu and Gordon, 1987)

남아프리카

(Griffin et

al., 1999)

에서도양식되고있으며특히국내김은일본과북미

포함하여

2017

수출량이

4

5000

달러에달하기도

였다

(KMI, 2017).

이들김의양식은바다양식장에김발을

치하고김발에붙은포자가성장하여김발설치

1.5-2

개월

이면수확할있을만큼성장한다

.

하지만바다환경은영양 등의조절이되기때문에

,

이들의성장과저해실험을

해서는조절된환경내에서실험밖에없다

.

연구에서 고염수처리제에차아염소산의첨가에따른김과김발부착 생물인파래의사세포율변화와고염수처리제에함유되어 차아염소산

,

칼슘그리고인산이엽체의성장에미치는 영향에대한조사를하였다

.

국내의경우

,

김의붉은갯병감염 여부에대한차아염소산과칼슘의영향에대한연구는있지만

(Kim, 2013),

파래구제효과또는엽체의성장등에대한연구

없다

. Mckenna and Davies (1988)

미생물을사멸시킬 있는차아염소산의최소농도를

0.104-0.156 ppm

으로제시하 있다

.

연구에서는차아염소산을

2%

함유한고염수처리제 사용하였는데

, 10

배에서

40

희석하는과정에서차아염소 산의최종농도는

0.1-0.4 ppm

으로희석된다

.

경우

Mckenna and Davies (1988)

제시한단세포생물인미생물살균의 소농도범위에해당하여단세포층엽체

(McArthur and Moss, 1977)

창자파래

(Ulva intestinalis)

대한살균력을예상할 있는농도가된다

.

결과는

Dukan and Touati (1996)

장균

(Escherichia coli)

대상으로했던연구에서도같은결과 보고되어단세포생물인대장균의사멸과양식장의해적 생물인파래의사세포율이유사한경향을나타내었다

.

창자파

Fig. 2. The death cell ratio of Laver Pyropia yezoensis and Green laver Ulva intestinalis by treatment with HS and HS+HOCl at early and harvest stage of laver. Values with different characters are significantly different (P<0.05). (a) early stage of laver; (b) harvest stage of laver; (c) early stage of green laver; (d) harvest stage of green Laver. HS, High salty solution.

Laver Treated laver by HS+HOCl

Treated green laver by HS+HOCl Green laver

a 0 20 40 60 80 100

0 20 40 60 80 100

c

10

c

10

a 40

ab

40 c

10

d

10

a

40

ab

40 b

20 HS

b

20 HS

b

20 HS +HOCl

c

20 HS +HOCl Control

(a)

a

Control (c)

a

10

d

10

a 40

b

40 a 10

d

10

a 40

d

40 Control

a

20 HS

c

20 HS

a

20 HS +HOCl

d

20 HS +HOCl a

Control (b)

a (d)

Early stage Harvest stage

Death ratio of green laver (%) Death ratio of laver (%)

(5)

권오남

윤영랑

신일식

686

래는단세포층엽체인반면

,

방사무늬김은다세포층엽체

(Ueki et al., 2008)

외부환경요인에의한영향을적게받는다

.

따라 고염수처리제에차아염소산을적정농도첨가하였을 에서는유엽기와수확기모두에서차아염소산을첨가하지 고염수처리제와사세포율에유의적인차이를보이지않았 지만

,

파래의경우희석농도에따라정도의차이는있었지만 아염소산의첨가에따른사세포율이유의적으로높게나타났

(Fig. 2).

엽체 성장률

소금과무기산만을 함유한고염수처리제

(control)

차아염 소산

,

칼슘

,

인산의첨가가김의엽체성장

(

면적증가

)

미치 영향을조사한결과는

Fig. 3

같다

.

배양초기의엽체

적은

100.0±0.00 mm

2이었으며차아염소산과인산의첨가는

엽체의면적증가에유의할만한효과를나타내지않았다

(P>0.05).

특히인산의경우

, 2.7%

이상첨가구

(solution 5, 6)

에서는대조구보다오히려면적이감소하는경향을나타내었

.

칼슘첨가군의경우

, 0.1%

첨가구

(solution 7)

0.3%

첨가

(solution 8)

엽체면적은각각

179.2, 204.7 mm

2으로 조구

(135.0 mm

2

)

보다유의적으로높은면적증가

(P<0.05)

보였으나

, 0.5%

첨가구

(solution 9)

대조구보다오히려면적 감소하였다

.

차아염소산

,

칼슘

,

인산의첨가가김의엽체성장

(

중량증가

)

미치는영향을조사한결과는

Fig. 4

같다

.

대조구의

중량은

1.7 mg/blade

이었으며차아염소산

(solution 1, 2, 3)

인산

(solution 4, 5, 6)

첨가하였을엽체의중량은

1.4-1.9 mg/blade

유의할만한증가효과를나타내지않았다

(P>0.05).

그러나칼슘첨가군의경우

,

첨가구

(solution 7, 8, 9)

엽체중량이각각

2,8, 3.0, 2.1 mg/blade

첨가구모두

대조구

(1.7 mg)

보다유의적으로중량이증가하였다

(P<0.05).

