J Appl Phycol (2018) 30:535–545 DOI 10.1007/s10811-017-1262-8
Seasonal variations in a polysaccharide composition of Far Eastern red seaweed Ahnfeltiopsis flabelliformis (Phyllophoraceae) A. O. Kravchenko 1 & A. O. Byankina Barabanova 1 & V. P. Glazunov 1 & I. M. Yakovleva 2 & I. M. Yermak 1
Received: 29 March 2017 / Revised and accepted: 29 August 2017 / Published online: 11 September 2017 # Springer Science+Business Media B.V. 2017
Abstract The quantitative and qualitative composition of polysaccharides from red alga Ahnfeltiopsis flabelliformis collected in the Amur Bay (Sea of Japan) from February to December 2009, depending on the water temperature and daily dose of photosynthetically active radiation (PAR), was analyzed. The highest content of polysaccharides was observed in February and November–December at low water temperature and daily PAR dose. The polysaccharide amount produced by the seaweed significantly decreased from March to September and increased from September to November. The main components of the polysaccharides were galactose and 3,6-anhydrogalactose containing sulfate groups. The galactose content was high throughout the studied period (28– 42%), 3,6-anhydrogalactose ones in the summer (17.4%) and did not depend on environmental factors. The sulfate group amount was high in April–May and July–August. A statistically significant positive correlation was found between the sulfate group amount in the polysaccharide and the average daily PAR dose in the habitat of seaweed. The dynamics of dry weight accumulation during the year was similar to the polysaccharides. According to IR spectroscopy, the polysaccharides mainly contained disaccharide units of kappacarrageenans and iota-carrageenans. The polysaccharide from
* A. O. Kravchenko
[email protected]
1
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 100 Let Vladivostoku Prosp., 159, Vladivostok, Russian Federation 690022
2
National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Palchevskogo, 17, Vladivostok, Russian Federation 690041
A. flabelliformis collected in June had more regular structure than that in other months. Keywords Ahnfeltiopsis flabelliformis . Rhodophyta . Sulfated polysaccharides . Chemical composition . Water temperature . Photosynthetically active radiation . IR spectroscopy
Introduction Marine algae have been used in the food industry and medical practice for more than 600 years, but it was not until the middle of the twentieth century when researchers started to actively search for and isolate natural products from them. Based on their composition, marine algae, and specifically red seaweeds, are good potential functional foods. Recently, it has been shown that the consumption of seaweed in Asian countries is associated with a low incidence of cancers compared to European and North American countries (Kumar et al. 2011). The ability to synthesize acid polysaccharides is the most interesting property of red seaweeds. The amorphous matrix of red algae consists of sulfated galactans, which are polysaccharides that contain multiple units of the galactose monosaccharide with sulfate ester, such as carrageenans, agars, and BDL-hybrid^ (Gomez-Ordonez and Ruperez 2011), and usually extend to intercellular spaces between adjacent cells. The interest in agar and carrageenan is increasing because their physico-chemical and biological properties make it necessary to search for new promising sources of these polysaccharides. Although red seaweeds are found in all latitudes, there is a marked abundance in equatorial regions. Larger species of red algae with massive thalli appear in cold and temperate areas, while in tropical seas, red algae are mainly
536
small filamentous plants. Rhodophyta have a greater ability to live at great depths than other algae groups, and they can grow at up to 200 m deep, a skill related to the presence of accessory pigments (Lee 2008). Red seaweeds of the genus Ahnfeltiopsis in the family Phyllophoraceae are widespread in the seas of temperate and tropical latitude. Ahnfeltiopsis flabelliformis grows throughout the year and forms extensive populations presented by attached and unattached forms in the Russian Far Eastern seas (Perestenko 1994). Data on the polysaccharide structures of the representatives of the genus Ahnfeltiopsis are primarily for macrophytes growing in the tropical seas. For example, polysaccharides with difficult structures containing kappa/iota units and L-galactose were isolated from Gymnogongrus torulosus (Estevez et al. 2001) and Gymnogongrus tenuis (Recalde et al. 2016), whereas polysaccharides from Gymnogongrus crenulatus and Ahnfeltiopsis devoniensis dwelling along the coast of Portugal were kappa/iota-carrageenans and iota/kappa-carrageenans, respectively (Pereira and Van de Velde 2011). It is known that variations in the structure of polysaccharides are observed not only for different species of red algae but also between those isolated at different life stages of the same species (McCandless et al. 1973; Craigie 1990; Stortz and Cerezo 1993; Falshaw and Furneaux 1994). So, we have previously established that the gelling polysaccharide from sterile form of A. flabelliformis, growing in the Sea of Japan, was kappa/beta-carrageenan (Kravchenko et al. 2014), but iota/ kappa-carrageenan was found in the reproductive (cystocarps) form (Kravchenko et al. 2016). It is known that the biosynthesis of polysaccharides in the cell wall of algae, their content, and structural characteristics depend on the conditions of the macrophyte habitat: season, water temperature, salinity, light intensity, and nutrient availability (Chapman and Chapman 1980; Craigie 1990; Zertuche-Gonzalez et al. 1993; Yakovleva et al. 2001; Hung et al. 2009). For example, it has been shown that tropical Gracilaria salicornia (Buriyo and Kivaisi 2003) and subtropical Gracilaria multipartita (Givernaud et al. 1999) produce high amounts of polysaccharides at a low nitrogen concentration, because high nitrogen content favors the biosynthesis of proteins rather than polysaccharides. On the contrary, a directly proportional relationship was found between the amount of polysaccharides and the nutrients’ availability for tropical Eucheuma isiforme (Freile-Pelegrin and Robledo 2006) and Eucheuma uncinatum from the Bay of California (ZertucheGonzalez et al. 1993). Several research groups have noted close relationship between the accumulation of polysaccharides and the growth processes of macrophytes caused by environmental conditions for a variety of representatives of red algae (Brown 1995; Reani et al. 1998; Yakovleva et al. 2001; Freile-Pelegrin and Robledo 2006). For example, it was shown that an increase in the growth rate of Gelidium crinale (Gelidiaceae) (Boulus
