Chemical Composition, Structural Properties, and Bioactivity of Carrageenan from Field-Cultivated Betaphycus gelatinus

Abstract

This study investigates seasonal biomass variations in Betaphycus gelatinus, a red alga cultivated in the field in Ninh Thuan, Vietnam, along with the chemical composition, structural properties, and bioactivity of its carrageenan. Monthly measurements over a one-year period revealed peak growth (2.02% per day) and carrageenan yield (59.61%) in June, identifying it as the optimal harvest period. FTIR and NMR analyses of carrageenan extracted from field-cultivated B. gelatinus showed hybrid κ- and β-carrageenan forms and a unique pyruvylated β-carrageenan structure not previously reported for this species. Bioactivity assays indicated high antioxidant potential, with a total antioxidant capacity equivalent to 48.30 mg ascorbic acid/g carrageenan and an ABTS radical scavenging IC50 of 3.64 µg/mL. Additionally, antibacterial tests demonstrated strong inhibition of Listeria monocytogenes (12.00 mm inhibition zone). These findings suggest that field cultivation is a sustainable approach for carrageenan production, yielding bioactive compounds with promising applications in pharmaceuticals, cosmetics, and food preservation as a viable alternative to wild harvesting.

1. Introduction

Red algae, particularly those within the division Rhodophyta, are notable for producing bioactive polysaccharides, with carrageenan standing out for its wide range of applications in the food, cosmetic, and pharmaceutical industries due to its gelling, thickening, and stabilizing properties [1,2]. Among carrageenan-producing algae, Betaphycus gelatinus (formerly Eucheuma gelatinum) is highly valued for its carrageenan [3]. This species, part of the family Solieriaceae and order Gigartinales, thrives in tropical and subtropical coastal regions across Southeast Asia, including the Philippines, Indonesia, southern China, and Vietnam [4,5]. In Vietnam, B. gelatinus is primarily found along the coast from Quang Ngai to Ninh Thuan provinces, favoring reef habitats exposed to strong wave action [6,7].

Carrageenan, a sulfated polysaccharide derived from red algae (Rhodophyta), is categorized into three primary types based on molecular structure: kappa (κ), iota (ι), and lambda (λ). These types exhibit distinct gelling behaviors attributed to variations in sulfate content and the presence of the 3,6-anhydro-D-galactose linkage. Kappa-carrageenan forms a rigid and brittle gel when combined with potassium ions, while iota-carrageenan generates a softer, elastic gel in the presence of calcium ions. In contrast, lambda-carrageenan, which has a higher sulfate content, lacks gelling ability but provides viscosity, making it effective as a thickening agent [8,9]. These structural variations enable carrageenan to be applied across diverse industries, including food, pharmaceuticals, and cosmetics [10,11].

Carrageenan has attracted significant interest due to its diverse biological activities, particularly its antioxidant and antimicrobial properties [12,13,14]. Its structure, rich in sulfate groups, enables carrageenan to neutralize free radicals, reduce oxidative stress, and protect cells from damage [15]. Different types of carrageenan, including kappa, iota, and beta, exhibit unique antioxidant activities based on the distribution of sulfate groups and specific molecular structures. For instance, iota-carrageenan has demonstrated protective effects on gastric tissue against ethanol-induced damage in rodents by inhibiting free radical formation and conserving sulfhydryl groups [16]. Additionally, when combined with compounds like tranexamic acid, iota-carrageenan produces antioxidant and antibacterial powders that are valuable in medical materials [17]. These properties underscore carrageenan’s potential as a natural antioxidant, enhancing health benefits and food preservation while paving the way for novel medical treatments.

In addition to its antioxidant effects, carrageenan exhibits significant antimicrobial properties against various pathogenic bacteria and fungi, making it a promising candidate for multiple industrial applications. This antimicrobial activity likely arises from carrageenan’s capacity to disrupt microbial cell membranes and prevent biofilm formation, leading to cell death or inhibition of bacterial growth. Studies have shown that certain forms of carrageenan, particularly low-molecular-weight and N-alkyl derivatives, are effective against strains such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [14,18]. Thus, carrageenan has been investigated as a natural preservative in food and a therapeutic agent for infections, underscoring its functional versatility in the food and pharmaceutical industries [11].

The carrageenan extracted from B. gelatinus exhibits a hybrid composition, containing beta-, kappa-, and gamma-carrageenan [3], making it highly promising for applications in various fields of life. However, sustainable production of B. gelatinus carrageenan faces challenges due to environmental pressures and overharvesting, highlighting the need for effective cultivation techniques. By assessing the biomass variations and carrageenan content of B. gelatinus throughout the year, this study aims to identify optimal harvest periods and analyze the structure and bioactive properties of carrageenan. This study provides a comprehensive analysis of the seasonal biomass dynamics, chemical composition, structural properties, and bioactivity of carrageenan obtained from field-cultivated B. gelatinus in Ninh Thuan, Vietnam. The findings contribute to the understanding of sustainable carrageenan production and offer insights into its potential applications in various industries.

