Methods of extraction, physicochemical properties of alginates and their applications in biomedical field – a review

Abstract In this paper, the current state-of-art of extraction of alginates and the determination of their physico-chemical properties as well as their overall applications focussing on biomedical purposes has been presented. The quality and quantity of the alginate obtained with a variable yield prepared from brown seaweeds as a result of many factors, such as type of algae, extraction methods, chemical modification and others. Alginates are mainly extracted by using conventional alkaline extraction. However, novel extraction techniques such as microwave and ultrasound assisted extractions have gained a lot of interest. The extraction parameters (e.g., temperature and time of extraction) have critical impact on the alginate physiochemical and mechanical properties and thus, their potential applications. By controlling a chemical process makes it possible get various forms of alginates, such as fibres, films, hydrogels or foams. It is important to characterise the obtained alginates in order to their proper applications. This article presents several techniques used for the analysis of alginate properties. These natural polysaccharides are widely used in the commercial production, as a food ingredient, in the pharmaceutical industry due to their antibacterial, anticancer and probiotic properties. Their gelling characteristic and absorbable properties enable using alginates as a wound management material. Moreover, they are also biocompatible, non-toxic and biodegradable, therefore adequate in other biomedical applications.


Introduction
Brown seaweeds (known also as macroalgae) are abundant in many coastlines and in some of them they represent an almost unexploited but very valuable marine resource. This biomass constitutes a production system of bioactive compounds such as polysaccharides, proteins, minerals, lipids including polyunsaturated fatty acids, pigments, vitamins, antioxidants, etc., which are known to have antibacterial, antifungal, antiviral, antioxidative, anti-inflammatory and antitumor properties [1]. In this review paper we describe alginates that are natural polysaccharides extracted mainly from brown seaweeds. They are composed of two hexuronic acids: β-Dmannuronic acid (M) and α-L-guluronic acid (G) linked by 1-4 bonds [2,3]. These units are randomly distributed in a linear chain. They can be also arranged as homogeneous blocks MM or GG and heterogeneous or alternating as MG, as shown in Figure 1 [3,4]. Alginates are salts of long-chain alginic acids, that in brown seaweeds are present mainly as the calcium salt of alginic acid, although magnesium, potassium and sodium salts are also present [5]. Among them, sodium alginates are water soluble polymers, which give highly viscous solutions [6]. In the presence of polyvalent cations, such as calcium, sodium alginate has the ability to form gel [2,7].
Alginates are used widely due to their rheological properties [8,9], as well as biocompatibility, biodegradability and lack of toxicity [10,11]. The proportion of the three types of blocks -MM, GG and MG determines the physical properties of alginates -alginates with high G have higher gelling properties, whereas those with high M have higher viscosity [3,4]. Assessment of the M/G ratio is also fundamental -for alginates with a high M/G ratios alginates provide elastic gels, whereas for alginate with a low M/G ratios generate brittle gels [4,12].
Alginates are also characterised by unique biological and pharmacological properties [11]. In the food and pharmaceutical industry they mainly act as a gelling agent, stabilizer and thickener [2,13]. Nowadays, several applications of alignates have been seen, mainly as food additives to stabilize and improve food consistency (e.g., baked food, fruit industry, jellies, jams, ice cream, mayonnaise) [11,[14][15][16]. Moreover, these biopolymers are commonly considered as healthy food elements due to their anticancer and prebiotic properties [17]. Alginates additives could also be used as a complementary cure for obesity. Furthermore, these hydrocolloids are a potential beverage ingredient (e.g., beer foam stabilizers, dairy products, drinks for lowering blood sugar level) [11,18]. Another use of alginates in food industry is product packaging [14].
The aim of the present paper is to present the current trends in extraction of alginates from brown seaweeds. Conventional and novel methods of alginate extraction have been described, as well as process parameters that enable you to receive the optimal amount of alginate. The process efficiency is dependent not only on extraction parameters, but also other factors, such as species and origin of algae, preparation of samples, as well as the biomass pre-treatment. The type and procedure of extraction is significant due to the impact of chemical modification on the alginates form and their mechanical properties. It is an important aspect, because mechanical and physicochemical properties determine the alginate application. In the present article, the characteristics of alginates has been described, as well as their application in biomedical fields, but also in a sector of wound management materials based on alginates and their influence on wounds healing. Figure 2 shows a graphical representation of the present paper.

