The method for producing seaweedfortified tempeh was based on that of (Dewi et al., 2025), with modifications in the source of E. spinosum. E. spinosum was obtained from seaweed farmers in Jepara, Central Java, Indonesia (Figure 1). White soybeans and Raprima tempeh starter were obtained from a traditional market in Semarang, Central Java, Indonesia. Raprima consist of rice flour 99% and Rhizopus oligosporus 1%. First, E. spinosum samples were prepared for analysis. The seaweed was washed thrice to remove impurities. The samples were then soaked in water at a ratio of 1:15 for 3 h. After soaking, the seaweed was blanched in boiling water for 4 min and then blended until smooth. Second, tempeh production was performed. Soybeans were washed and boiled at a ratio of 2:1 (water: soybeans) for 30 min.

The soybeans were then soaked in the boiled water residue for 24 h. The outer skin was removed by squeezing the soybeans until the skins separated, leaving the soybean kernels without the skin. The soybeans were washed and steamed for 30 min. Next, 0.1% tempeh starter was added based on the soybean weight and mixed thoroughly. E. spinosum was added at concentrations of 0% (T0), 10% (T1), 20% (T2), and 30% (T3), with three replicates. The seaweed was mixed thoroughly until fully incorporated, then wrapped in plastic, sealed, and punctured with a toothpick at intervals of approximately 1 cm. Fermentation was carried out by placing raw tempeh on a perforated rack at room temperature for 48 h. The fermented tempeh was tested.

This test was conducted using the Bradford method, with Coomassie Brilliant Blue (CBB) as the indicator. CBB binds to proteins, causing a color change to blue, which was measured using a visible spectrophotometer (PG Instruments Ltd., UK) at wavelengths of 465–595 nm, using bovine serum albumin (BSA) as the standard (Mardhika et al., 2020).

Antioxidant activity was assessed using the DPPH method and expressed as % inhibition and IC /ppm using α,α- diphenyl-β-picrylhydrazyl (DPPH). Samples were dissolved in methanol at diferent concentrations, and 0.1 mM DPPH reagent was added to the solution. The mixture was incubated at room temperature in the dark for 30 min. After incubation, the absorbance was measured using a spectrophotometer (T70 UV-Vis, PG Instruments Ltd., UK) at 517 nm. The percentage inhibition (PI) of DPPH radicals was calculated as follows:

Radical DPPH (PI) = [(Ab − As)/Ab] × 100 Where Ab refers to the absorbance of the control (without the sample) and As refers to the absorbance of the sample.

Antioxidant activity was reported in two parameters: % inhibition at a specific concentration and (ppm), defined as the sample concentration required to inhibit 50% of the DPPH radicals (Salem et al., 2017; Beyazen et al., 2017).

Total phenol was measured using the Folin-Ciocalteu method to evaluate the phenolic content in the samples. One milliliter of FC reagent was mixed with the sample for 5 min, and then 10 ml of 7.5% sodium carbonate solution was added and mixed. The final volume was adjusted to 25 ml with deionized water and allowed to stand for 1 h. Phenolic content was analyzed at 750 nm using a spectrophotometer (Shimadzu, Japan) based on a calibration curve prepared with standard compounds (gallic acid, 0–200 mg/ ml). The final quantitative result was expressed as mg gallic acid equivalents (GAE) per 100 g of fresh weight (Alkaltham et al., 2021).

Texture testing was performed to measure the hardness, cohesiveness, springiness, gumminess, chewiness, adhesiveness, and fracture force of tempeh. The measurements were performed using a texture analyzer by pressing the sample at a specific speed. The tempeh was placed in a prepared container and pressed with a round probe of 0.5 inch diameter (Rochmah et al., 2019).

This test was conducted using ultraperformance liquid chromatography (UPLC) (Waters Corporation, USA). The amino acid score was used to assess how well the amino acids in a food product can be absorbed and utilized by the body. The score was calculated by comparing the amino acid content of the sample with that of the standard essential amino acid pattern. The standard essential amino acid requirements were as follows: histidine (1.9), lysine (5.8), threonine (3.4), isoleucine (2.8), leucine (6.6), valine (3.5), methionine + cysteine (2.5), phenylalanine + tyrosine (6.3), and tryptophan (1.1).

SAA = AAE sample / AAE standard × 100% Where SAA is the Amino Acid Score and AAE is the Essential Amino Acid content (Riviani et al., 2020).