2. Materials and Methods

2.1. Materials

The red alga B. gelatinus was cultivated in Thai An village, Ninh Hai district, Ninh Thuan province, Vietnam, over a year-long period from March 2022 to February 2023. After harvesting, the alga was transported to the Hon Chong Advanced Research and Innovation Center at the Nha Trang Institute of Technology Research and Application (NITRA). Upon arrival, samples were thoroughly rinsed with distilled water to remove any mechanical debris, sand, and mud. Cleaned samples were then dried at 40 °C until reaching a constant weight, and then stored in nylon bags at −20 °C until further analysis, including IR spectroscopy.

All chemicals used for the extraction and chemical composition analysis and bioactivities assays of polysaccharides were of analytical grade, including anthrone, resorcinol, trichloroacetic acid (TCA), agarose, ammonium molybdate, ferric chloride, ABTS, and ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), as well as sulfuric acid, ethanol, hydrochloric acid, sodium sulfate, barium chloride, sodium phosphate, potassium ferricyanide, potassium peroxodisulfate, and Mueller–Hinton Agar (Merck, Darmstadt, Germany).

2.2. Methods

2.2.1. Biomass Measurement of Field-Cultivated B. gelatinus

The study commenced with the collection and experimental setup of B. gelatinus in Thai An, Ninh Thuan, from March 2022 to February 2023. Upon collection, the alga was carefully cleaned using soft brushes to remove epiphytes and organic debris. Excess moisture was blotted away with absorbent paper, and any coral fragments attached to the alga were removed. Healthy thalli weighing 20 g (fresh weight) were selected for the study and attached directly to coral substrates at the site using plastic zip ties. Each sample was individually labeled with a unique identification code, and a total of 20 samples were secured onto the coral using free-diving techniques [19].

Monthly, all samples were retrieved, cleaned, and weighed to determine fresh weight changes. After weighing, the samples were reattached to the coral substrate. The daily growth rate (DGR) of the cultivated alga was calculated following the formula from Yong et al, 2013 [20], allowing for precise monthly monitoring of growth dynamics across the study period.

$$\mathrm{D}\mathrm{G}\mathrm{R}=⌊\sqrt[d]{{\displaystyle \frac{W_t}{W_0}}}-1⌋\times 100$$

where

DGR is the daily growth rate as a percentage (%/day);

W₀ is the initial fresh weight of the seaweed (g);

W_t is the fresh weight of the seaweed after d days of cultivation (g);

d is the cultivation period (days).

2.2.2. Carrageenan Extraction Method from Raw Alga

Carrageenan was extracted following a modified version of Ohno’s method [21]: Initially, 10 g of dried alga was soaked in 200 mL of distilled water at room temperature for 12 h. To remove pigments and other soluble organic substances, the alga was then treated with a 200 mL methanol–acetone solvent mixture (1:1) for an additional 12 h. After this treatment, the algae were dried, weighed, and then prepared for polysaccharide extraction. The extraction was conducted in distilled water at 85 °C for 3 h, and an alga-to-water ratio (w/v) of 1:50 was used, with the pH maintained at 7. After extraction, the mixture was filtered to separate the extract from the solid residue, which was then used for a secondary extraction. The collected extract was concentrated using rotary evaporation to one-third of its initial volume. Polysaccharides were precipitated by adding ethanol at a sample-to-ethanol ratio of 1:3 (v/v) and then dried to constant weight to determine the extraction yield.

2.2.3. Chemical Composition Analysis of Carrageenan

Galactose content analysis: The galactose content in carrageenan was measured using a colorimetric assay with anthrone reagent, following a modified version of the method by Yaphe (1960) [22]. Galactose served as the calibration standard. A stock anthrone solution was prepared by dissolving 200 mg of anthrone in 100 mL of 83.6% sulfuric acid and then stored at 4 °C for stability. For each assay, 1 mL of the carrageenan solution was combined with 10 mL of freshly prepared anthrone reagent in a boiling tube. The reaction mixture was then heated in a boiling water bath for 11 min, followed by rapid cooling in an ice bath. Absorbance was measured at 630 nm.

3,6-Anhydrogalactose content analysis: The 3,6-anhydrogalactose content in carrageenan was determined using a modified version of Yaphe’s method (1960) [22]. A stock solution of resorcinol was prepared by dissolving 130 mg of resorcinol in 100 mL of absolute ethanol. For the assay reagent, 10 mL of this stock solution was combined with 100 mL of 12 M hydrochloric acid, freshly prepared and stored in a brown bottle to maintain stability. To perform the assay, 2 mL of the carrageenan solution was mixed with 10 mL of freshly prepared resorcinol reagent in a boiling tube, and then placed in an ice bath for 5 min. The sample was subsequently heated at 80 °C for 10 min and quickly cooled in an ice bath. Absorbance was recorded at 500 nm.

