The main material used was U. lactuca seaweed obtained from the coast of Seriwe Village, Jerowaru District, East Lombok Regency, Nusa Tenggara Barat Province (coordinates 8°53’18.6” S and 116°30’27.8” E). The sampling location map of U. lactuca is shown in Figure 1. Ulva lactuca seaweed was washed using seawater, drained, and stored in a styrofoam box equipped with ice flakes as a container during the transportation process, which was then washed again using freshwater to remove any remaining sand, dirt, and other impurities still attached to the thallus. U. lactuca was then dried using a Getra lowpressure gas oven (NFC-8Q) for 48 h at 35ºC (Moreira et al., 2017; Fithriani et al., 2017).

Ulva lactuca seaweed was dried and then mashed using a grinder (BRANDT brand), and then the sieving process sieved using a 40 mesh sieve, based on specifications from the industry. The size was then reduced to nanoparticles using a planetary ball mill (DECO-PBM-V-60L brand) with a topdown method for 12 h with 1 in total, each step includes 10-15 minutes (on) and 10-15 minutes (of), and the speed used was 400- 700 rpm. The milling speed and time were optimized according to the characteristics of the material. The process flow of the U. lactuca nanopowder is shown in Figure 2.

Proximate analysis was carried out to determine the moisture content by the gravimetric method (Nasional, 2015), ash content by the gravimetric method (Nasional, 2010), fat content by the Soxhlet method (Nasional, 2017), protein content by the Kjeldahl method (Nasional, 2006), and carbohydrate content by the Carbohydrate by Diference method.

Amino acid analysis was performed using high-performance liquid chromatography (HPLC) with fluorescence detection after pre-column derivatization using o-phthalaldehyde (OPA) at the Laboratory of the IPB University, Bogor. A total of 6 mg of protein from each sample was hydrolyzed with 2 mL of 6 N HCl in a screwcap tube under a nitrogen atmosphere and incubated at 110°C for 24 h. The hydrolysate was then evaporated to dryness using a rotary evaporator, reconstituted with 10 mL of 0.01 N HCl, and filtered through a 0.45 µm membrane filter before derivatization. The OPA reagent was prepared by mixing 25 mg of OPA, 2 mL of methanol, 0.02 mL of 2-mercaptoethanol, 0.05 mL of Brij-30, and 0.5 mL of 1 M borate bufer (pH 10.4), followed by dilution with borate bufer (1:2, v/v) prior to use. Subsequently, 5 µL of the sample solution was reacted with 25 µL of the OPA reagent for one minute at room temperature to ensure complete derivatization. After derivatization, 5 µL of the mixture was injected into the HPLC column, and chromatographic separation was performed for approximately 25 min. The analytical conditions for HPLC were as follows:

Mobile phase : Thermo Scientific ODS-2 Hyersil

Flow rate : Bufer A and bufer B using a gradient elution system

Detector : Fluorescence

Heavy metal analysis was conducted using inductively coupled plasma-mass spectrometry (ICP–MS) at the Saraswanti Indo Genetech Laboratory. Sample preparation for heavy metal analysis was performed by establishing a six-point standard calibration curve. For the determination of mercury (Hg), lead (Pb), and cadmium (Cd), approximately 0.25 g of the sample was subjected to wet digestion using a mixture of concentrated HNO₃ and H₂O₂ to ensure complete mineralization of the sample. For arsenic (As) analysis, the sample was treated with concentrated HCl, dilute hydrazine sulfate solution, and HBr-p solution, followed by chloroform extraction. The obtained extract was then evaporated with HNO₃ until nearly dry, redissolved in HCl, and treated with a reducing agent mixture of KI and ascorbic acid to stabilize the As species prior to instrumental analysis.

Characterization was carried out using a particle size analyzer (Beckman Coulter) to determine the size of the particles produced. Measurements were conducted under optimized dispersion conditions, employing deionized water containing 2% sodium polyphosphate as the dispersant, a sample concentration of 0.01 ppm, and 1-minute sonication at ambient temperature. Fourier Transform Infrared (FTIR) spectroscopy (Smart iTX Accessory type) was used to determine the functional groups present. Scanning Electron Microscopy (SEM; JEOL-6510 LA) and Transmission Electron Microscopy (TEM; Tundra 100 kV) were used to observe the surface morphology of the resulting powder. Approximately 2 g of U. lactuca powder and nanopowder were placed on circular copper plates (sample holders) before microscopic observation. The microstructure was examined at magnifications of 500×, 5,000×, and 10,000×, with the measurements performed under an accelerating voltage of 20 kV.