Fourier-transform infrared spectroscopy (FTIR) (Perkin Elmer, USA) was performed on dried and powdered tempeh. Eight scans were accumulated in the transmission mode with a resolution of 4 cm-1. The spectrum was recorded from 4,000 to 600 cm-1 (Gullon et al., 2017).

This study used a completely randomized design with a single factor, namely, the addition of E. spinosum at diferent concentrations. The study was performed in triplicates. Data were analyzed using analysis of variance (ANOVA). If a significant diference was found (p<0.05), Duncan’s post hoc test was performed. Data were processed using SPSS IBM 23.

Soluble proteins refer to simple protein components (oligopeptide group) consisting of amino acid chains shorter than ten, making them more water-soluble than larger proteins due to their shorter chain length (Prihatiningsih et al., 2021) .Table 1 shows that sample T3 had the highest soluble protein content at 24.07%, while the addition of 10% seaweed (T1) showed no significant diference from that of the control. T3 treatment significantly increased the soluble protein content, and higher concentrations of seaweed led to higher soluble protein levels. This result aligns with that of (Kusnandar et al., 2020), who found that tempeh made from red beans had soluble protein levels of approximately 20–27%.

Red seaweed has a higher protein content than green and brown seaweeds. Red seaweed contains approximately 150–200 g/ kg dry weight, green seaweed 90–180 g/kg dry weight, and brown seaweed 50–120 dry weight (Olsson et al., 2020). Fortification with E. spinosum increases the soluble protein content in tempeh due to the direct addition of protein/peptide fractions from seaweed (Brien et al., 2022). The increase in soluble protein content in tempeh is also due to fermentation. Microbial proteolytic activity during fermentation enhances the release of soluble peptides (Damongilala et al., 2021). During fermentation, Rhizopus produces proteolytic enzymes that hydrolyze proteins by breaking peptide bonds, resulting in amino acids and short-chain polypeptides. The breakdown of peptide bonds by this proteolytic activity is associated with an increase in soluble protein content (Puspitojati et al., 2019; Ishartani et al., 2021). The results of this study are consistent with those of (Ishartani et al., 2021) who produced tempeh from lamtoro seeds and found that the soluble protein content increased as the amount of seeds increased.

The antioxidant activity value decreased with increasing E. spinosum concentrations. The highest value was observed in T0 (146.9 ppm), whereas the lowest was observed in T3 (109.4 ppm). This indicates that the ability of the product to capture or neutralize free radicals increased. Lower values indicate a stronger antioxidant potential (Gulcin and Alwasel, 2023). This is supported by the increasing % inhibition, where higher E. spinosum concentrations led to higher radical scavenging activity. The results of this study were higher than those reported by (Evangelista & Surya, 2024), who found that tempeh with 0.7% butterfly pea flowers had an antioxidant activity of 82.94%. However, the results were lower than those reported by (Yudiono, 2023), who found that tempeh with Moringa leaf flour had an antioxidant activity of 93.67%. As shown in Table 1, the highest radical scavenging activity was observed in T3 (86.64%) and the lowest in T0 (63.25%). These values are consistent with the IC50 results, where lower values indicate that a smaller sample size produces strong antioxidant efects (Ziemlewska et al., 2021). Fortification of tempeh with E. spinosum improved its antioxidant activity. This enhancement is attributed to the presence of bioactive compounds in E. spinosum, such as phenolic compounds, flavonoids, and carotenoid pigments (Sofiana et al., n.d.). Additionally, soybeans produce isoflavones through fermentation. Isoflavones are known to act as antioxidants and can function synergistically with other antioxidants (Kuligowski et al., 2017). Fermentation of grains, vegetables, and other plants using starter fungi, lactic acid bacteria, or yeast increases the levels of phenolic compounds, flavonoids, vitamins, and bioactive peptides. Microbial growth produces enzymes such as β-glucosidase, esterase, and protease, which break down cell walls and hydrolyze related compounds, releasing bioactive compounds into the medium and enhancing the radical scavenging capacity (Hur et al., 2014). The combination of two diferent antioxidant sources has the potential to enhance the overall efectiveness of the product in neutralizing free radicals, making E. spinosum-fortified tempeh more efective than regular soybean tempeh in this regard. The antioxidant activity results of this study are consistent with the increased total phenol content. A high total phenol content has a linear efect on high antioxidant activity. Similar results were obtained by (Yudiono et al., 2021), who produced tempeh using various types of soybeans.

