International Journal of Transdisciplinary Knowlegde

ISSN : 2797-4251 (Online)

Journal Homepage : https://ijtk.iainkendari.ac.id/IJTK

Spectroscopic Analysis of Tempeh Protein Content during the

Production Process
Zul Arham1, Kurniawan2, Laode Anhusadar3, Ismaun4
Department of Mathematics and Natural Sciences, Faculty of Tarbiyah, Institut Agama Islam Negeri (1,4)
Department of Textile Chemistry, Politeknik STTT Bandung, 40272, West Java, Indonesia (2)
Department of Early Childhood Education Faculty of Tarbiyah, Institut Agama Islam Negeri (IAIN) Kendari (3)

DOI: 10.31332/ijtk.v2i1.17

 Corresponding author:
[arhamzul88@gmail.com]

Article Info Abstrak

Keywords: Tempeh is a local Indonesian food that is liked by the community, both locally
Indonesian local food; and internationally. The high protein content is the reason for choosing tempeh
Tempeh; as a complementary food. In its production, tempeh is made by two important
processes, including treatment and fermentation. During this process, the
Pretreatment; protein can be damaged, resulting in a decrease in the protein content of
Fermentation; tempeh. Based on these problems, this study aims to analyze the reduction of
Protein Content; tempeh protein content during the production process. The analysis used two
Spectroscopic Method; spectroscopic methods, including FTIR and UV-Visible. FTIR spectroscopy was
used to observe the reduction in total protein content, while UV-Vis
spectroscopy was used to observe the reduction in dissolved protein content.
The results of the study for FTIR analysis showed that at the pretreatment and
fermentation stages, both caused a decrease in the total protein content of
tempeh. This was observed with a decrease in the intensity of IR absorption at a
wavenumber of 1745 cm -1 which is identical to the C=O group and 1543 cm -1
which is identical to the N-H group. Based on the results of the UV-Visible study,
it shows that the fermentation time affects the amount of dissolved protein. This
situation is supported by the increasing pH of tempeh and decreasing water
content during fermentation.

1. INTRODUCTION
Spectroscopy is a modern analytical method based on the interaction of matter and
energy. There are several types of spectroscopic methods, including nuclear magnetic
resonance (NMR) (Capitani et al., 2017), infrared (IR) (McMullin et al., 2015), near infrared
(NIR) (Landau et al., 2018), middle infrared (MIR) (Song et al., 2016), Raman, fluorescence
(Acković et al., 2018), and UV-Vis spectroscopy (Van de Voort, 1992). In recent years, this
method has been widely reported for its use in the analysis of food products, both
qualitatively and quantitatively. Several advantages, such as easy sample preparation, fast
analysis, sensitivity with high accuracy, are the reasons why this method is widely used
(Baeten & Dardenne, 2002).
Among the types of spectroscopic methods above, FTIR and UV-Vis are two
spectroscopic methods commonly used in the analysis of a food sample. Generally, FTIR is
used in qualitative analysis by studying the chemical properties of the constituents of a
sample, such as functional groups (Durazzo et al., 2018; Lucarini et al., 2020). The analysis

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was carried out by observing the absorption band at a specific wave number. Where, each
sample will produce a different specific absorption band, so on this basis an analysis of the
chemical properties of the sample can be carried out. As for UV-Vis, it is used in quantitative
analysis. This analysis is intended to determine the level or concentration of a chemical
compound (da Silva-Buzanello et al., 2015; Zhang et al., 2015). In the analysis, a chemical
compound will produce a different maximum wavelength. This wavelength is then used in
determining the level or concentration of a chemical compound. Examples of food analysis of
these two methods include carbohydrate analysis of juice samples (Domingues et al., 2014;
Leopold et al., 2011), glucose analysis (Yu et al., 2012), lipid analysis of meat products
(Candoğan et al., 2021), and others.
The content of chemical compounds in a food product which is also important to
analyze is protein. Protein is a group of biochemical compounds that have many roles in the
body, for example as a source of energy, providing enzymes and hormones, and supporting
the immune system (Wycherley et al., 2012). Based on this, protein becomes one of the
important parameters in explaining the quality of a food product.
One food product that contains a lot of protein is tempeh. Tempeh is a processed
soybean product that is made through two important stages, including the pretreatment and
fermentation processes (Suwanto et al., 2013). Tempeh is not only consumed by local
community, but tempeh is widely consumed by the global community. Tempeh has an
economical selling price with a distinctive taste, so it is used as a favorite complementary
food. In the health sector, tempeh is reported to have many benefits, including as an
antioxidant, inhibitor and cholesterol-lowering, preventing the risk of cancer, prostate, and
many other benefits (Tahir et al., 2018). In the food industry, tempeh is used as a food
additive in the manufacture of fermented soy sauce (Rosmini et al., n.d.).
The minimum protein content in tempeh is 16% (w/w) (Lestari & Mayasari, 2016). This
level can be reduced during the production process. As previously explained, the tempeh
production process consists of two important stages, namely pretreatment and fermentation.
The pretreatment process consists of five stages, including boiling, grinding, washing,
soaking, and steaming (Ahnan‐Winarno et al., 2021; Damanik et al., 2018; Ferreira et al.,
2011). The presence of protein content in this process is strongly influenced by temperature.
In general, this process uses very high temperatures, so it will have an impact on damage to
the protein structure, and continue to decrease the protein content of tempeh (Schön et al.,
2017). Likewise with the fermentation stage. This step is reported to cause the breakdown of
complex proteins into simple proteins. The use of time is an important parameter that needs
to be considered during this process. The long fermentation time will gradually cause
damage to the protein structure (Babu et al., 2009; Chang et al., 2009; Reyes-Moreno et al.,
2000). This process will produce protease enzymes that play a role in breaking down the
structure of protein complexes into amino acid monomers or simple proteins (Handoyo &
Morita, 2006).
Although research on tempeh has been widely reported, the analysis of protein
content during the production process has not been reported. So in this work, we specifically
report the analysis of tempeh protein content during the production process using two
spectroscopic methods, including FTIR and UV-Visible.