The experimental data were analyzed using an Independent Samples t-test to determine significant diferences between the two treatments, U. lactuca powder (micro) and nanopowder (nano), in terms of chemica composition (proximate and amino acid profile) and heavy metal content (Hg, Pb, Cd, and As). The analysis was performed in two replicates. Statistical significance was assessed at a confidence level of to evaluate the existence of meaningful diferences between treatments. Prior to the t-test, the data were examined for normality and homogeneity of variance to ensure compliance with the parametric test assumptions. Statistical analyses were performed using the IBM SPSS Statistics software. The results are expressed as mean ± standard deviation (SD). Additionally, supporting data from PSA, SEM, TEM, and FTIR analyses were interpreted descriptively to evaluate the changes in morphological structure, particle size, and functional groups between the powder and nanopowder samples. The quantitative and qualitative results were comprehensively integrated to elucidate the relationship between particle size reduction, chemical composition variations, and functional potential of Ulva lactuca biomass.

The proximate composition between the particle size of U. lactuca powder and nanopowder (Table 1) exhibited statistically significant diferences (p<0.05) in several parameters, reflecting the compositional and structural modifications induced by the dry ball milling process.

In this study, dried U. lactuca was first ground to 40 mesh using a mechanical grinder, followed by high-energy dry ball milling without any solvent addition until the particle size reached the nanoscale. Unlike wet milling, the absence of a liquid medium during this process causes high-frequency particle–particle and particle–wall collisions, which generate localized frictional heat and strong shear forces. These mechanical stresses are known to induce changes in the physicochemical properties of biopolymers, such as fragmentation, surface oxidation, and molecular rearrangements (Loh et al., 2015).

The moisture content of U. lactuca showed a significant increase from 7.38±0.32% in micron particles (powder) to 14.27±0.17% after ball milling. This increase indicates a greater water retention capacity resulting from the finer particle size and more porous particle structure. The top-down milling process breaks down large aggregates into nanoscale particles, substantially expanding the specific surface area and increasing the number of microand nanopores in the material. These structural changes facilitate the easier penetration of water molecules into the particle matrix and enhance the overall water adsorption capacity (Monks et al., 2013).

(Zhao et al., 2024) reported that smaller hydrogel particles (with a higher specific surface area) significantly increased the water holding capacity (WHC) and physically adsorbed water (PAW) compared to larger particles. In addition to the increased surface area, the rise in moisture content in the U. lactuca nanopowder is closely related to the enhanced hygroscopicity and surface energy induced by structural transformations during dry milling. The high mechanical energy generated during ball milling leads to the partial amorphization of cell wall polysaccharides, such as ulvan, cellulose, and hemicellulose. This amorphous transformation exposes hydroxyl (-OH) and carboxyl (-COOH) functional groups that have a strong afinity for atmospheric water molecules (Jung et al., 2018).

The ash content represents the inorganic mineral fraction remaining after the complete combustion of the sample. In this study, U. lactuca nanopowder exhibited a lower ash content (28.77 ± 0.39%) than its powdered form . Rather than mineral volatilization, in the study by Chan et al. (2018), this reduction is more likely attributable to the mechanical redistribution and partial loss of loosely bound minerals during prolonged ball milling, as well as increased adhesion of ultrafine particles to the milling media and container surfaces, which may reduce mineral recovery during ash determination.

The lipid and protein contents were significantly higher in the U. lactuca nanopowder than in the powder. This increase was attributed to intensified cell wall disruption during prolonged ball milling, which enhanced the release and extractability of intracellular lipid fractions and previously inaccessible protein components. Mechanical forces, such as shear stress, impact, and friction during milling, promote the cracking of the polysaccharide-rich cell wall matrix, thereby exposing both hydrophobic domains associated with lipid bodies and hydrophilic protein structures (Spínola et al., 2023).

The lipid content in powder and nano forms was similar to the results of previous studies, which showed that Ulva lactuca composition generally contains 0.5-4% lipid and 5-27% protein, indicating that the pulverization process enriches the availability of these macro compounds (Metwaly et al., 2023). A notable decrease in the lipid content from to 1.30 ± 0.25% was observed after the transition from powder to nanopowder. This reduction is primarily attributed to the oxidative and thermal degradation of unsaturated fatty acids resulting from high-energy impact collisions during dry ball milling (Lam et al., 2001).