The total phenol content increased with higher concentrations of E. spinosum. This indicates that fortification with E. spinosum enriched the bioactive components of tempeh. Tempeh without E. spinosum (T0) had the lowest phenolic content at 9.34 mg GAE/g, while tempeh with 30% E. spinosum (T3) had a phenolic content of 11.01 mg GAE/g. Phenolic compounds act as antioxidants by donating hydrogen atoms to neutralize free radicals, thereby stopping chain reactions that could damage lipids or proteins in the body (Shahidi & Ambigaipalan, 2015). The increase in total phenol content in fermented products (tempeh) is directly related to higher antioxidant activity (Xiao et al., 2021). This study’s total phenol content was higher than that reported by (Šulc & Rysová, 2025), who found tempeh made from a 2:1 mixture of yellow peas and sorghum had total phenol of 10.14 mg GAE/g, and tempeh made from a 1:2 mixture of quinoa and sorghum had 4.68 mg GAE/g. This indicates that E. spinosum efectively increased the phenolic content of tempeh and enhanced its antioxidant properties. The increase in total phenol content was due to the high levels of phenolic compounds and various secondary metabolites, such as polyphenols, flavonoids, and tannins, which act as antioxidants (Syakri et al., 2024). During tempeh fermentation, total phenol content tends to increase due to the presence of β-glucosidase and esterase, which hydrolyze complex compounds into free phenolic forms (Zhao et al., 2021). The presence of E. spinosum may also exert a synergistic efect with soybean isoflavones, enriching the total phenol content. Therefore, the higher the concentration of E. spinosum used, the higher the total phenol content in the tempeh product.

Table 2 shows that the addition of E. spinosum significantly afected the tempeh texture. The hardness decreased with increasing seaweed concentration. The highest hardness was observed in T0 (0.65 kgf), and the lowest in T3 (0.13 kgf). This indicates that the tempeh texture became softer with increasing E. spinosum levels. The hardness values in this study were lower than those reported by (Sundari et al., 2024), where soybean tempeh combined with groundnut had hardness values of 12–14 kgf. Fresh tempeh is firm and soft. Increasing E. spinosum concentration improved cohesiveness and springiness, although these values were lower than those of the control. Cohesiveness measures product compactness, whereas springiness indicates the degree of elasticity or resilience, where a sample returns to its original shape (Danella, 2024). In this study, the cohesiveness and springiness values decreased. These values are consistent with those reported by (Wikandari et al., 2020), who reported that tempeh made from various legumes had cohesiveness values of 0.45–0.61 and springiness values of 0.85–0.91 mm. The gumminess, chewiness, and adhesiveness decreased in sample T3, with values of 0.06 kgf, 1.26 kgf·mm, and 0.03 kgf·mm, respectively, indicating that the sample was easier to chew and less sticky, whereas the fracture force remained relatively stable.

Overall, fortification with E. spinosum (Figure 2) reduced the hardness and springiness but increased the elasticity and improved the sensory properties. Texture testing afects the physical characteristics of fresh and processed tempeh. If the texture values are too low, the tempeh may easily break, become brittle, or lack firmness, making it dificult to cut. If the texture values are too high, the tempeh will have poor texture quality due to excessive hardness, making it dificult to consume (Sundari et al., 2024). The higher the concentration of E. spinosum, the softer, more elastic, and easier-to-chew the tempeh. This is due to the presence of natural hydrocolloids in E. spinosum, particularly carrageenan, which can bind water and form a gel structure, thereby increasing the moisture and elasticity of the product (Dewi et al., 2025b). In addition, the soluble fiber and polysaccharide components in seaweed can influence the functional characteristics of food, such as water-holding capacity, thereby reducing the density of the soybean matrix and resulting in a smoother and less brittle tempeh structure (Peñalver et al., 2020). Seaweed also helps retain moisture during fermentation, reduces mass loss, and creates a soft texture (Syakri et al., 2024).

The analysis of the amino acid profile is important because it can indicate whether a material meets the essential amino acid requirements for humans according to WHO standards and help in understanding changes in protein composition resulting from processing methods such as fermentation (Melini & Melini, 2021). In addition, several amino acids, such as glutamate and aspartate, play a role in developing the umami (savory) taste in fermented food products such as tempeh (Purwandari et al., 2025). The amino acid profiles of the tempeh samples are presented in Table 3.