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2. METHODS
1. Materials
Tempeh as a test sample, soybean as a comparison sample, phosphate buffer pH 8
(Sigma-Aldrich, CAS number : 9011-18-1) as a solvent, biuret reagent (Sigma-Aldrich, CAS
number : B3934) as a reagent, aquades as a solvent, and qualitative filter paper (Whatman,
Cat No. 1004.110) as a filter.
2. Methods
Tempeh sampling
Tempeh samples were obtained from the Baruga traditional market, Southeast
Sulawesi-Indonesia (Fig. 1). Sampling was done randomly, with the selected sample criteria
were new tempeh that had not undergone the fermentation process. The tempeh samples
obtained were then stored in a sterile room at room temperature. This storage is part of the
fermentation process.

Figure 1. Map of Bagura traditional market.

Analysis of protein content

The protein content of tempeh was analyzed by two spectroscopic methods, including
FTIR (IRprestige-21 fourier transform infrared spectrophotometer Shimadzu) and UV-Visible
(Agilent 8453). FTIR analysis begins with drying the sample in an oven at 80 oC for 24 hours.
Tempeh was mashed and filtered using a sieve with a pore size of ± 0.3 mm. Furthermore, it
is formed into thin pellets by adding KBr solids and measuring the absorbance of the wave
number. The UV-Visible analysis was carried out by weighing 1.0 g of fermented tempeh
samples (1 to 5 days), dissolved in 50 mL of phosphate buffer pH 8 and stirred with a
magnetic stirrer. Next, the mixture was centrifuged and filtered using filter paper. The filtrate
obtained was transferred to a 50 mL volumetric flask, then phosphate buffer pH 8 was added
to the mark. 1.0 mL of filtrate was taken, then 3.0 mL of Biuret reagent was added, vortexed
and incubated at room temperature for 30 minutes. Then the absorbance was measured at a
maximum wavelength (λmax) of 540 nm using a UV-Vis spectrophotometer.
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3. RESULT AND DISCUSSION

FTIR analysis of tempeh total protein
The decrease in the amount of total protein, both during the pretreatment and
fermentation processes, was observed based on the specific IR absorption of the protein
functional groups. Based on their chemical structure, proteins are composed of amino acid
monomers linked through amide bonds (Valdar & Thornton, 2001). This bond consists of
C=O and N-H groups which will absorb IR at wave numbers 1745 cm-1 (specific group for
C=O) and 1543 cm-1 (specific group for N-H). The structure of the protein amide bond is
shown in Figure 2.

Figure 2. Protein amide bond structure.

The amide bond has a tendency to resonate (see Fig. 2), this situation can describe the
stable nature of the peptide bond at high temperature. The resonance process produces a
planar structure that prevents the free rotation process around the C=O-N bond. The amide
bond will initiate the formation of a more complex protein structure. Through this bond,
protein monomers will bind to each other and form a distinctive structure (Fig. 3). Protein
monomers are amino acids, which can be grouped into 20 types of amino acids. Everything
is used by the body in the metabolic process.

Figure 3. The process of protein formation.