In contrast, the protein content exhibited a significant increase from 4.69 ± 0.11% to after milling treatment. The ball milling process promotes the solubilization of intracellular proteins and facilitates their interaction with analytical reagents, which may lead to higher protein concentrations. Additionally, compositional shifts caused by lipid loss and moisture absorption can increase the relative proportion of protein when expressed on a wet weight basis. The heat generated during milling can cause partial denaturation or mild Maillardtype reactions, as explained by (Khalid et al., 2023), who stated that these reactions can change the nitrogen content and cause a significant increase in the measured protein values.

Furthermore, the carbohydrate composition of nano and powder, which is the diference of 100% proximate results (by diference), shows that the smaller particles contain lower carbohydrates (49.19% nano and 53.94% powder). Polysaccharides are one of the constituents of carbohydrates, which are generally related to the fiber in a material (Nufus et al., 2017). Polysaccharides, particularly ulvan, remain the dominant carbohydrate fraction in U. Lactuca (approximately 48%), indicating that the main polysaccharide structure is largely preserved after milling (Li et al., 2023).

However, a lower carbohydrate content was observed in the nanopowder than in the powder. This decrease should not be interpreted as extensive polysaccharide degradation but is mainly associated with proximate analysis by diference, where increased protein and lipid extractability after intensive milling mathematically reduces the calculated carbohydrate fraction (Patrichi et al., 2023). Furthermore, prolonged ball milling promotes the mechanical disruption of the ulvan-rich cell wall matrix, which may induce partial mechanochemical depolymerization and structural rearrangement of polysaccharides. These efects can influence polysaccharide distribution, solubility, and analytical recovery without causing a complete loss of ulvan. The literature states that carbohydrates in Ulva range from 53-78 % depending on environmental factors and extraction methods (Hofmann et al., 2024).

Amino acid analysis of the powder and nano samples revealed 15 types of amino acids with diferent compositions. The total amino acid content increased from 4.7% in the powder to 6.2% in the nanopowder, aligning with the corresponding rise in protein content (from 4.69% to 6.47%).

The diference in composition or total amount of amino acids may arise due to the thermal or oxidative degradation of some amino acids during processing. The ball milling process, which involves intensive friction, generates heat, thus causing a decrease in the amino acid content in the nanopowder. (Jacoeb et al., 2012) mentioned that some amino acids are susceptible to heat damage, which results in a reduction in the amount of amino acids. The results of the amino acid analysis of U. lactuca powder and nanopowder are presented in Table 2.

U. lactuca seaweed contains essential (threonine, valine, isoleucine, leucine, phenylalanine, histidine, lysine, and arginine) and non-essential (aspartic acid, serine, glutamate, glycine, alanine, valine, tyrosine, and proline) amino acids. According to (Shuuluka et al., 2013), Ulva sp. generally has a total free amino acid content between 2-5%, depending on the harvest season and extraction method, so these results are in line. Research on the amino acid content of diferent Ulva species by (Meiyasa et al., 2023) showed that U. reticulata has the same amino acid content with diferent compositions.

Based on the data in Table 2, mechanical milling significantly influenced the total amino acid content of Ulva lactuca. As the amino acid analysis involved an acid hydrolysis step prior to HPLC quantification, the measured values represent total amino acids, including both protein-bound and free amino acid fractions. The observed increase in the total amino acid content is attributed to the enhanced accessibility and analytical recovery of amino acids resulting from cell wall disruption and protein matrix disintegration during milling.

This observation agrees with that of (Wang et al., 2024), who reported that when proteins are ball-milled, the protein particle size changes, and the particle size may be reduced under mechanical forces. As a result, the protein or particles through high-energy milling enhances the release of amino acidrich peptides and soluble nitrogen compounds through shear stress, localized heating, and oxidative microenvironments. However, some compositional diferences may also result from the thermal or oxidative degradation of heatsensitive amino acids (Pratama et al., 2013) during ball milling. (Sailah & Miladulhaq, 2021) noted that such conditions can reduce certain amino acid levels through degradation or Maillard-type reactions with reducing sugars.

The data show that glutamic acid has the highest content in nanopowder form (0.77 %), far exceeding other amino acids. Glutamic acid, together with aspartic acid, is responsible for the savory (umami) taste typical of seaweed. This is in line with (Meiyasa et al., 2023), who showed that Ulva sp. is rich in flavor-forming amino acids, especially aspartate and glutamate, which makes it a potential natural flavor enhancer. This decrease may afect the taste and flavor potential, although in terms of bioavailability, nanosized particles may increase amino acid absorption in the gastrointestinal tract.

