Sargassum sp. seaweed was collected from the waters of Lhok Bubon, West Aceh. The seaweed was soaked for 6 h (in two repetitions), rinsed with running water, and weighed 10,000 g for each treatment. The samples were dried using three diferent methods: (1) sun drying (SD) from 08:00 to 17:00 WIB (Western Indonesian Time) at a temperature of approximately 30 °C for 4 days; (2) air drying (AD) from 08:00 to 17:00 WIB at approximately 24 °C for 8 days; and (3) oven drying (OD) at 40 °C for 48 h (Larasati & Husni, 2021) with modifications. The dried samples were analyzed for moisture content (AOAC, 2012), total phenol, total tannin, and pH using a pH meter.

The starter culture used was Lactobacillus plantarum SK (5), isolated from bekasam of seluang fish originating from Kayu Agung, South Sumatra, and obtained from the Microbiology Laboratory, Department of Fisheries Product Technology, IPB University. A loopful of L. plantarum SK (5) from the glycerol stock culture was inoculated onto de Man Rogosa and Sharpe Agar (MRS-Agar) (Oxoid, United Kingdom) slants and incubated under semi-anaerobic conditions using a Thermocline type 4200 incubator at 37 °C for 48 h (Desniar et al., 2023). Cultures grown on MRS-Agar were verified through Gram staining, motility testing, catalase testing, and glucose fermentation tests.

The starter inoculum was prepared by transferring one loopful of culture from MRS-Agar to MRS-Broth (de Man Rogosa and Sharpe Broth) (Oxoid, United Kingdom), followed by incubation at 37 °C for 24 h under semi-anaerobic conditions. The inoculum was then analyzed for total lactic acid bacteria (LAB) count using the pour plate method (Total Plate Count) (Badan Standardisasi Nasional [BSN] 2009).

The dried Sargassum sp. from the three drying methods was blended into pieces of approximately ±0.5–1 cm in size. A total of 10 g of Sargassum sp. was mixed with approximately ±30 mL of water in a 100 mL Schott bottle. The samples were sterilized using an autoclave (Yamato SM 52, Japan) at for 15 min. After sterilization, each sample was inoculated with 1,5 mL of L. plantarum SK (5). Fermentation was performed at 37 for 4 days (Healy et al., 2023, modified). Observations were conducted on days 0, 1, 2, 3, and 4 of fermentation, with parameters including total lactic acid bacteria (LAB) count, total titratable acidity (TTA), and pH.

The fermented samples were dried using a dehydrator at for 5 h, followed by brewing. One gram of dried fermented seaweed was placed into an empty tea bag and infused with 100 mL of hot water (80 for 6 min (Sinurat & Suryaningrum, 2019). The resulting tea solution was analyzed to determine the total phenol and tannin contents.

Total titratable acidity (TAT) measurement refers to BSN (2009). A 5 g seaweed fermentation sample was weighed, and 45 mL of aquades was added and homogenized using a homogenizer. The homogeneous sample was then placed in a volumetric flask (Iwaki, Pyrex) and diluted with aquades to a 50 mL mark. The sample solution was filtered using filter paper, and 5 mL of the filtrate was transferred into an Erlenmeyer flask (Iwaki, Pyrex). Then, 1–2 drops of 1% phenolphthalein (PP) (Merck) indicator were added, and the sample was titrated with NaOH 0.1 N (Merck) until the color changed to pink.

The total LAB count was determined according to BSN (2015). Fermented seaweed (10 g) was weighed, ground in a mortar, and diluted in 90 mL of sterile physiological solution to obtain a dilution. Serial dilutions were prepared up to . The last three dilution levels were plated (in single replicates) on MRS Agar medium supplemented with 0.5% sterile (Merck). The plates were incubated at for 48 h under microaerophilic conditions. The colonies were counted using a colony counter.