Brown seaweeds as a source of alginates
Alginates are produced industrially from marine seaweeds that belong to the taxonomic group of brown algae (class Phaeophyceae) [11]. Alginate-yielding seaweeds, also called "alginophytes", are mainly harvested from wild populations, although they can also originate from the artificial cultivation of algae -for example kelp Saccharina japonica [11,32]. Nowadays, commercial alginates are produced mainly from brown seaweeds of the following genera Ascophyllum, Durvillaea, Ecklonia, Laminaria, Lessonia, Macrocystis and Saccharina [16,33]. A detailed list of species of brown seaweeds that are used in various countries for the alginate production is presented in a book chapter in a book chapter of Peteiro [11].
Alginates are a part of the cell wall and inter-cellular matrix of brown seaweeds and provide the flexibility and the mechanical strength in order to survive in the water reservoirs [3,6,34]. Seaweed alginates exist as an insoluble mixed salt of all cations that are found in a sea water such as calcium, sodium and magnesium [16,34]. The alginate biopolymer can represent up to 40% of dry matter of brown seaweeds [30]. The quantity and quality of the alginates in brown seaweeds depend on many factors such as algae species, season of their harvesting, the type and age of the tissues [14,25]. Kumar and Sahoo [35] revealed that the alginate content in a biomass fluctuated as a function of seasonality (the highest content was in Sargassum wightii collected from Mandapam, Tamil Nadu, India in March, amongst other periods -January, May, July, September, November), as well as different vegetative parts of the thallus (the highest was in the main axis, among other parts such as whole thallus, young and old blades). In the case of Sargassum filipendula collected from Cigarras beach in Brazil, the highest extraction yield of alginates was for algae harvested in fall -May and spring -November and the smallest during summer -February [27]. On the other hand, Zubia et al. [36] found that the alginate yield from Sargassum mangarevense (Arue, Tahiti, French Polynesia) and Turbinaria ornata (Punaauia, Tahiti, French Polynesia) displayed spatial variations, but no significant seasonal changes (algae collected in February and July). A detailed description of genera and species used for alginate production, as well as natural habitats of brown seaweeds, harvesting methods of wild seaweeds, their quantities and cultivation methods is presented in the work of McHugh [32].