Sulfate content analysis: The sulfate content in carrageenan was determined following the turbidometric method of Jackson and McCandless (1978) [23]. Samples were hydrolyzed by heating with 1N HCl at 100 °C for 2 h in screw-capped tubes to prepare for analysis. A fresh barium–agarose reagent was prepared by dissolving 0.02% agarose in a 0.5% barium chloride (BaCl₂) solution. For the assay, 1 mL of the hydrolyzed carrageenan sample was mixed with 1.2 mL of 8% trichloroacetic acid (TCA), followed by the addition of 600 µL of the barium–agarose reagent. This mixture was then incubated at room temperature for 30 min. Absorbance was measured at 360 nm, using a blank as a reference, and sodium sulfate served as the calibration standard.

FTIR analysis: The dried B. gelatinus samples were lightly ground to achieve homogeneity, with sample volumes limited to a maximum of 2 mm³ to ensure complete and even coverage of the IR diamond window. Both the alga and extracted carrageenan samples were analyzed using Fourier-transform infrared (FTIR) spectroscopy. Spectral data were collected with a Shimadzu Affinity-1S FTIR spectrometer equipped with a QATR detector, spanning a wavenumber range from 400 to 4000 cm⁻¹. Data acquisition and analysis were performed using LabSolutions IR software (Version 2.27).

NMR analysis [24]: Approximately 10 mg of carrageenan was dissolved in 500 μL of deuterium oxide, D₂O) for nuclear magnetic resonance (NMR) analysis. The spectra were acquired using a Bruker Avance III HD 500 MHz instrument equipped with a 5 mm TCI cryoprobe and an Oxford magnet. One-dimensional proton (¹H) NMR spectra were recorded with 16 transients across 16,384 complex data points, with a sampling duration of 1.7 s. For two-dimensional NMR analyses, ¹H-¹H COSY spectra were captured with 2048 × 512 complex data points, with direct and indirect dimension sampling times of 213 ms and 53 ms, respectively. Additionally, ¹H-¹³C heteronuclear multiple-bond correlation (HMBC) spectra were acquired with 2048 × 128 complex data points, at 256 ms and 6.3 ms sampling intervals. Multiplicity-edited ¹H-¹³C heteronuclear single quantum coherence (HSQC) spectra with adiabatic decoupling were recorded with 2048 × 512 complex data points, with sampling times of 213 ms and 15.5 ms. Further, ¹H-¹³C HMBC spectra were obtained with 1024 × 100 complex data points, sampled at 128 ms and 3 ms. Data processing, including baseline correction and zero-filling in all dimensions, was completed using Bruker Topspin 3.5 pl7 software, which was also used for data analysis. Spectra of high-molecular-weight carrageenan were recorded at 80 °C.

2.2.4. Methods for Determining Biological Activity

Determination of total antioxidant capacity (TAC): The total antioxidant capacity (TAC) was evaluated using the method outlined by Prieto et al. (1999) [25]. In this procedure, 1 mL of the carrageenan sample was combined with 3 mL of a reagent mixture consisting of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. This reaction mixture was then incubated at 95 °C for 90 min and subsequently allowed to cool to room temperature. Absorbance was measured at a wavelength of 695 nm, with ascorbic acid serving as the reference standard for TAC calculation.

Ferric-reducing antioxidant Power (FRAP) assay: A FRAP assay was conducted following the protocol outlined by Zhu et al. (2002) [26]. Specifically, 1 mL of phosphate buffer (pH 7.2) and 1 mL of 1% (w/v) potassium ferricyanide were added to 1 mL of the carrageenan solution. The mixture was then incubated at 50 °C in a water bath for 20 min. After incubation, 1 mL of 10% trichloroacetic acid was added, followed by mixing with 0.6 mL of distilled water and 0.16 mL of ferric chloride. The absorbance of the final solution was measured at 655 nm, with ascorbic acid used as the reference standard for TAC calculation.

ABTS Assay: The ABTS radical scavenging activity was evaluated using a modified version of the method by Leutou et al. (2016) [27]. The ABTS•+ solution was prepared by combining ABTS (7 mM) with potassium peroxodisulfate (2.45 mM) and allowing the mixture to activate for at least 16 h. Before the assay, the solution was diluted to an absorbance of 0.7 ± 0.02 at 734 nm. For testing, 100 µL of ABTS•+ solution was mixed with 100 µL of the sample (200 µg/mL) in a 96-well plate and incubated in the dark at 28 °C for 15 min. Absorbance was recorded at 734 nm using a MultiscanFC microplate reader (ThermoScientific, Waltham, MA, USA). L-ascorbic acid was used as a positive control, and each test was conducted in triplicate. The IC50 value, or the concentration needed to scavenge 50% of ABTS radicals, was calculated for the sample.

