김의 품종 및 채취시기별 성분 및 항산화 특성

The laver used in this experiment was provided by Jeollanam-do Institute of Ocean & Fisheries Science, Haenam office. P. dentata was harvested in November 2019 in Imhado, Haenam, and P. seriata was harvested in December 2019 in Imhado, Haenam. P. yezoensis was harvested in December 2019 and January and March 2020 in Eoran, Haenam. The samples were freeze-dried (PVTFD50R, Ilsin, Dongducheon, Korea) and stored at −40°C before analysis. The Folin-Ciocalteu‘s phenol reagent, sodium carbonate (Na₂CO₃), tannic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABST⁺), potassium persulfate, 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), iron (II) sulfate (FeSO₄), and ferric chloride hexahydrate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Mycosporine-like amino acids (shinorine and porphyra-334) used in this study were isolated from laver at our laboratory. Other organic solvents and reagents were high performance liquid chromatography (HPLC) grade or guaranteed reagent.

Total amino acid content was analyzed using the protocol used by Seo et al. (2007). 10 mL of 6 N hydrogen chloride (HCl) was added to 50 mg of freeze-dried laver, flushed with nitrogen gas, and heated at 105°C for 24 h using a heating block. The mixture was then filtered using a 3G-4 glass filter, and the filtrate was concentrated under reduced pressure using a rotary vacuum evaporator (N-1200B, Eyela, Tokyo, Japan). The concentrate was dissolved with sodium 10 mL of citrate buffer solution (pH 2.2), filtered through a 0.2 μm membrane filter (Satorius Stedim Biotech, Goettingen, Germany), and analyzed using an automated amino acid analyzer (S433-H SYKAM, Eresing, Germany) equipped with a cation separation column (LCA K06/Na, 4.6×150 mm, 7 μm, Sykam, Eresing, Germany). The flow rate of the buffer solution (pH 3.45-10.85) was set at 0.45 mL/min, and that of reagent was set at 0.25 mL/min. A fluorescence detector (Ex=440 nm, Em=570 nm) was used for amino acid detection.

Mineral content was analyzed in accordance with the Food Codex (Ministry of Food and Drug Safety, 2020). Ten milliliter of nitric acid was added to 0.3 g of freeze-dried laver sample in a Teflon-coated vessel, and the mixture was dissolved in microwave (MARS, CEM, Matthews, NC, USA). The dissolved solution was diluted to 50 mL using distilled water and analyzed with an inductively coupled plasma atomic emission spectrometer (Optima 8300, PerkinElmer, Waltham, MA, USA).

Extraction was performed for 24 h by adding 200 mL of 50% methanol (MeOH) to 3 g of freeze-dried laver at room temperature. The extract was filtered through a No. 2 filter paper (Whatman, Maidstone, England) to obtain the filtrate. Two hundred milliliter of 50% MeOH was added to the residual sample and a second filtrate was obtained via the same protocol. The two filtrates were mixed and concentrated under a vacuum to obtain 50% MeOH extract. This extract was dissolved by adding 20 mL of MeOH and filtered through a 0.45 μm membrane filter (Advantec, Tokyo, Japan). Shinorine and porphyra-334 were analyzed using a HPLC (SPD-M20D, Shimadzu) equipped with a Capcell pak C₁₈ AQ column (4.6×250 mm, 5 μm, Shiseido, Tokyo, Japan) and a photodiode array (PDA, 190-800 nm) detector. The mobile phase consisted of water containing 0.1% acetic acid (A) and acetonitrile (B) with using a gradient elution (0-5 min 0% B; 5-25 min 0-20% B; 25-30 min 20% B). The compounds were monitored at 330 nm and the flow rate of the mobile phase was set at 1 mL/min. Shinorine and porphyra-334 purified from laver were used as the external standards.