Conventional extraction of alginates from brown seaweeds
Generally, the protocol concerning extraction of alginates is composed of 5 steps: (1) acidification of the seaweeds, (2) alkaline extraction with sodium carbonate (Na 2 CO 3 ), (3) solid/liquid separation, (4) precipitation and (5) drying [5,8,11,37,38]. All these steps were described in details in a series of publications dedicated to the pilot plant scale extraction of alginates from Macrocystis pyrifera: effect of pre-extraction treatments on yield and quality of alginate [5], extraction conditions and methods of separating the alkaline-insoluble residue [39], precipitation, bleaching and conversion of calcium alginate to alginic acid [40] and conversion of alginic acid to sodium alginate, drying and milling [41]. The results of the experiments carried out in a pilot plant presented in the mentioned publications are especially important because they are closer to those that can be expected at an industrial scale [39]. Some of the procedures of the brown seaweeds pre-treatment, alginates extraction and their purification mainly in the laboratory scale are presented in Table 1.
Algal biomass before extraction of alginates can be pre-treated not only chemically (e.g., acidification), but also physically. First of all, the biomass used for the alginates extraction must contain 83% of a dried matter (17% moisture) and less than 3% sand [3]. Another important parameter is a biomass size. Reduction of the size of a raw material facilitates the subsequent processing of algae [38]. Fertah et al. [4] used for the extraction of alginates from Laminaria digitata ground biomass with the size <1 mm and 1-5 mm. It was shown that a higher yield was obtained for the biomass with lower particle size what can be explained by its higher surface area. Santagata et al. [42] showed also that the using a lowfrequency ultrasounds induced seaweed (Sargassum) cell wall disruption, causing the improvement of the extraction yield of alginates.
Several authors [4,12,13,27,29,36,39,43] before extraction of alginates from brown seaweeds performed soaking of algae in formaldehyde/formalin (usually 1-12 h) in order to soften the seaweed tissue, bleaching algal biomass and avoid alginate pigmentation [3,5,27]. The solution of formaldehyde reacts with algal phenolic compounds, polymerize and make the colouring substances insoluble [43]. Additionally, then the extraction yield of alginates can be higher [3]. In the work of Andriamanantoanina and Rinaudo [2] it was shown that the bleaching of brown algae before extraction (e.g.,    with chlorine) favoured the yield of extraction possibly due to the swelling of the algae cell walls. The treatment of seaweeds before extraction of alginates with diluted mineral acids is considered by many authors as an essential step that makes the alginate more readily soluble in an alkaline solution [5,8,9,13,23,35,36] etc., (see Table 1). More precisely, the acidification of algae can have several goals, for example it is necessary (1) to remove external salts and residual formaldehyde [3], (2) to remove non-target compounds such as polyphenols and other polysaccharides such as fucoidans and laminarins [27,44] and (3) to provide acidic condition up to pH 4 where alginate salts, for example with calcium, sodium, potassium are converted to alginic acid which is then more easily converted into soluble sodium alginate during extraction with sodium carbonate [3,13,44]. In order to remove non-target compounds such as pigments and lipids, Borazjani et al. [16] treated the milled algae with 85% ethanol overnight at room temperature. Youssouf et al. [10] used pre-treatment of Sargassum binderi and Turbinaria ornata with 80% ethanol at room temperature in order to eliminate other compounds such as pigments, polyphenols and fatty acids. Leal et al. [43] performed pre-treatment of algae (Lessonia flavicans, Desmarestia ligulata and Desmarestia distans) with petroleum ether to remove fats. Borazjani et al. [16] proposed pre-treatment of algal biomass (Sargassum angustifolium) before extraction with (1) distilled water: 65°C, 3 h, (2) HCl: 0.1M, pH 2, 65°C, 3 enzymes: (3) alcalase: 5% w/w, pH 8, 50°C, 24 h or (4) cellulase: 5% w/w, pH 4.5, 50°C, 24 h and examined its effect on the chemical composition and molecular properties of alginates.
Another approach was proposed by Lorbeer et al. [44] who used an acidic treatment (HCl solution) of the brown alga Ecklonia radiata in order to extract fucoidan (a sulphated polysaccharide found mainly in brown seaweeds) and at the same time to facilitate the efficient sequential extraction of alginates. In the case of the extraction of fucoidan from seaweeds, acid treatment disrupts hydrogen bonds between polysaccharides and liberates fucoidan. At the same time, alginate is converted into an insoluble alginic acid and do not contaminate the fucoidan extract [45]. This is an example of a sequential biorefinery extraction process of two polysaccharides [44,45]. Yuan and Macquarrie [46] also described a stepby-step biorefinery process of a seaweed Ascophyllum nodosum, which was designed to obtain fucoidan, alginates, sugars and biochar using microwave-assisted extraction. It was shown that brown seaweeds could be potentially used as a feedstock for a biorefinery process in order to produce valuable chemicals and fuels. By using an acid pre-treatment of brown seaweeds it is possible to extract separately alginate and fucoidan. Fenoradosoa et al. [12] found that the alginate extracted from Sargassum turbinarioides after its pre-treatment with HCl was a polymer of uronic acids (0.3% w/w). It was not associated with sulphated fucans which can occur together with alginates as matrix polysaccharides in Sargassum species.
The central step in the extraction protocol is the alkaline extraction which aims at the conversion of the original insoluble calcium and magnesium salts into soluble purified sodium alginate [5]. If the seaweeds (an original calcium alginate) are treated with alkali without prior acid pre-treatment of biomass, then the extraction is due to an ion-exchange [5]. In the extraction process it is necessary to optimize the temperature, extraction time, alkali concentration and consumption of solvent used for precipitation of alginates [15].
