Structural and Physical Properties of Alginate Pretreated by High-Pressure Homogenization

To develop a high-efficient extraction method, we investigated the use of high-pressure homogenization (HPH) as a novel pretreatment technology for the extraction of sodium alginate (SA) from Laminaria japonica. After the single-factor experiment, the results demonstrated that under the conditions of 100 MPa HPH pressure, 4 cycles, pH 6.0, and 0.5% EDTA for 3.0 h, the optimized extraction yield of HPH reached 34%. To further clarify the effect on the structural properties of HPH-extracted SA, we conducted comprehensive analysis using SEM, FTIR, MRS, NMR, XRD, TGA, and a T-AOC assay. Our findings revealed that HPH pretreatment significantly disrupted the structure of L. japonica cells and reduced their crystallinity to 76.27%. Furthermore, the antioxidant activity of HPH-extracted SA reached 0.02942 mgVceq∙mg−1. Therefore, the HPH pretreatment method is a potential strategy for the extraction of alginate.


Introduction
Marine seaweed have gained a lot of attention as a third-generation renewable feedstock in recent years due to its high growth rate and biomass output, as well as its ability to be cultivated on a wide scale, without the need for arable land [1]. Furthermore, because brown macroalgae do not contain lignin, simple biorefinery processes such as milling, leaching, and extraction can separate the sugars for conversion into bioactive oligosaccharides, biofuels, and renewable chemicals. Brown algae is currently the main source of alginate, a linear acidic polysaccharide carbohydrate composed of β-D-mannuronic acid (M) and its C5 epimer α-L-guluronic acid (G) [2]. The performance of alginate is revealed to be strongly correlated with the M/G ratio [3,4]. Alginate mainly exists in various types of brown algae, with the molecular weight sequence being Fucus vesiculosus < Ascophyllum nodosum < Sargassum fluitans < Laminaria japonica. Among these seaweed species, L. japonica is currently the main source for alginate extraction due to its high content, large molecular weight, and ease of extraction [5].
Commercial production of alginate began in the late 1920s and eventually dominated the food ingredient market in the mid-20th century [6]. Large-scale industrial production commenced in the United States in 1929. In 1938, it gained approval from the Food and Drug Administration (FDA) for use in the food and pharmaceutical industries. Today, the market size of alginate is expected to reach USD 923.8 million by 2025 due to its increasing use in food and biomedical industries [7]. Alginate has numerous proven properties, such as being non-toxic, water-soluble, biodegradable, film-forming, gelling, 1.
L. japonica was soaked in fresh water for 4 h and washed three times with distilled water to remove impurities. The plant material was then dried and ground into a powder.

2.
A solution of L. japonica was prepared by soaking 2.0 g of the plant material in 200 mL of pure water for 1 h. The L. japonica solution was then subjected to different pretreatment methods, including HPH, UAE, CE, and CE-UC. 3.
After adding 30 mL of a 2% (w/v) Na 2 CO 3 and EDTA (with or without) solution, the homogenate was incubated at 50 • C for 3 h. The mixture was then centrifuged, and the supernatant was adjusted to the desired pH using 1 M HCl. 4.
Following this, 20 mL of 10% (w/v) calcium chloride was added and the mixture was allowed to stand. The resulting precipitate was then filtered and washed twice with distilled water to obtain a yellow-white gelatinous precipitate. 5.
The precipitate was dissolved in 20 mL 15% (w/v) sodium chloride solution for ion exchange. The solution was then filtrated using medical gauze. Subsequently, 100 mL of anhydrous ethanol was added to induce precipitation. The resulting white flocculent precipitates were obtained through filtration. 6.
The precipitates were collected and frozen at −80 • C for 12 h, followed by freezedrying for 8 h using a vacuum freeze dryer. The dried precipitates were then crushed to obtain crude sodium alginate.
The yield of SA was calculated using the following equation: SA yield (%) = (m 1 /m 2 ) × 100 (1) where, m 1 is the dry weight of obtained SA, and m 2 is the dry weight of L. japonica. All experiments were performed in triplicate. The standard deviations are illustrated as error bars in the figures. Above detailed steps were shown in Figure 1. Of the L. japonica powder with a 100-mesh size, 2.00 g was taken and tap water was added in a 1:50 ratio to obtain a total volume of 100 mL.  Of the L. japonica powder with a 100-mesh size, 2.00 g was taken and tap water was added in a 1:50 ratio to obtain a total volume of 100 mL.