The total EAA content slightly decreased in the nanopowder (2.68%) compared to that in the powder (3.02%), whereas the NEAA content increased markedly from 1.7% to 3.57%. This trend suggests that while some EAAs, such as valine, isoleucine, and lysine, were partially degraded due to heat or oxidation, other amino acids, especially aspartic acid, glutamic acid, alanine, and serine, increased due to enhanced protein breakdown and hydrolysis. Interestingly, some essential amino acids, such as arginine, threonine, and histidine, increased in the nanopowder form. This increase could be due to the efect of greater protein release from the Ulva lactuca cell matrix when pulverized to nanosized particles, making certain amino acids more detectable in the analysis. These essential amino acids play important roles in physiological functions; for example, threonine plays a crucial role in maintaining the integrity and function of the gastrointestinal mucosal layer (Mao et al., 2011). This decrease, along with the decrease in total amino acids in the nanopowder, indicates the degradation of some proteins due to heat, oxidation, or changes in molecular structure during the refining process.

Nutritionally, Ulva lactuca in both powder and nanopowder forms ofers a complete spectrum of amino acids, making it a potential candidate as a raw material for food, pharmaceutical, and health applications. Similar essential amino acid profiles of U. lactuca have been reported by (Pratiwi et al., 2021), supporting its nutritional relevance as a plant-based protein source.

Heavy metal analysis of Ulva lactuca revealed distinct diferences in concentration between the powder and nanopowder forms (Table 3). The analyzed parameters included mercury (Hg), lead (Pb), arsenic (As), and cadmium (Cd). The mercury content decreased from 0.05±0.00 mg/kg in the powder to 0.03±0.00 mg/kg in the nanopowder, whereas the Pb and As concentrations increased from 0.72±0.01 mg/kg to 1.06±0.03 mg/kg and from 0.28±0.00 mg/kg to 0.82±0.01 mg/kg, respectively.

Meanwhile, Cd levels remained relatively stable between 0.07–0.08 mg/kg. Despite these variations, all measured values were well below the maximum permissible limits established by SNI 7387:2009, namely, 0.5 mg/kg for Hg, 10 mg/kg for Pb, 1.0 mg/kg for As, and 0.2 mg/kg for Cd. Therefore, both Ulva lactuca powders and nanopowders are considered safe for consumption and comply with national food safety standards.

The reduction of mercury (Hg) in the nanopowder is likely due to its high volatility during ball milling. According to (Vassilev et al., 2024), mercury exhibits a high volatilization rate (83–99%) during thermal treatment. Frictional heat and localized temperature elevation inside the milling chamber may induce partial vaporization or transformation of Hg into a gaseous form, thereby reducing its detectable concentration in the final product. This finding aligns with (Liu et al., 2024), who reported that mechanical and thermal processing can promote the volatilization of mercury from marine biomass. Furthermore, the physicochemical nature of mercury supports this behavior, as it exists as a liquid metal with a low vapor pressure (2 mmHg) and a low latent heat of evaporation (295 kJ/ kg), making it susceptible to vaporization even at ambient temperatures (Veeraswamy et al., 2023). Consequently, frictional heating or localized thermal stress during milling can significantly enhance mercury volatilization from the biomass matrices.

Conversely, the increase in Pb and As concentrations in the nanopowder may be attributed to the greater specific surface area generated after the particle size reduction. Nanoparticles exhibit a higher adsorptive capacity, allowing heavy metal ions such as and to bind more efectively to negatively charged functional groups, particularly carboxyl (-COOH) and hydroxyl (-OH) groups, on cell wall polysaccharides (Safitri, 2020). Moreover, the broader dispersion of mineral fractions in the nanopowder can facilitate the detection of heavy metals previously entrapped within the cell wall matrix during ICP-MS analysis. I (Indah et al., 2021) showed that the particle size of the adsorbent afects the adsorption process; the smaller the size of the adsorbent, the higher the absorption of metals. These results are reinforced by (Raya & Rahmah, 2012), who stated that a wider specific surface of the adsorbent can provide more active sites to bind metal ions. A large surface area is generally associated with a fine pore structure; the smaller the pore size, the greater the total surface area, and the greater the adsorption ability of magnesium ions.

Overall, these findings indicate that ball milling not only alters the physical and chemical properties of Ulva lactuca particles but also afects the behavior of heavy metals in the biomass matrix. The reduction in Hg reflects partial volatilization due to mechanicalthermal efects, whereas the increase in Pb and As represents a redistribution of metals from bound to more detectable forms. Nevertheless, all values complied with (Nasional, 2009) and FAO/WHO (2019) international safety standards, confirming the safe utilization of Ulva lactuca nanopowder for food and nutraceutical applications.