The conventional extraction process of alginates is based mainly on thermal treatment [38]. During extraction  process, seaweeds together with sodium carbonate are heated to higher temperature (from room temperature till 100°C) [3]. A slight increase of treatment temperature can lead to the rise in the extraction yield but also to the decrease of viscosity. Normally, the extraction temperature ranges between room temperature and 50°C [47]. Fertah et al. [4] showed that among three tested temperatures -25, 40 and 60°C, the highest extraction yield of alginates from Laminaria digitata was achieved for 40°C. However, alternative results were obtained by Mazumder et al. [15] who extracted alginates from Sargassum muticum at following temperatures -50, 60, 70, 80, 90 and 100°C. It was found that the extraction yield increased significantly above 70°C and the highest yield was observed when the temperature was between 90 and 100°C. Hernández-Carmona et al. [39] tested the effect of temperature on the extraction of alginates from Macrocystis pyrifera at 70, 80 and 90°C. It was found that the yield at 90°C -21.9 % was significantly higher than the yield at 70°C -19.4%. There is also an important role of extraction time acting as a critical variable that influences the yield of alginate [15]. The extraction process usually requires several hours (Table 1). In the work of Mazumder et al. [15] it was shown that there was a direct relationship between extraction time -1, 1.5, 2, 2.5, 3 and 3.5 h of alginates from Sargassum muticum and the extraction yield. Maximum yield was reached for 3 hours. Also Hernández-Carmona et al. [39] examined the effect of time -from 1 to 9 h of the alkaline extraction of Macrocystis pyrifera on the alginate yield. It was found that the yield of alginate increased with time and maximum was reached after 3.5 h of extraction.
In the work of Vauchel et al. [8] it was shown that the rheological properties of alginates (e.g., dynamic viscosity) can be decreased after 2 hours of alkaline treatment. Also Truus et al. [48] indicated that longer the extraction process and higher the temperatures will cause the production of alginates with lower viscosity, because of depolymerisation of the polymeric chain of the alginate. In the work of Torres et al. [49] alginates extracted from Sargassum vulgare for 1 h at 60°C were denoted as "low viscosity", whereas for 5 h at 60°C as "high viscosity"increase in the temperature during alginates extraction resulted in higher viscosity of solution, probably due to the dissolution of macromolecules with a high molar mass. The M/G ratio values were 1.56 and 1.27, respectively, what is higher than the ratio for most Sargassum spp. alginates which is in a range 0.19-0.82.
For the extraction of alginates from brown seaweeds, alkali are used and they change the alginic acid to soluble sodium alginate that appears like a viscous gel [12,15,29]. Alginates are extracted mainly with sodium carbonate which is also added to reach pH ~10 [3,15]. Rahelivao et al. [26] used the aqueous solution of 1M Na 2 CO 3 and EDTA in order to increase pH to 11. The basic pH leads to the formation of a water soluble sodium alginate [10]. Mazumder et al. [15] examined the effect of alkali concentration (0.5-5%) on the extraction yield of alginates (for constant other parameters -0.2M HCl, 3 h extraction time, 100°C, that also influence extraction yield) from Sargassum muticum. It was noted that the extraction yield increased rapidly up to 3% of alkali and decreased significantly after this concentration, probably due to the depolymerization of the alginate structure.
The last stage in the production of alginates is their precipitation and purification. Gomez et al. [6] studied three routes of precipitation of sodium alginate from the extract obtained from Macrocystis pyrifera -with ethanol, HCl and CaCl 2 . In the first route, sodium alginate is directly precipitated from the extract wit ethanol. In the HCl route, sodium alginate extract is mixed with HCl. The obtained precipitate of alginic acid is separated by centrifugation and mixed with distilled water and Na 2 CO 3 to obtain soluble sodium alginate. It is precipitated then with ethanol. CaCl 2 route involves its utilization for the precipitation of sodium alginate from the extract. The obtained calcium alginate after washing with water is mixed with HCl to obtain insoluble alginic acid, which is separated by centrifugation. Later, the procedure is the same as for HCl route. Gomez et al. [6] showed that ethanol route had the lowest number of steps and displayed the best performance -the highest yield and rheological properties of the obtained sodium alginate. Treatment of the sodium alginate extract with HCl caused degradation of the polymer chain. In this research it was also shown that CaCl 2 route gave alginates with the lowest molecular weight and poor mechanical properties [6]. As can be seen from Table 1, ethanol route is the most often used for the precipitation of sodium alginate.
The effect of the ethanol concentration on the precipitation of alginates from the solution after extraction is randomly studied. Mazumder et al. [15] presented interesting data which showed that the extraction yield of alginate from Sargassum muticum was negligible for 50 and 60% ethanol. The precipitation range increased slightly at 70% and reached a maximum between 90 and 100%.
Taking into account the application of alginates for some food/pharmaceutical/medical applications, it is also necessary to bleach the obtained product. Part of the compounds that impair the colour of alginates is removed in the previous steps -treatment with formalin/ formaldehyde and acid treatment, but the final colour of alginate can be still dark brown. In the literature it was shown that the best stage to bleach the product is as calcium alginate (CaCl 2 route) since it is more resistant to the degradation than alginic acid (HCl route) [50]. Usually, for bleaching a sodium hypochlorite solution (12%) is added to a suspension of the calcium alginate in water [39].

