Only mature brunok specimens (average wet weight 250–300 g) with intact body structures were used in this study. Fresh samples were obtained from the Tanjung Balai Karimun waters, Riau Islands, Indonesia. The samples were immediately washed with freshwater to remove sand and impurities, followed by evisceration and cleaning before further processing. The yield of fresh brunok was approximately 25%, calculated as the ratio of the edible flesh weight after cleaning to the initial whole-body weight. This yield value was used as the basis for the formulation calculations.

Cleaned brunok flesh was cut into small pieces and blended with water (1:1, w/v) for 10–30 min to extract the juice. The homogenate was heated at 90°C for 8–10 min and filtered through a muslin (blacu) cloth to obtain the aqueous extract. No additional evaporation step was applied at this stage, and the filtrate was directly used for subsequent formulation and processing. The extraction procedure was adapted from the method described by (Putri et al., 2013).

The formulation was developed based on experimental evaluations of the organoleptic quality attributes of instant brunok powder beverages, resulting in a product with high taurine content and favorable sensory acceptance by the panelists. Fresh ginger and Curcuma xanthorrhiza were peeled, washed with potable water, and blended for 10–20 min to obtain smaller particle sizes, with the addition of water at a 1:1 (b/v) ratio. The mixture was subsequently filtered using a muslin cloth to obtain the extract, followed by thermal processing at 90°C for 6–10 min. The incorporation of ginger and temulawak extracts aimed to reduce the fishy odor inherent to brunok and enhance the synergistic efect on taurine functionality. Based on the study conducted by (Putri et al., 2013), the combination of Discodoris sp. and Curcuma xanthorrhiza can produce a synergistic efect on taurine, thereby increasing the taurine content in functional beverages. Fresh lemons were squeezed, and the resulting juice was diluted with water at a 1:1 (v/b) ratio to obtain a fresh lemon extract.

The primary ingredients and supplementary components were mixed according to a predetermined formulation. Maltodextrin, amounting to 10% of the total weight of the beverage solution, was added during the spray-drying process to prevent nutrient degradation caused by heat exposure. The spray drying was carried out at an inlet temperature of 180 °C and an outlet temperature of for 45 min, ensuring proper drying while minimizing nutrient loss. Following dehydration, sucrose (1:1, w/w) and seaweed-derived carrageenan (1%) were added. The final product was obtained as an instant powdered beverage (Modified from Putri et al., 2013). The formulation was conducted in three independent replicates for each sample. The compositions of the functional brunok instant powdered beverages formulated at diferent ratios are shown in Table 1.

Proximate composition (moisture, protein, ash, lipid, and carbohydrate content) of fresh brunok and its extract. Moisture content was determined using the oven-drying (thermogravimetric) method, and protein content was measured using the Kjeldahl method. The ash content was determined by incineration in a mufle furnace, and the lipid content was analyzed using the Soxhlet extraction technique. The carbohydrate content was calculated using the diference method by subtracting the combined percentages of moisture, ash, protein, and lipids from 100%. All proximate analyses were conducted in accordance with the standard procedures of the AOAC (2005).

Amino acid profiles were determined according to AOAC Oficial Method 982.30 E (a,b,c) using high-performance liquid chromatography (HPLC). Samples were homogenized and hydrolyzed with 6 N HCl at 110°C for 24 h under vacuum to release protein-bound amino acids. After cooling, the hydrolysates were filtered and neutralized before derivatization. Precolumn derivatization was performed using phenylisothiocyanate (PITC) to form phenylthiocarbamyl (PTC) amino acid derivatives. Chromatographic separation was performed on a reversed-phase C18 column (250 mm × 4.6 mm i.d., 5 μm particle size). The mobile phase consisted of (A) 0.1 M sodium acetate bufer (pH 6.5) and (B) acetonitrile– water (60:40, v/v) and was subjected to gradient elution at a flow rate of 1.0 mL/min. The column temperature was maintained at 30°C. The detection was performed using a UV–Vis detector at 254 nm. Quantification was achieved using external calibration with certified amino acid standards.

Taurine concentration was determined according to the AOAC Oficial Method 997.05 using high-performance liquid chromatography (HPLC). Samples were prepared and derivatized prior to analysis, and chromatographic separation was performed on a reverse-phase column under controlled conditions. Taurine was identified based on its retention time by comparison with a certified taurine standard (≥99% purity). Quantification was performed using an external calibration curve constructed from standard solutions at various concentrations. The results were expressed as mg per 100 g of sample.

