Variation in the Chemical Composition of Saccharina Japonica with Harvest Area and Culture Period

China, Japan, and the Republic of Korea are the largest consumers of edible seaweeds [1]. Seaweeds include high alginic acid, fucoidan, and laminara contents, so it is effective for hematocele and lipid metabolism improvement such as lowering blood pressure and cholesterol in the blood, and anti-cancer [2]. According to a survey conducted on worldwide production of aquatic plants, there are approximately 16 million tons of annual aquatic plants, of which 14.9 million tons produced by aquaculture [3]. Algal production in Korea is mainly limited to Porphyra tenera, Saccharina japonica, and Undaria pinnatifida, which comprise 94% of the total harvested seaweed [4]. S. japonica is very popular as a healthy food because of low calorie and abundant vitamin, mineral, dietary fiber, calcium, potassium, magnesium, phosphoric acid, and microelements and high iodine content as compared with other seaweeds [5].


Sampling
In order to observe variations in chemical composition during the harvest time, S. japonica was collected from an environmentally quite different seaweed farm at Kijang and Wando located on the southern coast of the Republic of Korea once a month from February to July 2011 ( Figure 1). Both sporophytes of S. japonica transferred to the ocean at 0.5 m water depth in the same time (December 2010), and 5-20 individuals of whole S. japonica (blade, stem, and root) collected during the 2011 culture season. Freshly collected plants wrapped in paper towels with seawater, sealed in plastic bags, kept in an icebox, and transport to the laboratory where they washed with distilled water twice and freeze-dried. Each powered S. japonica (about 500 g) used for triplicate analysis. 2560 capillary columns (0.20-μm stationary phase thickness, 100 mm (length)×0.25 mm (i.d.); Supelco, Inc., USA). The sample (0.5 μl) was injected in the split mode using an automatic injection system (AOC-20i, Shimadzu Co., Japan). The oven temperature was programmed to increase from 160 to 220°C at 1°C min −1 with an initial hold of 5 min and final hold of 40 min. The other operation parameters were as follows: injector temperature, 250°C; detector temperature, 250°C; helium carrier gas flow, 20 cm s −1 ; split ratio, 1:50. The peak areas for the calibration curves and for calculation of fatty acid composition of oil samples were measured using a GC Solution system (Shimadzu Co., Japan).

Mineral contents
For the determination of mineral elements (calcium, copper, iron, potassium, magnesium, manganese, sodium, and zinc), samples were digested by dry ashing and dissolved in 1 M HCl [12]. The final diluted solution for calcium contained 1% lanthanum to overcome interferences. The concentration of the elements in S. japonica were determined with atomic absorption spectrophotometry (Perkin-Elmer, model 3110). Triplicate determinations for each element were carried out. The concentration of the elements were determined from calibration curves of the standard elements.

Amino-acid analysis
Samples (0.5 g) were acid-hydrolyzed with 3 mL of 6 N HCl in vacuum-sealed hydrolysis vials at 121°C for 24 h. Tubes were cooled after hydrolysis, opened, and placed in a rotary evaporator at 50°C to remove HCl from the sample. The residue was then adjusted to pH 2.2 with 0.2 M sodium citrate loading buffer (pH 2.2), diluted to a final volume of 10 mL with water, filtered through a Millipore membrane (0.2 μm pore size), and analyzed for amino acids using an amino-acid analyzer (Pharmacia Biochrom 20, Biochrom Ltd., UK).

Statistical analysis
All mean values were analyzed by one-way analysis of variance (ANOVA, SPSS 1999). Values are expressed as mean ± standard deviation (SD; n=3 replicates). Group means were considered to be significantly different at p<0.05.

Changes in proximate composition with harvest area and culture period
The proximate compositions of Kijang and Wando samples are shown in Tables 2 and 3. There was a high variation in moisture, crude protein, ash and crude lipid content with culture period and harvest area among the Kijang and Wando samples collected at different months from February to July. S. japonica in Kijang and Wando showed the highest crude protein content in February and the highest carbohydrate content in July. In the crude lipid content, February samples in Kijang and Wando generally tended to decrease until July. There was a high variation in ash content with culture period and harvest area, ranging from 14.29 ± 1.47% to 19.39 ± 0.75% (Tables 2 and 3).

Changes in component sugar and fatty acid composition with harvest area and culture period
Component sugar compositions of Kijang and Wando samples are shown in Tables 4 and 5. Fucose was the most abundant and galactose the second most abundant in the monosaccharide composition profiles. Mannose, glucose, xylose, ribose, and rhamnose were present at low

General component analysis
Moisture, crude protein, crude lipid, and ash content were determined using the standard methods described by the Association of Official Analytical Chemists [12]. Protein content analyzed using the semi-Kjeldahl method. Lipids extracted with anhydrous diethyl ether using a Soxhlet apparatus. Moisture quantified by oven drying the samples at 105°C for 12 h. Ash was determined after incineration in a furnace at 550°C. Total carbohydrate content calculated by subtracting the sum of moisture, crude protein, crude lipid, and ash mass from that of the total sample [13].

Component sugar analysis
In order to extract component sugar, a test sample (100 mg) mixed in the 15 mL test tube with 5 mL of 2M HCl. The oxygen in the test tube replaced by nitrogen gas, sealed, and placed in a heating mantle at 100°C for 5 h [14]. Hydrolyzed sample cooled, neutralized by adding 5 mL of 2M NaOH, and centrifuged at 650 g for 30 min. 3 mL supernatant filtered through a Millipore membrane (0.45 μm pore size), and analyzed by operating conditions (Table 1) using HPLC (Prominence HPLC, Shimadzu Co, Ltd. Kyoto, Japan).