Novel extraction techniques of alginates from brown seaweeds
Nowadays, there is a demand for new, eco-friendly methods which will improve extraction process (e.g., yield, experimental conditions). Such a technique can be ultrasound assisted extraction (UAE) which limits energy consumption by the reduction of the extraction time, as well as the volume of solvents used, thus making it a "greener" process [10,42]. Youssouf et al. [10] showed that the extraction yield of alginates from Sargassum binderi and Turbinaria ornata depended on algae/water ratio, pH and the time of exposure to ultrasounds. The highest extraction yield of alginates equal to 54% was obtained for the following experimental conditions: algae/ water ratio of 10 g/L, pH 12, ultrasounds for 40 minutes and ultrasound power 150 W. Conventional extraction methods permit to obtain 25% alginate yield in 2 h [10]. As can be seen, the time of the extraction of alginates was significantly reduced -from about 2 h for a conventional method to 15-30 minutes with ultrasound assistance. Also microwave-assisted extraction (MAE) is proposed as a novel technique for the extraction of alginates since it possesses enormous potential to overcome major drawbacks that accompany the conventional extraction -thermal and/or solvent based techniques [38,46]. In the work of Silva et al. [38] it was demonstrated that the determination of the optimal acid pre-treatment conditions ( Table 2.

Characteristic of alginates obtained from brown seaweeds
What is important, the extracted alginates should be characterised in details -their morphology, content of chemical elements and functional groups, etc., in order to use them appropriately for several applications. The techniques that can be used for the analysis of properties of extracted alginates are briefly described below. 1 H-NMR (nuclear magnetic resonance) is used to determine composition, structure, M/G ratio and block distribution of alginates (e.g., 2,4,6,10,12,13,15,16,24,26,27,29,34,46,49). This analysis is considered to be the most reliable method to determine composition and block structure of the alginate [12]. Additionally, it provides insight into the relation between rheological functionality and chemical structure of alginates [13].
Rheological characteristic is an important parameter for the application of polysaccharides (including alginates) in the industry [12] and is considered in terms of: (a) intrinsic viscosity (capillary viscosimetry) (e.g., 4,9,12,24,26,34,47,49). The intrinsic viscosity is directly linked to the average molecular weight -the higher viscosity, the higher molecular weight [8]. This parameter depends also on the extraction technique -for example, for the sodium alginate obtained by batch extraction it was 0.3 L/g, whereas for reactive extrusion extraction 0.8 L/g [8]. Additionally, bleaching of seaweeds before extraction can decreases the intrinsic viscosity of alginate [2]. (b) gel strengths of the alginate (gel cylinders) (e.g., 2,34). This technique is used for gel characteristic in the presence of calcium ions [2]. Bleaching of seaweeds before extraction process can favour the gel formation even if the molecular weight is decreased [2]. (c) oscillatory rheology (gelation kinetics of hydrocolloids -rheometer) (e.g., 6,13,26).
Another important parameter in terms of application in food and biomedical field is alginate purity. It can be determined by measuring the level of phenolic compounds (Folin-Ciocalteu reagent e.g., 16; fluorescence spectroscopy e.g., 4, 15) and proteins (Lowry method) introduced to the extracted alginate [16]. Fluorescence spectroscopy can be applied to alginates, because they are strongly fluorescent due to small amounts of polyphenolic residues [4]. Protein content in the alginate can be reduced by the pre-treatment of seaweeds before extraction with HCl or protease and carbo-hydrase enzymes [16]. Other analyzes include determination of the uronic acid content (spectrophotometry) (e.g., 9,12) and the chemical elements in alginates (SEM-EDX) (e.g., 31). Some impurities (e.g., presence of Al) can be associated with the alginate extraction [31]. Structural characteristic of extracted alginates can be performed using SEM, XRF and FTIR techniques. SEM (Scanning Electron Microscopy) is used to study the morphology of alginates (e.g., 16,23,27,28,30,31,42). XRF (X-ray Fluorescence) is applied to quantify chemical elements in the alginate, for example Na, Ca, Si, Fe, S (e.g., 23). The major elements in alginates from Sargassum duplicatum and S. crassifolium were Na and then Ca [23]. The presence of sulphur can be associated with residues of fucoidan present in the brown seaweeds, from which alginate is extracted [31]. To confirm the existence of functional groups in the alginate FTIR (Fourier Transform Infra-Red Spectroscopy) analysis is used (e.g., 4,10,12,13,16,22,23,28,30,42,43). FTIR spectra of alginates are usually performed in the band range of 4000-450 cm -1 . Carboxyl (COO -) at about 1626-1623 cm -1 and at 1421 cm -1 and hydroxyl groups (O-H bending of guluronic acid  [10] units) at about 1025 cm -1 are evident in the FTIR spectra of alginates [13,23]. A band around 1100 cm -1 is typical for mannuronic acid units [13]. Carboxyl groups are from the mannuronate and guluronate moieties in alginate present in the sodium salt form [13].
Alginates are also examined in terms of their biological properties such as phenolic content (the Folin-Ciocalteu reagent) (e.g., 15,36), antioxidant activity determined using DPPH radical scavenging activity (1,1-diphenyl-2picrylhydrazyl) (e.g., 16,22,36) and reducing power (TCA; tricarboxylic acid) (e.g., 15,16), as well as antimicrobial activity (bacteria, yeast, mould) (e.g., 22). Borazjani et al. [16] showed that alginates obtained from S. angustifolium produced the stable form of DPPH in a dose-dependent manner and additionally were able to reduce ferric to ferrous in a redox linked colorimetric reaction. In the work of Janarthanan and Kumar [22] the antibacterial activity (against Staphylococcus aureus and Escherichia coli) of the cotton fabric coated with Sargassum wightii alginate film was tested. Alginate has good antibacterial activity.

