Tempeh is a popular conventional fermented food from Indonesia. The process of producing tempeh involves soaking, boiling, and fermenting with a starter culture such as Rhizopus sp. (Egounlety and Aworh, 2003). Originally, soybeans were the primary ingredient used to produce tempeh. However, advancements in science have enabled tempeh production from several types of beans, grains, and legumes (Dewi et al., 2014; Puspitojati et al., 2019a). During fermentation, Rhizopus sp. creates a thick, white mycelium that holds the beans together. Rhizopus mycelia develop on cooked beans and release many enzymes, including α-amylase, esterase, cellulase, protease, and β-glucosidase (Radiati and Sumarto, 2016). These enzymes induce chemical modifications in the beans, improving protein digestibility (Jayanti, 2019), reducing antinutritional factors (Puspitojati et al., 2019a), enhancing the flavor, and generating bioactive peptides (Puspitojati et al., 2019b). However, non-soy tempeh remains relatively unpopular. Therefore, the functional characteristics of non-soy tempeh need to be investigated to enhance consumer interest.

Jack bean [Canavalia ensiformis (L.) DC.] has been cultivated in Indonesia as a feasible protein alternative to soybeans. The germination process enhances protein digestibility by causing protein breakdown, forming amino acids and short-chain peptides (Agustia et al., 2023a). According to Agustia et al. (2023b), jack bean sprouts that were germinated for 60 h had a protein content of 33.60% (dry basis, db), a dietary fiber content of 18.60% (db), an essential amino acid content of 23.89 g/100 g of protein, and a hydrogen cyanide (HCN; an antinutritional chemical) content of 9.62 ppm. Jack bean sprouts can be transformed into a valuable source of vegetable proteins. However, additional processing, including tempeh fermentation, is needed to decrease the HCN content to safer levels. Germination following fermentation affects the physicochemical properties and sensory profiles of velvet bean (Mucuna pruriens) and soybean tempeh (Astawan et al., 2024). Using jack bean sprouts in tempeh production shows promise in enhancing nutritious components, reducing antinutritional components, and serving as an alternative substitute for soybean tempeh in Indonesia.

The nutritional quality of tempeh is affected by several factors, including temperature, humidity, fermentation time, and packaging (Sayuti, 2015). The protein, moisture, and fiber contents of tempeh can be enhanced by increasing the fermentation time to a certain extent. Nevertheless, if the fermentation is prolonged, the tempeh may become soft, emit a foul odor, and have a lower nutritional value (Anyiam et al., 2023). Wulan et al. (2021) reported that the protein content of red bean (Phaseolus vulgaris L.) tempeh was 13.64% at 48 h of fermentation, but it decreased to 9.64% after 96 h of fermentation. The fermentation process of tempeh requires moist conditions and good oxygen circulation (Suknia and Rahmani, 2020). Aside from fermentation, the characteristics of the packaging materials of tempeh influence the mycelia growth and quality of tempeh, especially the protein content, because they directly influence the protein hydrolysis process (Salim et al., 2017; Umami et al., 2018). Mycelia can grow better in leaf packaging because it is lightproof and has good aeration, thereby maintaining humidity during fermentation (Sayuti, 2015). The tempeh packaging material is determined by the availability of ingredients and the sociocultural aspects of each region (Kristiadi and Lunggani, 2022). Sulistiyono et al. (2016) reported that banana leaf (Musa balbisiana Colla), teak leaf (Tectona grandis), and Hibiscus tiliaceus leaf are commonly used as natural leaf packaging to wrap tempeh. The leaf packaging gives off a distinctive aroma because the leaves contain polyphenols. According to Kristiadi and Lunggani (2022), people prefer plastic packaging because it is more practical. However, plastic packaging requires holes because it has low air, vapor, and heat permeability. Andriati et al. (2018) reported that the packaging material impacts the dissolved protein, fat, pH, and color of jack bean tempeh.

The functional characteristics and nutritional and antinutritional aspects of tempeh fermentation in jack bean tempeh have been widely studied (Puspitojati et al., 2019b; Ramli et al., 2021; Purwandari et al., 2024). However, no studies have reported the effects of the fermentation time and packaging material on tempeh produced from jack bean sprouts. Therefore, the present study aims to determine the amino acid concentration, proximate content, and antinutritional components of tempeh from jack bean sprouts made using various fermentation times and packaging materials. This study provides insights into the impact of fermentation time and packaging material on the nutritional and antinutritional components of jack bean sprout tempeh.

Materials

Jack bean seeds were obtained from Wonogiri, Central Java, Indonesia. Commercial tempeh inoculum (brand “Raprima”) containing Rhizopus oligosporus was obtained from the local market. The packaging materials for tempeh included plastic (polyethylene, 0.1 mm thickness), banana leaves (M. balbisiana Colla), and teak leaves (T. grandis L.f). All chemicals used in this study were of analytical grade. This study used experimental methods with two factors: fermentation time (36, 48, 60, and 72 h) and packaging material (plastic, banana leaf, and teak leaf). Thus, 12 treatment samples were obtained. These samples were replicated three times, resulting in 36 experimental units.

