FAA Composition and Content Variation in Different Pyropia yezoensis Cultivar in Harvest Period

Chuanming HU，Qinqin LU，Meiru ZHANG，Jianyi ZHU

1.Jiangsu Marine Fisheries Research Institute，Nantong 220067，China；

2.Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm，Nanjing 210014，China；

3.College of Food Science&Technology，Shanghai Ocean University，Shanghai 201306，China；

4.Changshu Institute of Technology，Changshu 215500，China

FAA Composition and Content Variation in Different Pyropia yezoensis Cultivar in Harvest Period

Chuanming HU1，2，3*，Qinqin LU1，Meiru ZHANG1，Jianyi ZHU4

1.Jiangsu Marine Fisheries Research Institute，Nantong 220067，China；

2.Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm，Nanjing 210014，China；

3.College of Food Science&Technology，Shanghai Ocean University，Shanghai 201306，China；

4.Changshu Institute of Technology，Changshu 215500，China

The contents of 16 kinds of free amino acids （FAAs）in 7 Pyropia yezoensis cultivars were determined by pre-column derivatization high performance liquid chromatographyin early，middle and late harvest period.The results showed that the total FAA content ranged from 48.71 to 62.05 mg/g among the 7 test materials.Among the 16 kinds of FAAs，Ala，Arg，Glu and Asp，four kinds of flavor amino acids，showed the highest contents，and their total content ranged from 36.05 to 53.96 mg/g，accounting for 69.11%-86.47%of the total FAA content.The total contents of flavor amino acids and FAAs all reached the peaks in all the test materials，except the control cultivar，at middle harvest period.The essential amino acid content was trended to be increased in all the test materials with the extended harvest period，and it reached the peak at the late harvest period.Sutong showed the highest flavor amino acid content and FAAs content，followed by Y-H002，Suyan and Sulian，while the control cultivar showed the lowest contents.

Pyropia yezoensis；Free amino acid；Harvest period；Cultivar

P yropia is a kind of original large red algae，and it is widely grown and distributed.Pyropia yezoensis and Pyropia haitanensis are important economic algae cultivars in China.Their life cycle consists of macroscopic frond （gametophyte）phase and microscopic filament（sporophyte）phase［1-2］.In 1953，Kurogi cultured filaments in a laboratory and obtained conchospores，and then obtained young fronds［3-4］.In the late 1950s，foreign scholars proposed the concepts of Pyropia cultivar and cultivated characters.Since then，the artificial cultivation techniques of Pyropia have been established，and the breeding of P.yezoensis has also been widely developed.In 1969，Miura bred out P.yezoensis f.narawaensis with stable genetic traits after years of breeding.Thereafter，new P.yezoensis cultivar Xiaoguang with excellent color and high yield was successfully bred out by haploid breeding method，and it was a popular P.yezoensis cultivar in Japan in the 1990s［5］.In the early 1980s，a long-shaped P.yezoensis cultivar was bred by Chinese scholars with the basically same approach［2］. Currently，haploid breeding technology has made great progress，and a large number of new P.yezoensis cultivars have been bred by intraspecific and interspecific hybridization，directional breeding and mutation breeding.

P.yezoensis belongs to a class of flavor food.In addition to yield，morphological characteristics［6］，chlorophyll fluorescence characteristics，fatty acidcomposition and volatile matter content［7-11］，flavor is also an important indicator，and it can reflect the eating quality and commercial value of certain cultivar.Flavor substances include free amino acids，5’-nucleotides，reduction sugar and organic acids［12］.In P.yezoensis，Glu，Ala，Asp and Arg have the highest contents，endowing P.yezoensis with specific flavor［13］. FAA composition and content are not only related to cultivar，but also related to cultivation environment and harvest time of P.yezoensis［13-16］.In this study，total 7 P.yezoensis cultivars，including one hybrid cultivar，one high-intensity light-induced cultivar，one mutation-induced cultivar，one naturally bred cultivar and three cultivation cultivars，were selected as test materials，and the FAAs contents in the 7 test materials were determined in the early，middle and late harvest period.The FAA composition and content variation in each test material was analyzed，thereby providing some reference for evaluation of various P.yezoensis cultivars.