Fourier transform infrared (FT-IR) analysis was used to evaluate whether the particle size reduction altered the main functional groups of U. lactuca, allowing a comparison between the powder and nanopowder samples. The FTIR characterization results are presented in Figure 3 and Table 4.

The FTIR spectra of U. lactuca powder and nanopowder exhibited characteristic ulvan-related bands, including sulfate ester, carboxylate, and glycosidic vibrations. The slight peak shifts and intensity variations observed after milling reflect changes in the intermolecular interactions and molecular organization induced by particle size reduction, without evidence of covalent bond degradation. (Ramadhan et al., 2022) reported that hydroxyl (O–H) and C–H stretching vibrations are the principal absorption bands observed in polysaccharides. Table 4 shows a broad absorption band at 3,211 cm⁻¹ (powder) and 3,257 cm⁻¹ (nanopowder), corresponding to O–H stretching vibrations, which are the main features of the polysaccharide backbone owing to extensive hydrogen bonding among hydroxyl and carboxyl groups. The slight shift to a higher wavenumber indicates an increase in the O–H bond energy caused by alterations in the hydrogen bonding environment following nanopowder formation.

This phenomenon can be attributed to the increase in surface area and the partial disruption of intramolecular hydrogen bonds. The absorption band at 2,926-2,927 cm⁻¹, assigned to C–H stretching vibrations (–CH₂, –CH₃), remained stable after the size reduction from powder to nanopowder. This stability is expected because these aliphatic C–H bonds are part of the polysaccharide backbone and side chains of ulvan, which are covalent bonds with high bond dissociation energies and are not readily cleaved under ambient mechanical milling conditions (Barakat et al., 2022). Mechanical milling primarily afects particle size and intermolecular interactions rather than breaking stable covalent C-H linkages, resulting in a preserved aliphatic fingerprint in the FTIR spectra (Mao et al., 2011).

The absorption bands at 1,631 cm (powder) and 1,628 cm⁻¹ (nanopowder) are attributed to the asymmetric stretching vibrations of deprotonated carboxylate groups (–COO⁻) arising from uronic acid residues, such as glucuronic and iduronic acids, in the ulvan polysaccharides. This assignment is consistent with the FTIR analyses of ulvan from Ulva species, where the bands near 1640 cm⁻¹ were attributed to the COO asymmetric stretch and weaker bands around 1,438 cm⁻¹ to the COO⁻ asymmetric stretch, reflecting the presence of uronic acid residues in the polysaccharide structure (Garcia et al., 2023).The relatively minor shift observed in this region indicates a slight change in the hydrogen bonding environment between the carboxylate and neighboring hydroxyl groups, rather than any cleavage of covalent bonds. This is more appropriately attributed to conformational rearrangements of uronic acid residues induced by particle size reduction during nanopowder formation, and such mechanical processing primarily afects interand intramolecular hydrogen interactions while preserving the integrity of the polysaccharide carbon backbone (Tran et al., 2023). These results support the conclusion that nanoparticle processing can modify intramolecular interactions without destroying the main carbon framework of polysaccharides.

The most significant change was observed in the absorption bands corresponding to the C-O and C-O-C stretching vibrations (the polysaccharide fingerprint region), which shifted from 1,086 cm⁻¹ (powder) to 1,028 cm⁻¹ (nanopowder). This region is characteristic of glycosidic linkages and secondary alcohol (C–O) vibrations from rhamnose and glucuronic acid residues (Ramadhan et al., 2022). The observed shift to a lower wavenumber did not provide direct evidence of glycosidic bond cleavage. Instead, such minor FTIR band shifts are more appropriately interpreted as changes in the hydrogen bonding environment and the conformation of glycosidic linkages due to physical processing, such as particle size reduction during the formation of the nanopowder. Infrared spectroscopy reflects alterations in interand intramolecular interactions and molecular packing rather than covalent bond breakage, and small wavenumber shifts within the glycosidic region often correlate with conformational adjustments or hydrogen bond rearrangements in polysaccharide chains without actual cleavage of the primary glycosidic bonds (Hong et al., 2021). (Tran et al., 2023), who observed that physical treatments such as sonication or grinding caused downward shifts in C–O stretching peaks of ulvan due to an increased number of free terminal hydroxyl groups (–OH). Therefore, this shift cannot be attributed to instrumental variation, as it is accompanied by changes in sulfate and uronate band intensities, suggesting genuine alterations in the micromolecular structure of ulvan.