The total phenolic content was determined according to (Ramamoorthy & Bono, 2007). One gram of the fermented seaweed sample was dissolved in 100 mL of hot demineralized water (80°C). The diluted solution was 1 mL, then 0.5 mL of 96% ethanol (Merck), 2.5 mL of aquadest, and 0.5 mL of 50% Folin-Ciocalteau (Merck) reagent. The mixture was allowed to sit for 5 min, and then 2 mL of 5% Na ₂ CO ₃ (Merck) was added. The mixture was homogenized and incubated in the dark conditions for one hour. Standard gallic acid (Merck, Germany) was used at concentrations of 0, 5, 10, 15, 20, 30, and 40 ppm. The results are expressed in milligrams of gallic acid equivalent per gram (mg GAE/g). The absorbance of the standard solution and sample was measured using a UV-Vis spectrophotometer at a wavelength of 725 nm.

The total tannin test was performed as described by (Maulida et al., 2020). One gram of the fermented seaweed samples was dissolved in 100 mL of hot demineralized water at Next, 50 mL of the sample solution was placed into a 100 mL measuring flask, and 2.5 mL of indigocarmine (Merck) and water were added until it reached the impression mark. The solution was titrated with (Merck) until golden yellow. The same procedure was used to prepare the blank solution without adding the samples. It is known that 1 mL of KMnO₄ 0.1 N is equivalent to 0.004157 g of tannins.

This study consisted of two stages. The first stage used a completely randomized design (CRD) to test the characteristics of raw materials with diferent drying method treatments (sun-dry, air-dry, and oven-dry), each carried out three times. The second stage used a completely randomized factorial design (CRFD) with two factors. The first factor was the drying method, which consisted of three stages, and the second factor was the fermentation time, which consisted of four stages (0, 1, 2, 3, and 4 days). Each treatment was performed in triplicate. Observational data were first tested for normality and homogeneity, and then analyzed using oneway analysis of variance (ANOVA) at a confidence level of 95% (α=0.05). If there was a significant efect , a follow-up test was performed using Duncan’s Multiple Range Test (DMRT) to determine the diference between treatments. All data processing was performed using Microsoft Excel 2021 and SPSS version 25.0.

The study utilized a single-strain lactic acid bacteria starter, L. plantarum SK (5), with a cell count of 9.07 log CFU/mL CFU/mL). The number of starter cells in this study met the required standard, aligning with the Indonesian National Standard (SNI), which stipulates that probiotic bacteria must have cell counts exceeding 10⁷ CFU/mL to exert health benefits (BSN, 2009).

The characteristics of Sargassum sp. as a herbal tea raw material, including moisture content, total phenols, total tannins, and pH, are presented in Table 1. Based on the ANOVA test, diferences in drying methods during the extraction of Sargassum sp. showed a significant efect (p<0.05) on the moisture content, total phenol, and pH of the raw material, but no significant efect (p>0.05) on total tannin. The lowest moisture content was obtained from seaweed dried using the sun-drying method (7.38%), compared to the air-drying and oven-drying methods. The moisture content obtained in this study was lower than that reported by Gazali et al. (2018), which was 10.54%. A lower moisture level reflects better material quality (Tamaheang et al., 2017). In addition, dried Sargassum sp. simplicia can have a longer shelf life if its moisture content is below 10% (Gazali et al., 2018). All three drying methods produced moisture content values that complied with the SNI 2690:2018 standard, which sets a maximum limit of 18%.

The highest total phenol content was obtained from air drying, reaching 33.97 mg GAE/g, compared to oven drying at 31.24 mg GAE/g. This finding is consistent with that of (Dhakal et al., 2024), who reported that high temperatures can significantly reduce phenolic content. (Sedjati et al., 2017) reported that the total phenol content in seaweed dried by air and extracted with infused hot water was 0.95 mg GAE/g. Masduqi et al. (2014) reported total phenolic contents obtained through sundrying, air-drying, and oven-drying methods, with respective values of 1,179.7, 1,656.3, and 1,274.4 ppm. The extraction of phenolic compounds from plants or fruits is strongly influenced by the polarity of the solvent and solubility of the extracted compounds. Highly polar solvents are more efective in dissolving phenolic compounds in polar media (Dip et al., 2024). Water-based solvents can extract more phenolic compounds than alcoholbased solvents can (Sedjati et al., 2017).