In accordance with Son’s (2014) method, extraction was performed for 24 h by adding 50 mL of distilled water to 2 g of freeze-dried laver and filtered through a suction filter (No. 2, Whatman, Maidstone, England) to obtain the filtrate. The filtrate was concentrated under reduced pressure and prepared to a concentration of 200 mg/mL by adding distilled water. Total phenolic content was measured using the Folin-Denis method (Folin and Denis, 1912), which utilizes the principle in which the reduction of the Folin-Ciocalteu’s phenol reagent by phenolic compounds generates the molybdenum blue. In other words, 20 μL of Folin-Ciocalteu’s reagent was added to 20 μL of the sample and incubated in a dark room for 3 min. After adding 200 μL of 10% Na₂CO₃ to this solution and incubating in a dark room for 15 min, absorbance was measured at 765 nm using a microplate reader (Infinite M200 PRO, Tecan, Salzburg, Austria). The total phenol content of the sample was quantified from the absorbance value using a standard curve obtained using tannic acid and was expressed as mg tannic acid equivalent per gram of laver sample (mg TAE/g dry wt.).

The ABTS⁺ radical-scavenging activity of the aqueous laver extract prepared above was measured using the method delineated by Pellegrin et al. (1999). In other words, 5 mL of 7 mM ABTS⁺ solution was mixed with 88 μL of 140 mM potassium persulfate solution and incubated in dark for 14-16 h. Then, absolute ethanol was added to prepare an ABTS⁺ radical solution with an absorbance of 0.70±0.02 at 734 nm. 20 μL of the sample solution was mixed with 180 μL of ABTS solution and incubated in dark for 3 min, and absorbance was read at 734 nm using a microplate reader (Infinite M200 PRO, Tecan). The ABTS⁺ radical-scavenging activity was measured using the equation below with the absorbance values of the non-treated and treated samples. Further, the concentration at 50% ABTS⁺ radical-scavenging activity (SC₅₀, mg/mL) was calculated using the free radical-scavenging activity curve by concentration for each sample.

FRAP activity was measured using the method proposed by Benzie and Strain (1996). The FRAP reagent was prepared by heating 40 mL acetate buffer (300 mM, pH 3.6) at 37°C and mixing with 4 mL of 10 mM TPTZ dissolved in 40 mM HCl and 4 mL of 20 mM ferric chloride hexahydrate (FeCl₃․6H₂O). 20 μL of the aqueous laver extract was added to 180 μL of the prepared FRAP reagent and incubated for 10 min at 37°C. Absorbance was measured at 593 nm using a microplate reader (Infinite M200 PRO, Tecan). The reducing power was computed from these absorbance values using a standard curve for iron (II) sulfate at concentrations of 0.156, 0.313, 0.625, 1.25, and 2.5 mM.

All experiments were conducted three times, and the data were statistically analyzed using IBM SPSS statistics (24, IBM Corp., Armonk, NY, USA). The results were presented as mean and standard deviation. One-way ANOVA and post-hoc test (Scheffe test) were performed to analyze the differences among the samples, and significance was set at p<0.05.

Table 1 shows the total amino acid content of laver samples with varying laver varieties and harvest periods. Total amino acid content wss the highest in P. yezoensis (PY) harvested in December (PY12, 40,914.26 mg/100 g dry wt.) and January (PY01, 41,009.52 mg/100 g dry wt.), respectively and the lowest in P. dentata (PD11, 33,762.75 mg/100 g dry wt.). Total amino acid content of laver varied greatly not only according to the variety but also according to the harvest times. In our analysis of the total amino acid contents of PY harvested at different times, the sample collected in March (PY03, 32,906.54 mg/100 g dry wt.) had a lower total amino acid content than the samples collected in December (PY12, 40,914.26 mg/100g dry wt.) and January (PY01, 41,009.52 mg/100 g dry wt.). This is consistent with previous results that the total amino acid contents of laver varies widely according to the growing environment, such as season, habitat, and oceanic conditions (Noda, 1993). In particular, PY had a higher total amino acid contents when harvested in December or January compared to March, suggesting that the abundance of amino acids in the growing environments of December and January is higher than that in March.