The experimental data were analyzed using a Completely Randomized Design and processed using SPSS Version 33. A one-way analysis of variance (ANOVA) was performed to evaluate the efect of treatments, and when significant diferences (p < 0.05) were detected, the means were compared using Tukey’s Honestly Significant Diference (HSD) test. For proximate, amino acid, and taurine parameter comparisons between fresh brunok and brunok extract, a t-test was applied to ensure appropriate statistical evaluation. All results are presented as mean ± standard deviation (SD).

Chemical composition is one of the most important parameters for determining the quality of raw materials or processed products. Proximate analysis was performed on fresh brunok and its extract to identify changes during extraction. Thermal treatment of fresh brunok at 90°C resulted in significant changes in the proximate composition of the bruno extract, as indicated by a decrease in moisture content and a relative increase in protein and lipid levels. This finding is consistent with that of (Panayotova et al., 2025) on bluefish (Pomatomus saltatrix), who reported that cooking processes such as grilling, pan-frying, and smoking reduced moisture content by 7–18% and increased total lipid content by 26–80% due to moisture loss during heating. The results of the proximate analysis are presented in Table 2.

As shown in Table 2, significant diferences (p<0.05) were observed in the ash, fat, and carbohydrate contents on a dry basis between fresh brunok and its extract, as indicated by diferent superscript letters within the same row. In contrast, the protein content did not difer significantly after heatassisted extraction. These findings indicate that thermal processing primarily afects mineral and lipid fractions through leaching and thermal degradation, whereas the total protein content remains relatively stable. The apparent increases observed on a wet basis were mainly attributable to moisture reduction rather than to actual nutrient enrichment.

The chemical composition of fresh brunok showed high nutritional value, particularly in terms of protein (17.12%) and lipid (5.42%) content. After heating at 90°C for 8–10 min, the brunok extract exhibited an increase in protein (46.72%) and lipid (7.18%) contents, largely due to the reduction in moisture content (from 72.18% to 18.17%). This phenomenon is consistent with studies on Holothuria sp., which demonstrated that heating causes water loss (cooking loss), resulting in a relative increase in the protein and lipid concentrations (Bilgin & Tanrikulu, 2018).

The moisture content of fresh brunok (72.18%) decreased significantly to 18.17% in the extract after heating at 90°C for 8–10 min. This decline is consistent with the cooking loss phenomenon, which leads to the loss of water and soluble substances in the meat. Similar findings were reported by (Bilgin & O, 2018) for Holothuria atra, where moisture content significantly decreased after cooking, afecting both texture and shelf life. (Zhang et al., 2022) also reported that heat treatment can reduce moisture content by up to 60–70%. The protein content in brunok increased from 17.12% in fresh flesh to 46.72% in the extract. This increase occurred as a result of the reduced water content, which led to a relative increase in protein concentration. A similar phenomenon was observed in Holothuriascabra, where protein levels increased from 38% to 50% after boiling (Bilgin & Tanrikulu, 2018). Although the protein content increased, its quality may decline due to damage to the amino acid structures.

The results of this study revealed a significant decrease in the ash content. This significant reduction in ash content indicates that the loss of soluble components, including minerals and water-soluble carbohydrates, during thermal processing exceeds the concentration efect caused by water loss. (Zhang et al., 2022) reported that boiling induces the leaching of potassium and sodium into the cooking medium. (Singh et al., 2023) further emphasized that minerals such as calcium are relatively stable, whereas iron is highly susceptible to loss owing to oxidation during heating. The lipid content of fresh brunok flesh was 5.42%, which increased to 7.18% after thermal processing during extraction. This increase was primarily attributed to the substantial reduction in moisture content from 72.18% to 18.17%, thereby elevating the proportion of lipids within the total solids. Similar phenomena have been reported in various marine organisms, such as fish and mollusks, where boiling leads to water evaporation and an increase in the concentration of lipid components (Tan et al., 2023). Although the relative lipid content increased, the lipid quality was significantly afected by heating. Lipids, particularly those containing unsaturated fatty acids, are susceptible to thermal oxidation. This process produces secondary compounds, such as aldehydes and ketones, which can impair sensory quality and reduce the nutritional value of the product (Fan et al., 2025). Several studies have shown that heating at temperatures above 100°C can accelerate the degradation of polyunsaturated fatty acids (PUFAs), particularly EPA and DHA, which are crucial for cardiovascular health (Bae et al., 2022).

