ABSTRACT: Glycerol galactoside (GG; floridoside + isofloridoside) and porphyra-334 (P-334) are contained in nori (Susabinori Porphyra yezoensis and Asakusanori Porphyra tenera). Glycerol galactoside has been found to have bifidogenic growth stimulator activity and P-334 is known to have ultraviolet-absorbing activity in the UVA region of sunlight. These substances have, respectively, potential for application to pre-biotic foods and in cosmetics as a sunscreen. In the present study, to investigate the relationships between GG and P-334 contents and the quality of nori, we measured the GG and P-334 contents with other components (total protein, chlorophyll-a, β-carotene and phycobillins) that are related to the quality of nori samples produced from different production areas and with different qualities. We found that the GG content was closely negatively correlated with the contents of other components, whereas P-334 was positively correlated with the other components. From these results, it is suggested that low-quality nori is a potential source of GG, and as a source for P-334, scraps of nori produced during nori processing should be suitable.
The red algae Susabinori Porphyra yezoensis and Asakusanori Porphyra tenera are processed into a sheet-like dried food called nori. Nori is popularly consumed in East and South-East Asia, and its popularity is spreading further in the world as a component of sushi. Nori is an important part of the Japanese diet. Nori contains nutritionally and physiologically functional components, such as β-carotene, porphyran and vitamin B12.1 Therefore, the physiological functions of nori and its components have been extensively studied, and the antitumor,2 immunostimulating,3 anti-allergic,4 anti-oxidant,5 enhancing dioxin excretion6 and antimutagenic7 effects of nori and its components have been previously reported.
Glycerol galactoside (GG; floridoside + isofloridoside) is known to be a component of nori.8 Recently, GG has been found to have bifidogenic growth stimulator activity and is potentially a new pre-biotic material.9 Porphyra-334 (P-334), a type of mycosporine-like amino acid, was isolated from Porphyra tenera10 and has been found to be widely distributed in marine organisms.11,12 Porphyra-334 has strong absorption characteristics in the ultraviolet (UV) region around 334 nm and is considered to protect marine organisms from the UV rays of sunlight.12 From these properties, the potential application of P-334 to cosmetics and toiletries can be expected.
There are many reports describing the relationships between the quality and components of nori.8,13–20 Glycerol galactoside has been reported to be contained in low-quality nori.8,9 However, these reports analyzed only limited sample numbers. Studies about the content of P-334 and relationships with quality and other components have not been published yet. Therefore, in the present study, we measured the GG and P-334 contents, along with other various components that are related to the quality of nori, in nori samples produced from different production areas and with different qualities and analyzed their relationships. These results provide basic data to enhance the effective utilization of nori resources.
MATERIALS AND METHODS
Nori samples were kindly donated from the Kumamoto Prefectural Federation of Fisheries Cooperatives, Mr Yamada, the Federation of Japan Fisheries Cooperatives and Dr Fujiwara, Kagawa Prefectural Fisheries Experimental Station. Nori samples were stored at 5°C with desiccant until analysis. Sample number, areas of production and major grade [commercially judged quality (Hon-tokyu' in Japanese)] are shown in Table 1.
Table 1. Places of production, grade and analytical results of the nori samples
Three sheets of each nori sample were freeze-dried. The water content was measured by weighing the sheets before and after freeze-drying. Lyophilized samples were ground using a chemical mill (R-8; Nippon Rikagaku Kikai, Tokyo, Japan) and used for further analyses. All measurements were carried out twice and the results are expressed as the mean of the two measurements.
Chlorophyll-a content was measured essentially according to the method reported by Amano and Noda.17 In brief, approximately 0.2 g of the ground nori sample was weighed and 1.5 mL of water was added and the sample was humidified by allowing to stand for 10 min. Acetone (6 mL) was added and extracted by treating with an ultrasonic disruptor (UR-200P; Tomy Seiko, Tokyo, Japan) for 60 s (15 s × 4). After centrifugation at 1500 ×g for 15 min, the supernatant was gathered. Precipitant was extracted with 8 mL of 80% aqueous acetone three times. Extracts were combined to make 50 mL. The absorbance of the extract at 663 nm was measured and the chlorophyll-a content was calculated using E1%663 = 84.0 for chlorophyll-a.