Materials and Methods

Test materials

The samples were collected from the algae cultivation area in Nantong City on January 1 （early period），February28 （middle period）and March 28（late period），2013，respectively.The algae samples （Table 1）were washed with seawater，and the impurities and algae base were removed.Subsequently，the algae samples were rinsed rapidly with clean water to remove salt.They were dried in natural wind，and then preserved at -20℃for use.

Determination of FAA content

The samples were ground and passed through a 100-mesh sieve.A certain amount（0.30 g）of algae powder of each test material was dissolved in a certain volume （50 ml）of 75% ethanol.The solutions were bathed in boiling water for 15 min and evaporated to dryness at 40℃，which was repeated once.The collected solid substances were dissolved in certain volumes of citrate buffer（pH 2.2）.The solutions were centrifuged at 3 000 r/min for 5 min.The supernatants were collected and diluted to 50 ml，respectively.The dilutions were sieved and the supernatants were collected for determination.The derivation was carried out on line automatically by OPA-FMOC. After the first-level derivation by OPA and second-level derivation by FMOC，the FAA contents in the samples were determined by HPLC，and calculated by area normalization method.The concentration of the standard was arranged as 9，90 and 900 pmol，respectively.

Data statistics and analysis

The data were processed and analyzed using Excel and SPSS 19.0.

Table 1 P.yezoensis cultivars

Results and Analysis

FAA composition in P.yezoensis

As shown in Fig.1，total 16 kinds of free amino acids were detected in the 7 P.yezoensis cultivars，including 7 kinds of essential amino acids（Thr，Tyr，Val，Met，Phe，Ileand Leu），2 kinds of semi-essential amino acids（His and Arg）and 7 kinds of non-essential amino acids（Ala，Asp，Glu，Ser，Gly，Cys and Pro）.

Table 2 showed that among the 21 samples，the total FAA contents ranged from 48.71 to 62.05 mg/g.In the early harvest period，the total FAA content was highest in Y-H002（56.87 mg/g），but was lowest in Y-008（50.83 mg/g）；in the middle period，the total FAA content was highest in Sulian（62.05 mg/g），but was lowest in the control cultivar（53.69 mg/g）；in the late harvest period，the total FAA content was highest in Y-H002 （58.86 mg/g），but was lowest in the control cultivar（48.71 mg/g）.During the harvest period，the average total FAA content was highest in Sutong，followed by Y-H002，but was lowest in the control cultivar.

Among the 16 kinds of free amino acids，the four kinds of flavor amino acids，such as Ala，Arg，Glu and Asp，showed the highest contents.The total content of the four flavor amino acids ranged from 36.05 to 53.96 mg/g，accounting for 69.11%-86.47%of the total FAA content.Among them，the content of Ala accounted for 23.4%-32.79%，the content of Arg accounted for 23.51%-32.65%，the content of Glu accounted for 9.51%-18.94%，and the content of Asp accounted for 6.68%-10.05%of the total FAA content.In the early harvest period，thetotal content of the four kinds of flavor amino acids was highest in the control cultivar（45.51 mg/g），followed by Sutong（45.05 mg/g），but was lowest in YH002（41.70 mg/g）；in the middle period，the total content of the four kinds of flavor amino acids was highest in Sutong（53.96 mg/g），followed by Y-H002（51.81 mg/g），but was lowest in the control cultivar（42.97 mg/g）；in the late harvest period，the total content of the four kinds of flavor amino acids was highest in Sutong （43.60 mg/g），followed by Y-2011 （43.84 mg/g），but was lowestin the controlcultivar（36.05 mg/g）.

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As shown in Table 3，among the 16 kinds of free amino acids，the contents of essential amino acids，including 7 kinds of essential amino acids and 2 kinds of semi-essential amino acids，were relatively high.The total content of the essential amino acids ranged from 21.3 to 31.36 mg/g，accounting for 40.55%-53.27%of the total FAA content.In the late harvest period，the total content of essential amino acids was all higher than 50%of the total FAA content，and was much higher than those in the early and middle harvest periods.