Fatty acid composition analysis
Bligh and Dyer extraction was performed using the following method [15]: Briefly, lipids were extracted from 5-g samples by homogenization with 100 mL of chloroform and 200 mL methanol. The samples were then filtered and evaporated to remove solvent. Fatty acid methyl esters (FAME) were prepared using boron trifluoride (BF3) according to a method described by the AOAC [12]. Quantitative analysis of FAME was carried out on a GC-2010 gas chromatograph(Shimadzu Co., Japan) equipped with a split/splitless capillary inlet system and a flame ionization detector (FID) using SP-

Changes in mineral content and total amino acid composition with harvest area and culture period
The mineral contents of Kijang and Wando samples are shown in Tables 8 and 9. The results show that S. japonica is rich in K and Na with moderate amounts of Ca and Mg whereas Cu, Fe, Mn, and Zn are present in small quantities. The total amino acid (TAA) compositions of Kijang and Wando samples are shown in Tables 10 and 11. Glutamic acid, aspartic acid, alanine, and leucine were the most common amino acids in all samples, while the percentage of cysteine was the lowest in the TAA profile. TAA of Kijang samples decreased during the harvest time from April to July while TAA of Wando samples decreased from March to July. quantities, and lactose, mannitol, and arabinose were not detected.

Discussion
There are big environmental differences between Wando and Kijang. Wando is semi-closed sea, and affected by big tide and fresh water inflow from many rivers around. Kijang has a small tide, but high temperature high salinity Tsushima current and low temperature low salinity North Korea current meets in this area. To our knowledge, this is the first study that evaluated differences in the nutritional composition of S. japonica with harvest area and culture period. We found that protein content of S. japonica was highest in February and the carbohydrate content was highest in July for the Kijang and Wando samples over the culture period from February to July 2011. A similar pattern was previously reported for the collection of Laminaria japonica [16]. Rosemberg and Ramus [17] found inverse relationships between carbohydrate and protein content in the red seaweed Gracilaria cervicornis during collection from July 2000 to June 2001. The seaweed protein content was lowest when photosynthetic activity and carbohydrate synthesis were highest. Shin et al. [18,19] found that carbohydrate content of Porphyra yezoensis increased with late culture period: Dec (39.4%), Feb (47.2%). However, the protein content decreased with late culture period: Dec (39.4%), Feb (34.6%). Lipid content was not affected by culture period. A positive correlation was also detected between carbohydrate and temperature, along with correlations with salinity and solar radiation, which indicated that carbohydrate synthesis and protein concentration are affected by several seasonal factors, including water temperature, nitrogen content, and light intensity [16,18]. The lipid content was low relative to the other chemical constituents. However, the lipid content observed   in this study was similar to the content observed in other seaweeds, comprising from 1% to 3% of dry matter [20,21]. The ash content varied from 14.3% to 18.4% in our samples. It has been reported that the ash content fluctuates depending on the species, geographical location, and season investigated [22,23].
Component sugar compositions of Kijang and Wando samples were high in the following order: fucose, galactose, glucose, mannose, and so on. Polysaccharide of seaweed generally classified into cytoskeleton, intercellular mucoid, and storage polysaccharides, most of 2M HCl hydrolyzed polysaccharide from S. japonica in this study originated from storage polysaccharide [24]. In the Kijang and Wando samples, there was a variation in the sugar content depending on culture period (P<0.05). Galactose content of Kijang samples were higher than that of Wando in the all culture period (p<0.05).
In the mineral contents of Kijang and Wando S. japonica samples, the results show that S. japonica is rich in K and Na with moderate amounts of Ca and Mg whereas Cu, Fe, Mn, and Zn are present in small quantities.
Major amino acid of Kijang and Wando S. japonica samples are glutamic acid, aspartic acid, leucine, alanine, glycine, valine, phenylalanine, but cystine was not detected. Glutamic acid and aspartic acid occupied over 20% in the total amino acid. It is known that amino acid of seaweed is generally composed of high contents in neutral and acidic amino acids such as alanine, aspartic acid, glycine, and proline [24], but S. japonica contained low glycine and proline contents. Sulfur amino acid, cysteine and cysteine, was not detected, methionine, histidine, and tyrosine were included in small amount. The average percentages of essential amino acids (EAA) in Kijang and Wando S. japonica samples were 39.5%, 37.8%, which is higher than the EAA requirement (32.3%) suggested by the Food and Agriculture Organization [35]. The amino acid composition observed in this study was similar to previous studies [36], where the sum of the average percentage of three amino acids, glutamic acid (13.5%), aspartic acid (11.9%), and alanine (7.9%), comprised the greatest proportion (33.3%) of TAA composition. Noda [37] suggested that the former three amino acids (glutamic acid, aspartic acid, and alanine) might produce the flavors specific to Nori (Porphyra). TAA content decreased at the end of the culture period. This phenomenon has also been observed in other seaweeds such as Enteromorpha prolifera, C. fulvescens, and Codium fragile [38].
In conclusion, we have ascertained that the monthly nutritional composition of S. japonica affected by harvest area and culture period from February to July 2011. S. japonica in Kijang and Wando showed the highest crude protein content in February and the highest carbohydrate content in July. Fucose was the most abundant and galactose the second most abundant in the monosaccharide composition profiles. Significant increases of the major fatty acids in Kijang (C18:2 n-6 and C20:4 n-6) and Wando (C18:3 n-6) were observed as the culture period progressed. The highest mineral content of both Kijang and Wando samples is potassium and followed by sodium, calcium, magnesium, and so on. In the total amino acid contents, Kijang samples increased from February to April but decreased from May to July, while Wando samples increased on March but decreased from April to July.