Production of tempeh from jack bean sprouts

The jack bean sprouts were germinated for 60 h in accordance with the method of Agustia et al. (2023b). The seeds were sorted, cleaned, and soaked in warm water (50°C) with 1% w/w NaHCO₃ for 24 h at a seed-to-water ratio of 1:5 w/v to start the germination process of jack beans. The water was substituted with tap water every 6 h, and the seeds were subsequently washed and drained before germination. The seeds were germinated at room temperature (25°C－28°C) and approximately 100% relative humidity. The sprouts were harvested after 60 h.

Tempeh was produced in accordance with the method of Puspitojati et al. (2019b) with slight modifications. The initial process of tempeh production included peeling, washing, and then boiling the sprouts for 30 min. The ratio of water-to-jack-bean-sprouts used was 4:1. Subsequently, the sprouts were cut into five to six slices and soaked in tap water for 24 h using a water-to-jack-bean-sprout ratio of 4:1. The water used for soaking was replaced every 6 h. Next, the sprouts were cooked in boiling water for 30 min. Afterward, they were drained until their temperature reached 30°C. A tempeh inoculum (R. oligosporus) concentration of 0.2% was applied to the sprouts. They were thoroughly mixed and subsequently packaged with plastic, banana leaf, and teak leaf. The incubation was conducted at a constant temperature of 30°C for 24, 36, 48, 60, and 72 h. The obtained tempeh was then oven-dried (50°C) for 24 h, ground, passed through a 60-mesh sieve, and stored at −18°C until further examination.

Analysis method

Determination of proximate content: The moisture (gravimetric, AOAC Official Method 930.15), ash (gravimetric, AOAC Official Method 942.05), protein (Kjeldahl, AOAC Official Method 990.03), fat (Soxhlet, AOAC Official Method 2003.05), and crude fiber contents (AOAC method 978.10) of the tempeh were tested in accordance with the Association of Official Analytical Chemists (AOAC) standard procedures. The carbohydrate contents were determined using the carbohydrate-by-difference method (100 minus water, ash, protein, and fat contents). All analyses of tempeh samples were performed in triplicate.

Determination of soluble protein content: The soluble protein content was determined using the Lowry method. Lowry A was prepared by diluting 2 g of NaKC₄H₄O₆･4H₂O, 100 g of Na₂CO₃, and 500 mL of 1 N NaOH to a final volume of 1 L. Meanwhile, Lowry B was formed using 2 g of NaKC₄H₄O₆･4H₂O, 1 g of CuSO₄･5H₂O, 10 mL of 1 N NaOH, and 90 mL of distilled water. Finally, Lowry C was produced by combining Folin-Ciocalteu reagent and distilled water in a 1:15 (v/v) ratio. In a reaction tube, 0.9 mL of Lowry A and 1 mL of the protein extract were mixed. After 10 min of incubation at 50°C and 80 rpm in a waterbath (Memmert Waterbath WNB 29, Memmert), the solution was cooled to room temperature before adding 0.1 mL of Lowry B. Following a 10-min room temperature incubation period, the solution was added with 3 mL of Lowry C. After being incubated for 10 min at 50°C and 80 rpm, the mixture was cooled to 25°C to 28°C. The absorbance at 650 nm was measured using an ultraviolet-visible spectrophotometer (GENESYS 10S, Thermo Scientific). The standard stock solution was made of bovine serum albumin (100 mg/L).

Determination of tannin content: The tannin content was assessed in accordance with the method of Chanwitheesuk et al. (2005). Distilled water (62.5 mL) was used to dilute 0.3125 g of tempeh samples before being boiled for 2 h at 100°C. Then, the cooled extract was filtered using Whatman filter paper No. 1. A mixture comprising 1 mL of the extract, 0.5 mL of Folin-Ciocalteu reagent, and 2 mL of 20% Na₂CO₃ was prepared. After the solution was incubated for 30 min at room temperature (25°C－28°C), its absorbance at 748 nm was measured using an ultraviolet-visible spectrophotometer (GENESYS 10S, Thermo Scientific). In the same way as the samples, a standard curve was created using tannic acid standards (0.00, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL).

Determination of phytic acid content: The phytic acid content was determined in accordance with the method of Fitriani et al. (2021). To create the sample solution, 0.1 g of the sample was added to 20 mL of 0.5 M HNO₃. The solution was then transferred into a water bath shaker (Memmert Waterbath WNB 29, Memmert) and agitated for 4 h at 28°C to 30°C. Subsequently, the extract was filtered using Whatman filter paper No. 1. After mixing 1 mL of the extract with 0.4 mL of distilled water and adding 1 mL of 0.005 M FeCl₃, the mixture was placed in boiling water (100°C) for 20 min. Once the solution had cooled, it was added with 5 mL of amyl alcohol and 0.1 mL of 0.1 M ammonium thiocyanate. The mixture was centrifuged for 10 min at 1,008 g (Sorvall^TM ST 8 Centrifuge, Thermo Scientific). The absorbance of the solution at 495 nm was measured using an ultraviolet-visible spectrophotometer (GENESYS 10S, Thermo Scientific). A standard curve for phytic acid was created by combining sodium phytate solutions (50, 100, 150, and 200 ppm) with HNO₃. The phytic acid concentration was expressed as a percentage of milligram per gram of dry matter.