J Appl Phycol (2018) 30:535–545
et al. 2007), Gelidium robustum (Gelidiaceae) (Hurtado et al. 2011), and Gracilaria multipartita (Gracilariaceae) algae (Givernaud et al. 1999) from subtropical regions was accompanied by a reduction in the polysaccharide content. Similar dependence was observed for Calliblepharis jubata (Cystocloniaceae) seaweed, growing in the temperate latitudes (Zinoun and Cosson 1996). The maximum growth of C. jubata was recorded in the winter months, characterized by minimal lighting. The accumulation of polysaccharides in Cystoclonium purpureum (Cystocloniaceae) from the temperate latitudes was observed when light and water temperature decreased and was accompanied with a fall in the growth rate (Reani et al. 1998). In contrast, the highest growth rate of Tichocarpus crinitus (Tichocarpaceae) from temperate latitudes was registered in August at a water temperature of 24 °C and 10–15% photosynthetically active radiation (PAR), and coincided with the period of polysaccharide accumulation (Barabanova et al. 2004). A positive correlation between the growth processes of macrophytes and carrageenan content was explained in this case by the fact that T. crinitus was slowly growing and its growth rate did not adversely affect the polysaccharide amount (Yakovleva et al. 2001). There is little information about the effect of environmental conditions on the polysaccharide composition of Phyllophoraceae family members. In early work, it was shown that the decrease in the water temperature to negative values coincided with a reduction in the amount of extracted polysaccharides for Gymnogongrus crenulatus, Phyllophora pseudoceranoides, and Phyllophora truncata, growing in temperate latitudes (Mathieson et al. 1984). The maximum content of polysaccharides from Mastocarpus stellatus, dwelling along the Atlantic coast of Spain (Laxe, Mogas), was observed at high light levels and water temperature (Tasende et al. 2013). For Gymnogongrus griffithsiae from North Carolina, it was shown that the amount of synthesized polysaccharide did not depend on light intensity and temperature, but at the same time varied between seasons (Breden and Bird 1994). There is no information on the seasonal dynamics of polysaccharides from A. flabelliformis inhabiting the Russian Far Eastern seas. Since A. flabelliformis may be a new potential source of sulfated polysaccharides, information obtained from algae grown under controlled conditions can be useful in selecting cultivation techniques leading to higher polysaccharide content in seaweed. However, identification of possible regulatory factors is usually inconclusive because the environmental interactions and physiological history of experimental algae collected from their natural habitats are unknown. In particular, some difficulties have been experienced in separating the influence of environmental factors from the effects of algae life history stages (Rivera-Carro et al. 1990; Moseley 1990; Chopin et al. 1994). The present experiments were conducted when algae were presented by the reproductive form.
J Appl Phycol (2018) 30:535–545
This limited the factors influencing changes to the quantitative and qualitative composition of polysaccharide from seaweed by the elimination of a very important one—the status of the plant. The aim of this study was to determine the influence of environmental parameters, such as light and water temperature, on the quantitative and qualitative composition of the polysaccharides produced by the Far Eastern alga A. flabelliformis.
Materials and methods Algal samples The attached form of Ahnfeltiopsis flabelliformis (Harv.) Masuda, inhabiting the Amur Bay near Cape Red (43° 11′ 52″ N, 131° 55′ 09″ E) (Sea of Japan), was collected monthly during 2009. Samples of unshaded, this year, seaweed growing on top of boulders at a depth of 2 m represented by the reproductive form (cystocarps) were selected from the A. flabelliformis population for analysis. Medium size and intact fronds whose growth portion in the apical part did not exceed 5% of the total algal biomass were used. Seaweed was washed with running water for epiphyte and soluble salt removal. A portion of this material was used for the analysis of dry weight and total soluble protein in the tissue of algae. The remaining samples were air dried and stored in sealed plastic bags before the analysis of polysaccharides. Environmental parameter measurements Water temperature, salinity, and intensity of PAR in the habitat of A. flabelliformis were recorded every month during the year. Water temperature was measured with a mercury thermometer. Salinity was measured with a salinometer refractometer (Atago, Japan). The range between measurements of water temperature and salinity was 3–5 days. PAR intensity penetrating through the water column at the site of the collection of biological material was detected with a LI-COR 189 quantum photometer equipped 2D underwater sensor UWQ, LI-COR 7254 (Biospherical Instruments, USA). PAR intensities were measured every 3–5 days during the research period. Measurements were performed from 8:00 to 20:00 h, and the time between measurements was 30 min. Data obtained for the day were integrated, and daily doses of PAR reaching the surface of algal thallus were calculated. Dry algal weight analysis A. flabelliformis samples were cleaned of epiphytes, washed with running water, dried by filter paper, and weighted (n = 3) using the CAS ME-210 scales (CAS Corp., Korea). Then, the samples were dried in an oven at 60 °C for 24 h, and the weight was measured again. Seaweed weight, allowing determination of monthly changes of algal dry weight, was calculated as the ratio of the dry algal weight to the wet seaweed weight.