The most dominant amino acids in soybeans and E. spinosum were L-glutamic acid at 5,7941.98 mg/kg and 1,289.46 mg/kg, respectively, followed by L-aspartic acid at 32,649.19 mg/kg in soybeans and 1,226.22 mg/ kg in E. spinosum. L-Glutamic and L-aspartic acids contribute to the umami flavor of food products (Amaliah et al., 2024). Based on Table 3, the amino acid levels in tempeh with added E. spinosum (T1, T2, and T3) were generally lower than those in tempeh without E. spinosum (T0). T0 had L-glutamic acid at 18850.1 mg/kg, which decreased progressively with the addition of E. spinosum, reaching 10,258.2 mg/kg in T3. T0 in this study had higher L-glutamic acid than (Wikandari et al., 2020), who reported 14,241.77 mg/kg in green bean tempeh, but lower than tempeh made from peanuts (53,370.99 mg/kg).

The decrease in amino acids may be due to the degradation of soybean protein during fermentation and interactions with sulfate polysaccharides from seaweed, which may inhibit the release of free amino acids (Ghelichi & Jacobsen, 2025). Another possibility is that during fermentation, the addition of E. spinosum catalyzes increased microbial growth, thereby boosting the production of proteolytic enzymes. This leads to a greater breakdown of peptide bonds, resulting in an increased quantity of short-chain peptides. This is related to the increasing levels of soluble proteins (Surya et al., 2024). Soluble proteins are important components of food products because they play a role in texture formation, afect the functional properties of proteins, and influence the digestibility of food (Anyiam et al., 2025; Grossmann & McClements, 2023). Although it decreases the amino acid content of tempeh, the addition of E. spinosum results in higher polyphenol and flavonoid content, which enhances antioxidant activity. Other studies have also shown that adding E. spinosum increases the dietary fiber content of tempeh (Dewi et al., 2025b).

Fourier Transform Infrared Spectroscopy (FTIR) is a vibrational spectroscopy technique that measures the interaction between infrared radiation and the molecules in a sample, producing an absorption spectrum that reflects the chemical functional groups and molecular bonds present in the substance (Li et al., 2019). The FTIR spectra of tempeh with the addition of seaweed are presented in Figure 3.

The FTIR spectrum of soybeans showed characteristic peaks at 1,650 cm⁻¹ (Amide I), 1,540 cm-1 (Amide II), and 1,240 cm-1 (Amide III) from the protein, while E. spinosum showed a strong peak at \~1,220–1,260 cm-1 (S=O group from sulfated carrageenan). Soybeans were dominated by protein, whereas E. spinosum was dominated by sulfated polysaccharides. Tempeh with E. spinosum fortification showed that in the O-H and N-H stretching region (3,400–3,200 cm-1), sample T0 had a strong peak around 3,400 cm 1, indicating hydrogen bonding in hydroxyl (O-H) and amino (N-H) groups. In samples T1, T2, and T3, the peak intensity decreased, indicating a reduction in free hydroxyl and amino groups, likely due to their interactions with seaweed components. According to (Kedang et al., 2024), a strong absorption band around \~3,300 cm⁻¹ indicates the presence of –OH and –NH stretching vibrations derived from the hydroxyl and amide groups of polysaccharides and proteins, respectively (amide A band). In the C-H stretching region (3,000–2,800 cm-1), peaks around 2,920 cm-1 and were observed, which were attributed to methyl and methylene groups, respectively. In T1, T2, and T3, slight shifts and a decrease in intensity were observed, indicating modifications to the aliphatic structure of tempeh. In the Amide I and II region (1,700–1,500 cm-1), peaks around 1,650 cm-1 (Amide I) and 1,550 cm-1 (Amide II) indicate C=O and N-H bonds from the protein.

The addition of seaweed caused shifts and changes in peak intensity, indicating interactions between tempeh protein and seaweed components, possibly forming new complexes or degrading peptide bonds. In the C-O stretching region (1,250–1,000 cm-1), strong peaks in the 1,100–1,000 cm-1 range indicate ether (C-O-C) and alcohol (C-OH) groups. In T1 to T3, the peak intensity decreased, indicating chemical reactions between the carbohydrates in tempeh and seaweed components. All four samples shared the same main functional groups: O-H, C=O, and C-H groups. The diferences were in the peak intensity, indicating varying amounts or compositions. These diferences arose from the interactions between seaweed and tempeh, which altered the tempeh structure. Thus, tempeh with seaweed addition showed improved bioactive properties, such as higher antioxidant activity and phenolic content, despite slight reductions in some amino acid levels. This finding aligns with that of (Arham et al., 2021). where a decreased intensity at 1,745 and 1,543 cm-1 indicated protein degradation, showing reduced intensity in the amide range.