The results also showed that variations in the drying methods did not significantly afect the total tannin content. The total tannin values obtained in this study were lower than those reported by Paga et al. (2021), which were 0.77% for oven-dried drying and 0.89% for sun-dried samples. The pH values of the dried seaweed difered across the various drying methods. Sunand air-dried samples showed pH values of 4.90 and 4.91, respectively, whereas oven drying produced a higher pH of 5.26. These findings indicate that, regardless of the drying technique applied, the dried seaweed remained within an acidic pH range. (Kumesan et al., 2017) similarly observed variations in pH resulting from two diferent drying approaches, namely sun drying and cabinet drying, which yielded pH values of 5.58 and 4.93, respectively. The increase in pH may be associated with the microbial production of nitrogenous compounds and the formation of peptides and amines derived from protein degradation during the drying process (Delbarre-Ladrat et al., 2006).

Lactic acid bacteria are natural microorganisms commonly used as fermentation agents in the food industry. The verification results showed that L. plantarum SK (5) belongs to the homofermentative group of bacteria. The total lactic acid bacteria (LAB) count of fermented Sargassum sp. is presented in Figure 1.

The analysis of variance, the drying method, fermentation duration, and their interaction had a significant efect (p< 0.05) on the total count of lactic acid bacteria. During fermentation, the total LAB in the sundrying and air-drying treatments remained relatively stable until day 2, with counts of 7.7 and 7.6 log₁₀ CFU/g, respectively. This suggests that these two drying methods can maintain conditions that support microbial activity throughout fermentation. In contrast, the oven-drying treatment decreased the total LAB count to on the same day, which was significantly diferent (p<0.05) from the other two methods. High temperatures during oven drying may degrade bioactive compounds that play an important role in support the growth of lactic acid bacteria during fermentation.

The significant interaction (p<0.05) between the drying method and fermentation duration indicates that the pattern of LAB growth over time was influenced by the drying method used. Accordingly, natural drying at lower temperatures tends to maintain LAB growth during fermentation more efectively. (Lein et al., 2025) stated that simple compounds’ environmental conditions and nutrient availability determine bacterial growth phases. These findings contrast with those of (Ambarsari et al., 2018), who reported that during the fermentation of dried Ulva lactuca seaweed, LAB counts increased over fermentation time, from 8.31 to 9.10 log₁₀ CFU/g.

Airand sun-drying tended to maintain more stable LAB counts than oven drying. This diference is likely not solely related to the heating temperature, as all three methods can, in principle, be set to similar temperature ranges. Other factors, such as temperature and humidity fluctuations during sun-drying, as well as the lower thermal stability of the raw material, may influence the availability of bioactive compounds that support LAB growth during fermentation. Meanwhile, the more consistent heating conditions during oven-drying may accelerate the degradation of heat-sensitive compounds, leading to a diferent response during fermentation. LAB may enter the stationary phase due to nutrient limitation in the medium or a decrease in pH caused by lactate accumulation during fermentation (Allahgholi et al., 2023). Lactic acid fermentation is an anaerobic respiration process in which glucose is fermented to produce lactic acid as an end product (Carr et al., 2002). LAB consortia can utilize most monosaccharides that make up brown seaweed polysaccharides, including glucose, mannitol, galactose, xylose, and mannose, except for fucose from fucoidans. Glucose and mannitol are the preferred substrates, serving as the primary sources of carbohydrates and energy in the fermentation medium (Allahgholi et al., 2023).