Determination of HCN content: The HCN content was determined in accordance with the method of Nwokoro et al. (2010) with slight modifications. The samples were prepared by incubating tempeh samples for 4 h after adding distilled water. A mixture comprising 1 mL of 2% potassium hydroxide and 5 mL of alkaline picrate was used to dilute the filtrate. The solution was heated at 100°C for 15 s. Using potassium cyanide as the standard, the absorbance was examined at 510 nm using an ultraviolet-visible spectrophotometer (Dynamica Scientific, Halo SB-10).

Determination of amino acid concentration: The concentrations of amino acids, except cystine, methionine, and tryptophan, were determined using the Acquity ultra-performance liquid chromatography (UPLC) H-Class amino acid analysis method from Waters Corp. (2012). Meanwhile, the concentrations of cystine, methionine, and tryptophan were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS; LCMS-8060 NX, Shimadzu) because of their unique chemical properties and the need for high sensitivity and specificity. The Acquity UPLC H-Class amino acid analysis method started with the preparation of 0.1 g of powdered tempeh samples. Subsequently, these samples were hydrolyzed for 24 h at 105°C using 10 mL of 6 N hydrochloric acid (HCl). Then, the hydrolysis solution was placed in a 50 mL volumetric flask, diluted with distilled water, and filtered through a 0.2 µm syringe filter (Sartorius Minisart). The filtrate was supplemented with an internal standard (specifically 2.5 mM alpha-aminobutyric acid). The solution was derivatized by adding AccQ-Tag Ultra reagent and borate buffer and vortexing for 1 min. Subsequently, the solution (1 µL) was injected at 49°C into a UPLC system using an Acquity UPLC BEH C18 (2.1×100 mm², 1.7 µm) column, and its absorbance at 260 nm was measured using an Acquity UPLC photodiode array detector (Waters). The flow rate was 0.7 mL/min. The mobile phases used were labeled as A, B, C, and D. The eluents used in this experiment were as follows: (A) 100% concentrated AccQ-Tag Ultra eluent A, (B) 10% AccQ-Tag Ultra eluent B, (C) double-distilled water, and (D) 100% AccQ-Tag Ultra eluent B. The gradient elution scheme is shown in Table 1.

Table 1 . Gradient elution used to determine the amino acid concentration.

Time (min)	Eluent A (%)	Eluent B (%)	Eluent C (%)	Eluent D (%)
0.00	10.0	−	90.0	−
0.29	9.9	−	90.1	−
5.49	9.0	80.0	11.0	−
7.30	8.0	15.6	57.9	18.5
7.69	7.8	−	70.9	21.3
7.99	4.0	−	36.3	59.7
8.59	4.0	−	36.3	59.7
8.68	10.0	−	90.0	−
10.20	10.0	−	90.0	−

The eluents used in this experiment were as follows: 100% concentrated AccQ-Tag Ultra eluent A, 10% AccQ-Tag Ultra eluent B, double-distilled water, and 100% AccQ-Tag Ultra eluent B..

−, not available..

LC-MS/MS analysis was used to analyze cystine, methionine, and tryptophan. Powdered samples (0.5 g) were kept cold for 15 min at −15°C in a cooling water bath. After being diluted with 5 mL of an oxidizing solution (9 mL formic acid and 1 mL 30% hydrogen peroxide), the sample was incubated at −15°C for 16 h. The oxidized sample was mixed with 0.84 g of sodium bisulfite and allowed to sit at room temperature for 3 h. Then, the material was hydrolyzed for 24 h at 110°C using 5 mL of HCl (6 M with 0.1% phenol). The sample volume was reduced to 25 mL using distilled water. After adjusting the pH of the solution in a cooling water bath to 2.20±0.05, aquabides were added to a 50 mL volumetric flask. Subsequently, the solution was centrifuged for 3 min at 21,952 g. Then, it was filtered through a 0.2 µm regenerated cellulose membrane filter and injected (2 µL) into the LC-MS/MS devices. Mobile phase A was prepared by dissolving 0.1% formic acid in acetonitrile, whereas mobile phase B was made by dissolving 100 mM ammonium formate. A gradient of 14% B was maintained after 3 min, and it was increased to 100% B after 10 min. The gradient reached a value of 14% B at the 12th min. The flow rate through the Imtakt Intrada amino acid column (50×3 mm², 3 µm; Imtakt) was 0.4 mL/min, and the column was adjusted at 37°C (Dahl-Lassen et al., 2018).

Statistical analysis

The data were analyzed using analysis of variance. The average difference between treatments was analyzed using Tukey’s test, and statistical significance was considered at P<0.05. All data analyses were performed using IBM SPSS Statistics 26 developed by IBM Corp.