In addition to hydroxyl and carboxyl groups, sulfate groups (–OSO₃⁻) are the most distinctive functional features of ulvan and warrant an in-depth discussion. The strong bands observed at approximately 1,220–1,260 cm⁻¹ (S=O stretching, sulfate ester) and 843– 846 cm⁻¹ (C–O–S stretching) confirm the presence of sulfate groups linked to rhamnose or iduronic backbones (Figueira et al., 2020). The relative intensity of sulfate to sugar bands correlates with the degree of sulfation (DS). In the nanopowder spectrum, the sulfate bands appeared slightly weakened and shifted to lower wavenumbers. Such changes in intensity and position are more appropriately interpreted as reflections of alterations in the local bonding environment and intermolecular interactions of the sulfate ester groups rather than evidence of sulfate group removal or cleavage of covalent C-O-S bonds (Je et al., 2021).Infrared spectroscopy is sensitive to changes in molecular orientation, hydrogen bonding, and packing, which can influence the vibrational characteristics of sulfate esters in sulfated polysaccharides, as reported in studies of fucoidan and other sulfated polysaccharides, where shifts and intensity variations of the S=O stretching bands occur without implying desulfation (Ptak et al., 2021). As sulfate groups play a critical role in determining the biological activity of ulvan, particularly its antioxidant and antidiabetic efects, these subtle changes should be noted as potential indicators of bioactivity modification following nanoparticle processing.

Overall, the FTIR spectral pattern of U. lactuca in this study was consistent with previous reports describing the characteristic bands of ulvan at approximately 3,400 cm⁻¹ (O–H), 2,920 cm⁻¹ (C–H), 1,620 cm⁻¹ (C=O), 1,415 cm⁻¹ (COO⁻), 1,250 cm⁻¹ (S=O), 1,060–1,030 cm⁻¹ (C–O/C–O–C), and 845 cm⁻¹ (C–O–S) (Barakat et al., 2022; Ibrahim et al., 2022). Minor variations in the peak position and intensity after processing indicate that the fundamental polysaccharide structure remained intact, although modifications in substituent groups and hydrogen bonding interactions occurred.

The PSA characterization results showed that the powder sample was not detected because the particle size (±100 µm) exceeded the maximum limit of the device (10 µm). During the griding process, U. lactuca is dried in a grinder to a size of 40 mesh or 420 microns in accordance with industry demand standards as a raw material for seasonings in both snack and non-snack products (PT. Indesso Culinaroma). Figure 4 shows the images of Ulva lactuca seaweed that were ground into powder using a grinder and subsequently subjected to further size reduction using a Planetary Ball Mill (PBM). The particle size data of the resulting nanopowder samples are presented in Table 5.

Particle size analysis performed using a Particle Size Analyzer (PSA) revealed that Ulva lactuca samples subjected to ball milling exhibited an average hydrodynamic diameter (Z-average) of 728.1±190.9 nm, with a polydispersity index (PDI) of 0.53±0.13. The zeta potential of the dispersed nanoparticles was recorded as −231±1.62 mV for the overall population, with Peak 1 corresponding to −32.79 mV. These parameters collectively indicate that the ball-milled U. lactuca particles exhibit a broad size distribution with high heterogeneity, where partial aggregation or agglomeration likely occurred during dispersion.

The Z-average of 728 nm suggests that the intensity-weighted mean particle size is within the submicron range. However, this parameter is highly influenced by the presence of larger particles, as the intensity of light scattering scales proportionally to the sixth power of the particle diameter (d⁶) under the Rayleigh scattering regime (Hantke et al., 2018). Consequently, even though the measured Z-average appears relatively large, the presence of smaller nanoparticles (<100 nm) can be masked by the dominant scattering signal from the larger aggregates (Stetefeld et al., 2016). In other words, a high Z-average value does not necessarily preclude the existence of nanosized primary particles but rather reflects a mixed system comprising nanoscale primary and aggregated entities.