537
Total soluble protein analysis The content of total soluble protein in algal tissues was determined on the raw material according to the Bradford method (Bradford 1976). Fresh samples (n = 3) weighing 1 g were powdered in liquid nitrogen and extracted with 2–3 mL of 50 mM phosphate buffer (pH 7.4) containing 4% PVP-10, 0.1 mM EDTA, and 0.25% Triton X-100. The extracts were centrifuged at 11,500×g for 15 min at 4 °C. The supernatant was used to determine the total soluble protein content in the tissue of algae. Bovine serum albumin (Sigma-Aldrich, USA) was used as a standard. The amount of protein in milligrams was calculated and expressed as the percentage of the dry algal weight. Polysaccharide extraction Dried (3 g) and milled seaweeds (3 g) (n = 3) were suspended in distilled water at a 1:30 (w/v) ratio, and polysaccharides were extracted at temperature of 20 °C for 12 h. Obtained extract was filtered, centrifuged at 3500×g at 4 °C for 30 min to remove cell wall residues, and concentrated with rotor evaporator, and polysaccharides were precipitated with 96% ethanol at a 1:3 ratio. Then, precipitate was separated by centrifugation at 3500×g at 4 °C for 30 min and lyophilized. Algal residue was re-suspended in distilled water at a 1:60 (w/v) ratio, and polysaccharides were extracted three times at 80° С for 3 h in a boiling water bath with constant stirring. After that, three hot water extracts were combined and filtered, and polysaccharides were precipitated as described above and lyophilized. Polysaccharide yields were expressed as a percentage of dry algal weight. Polysaccharide analysis The monosaccharide composition was determined by total reductive hydrolysis (Englist and Cummings 1984; Usov and Elashvili 1991). Neutral monosaccharides were analyzed as alditol (Englist and Cummings 1984) and aldononitrile (Usov and Elashvili 1991) acetate derivatives by gas-liquid chromatography with a 6850 chromatograph (Agilent Technologies, USA) equipped with a capillary column HP-5MS (30 m × 0.25 mm, 5% phenyl methyl siloxane) and a flame ionization detector. The analyses were carried out using a temperature gradient program from 175 to 225 °C; the rate of temperature change was 3 °C min−1. The sulfate ester content of polysaccharides was determined by the turbidimetric method (Dodgson and Price 1962). Fourier transform-infrared spectroscopy (FT-IR) IR spectra of the studied polysaccharides were recorded in films on a Vector 22 Fourier transform spectrophotometer Equinox 55 (Bruker Corp., USA) with 4 cm−1 resolution. For the sample preparation, compound (8 mg) was dissolved in H2O (1 mL) and was heated at 37 °C on a polyethylene substrate until a dry film was produced. Then, the film was clamped between two NaCl plates, and the IR spectra were recorded in the 4000– 600 cm−1 region. The spectra were normalized by the absorption of the monosaccharide ring skeleton at ~1074 cm−1
538
(A1074 ≈ 1.0). The kappa-carrageenan and iota-carrageenan (Sigma-Aldrich) were used as a standard. Statistical analysis To evaluate statistical discrepancies in dry weight, total soluble protein content, polysaccharide yields, and its monosaccharide composition between months data were processed using one-way dispersion analysis (one-way ANOVA) and multirank LSD test. Submitted data corresponded to average values and their standard deviations. Correlations were considered significant at P < 0.05. Spearman nonparametric analysis (Spearman Rank test) was used to evaluate the interrelation between dry weight, content of total soluble protein, polysaccharide yields, its monosaccharide composition, and environmental factors (average daily PAR dose, water temperature) during the year. The relationship was considered significant at P < 0.05.
J Appl Phycol (2018) 30:535–545 Table 1 Average monthly values of water temperature (T, °C) and average, maximal, and minimal values of daily doses of photosynthetically active radiation (PAR, 400–700 nm) penetrating in the water column at the site of A. flabelliformis collection in the Amur Bay (Sea of Japan) from February to December 2009 Month
Max
Min
− 1.0 ± 0.5 1.1 ± 1.2
2.02 4.53
2.86 7.25
0.23 0.32
5.5 ± 1.3 7.6 ± 1.6
21.34 10.75
31.40 37.16
2.89 4.42
June July
11.6 ± 0.9 18.1 ± 1.8
7.21 14.44
36.69 38.12
6.19 4.29
August
22.7 ± 1.6
19.47
29.91
4.55
September November
20.6 ± 1.7 2.8 ± 0.4 1.3 ± 1.4
17.24 3.88 0.78
23.49 5.83 2.42
2.24 0.36 0.11
Februarya Marcha April May
Mean ± standard deviation (n = 8) a
Seasonal changes in algal dry weight, content of total soluble protein, and the qualitative and quantitative composition of the polysaccharides from A. flabelliformis were studied. Seaweed samples were collected in natural habitats in the Amur Bay (Sea of Japan) at a depth of 2 m from February to December 2009. Collected algae were represented by cystocarpic plants during the year to eliminate the influence of macrophyte life cycle stage on polysaccharide composition.
Daily PAR dose (mol photons m−2 day−1) Average
Decembera
Results
Т (°С)
Ice cover was observed
accumulation of DW (28% of wet weight) was recorded in the winter months (Fig. 1a). A statistically significant (LSD test, P < 0.05) DW decrease in the spring and summer to the minimum value (16.8% of wet weight) in September was recorded. The Spearman correlation coefficient characterized the relationship between the DW of the algae, and
Environmental parameters The maximum water temperature in 2009 was recorded in August, and the minimum from December to March (Table 1). Average daily PAR doses were used to study the effect of light on algal dry weight, content of total soluble protein, polysaccharide yields, and its monosaccharide composition. The maximum average daily dose of PAR reaching the surface of algal thallus was reported in April (Table 1). It is probably related to an increase in solar activity and ice melting in the spring. High average daily PAR dose was registered in the period from July to September, which is apparently due to an increase in the air transparence and the completion of the monsoon period typical for the Far Eastern region from May to early July. The water salinity in the habitat of A. flabelliformis varied slightly and averaged 32–33‰ during the studied period. Variation in dry weight and total soluble protein content in A. flabelliformis thallus According to the analysis, the dry weight (DW) of A. flabelliformis significantly (ANOVA, F2,3 = 45.566; P < 0.05) varied during the year (Fig. 1a). The maximum
Fig. 1 Seasonal variations of dry weight (DW) (a), polysaccharide (PS) yield (a), and total soluble protein (TSP) (b) content in A. flabelliformis thallus. Bars show standard deviation (n = 3)
J Appl Phycol (2018) 30:535–545
539
environmental factors indicated a significant (Spearman test, P < 0.001) negative effect of water temperature and average daily PAR dose on the content of the dry weight of A. flabelliformis during the year (Table 2). The content of the total soluble protein (TSP) in tissues of algae (ANOVA, F2,3 = 45.566; P < 0.05) also varied between seasons (Fig. 1b). The TSP amount in the tissues of A. flabelliformis reached a maximum value in June and was negligible in the period from September to March (Fig. 1b). According to the Spearman correlation coefficient, water temperature and average daily PAR dose did not affect the protein synthesized in the algal thallus during the year (Spearman test, P > 0.05) (Table 2).