Visual appearance of tempeh from jack bean sprouts

The visual appearance of mycelia in tempeh from jack bean sprouts can be seen after 24 h of fermentation. As shown in Fig. 1, the mycelia were fully formed after 36 h of fermentation for all tempeh treatments with three different packaging materials. The increasing number of mycelia indicated an increase in the synthesis of hydrolytic enzymes, such as protease, lipase, and amylase, which can break down proteins, lipids, and carbohydrates into simpler compounds (Radiati and Sumarto, 2016). Thus, the tempeh samples in this study were tested for nutritional and antinutritional components beginning at 36 h of fermentation. Tempeh from jack bean sprouts and soybean had a compact structure at the same fermentation time. In soybean tempeh, the cottony mycelia covered the bean entirely after 36 h of fermentation (Ihtifazhuddin et al., 2016). Puspitojati et al. (2019b) reported that the mycelia of tempeh jack bean were compactly bound at 72 h. In the present study, the tempeh from jack bean sprouts grew mycelia faster than jack bean tempeh. This is probably because the texture of jack bean sprouts, as the raw material for tempeh, is softer compared with that of jack bean seeds, so they are easier Rhizopus sp. to break down. According to Astawan et al. (2024), germinated velvet bean tempeh exhibited lower hardness than nongerminated velvet bean tempeh.

Figure 1. Tempeh from jack bean sprouts fermented at different fermentation times in different packaging materials.

Proximate components of tempeh from jack bean sprouts

The proximate components of tempeh derived from jack bean sprouts are shown in Table 2. Statistical analysis revealed significant alterations (P<0.05) in the moisture, protein, and fat contents during the fermentation of tempeh packaged in various packaging materials. However, the ash and carbohydrate contents showed no significant changes. Table 2 shows that the moisture content of tempeh in each packaging material increased throughout the tempeh fermentation process. The increase in the moisture content is believed to be a result of R. oligosporus respiration activity. During fermentation, R. oligosporus releases water vapor because of the degradation of complex molecules trapped by the packaging. As the fermentation time increases, the intensity of macromolecular breakdown increases, resulting in an increase in the moisture content. According to Steinkraus (2002), microorganisms break down carbohydrates, resulting in water production. Table 2 also shows that variations in packaging materials do not affect the moisture content regardless of the fermentation time. The moisture content of tempeh packaged in plastic was generally similar to that of tempeh packaged in teak and banana leaves. This is likely because of the small perforations in the plastic packaging, which facilitate proper air circulation to maintain optimal humidity levels during fermentation. The findings of this study are consistent with those of Andriati et al. (2018), which found no statistically significant alteration in the moisture content of tempeh packaged with either plastic or banana leaves. However, Radiati and Sumarto (2016) found that packaging tempeh in banana leaves increased the moisture content compared with plastic. This is because banana leaves can preserve the moisture content in tempeh.

Table 2 . Proximate components of tempeh from jack bean sprouts fermented at different fermentation times in different packaging materials.

Fermentation time (h)	Packaging material	Moisture content (% wb)	Ash content (% db)	Protein content (% db)	Fat content (% db)	Carbohydrate by diff (% db)
36	Plastic	62.52±0.01aC	1.92±0.15	29.24±0.10aD	4.45±0.25aA	57.30±0.39
	Banana leaf	62.45±0.32aC	1.92±0.08	26.75±0.12bC	4.24±0.03aB	57.70±0.05
	Teak leaf	62.40±0.07aB	1.89±0.12	26.76±0.23bD	4.42±0.22aA	57.87±0.20
48	Plastic	63.69±0.27aB	1.87±0.15	30.88±0.01bA	4.26±0.03bAB	57.53±0.36
	Banana leaf	63.46±0.12aB	1.88±0.13	30.56±0.05cA	4.47±0.16aA	57.63±0.20
	Teak leaf	63.48±0.52aA	1.87±0.22	31.84±0.01aA	4.39±0.05abA	57.46±0.10
60	Plastic	63.78±0.04aB	1.88±0.09	30.38±0.01aC	4.22±0.09aAB	57.85±0.35
	Banana leaf	63.50±0.13aB	1.90±0.03	30.28±0.01bB	4.27±0.03aB	57.12±0.17
	Teak leaf	63.48±0.62aA	1.88±0.17	30.27±0.01bC	4.27±0.06aAB	57.10±0.16
72	Plastic	64.20±0.35aA	1.91±0.12	30.54±0.04bB	4.08±0.01aB	57.80±0.20
	Banana leaf	64.02±0.13aA	1.87±0.16	30.34±0.15bB	4.12±0.05aB	57.05±0.37
	Teak leaf	64.16±0.43aA	1.89±0.15	30.80±0.05aB	4.08±0.08aB	57.40±0.58
SNI		Max 65.0	−	Min 15.0	Min 7.0	−

Values are presented as mean±SD..

In the same column, different lowercase letters (a, b) in the same fermentation time but different packaging materials indicate a significant difference (Tukey’s test, P<0.05). In the same column, different uppercase letters (A-C) in the same packaging material but different fermentation times indicate a significant difference (Tukey’s test, P<0.05)..

wb, wet basis; db, dry basis; SNI, Indonesian National Standard; −, not available..