Figure 4 generally shows the IR spectra of samples before tempeh production (Fig.
4a), samples during pretreatment (Fig. 4b) and samples after fermentation (Fig. 4c).

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Figure 4. IR spectra of the sample: a) before tempeh production, during pretreatment, and c)
after fermentation.
The sample in Figure 4 is observed based on the difference in the intensity of IR
absorption at wavenumbers 1745cm-1 and 1543 cm-1. The high absorption intensity
correlated with the total protein amount contained in each sample. Based on Table 1, it can
be seen that there was a decrease in the absorption intensity of the protein during the
pretreatment process, both boiling and steaming (IR spectra can be seen in Fig. 4b).

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Table 1. Intensity of IR absorption during the tempeh production process.

Transmitance (%)
Wave number Before
Pretreatment
(cm-1) pretreatment Fermentation
process
and Process
fermentation
Boiling Steaming Day 3 Day 4

1543 26.69 ± 0.1 25.45 ± 14.80 ± 13.85 ± 13.14 ±

0.1 0.1 0.1 0.1

1745 25.51 ± 0.1 22.29 ± 14.21 ± 14.11 ± 13.25 ±

0.1 0.1 0.1 0.1

The same thing was also shown in the samples after fermentation, both on the third
and fourth days (IR spectra can be seen in Figure 4c). In the boiling stage there was a
decrease in % Transmittance (% T) in the two typical wave numbers of proteins by 4.64 %
(for 1543 cm-1) and 12.62 % (for 1745 cm-1). Similar to the boiling stage, in the steaming
stage there was also a decrease in % T of 44.54 % (for 1543 cm-1) and 44.29 % (for 1745
cm-1). Meanwhile, during the fermentation process, it was seen that the fermentation time
affected the total protein content of tempeh. The decrease in % T in fermentation for 3 days
was 48.10 % (for 1543 cm-1) and 44.29 % (for 1745 cm-1), while in fermentation for 4 days it
was 50.76 % (for 1543 cm-1) and 48.05 % (for 1745 cm-1). The percentage reduction in % T
during the pretreatment and fermentation processes is shown in Figures 5 and 6.

Figure 5. Percentage of % T reduction in the pretreatment process : a) boiling stage, b)

steaming stage.
In general, Figure 5 shows that the decrease in % T, both during the boiling and steaming
stages, correlates with a reduction in the protein content of tempeh. In addition, based on the

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figure, the rate of protein reduction in both pretreatment processes was relatively the same.
However, the initial protein reduction occurs more rapidly at the steaming stage.

Figure 6. Percentage of % T reduction in the fermentation process.

As in Figure 5, the decrease in % T during the fermentation stage (Fig. 6) correlated with a
reduction in the protein content of tempeh. The rate of protein reduction during this process is
relatively the same. However, when compared to the pretreatment stage, the rate of protein
reduction at this stage is relatively slower and occurs constantly.

UV-Vis analysis on tempeh dissolved protein content

In evaluating the dissolved protein content using UV-Vis spectroscopy, we observed
the effect of pretreatment temperature and fermentation time. The use of Benedict's Reagent
will cause the formation of a complex between the peptide bond and Cu 2+ ions, as shown in
Figure 7a. The formation of the complex is based on the presence of an electron pair in the
peptide bond which is not shared with nitrogen and water oxygen. The formation of this
complex produces a purple color with a maximum wavelength of 540 nm (Fig. 7b).

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Figure 7. (a) Complex structure of the peptide bond with Cu2+; (b) Maximum wavelength
(λmax) of the peptide complex and Benedict's Reagent.

Figure 8 shows the effect of pretreatment temperature and fermentation time on

dissolved protein. Based on Figure 8a, it can be seen that the high pretreatment temperature
(boiling and steaming) causes the peptide bonds to break, thereby increasing the amount of
dissolved protein. These results are correlated with the results of the FTIR analysis as shown
in Figure 4b. Based on this data, tempeh pretreatment needs to pay attention to temperature,
so as to produce tempeh that meets BSN standards. The results of observations for soluble
protein content of tempeh which fermented for 1 to 5 days are shown in Figure 8b. Based on
the figure, it can be seen that there is a correlation between fermentation time and the
amount of dissolved protein. The amount of dissolved protein will decrease along with the
length of fermentation time. This decrease can be attributed to the activity of the protease
enzyme produced by Rhizopus sp. in breaking the peptide bonds of tempeh protein into
amino acids and short chain peptides.

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Figure 8. Correlation of dissolved protein to: a) temperature; b) fermentation time.