Quantitative composition of the polysaccharides The amount of polysaccharide extracted from dried A. flabelliformis at 20 °C was minor (0.7–4.7%). The content of the polysaccharide (PS) extracted at 80 °C ranged from 5.9 to 26.7% of dry weight (Fig. 1a). The total polysaccharide yield from A. flabelliformis and its relationship with the time of macrophyte collection, average daily PAR dose, and water temperature are shown in Fig. 1a and Table 2. The character of seasonal changes of the content of the polysaccharide from A. flabelliformis was similar to the dynamics of the dry weight. The maximum yields of the PS have been observed in the cold time of the year, February and November– December (Fig. 1a, Table 1), when the water temperature did not exceed 3 °C and average daily PAR dose was minimum. No statistically significant (LSD test, P > 0.05) variations in the polysaccharide content were detected during this Table 2 Spearman correlation coefficients characterized the relationship of dry weight, total soluble protein amount, polysaccharide content, and the chemical composition of the polysaccharides with environmental factors of A. flabelliformis from February to December 2009 Water temperature
Average daily PAR dose
DW TSP PS Gal
− 0.858*** 0.025 − 0.841*** − 0.085
− 0.615*** 0.029 − 0.651*** − 0.260
AnGal Glc Xyl SO3Na
0.379 − 0.516* − 0.774*** 0.297
0.092 0.012 − 0.414 0.544**
Significant values of impact of the environmental factors on DW, the content of TSP, polysaccharides, and their chemical composition at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) are highlighted in ital DW dry weight, PS polysaccharide, TSP total soluble protein, PAR photosynthetically active radiation, Gal galactose, AnGal 3,6anhydrogalactose, Glc glucose, Xyl xylose
period. The polysaccharide yields significantly decreased by 1.5 and 2-fold (ANOVA, F2,3 = 45.566, LSD test, P < 0.05) in the spring and summer months, respectively, and reached a minimum value in September (Fig. 1a). The water temperature and average daily PAR dose had a statistically significant (Spearman test, P < 0.001) negative correlation with the amount of produced polysaccharide (Table 2).
The qualitative composition of polysaccharides The analysis of the monosaccharide composition of the polysaccharide from A. flabelliformis showed that the major monosaccharides are galactose and 3,6-anhydrogalactose, which are structural components of carrageenan and agar (Table 3). The amount of these monosaccharides varied slightly within 1 year. According to the Spearman coefficient, a statistically significant (Spearman test, P > 0.05) correlation between the content of galactose, 3,6-anhydrogalactose, and environmental factors (water temperature, average daily PAR dose) was not observed for 1 year (Table 2). The polysaccharide from A. flabelliformis also contained minor amounts of glucose and xylose that may be structural units of floridean starch (Meeuse et al. 1960) and xylan (Craigie 1990), respectively (Table 3). The maximum content of these monosaccharides was recorded in the period from February to May, and the minimum ones in the summer months. The xylose content was partially recovered in November–December to the level (ANOVA, F2,3 = 45.566, LSD test, P < 0.05) recorded in the spring. The water temperature had a statistically significant negative effect on the content of both glucose (Spearman test, P < 0.05) and xylose (Spearman test, P < 0.001) in the polysaccharide from A. flabelliformis throughout the year (Table 2). The high content of sulfate groups in the polysaccharide from A. flabelliformis was observed in April–May and July– August (Table 3). A statistically significant (Spearman test, P < 0.01) positive correlation between the sulfate group amount in the polysaccharide and the average daily PAR dose in the habitat of seaweed was shown (Tables 1 and 2). The molar ratio of the main monosaccharide residues (galactose and 3,6-anhydrogalactose) and sulfate groups of the polysaccharides (Table 3) allowed us to make the assumption about the structural features of the polysaccharides synthesized in June and February. The polysaccharide from A. flabelliformis collected in June was characterized by a regular structure (molar ratio AnGal/Gal 1.0:1.4) typical for the gelling carrageenan when the AnGal/Gal ratio was 1.0:1.0. At the same time, this regularity was not observed (molar ratio AnGal/Gal 1.0:2.9) for the polymer from the algae collected during the winter months. This fact indicated that the polysaccharide had a hybrid structure or presented as a mixture of polysaccharides.
540 Table 3 The chemical composition of the polysaccharides extracted from dried A. flabelliformis (80 °С) collected in February–December 2009 in the Amur Bay (Sea of Japan)
J Appl Phycol (2018) 30:535–545
Month
Content (% of sample weight)
Molar ratio AnGal/Gal/SO3Na
Gal
AnGal
Glc
Xyl
SO3Na
February
42.2 ± 2.1
12.7 ± 0.2
4.7 ± 0.2
4.3 ± 0.6
23.2 ± 0.6
1.0:2.9:2.6
March
29.1 ± 1.1
12.4 ± 1.0
3.5 ± 0.1
3.2 ± 0.4
25.3 ± 0.5
1.0:2.1:2.9
April May
28.3 ± 1.6 30.2 ± 0.7
13.1 ± 0.3 14.0 ± 1.2
4.0 ± 0.3 3.1 ± 0.4
2.8 ± 0.1 3.5 ± 0.3
26.7 ± 0.2 27.0 ± 0.2
1.0:1.9:2.8 1.0:1.9:2.7
June
27.6 ± 0.8
17.4 ± 0.6
1.2 ± 0.1
1.6 ± 0.2
25.6 ± 0.4
1.0:1.4:2.1
July August
32.6 ± 1.3 32.2 ± 2.3
15.6 ± 0.6 14.8 ± 1.0
1.6 ± 0.1 2.0 ± 0.4
1.7 ± 0.4 1.6 ± 0.2
26.6 ± 0.2 26.1 ± 0.2
1.0:1.9:2.4 1.0:1.9:2.5
September November
34.3 ± 1.1 28.1 ± 1.9
11.5 ± 0.7 13.0 ± 1.4
2.1 ± 0.1 1.5 ± 0.5
2.3 ± 0.4 2.8 ± 0.1
21.7 ± 0.5 25.1 ± 0.6
1.0:2.6:2.6 1.0:1.9:2.7
December
32.5 ± 1.8
14.3 ± 0.6
2.0 ± 0.4
2.7 ± 0.2
24.2 ± 0.6
1.0:2.0:2.4
Mean ± standard deviation (n = 3) Gal galactose, AnGal 3,6-anhydrogalactose, Glc glucose, Xyl xylose
FT-IR spectroscopy analysis of polysaccharides Since polysaccharides extracted from A. flabelliformis collected in February and June significantly differed in the ratio of AnGal/Gal (Table 3), FT-IR spectroscopy was used for comparative structural analysis of these polysaccharides. Its spectra were compared with that of carrageenans with known structures. As shown in Fig. 2, spectra of these polysaccharides were identical. The IR spectra of the polysaccharides showed a very intense absorption band at about 1240– 1250 cm−1 (O=S=O asymmetric stretching), which indicated the presence of a large number of sulfated ester groups (Pereira et al. 2009). This is in agreement with the results of the chemical analysis (Table 3). The ratios of the intensities of absorption bands A1240/A1070 in the spectra of the polysaccharides isolated from samples of algae collected in February and June did not vary significantly and were 0.84 and 0.87, Fig. 2 FT-IR spectra of polysaccharides from A. flabelliformis collected in February (a) and June (b)