The fermentation time influences the increase in the protein content (Table 2). The protein content of tempeh packed in each packaging material increased during fermentation (P<0.05), reaching its highest value after 48 h. The increase in protein content in samples with prolonged fermentation time can be attributed to the metabolic activity of R. oligosporus, which generates protease enzymes that hydrolyze proteins into free amino acids. The presence of an N group in these free amino acids enhances the protein content (Dewi et al., 2014). Similar findings were also observed using Saccharomyces cerevisiae and R. oligosporus in the fermentation of soybean tempeh (Rizal et al., 2022). Table 2 shows that differences in the packaging materials influence the protein content at each fermentation time. The packaging material may affect hydrolysis during tempeh fermentation (Sayuti, 2015). According to Munir et al. (2023), sorghum (Sorghum bicolor L. Moench) tempeh packaged in banana leaves had a higher protein content than that packaged in plastic. Meanwhile, Andriati et al. (2018) reported that tempeh packaged in plastic or banana leaves had the same protein content.

As the fermentation time increased, the fat content of tempeh decreased significantly (Table 2). The decrease in lipid content could be because of biochemical and physiological changes that require energy, which causes some lipids in the samples to be used for energy production (Ogodo et al., 2017). In the present study, the carbohydrate levels remained stable (P>0.05), suggesting that R. oligosporus utilizes fat as an energy and carbon source during growth. The reduction in fat content might also result from the metabolism of fatty acids and glycerol by the fermentation organisms, which improves aroma, flavor, and texture (Ojokoh et al., 2014). The findings of the present study corroborated those reported by Sayuti (2015), who observed a decrease in the fat content of pigeon pea tempeh. Table 2 shows that different packaging materials resulted in varying fat content. Tempeh packaged in banana and teak leaves had a higher fat content than tempeh packaged in plastic. This might be attributed to the higher lipase activity in packaged tempeh leaves (Andriati et al., 2018). The leaf packaging is lightproof, allows adequate air circulation through the holes in the leaves, facilitates oxygen access into the packaging, and maintains a humidity level that is favorable for microbial growth during fermentation (Sayuti, 2015). In addition, Indreswari et al. (2022) indicated that an elevated humidity level can inhibit mold growth because it promotes the proliferation of spoilage bacteria.

The present study evaluated tempeh in accordance with the established Indonesian National Standard (SNI). All tempeh treatments contained comparable moisture content (ranging from 62% to 64%), thereby satisfying the SNI criteria, which requires a maximum of 65%. In the present study, tempeh from jack bean sprouts met the protein content required by SNI (i.e., a minimum of 15%). Kadar et al. (2020) indicated that the protein content is a critical indicator in tempeh as it is recognized as a protein source. In the present study, jack bean sprout tempeh had a lower fat content. This was because there was less fat in the initial raw components. The jack bean sprouts used in this study contained 3.6% fat. This low fat value failed to fulfill the minimum fat content of 7.0%.

Crude fiber content of tempeh from jack bean sprouts

Fig. 2 shows that a prolonged fermentation time is correlated with an increased crude fiber content in tempeh (P<0.05). According to Widoyo et al. (2015), Rhizopus sp. grows faster during the 54-h tempeh fermentation by creating mycelia on the surface of the soybean seeds. These mycelia eventually become denser and form a more compact tempeh mass. Mycelia are hyphae that contain the protoplasm and cell walls made of chitin and cellulose. Prasetyo et al. (2022) indicated that cellulose is a constituent component of crude fiber. Thus, the longer the fermentation process, the more hyphae are converted into mycelia, the more cellulose is produced, and the higher the crude fiber content. Fig. 2 also shows that the variations in packaging material influence the crude fiber content of tempeh. During specific fermentation periods, the tempeh packaged in plastic had a greater crude fiber content than those packaged in leaves. This was most likely because we provided a lot of holes, which allowed the tempeh packaged in plastic to have improved air circulation and water evaporation. This condition could lead to an increased mycelial growth accumulation during fermentation, increasing the amount of crude fiber. This finding is consistent with that of Irvan et al. (2021), who confirmed that peanut tempeh packaged in plastic had a higher crude fiber content than that packaged in banana leaves.

Figure 2. Crude fiber content of tempeh from jack bean sprouts fermented at different fermentation times in different packaging materials. Different lowercase letters (a, b) in the same fermentation time but different packaging materials indicate a significant difference (Tukey’s test, P<0.05). Different uppercase letters (A-D) in the same packaging material but different fermentation times indicate a significant difference (Tukey’s test, P<0.05).