The particle size distribution obtained from PSA displayed two prominent peaks at approximately 30 and 261 nm, signifying the coexistence of two distinct particle populations. The peak at approximately 30 nm likely represents primary nanosized particles generated through the high-energy mechanical milling process, whereas the 261 nm peak corresponds to secondary particles formed by the agglomeration of nanoscale fragments. This observation supports the interpretation that, although a fraction of the particles remains within the nanoscale range (<100 nm), aggregation and agglomeration phenomena contribute significantly to the overall intensity-weighted size. This behavior aligns with the known tendency of biopolymeric nanomaterials to form agglomerates owing to surface charge interactions and hydrogen bonding, particularly in hydrophilic matrices such as Ulva lactuca, which is rich in polysaccharides and proteins (Nemeth et al., 2022). Hence, the PSA results suggest that ball milling produces a heterogeneous mixture of primary nanoparticles and larger agglomerated structures.

Furthermore, the PDI value of 0.53 indicates a polydisperse system, as PDI values greater than 0.3 typically denote a broad size distribution and non-uniformity in particle dispersion. This result implies that not all particles were evenly dispersed within the medium, and a significant fraction existed as loosely bound agglomerates. Meanwhile, the zeta potential of −23.1 mV reflects a moderately negative surface charge, suficient to impart partial electrostatic repulsion among particles, although not strong enough to ensure long-term colloidal stability, which is commonly associated with absolute zeta potential values ≥30 mV. Therefore, the Ulva lactuca nanopowder exhibits intermediate electrostatic stability, indicating that although some electrostatic repulsion is present, the particles remain susceptible to agglomeration under changes in pH, ionic strength, or colloidal concentration (Nemeth et al., 2022).

Taken together, the PSA data indicate that although the Z-average exceeds 100 nm, the presence of a distinct population peak at 30 nm confirms that a fraction of the particles falls within the nanoscale range. According to the European Food Safety Authority (EFSA) (More et al., 2021)and the International Organization for Standardization (ISO/ TR 18401:2017), the classification of nanomaterials should not rely solely on intensity-based mean size but also on the number-based distribution and fraction of particles below 100 nm. Therefore, the PSA findings substantiate that the ball-milled Ulva lactuca material can be classified as a nanopowder, given the coexistence of primary nanosized particles and larger agglomerates formed during dispersion.

Nevertheless, to confirm the presence of nanoscale primary particles and visualize the interparticle boundaries, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analyses are required. These complementary techniques provide direct morphological evidence, surface structure details, and actual particle size distributions, thereby corroborating the PSA results. A detailed discussion of these findings is presented in the following section.

SEM characterization was performed to observe the surface morphology of Ulva lactuca powder and nanopowder. The analysis of Scanning Electron Microscopy (SEM) images before and after the nanosizing process can describe the surface morphology and microstructure of the samples in detail through various magnifications (500×, 5,000×, and 10,000×), which are presented in Figures 5 and 6.

Scanning electron microscopy (SEM) analysis revealed a significant morphological diference between Ulva lactuca in powder and nanopowder forms. In the powder sample, SEM micrographs (at 500×, 5000×, and 10,000× magnifications) exhibited a relatively compact and layered surface structure, which retained fragments of cellulose polysaccharide sheets that were not completely degraded. This morphology reflects the natural characteristics of Ulva lactuca, which is rich in a matrix of ulvan polysaccharides, cellulose microfibrils, and structural proteins (Putra et al., 2024). This morphology is consistent with the SEM results reported in a study of ulvan extraction from U. fasciata, where researchers also found an amorphous architecture characterized by particle aggregation and high porosity due to calcium bonds that are dificult to break (Moustafa et al., 2024).

Conversely, the SEM micrographs of the nanopowder produced through ball milling showed a more porous and irregular surface, composed of micro-to-sub-micrometer-sized fragments, indicating intense mechanical degradation. These fragments formed loosely bound agglomerates with a rough texture and fine protrusions on their surfaces. This observation suggests the formation of primary nanoparticles that subsequently aggregate into larger clusters (agglomerates), as further supported by Particle Size Analysis (PSA) results showing two major peaks at 30 nm and 261 nm, with a Z-average diameter of 728 nm. This relationship confirms that the larger particle size detected by PSA mainly originates from the aggregation of nanoscale primary particles rather than from inherently large individual particles (More et al., 2021). Such behavior is commonly observed in biomaterials subjected to dry ball milling, where high-impact collisions generate nanoparticles that tend to re-agglomerate owing to the elevated surface energy, which is often driven by Van der Waals forces or hydrogen bonding (Nemeth et al., 2022).