respectively. The absorption band at about 930 cm −1 corresponded to the 3,6-anhydrogalactose, which was characteristic for all cyclic carrageenans and agarose. The band at 848 cm−1 corresponded to axial sulfate at C4 of the 1,3-linked β-D-galactose residue (the stretching vibrations of C4-O-S) and pointed to the presence of kappa-carrageenan and iotacarrageenan (Stancioff and Stanley 1969; Rees et al. 1993). The absorption band at 805 cm−1, characteristic of the stretching vibrations of C2-O-S of the 3,6-anhydro-α-D-galactose, could indicate the presence of iota-type disaccharide units in the structure of the polysaccharide (Prado-Fernandez et al. 2003). The weak band at 890 cm−1 evinced the presence of unsulfated galactose residues, typical for both beta and alpha types of carrageenan (Pereira et al. 2009) and also agarose (Andriamanantoanina et al. 2007). It can be seen from the IR spectra of the polysaccharides that the intensity of the absorbance band at 930 cm−1 was
J Appl Phycol (2018) 30:535–545
541
higher for the polysaccharide from algae collected in June than that in February (Fig. 2). This finding indicated that the 3,6-anhydrogalactose content was higher in polysaccharides in summer months than in winter months, that was consistent with the results of chemical analysis (Table 3). Therefore, the ratios of the area of the S930/S848 and S930/S805 absorption bands were calculated (Fig. 3). The S930/S848 ratios were 1.23 and 1.30, and the ratios of S930/S805 were 1.07 and 1.18 for polysaccharides from algae collected in February and June, respectively. This fact indicated that the polysaccharide from algae collected in February had a less regular structure and, likely, contained biosynthetic precursors or non-gelling polysaccharides. The decomposition of the IR spectra into the individual components allowed for estimation of the content of kappa and iota units in the studied polysaccharides (Fig. 3). The ratios of the absorption bands areas at 848 and 805 cm−1 were calculated. The S848/S805 ratios were 0.87 and 0.91 for
Discussion It is known that the variation of environmental factors (light, temperature, and the concentration of nutrients in seawater) causes adaptive adjustments in algae, which are accompanied
a
0.8
0.9
930
0.4
0.5
0.6
805
0.0
0.1
0.2
0.3
Absorbance Units
0.7
848
1000
950
900
850
800
750
700
-1
cm
0.9
b
0.6
848
0.2
0.3
0.4
0.5
805
0.1
Absorbance Units
0.7
0.8
930
0.0
Fig. 3 The decomposition of FTIR spectra of polysaccharides from A. flabelliformis collected in February (a) and June (b)
polysaccharides from seaweed collected in February and June, respectively. It should be noted that the ratios were smaller than a similar ratio for the standard of iotacarrageenan (Sigma-Aldrich) (S848/S805 = 1.2). This may be due to the presence of nu-carrageenan in the polymer chain of the studied polysaccharide, and the nu-carrageenan amount was higher in the algae collected in February than in June. This fact confirmed to the chemical analysis (Table 3). Thus, according to the IR spectroscopy, isolated polysaccharides contained disaccharide units kappa-carrageenan, iota-carrageenan, nu-carrageenan, and unsulfated galactose.
1000
950
900
850
800
750
700
cm-1
542
by changes to the polysaccharide composition of seaweed (Brown 1995; Buriyo and Kivaisi 2003; Reis et al. 2008; Hung et al. 2009). Seasonal changes in the qualitative and quantitative composition of the polysaccharides from A. flabelliformis were studied. The dynamics of the polysaccharide accumulation in algal tissue during a year is known to be determined not only by direct action of environmental factors but also by indirect influence of the seaweed growth rate on the biosynthetic processes in cells. The changes to the dry weight can indirectly reflect periods of seaweed growth. Thus, the low values of dry weight correspond to active growth of the plants. On the contrary, the increase of the dry matter content is accompanied by a reduced growth rate (Freile-Pelegrin and Robledo 2006). Moreover, protein synthesis predominates in the periods of active seaweed growth, whereas the polysaccharide accumulation is minimal (Hurtado et al. 2011). Thereby, dry weight and content of the total soluble protein depending on the season of macrophyte collection, water temperature, and average daily PAR dose were determined along with the polysaccharide amount. Negative effect of water temperature and average daily PAR dose on the content of the dry weight of A. flabelliformis during the year was shown. The maximum accumulation of DW (28% of wet weight) in the winter months is likely connected with moderation of the growth rate of macrophytes at low water temperatures (Freile-Pelegrin and Robledo 2006). Total soluble protein content is not associated with changes in environmental parameters. In this study, the water temperature and average daily PAR dose had a statistically significant (Spearman test, P < 0.001) negative correlation with the amount of produced polysaccharide (Table 2). Probably, high average daily PAR dose and water temperature increased the growth rate of the algae, and photoassimilates were used not only to produce the reserve components in the cell wall but also for growth (Bird 1988). It means that reproduction occurred in cells and the cellular volume increased. As for A. flabelliformis, a negative correlation between the amount of the polysaccharide, water temperature, and irradiance was recorded earlier for seaweed from temperate latitudes, including Cystoclonium purpureum (Cystocloniaceae) (Reani et al. 1998), subtropical Hypnea musciformis (Cystocloniaceae) (Mtolera and Buriyo 2004), Gelidium crinale (Gelidiaceae) (Boulus et al. 2007), Kappaphycus alvarezii (Solieriaceae) (Hung et al. 2009), and tropical Eucheuma isiforme (Solieriaceae) (FreilePelegrin and Robledo 2006). In contrast, a positive correlation between the polysaccharide content and irradiance level was observed for Solieria chordalis (Solieriaceae) from temperate latitudes (Brown 1995). The largest accumulation of the polysaccharides in another member of the temperate latitudes Tichocarpus crinitus (Tichocarpaceae) was recorded at a high temperature and low PAR level reaching the thallus surface (Yakovleva
J Appl Phycol (2018) 30:535–545