해조류의배양촉진을위해서칼슘

,

인과같은무기질을 지에 포함하는것은 이미알려져있다

(Kuffner and Paul, 2001). Iwasaki (1967)

0.1-5 mg%

인산과

10-100 mg%

칼슘첨가가김의성장을극대화되는것으로보고한있는데

,

연구의결과도이와유사한경향을나타내었다

.

하지만인산 경우

, 2.7%

이상첨가구

(solution 8, 9)

에서오히려성장저해 확인되었는데

,

이는주로식물체배양에는

Na

2

HPO

4형태 인산이제공되지만연구에서

H

3

PO

4형태로제공되었 것에기인한것으로판단된다

.

하지만

(P)

공급원에따른 성장촉진혹은저해와관련된다양한실험들이없기에향후 많은실험을필요가있을것으로판단된다

.

또한고염수처리제에기능성을부여하기위해차아염소산

(

아염소산에의한파래의선택적괴사와병원성세균의살균

),

슘과

(

김엽체영양공급

)

추가하였는데이들의과도한첨가 시험용액에침전이생기는결과를초래하여경우의수를 한정많게수가없었다

.

이에

5

가지원료

2

가지

(

정제염

,

)

농도를고정하고

, 3

가지

factor (

차아염소산

,

염화칼슘

,

)

영향을

one-way ANOVA test

측정하였다

.

추후인산의 양을줄이고

,

칼슘의양을늘려김엽체성장에도움을주는최적 배합비를찾는연구가필요할것으로사료된다

.

영양염의부족과과잉모두

(Pyropia umbilicalis)

광합성 호흡에저해를가져올있다

(Wiencke and Läuchli, 1980).

특히지나친농도의영양염은세포액포

(vacuoles)

크기

줄여서성장에저해를주게되어배지를사용하는경우지나 치게과도한영양염공급을지양하도록하고있다

.

또한

Tait et al. (1990)

(Pyropia umbilicalis)

성장에있어서질소부족 피해야하며

,

최종수확기에있어서탄소원공급을위한 기완충용액

(organic buffer)

추가가수확량증가에도움이

Fig. 4. The effects of addition of HOCl, CaCl

2

and H

3

PO

4

to HS on increase of Laver Pyropia yezoensis blade weight.

Values with different characters are significantly different (P<0.05). HS, High salty solution.

b b

ab b b

ab ab

c d

a

0 50 100 150 200 250

Initial ControlSol. 1 Sol. 2 Sol. 3 Sol. 4 Sol. 5 Sol. 6 Sol. 7 Sol. 8 Sol. 9 Blade area (mm

2

)

Tested solution

ab ab

ab ab ab

ab a

c d

b

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Initial ControlSol. 1 Sol. 2 Sol. 3 Sol. 4 Sol. 5 Sol. 6 Sol. 7 Sol. 8 Sol. 9

Blade weight (mg)

Tested solution

Fig. 3. The effects of addition of HOCl, CaCl

2

and H

3

PO

4

to HS on increase of Laver Pyropia yezoensis blade area. Values with different characters are significantly different (P<0.05).

HS, High salty solution.

b b

ab b b

ab ab

c d

a

0 50 100 150 200 250

Initial ControlSol. 1 Sol. 2 Sol. 3 Sol. 4 Sol. 5 Sol. 6 Sol. 7 Sol. 8 Sol. 9 Blade area (mm

2

)

Tested solution

ab ab

ab ab ab

ab a

c d

b

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Initial ControlSol. 1 Sol. 2 Sol. 3 Sol. 4 Sol. 5 Sol. 6 Sol. 7 Sol. 8 Sol. 9

Blade weight (mg)

Tested solution

(6)

차아염소산, 칼슘 및 인산의 첨가가 김의 성장에 미치는 영향

687

다고하였다

.

따라서김의성장촉진을위해서고염수처리제 아니라김발에처리하게되는영양제에대한과학적접근도 필요할것으로사료된다

.

하지만국내에서양식되고있는김은 연구에서사용한방사무늬김뿐만아니라전라남도를중심 으로개량되고있는종과지역별로김의종류와특성이다르기 때문에모든지역과모든종류에서같은결과를보이는것은 아니기때문에보다다양한연구결과들이추가적으로필요하 다고판단된다

.

사 사

논문은

2017

년도강릉원주대학교전임교원연구년지원에

의하여수행되었으며

,

이에감사드립니다

.

References

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erichia coli: resistance, DNA damage, and comparison

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수치

Table 1. Composition (%) of high salty complex solution used  for measurement of death cell ratio of Laver Pyropia  yezoen-sis and Green laver Ulva intestinalis blade
Fig. 1. Change of Laver Pyropia yezoensis and Green laver Ulva intestinalis cells by treatment with HS+HOCl on early and harvest  stage of Laver
Fig. 3. The effects of addition of HOCl, CaCl 2  and H 3 PO 4  to  HS on increase of Laver Pyropia yezoensis blade area

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