The morphological transformation induced by planetary ball milling is also correlated with an increase in the specific surface area and disruption of cell wall structures, thereby enhancing the solvent accessibility of bioactive components such as polysaccharides, proteins, and photosynthetic pigments. (Ibrahim et al., 2022) reported microstructural observations where cell wall degradation and increased extractability of ulvan were associated with mechanical treatments. Several studies on biomass ball milling (Wang et al., 2021; Mohamed et al., 2023) have documented that high-energy milling reduces particle size, introduces surface defects, and increases surface area, while sometimes promoting partial depolymerization of polysaccharides.

From a chemical perspective, the mechanochemical processes occurring during milling may lead to the cleavage of glycosidic bonds within the ulvan and cellulose chains, producing low-molecular-weight oligosaccharide fragments (Nemeth et al., 2022). This degradation improves solubility and enhances particle reactivity toward polar solvents, supporting its functional application in food and pharmaceutical systems.

Overall, the SEM comparison demonstrated that the ball milling process efectively altered the morphology of Ulva lactuca particles into a more porous and heterogeneous structure, indicating the formation of nanoscale primary particles aggregated into agglomerates. These findings align with the PSA results, which show a bimodal size distribution (30 nm and 261 nm) and a zeta potential value of − 23, 1 mV, indicating moderate electrostatic stability. Thus, the resulting nanopowder morphology reflects the typical characteristics of biomassderived nanomaterials, consisting of nanoscale primary particles physically joined into agglomerated structures. According to the European Commission (2011/696/EU) and EFSA (2021) definitions, this material can be classified as a “nano structured agglomerate” and should be functionally evaluated as a nanomaterial.

The analysis of Transmission Electron Microscopy (TEM) images before and after the ball milling process can explain the surface morphology and microstructure of the samples in detail through various magnifications, as presented in Figures 7 and 8.

In the U. lactuca powder sample revealed a layered lamellar structure with large fragments and cellulose fibril networks embedded within the ulvan polysaccharide matrix. The contrast variations, both high and low, indicate compositional heterogeneity between the crystalline (cellulose) and amorphous (ulvan and structural protein) domains. This observation is consistent with the findings of (Piras et al., 2019), who reported that cellulose chains can organize into either irregular amorphous regions or highly ordered crystalline structures. This morphology indicates that the cell wall integrity remains largely preserved and has not undergone significant structural disruption due to the initial mechanical grinding. The lamellar and fibrillar architectures observed in this study are comparable to those described by (Wahlstrom et al., 2020), who demonstrated that the Ulva lactuca cell wall consists of randomly oriented cellulose fibrils embedded in an amorphous ulvan matrix. Similar fibrillar arrangements are commonly found in green macroalgae, where they contribute to the rigidity and mechanical stability of the cell walls (Pari et al., 2025).

Conversely, the TEM micrographs of the nanopowder sample exhibited pronounced morphological changes. The previously intact lamellar structures in the powder were fragmented into smaller particles ranging from several tens to hundreds of nanometers, suggesting that the ball milling process efectively caused cell wall disintegration and disruption of the internal fibrillar network of the cellulose. The high-contrast regions indicate the presence of spherical particles sized 20–80 nm and loosely aggregated clusters (200–300 nm), representing agglomerated primary nanoparticles. The appearance of fine pores and internal voids in the TEM images demonstrates intraparticle porosity, which can enhance the surface area and facilitate solvent difusion during bioactive extraction (Ganeshan et al., 2025). Such porosity not only accelerates extraction processes but can also shorten enzymatic reaction times for polysaccharides (Qu et al., 2017). These findings support the PSA results, which show a bimodal size distribution (30 nm and 261 nm) with a Z-average of 728 nm and a PDI of 0.53.

This suggests that the nanopowder consists of nanometer-scale primary particles weakly bound together into larger aggregates by Van der Waals forces and hydrogen bonding among ulvan and cellulose molecules (Nemeth et al., 2022). Comparable results have been reported in studies on Turbinaria (Mohamed et al., 2023) and Ulva (Madany et al., 2021), where planetary ball milling transformed fibrous or sheet-like structures into nanoscale spheroidal fragments, increased porosity, and reduced the polysaccharide molecular weight.

Chemically, the morphological transformation observed in the nanopowder also indicates a mechanochemical efect, whereby the impact energy during ball milling breaks glycosidic bonds in ulvan and cellulose, yielding more soluble oligomeric fragments. The thin lines or short rod-like fragments observed under TEM may represent cellulose nanofibrils generated through mechanical cleavage (Piras et al., 2019). Similarly, (Li et al., 2023) reported that ulvan appears as an amorphous matrix occupying interfibrillar spaces and that mechanical treatment facilitates the release of oligosaccharide fragments.