Variation in flavor amino acid content in P.yezoensis during harvest period

As shown in Fig.2，the total FAA contentswere all highest in the middle harvest period in the test materials，except the control cultivar.The total FAA contents ranked second in the late harvest period，and ranked last in the early harvest period.Fig.3 showed that the flavor amino acid composition differed among different harvest periods. In the 7P.yezoensis cultivars，the Ala contents all reached the peaks in the middle harvest period，and were low-est in the late harvest period；the Arg contents did not differed significantly among the harvest periods；the Glu contents and Asp contents were all highest in the middle harvest period except in the control cultivar，and significant differences were observed in Glu contents and Asp contents between the early and late harvest periods；the Glu contents in the early harvestperiod were higher than those in the late harvest period except in Y-2011，and the Asp contents in the early harvest period were lower than in the late harvest period except in Sulian.

Table 3 Contents of flavor amino acids，essential amino acids and semi-essential amino acids and their proportionsin total FAA content

Conclusions and Discussion

FAA composition and content in P.yezoensis

Differentseawatertemperature and nutrient level will produce different effect on growth of P.yezoensis［14-15］. In addition to effects of germplasm and harvest time，there are great differences in total FAA content in P.yezoensis.It has been reported that the total FAA content ranged from 25 to 50 mg/g in P.yezoensis in Japan［15］，but ranged from 20 to 50 mg/g in China［17-18］.In this study，the samples of the 7 P.yezoensis cultivars were all collected from the same sea area of radial sand ridge of Haimen City，avoiding the differences in total FAA content caused by different sea areas.The result showed that the total FAA contents ranged from 48.71 to 62.05 mg/g，which were significantly different with the study results of Li et al［17］.This might be caused by the difference in treatment method of samples.

Glu，Asp，Arg and Ala are important flavor amino acids in P.yezoensis［17］.Among them，Ala and Glue are important amino acids affecting the flavor of P.yezoensis［12，19］.The study results showed that the contents of the four kinds of amino acids were highest inP.yezoensis，and their total contents ranged from 36.05 to 53.96 mg/g，accounting for 69.11%-86.47%of the total FAA contents.This explains why P.yezoensis has great taste.Yoshie et al.［20］studied the FAA contents in 8 kinds of dry products of P.yezoensis produced in Japan.They found that Ala content ranged from 3.08 to 16.02 mg/g；Glu content ranged from 3.22 to 18.44 mg/g；Asp content ranged from 0.66 to 2.82 mg/g.Ji et al.［14］detected FAA composition in P.yezoensis originating from Qingdao.The results showed thatin high-fertilizerlevel area，the contents of Ala，Glu and Asp were 7.3-30.2，2.9-9.3 and 0.5-2.7 mg/g，respectively.This study found that among the 7 cultivars，the Ala，Glu and Asp contents ranged from 11.40-19.42，4.63-11.63 and 3.59-5.28 mg/g，respectively.The Asp content was significantly higher than those determined by Yoshie et al.and Ji et al. The Ala and Glue contents were equivalent to those determined by Yoshie et al.and Ji et al.The lowest values were higherthan those determined by Yoshie et al.and Ji et al.

The total FAA content was highest in Y-H002 in the early harvest period，was highest in Sutong and Sulian in middle harvest period，and was highest in Y-H002 and Sulian in late harvest period.Among the cultivars，the average total FAA content was highest in Sutong and Y-H002，and was lowest in the control.In terms of total contentof the four kinds of flavor amino acids，the control cultivar and Sutong showed the highest content in early harvest period，Sutong and Y-H002 showed the highest content in the middle harvest period，and Y-2011 and Sutong showed the highest content in the late harvestperiod.Among the 7 P. yezoensis cultivars，Sutong had the highest total flavor amino acid content and total FAA content，followed by YH002，and Suyan，Sulian and control cultivar had the lowest total contents.