Antibacterial Activity: The antibacterial activity of the extract was evaluated using an agar well diffusion assay. Eight bacterial strains were tested, including four Gram-positive bacteria (Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19111, Streptococcus faecalis ATCC 19433, and Staphylococcus aureus ATCC 25923), and four Gram-negative bacteria (Escherichia coli ATCC 25923, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, and Salmonella Typhimurium ATCC 14028). The bacterial strains were cultured in Mueller–Hinton Broth (MHB), and their suspensions were adjusted to a turbidity of 10⁵ CFU/mL. Subsequently, 100 μL of each bacterial suspension was spread evenly onto Mueller–Hinton Agar plates (Merck KGaA, Darmstadt, Germany). The extract, prepared in distilled water at a concentration of 2 mg/mL, was applied to wells (8 mm diameter) punched into the agar using a sterile cork borer, with 100 μL of the extract loaded into each well. Cefotaxime (30 µg/disk) was used as a positive control. The plates were incubated at 37 °C for 24 h, and the zones of inhibition were measured in millimeters.

3. Results

3.1. Biomass and Carrageenan Content Fluctuations in Field-Cultivated B. gelatinus

The results show the Dry Growth Rate (DGR) and extraction yield (%) of B. gelatinus throughout the year, with data collected across the four seasons: spring, summer, autumn, and winter (Figure 1)

In spring, the daily growth rate (DGR) of B. gelatinus increased from 1.53% in March to 1.98% in May, alongside a rise in carrageenan yields, which peaked at 40.54% in April before slightly declining to 38.77% in May. The DGR reached its highest rate of 2.02% per day in June, with carrageenan yields peaking at 59.61%, indicating June as the optimal month for harvesting due to favorable growth conditions. Yields stayed relatively high in July (54.1%) and August (54.6%), though growth rates began to decline. In autumn, the DGR gradually fell from 1.53% in September to 1.39% in November, with carrageenan yields slightly lowering from 47.56% to 48.86%. By winter, the DGR and yields further dropped, with the lowest DGR of 1.16% in January and yields decreasing from 53.32% in December to 36.62% in February. The data suggest that harvesting in early summer, particularly in June, maximizes both growth rate and carrageenan yield, ensuring efficient biomass collection and polysaccharide extraction. The carrageenan yield obtained in our study is lower than that reported in previous studies, where carrageenan yields from B. gelatinus ranged from 68.2% to 73.1% of the dry algal weight for native carrageenan. This difference is attributed to variations in extraction methods. In this study, we extracted carrageenan using water and precipitated it with ethanol, omitting the NaHCO₃ treatment used in previous methods [3]. The choice of extraction method can affect both yield and the structural composition of carrageenan types, so to determine these structural forms, we conducted IR spectral analysis on the raw algal powder. This approach allowed us to observe the original structural forms of carrageenan.

The IR spectra of algal samples showed characteristic absorption bands at 845 cm⁻¹, 930 cm⁻¹, 1260 cm⁻¹, and 890 cm⁻¹, indicating a carrageenan composition that includes both κ- and β-carrageenan types [28,29]. The β/κ-carrageenan ratio was determined by calculating the intensity ratio of the 890 cm⁻¹ and 845 cm⁻¹ bands, as this approach accurately reflects the sulfate content at the C4 position of galactose residues, a primary structural distinction between β- and κ-carrageenan [30]. In this study, to obtain the most precise β/κ ratio, IR measurements were conducted directly on raw seaweed material rather than on extracted carrageenan. This method minimizes potential alterations in carrageenan structure that may arise during the extraction process, ensuring that the IR spectra accurately represent the native β/κ-carrageenan composition in B. gelatinus samples. Consistent with previous findings in the Betaphycus genus, B. gelatinus samples exhibited a predominance of β-carrageenan, though seasonal IR data indicated slight fluctuations in the β/κ ratio. The ratio remained low during spring (1.036–1.058) and reached a peak in July (1.067), with intermediate values in June (1.060) and August (1.045). During autumn, the ratio stabilized (1.036–1.054), and by winter, it showed minimal variation (1.046–1.054), indicating stable structural integrity of the carrageenan across seasonal changes.

This study identifies June as the optimal harvest month for B. gelatinous, as it maximizes both growth rate and carrageenan yield. Seasonal IR analysis also shows that the structural characteristics of κ- and β-carrageenan remain stable throughout the year, despite fluctuations in biomass and yield.

3.2. Structural Characterization of Carrageenan Extracted from Field-Cultivated B. gelatinus

3.2.1. Chemical Composition of Carrageenan

The chemical composition analysis of carrageenan extracted from field-cultivated B. gelatinus in Thai An, Ninh Thuan, identified three primary components: galactose (58.93%), 3,6-anhydrogalactose (25.22%), and sulfate (SO₄) (12.35%). This composition aligns with the typical structure of polysaccharides in the Betaphycus genus, which is known to predominantly yield β-carrageenan, characterized by its high galactose content. However, the simultaneous presence of 3,6-anhydrogalactose and sulfate groups suggests that the extracted carrageenan also includes κ-carrageenan, supporting previous findings on native B. gelatinus carrageenan from the coastal waters of Ninh Thuan, Vietnam [3]. Furthermore, a molar comparison of sulfate (SO₄) to 3,6-anhydrogalactose yielded a ratio of approximately 2:1 (0.229:0.125), providing further evidence for the presence of both β- and κ-carrageenan in this species.