Soluble protein content of tempeh from jack bean sprouts

Fig. 3 shows a considerable increase in the soluble protein content during the 72-h fermentation process (P<0.05). The percentage of soluble proteins in tempeh varied from 2.15% (db) to 3.53% (db). Proteins are hydrolyzed and decomposed into peptides or free amino acids and other byproducts during fermentation, which are more soluble than proteins in their complex form. Consequently, a longer fermentation time will yield more soluble proteins (Puspitojati et al., 2019b). The findings of the present study are consistent with those of Pebrianti et al. (2020), who found an increase in soluble protein levels over 96 h of pigeon pea tempeh fermentation. The variation in the packaging materials affected the soluble protein concentration of tempeh (P<0.05). Tempeh packaged in leaves had a higher soluble protein content than that packaged in plastic. Sayuti (2015) indicated that natural leaves have pores and holes that facilitate oxygen entry and maintain humidity in packaging, promoting the proliferation of Rhizopus. Thus, natural leaf packaging will enhance protein hydrolysis and the soluble protein content. Indreswari et al. (2022) added that tempeh packaged with banana and teak leaves has greater permeability than that packaged with plastic. Leaf packaging facilitates the proliferation of mold during tempeh fermentation, which allows tempeh mold to efficiently utilize the organic materials in jack bean sprouts as a source of nutrition.

Figure 3. Soluble protein content of tempeh from jack bean sprouts fermented at different fermentation times in different packaging materials. Different lowercase letters (a, b) in the same fermentation time but different packaging materials indicate a significant difference (Tukey’s test, P<0.05). Different uppercase letters (A-D) in the same packaging material but different fermentation times indicate a significant difference (Tukey’s test, P<0.05).

Antinutritional component of tempeh from jack bean sprouts

The antinutritional components of tempeh derived from jack bean sprouts are presented in Table 3. Statistical analysis revealed substantial alterations (P<0.05) in tannin, phytic acid, and HCN contents in tempeh packaged in varying packaging materials throughout fermentation. The fermentation time significantly influenced the decline of tannin levels in tempeh (P<0.05). In this study, the jack bean sprouts had a tannin content of 1.76 mg/g (db). The tannin content in jack bean sprout tempeh packaged in plastic, banana leaves, and teak leaves significantly decreased by 50%, 44.89%, and 60.23%, respectively, following 72 h of fermentation. This decreasing trend is consistent with the results of previous studies and may be related to the hydrolysis of enzymes, including polyphenol oxidase or other catabolic enzymes (Shang et al., 2019). Furthermore, variations in tempeh packaging did not result in significant changes in the tannin content. The tannin content in tempeh packaged in plastic is similar to those in tempeh packaged in leaves. This may be because of the small impact of packaging materials on alterations in bacterial community composition (Erdiansyah et al., 2022). Thus, the process of enzymatic hydrolysis in each packaging material is essentially the same.

Table 3 . Antinutritional components of tempeh from jack bean sprouts fermented at different fermentation times in different packaging materials.

Fermentation time (h)	Packaging material	Tannin (mg/g db)	Phytic acid (mg/g db)	HCN (ppm)
36	Plastic	1.32±0.20aA	0.24±0.04aA	1.13±0.10aA
	Banana leaf	1.27±0.10aA	0.30±0.03aA	1.06±0.02aA
	Teak leaf	1.27±0.01aA	0.27±0.03aA	1.07±0.01aA
48	Plastic	1.31±0.30aA	0.21±0.03aA	0.97±0.01aB
	Banana leaf	1.24±0.11aA	0.22±0.04aB	0.98±0.02aB
	Teak leaf	1.10±0.30aAB	0.25±0.05aA	0.94±0.01bB
60	Plastic	1.09±0.26aAB	0.22±0.03aA	0.89±0.03bB
	Banana leaf	1.00±0.10aA	0.22±0.03aB	0.96±0.01aB
	Teak leaf	1.07±0.06aAB	0.23±0.02aA	0.95±0.02aB
72	Plastic	0.88±0.10aB	0.21±0.00aA	0.85±0.02aB
	Banana leaf	0.97±0.17aA	0.20±0.02aB	0.84±0.02aC
	Teak leaf	0.70±0.26aB	0.21±0.02aA	0.81±0.03aC

Values are presented as mean±SD..

In the same column, different lowercase letters (a, b) in the same fermentation time but different packaging materials indicate a significant difference (Tukey’s test, P<0.05). In the same column, different uppercase letters (A-C) in the same packaging material but different fermentation times indicate a significant difference (Tukey’s test, P<0.05)..

db, dry basis; HCN, hydrogen cyanide; ppm, part per million..

Table 3 shows the reduction in phytic acid content with prolonged tempeh fermentation time, and the differences were significant (P<0.05). In this study, the jack bean sprouts had a phytic acid concentration of 0.31 mg/g (db). Fermenting jack bean sprouts for 72 h reduced the phytic acid concentration by 32.26%, 35.48%, and 32.26% for tempeh packaged in plastic, banana leaves, and teak leaves, respectively. These findings are consistent with those of Egounlety and Aworh (2003), who found a 30.7% reduction in phytic acid content in soybeans following 48 h of fermentation. Mohamed et al. (2011) reported that treatments, including soaking, dehulling, boiling, autoclaving, microwave cooking, germination, and fermentation with L. plantarum, could decrease phytic acid levels in soybeans by 77%. Moreover, during soaking, boiling, germination, and fermentation processes, the phytic acid content leaches out (Egounlety and Aworh, 2003). The reduction of phytic acid content significantly enhances the availability of minerals, including calcium, zinc, and iron (Gupta et al., 2015). Table 3 shows that variations in packaging materials do not affect the phytic acid content of tempeh. Tempeh packaged in plastic contained an equivalent amount of phytic acid as that packaged in leaves. The three different types of packaging materials provided the same fermentation conditions, making it possible for the phytic acid to be hydrolyzed by phytase into inositol and orthophosphate (Rokhmah et al., 2009).