2.
The pH value was adjusted to 6 and 3% (w/v) cellulase of the L. japonica powder, 3% (w/v) pectinase, and 1% (w/v) papain were added. The mixture was stirred well and transferred to a 50 • C water bath for 3 h. After the reaction, the enzyme solution was inactivated by boiling in water for 15 min.

3.
Of a 2% (w/v) sodium carbonate solution, 24 mL was added and the mixture digested in a 50 • C water bath for 3 h. The digested solution was centrifuged at 8500 r/min for 10 min and the supernatant removed. The pH of the supernatant was adjusted to 6. The subsequent operations were continued as described in Section 2.2 from step (4) to step (6).

1.
Of the L. japonica powder with a 100-mesh size, 2.00 g was taken and stirred into tap water at a material-to-liquid ratio of 1:50.

2.
An ultrasonic cell crusher was used to break the samples for 10 min with the following conditions: 350 W of output power, a temperature of 30 • C, and a working time and interval of 2 s.

3.
A 2% (w/v) sodium carbonate solution was added (24 mL) and digested in a water bath at 50 • C for 3 h. After digestion, the enzymolysis solution was centrifuged at 8500 r/min for 10 min. The supernatant was collected and its pH adjusted to 6. The subsequent operations were the same as in Section 2.2, from (4)-(6).

1.
Of the 100-mesh size L. japonica powder, 2.00 g was taken and stirred into tap water at a material-to-liquid ratio of 1:50. The samples were then subjected to ultrasonic cell crushing for 10 min using a 350 W power, 30 • C temperature, and 2 s working time and intervals. 2.
The pH value was adjusted to 6, and L. japonica powder with 3% (w/v) cellulase, 3% (w/v) pectinase, and 1% (w/v) papain were added. The mixture was stirred well and placed in a 50 • C constant temperature water bath for enzymolysis for 3 h. After the enzymolysis reaction, the enzyme solution was inactivated by boiling and heating for 15 min.

3.
Of the L. japonica powder in a 2% (w/v) sodium carbonate solution, 24 mL was added and digested in a 50 • C water bath for 3 h. After digestion, the enzymolysis solution was centrifuged at 8500 r/min for 10 min, and the supernatant was collected and adjusted to pH 6. Subsequent operations were the same as in Section 2.2, steps (4)-(6).

Single-Factor Experiment of the HPH Method
In order to optimize the extraction conditions using the HPH method, various factors were investigated that have an impact on the yield of sodium alginate. These factors include pressure, cycle times, pH, EDTA, and digestion time.
Homogeneous pressure (bar): Pressures of 400, 600, 800, 1000, 1200, and 1400 bar were selected, respectively, and the yield of sodium alginate was determined while keeping the other steps unchanged.
Homogenization times: Under the aforementioned optimal conditions, extraction was conducted with homogenization performed 2, 3, 4, 5, and 6 times, respectively, while keeping the other steps unchanged. The yield of sodium alginate was determined.
pH Adjustment: Under the aforementioned optimal conditions, pH values of 5, 5.5, 6, 6.5, and 7 were utilized to adjust the extraction process. The remaining steps of the procedure were kept constant in order to determine the yield of sodium alginate. EDTA addition amount (%): 0%, 0.25%, 0.5%, 0.75% and 1% (w/v) EDTA were added to the extraction process without changing any other steps. The yield of sodium alginate was determined under these conditions. Digestion time (h): Under the aforementioned optimal conditions, digestion was conducted for 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and 3.5 h, respectively. Extraction was carried out following all other steps, unchanged, in order to determine the yield of sodium alginate.