The formation of short chain amino acids and peptides was indicated by an increase in
pH during the fermentation process (Table 2). Gradually, the fermentation time caused the
tempeh pH to be higher until it approached neutral pH (pH = 7.0). This property is correlated
with the amount of protein that decomposes and the decreased activity of the protease
enzyme from Rhizopus sp.. The activity of the protease enzyme is strongly influenced by pH,
where at an increasingly alkaline pH, the activity of the protease enzyme will decrease. An
increase in pH is also associated with the formation of free amino acids (Churchill et al.,
2004). Table 2 also shows that another phenomenon regarding the decrease in the amount
of dissolved protein can be associated with a decrease in the amount of water content in
tempeh. Other enzymes from Rhizopus sp. can damage hydrogen bonds, both those formed
between the amide group and water molecules or the amide group with other amide groups.
Another factor that causes a decrease in water content is the activity of microorganisms that
damage the starch component thereby reducing the OH bond in its structure (Kurniadi et al.,
2019).
Tabel 2. Correlation of time to pH.

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Time
Water content
(hours) pH
(%)

1 4.67 (±0.2) 6.40 (±0.1)

2 5.07 (±0.2) 4.17 (±0.1)

3 5.40 (±0.2) 3.09 (±0.1)

4 5.50 (±0.2) 2.74 (±0.1)

5 5.65 (±0.2) 2.04 (±0.1)

4. CONCLUSION
This study succeeded in analyzing the reduction of tempeh protein content during the
production process. This analysis is important to do in describing the quality of tempeh. The
use of two spectroscopic methods, including FTIR and UV-Vis, was able to effectively
analyze tempeh proteins, both total and dissolved proteins. Specific IR absorption of tempeh
protein was observed at two wave numbers (1/λ) namely 1543 cm-1 and 1745 cm-1. These
absorptions are derived from the N-H and C=O groups, respectively, which are connected via
amide bonds. Based on the results of the analysis, both FTIR and UV-Vis explained that the
tempeh production process was influenced by two conditions, namely pretreatment and
fermentation. Both have an effect on the availability of protein, both total protein and soluble
protein. The results of this study can then be used as a reference for protein analysis in
various other food samples based on FTIR and UV-Vis spectroscopy.

5. REFERENCES
Acković, L. L., Zeković, I., Dramićanin, T., Bro, R., & Dramićanin, M. D. (2018). Modeling food
fluorescence with PARAFAC. In Reviews in Fluorescence 2017 (pp. 161–197). Springer.
Ahnan‐ Winarno, A. D., Cordeiro, L., Winarno, F. G., Gibbons, J., & Xiao, H. (2021). Tempeh:
A semicentennial review on its health benefits, fermentation, safety, processing,
sustainability, and affordability. Comprehensive Reviews in Food Science and Food
Safety, 20(2), 1717–1767.
Babu, P. D., Bhakyaraj, R., & Vidhyalakshmi, R. (2009). A low cost nutritious food “tempeh”-a
review. World J Dairy Food Sci, 4(1), 22–27.
Baeten, V., & Dardenne, P. (2002). Spectroscopy: Developments in instrumentation and
analysis. Grasas y Aceites, 53(1), 45–63.
Candoğan, K., Altuntas, E. G., & İğci, N. (2021). Authentication and quality assessment of
meat products by fourier-transform infrared (FTIR) spectroscopy. Food Engineering
Reviews, 13(1), 66–91.
Capitani, D., Sobolev, A. P., Di Tullio, V., Mannina, L., & Proietti, N. (2017). Portable NMR in
food analysis. Chemical and Biological Technologies in Agriculture, 4(1), 1–14.
Chang, C., Hsu, C., Chou, S., Chen, Y., Huang, F., & Chung, Y. (2009). Effect of
fermentation time on the antioxidant activities of tempeh prepared from fermented
soybean using Rhizopus oligosporus. International Journal of Food Science &
Technology, 44(4), 799–806.
Churchill, H., Teng, H., & Hazen, R. M. (2004). Correlation of pH-dependent surface