To gain deeper insights into the structural characteristics of the carrageenan from cultivated B. gelatinus, we utilized infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) analyses. These techniques offer precise information on the functional groups, sulfation patterns, and carrageenan types present, thus enhancing our understanding of the bioactive polysaccharide composition in this species.

3.2.2. IR Spectral Analysis of Carrageenan Extracted from Field-Cultivated B. gelatinus

The IR spectrum of carrageenan extracted from field-cultivated B. gelatinus (Figure 2) shows the presence of a signal at 845 cm⁻¹, which is assigned to D-galactose-4-sulfate (G4S). A strong signal at 930 cm⁻¹ indicates the presence of 3,6-anhydro-D-galactose (DA). The broad signal observed between 1220 cm⁻¹ and 1260 cm⁻¹ corresponds to the stretching vibration of S=O in sulfate groups, reflecting the sulfate content in the carrageenan samples [30]. Furthermore, all samples exhibit an absorption band at 890 cm⁻¹, which is associated with the presence of non-sulfated galactose units [28]

Small to medium absorption bands in the 1600 cm⁻–1300 cm⁻ region correspond to the vibrations of carboxylic acid groups, while the bands in the 1570 cm⁻–1540 cm⁻ region are associated with amide (N-H) bond vibrations [31]. The presence of an absorption band at 1432 cm⁻ in the IR spectrum (Figure 2) indicates the presence of carboxylic acid, suggesting that the carrageenan may contain pyruvylated β-carrageenan.

To further investigate this, we conducted a structural analysis of carrageenan extracted from B. gelatinus using NMR spectroscopy.

3.2.3. Results of Analyzing NMR Spectroscopy

The ¹H-NMR spectrum of carrageenan extracted from B. gelatinus (Figure 3) displays key signals in the anomeric region, confirming the presence of both κ-carrageenan and β-carrageenan. A signal at 5.09 ppm corresponds to the α-D-AnGal residue of κ-carrageenan, while the signal at 5.07 ppm is assigned to the α-D-AnGal residue of β-carrageenan. Additional signals between 4.58 ppm and 4.65 ppm are associated with the β-D-Gal residues of both κ- and β-carrageenans. Resonances observed in the 3.6–4.8 ppm range are characteristic of methylene and methine protons in the polysaccharide backbone. These signals provide further confirmation of the structural composition and hybrid nature of the extracted carrageenan, with both κ- and β-carrageenan contributing to its overall structure [32].

The ¹³C-NMR spectrum of carrageenan extracted from B. gelatinus (Figure 4) reveals distinct signals confirming the structural composition of the polysaccharide.

Anomeric carbons display chemical shifts at δ = 104.94, 104.77, 104.65, 97.28, and 96.75 ppm, while signals between 67.79 and 82.55 ppm correspond to pyranose ring carbons. The resonances at δ = 63.48, 63.59, and 63.48 ppm are characteristic of C-6 carbons in the CH₂ groups of galactose residues. Furthermore, the spectrum shows additional signals at 179.0 ppm (low-field region) and 22.5, 22.34, and 22.18 ppm (high-field region), along with a low-intensity signal around 105 ppm, confirming the presence of pyruvate units. These signals correspond to the characteristic resonances of carboxyl, methyl, and acetal groups, indicating the incorporation of pyruvylated residues into the carrageenan structure [33,34]

Based on the anomeric region of the HSQC spectrum (Figure 5), in combination with chemical analysis and IR spectroscopy, and in comparison with previously published carrageenan data, we assigned the correlated C/H signals as follows: (104.77/4.62 ppm) and (97.28/5.09 ppm) correspond to κ-carrageenan; (104.65/4.62 ppm), (96.75/5.07 ppm), (104.94/4.58 ppm), and (96.75/5.07 ppm) are assigned to β-carrageenan or pyruvylated β-carrageenan, labeled as A, B, and B′, respectively.

The proton signals of the disaccharide units A, B, and B′ were identified through cross-peaks in the COSY spectrum (Figure 6), while the carbon chemical shifts were determined based on the proton shifts via the HSQC spectrum (Figure 5). The complete spectral assignments are summarized in

Table 1. ¹H and ¹³C NMR data for the carrageenans from B. gelatinus.