Characterization of Sodium Alginate
For the SEM analysis, the untreated and pretreated L. japonica power samples were examined to analyze the effect of pretreatment on their structure and morphological properties. To do this, small amounts of dried L. japonica samples were placed on a double-sided carbon tape adhered to aluminum stubs. These stubs were then coated with a layer of gold using sputter-plating. Finally, the samples were observed using an S-3400N electron microscope from Hitachi, Japan.
For the FTIR analysis, sodium alginate samples were analyzed using the Nicolet IS 10 Fourier Transform Infrared spectrometer (Thermo Fisher Scientific, MA, USA). The samples were scanned from 4000 to 600 cm −1 32 times, at a resolution of 4 cm −1 . This was performed to investigate any changes in the functional groups.
For the MRS analysis, the sodium alginate powder was initially spread evenly on the groove of a glass slide. The Raman spectrum (Thermo Fisher Scientific, MA, USA) in the range of 3378 cm −1 to 50 cm −1 was measured, with an exposure time of 10 s for 30 repetitions.
For the NMR analysis, the procedure was performed following the guidelines of ASTM-F2259-10 (2012) using an Agilent NMR Systems 800 MHz NMR Spectrometer (Agilent, CA, USA). Initially, the sodium alginate underwent depolymerization through acid hydrolysis. A sodium alginate solution of 100 mL with a concentration of 1 mg/mL was prepared, and the pH was adjusted to 5.6 using 1 M hydrochloric acid. The solution was then heated in a water bath at 100 • C for 1 h. Subsequently, the pH was readjusted to 3.8 using 1 M hydrochloric acid, and again, heated in a water bath at 100 • C for 30 min. The pH was then neutralized to 7.2 with 1 M sodium hydroxide, and the sample was freeze-dried overnight. The resulting freeze-dried sample was dissolved in 5 mL of 99.9% D 2 O and then freeze-dried once more. Next, 10-12 mg of the sample was dissolved in 1 mL of 99.9% D 2 O. Subsequently, 0.7 mL of the alginate solution, along with 20 µL of 0.3 M TTHA (triethylenetetraminehexaacetic acid) was added to an NMR tube. The pH of the TTHA was adjusted to 5.2 using sodium deuterium chloride (DCl) and deuteroxide (NaOD), as the TTHA served as a chelator to hinder the reaction of divalent cations with sodium alginate. For the XRD analysis, the sodium alginate samples underwent various pretreatment and were commercially recorded using an X-ray diffractometer Ultima IV (Rigaku, Tokyo, Japan). The diffraction pattern was obtained by scanning the samples at a rate of 5 • /s within a range of 2θ angles of 5 • to 80 • . Cu radiation (40 mA, 40 kV) was used during the analysis.
TGA analysis was performed on various sodium alginate samples using a TGA Q50 Thermogravimetric Analyzer instrument (TA, DE, Waltham, MA, USA). To obtain thermograms, 10 mg of sodium alginate samples were placed in an alumina pan and was purged with nitrogen at a rate of 30 mL/min. The temperature was then increased from 20 • C to 600 • C at a heating rate of 20 • C/min.
For T-AOC analysis, the assay was based on the reduction of molybdate-IV (Mo IV) to molybdate-V (Mo V) by the extracts, and the subsequent formation of a green phosphate/Mo V complex in an acidic pH. In this study, the total antioxidant capacities (T-AOC) of different sodium alginate samples were evaluated using the ammonium molybdate method, as previously described [41]. Specifically, 0.5 mL of the sample (10 mg/mL) was dissolved in 10% double-distilled water and mixed with 5 mL of the reagent, which consisted of 28 mM sodium phosphate, 0.6 M sulfuric acid, and 4 mM ammonium molybdate. The mixture was then incubated at 95 • C for 90 min and subsequently cooled. The ab-Polymers 2023, 15, 3225 6 of 16 sorbance was measured at 695 nm against the blank. The antioxidant activity was expressed as the vitamin C equivalent antioxidant capacity (VCEAC) using a standard plot.
Statistical analysis and graphing were performed using the GraphPad software program.