Alginates applications in a biomedical field
The introduction of this review paper contains a description of general applications of alginates. The following section characterizes in details applications of alginates in bioengineering sectors including medical textiles thereby supporting healing wound.
Alginates are increasingly used in a health care industry due to their favorable properties. This compound of brown seaweeds has antimicrobial ability, strong free radical scavenging and antioxidant activity, renoprotective effect, anticancer and immunostimulatory properties [13,15,22]. These properties are result of the structure, as well as the presence of bioactive compounds such as amino acid, alkaloids, tannins, flavonoids and phenols [22] which contribute higher healing of skin wound. Beneficial alginates properties give the possibility to build skin graft and deliver medications in a controlled manner. They can be used in a treatment of patients suffering from diabetes mellitus, liver and parathyroid disease, as well as in repair and regeneration of tissues, certain cartilages and organs (e.g., liver) in the case of loss or failure of tissues or organs. It is possible to construct alginate-based scaffolds made for cells growth matrix, but also, for other health cases [22,23,[51][52][53].
Low-cost, wetting and visco-elastic properties make a possibility to use alginate in dental area, especially as impression materials [54,55]. Non-toxicity and nonirritating characterisitic makes them a popular choice to using them in preliminary impressions, for fullarch impressions, partial removable dental prosthesis frameworks, provisional crown-and-bridge impressions and more [55,56]. Alginate impressions have good qualities of reproduction surface detail and they are easier to remove compared with elastomeric materials. Unfortunately, they have low tear strength, thus they might tear upon removal over deep undercuts [56]. Most of dental applications consist of using alginate as an irreversible hydrocolloid, which are composed of 80% of water, but they are dimensionally unstable on storing because of evaporation, likewise alginate powder are not safe in the presence of humidity and higher temperatures due to its hydrophilic nature. Hydrophilicity of the alginate impression materials allows to be used in the presence of saliva or blood [55,56,57,58].
In tissue engineering, alginates are utilized mainly in the hydrogel form since several decades [59]. Moreover this field is still being developed due to possibilities of future applications, as well as healing properties of alginates and advancement of technology in medicine. These hydrocolloids are utilized in alginates scaffolds as a three-dimensional structure for cells growth matrix, to transport cells or proteins to the desired location, which can control the process of engineering or regeneration of tissues and organs. Furthermore, alginate scaffold can provide a protection of a drug or cell from the biological environment and it is a temporary skeleton supporting the formation of a new tissue, then the biodegradability ensures absorption of alginate scaffold by tissues in the nearby environment [23,53,60,61]. Alginates are applied in tissue engineering due to their structural similarities to the macromolecular-based human tissues. Moreover they are characterized by an easy injectability and they have a hydrophilic nature [11]. Another aspect is the ability to avoid the stimulation of a chronic inflammation or immunological reactions and the ability to biodegrade [23,30,53,62].
Regarding the damage of the external tissues of patients, nowadays, there are many professional medical textiles on different injury and there is no universal wound dressing. Wound healing is a complex process and each wound requires other procedure and a proper care, because this process may be compromised by a numerous factors (e.g., improper oxygenation, infections, inadequate dressing choice to type of wound) [52,63]. However, wound management material based on alginate has been created to enable faster heal from injuries and thus, has an positive effect on most of the wounds, especially traumatic, chronic and surgical wounds created to enable faster humans' recovery [14,19,52]. For instance, professional medical alginate fibres, biodegradable alginates films, which could control drug delivery on injury place by the slow disappearance of the material or could be used for making skin graft and even alginate agent applied to ordinary gauze [22,64]. Alginate bandage ensures a beneficial, moist and occlusive microenvironment, what can prevent the wound bed from drying out, accelerates and facilitates the process of wound healing due to minimizing of bacterial infection at injury site [22,53,59,60]. Furthermore they absorb exudate or serous fluid and form a hydrophilic gel by a chemical reaction with it. Alginate wound dressings are able to absorb of 15-20 times their own weight of liquid [19]. These bandages are characterized by a flexibility, thus, they are comfortable in daily using, moreover they are water and air permeable [22,53].
Alginates bandages are suitable for burn patients due to reduction of pain. They are also painless during their application and removal [52]. Their absorbable properties make them widely used in the treating of wounds with a high exudation, due to the hemostatic properties of calcium ions released during their use, as well as for patients with amputations, lacerations, chronic wound, diabetic and leg ulcers, diabetic foot lesions, pressure sores and many others [14,19,20,65]. Researches confirmed a significantly reduced area of chronic and acute wounds in a faster period after use of alginate bandage when compared with an ordinary gauze. It is also a result of the debridement of the necrotic tissue from a chronic wound [20,66].
In the case of the application of alginates in the biomedical field it is important to cleanse them before extraction, because this natural polymer can be impure. These contaminations can lead to the development of fibrotic cell overgrowth around alginate microcapsules, what might be a collateral damage on human health. Main alginate contaminants are endotoxins, polyphenols and proteins [4].
Wound management is an intricate process, in particular with regard to acute and chronic wounds. Chronic wounds such as ulcers, diabetes, burns, cancer etc. require recovery time more than 12 weeks [67]. The healing process consists of four phases: (a) coagulation and hemeostasis, (b) inflammation, (c) proliferation and (d) wound remodeling with a scar tissue formation. It is important to choose a proper approach to wound healing, adequate dressing to the type of injury since they may impact on the clinical outcome [68]. The most common available wound dressings are based on alginates, collagen, chitosan, hyaluronic acid and silicon, but also cellulose, gelatin and heparin [69]. Due to the variety of injuries, alginate-based bandages are supplied in different forms such as alginate fibre, hydrocolloids, hydrogels, films, foams and also combination dressings, which contain several ingredients supporting the healing process [60,70,71]. Commercially used alginate wound dressings with their purpose of application are presented in Table  3. The particular types of alginate wound dressings are characterized below.

Fibre
The majority of commercially available bandages are based on calcium alginate fibre. They absorb a large amount of exudate to create a gel-like covering over the wounds, what ensures moist environment and prevents wound from drying wound bed [72]. These dressings don't require frequent changing. Depending on the amount of exudate, wearing time is 2-7 days. They are suitable for wounds with moderate to heavy exudation, partial and full-thickness wounds, surgical wounds, infected wounds, pressure ulcers, diabetic and venous ulcers [73,74]. Alginate fibres are not recommended for wounds with little or no exudate, due to the possibility of drying the wound bed and separating or sticking fibres on wound bed. Moreover, alginate fibres always require a secondary dressing [75].