et al. 2001). A positive correlation between the polysaccharide content, light level, and water temperature was observed for subtropical Mastocarpus stellatus (Phyllophoraceae) (Tasende et al. 2013). At the same time, necessary conditions for the accumulation of the polysaccharides from subtropical Gracilaria multipartita (Gracilariaceae) (Givernaud et al. 1999) and tropical H. musciformis (Cystocloniaceae) (Reis et al. 2008) were high irradiance and high water temperature, respectively. The character of seasonal changes of the content of the polysaccharide from A. flabelliformis was similar to the dynamics of the dry weight (Fig. 1a), whereas the correlation between the amount of protein and polysaccharide was absent during the studied period. However, it should be noted that there was a gradual decrease of the dry weight and polysaccharide content and protein accumulation in the first half of the year (February–May). It can be explained by a so-called Neish effect (Hurtado et al. 2011), when the active growth of plants is accompanied by an increase in protein synthesis and reduction in polysaccharide accumulation. An analogous relationship between the content of the protein and polysacchar i de w a s pr ev i o us l y m a r k e d fo r H . m u s c i f o r m i s (Cystocloniaceae) (Durako and Dawes 1980), E. isiforme (Solieriaceae) (Freile-Pelegrin and Robledo 2006), G. crinale (Gelidiaceae) (Boulus et al. 2007), and G. robustum (Gelidiaceae) (Hurtado et al. 2011) growing in subtropical and tropical latitudes. It was registered that the content of the polysaccharide from A. flabelliformis decreased to the minimum annual values in July–September (Fig. 1a). This phenomenon was probably connected with a total loss of algal biomass at combined adverse effects of high average daily PAR dose and water temperature, but not with the active seaweed growth. It was indirectly confirmed by the fall of the protein content, reducing the amount of dry weight (Fig. 1) and the photoinhibition process that was observed for A. flabelliformis in this period (Kravchenko et al. 2011). The high polysaccharide yield from A. flabelliformis collected in April was observed despite the dramatic increase of average daily PAR dose. This was likely due to the fact that the water temperature, which had a greater influence on the polysaccharide accumulation (Table 2) (Spearman coefficient of water temperature was higher than average daily PAR dose), remained low during this period (+5.5 °C). There were generally favorable conditions for the polysaccharide biosynthesis, whose content did not change significantly compared to March (ANOVA, F2,3 = 45.566, LSD test, P > 0.05) (Fig. 1a). The polysaccharide content in the tissues of A. flabelliformis was lower in summer than in the winter months (Fig. 1a), which does not seem to be associated with a reduction in the carbohydrate synthesis in this period, as algal growing conditions were not stressful (Table 1). This phenomenon can be explained by the prevalence of young
J Appl Phycol (2018) 30:535–545
growing tissue since the growth conditions were favorable. Thus, the rate of the polysaccharide accumulation could remain the same, but its amount per unit of plant biomass would be less. In our research, a statistically significant (Spearman test, P > 0.05) correlation between the content of galactose, 3,6anhydrogalactose, and environmental factors (water temperature, average daily PAR dose) was not observed for 1 year (Table 2). The absence of influence of environmental factors and the galactose and 3,6-anhydrogalactose contents was also noted for the representatives of red algae from subtropical latitudes, such as H. musciformis (Aziza et al. 2008) and E. uncinatum (Zertuche-Gonzalez et al. 1993). In contrast, a positive correlation between the amount of 3,6anhydrogalactose and irradiance was recorded for polysaccharides from the tropical E. isiforme (Freile-Pelegrin and Robledo 2006). According to chemical analysis, high galactose content was observed in the polysaccharide from A. flabelliformis collected in February, and high 3,6-anhydrogalactose content in June (ANOVA, F2,3 = 45.566, LSD test, P < 0.05) (Table 3). There was a gradual reduction of dry weight in the summer months compared to the winter-spring period (Fig. 1a) that indirectly indicated an increase in the growth rate of macrophytes. In the same period, the content of 3,6-anhydrogalactose, a monosaccharide determining the gelling properties of the polysaccharide, was increased (Table 3). This could be connected with the fact that the gelling polysaccharides were mainly synthesized in young algal parts. Similar dependence was observed earlier in the tropical K. alvarezii (Hung et al. 2009). The polysaccharide from A. flabelliformis contained minor amounts of xylose (Table 3). The maximum content of this monosaccharide was recorded in the period from February to May. Previously, we showed that the xylose was a substituent of the hydroxyl group at C-6 of the 1,3-linked β-D-galactose in the total polysaccharide from A. flabelliformis (Kravchenko et al. 2016). This explains the fact that the polysaccharide from the seaweed collected in February contained a higher galactose amount than in the other months. In this study, a statistically significant (Spearman test, P < 0.01) positive correlation between the sulfate group amount in the polysaccharide and the average daily PAR dose in the habitat of seaweed was shown (Tables 1 and 2). Analogical dependence has been established previously for S. chordalis from the temperate latitudes (Fournet et al. 1999) and subtropical E. uncinatum (Zertuche-Gonzalez et al. 1993). A protective role of sulfated polysaccharides from algae under oxidative stress and their ability to remove reactive oxygen species produced in plant cells at high light has been previously observed (Xue et al. 2000; Tannin-Spitz et al. 2005). It was believed that the antioxidant mechanism involved sulfated polysaccharides as the source of hydrogen, which was capable of binding to active radicals, and as a
543
result, the radical chain reaction was broken (Mohamed 2008). Ahnfeltiopsis flabelliformis was subjected to oxidative stress in April and July–August at high average daily PAR dose, as evidenced by the high level of malondialdehyde (Kravchenko et al. 2011). It can be assumed that the production by seaweed of highly sulfated polysaccharide in the period of high irradiance was associated with the adaptive reaction of the plant to oxidative stress in order to preserve its viability. According to the IR spectroscopy, polysaccharides from A. flabelliformis mainly contained disaccharide units of kappa-carrageenan and iota-carrageenan. In addition, the polysaccharide from seaweed collected in February had a less regular structure than polysaccharide from alga collected in June. Structural analysis of the gelling polysaccharide from A. flabelliformis collected in April showed that the polysaccharide represented hybrid iota/kappa-carrageenan with iota and kappa-type units in a 2:1 ratio, containing betacarrageenan units and minor amounts of nu-carrageenan and mu-carrageenan (Kravchenko et al. 2016). The HPLC and ESI MS/MS data of enzymatic hydrolysis products revealed that the main components of the polymer chain were iotacarrabiose, iota-carratetraose, and hybrid tetrasaccharide and hexasaccharide consisting of kappa and iota units. In conclusion, a period characterized by low water temperature and average daily PAR dose (November–February) was the most favorable for collecting A. flabelliformis in 2009 because polysaccharide yields were highest at this time. At the same time, galactans with a high sulfation degree were synthesized in the cell wall of macrophytes at high average daily PAR dose (April, July, and August). However, the question concerning the reproducibility of obtained results in other years is the subject of further research.
References Andriamanantoanina H, Chambat G, Rinaudo M (2007) Fractionation of extracted Madagascan Gracilaria corticata polysaccharides: structure and properties. Carbohydr Polym 68:77–88 Aziza M, Givernaud T, Chikhaouikhay M, Bennasser L (2008) Seasonal variation of the growth, chemical composition and carrageenan extracted from Hypnea musciformis (Wulfen) Lamouroux harvested along the Atlantic coast of Morocco. Sci Res Essay 2:509–514 Barabanova AO, Yermak IM, Glazunov VP, Yakovleva IM, Kim YH, Solov’eva TF (2004) Influence of life-history stage and photon irradiance on yield and quality of carrageenan in Tichocarpus crinitus (Rhodophyta, Tichocarpaceae). Jpn J Phycol 52:61–65 Bird KT (1988) Agar production and quality from Gracilaria sp. strain G-16: effects of environmental factors. Bot Mar 31:33–39 Boulus A, Spaneir E, Friedlander M (2007) Effect of outdoor conditions on growth rate and chemical composition of Gelidium crinale in culture. J Appl Phycol 19:471–478 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
544 Breden PC, Bird KT (1994) Effects of environmental factors on carrageenan from Gymnogongrus griffithsiae (Gigartinales, Rhodophyta). J Appl Phycol 6:371–380 Brown MT (1995) Interactions between environmental variables on growth rate and carrageenan content of Solieria chordalis (Solieriaceae, Rhodophyceae) in culture. J Appl Phycol 7:427–432 Buriyo AS, Kivaisi AK (2003) Standing stock, agar yield and properties of Gracilaria salicornia harvested along the Tanzanian coast. West Indian Ocean. J Mar Sci 2:171–178 Chapman VJ, Chapman DJ (1980) Seaweeds and their uses. Chapman and Hall, London, pp 98–148 Chopin T, Hanisak MD, Craigie JS (1994) Carrageenans from Kallymenia westii (Rhodophyceae) with a review of the phycocolloids produced by the Cryptonemiales. Bot Mar 37:433– 444 Craigie JS (1990) Cell wall. In: Cole KM, Sheath RG (eds) Biology of the red algae. Cambridge University Press, Cambridge, pp 221–257 Dodgson KS, Price RG (1962) A note on the determination of the ester sulfate content of sulfated polysaccharides. Biochem J 84:106–110 Durako MJ, Dawes CJ (1980) A comparative seasonal study of two populations of Hypnea musciformis from the east and west coasts of Florida, USA I. Growth and chemistry. Mar Biol 59:151–156 Englist HN, Cummings JH (1984) Simplified method for the measurement of total non-starch polysaccharides by liquid chromatograph of constituent sugars as alditol acetates. Analyst 109:937–942 Estevez JM, Ciancia M, Cerezo AS (2001) DL-Galactan hybrids and agarans from gametophytes of the red seaweed Gymnogongrus torulosus. Carbohydr Res 331:27–41 Falshaw R, Furneaux RH (1994) Carrageenan from the tetrasporic stage of Gigartina decipiens (Gigartinaceae, Rhodophyta). Carbohydr Res 252:171–182 Fournet I, Zinoun M, Deslandes E, Diouris M, Floc’k JY (1999) Floridean starch and carrageenan contents as responses of the red alga Solieria chordalis to culture conditions. Eur J Phycol 34:125– 130 Freile-Pelegrin Y, Robledo D (2006) Carrageenan of Eucheuma isiforme (Solieriaceae, Rhodophyta) from Yucatan, Mexico. II. Seasonal variations in carrageenan and biochemical characteristics. Bot Mar 49: 72–78 Givernaud T, Gourji AE, Mouradi-Givernaud A, Lemoine Y, Chiadmi N (1999) Seasonal variations of growth and agar composition of Gracilaria multipartita harvested along the Atlantic coast of Morocco. Hydrobiologia 398(399):167–172 Gomez-Ordonez E, Ruperez P (2011) FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds. Food Hydrocoll 25:1514–1520 Hung D, Hori K, Nang HQ, Kha T, Hoa LT (2009) Seasonal changes in growth rate, carrageenan yield and lectin content in the red alga Kappaphycus alvarezii cultivated in Camranh Bay, Vietnam. J Appl Phycol 21:265–272 Hurtado MA, Manzano-Sarabia M, Hernandez-Garibay E, Pacheco-Ruiz I, Zertuche-Gonzalez JA (2011) Latitudinal variations of the yield and quality of agar from Gelidium robustum (Gelidiales, Rhodophyta) from the main commercial harvest beds along thee western coast of the Baja California peninsula, Mexico. J Appl Phycol 23:727–734 Kravchenko AO, Belotsitsenko ES, Yakovleva IM, Barabanova AO, Yermak IM (2011) Seasonal changes of photosynthetic pigments content in red alga Ahnfeltiopsis flabelliformis from the Japan Sea. Izv TINRO 166:138–148 Kravchenko AO, Anastyuk SD, Isakov VV, Sokolova EV, Glazunov VP, Yermak IM (2014) Structural peculiarities of polysaccharide from sterile form of Far Eastern red alga Ahnfeltiopsis flabelliformis. Carbohydr Polym 111:1–9 Kravchenko AO, Anastyuk SD, Sokolova EV, Isakov VV, Glazunov VP, Helbert W, Yermak IM (2016) Structural analysis and cytokine-