Variation in FAA content in P.yezoensis during harvest period

The conchospore seedling collection，spore release and vegetative growth of P.yezoensis are all closely related to sea temperature and water temperature.The harvest period of P. yezoensis also differs among sea areas with differenttemperature.The FAA composition is different among different P.yezoensis cultivar.Ji et al. studied FAA composition in P.yezoensiscollected from January to April.Although contents ofsome FAAs reached the peaks in middle harvest period，total FAA content was decreased overall with the extension of harvest period［14］.Sakai et al.［19］investigated FAA contents in P.yezoensis originating from Hokkaido，Japan during Novemberto nextJanuary.They found that the contents of FAAs，especially Asp，Ser，Glu and Ala，were trended to be increased with extension of harvest period.Niwa et al.［13］studied effects of harvest time on FAAs in two P.yezoensis cultivars，HG-4 and HG-5.Among Ala，Asp，Glue and Tau，only the content of Glu was reduced with the delay of harvest period.The contents of the other three kinds of amino acids in HG-4 reached the peaks at the third harvest，and then decreased. Similarly，during conchospore seedling collection to harvest（over 70 d），the contents of four major flavor amino acids（Ala，Asp，Glu and Arg）in all the test cultivars，except control cultivar，ranged from 41.70 to 45.51 mg/g in the early harvest period，from 42.97 to 53.96 mg/g in the middle harvest period，and from 36.05 to 43.84 mg/g in the late harvest period.

Different from flavor amino acids，the contents of essential amino acids in the 7 P.yezoensis cultivars were all trended to be increased with the delay of harvest period.The contents of essential amino acids determine the balance degree and utilization value of nutrients.There was a complementary role between changes in flavor amino acid and essential amino acid contents in P.yezoensis.

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Responsible editor：Tingting XU

Responsible proofreader：Xiaoyan WU

不同品系条斑紫菜采收期游离氨基酸组成与含量变化特征

胡传明1，2，3*，陆勤勤1，张美如1，朱建一4
（1.江苏省海洋水产研究所，江苏南通220067；2.江苏省农业种质资源保护与利用平台，江苏南京210014；3.上海海洋大学食品学院，上海201306；4.常熟理工学院，江苏常熟215500）

以7个条斑紫菜栽培品系为材料，利用柱前衍生高效液相色谱法，对其在采收期前期、中期与后期的16种游离氨基酸含量进行测定。结果显示，试样游离氨基酸总含量在48.71～62.05 mg/g之间，其中，Ala、Arg、Glu与Asp 4种呈味氨基酸含量最高，总含量在36.05～53.96 mg/g之间，占总游离氨基酸的69.11%～86.47%，除对照品系外，其他6个品系的呈味氨基酸与游离氨基酸总量均在采收中期达到最高；所有品系的必需氨基酸含量均随采收时间延迟呈增长趋势，在采收后期达到最高；苏通条斑紫菜品系在呈味氨基酸与总游离氨基酸含量组成水平上最高，其次为YH002、苏研与苏连，对照品系最低。

条斑紫菜；游离氨基酸；采收期；栽培品系

国家高技术研究发展计划（2012AA10A406-5）；国家自然科学基金项目（31272664）；江苏省自然科学基金青年基金项目 （BK20140439）；省科技厅支撑项目（BE2012420）；江苏省农业科技自主创新资金项目（CX（14）2081）。

胡传明（1980-），男，江苏新沂人，博士，助研，主要从事海洋水产与生物化学研究。*通讯作者，E-mail：hucharming@163.com。

2015-09-09

Supported by National High-tech R&D Program of China（2012AA10A406-5）；National Natural Science Foundation of China （31272664）；Natural Science Foundation of Jiangsu Province （BK20140439）；Jiangsu Province Science and Technology Support Program，China （BE2012420）；Independent Innovation Foundation of Agricultural Science and Technology of Jiangsu Province（CX（14）2081）.

*Corresponding author.E-mail:hucharming@163.com

Received:September 9，2015 Accepted:November 4，2015

修回日期 2015-11-04