Hydrofibre
Another type of alginate bandage is hydrofibre dressing, which is a non-woven ribbon or pad composed of hydrocolloid fibres -sodium carboxymethylcellulose [80]. Alginate hydrofibre dressings are highly sorptive and form gel in combination with exudate to maintain a moist environment, what is an advantage in a fast wound healing and in a painless removal without harming fragile granulation tissue [80,81]. These dressings don't require frequent changing -depending on the amount of exudate wearing time is 2-7 days. Hydrofibre dressing, such as AQUACEL Hydrofibre Wound Dressing (Conva Tec), is a proper to acute or chronic wounds with large amount of exudate, partial and full-thickness wounds, donor sites, surgical wounds, pressure ulcers, venous and diabetic    ulcers [80,82]. However, they demand a secondary dressing. These bandages are contraindicated in wounds with low amount of exudate, due to the fact that they may induce drying of wound bed.

Hydrogels
Hydrogel dressing based on alginate contains 70-90% water, in consequence it provides a moist environment. Furthermore, this promotes a self-cleaning wound process, as the result of liquifying necrotic tissue on the wound surface [81,83]. The wearing time is 1-3 days [71].

Hydrocolloids
Wafer dressings fused adhesive elastomers with absorbent colloidal materials are called hydrocolloids [81]. They create gel together with little to moderate amount of exudate, what can provide a moisture environment to protect wound bed against drying [84]. Hydrocolloids are comfortable in the application and painless removal, more importantly, they do not require a secondary wound dressing [80]. Wear time is up to 7 days. They are waterproof, thus they protect from bacterial contamination. These dressings are oxygen impermeable which can cause unattractive odor during removal, and then they are not recommended for infected wounds [83]. Hydrocolloids based on alginate are the most common applied on granulating and epitheliazing wounds, partial and full-thickness wounds, surgical wounds, necrotic wounds, pressure ulcers, diabetic and venous ulcers [80,81]. They are not proper for highly exudation wounds, because large amount of fluid may contribute to more frequent change of dressings [80].

Films
Film is a transparent, semi-permeable membrane dressing, which is impermeable to liquid and bacteria contamination from outside, but permeable to water vapor, carbon dioxide and oxygen [71,81,87]. It creates a moist environment in conjunction with a small amount of exudate what provides removal of necrotic tissue and enables a cellular migration to promote wound healing [80]. Films based on alginate have some advantages, for example they prevent or reduce friction, wound healing progress may by controlled by the transparent film and they are economical. Dressing like Tegaderm™ (3 M Health Care) is suitable for lightening exudation wounds, superficial wounds, blisters, wounds on heels, elbows, flat surfaces [79,81]. These bandages are not recommended for wounds with a heavy exudation [80].

Foams
Foam dressings are made from hydrophilic polyurethane foam and they are proper for highly exudation wounds, due to their highly sorptive properties, what promote autolysis of necrotic tissue [80]. Moreover, they are appropriate for partial and full-thickness wounds, surgical wounds, pressure ulcers, venous and diabetic ulcers [71]. They are comfortable for using and conform to wounds. Wearing time is up to 7 days, in most cases 3-5 days [80]. Alginate based foam dressings are not intended for wounds with

Antibacterial dressings
Alginate dressings with additives, such as nanoparticles of silver, charcoal or other medical care ingredients have antibacterial, antifungal and antiviral properties and reduce unpleasant odors [80,81]. They are also able to absorb a large amount of exudate, although they should be used short-term only and often requires a secondary dressing. These alginate dressings maintain a favorable moist wound environment to help to put through autolysis process and to accelerate wound healing [80].