The most abundant amino acids in all laver varieties were aspartic acid, glutamic acid, leucine, arginine, and alanine, and these five amino acids accounted for 46.24-50.78% of the total amino acid content. This is similar to the major amino acid composition of laver reported in the study on dried laver produced in Korea by Lee et al. (1987) and that of the study on the nutritional composition of commercial laver products by Kim et al. (2014). Essential amino acids, such as threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine, were some of the major ingredients of laver. The percentage of essential amino acids from the total amino acid content was 37.68-38.61% in PY, 37.53% in PS12, and 35.64% in PD11, with no marked differences across varieties. Leucine, which is a branched chain amino acid (BCAA), was the most abundant in all laver varieties, and laver contained a rich amount of essential BCAAs other than leucine, such as valine and isoleucine. In other words, BCAA content was 6,395.39-8,203.72 mg/100 g dry wt. in PY, 7,016.49 mg/100 g dry wt. in PS12, and 6,168.74 mg/100 g dry wt. in PD11, accounting for 17.65-21.05% of total amino acid contents. BCAAs is not only an important regulator of insulin signaling and lipid metabolism but also is the largest storage of muscle proteins that plays a crucial role in protein metabolism (Yao et al., 2016). Particularly, leucine has been reported to enhance protein synthesis and inhibit protein degradation (Kimball and Jefferson, 2001). Laver is an important source of amino acids, and with its rich content of BCAAs, including leucine, valine, and isoleucine, it has benefits in terms of insulin signaling, lipid metabolism, enhanced protein synthesis, and suppressed protein degradation.

Table 2 shows the mineral contents of laver samples with varying laver varieties and harvest periods. The major minerals found in laver were calcium (Ca), potassium (K), magnesium (Mg), and sodium (Na), all of which accounted for 96.0-99.2% of total mineral content. This is consistent with previous findings that Ca, K, Mg, and Na were the primary minerals found in commercial laver products and PY in different regions (Kim et al., 2014; Mok et al., 2011).

The results of this study showed that the types and contents of minerals varied widely according to variety and harvest time. Although the mineral content could not be compared among laver varieties due to the varying harvest periods, PD11 had the richest K content, while PS12 had relatively higher amounts of Ca and Mg. PY had high contents of Ca, K, Mg, and Na at all harvest periods, although the specific contents of minerals differed slightly depending on the harvest period.

Regarding trace minerals in laver, iron (Fe) was found in the highest concentration at 7.50-24.49 mg/100 g dry wt. In particular, Fe content was twofold higher in PS12 (24.49 mg/100 g dry wt.) and PY (17.51-20.20 mg/100 g dry wt.) than in PD11 (7.50 mg/100 g dry wt.), with no statistically significant difference in Fe content between PS12 and PY (p>0.05). Fe content also did not statistically significantly differ in PY samples according to the harvest period. Interestingly, zinc (Zn) content varied more greatly according to laver variety and harvest period. Zn content was more than 1.8 times higher in PS12 (10.55 mg/100 g dry wt.) than in PD11 (4.69 mg/100 g dry wt.) and PY (4.58-5.59 mg/100 g dry wt.). Zn content in PY was similar to that in PD, and although there were significant differences in Zn content according to harvest period, the content was very low compared to that in PS12 and thus was insignificant. Manganese (Mn) content was the highest in PD11 (2.47 mg/100 g dry wt.), while copper (Cu) content was the highest in PS12 (1.56 mg/100 g dry wt.).

Kim et al. (2014) and Jung et al. (2016) reported that mineral contents differ even in the same variety depending on the environment of the farming region. Although the exact order of mineral content differed, they found that the major minerals were Ca, K, Mg, and Na, with Fe being the most abundant trace mineral. Baek et al. (2019) reported that PY and PD differ in their mineral contents, and similar to the results of this study, the major minerals were Ca, K, Mg, and Na, with Fe being the most abundant trace mineral. In contrast with our findings, Fe content was 1.7 times higher in PD. This may be attributable to the differences in the farming environments.