Approximately 0.1 g of the ground nori sample was weighed and 4 mL of 3% pyrogallol in 10 N KOH : ethanol (1:10) was added. The samples were saponified by incubating at 70°C for 1 h with occasional mixing. The reactant was extracted with 5 mL of n-hexane four times. Extracts were combined to make 50 mL. The absorbance of the extract at 447 nm was measured and β-carotene content was calculated using E1%447 = 2080 for β-carotene.
Total nitrogen content was measured using the Kjehldahl method. Total protein content was calculated by multiplying the total nitrogen content by 6.25.
Approximately 0.2 g of ground nori sample was weighed and 10 mL of ice-cold 10 mM sodium phosphate buffer (pH 6.5) was added. The sample was treated with an ultrasonic disruptor for 60 s (15 s × 4) on ice and extracted by shaking (30 shakes/min) overnight at 10°C. After centrifugation at 1500 ×g for 15 min, the supernatant was removed and gathered into another flask. Precipitate was further extracted with 10 mL of the buffer two times (shaking time was shortened to 1 h). Extracts were combined to make 50 mL and filtered with 0.2 µm membrane filters (Advantec, Tokyo, Japan). The absorbance of the extract at 656 nm and 615 nm was measured and the content of phycobilins (phycoerythrin and phycocyanin) was calculated using the following equations:17
An approximately 0.5 g portion of nori sample was added to 10 mL of water and 0.5 mL of 6% sucrose solution (internal standard). The sample was incubated at 95°C for 1 h with occasional mixing. After centrifugation at 1500 ×g for 15 min the supernatant was gathered. Precipitate was further extracted with 5 mL of water three times. Extracts were combined to make 30 mL and filtered through an ultrafiltration cartridge, Ultrafree (NMCO5K; Millipore, Billerica, MA, USA). The filtrate (20 L) was applied to a high-performance liquid chromatography system (System 305 Programmable Pump; Gilson, Middleton, WI, USA) equipped with a Coregel 87 MM column (7.8 × 250 mm; Transgenomic, Omaha, NE, USA). The column temperature was set at 85°C and water was used as a solvent (0.6 mL/min). The eluent was monitored with a RID-6A refractive index monitor (Shimadzu, Kyoto, Japan). The GG was quantified by comparing the relative peak area of the internal standard with that of an authentic standard purified as previously described.9
Approximately 0.1 g of the sample was weighed and added to 5 mL of 80% aqueous methanol. The sample was treated with an ultrasonic disruptor for 60 s (15 s × 4) and incubated at 95°C for 1 h. After centrifugation at 1500 ×g for 15 min, the supernatant was gathered. The precipitant was further extracted twice with 5 mL of 80% methanol. Extracts were combined and made up to 25 mL with water. The extract was passed through a SepPak C18 cartridge (Millipore) to remove the lipids. After appropriate dilution with water, the optical density of the eluate was measured at 334 nm. The P-334 content was calculated using a molecular absorption coefficient of P-334 at 334 nm, ε334nm = 42 300 nm.21
Results of all analyses are shown in Table 1. The GG content ranged from 0 to 11.9 g/100 g, whereas that of porphyra-334 ranged from 0.5 to 1.5 g/100 g (Table 1).
For an objective estimation of nori quality, the relationships between quality and the ingredients of nori, such as protein, free amino acids, chlorophyll-a, β-carotene, phycobilins and minerals,13–17,19,20 have been reported. In these reports, the quality of nori (sensory score, major grade, price, classification by a judge of a prefectural fisheries cooperative) is positively correlated with the total protein, chlorophyll-a, β-carotene, phycobillins and free amino acid contents. Indeed, a strong correlation between total protein content and quality of nori has been reported.13 Thus, total protein content was shown in order of the major grade within the same production site (Fig. 1).