Properties of alginate hydrogel used in biomedical field
Bioengineering use alginate in different form, such as hydrogels, microspheres, microcapsules, foams, sponges and fibres [60]. Hydrogel is the most common form of this natural polymer used in a medical industry. They are three-dimensionally cross-linked networks constructed of hydrophilic polymers with a high water content [53,60]. Creation of gels from alginate depends primarily on the inherence of zones rich in GG block [2]. Alginate creates gels in the presence of divalent cations, mainly calcium ions, which bind GG blocks of aligned alginate chains. This process is giving rise gel-network, commonly called "egg-box", as shown in Figure 3 [4,12,13,60].
Alginates are also found in bioengineering as thermoresponsive and thermo-reversible hydrogels. Thermoreversible alginate hydrogel has a possibility to form gel in response to variation of at least two physical parameters at the same time (e.g., pH, temperature or ionic strength) with ability to convert to the previous consistency. They can be used as a smart delivery of bioactive agents. Thermoresponsive alginate hydrogel has potential application in tissue engineering as injectable cell scaffold [38,60].
On the chemical and physical properties of alginates have considerable impact their molecular characteristic, especially uronic acid ratio (M/G), gelation agent concentration (e.g., calcium ions concentration), molecular weight, degree of polymerization and blockstructure of alginate backbone. The monomer sequence (M and G) may be individual for different algal species, as well as in various tissues of identical species [2,4,13,19]. Content of guluronic acid from alginate gels decides about their fragility and strength, whereas amount of mannuronic acid determines their flexibility and weaker form of gel [11,49]. Pre-gel solution viscosity and post-gelling stiffness can be individually regulated by manipulation of the molecular weight and its distribution. Molecular weight is dependent upon extraction method, it can be increased by using cold method. Assessment of alginate molecular weight is determined by viscosity measurement [14,47,53]. Increasing alginates molecular weight can improve the physical properties of gels, although excessively high molecular weight in alginates solution can lead to extreme viscosity, which could be unacceptable in some process. Using fusion of a high and low molecular weight alginate polymers creates opportunity to increase elastic modulus and minimally raise the viscosity of the solution [53]. Biopolymers with higher amounts of G can be used to get more resistant gels and find applications in cosmetic and food industries. However, gels more flexible, with higher block M, can be applied in paper industries and can be used for the production of micro-and nanoparticles of drug [27,47]. The physical properties substantially control the stability of the gels, what can determine applications of alginate, for example in drug release from gels [14].
The mechanical characteristics of alginate hydrogels depend on, inter alia, gel uniformity, which may be controlled by gelation rate. Slower gelation give a possibility to obtain more uniform structures, thereby better mechanical properties. Important parameter, which has influenced on gelation rate, is temperature of gelation. Slower gelation proceed at lower temperature (reactivity reduction of ionic cross-linkers (e.g., Ca 2+ ) and slower cross-linking) [53]. The mechanical strength of alginate hydrogel is comparable with a soft tissue elasticity, what makes it a proper component for soft tissue reconstruction [62]. In order to improve or change physical and mechanical properties of alginates hydrogels, they can be combined with other biomaterials [60]. Stability of an alginate molecule is highly influenced by the environment conditions (e.g. pH, temperature and amount of contaminants) [11].

Conclusions
Alginates are one of the compounds of brown seaweeds, which characterise of favourable properties. There are known many methods to obtain these natural polysaccharides from algae, but quantity and quality of achieved yield depend on many aspects [3]. Algae species, season and place of their harvesting are important, but on the yield rate have an impact also process parameters (temperature, time of extraction, alkali concentration, pre-treatment) and method of extraction (conventional or novel method) [3,14,15,25]. The higher alginate yield was attained by higher temperatures and longer time of extraction. Moreover, using a novel technique of alginate extraction (e.g., ultrasound assisted extraction) allow to reduce the extraction time, but also solvents volume used and to increase the efficiency of the process [10,42].
These natural polysaccharides are widely applied in everyday life sectors such as food and beverage industry, textile manufacturing, pharmaceutical industry, but also in bioengineering technology with the area of biomedical field [3,11,14,15,[17][18][19][20][21][22][23]. Extensive applications are result of alginate rheological properties, but also due to biocompatibility, biodegradability and absence of toxicity [8][9][10][11]. Their biological and pharmacological properties, such as antimicrobial ability, antioxidant activity, anticancer and immunostimulatory properties, strong free radical scavenging and renoprotective effect, have contributed to the development of alginate research in the biomedical area [11,13,15,22]. The using of alginates in medicine assists in a faster healing recovery from inside, as a medicine for diabetes mellitus, liver and parathyroid disease, but also as a treatment for external areas of human body, especially in tissue engineering, tissue regeneration, wound dressings. Technological development enabled also the application of alginates in repair and regeneration of certain cartilages and organs, moreover, it enabled to construct alginate-based scaffolds. The biodegradation ability of alginates gives the possibility for the release of medications in a controlled manner [22,23,[51][52][53].
In this review paper, in particular management of wound healing and type of wound dressings have been described. Alginates are applied in the wound management material sector due to their therapeutic properties, faster healing and positive effect on most of the wounds, especially traumatic, chronic and surgical wounds, when compared to traditional bandages [14,19,52]. Alginates extracted from marine seaweeds have to be purified to achieve medical grade [4]. Moreover, medical sector requires to obtain alginates in reproducible way to achieve obtain an equal structure. It is important, because chemical modifications may change alginate form and also their mechanical properties [60]. Depending on the purpose for which the alginates are applied, there are different forms of alginate, such as hydrogels, hydrocolloids, fibres, films, foams, microspheres or microcapsules [60]. Continued technical development and researches will widen up perspectives and potential uses in the future.
There is many articles regarding conventional extraction methods of alginates and there are increasing novel techniques of extraction which are using more eco-friendly equipment, improving extraction yield of alginates and reduction time of the extraction. During write this review, it is noted that there is lack of information about combination or composite alginate wound dressings. Using of properties such as biodegradability might contribute to creation of a new generation of drugs or wound dressings enabling slow medicament release in a controlled manner. This is important topic, due to the possibility of connection several medical care ingredients in alginate dressing to accelerate healing wounds. This make perspectives for the future research and innovation.