Disaccharide (Carrageenan Form)	Unit	C1 H1	C2 H2	C3 H3	C4 H4	C5 H5	C6 H6
A Kappa carrageenan	β(1,3)-D-galactose -4- sulfate	104.77 4.62	71.67 3.62	80.49 3.97	76.19 4.82	77.51 3.75	63.59 3.79
	α (1,4)-3,6-anhydro galactose	97.28 5.09	72.06 4.119	81.25 4.502	78.92 4.61	79.03 4.65	71.67 4.119
B Beta carrageenan	β(1,3)-D-galactose	104.65 4.62	72.06 3.602	82.5 3.85	68.52 4.073	76.94 3.826	63.59 3.81
	α (1,4)-3,6-anhydro galactose	96.75 5.07	70.48 4.039	81.58 4.514	80.49 4.59	78.92 4.61	71.28 4.17
B′ pyruvylated β-carrageenan	β(1,3)-D-galactose-P	104.94 4.58	71.28 3.618	80.49 3.95	~71.67 4.064	68.52 4.068	67.79 4.073
	α (1,4)-3,6-anhydro galactose	96.75 5.07	71.67 4.064	81.25 4.52	81.37 4.526	79.03 4.63	70.48 4.124

.

The chemical shifts of anomeric protons from D-β-galactose residues in all three disaccharides (A, B, B′) cluster around 4.6 ppm, indicating that the glycosidic linkage is in the α-configuration. In contrast, the anomeric proton signals of α-D-AnGal residues appear at 5.09 ppm and 5.07 ppm, confirming the glycosidic linkage in/of the β- configuration). The primary structural difference between κ-carrageenan, β-carrageenan, and pyruvylated β-carrageenan lies in the nature of the α-D-AnGal linkage to the following residues: D-galactose-4-sulfate (G4S), D-galactose, or galactopyranose substituted with a pyruvate acetal in the form of 4,6-O-(1-carboxyethylidene)-Galp (GP). The structural assignment was confirmed by analyzing the carbon chemical shifts of the galactose residues.

Comparison with reference NMR data reveals that the chemical shifts of H4 and C4 in the galactose residue of disaccharide A increase from 4.1 ppm to 4.8 ppm and from ~68 ppm to 76.19 ppm, respectively. This shift occurs due to sulfation at the O-4 position, which alters adjacent bonds and causes the resonances to shift downfield, confirming that the galactose residue in disaccharide A is D-galactose-4-sulfate (G4S). Thus, disaccharide A is identified as κ-carrageenan.

The ¹³C-NMR spectrum also shows evidence of pyruvate units in the carrageenan sample, particularly through changes in the chemical shifts of carbon atoms in disaccharides B and B′. The shifts at C4 and C6 increase by 3.15 ppm and 4.2 ppm, respectively, while shifts at C3 and C5 decrease by 2.0 ppm and 8.42 ppm. These changes are explained by pyruvate acetal substitution at the C4 and C6 positions of the galactose residue. This substitution causes α-effects (upfield shifts) at the substituted carbons and β-effects (downfield shifts) at adjacent carbons, confirming that disaccharide B′ is pyruvylated β-carrageenan, while disaccharide B is β-carrageenan.

The HMBC spectrum (Figure 7) provides further insights into the glycosidic linkages. It shows interactions between the H1 proton of β-D-galactose-4-sulfate (G4S) and the C4 carbon of α-D-AnGal in disaccharide A, as well as between the H1 proton of α-D-AnGal and the C3 carbon of β-D-galactose-4-sulfate. These interactions confirm that disaccharide A consists of β-D-galactose-4-sulfate (1→4) α-D-AnGal and α-D-AnGal (1→3) β-D-galactose-4-sulfate. Similarly, disaccharide B consists of β-D-galactose (1→4) α-D-AnGal and α-D-AnGal (1→3) β-D-galactose, while disaccharide B′ consists of 4′,6′-O-(1-carboxyethylidene)-β-D-galactopyranosyl (GP) (1→4) α-D-AnGal and α-D-AnGal (1→3) 4′,6′-O-(1-carboxyethylidene)-β-D-galactopyranosyl (GP).

The results of the NMR analysis suggest that the carrageenan extracted from B. gelatinus is a mixture of three structural forms (Figure 8), including the following:

A. κ-carrageenan: repeating disaccharide units of [→4) α-D-AnGal (1→3) β-D-galactose-4-sulfate (1→]n;

B. β-carrageenan: repeating disaccharide units of [→4) α-D-AnGal (1→3) β-D-galactose (1→]n;

B′. Pyruvylated β-carrageenan: repeating disaccharide units of [→4) α-D-AnGal (1→3) 4′,6′-O-(1-carboxyethylidene)-β-D-galactopyranosyl (1→]n.