Optimization of Single-Factor Extraction Conditions for the HPH Extraction
The effect of HPH pressure on yield: The effect of HPH pressure (40-140 MPa) on the yield of sodium alginate was investigated while keeping other parameters constant, including 3 cycles, pH 6.0, 0.25% (w/v) EDTA concentration, and 4.0 h digestion time. The results showed that the yield of sodium alginate increased significantly with increasing pressure from 40 to 100 MPa, but decreased after reaching 100 MPa at 1% SA concentration ( Figure 2A). This observed trend is likely due to the cavitation, turbulence, and collision phenomena induced by HPH. These mechanical forces cause the material to break down and achieve super refinement, facilitating the dissolution of sodium alginate. However, at higher pressures (>100 MPa), the yield decreased as the cells are severely damaged. This may result in sodium alginate molecules being damaged into smaller sizes, which hinders the extraction process. Effect of digestion time on the yield: In order to investigate how digestion time affects the yield of sodium alginate, the extraction process was carried out under the aforementioned optimal conditions ( Figure 2B). It was observed that the yield increased as digestion time was extended, reaching its highest value (33.5%) at 3.0 h. This remarkably reduced the production cycle. However, excessively short digestion times might result in an incomplete destruction of L. japonica. On the other hand, if the digestion time is too long, the alginate might degrade, leading to a reduction in the yield and making the subsequent separation process more challenging. Therefore, a digestion time of 3.0 h was taken as the optimal condition.
The effect of pH on yield: The influence of pH values ranging from 5 to 7 on the yield of sodium alginate was investigated under fixed conditions of HPH pressure at 100 MPa, 3 cycles, EDTA concentration at 0.25% (w/v), and digestion time of 3.0 h. The results showed that the yield of sodium alginate initially increased and then then decreased as the pH increased ( Figure 2C). Sodium alginate demonstrated excellent stability within the pH range of 6-11. When the pH was lower than 6, alginate precipitated and the yield Effect of digestion time on the yield: In order to investigate how digestion time affects the yield of sodium alginate, the extraction process was carried out under the aforementioned optimal conditions ( Figure 2B). It was observed that the yield increased as digestion time was extended, reaching its highest value (33.5%) at 3.0 h. This remarkably reduced the production cycle. However, excessively short digestion times might result in an incomplete destruction of L. japonica. On the other hand, if the digestion time is too long, the alginate might degrade, leading to a reduction in the yield and making the subsequent separation process more challenging. Therefore, a digestion time of 3.0 h was taken as the optimal condition.
The effect of pH on yield: The influence of pH values ranging from 5 to 7 on the yield of sodium alginate was investigated under fixed conditions of HPH pressure at 100 MPa, showed that the yield of sodium alginate initially increased and then then decreased as the pH increased ( Figure 2C). Sodium alginate demonstrated excellent stability within the pH range of 6-11. When the pH was lower than 6, alginate precipitated and the yield decreased. On the other hand, when the pH exceeded 11, the alginate coagulated again.
The effect of EDTA concentration on yield: The influence of EDTA concentration on the yield of sodium alginate was evaluated under the following conditions: 100 MPa HPH pressure, 3 cycle times, pH 6.0, and 3.0 h of digestion time. The data shown in Figure 2D clearly revealed that increasing the concentration of EDTA resulted in an increase in the yield of sodium alginate, reaching a maximum of 33.5% when using a concentration of 0.5% (w/v) EDTA. This finding was significantly higher than previous reports on the extraction of sodium alginate from brown algae. The extraction process heavily relied on the complexation of calcium in the cell wall by EDTA. The optimal concentration of 0.5% (w/v) EDTA for maximizing sodium alginate yield was consistent with previous studies [42]. Additionally, including 0.5% (w/v) EDTA during the digestion stage facilitated pH adjustment to 6, reducing the amount of acid and base required for pH adjustment. However, higher concentrations of EDTA led to higher pH values, which was detrimental to the extraction process of sodium alginate. Therefore, a concentration of 0.5% (w/v) was determined to be the most effective.
The effect of HPH cycle times on yield: As depicted in Figure 2E, the yield of sodium alginate demonstrated a gradual increase with increasing cycle times. The maximum yield was achieved after 4 cycles. It should be noted that exceeding 4 cycle times would be likely to affect the degradation of sodium alginate molecules a little, due to the heating machine to a certain extent. As a result, the extraction of sodium alginate would be impeded. Hence, 4 cycle times were identified as the optimal homogenization number.
Subsequently, the optimal conditions for sodium alginate extraction were determined to be 100 MPa HPH pressure, 4 cycle times, a pH of 6.0, a 0.5% concentration (w/v) of EDTA, and a digestion time of 3.0 h. With the optimum parameters, the yield was 34%. A comparison with other reported extraction methods is necessary. The yields of different methods are shown in Table 1.