MAAs, mycosporine-derived compounds found in various marine organisms, including red algae, consist of a cyclohexenimine or cyclohexenone ring with various substituents such as amino acids and amino alcohol (Carreto et al., 2011). MAAs have been reported to have antioxidant effects and protective effects against ultraviolet rays and photooxidative stress (Shick et al., 2002; Venkatraman et al., 2019; Ying et al., 2019). Particularly, shinorine and prophyra-334 isolated from microalgae not only protects against cytotoxicity caused by UV irradiation but also suppresses the expression of COX-2, an inflammation-related gene (Cho et al., 2014; Jung et al., 2014). A recent study reported that laver contains MAAs, including shinorine and porphyra-334 (Ying et al., 2019), and these compounds have surfaced as some of the beneficial components of laver. Thus, the shinorine and porphyra-334 contents of three laver varieties were analyzed via ODS-HPLC. As shown in Table 3, the shinorine content was markedly higher in PD11 (2,090 mg/100 g dry wt.) harvested in November compared to that of PY (1.56-19.00 mg/100 g dry wt.) and PS12 (1.62 mg/100 g dry wt.). Further, the porphyra-334 content was also markedly higher in PD11 (3,128 mg/100 g dry wt.) compared to that of PY (3.02-438.31 mg/100 g dry wt.) and PS12 (2.17 mg/100 g dry wt.). Among PY samples with varying harvest periods, the sample collected in March (PY03) had markedly higher shinorine (19.00 mg/100 g dry wt.) and porphyra-334 content (438.31 mg/100 g dry wt.) compared to samples collected in December (PY12) and January (PY01). In essence, shinorine and porphyra-334 have various biological activities, including skin protection, anti-inflammatory action, and antioxidant activities, as previously mentioned, and PD that contains large amounts of these compounds is promising as an ingredient for functional food compared to other laver varieties. Furthermore, PY03 had a higher shinorine and porphyra-334 content than PY12 and PY01, so it would be a more useful ingredient in various aspects.

Phenolic compounds, such as flavonoids, anthocyans, nignans, and stilbenes, are secondary metabolites widely found in plants (Lin et al., 2016). Phenolic compounds exhibit an array of biological activities, including antioxidant activity, dental caries prevention, hypertension prevention, and anti-cancer effects (Yu et al., 2006). While laver was not a rich source of various phenolic compounds, it has been reported to contain catechol, rutin, catechin, and quercetin (Kazlowska et al., 2010). In addition, it was reported to have increased the total phenol content of steamed fish cake and crispy rice snacks when adding laver powder (Kim et al., 2020a; Kim et al., 2020b). Phenolic compounds are expected to contribute to the health functionalities of laver. Therefore, this study investigated the total phenol content of laver samples from different varieties and harvested at varying periods, and the results are shown in Table 4. By variety, PD11 had the highest total phenolic content (702.99 mg TAE/100 g dry wt.), followed by PY (136.82-184.14 mg TAE/100 g dry wt.), and PS12 had the lowest phenol content (50.12 mg TAE/100 g dry wt.). In terms of harvest periods, PY12 had significantly higher phenolic content (184.14 mg TAE/100 g dry wt.) compared to PY01 (136.82 mg TAE/100 g dry wt.) and PY03 (144.32 mg TAE/100 g dry wt.). These results supported that total phenol content of laver harvested in December had a higher total phenol content than laver harvested in January to March (Kang et al., 2018). These results show that the total phenol content of laver varies according to the variety and harvest period. Particularly, PD can be utilized to develop food products due to a higher total phenol content than PS and PY, which enhanced functional ingredients, such as phenolic compounds. However, the structures of these phenolic compounds need to be confirmed even though PD is suggested to contain diverse phenolic compounds, including catechol, rutin, and hesperidin found in PY.