The carbohydrate content of fresh brunok flesh was 3.85%, which significantly increased to 26.81% in the extract. Similar to proteins and lipids, this increase was not due to new biosynthesis but rather to the relative concentration of solid components following water reduction. This mechanism is known as the solid-concentration efect (Jiang et al., 2025). Carbohydrates in marine organisms, particularly Holothuroidea, are generally present as glycogen, mucopolysaccharides, and dietary fibers (Senadheera et al., 2023). Some of these, especially sulfated polysaccharides, are known to possess bioactive properties, such as antioxidant, immunomodulatory, and anticoagulant activities (Hossain et al., 2023). Carbohydrates are highly susceptible to Maillard reactions during heating, particularly when they interact with proteins or amino acids. These reactions may lead to the formation of melanoidin compounds, which influence the color and flavor of the product while also reducing the availability of essential amino acids (Shakoor et al., 2022). In the context of instant powdered beverage development, a high carbohydrate content can provide advantages as a natural carrier agent, enhancing the solubility and improving the texture and sensory quality of the product. Additionally, maltodextrin added to the formulation serves as a nutrient stabilizer and prevents bioactive degradation caused by heat.

Amino acids are essential components that determine the nutritional and functional qualities of protein-rich foods. Changes in amino acid composition may occur during thermal processing due to protein degradation, leaching, or structural modification. Therefore, evaluating the amino acid profile is important for assessing the impact of processing on the nutritional quality of brunok. The amino acid composition of brunok is shown in Table 3.

As shown in Table 3, significant diferences (p<0.05), as indicated by diferent superscript letters within the same row, were observed for most individual amino acids between fresh brunok and its extract. Both essential and non-essential amino acids exhibited significant changes following heat-assisted extraction, indicating that the extraction process substantially modified their amino acid profile. These alterations may be attributed to protein structural modifications, diferences in amino acid solubility, and leaching during extraction.

The amino acid profile of brunok showed notable changes after its processing. In the fresh sample, the total essential amino acids reached 6.19 g/100 g sample and nonessential amino acids 11.09 g/100 g sample. After extraction with heat treatment, these values decreased drastically to 1.35% (essential amino acids) and 1.98 g/100 g sample(nonessential amino acids). This decline in amino acid content observed in this study is consistent with the findings of (Li et al., 2023), who reported that thermal treatment reduces amino acids through oxidation, deamination, and other forms of thermal degradation, resulting in the loss of free amino acid residues in the protein. Amino acids, such as lysine, histidine, and threonine, are particularly heat-sensitive because of their reactive amino groups. In this study, the Brunok extract was processed at for 8–10 min.

Taurine is an organic compound classified as a sulfonic amino acid (β-amino acid). Although often referred to as an amino acid, taurine is non-proteinogenic because it is not incorporated into proteins and contains a sulfonate group instead of a carboxylate group. In aquatic organisms, taurine plays vital physiological roles in metabolism, antioxidant defense, and regulation of various body functions (Bae et al., 2022)(Tzang et al., 2024). The taurine content (g/100 g sample) was for fresh Brunok and 81.73±0.03 for Brunok extract. The paired samples t-test revealed a statistically significant diference in taurine content between fresh Brunok and Brunok extract. These results suggest that heat-assisted extraction at significantly increased taurine levels compared to the fresh conditions.

Taurine is a key bioactive component of brunok, functioning as an antioxidant, osmoregulatory agent, and supporter of cardiovascular and nervous system health. Based on the analysis, the taurine content in fresh brunok flesh was 75.05 mg/kg and increased to 81.73 mg/kg after heating during the extraction process. This increase is relative and results from moisture reduction, which leads to a higher concentration of soluble compounds, including taurine. A similar observation was reported by (Hossain et al., 2023), who found that boiling can release free amino acids previously bound within the tissue, making them more detectable in extracts.

Although nutritional composition generally includes a wide range of macroand micronutrients, the present study specifically focused on the amino acid profile and taurine content as the main nutritional components of the brunok beverage. These parameters were selected because of their important physiological roles, sensitivity to thermal processing, and relevance to the functional characteristics of marine-based beverages. The amino acid composition of the Brunok instant beverage powder is presented in Table 4, with changes in essential and non-essential amino acids discussed in relation to controlled thermal processing and formulation variations. The taurine content of the Brunok instant beverage powder is presented in Table 4.

Table 4 shows the amino acid compositions of the brunok instant beverage powders formulated as MFB1–MFB3. Both essential and non-essential amino acids were detected in all samples, with significant differences among the formulations (p<0.05). Leucine and methionine were the dominant essential amino acids in all formulations, with leucine ranging from 3.01% to 3.21% and methionine ranging from 2.28% to 2.62%. Lysine was present at lower levels (0.48–0.73%) in all samples. The total essential amino acidcontent ranged from 5.99% to 6.72%, with the highest value observed in MFB3.