Compared Yield with That of Other Extraction Methods
Numerous pretreatment methods have been reported for the extraction of SA. However, most of them have certain deficiencies, such as harsh reaction conditions and high costs. In this study, we applied the HPH method to extract SA from L. japonica and compared it with four other methods, namely, UAE, CE, and CE-UC. The results showed that the UAE method produced the lowest yield at 30.2%, while the HPH method produced the highest yield at 34%, as shown in Figure 3. These findings demonstrated that the HPH method significantly improved the extraction yield.

Scanning Electron Microscopy (SEM) Analysis
The scanning electron micrographs of untreated and pretreated L. japonica power samples were taken at a magnification of 500× and a resolution of 100 µm. By comparing these micrographs, significant differences in surface morphology could be observed.
The pretreated samples appeared to noticeably change in the surface condition compared to the original sample ( Figure 4). The untreated sample exhibited a tightly packed structure with intact cell walls, possibly due to the presence of strong inter-and intramolecular hydrogen bonds in the cell wall. In contrast, the pretreated samples, including those treated with UAE, CE, CE-U, and HPH, exhibited a greater degree of surface decomposition compared to the untreated L. japonica powder. This observation indicated that the cell wall was significantly damaged by these pretreatment methods. Among these four methods, the sample pretreated with HPH exhibited a highly porous structure, which is likely to result in a higher extraction yield.

Scanning Electron Microscopy (SEM) Analysis
The scanning electron micrographs of untreated and pretreated L. japonica power samples were taken at a magnification of 500× and a resolution of 100 µm. By comparing these micrographs, significant differences in surface morphology could be observed.
The pretreated samples appeared to noticeably change in the surface condition compared to the original sample ( Figure 4). The untreated sample exhibited a tightly packed structure with intact cell walls, possibly due to the presence of strong inter-and intramolecular hydrogen bonds in the cell wall. In contrast, the pretreated samples, including those treated with UAE, CE, CE-U, and HPH, exhibited a greater degree of surface decomposition compared to the untreated L. japonica powder. This observation indicated that the cell wall was significantly damaged by these pretreatment methods. Among these four methods, the sample pretreated with HPH exhibited a highly porous structure, which is likely to result in a higher extraction yield.

Scanning Electron Microscopy (SEM) Analysis
The scanning electron micrographs of untreated and pretreated L. japonica power samples were taken at a magnification of 500× and a resolution of 100 µm. By comparing these micrographs, significant differences in surface morphology could be observed.
The pretreated samples appeared to noticeably change in the surface condition compared to the original sample ( Figure 4). The untreated sample exhibited a tightly packed structure with intact cell walls, possibly due to the presence of strong inter-and intramolecular hydrogen bonds in the cell wall. In contrast, the pretreated samples, including those treated with UAE, CE, CE-U, and HPH, exhibited a greater degree of surface decomposition compared to the untreated L. japonica powder. This observation indicated that the cell wall was significantly damaged by these pretreatment methods. Among these four methods, the sample pretreated with HPH exhibited a highly porous structure, which is likely to result in a higher extraction yield.

