Relationship between molecular weights and biological properties of alginates extracted under different methods from Colpomenia peregrina

Highlights

• Cellulase-assisted extraction produced alginate with the highest purity from C. peregrina.

• The use of various extraction methods resulted in alginates with different molecular weights and conformations.

• Enzyme-extracted alginates showed higher immunostimulatory activity.

• Using enzymes in the extraction of alginates enhances their antioxidant properties.

Abstract

The effect of extraction methods using water, acid, Alcalase and Cellulase was studied on structure–bioactivity relationship of alginates from Colpomenia peregrina. Enzyme-assisted extractions produced alginates with the lowest protein (<0.7%) and polyphenol (<5 mg gallic acid/g sample) contaminations. The molecular weight (M_w) of alginate extracted after acid treatment was 386.4 × 10³ g/mol. The minimum M_w of 247.9 × 10³ g/mol was determined for alginates extracted after Cellulase treatment. The specific volume of gyration (SV_g) increased to >0.57 cm³/g in enzymatically treated alginates. Alginate isolated after Cellulase treatment was nontoxic and stimulated RAW264.7 macrophage cells to release nitric oxide and inflammatory cytokines such as TNF-α, IL-1, IL-6, IL-10 and IL-12. The DPPH radical scavenging activity and reducing power were shown to be the highest for enzyme-treated. This study demonstrates the lab scale feasibility of extraction of alginate with the highest immunomodulatory and antioxidant properties.

Introduction

The cell wall of seaweeds is composed of a complex matrix of polysaccharides, proteins and low molecular weight compounds bound with each other through ionic interactions and hydrogen bonding [1]. Alginates are widely found in the cell wall of brown seaweeds in an insoluble form bound with Na⁺, Mg²⁺ and Ca²⁺ [2]. The presence of alginates in the structure of seaweeds provides them a relatively high level of softness and flexibility against currents [3]. Alginates are linear polymers composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G), linked by 1,4-linked residues [4]. The ratios of G and M blocks vary depending on seaweed species and their pattern determines the physicochemical properties of alginates [1]. For instance, alginates with low M:G ratios produce dense and brittle gels, whereas high M:G ratios of alginates form elastic gels [5].

In terms of application, ever since the alginic acid discovered by Stanford [6], a mounting number of studies investigated its peculiar physical characteristics and potential utilization in food industry. For instance, sodium alginate was found to be highly viscous when dissolved in water and thus can be used as stabilizer and thickener in suspensions [7]. Besides, the gel formation of sodium alginates in the presence of polyvalent cations such as Ca²⁺, which forms an egg-box structure, extended its utilization in a wide range of food systems [8].

Conversely, alginates have not been widely utilized as bioactive compounds in food and pharmaceutical industries. However, on the contrary to many natural bioactive polysaccharides, alginates are already widely accessible in the market, easy to isolate and inexpensive to extract [4]. The previous reports have proposed the potential applications of alginates as antioxidant and antidiabetic agents [9], [10]. It is reported that the biological properties of polysaccharides are closely related to their chemical structure and it could be totally vanished if the structure changes [11]. The chemical variations of polysaccharides might be originated from phylogenic differences or caused by growth environment of seaweeds and extraction methods [12]. In addition to the fact that there is limited information on the bioactivities of alginates [10], less attention has been paid to structure–activity relationship of polysaccharides as affected by extraction methods [3]. Essentially, the current extraction method to isolate alginate for food industry consists of two major stages. Primarily, it begins with a pretreatment using hydrochloric acid to remove undesirable compounds such as fucoidans, laminarins and polyphenols [13]. In the next step, the extraction process is conducted by solubilization of alginate molecules using Na⁺ and successive dissolutions and precipitations to eliminate remaining impurities [3].

The use of diluted acid during pretreatments or extractions disrupts the intermolecular bonding and facilitates the liberation of polysaccharides, although a low pH might compromise the structural integrity of target polysaccharides [14]. To overcome the degrading effect of acids, commercial carbohydrase and protease enzymes could be employed in the extraction process [15]. However, the digestion of cell wall structure by enzyme hydrolysis could lead to the release of other compounds after which expensive and tiresome purification steps are required in order to remove them [16].

The purpose of present study was to evaluate the effect of various pretreatment methods on the chemical composition and molecular properties of alginates extracted from Colpomenia peregrina. The immunostimulatory and antioxidant properties were also examined for alginates obtained under different extraction procedures.