The ratio between κ-carrageenan and β-carrageenan (including pyruvylated β-carrageenan) is approximately 1:1, which is consistent with the 890/845 cm⁻¹ absorption ratio in the FTIR spectra, measured at 1.047. The presence of these mixed carrageenan structures suggests that the extracted carrageenan may exhibit biological activity. The mixture of κ- and β-carrageenan has been previously reported in various agal species, including Furcellaria lumbricalis (also known as furcellaran), Eucheuma gelatinae, E. speciosa, Endocladia muricata [35], and Tichocarpus crinitus [36]. However, studies on pyruvylated carrageenans are relatively scarce, with most reports focusing on pyruvylated α-carrageenan.

One notable example is the work by Chiovitti et al. 1997, who identified highly pyruvylated carrageenans extracted from red alge of the genus Callophycus [37]. These carrageenans predominantly consisted of 3-linked 4′,6′-O-(1-carboxyethylidene)-β-D-galactopyranosyl (GP) units alternating with 4-linked 3,6-anhydro-α-D-galactopyranosyl 2-sulfate (DA2S) units, forming a pyruvylated α-carrageenan. Additionally, a small proportion of 3-linked β-D-galactopyranosyl (G) units alternating with 4-linked 3,6-anhydro-α-D-galactopyranosyl 2-sulfate (DA2S) units was also detected.

Our findings on the presence of pyruvylated β-carrageenan in B. gelatinus represent a rare report of this carrageenan type, expanding the knowledge of structural diversity among carrageenans and suggesting the potential for novel biological activities.

3.3. Bioactivities of Carrageenan from B. gelatinus

To evaluate the biological potential of carrageenan extracted from B. gelatinus, we examined its antioxidant activities, including total antioxidant capacity, iron-reducing activity, and ABTS radical scavenging ability, along with its antibacterial properties (

Table 2. Biological activities of carrageenan from field-cultivated B. gelatinus.

Biological Activity	Unit	Carrageenan from Field-Cultivated Alga
Total Antioxidant Capacity	mg ascorbic acid/g carrageenan	48.30 ± 1.12
Iron-Reducing Ability	mg ascorbic acid/g carrageenan	1.47 ± 0.21
ABTS	IC50 (µg/mL)	3.64 ± 0.19
Antibacterial Activity	Strain (mm)	L. mono—12.00 ± 1.00

). These results will provide crucial insights into carrageenan’s role in protecting cells from oxidative stress and inhibiting bacterial growth, further highlighting its potential applications in the health and food industries.

The carrageenan extracted from field-cultivated B. gelatinus exhibits notable antioxidant and antibacterial activities. The total antioxidant capacity reached 48.30 ± 1.12 mg ascorbic acid equivalent per gram of carrageenan, indicating a strong potential for scavenging free radicals. The iron-reducing ability, measured at 1.47 ± 0.21 mg ascorbic acid equivalent per gram carrageenan, further supports the antioxidant capacity of the carrageenan by showing its effectiveness in reducing Fe ions.

In the ABTS assay, the carrageenan displayed an IC₅₀ value of 3.64 ± 0.19 µg/mL, demonstrating its potent radical scavenging capability at low concentrations. This low IC50 value suggests high efficiency in neutralizing ABTS radicals, a key indicator of antioxidant strength.

Additionally, the carrageenan exhibited antibacterial activity against L. monocytogenes, with an inhibition zone diameter of 12.00 ± 1.00 mm. This result implies the potential use of carrageenan as an antimicrobial agent, especially in applications where L. monocytogenes control is crucial, such as in food preservation and safety.

4. Discussion

This study highlights the growth and carrageenan production of B. gelatinus cultivated in the field in Ninh Thuan, Vietnam. The coastal waters in this region, with favorable temperatures (27–30 °C) and high light exposure in early summer—especially in June—significantly enhance biomass accumulation and improve carrageenan quality. As a result, B. gelatinus is primarily distributed along the central coast of Vietnam, from Quang Ngai to Ninh Thuan, where environmental factors align with its growth needs. By synchronizing harvesting schedules with these peak seasonal conditions, we can improve productivity and ensure high-quality carrageenan yield. This approach not only provides a sustainable alternative to wild harvesting but also mitigates ecological impacts and supports resource conservation efforts [38]

The structural analysis of B. gelatinus carrageenan using FTIR and NMR revealed a hybrid composition of κ- and β-carrageenan, with a distinctive β-pyruvylated form that has not been previously reported in this species. The β-pyruvylated structure, identified by spectral signals at 890 cm⁻¹ and 845 cm⁻¹, includes a sulfate group at the C4 position of β-galactose, as well as unique carboxyl and acetal functional groups indicative of pyruvylation. The ¹³C and ¹H NMR spectra further confirm the presence of disaccharide units typical of κ- and β-carrageenan, alongside the 4,6-O-(1-carboxyethylidene)-β-D-galactopyranosyl unit unique to pyruvylated carrageenans. Pyruvylation is a rare modification that contributes to the structural diversity of red algal polysaccharides and may have significant implications for the biological activities of carrageenan [37].