Fourier Transform Infrared (FTIR) Spectrum Analysis
The FTIR spectroscopy results of various sodium alginate samples are shown in Figure 5, with characteristic bands consistent with those previously reported [23,45,46]. These

Fourier Transform Infrared (FTIR) Spectrum Analysis
The FTIR spectroscopy results of various sodium alginate samples are shown in Figure 5, with characteristic bands consistent with those previously reported [23,45,46]. These bands confirmed the presence of the main functional groups within all samples, confirming the identity of the extracted sample as sodium alginate.  The broad band that shifted from 3400.0 to 3200.0 cm −1 was assigned to the O-H stretching vibration, while the band at around 2921.80 cm −1 was attributed to C-H stretching vibrations, including the CH, CH2, and CH3 groups. The band that shifted from 1620 to 1590 cm −1 , as well as the band that shifted from 1420 to 1400 cm −1 , was derived from the The broad band that shifted from 3400.0 to 3200.0 cm −1 was assigned to the O-H stretching vibration, while the band at around 2921.80 cm −1 was attributed to C-H stretching vibrations, including the CH, CH 2 , and CH 3 groups. The band that shifted from 1620 to 1590 cm −1 , as well as the band that shifted from 1420 to 1400 cm −1 , was derived from the symmetrical stretching of the carbonyl (C=O) vibration. The band that shifted from 1200 to 1000 cm −1 was attributed to C-O-C and C-O-H vibrations. Additionally, the characteristic ring stretching band of a β-1,4-glycosidic bond was observed at around 886 cm −1 . It could be observed that the intensity of the bands was reduced in the HPH method, indicating that the HPH method effectively destroyed β-1,4-glycosidic bonds and improved the yield of sodium alginate.

Raman Microscope Spectrometer (MRS) Analysis
The characteristic peaks of SA in Figure 6 exhibited asymmetric and symmetrical stretching vibration absorption peaks corresponding to the COO-bond at about 1612 cm −1 and 1415 cm −1 . The stretching vibration absorption peaks assigned to the deformed C-O-H bond, C-O bond, and C-C bond could be observed within the range of 1200-900 cm −1 . Furthermore, the deformation vibration absorption peak of the pyranose ring and the C-O-C glycosidic bond was observed below 700 cm −1 [47].  Figure 7 presents the NMR spectra of the sodium alginate sample that was extracted using HPH. The peaks observed in the spectra were indicative of the presence of anomeric and other protons located at different carbon positions within the uronic acid sequence. The chemical shifts were observed at approximately 5.02, 4.62, 4.40, 4.17, 4.01, 3.96, 3.82, and 3.58 ppm, which could be attributed to A (anomeric proton of guluronic acid), B1 (H-5 proton of the central guluronic acid residue in a GGM traid), B2 (H-5 proton of the central guluronic acid residue in a MGM traid), B3 (anomeric proton of the mannuronic acid residue neighboring a mannuronic acid), B4 (anomeric proton of the mannuronic acid residue neighboring a guluronic acid), and C (proton 5 of guluronic acid), respectively. It is important to note that these chemical shifts differed slightly from those observed in the alginate extracted using the ASTM standard. This variation could be attributed to the presence of trace amounts of metal ions in the extracted alginate and the differences in its chemical composition, properties, and molecular weight. The presence of metal ions, such as calcium, could lead to a broadening of the signal lines and selective loss of signal intensity.  Figure 7 presents the NMR spectra of the sodium alginate sample that was extracted using HPH. The peaks observed in the spectra were indicative of the presence of anomeric and other protons located at different carbon positions within the uronic acid sequence. The chemical shifts were observed at approximately 5.02, 4.62, 4.40, 4.17, 4.01, 3.96, 3.82, and 3.58 ppm, which could be attributed to A (anomeric proton of guluronic acid), B1 (H-5 proton of the central guluronic acid residue in a GGM traid), B2 (H-5 proton of the central guluronic acid residue in a MGM traid), B3 (anomeric proton of the mannuronic acid residue neighboring a mannuronic acid), B4 (anomeric proton of the mannuronic acid residue neighboring a guluronic acid), and C (proton 5 of guluronic acid), respectively. It is important to note that these chemical shifts differed slightly from those observed in the alginate extracted using the ASTM standard. This variation could be attributed to the presence of trace amounts of metal ions in the extracted alginate and the differences in its chemical composition, properties, and molecular weight. The presence of metal ions, such as calcium, could lead to a broadening of the signal lines and selective loss of signal intensity. The block structure and M/G ratio were calculated following the calculation method outlined in the ASTM standard F2259-10 (2012). Table 2   The physicochemical properties of alginate were greatly influenced by its structure, including factors such as the M/G ratio and the arrangement of the M and G residues [49]. The uronic acid sequence played a crucial role in determining the gel-forming ability of alginate. Alginates with a high M/G ratio tended to form gels that were soft and elastic [50]. Therefore, as the extracted alginate in this study had a high mannuronic acid content of 67%, it was likely to form soft and elastic gels.