60
 International Journal of Transdisciplinary Knowlegde

interaction forces to amino acid adsorption: Implications for the origin of life. American
Mineralogist, 89(7), 1048–1055.
da Silva-Buzanello, R. A., Ferro, A. C., Bona, E., Cardozo-Filho, L., de Araújo, P. H. H.,
Leimann, F. V., & Gonçalves, O. H. (2015). Validation of an Ultraviolet–visible (UV–Vis)
technique for the quantitative determination of curcumin in poly (l-lactic acid)
nanoparticles. Food Chemistry, 172, 99–104.
Damanik, R. N. S., Pratiwi, D. Y. W., Widyastuti, N., Rustanti, N., Anjani, G., & Afifah, D. N.
(2018). Nutritional composition changes during tempeh gembus processing. IOP
Conference Series: Earth and Environmental Science, 116(1), 12026.
Domingues, D. S., Pauli, E. D., de Abreu, J. E. M., Massura, F. W., Cristiano, V., Santos, M.
J., & Nixdorf, S. L. (2014). Detection of roasted and ground coffee adulteration by HPLC
by amperometric and by post-column derivatization UV–Vis detection. Food Chemistry,
146, 353–362.
Durazzo, A., Kiefer, J., Lucarini, M., Camilli, E., Marconi, S., Gabrielli, P., Aguzzi, A.,
Gambelli, L., Lisciani, S., & Marletta, L. (2018). Qualitative analysis of traditional italian
dishes: FTIR approach. Sustainability, 10(11), 4112.
Ferreira, M. P., Oliveira, M. C. N. de, Mandarino, J. M. G., Silva, J. B. da, Ida, E. I., & Carrão-
Panizzi, M. C. (2011). Changes in the isoflavone profile and in the chemical composition
of tempeh during processing and refrigeration. Pesquisa Agropecuária Brasileira, 46(11),
1555–1561.
Handoyo, T., & Morita, N. (2006). Structural and functional properties of fermented soybean
(tempeh) by using Rhizopus oligosporus. International Journal of Food Properties, 9(2),
347–355.
Kurniadi, M., Arsyad, M. F., Sar, A. M., Nurhayati, R., & Wiyono, T. (2019). The effect of
fermentation duration and starter concentration on physicochemical properties of
modified sorghum flour by Lactobacillus plantarum FNCC 0027. Food Res, 3, 441–447.
Landau, S. Y., Dvash, L., Yehuda, Y., Muklada, H., Peleg, G., Henkin, Z., Voet, H., & Ungar,
E. D. (2018). Impact of animal density on cattle nutrition in dry Mediterranean
rangelands: a faecal near-IR spectroscopy-aided study. Animal, 12(2), 265–274.
Leopold, L. F., Leopold, N., Diehl, H.-A., & Socaciu, C. (2011). Quantification of
carbohydrates in fruit juices using FTIR spectroscopy and multivariate analysis.
Spectroscopy, 26(2), 93–104.
Lestari, O. A., & Mayasari, E. (2016). Potensi gizi tempe berbahan dasar jagung. Jurnal
Ilmiah Teknosains, 2(2/Nov).
Lucarini, M., Durazzo, A., Kiefer, J., Santini, A., Lombardi-Boccia, G., Souto, E. B., Romani,
A., Lampe, A., Ferrari Nicoli, S., & Gabrielli, P. (2020). Grape seeds: Chromatographic
profile of fatty acids and phenolic compounds and qualitative analysis by FTIR-ATR
spectroscopy. Foods, 9(1), 10.
McMullin, D., Mizaikoff, B., & Krska, R. (2015). Advancements in IR spectroscopic
approaches for the determination of fungal derived contaminations in food crops.
Analytical and Bioanalytical Chemistry, 407(3), 653–660.
Reyes-Moreno, C., Romero-Urias, C., Milán-Carrillo, J., Valdez-Torres, B., & Zárate-
Márquez, E. (2000). Optimization of the solid state fermentation process to obtain
tempeh from hardened chickpeas (Cicer arietinum L.). Plant Foods for Human Nutrition,
55(3), 219–228.
Rosmini, R., Arham, Z., & La Ode Anhusadar, N. (n.d.). Quality of Coconut Water Soy Sauce
in the Presence of Powder Tempeh as an Additive.
Schön, A., Clarkson, B. R., Jaime, M., & Freire, E. (2017). Temperature stability of proteins:
Analysis of irreversible denaturation using isothermal calorimetry. Proteins: Structure,
Function, and Bioinformatics, 85(11), 2009–2016.
Song, S. Y., Lee, Y. K., & Kim, I.-J. (2016). Sugar and acid content of Citrus prediction
modeling using FT-IR fingerprinting in combination with multivariate statistical analysis.

61
International Journal of Transdisciplinary Knowlegde

Food Chemistry, 190, 1027–1032.

Suwanto, A., Rahayu, G., & Nuraida, L. (2013). Population dynamics of yeasts and lactic acid
bacteria (LAB) during tempeh production. Hayati Journal of Biosciences, 20(2), 57–64.
Tahir, A., Anwar, M., Mubeen, H., & Raza, S. (2018). Evaluation of Physicochemical and
Nutritional Contents in Soybean Fermented Food Tempeh by Rhizopus oligosporus.
Journal of Advances in Biology & