Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)

Ulvan is a sulfated heteropolysaccharide present in the cell wall of Ulva species with unique structural properties and technological potential. Here we characterized by FTIR and NMR analysis the structure of ulvan from Ulva fasciata collected in natural environment (SEA) and after in vitro biomass cultivation in nutrient enriched water (CULT). FTIR spectrum of CULT ulvan presented stronger signals of sulfate groups than SEA. 1 H and 13 C NMR showed that both ulvan are composed mainly of ulvanobiuronic acid 3-sulfate type A and type B. SEA ulvan presented signals characteristics of xylose, suggesting the presence of ulvanobiose in its structure, while CULT presented most signals of type A disaccharide. The cultivation of Ulva could be an al-ternative to suffice the emerging demand for ulvan meeting requirements of quality and quantity.

Species of the genus Ulva are the most abundant and cosmopolitan macroalgae in the Chlorophyta Division being able to adapt across diverse geo-climatic conditions with high productivity and opportunistic growth. Ulva cultivation is increasing worldwide due to its potential as functional foods, feed and biofuel [3] [4]. Although polysaccharides from the red (carrageenan and agar) and brown (alginate) macroalgae have been used in the food industry, polysaccharides from green macroalgae remain largely unexploited. The main polysaccharide of Ulva species is ulvan, corresponding to 29% of dry weight with a promising technological application.
Ulvan is homogeneously distributed within the intercellular space and in the fibrillar wall [5] [6] being responsible for maintaining the osmolar stability and protecting the thallus from marine bacterial attack [2] [7]. Ulvan is composed of variable amounts of rhamnose, glucuronic acid, iduronic acid, xylose and sulfate [8] [9]. This sulfated heteropolysaccharide is built on sequences of two major repeating disaccharides unities designated as ulvanobiuronic acid 3-sulfate type [11]. Minor sulfated residues with xylose can also occur in place of uronic acids [12].
L-rhamnose used in a variety of anti-aging cosmetics [21] [22] is specifically recognized by a number of mammalians lectins [6]. Iduronic acid, which has never been identified in algal polysaccharides [5] [23] is required in the synthesis of heparin analogs being used against respiratory syncytial virus infection and antithrombotic activities [24] [25]. Currently, this substance is obtained through several steps that could be avoided using ulvan [5] [25].
Although the biotechnological applications of ulvan are promising, structural variations may occur due to ecophysiological factors acting on Ulva [26] [27].
The commercial use of polysaccharides requires ulvan with predictive structure and functional properties, which could be obtained by the controlled cultivation of Ulva. To determine the potential of cultivated Ulva for ulvan production in this work we characterized (FTIR and NMR) the ulvan extracts from Ulva fasciata Delile (Chlorophyta) after in vitro biomass cultivation in nutrient enriched water and compared it against ulvan from biomass collected in an oligotrophic natural environment, to enhance potential structural differences.

Algal Material
Healthy thalli of Ulva fasciata were collected in the intertidal zone at Prainha The species-level identification as U. fasciata was determined by molecular studies (barcoding using tuƒA markers) [28]. Voucher specimens were deposited in the Institute of Bioscience Herbarium, at the University of São Paulo, Brazil (SPF-57877). Part of the fresh biomass (SEA) was oven dried at 50˚C until constant weight and stored in desiccator until ulvan extraction. The remaining material was used in the cultivation experiment (CULT). In sequence biomass from natural environment and cultivation experiment (SEA and CULT, respectively) were used for ulvan extraction ( Figure 1).

Ulva Cultivation
To ensure that all individuals presented comparable initial physiological conditions, U. fasciata thalli underwent a seven-day acclimatization period to the la-   [29]. The nutrient concentrations were chosen with reference to the mean maximum nutrient concentration after a five years monitoring of one of the points of an important Brazilian bay [30] with potential for Ulva cultivation. The same light, photoperiod and temperature conditions of the acclimatization and starvation period were maintained. The experiment lasted five days.

Ulvan Extraction
After the cultivation, individuals were washed with distilled water to remove salts and oven dried at 50˚C until constant weight. The polysaccharide was extracted according to method described by [31]. Dried algal biomass (SEA and CULT) were grinded into a powder, suspended in ultrapure water (Milli-q ® ) (100 ml/10g) and autoclaved at 120˚C for 40 min. The supernatant was centrifuged at 10,000 g and 4˚C for 10 min (Eppendorf centrifuge 5810 r). Ulvan was precipitated with three volumes of ultrapure ethanol (Merck ® ), cooled at -20˚C for 48 hours and further centrifuged at 3500 g at 4˚C for 5 min. The recovered pellet (ulvan) was freeze-dried. Ulvan extraction yield was 16.29% ± 0.93% calculated using formula proposed by [32]: where, W e is the dry ulvan weight extracted and W f is the macroalgae dry weight.

Fourier-Transform Infrared Spectroscopy (FT-IR) Analysis
Ulvan (SEA and CULT) infrared spectra with Fourier transform (FT-IR) were recorded on a spectrophotometer (IR Prestige_21, Shimadzu) at room temperature. The FT-IR spectra were obtained in the transmission mode at 400-4000 cm −1 . The transmission spectra were recorded using KBr (Merck ® ) pellets containing 2.5 mg of ulvan powder.

Results and Discussion
FTIR and NMR spectroscopy are rapid and non-destructive analysis that provide IR Spectra of SEA and CULT ulvan are presented in Figure 2 with the signals assignment provided by comparison with published data [32] [33] [34] [35].
CULT and SEA spectra presented all the characteristics peaks described in literature [34] [36] [37], confirming that the extracted polysaccharides are ulvan.
According to FTIR spectra, there was no visible difference between the two ulvan extracts and those reported in the literature [14] [17] [33].
The absorption band at around 3300 cm −1 was attributed to a stretching of  Noise observed in the 13 C NMR spectra is related to sample dilution increased by the high molecular weight of the polymer and solution viscosity. Ulvan molecular weight can vary from 1.8 × 10 5 to 2 × 10 6 depending on extraction methods, specie and polydispersity of the samples [32] [43]. According to [6] ulvan  extracted with temperatures between 80˚C -90˚C, close to the used in this study, tend to present higher molecular weight.
In 13 C and 1 H NMR spectra of SEA ulvan we could observe signals of xylose, suggesting the presence of ulvanobiose (U3s) in this ulvan structure. In CULT we could not detect U3s, but peak characteristic to C-1 of rhamnose in A3s disaccharide (4.82 ppm) was present [6]. According to [27] during the active growth of Ulva the macroalgae tends to synthesize more ulvanobiuronic acid type A, with the production of ulvanobiose being developmentally regulated. In this study, U. fasciata presented an average growth rate of 5.7% day −1 and active nutrient uptake throughout the cultivation experiment (data not shown), an indication that individuals had not reach their growth plateau when collected.
In this study both ulvan presented similar global structure, but ulvan from cultivated U. fasciata presented stronger signals of sulfate and ulvan from natu-

Conclusion
The production of ulvan with predictive structure and in necessary amounts is one of the hindrances for the ulvan market development. The results gathered here show that ulvan from cultivated U. fasciata is similar to those reported in literature and could be a source for obtaining this polysaccharide. By controlling abiotic conditions ulvan production could be maximized meeting commercial requirements.