Table 3 shows a significant decrease in the HCN content (P<0.05) during the tempeh fermentation process. In the present study, jack bean sprouts contained 9.62 ppm of HCN. The soaking and boiling processes during pre-fermentation, followed by 72 h of fermentation, effectively decreased the HCN content in tempeh packaged in plastic, banana leaves, and teak leaves by 91.16%, 91.27%, and 91.58%, respectively. Boiling can effectively remove HCN, as it is volatile and evaporates rapidly at temperatures over 28°C (Modesto Junior et al., 2019). Wahono et al. (2016) found a reduction in the HCN content (specifically from 7.61 ppm to 1.89 ppm) in jack beans fermented over three days with a 1% tempeh inoculum. In the present study, the HCN concentrations (0.81－0.85 ppm) in tempeh derived from jack bean sprouts fermented for 72 h were slightly lower than those reported by Wahono et al. (2016). The typical threshold level of HCN generated by cyanogenic glycogen in plants (tubers, nuts, and seeds) is 50 ppm. The Codex Alimentarius Commission has established a maximum of 10 ppm HCN content for human-consumed products. In the present study, the HCN content in tempeh derived from jack bean sprouts varied from 0.81 ppm to 1.13 ppm, indicating that it is safe for consumption.

Amino acid concentration of tempeh from jack bean sprouts

Tempeh derived from jack bean sprouts fermented for 48 h in three packaging materials exhibited a desirable texture with complete mycelial coverage and the highest levels of crude and soluble proteins. The amino acid concentration in this sample was then measured to determine whether the packaging material affected the amino acid concentration of tempeh. Table 4 shows the concentrations of essential and nonessential amino acids in tempeh. The study found 18 amino acids (nine essential and nine nonessential). The concentration of nonessential amino acids was higher than that of essential amino acids. Tempeh packaged in teak leaves contained the highest levels of essential and nonessential amino acids, followed by those packaged in plastic and banana leaves. This might be attributed to the natural antibacterial chemicals in teak leaves, including anthratectone and naphthotectone (Lankaa and Parimala, 2017). These antibacterial properties of teak leaves foster an environment that encourages the proliferation of beneficial fermentation microorganisms, including lactic acid bacteria and yeast, while suppressing pathogens (e.g., Escherichia coli, Salmonella spp., and Clostridium spp.) that compete with fermentative bacteria. Moreover, this condition establishes a more conducive environment for R. oligosporus to ferment jack bean sprouts into tempeh, thereby facilitating the degradation of proteins into amino acids.

Table 4 . Essential and nonessential amino acid concentrations of tempeh from jack bean sprouts fermented at 48 h in different packaging materials.

Amino acid	Amino acid concentration (g/100 g protein dry weight)
	Plastic	Banana leaf	Teak leaf
Essential amino acid
L-histidine	2.25±0.02c	2.73±0.02b	3.46±0.04a
L-isoleucine	2.90±0.00b	2.56±0.02c	2.99±0.03a
L-leucine	7.39±0.02b	7.09±0.08c	7.96±0.03a
L-lysine	5.31±0.02a	4.14±0.05c	4.31±0.01b
L-methionine	0.70±0.00c	0.74±0.00b	0.80±0.00a
L-phenylalanine	4.03±0.01c	4.48±0.03b	6.50±0.05a
L-threonine	3.36±0.02b	3.18±0.04c	3.74±0.00a
L-tryptophan	1.07±0.00c	1.14±0.00b	1.20±0.00a
L-valine	3.17±0.01b	2.77±0.03c	3.31±0.01a
Total	30.18	28.83	34.27
Nonessential amino acid
L-arginine	4.40±0.02c	4.95±0.07b	6.09±0.02a
L-aspartic acid	11.37±0.05a	9.39±0.06c	11.04±0.06b
L-glutamic acid	11.36±0.07a	9.79±0.14b	11.48±0.06a
L-tyrosine	2.63±0.01c	3.37±0.04b	4.18±0.01a
Glycine	4.17±0.02c	4.61±0.07b	5.49±0.03a
L-alanine	5.30±0.02b	5.01±0.07c	5.54±0.04a
L-serine	6.27±0.03c	6.62±0.08b	7.74±0.05a
L-proline	4.28±0.02b	4.23±0.06b	4.77±0.00a
L-cysteine	3.45±0.00a	1.21±0.00c	2.64±0.00b
Total	53.23	49.18	58.97
Total (essential and nonessential amino acids)	83.41	78.01	93.24

Values are presented as mean±SD..