X-Ray Diffraction (XRD) Analysis
XRD analysis was conducted on various sodium alginate samples to investigate their crystallinity and recalcitrance properties. The crystallinity of sodium alginate was a key attribute in determining its efficiency for enzymatic hydrolysis. A comparative X-ray diffractogram of the different sodium alginate samples was shown in Figure 8. The HPH method resulted in a crystallinity of 76.26%, which was the lowest among all the samples, excluding commercial sodium alginate. The diffraction pattern of commercial sodium alginate only showed a peak at around 13.3° and a very weak diffraction peak at 22.5° [51]. The other samples exhibited characteristic strong peaks at 2θ values of 31.8°, 45.4°, 56.6°, and 66.2° [52]. Notably, the sodium alginate extracted using the HPH method displayed The block structure and M/G ratio were calculated following the calculation method outlined in the ASTM standard F2259-10 (2012). Table 2   The physicochemical properties of alginate were greatly influenced by its structure, including factors such as the M/G ratio and the arrangement of the M and G residues [49]. The uronic acid sequence played a crucial role in determining the gel-forming ability of alginate. Alginates with a high M/G ratio tended to form gels that were soft and elastic [50]. Therefore, as the extracted alginate in this study had a high mannuronic acid content of 67%, it was likely to form soft and elastic gels.

X-ray Diffraction (XRD) Analysis
XRD analysis was conducted on various sodium alginate samples to investigate their crystallinity and recalcitrance properties. The crystallinity of sodium alginate was a key attribute in determining its efficiency for enzymatic hydrolysis. A comparative X-ray diffractogram of the different sodium alginate samples was shown in Figure 8. The HPH method resulted in a crystallinity of 76.26%, which was the lowest among all the samples, excluding commercial sodium alginate. The diffraction pattern of commercial sodium alginate only showed a peak at around 13.3 • and a very weak diffraction peak at 22 [52]. Notably, the sodium alginate extracted using the HPH method displayed decreased intensities of characteristic peaks, suggesting that the HPH method induced amorphousness in sodium alginate, enhancing the accessibility of alginate lyase, and promoting the production of alginate oligosaccharides. decreased intensities of characteristic peaks, suggesting that the HPH method induced amorphousness in sodium alginate, enhancing the accessibility of alginate lyase, and promoting the production of alginate oligosaccharides.

Thermal Gravimetric Analysis (TGA) Analysis
Thermal gravimetric analysis (TGA) was performed to investigate the thermal stability of various sodium alginate samples. The TGA curve for sodium alginate displayed three distinct weight loss steps during thermal decomposition ( Figure 9). In contrast to the commercial sodium alginate, TGA curves of the extracted samples exhibited a tendency toward higher temperatures, indicating an enhanced thermal stability. The removal of water molecules from sodium alginate occurred within the temperature range of room temperature to 120 °C. During this interval, the sodium alginate extracted using the HPH method exhibited the highest weight loss (17%), while the sodium alginate extracted using the ultrasonic method underwent the lowest weight loss (8%).