Non-essential amino acids were present in higher proportions than those of essential amino acids. Aspartic and glutamic acids were the major non-essential amino acids, although their concentrations decreased from MFB1 to MFB3. Proline showed relatively higher values (2.00–2.53%) than other non-essential amino acids. The total non-essential amino acid content ranged from 6.54% to 8.51% in the samples.Table 4 shows the amino acid compositions of the brunok instant beverage powders formulated as MFB1–MFB3. Both essential and non-essential amino acids were detected in all samples, with significant diferences among the formulations (p<0.05). Leucine and methionine were the dominant essential amino acids in all formulations, with leucine ranging from 3.01% to 3.21% and methionine ranging from 2.28% to 2.62%. Lysine was present at lower levels (0.48–0.73%) in all samples. The total essential amino acid

The increase observed in methionine and leucine levels may be explained by several factors, including the contribution of added ingredients such as ginger (Zingiber oficinale) and Curcuma xanthorrhiza, which are known to possess rich and diverse amino acid profiles (Ajayi et al., 2013). In addition, the drying process plays a role, as the loss of water and non-protein components produces a concentration efect, resulting in relatively high amino acid levels (Li et al., 2023). The stability of proline against thermal degradation makes it more resistant to heating than other amino acids (Li et al., 2023). This stability is further reinforced by the fact that proline rarely participates in the initial stages of the Maillard reaction, allowing it to persist after food processing. In contrast, glutamic acid showed a decreasing trend among the powdered beverage samples, declining from 1.93% in MFB1 to 0.91% in MFB3, which may be attributed to its sensitivity to heat-induced reactions and involvement in flavor-forming processes.

From a nutritional perspective, although reductions were observed in several essential amino acids, such as lysine, the high levels of leucine and methionine still provide positive value. Leucine, for instance, is essential for muscle protein synthesis, whereas methionine functions as a methyl group donor and plays a key role in sulfur metabolism. Nevertheless, the low lysine content represents a limitation, as powdered brunok may have reduced protein biological value, particularly if lysine becomes the limiting amino acid in the diet (Zhang et al., 2022). The Maillard reaction not only afects nutritional quality but also impacts sensory properties, as melanoidin formation contributes to color and characteristic aroma; however, in advanced stages, it may yield undesirable compounds (El Hosry et al., 2025).

Based on the amino acid profiles observed in Table 4, further eforts to preserve amino acid quality in brunok-based instant beverages may benefit from the application of more nutrient-friendly processing methods.

Previous studies have shown that freezedrying is more efective in maintaining essential amino acid content than spray drying or direct heating (Li et al., 2023). Incorporating carriers or excipients during spray drying has been reported to protect proteins and amino acids from degradation, thereby enhancing their stability during storage (Dieplinger et al., 2023). Therefore, although brunokbased instant powdered beverages have demonstrated potential as functional drinks rich in amino acids, optimizing processing conditions and selecting appropriate drying methods are essential steps to further improve their nutritional quality. The taurine content of an instant brunok beverage powder was evaluated to determine the efect of the formulation on taurine retention. The results are shown in Table 5.Table 5 shows the taurine content of the brunok instant beverage powder formulated as MFB1–MFB3. The taurine content ranged from 65.63 to 70.06 g/100 g sample and increased with higher proportions of brunok extract in the formulation. Formulation MFB3 exhibited the highest taurine content and difered significantly from MFB1 and MFB2 (p<0.05). This trend suggests that the formulation composition, particularly the proportion of brunok extract, along with controlled processing conditions, plays a key role in determining taurine retention in the instant beverage powder.

The taurine content of the brunok instant beverage powder ranged from 66 to 70 g/100 g sample, as shown in Table 5. These values indicate relatively low taurine levels following the conversion of the extract into a powdered form using spray drying. Although taurine is considered relatively stable under moderate heating, high drying temperatures and prolonged exposure during spray drying may promote degradation and loss due to its high water solubility and hygroscopic nature. (Haas et al., 2024) reported that taurine is highly susceptible to loss during thermal processing, particularly during drying, because it can difuse with evaporating moisture. Previous studies have also shown that short-term heating during extraction may increase the detectable taurine content due to concentration efects, whereas subsequent spray-drying can reduce the initial taurine content by up to 20–30% (Tzang et al., 2024).

In the present study, formulation MFB3, which contained the highest proportion of brunok extract, exhibited the highest taurine content, indicating that the formulation composition plays a critical role in taurine retention during processing. Increasing the proportion of Brunok extract while reducing the concentration of Curcuma xanthorrhiza significantly influenced the taurine content of the instant beverage powder. This finding suggests that Brunok, as the primary taurine source, contributes more substantially to taurine levels than the plantbased additive, which primarily afects the formulation through dilution. During spray drying, matrices with higher marine-derived protein content may facilitate better taurine retention, whereas C. xanthorrhiza appears to contribute only indirectly to taurine stability.