The discovery of β-pyruvylated carrageenan in B. gelatinus opens new avenues for understanding how structural modifications affect the bioactivity of red algae-derived polysaccharides. Pyruvylation may enhance both the stability and bioactivity of carrageenan, as it introduces functional groups that interact with microbial cell membranes and increase antibacterial efficacy. This structural adaptation may account for the strong antibacterial activity observed in this study, especially against L. monocytogenes, a common foodborne pathogen. Previous research suggests that pyruvylated carrageenans from other algal species exhibit enhanced membrane-disruptive properties, likely due to the carboxyl and acetal groups that facilitate interactions with microbial membranes, disrupt biofilm formation, and inhibit bacterial growth [34,39].

In the present study, carrageenan extracted from B. gelatinus was tested for antibacterial activity against both Gram-positive and Gram-negative bacterial strains. Notably, activity was only observed against L. monocytogenes, suggesting selective antibacterial effects. This specificity may be attributed to key structural features such as sulfate content, polysaccharide chain length, and, importantly, molecular weight [13,40]. To better elucidate the structure–activity relationship of this carrageenan, further studies are necessary. These should include the preparation of low-molecular-weight carrageenan, detailed structural analysis, and an in-depth investigation of how specific structural elements influence antibacterial activity. Additionally, further testing against a wider range of Gram-negative bacteria is recommended to assess the potential for broader-spectrum antibacterial applications.

In addition to its antibacterial properties, B. gelatinus carrageenan demonstrates antioxidant activities through total antioxidant capacity, iron-reducing ability, and ABTS radical scavenging assays. These assays collectively highlight the role of the structural features of carrageenan, particularly its sulfate groups, in determining its antioxidant potential. The antioxidant capacity of carrageenan is primarily attributed to its sulfate groups, which enhance electron donation and free radical neutralization. Bener et al. (2018) demonstrated that these groups stabilize radical intermediates via resonance, amplifying antioxidant activity [41]. Similarly, Barahona et al. (2012) found that higher sulfate content and strategic positioning on sulfated galactans significantly improved antioxidant performance [42]. Acid hydrolysis of κ-carrageenan further confirmed this, showing that reduced sulfate content leads to diminished ROS scavenging ability [43]. The antioxidant properties of the carrageenan extracted from field-cultivated B. gelatinus are noteworthy and align well with the bioactivities commonly observed in sulfated polysaccharides derived from red algae like Eucheuma gelatinae [12]. The ABTS radical scavenging assay yielded an IC50 value of 3.64 ± 0.19 µg/mL, illustrating potent scavenging activity at low concentrations. This result underscores the efficiency of B. gelatinus carrageenan in neutralizing radicals, attributed to its sulfated backbone and possible synergistic effects between sulfate groups and other structural elements, such as 3,6-anhydrogalactose residues [15]. The unique β-pyruvylated structure may further enhance the antioxidant potential by providing additional reactive sites for electron transfer, thereby improving radical scavenging ability. This antioxidant property enhances the value of B. gelatinus carrageenan as a natural food preservative and broadens its application in the cosmetics industry, where antioxidant-rich ingredients are essential for anti-aging and skin protection. The stable β/κ ratio and strong antioxidant profile of B. gelatinus carrageenan make it an attractive candidate for both pharmaceutical and cosmetic formulations. To better understand the structure–function relationship, future studies should focus on generating low-molecular-weight derivatives and evaluating their activities. Additionally, exploring the interactions of β-pyruvylated carrageenan with a broader range of biological targets could reveal further insights into its potential for pharmaceutical and cosmetic applications. Such investigations would address existing gaps and provide a more comprehensive understanding of the impact of specific structural modifications on bioactivity.

The observed seasonal growth patterns in B. gelatinus indicate that June is the optimal harvest month for maximizing growth rates and carrageenan yield. Targeted harvesting during this period optimizes production efficiency while aligning with sustainable management practices that reduce ecological impact on marine ecosystems. Establishing a seasonal harvesting schedule based on these findings could support the development of a scalable and sustainable cultivation model for carrageenan production. Such a model would enable B. gelatinus to be a reliable, high-quality source of carrageenan for industrial applications, reducing dependency on wild stocks and supporting environmental conservation.

5. Conclusions

This study underscores the potential of open-sea cultivation as a sustainable approach for producing high-quality carrageenan from B. gelatinus. The results show that field cultivation not only achieves carrageenan yields comparable to wild samples but also retains a stable structural profile, essential for its bioactive properties. The discovery of pyruvylated β-carrageenan in B. gelatinus adds to the structural diversity and enhances its antioxidant and antibacterial efficacy. Seasonal analysis identifies June as the optimal harvest period, maximizing yield and biomass quality. The findings suggest that carrageenan derived from field-cultivated B. gelatinus has promising applications in the health and food industries, supporting its development as a natural preservative and antioxidant agent. Establishing seasonal harvesting and optimizing field cultivation methods can further improve productivity and contribute to the sustainable management of marine resources.