J Appl Phycol (2018) 30:535–545 induced activity of gelling sulfated polysaccharide from the cystocarpic plants of Ahnfeltiopsis flabelliformis. Carbohydr Polym 151:523–534 Kumar M, Kumari P, Trivedi N, Shukla MK, Gupta V, Reddy CRK, Jha B (2011) Minerals, PUFAs and antioxidant properties of some tropical seaweeds from Saurashtra coast of India. J Appl Phycol 23:797–810 Lee RE (2008) Phycology. Cambridge University Press, Cambridge Mathieson AC, Penniman CE, Tveter-Gallagher E (1984) Phycocolloid ecology of underutilized economic red algae. Hydrobiologia 116(117):542–546 McCandless EL, Craigie JS, Walter JA (1973) Carrageenans in the gametophyte and sporophyte stage of Chondrus crispus. Planta 112: 201–212 Meeuse BJD, Andries M, Wood JA (1960) Floriden starch. J Exp Bot 11: 129–140 Mohamed ZA (2008) Polysaccharides as a protective response against microcystininduced oxidative stress in Chlorella vulgaris and Scenedesmus quadricauda and their possible significance in the aquatic ecosystem. Ecotoxicology 17:504–516 Moseley CM (1990) The effect of cultivation conditions on the yield and quality of carrageenans in Chondrus crispus. In: Akatsuka I (ed) Introduction to Applied Phycology. SPB Academic Publishing, The Hague, pp 565–574 Mtolera MSP, Buriyo AS (2004) Studies on Tanzanian Hypneaceae: seasonal variation in content and quality of kappa-carrageenan from Hypnea musciformis (Gigartinales, Rhodophyta). West Indian Ocean. J Mar Sci 3:43–49 Pereira L, van de Velde F (2011) Portuguese carrageenophytes: carrageenan composition and geographic distribution of eight species (Gigartinales, Rhodophyta). Carbohydr Polym 84: 614–623 Pereira L, Amado AM, Critchley AT, Van de Velde F, Ribeiro-Claro PJA (2009) Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy (FTIR-ATR and RTRaman). Food Hydrocoll 30:1–7 Perestenko LP (1994) The red algae of the far eastern seas of Russia. Nauka, St. Petersburg [In Russian] Prado-Fernandez J, Rodriguez-Vazquez JA, Tojo E, Andrade JM (2003) Quantitation of κ-, ι- and λ-carrageenans by mid-infrared spectroscopy and PLS regression. Anal Chim Acta 480:23–37 Reani А, Cosson J, Parker A, Zaoui D (1998) Influence of culture conditions on growth and rheological properties of carrageenans in Cystoclonium purpureum. Bot Mar 41:299–304 Recalde MP, Canelon DJ, Compagnone RS, Matulewicz MC, Cerezo AS, Ciancia M (2016) Carrageenan and agaran structures from the red seaweed Gymnogongrus tenuis. Carbohydr Polym 136:1370–1378 Rees DW, Santos GA, Dumont LE, Parent CA, Stanley NF, Stancioff DJ, Guiseley KB (1993) β-Carrageenan: isolation and characterization. Carbohydr Polym 22:247–252 Reis RP, Yoneshigue-Valentin Y, Cesar PS (2008) Spatial and temporal variation of Hypnea musciformis carrageenan (Rhodophyta, Gigartinales) from natural beds in Rio de Janeiro state, Brazil. J Appl Phycol 20:1–8 Rivera-Carro H, Craigie JS, Shacklok P (1990) Influence of tissue source and growth rates on dry weight and carrageenan composition of Chondrus crispus (Gigartinales, Rhodophyta). Hydrobiologia 204205:533–538 Stancioff DJ, Stanley NF (1969) Infrared and chemical studies on algal polysaccharides. Proc Int Seaweed Symp 6:595–609 Stortz CA, Cerezo AS (1993) The systems of carrageenans from cystocarpic and tetrasporic stages from Iridaea undulosa: fractionation with potassium chloride and methylation analysis of the fractions. Carbohydr Res 242:217–227
J Appl Phycol (2018) 30:535–545 Tannin-Spitz T, Bergman M, van-Moppes D, Grossman S (2005) Antioxidant activity of the polysaccharide of the red microalga Porphyridium sp. J Appl Phycol 17:215–222 Tasende MG, Cid M, Fraga MI (2013) Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain). J Appl Phycol 25:587–596 Usov AI, Elashvili MY (1991) Quantitative-determination of 3,6anhydrogalactose derivatives and specific fragmentation of the red algal galactans under reductive hydrolysis conditions. Bioorg Khim 17:839–848 Xue CH, Hang Y, Lin H, Chen L, Li ZJ, Deng D, CX L (2000) Chemical characters and antioxidant properties of sulfated polysaccharides from Laminaria japonica. J Appl Phycol 13:1–5
545 Yakovleva IM, Yermak IM, Titlynov EA, Barabanova AO, Glazunov VP, Skriptsova AV (2001) Changes in growth rates, anatomy and polysaccharide content of a sterile form of Tichocarpus crtinitus (Gmel.) Rupr. (Rhodophyta, Tichocarpaceae) under differing photon irradiances in the Sea of Japan (Russia). Bot Mar 44:493–500 Zertuche-Gonzalez JA, Pacheco-Ruiz I, Soria-Mercado IE (1993) Carrageenan yield and properties of Eucheuma uncinatum (Seth. & Gard.) Daw. cultured under natural conditions. Hydrobiologia 260(261):601–605 Zinoun M, Cosson J (1996) Seasonal variation in growth and carrageenan content of Calliblepharis jubata (Rhodophyceae, Gigartinales) from the Normandy coast, France. J Appl Phycol 8:29–34