ABTS⁺ radical-scavenging activities of laver extracts were examined by concentration, and the concentrations at 50% radical-scavenging activity (SC₅₀) were computed (Table 5). PD11 had the lowest SC₅₀ (55.40 mg/mL), followed by PY (68.58-144.51 mg/mL) and PS12 (192.76 mg/mL), which shows that PD has the highest radical-scavenging potential. In addition, PY samples showed varying levels of ABTS⁺ radical-scavenging depending on the harvest period. This means that PY12 had the highest radical-scavenging activity at SC₅₀=68.58 mg/mL, while PY3 had the lowest radical-scavenging activity at SC₅₀=144.51 mg/mL. Kim et al. (2015) demonstrated that marine algae with a high total phenolic content also had superior ABTS radical-scavenging activity, which was similar to this study.

FRAP was measured for laver extracts with varying varieties and harvest periods, and the results are shown in Table 6. The FRAP results were similar to the ABTS⁺ radical-scavenging activities. In other words, PD11 showed the highest ABTS⁺ radical-scavenging activity and also had the highest FRAP (1.47 mM), while PS12 had the lowest FRAP (0.29 mM). Further, the FRAP of PY varied according to the harvest period, similar to the results of ABTS⁺ radical-scavenging activity. PY12 had the highest FRAP (1.19 mM), while there were no significant differences in FRAP between PY01 (0.75 mM) and PY03 (0.70 mM).

Antioxidant in laver include pigments such as chlorophyl and carotenoids as well as polyphenols and porphyrin, and the level of antioxidant activity differs according to the production region and antioxidant compounds (Kang et al., 2018; Oh et al., 2013). PD has superior antioxidant activities to PS and PY and thus is promising as an ingredient for natural antioxidants and health functional products.

In conclusion, we examined the physicochemical properties and antioxidant activities of Pyropia yezoensis (PY), Pyropia seriata (PS), and Pyropia dentata (PD), primarily produced in Jeonnam. PY produced in December (PY12) and January (PY01) had higher total amino acid content (40,914.26 mg/100 g dry wt.) than PS12 (35,720.29 mg/100 g dry wt.) and PD 11 (33,762.75 mg/100 g dry wt.). According to harvest time, PY01 had the highest total amino acid content (41,009.52 mg/100 g dry wt.), while PY03 had the lowest total amino acid content (32,906.54 mg/100 g dry wt.). This is consistent with previous reports that the total amino acid content of laver varies greatly according to the growing environment, such as season, habitat, and oceanic conditions. The major minerals found in laver were Ca, K, Mg, and Na, and Fe was the most abundant trace mineral (7.50-24.49 mg/100 g dry wt.). Mineral contents also seem to differ according to the variety and harvest period which depend on the environments of the cultivation regions. Shinorine and porphyra-334 contents were markedly higher in PD11 (2,089.84 mg/100 g dry wt. and 3,127.96 mg/100 g dry wt., respectively) compared to the other two varieties. As shinorine and porphyra-334 have skin-protective, anti-inflammatory, and antioxidant effects, PD11 is considered a promising ingredient for functional foods. By the harvest time, the shinorine and porphyra-334 contents were markedly higher in PY03 (19.00 mg/100 g dry wt. and 438.31 mg/100 g dry wt., respectively) than PY12 or PY01, suggesting that PY harvested in March should be utilized if PY is used as a functional food ingredient. PD11 showed the highest total phenol content (702.99 TAE mg/100 g dry wt.), followed by PY (136.82-184.14 TAE mg/100 g dry wt.), and PS12 (50.12 TAE mg/100 g dry wt.). PD11 also exhibited higher ABTS radical-scavenging activity and FRAP than PS12 and PY, suggesting that it has superior antioxidant activities to the other two varieties.