Different superscript letters (a-c) in the same row indicate a significant difference (Tukey’s test, P<0.05)..

In this study, jack bean sprouts contained 23.89 g of essential amino acids and 24.89 g of nonessential amino acids per 100 g of protein dry weight. During the 48-h fermentation process using plastic, banana leaf, and teak leaf packaging, the essential amino acid concentration increased by 26.33%, 20.68%, and 43.45%, whereas the nonessential amino acid concentration increased by 113.86%, 97.59%, and 136.92%, respectively. These findings verify that tempeh packaged in teak leaves contained higher amounts of soluble proteins than that packaged in plastic or banana leaves, resulting in elevated amino acid levels. According to Widiany et al. (2023), the concentrations of essential and nonessential amino acids in tempeh made from local Indonesian soybeans were 19.32% and 28.55%, respectively, which were less than those in our study. Pilco et al. (2019) reported that each amino acid in tempeh derived from beans (Phaseolus vulgaris L.) and quinoa (Chenopodium quinoa) fermented with R. oligosporus for 48 h at 35°C exhibited a concentration of approximately 1% to 4%.

Fig. 4 shows the concentrations of hydrophobic and hydrophilic amino acids in tempeh packaged in different packaging materials. All tempeh samples had high amounts of hydrophilic amino acids, including L-aspartic acid and L-glutamic acid. These negatively charged hydrophilic amino acids can function as angiotensin I-converting enzyme (ACE) inhibitors because of their ability to establish electrostatic interactions with Zn²⁺ ions at the active site of ACE (Fitriani et al., 2022). Agustia et al. (2024) observed that positively charged hydrophilic amino acids, including lysine and arginine, can form salt bridges and electrostatic interactions with Glu206, Glu205, and Tyr662 residues at the binding site of dipeptidyl peptidase-IV (DPP-IV), thereby functioning as DPP-IV inhibitors. Furthermore, the present study found elevated levels of hydrophobic amino acids, including L-leucine, L-alanine, L-proline, L-phenylalanine, L-valine, and L-isoleucine. The elevated concentrations of these hydrophobic amino acids enable jack bean sprout tempeh to function as a bioactive peptide.

Figure 4. Hydrophobic and hydrophilic amino acid concentrations of tempeh from jack bean sprouts fermented at 48 h in different packaging materials. Different letters (a-c) in the same amino acid indicate a significant difference (Tukey’s test, P<0.05). Ile, L-isoleucine; Leu, L-leucine; Val, L-valine; Phe, L-phenylalanine; Met, L-methionine; Pro, L-proline; Ala, L-alanine; Trp, L-tyrosine; Arg, L-arginine; His, L-histidine; Asp, L-aspartic acid; Glu, L-glutamic acid; Lys, L-lysine; Thr, L-threonine; Ser, L-serine; Cys, L-cysteine; Gly, glycine; Try, L-tryptophan.

Hydrophobic amino acids can be transformed into bioactive peptides that inhibit ACE, DPP-IV, α-amylase, and α-glucosidase (González-Montoya et al., 2018; Puspitojati et al., 2019b; Agustia et al., 2024). Proline can form hydrophobic interactions with hydrophobic residues at the active site of ACE, making it may an ACE inhibitor (Fan et al., 2019). L-methionine and L-tryptophan were present in minimal quantities among tempeh samples. Syida et al. (2018) found that methionine is one of the limiting amino acids in soybean tempeh. Meanwhile, Widiany et al. (2023) observed that tempeh made from Indonesian local soybean contains 0.03% methionine, but no tryptophan.

The nutritional content (e.g., protein, soluble protein, and crude fiber) and the functional characteristics (e.g., amino acid concentration) of jack beans can be enhanced through germination and fermentation in various packaging materials, as demonstrated by previous studies. Tempeh derived from jack bean sprouts fermented for 48 h in teak leaf packaging exhibited the highest nutritional content. This finding is likely because of the presence of natural antibacterial chemicals, including anthratectone and naphthotectone, in teak leaves that prevent the proliferation of undesirable bacteria and pathogens during fermentation, creating a more favorable fermentation environment. Consequently, the degradation of proteins into amino acids is accelerated. Further studies are needed to investigate the microbial properties and the use of incubators for the mass production of tempeh derived from jack bean sprouts packaged in teak leaves.

The authors are grateful to Rafida Salma and Salma Azra Hamidah for their assistance in providing the tempeh samples.

This project was financed by Directorate of Research Technology and Community Service (DRTPM), from the Directorate General of Higher Education, Research, and Technology, Ministry of Education, Culture, Research, and Technology, Republic of Indonesia, Grant Number: 20.21/UN23.35.5/PT.01.00/IV/2024 with a Fundamental Reguler (FR) Research Scheme.

The authors declare no conflict of interest.

Concept and design: FCA, AF. Analysis and interpretation: FCA, HW. Data collection: FCA, HW, AF, NL. Writing the article: FCA. Critical revision of the article: HW, AF, NL. Final approval of the article: all authors. Statistical analysis: AF, NL. Obtained funding: FCA, HW, AF, NL. Overall responsibility: FCA.