Total protein content is known to be highly correlated with the quality of nori.13,16,18 It seems that total protein content is a primary index for the quality of nori. Thus, to know the relationships between the primary quality index of nori and other constituents, correlations between the total protein content and other constituents are shown in Figure 2. Chlorophyll-a, phycoerythrin, phycocyanin, phycobilins, β-carotene and P-334 positively correlated with the total protein content. Correlations of these constituents were relatively strong (R > 0.75), whereas the correlation of phycocyanin was relatively weak (R = 0.581). Glycerol galactoside content was strongly negatively correlated with the total protein content (R = −0.8979). The GG was also negatively correlated with the content of the other constituents excluding water (data not shown).
Noda et al.8 analyzed free sugars in different quality nori samples and found that the GG (floridoside + isofloridoside) content ranged from 0.5 to 1 g/100 g, whereas in our results, GG content had a wider range, up to approximately 11 g/100 g (Table 1). This might indicate that our nori samples have a wider range of quality. The content of P-334 in the nori samples was shown to range from 0.5 to 1.5 g/100 g. The P-334 content of various samples of nori has not previously been investigated.
A strong correlation between total protein content and the quality of nori has been reported,13 and has been used for quality evaluation of nori.16,18 It seems that total protein content is a primary index for the quality of nori. Total protein content of nori tended to decrease with a reduction in the major grade within the same production site, but this tendency was not consistent among production sites. Fluctuations in the total protein content between production sites was larger than that within a production site (Fig. 1). From this result, it was suggested that the major grade can be used only for estimation of the quality of nori produced within the same production site. Ishihara et al. found that the major grade of nori is not applicable beyond a production site,16 and the present results show close agreement with this.
In Figure 2, correlations between the total protein content and the other constituents are shown. Amano and Noda reported that photosynthetic pigments, such as chlorophyll-a, β-carotene, phycoerythrin and phycocyanin, showed parallel changes during the cultivation season.17 Those changes might reflect changes in the culture conditions and quality of nori. The strong correlation between the contents of photosynthetic pigments and the total protein shown in the present results suggests that these photosynthetic pigments can be used as indices of the quality of nori. Porphyra-334 is thought to act as a protector from the UV rays of sunlight during photosynthesis.22 Therefore, it seems to be rational that the P-334 content showed a parallel change with photosynthetic pigments in the present study. Originally P-334 was isolated from the red alga Porphyra tenera.10 There are papers examining the UV-absorbing characteristics,23 distribution in marine organisms11,12 and sunscreen properties21 of P-334. However, we could not find reports describing the content of P-334 in nori and its relationship with the quality of nori. Thus, this is the first report detailing the P-334 content and its relationship with the quality of nori.
From these results, it can be concluded that GG is negatively correlated with the quality of nori. Noda reported that the contents of protein and carbohydrates in nori change in complement to each other, hence, the sum of the contents of proteins and carbohydrates keeps a constant value regardless of the fluctuation of quality.13 From an equation of the linear regression curve of the protein and GG contents in the present study [GG content (g/100 g) = 16.54–0.37 × total protein content (g/100 g)], GG accounts for approximately 37% of carbohydrate, which increases in complement with the reduction in protein content.
In the present study, we found that low-quality nori contains large amounts of GG and that high-quality nori has a high P-334 content. Glycerol galactoside has been found to have bifidogenic growth stimulator activity and is expected to have potential as a pre-biotic.9 Porphyra-334 is known to have strong UV-absorbing activity and may be used in sunscreen.21 Our results indicate that nori can be used as a potential source for GG and P-334. Every year, a significant amount, exceeding 2% in some years, of cultivated nori is discarded because of its low quality. This disposed nori can potentially be used as a GG source. As a source for P-334, scraps of nori produced during nori processing are potentially suitable.
The authors express their gratitude to Mr T. Yamada, the Federation of Japan Fisheries Cooperatives, and Dr M. Fujiwara, Kagawa Prefectural Fisheries Experimental Station, for their kind donation of nori samples.