Total titratable acidity (TTA) is a parameter used to determine the overall concentration of acids in materials. The TTA levels during fermentation are shown in Figure 2. Based on the ANOVA results, the total titratable acidity (TTA) of the fermented seaweed was significantly influenced by the drying method, fermentation duration, and their interaction (p<0.05). The TTA values after four days of fermentation increased significantly compared to the initial values (day 0) in the sun and air-drying treatments. In the sun-drying treatment, there was no significant diference between days 3 and 4 (p>0.05). Similarly, the air-drying treatment on day 4 did not difer significantly from sun-drying, indicating that both methods had similar efects on acid formation during fermentation (Figure 3).

During fermentation, the TTA values of the seaweed ranged from 0.09% to 0.37%. The highest TTA was observed in the airdrying treatment on day 4 (0.37%), while the lowest was found in the oven-drying treatment on the same day (0.09%). The increase in total acidity was influenced by the activity of lactic acid bacteria, which break down substrates into organic acids, primarily lactic acid, as a metabolic product of glucose utilization during fermentation (Lengkey & Belia, 2014; Nuraini et al., 2014). Lactic acid bacteria ferment glucose into organic acids, contributing to increased TTA and decreased pH (Desniar et al., 2023).

These results indicate that the drying method and fermentation duration afect this process. Airor sun-dried Sargassum sp. was more efective as a fermentation medium than oven-dried Sargassum sp. This is likely because natural heat drying better preserves bioactive compounds, whereas oven drying can accelerate the degradation of essential compounds owing to continuous heat exposure. (Kadam et al., 2015) reported that oven-drying typically involves high temperatures for extended periods, which can damage heat-sensitive bioactive compounds and reduce nutrient content. The higher total titratable acidity in fermented seaweed may be attributed to the accumulation of organic acids during L. plantarum fermentation (Yue et al., 2021).

The pH value is an important parameter for indicating the acidity level of a material and serves as a key indicator for assessing the success of fermentation. An increase in the acid concentration leads to a decrease in pH. Generally, pH values are inversely related to total titratable acidity (TTA), where an increase in TTA is accompanied by a decrease in pH (Desniar et al., 2023), and vice versa. The changes in pH during fermentation are shown in Figure 3.

Based on the ANOVA results, the pH of the fermented seaweed was significantly influenced by the drying method, fermentation duration, and their interaction (p<0.05). In the air-drying and sun-drying treatments, no significant diferences were observed between days 3 and 4 (p>0.05), indicating that both methods produced relatively similar fermentation conditions regarding acid production. The pH decreased from day 0 to day 4, ranging from 5.25 to 4.4 (except for the oven-drying treatment). The lowest pH was observed in the air-dried seaweed (4.4) on day 4, whereas the highest was found in the ovendried seaweed (5.47) on the same day. This decrease in pH indicates that fermentation proceeded efectively in seaweed.

The pH reduction observed during air drying reflects a more eficient fermentation process. This decrease was caused by the accumulation of organic acids produced by bacteria during fermentation. Desniar (2012) reported that L. plantarum SK (5) produces lactic and acetic acids as the main organic acids responsible for lowering the environmental pH during fermentation. The greater the lactic acid production during fermentation, the higher the concentration of hydrogen ions (H+) accumulated in the medium, resulting in a further decrease in pH (Silitonga et al., 2022). (Pratomo et al., n.d.) also stated that the increase in titratable acidity and the decrease in pH during fermentation are due to the acidification activity of lactic acid bacteria.

A low pH is also important for inhibiting the growth of contaminating microbes and enhancing the stability of the fermented product. A pH <4.6 is ideal for fermented beverages, as it inhibits the growth of pathogens such as Clostridium botulinum, whereas a pH >5 is considered less safe because it can still support the growth of proteolytic Clostridium botulinum and Listeria monocytogenes(Sørensen et al., 2021). In this study, sun-drying and air-drying treatments achieved a pH < 4.6, while the oven-drying treatment remained >5.