Thermal Gravimetric Analysis (TGA) Analysis
Thermal gravimetric analysis (TGA) was performed to investigate the thermal stability of various sodium alginate samples. The TGA curve for sodium alginate displayed three distinct weight loss steps during thermal decomposition ( Figure 9). In contrast to the commercial sodium alginate, TGA curves of the extracted samples exhibited a tendency toward higher temperatures, indicating an enhanced thermal stability. The removal of water molecules from sodium alginate occurred within the temperature range of room temperature to 120 • C. During this interval, the sodium alginate extracted using the HPH method exhibited the highest weight loss (17%), while the sodium alginate extracted using the ultrasonic method underwent the lowest weight loss (8%).
After the loss of water, sodium alginate decomposed at temperatures ranging from 210 • C to 300 • C. For commercial samples, decomposition occurred at 220 • C, with completion at 260 • C. Sodium alginate extracted through HPH degraded within a temperature range of 240 • C to 280 • C. The result of TGA displayed a strong correlation with the result of XRD, indicating that the reduction in crystallinity facilitated the degradation of the HPH-extracted sample. These findings were consistent with previous studies [53]. After the loss of water, sodium alginate decomposed at temperatures ranging from 210 °C to 300 °C. For commercial samples, decomposition occurred at 220 °C, with completion at 260 °C. Sodium alginate extracted through HPH degraded within a temperature range of 240 °C to 280 °C. The result of TGA displayed a strong correlation with the result of XRD, indicating that the reduction in crystallinity facilitated the degradation of the HPH-extracted sample. These findings were consistent with previous studies [53].

Total Antioxidant Capacity Assay (T-AOC)
The results of the antioxidant capacity of various extracted sodium alginate samples are presented in Figure 10, and their antioxidant activities were evaluated as vitamin C equivalent antioxidant capacity (VCEAC). Among these samples, sodium alginate extracted using UAE showed the lowest antioxidant capacity (0.01264 mgVceq·mg −1 ), whereas the sodium alginate extracted using HPH displayed the strongest antioxidant capacity (0.02942 mgVceq·mg −1 ), second only to commercially available sodium alginate. These findings could be partially explained by the different molecular weights of sodium alginate extracted using different methods. It is well-known that polysaccharides with lower molecular weights exhibit better antioxidant activity.

Total Antioxidant Capacity Assay (T-AOC)
The results of the antioxidant capacity of various extracted sodium alginate samples are presented in Figure 10, and their antioxidant activities were evaluated as vitamin C equivalent antioxidant capacity (VCEAC). Among these samples, sodium alginate extracted using UAE showed the lowest antioxidant capacity (0.01264 mgVceq·mg −1 ), whereas the sodium alginate extracted using HPH displayed the strongest antioxidant capacity (0.02942 mgVceq·mg −1 ), second only to commercially available sodium alginate. These findings could be partially explained by the different molecular weights of sodium alginate extracted using different methods. It is well-known that polysaccharides with lower molecular weights exhibit better antioxidant activity.

Conclusions
In this paper, we investigated the successful application of calcium coagulation exchange combined with high-pressure homogenization for extracting sodium algin from brown seaweed. We found that HPH effectively broke the cell wall of the L. japon

Conclusions
In this paper, we investigated the successful application of calcium coagulation ion exchange combined with high-pressure homogenization for extracting sodium alginate from brown seaweed. We found that HPH effectively broke the cell wall of the L. japonica, resulting in an improved yield, faster production, and reduced costs. Moreover, the sodium alginate extracted using this method exhibited stronger antioxidant activity. These findings present a promising new strategy for the industrial extraction of sodium alginate.
Furthermore, alginate oligosaccharides, which were the degradation products of alginate, have attracted considerable attention owing to their notable physiological characteristics. These include immune regulation, antimicrobial properties, antioxidant activity, antitumor effects, anti-inflammatory properties, and the promotion of plant growth. The low crystallinity of sodium alginate extracted in this work was advantageous for the production of AOS and effectively supported the advancement of their industrial production.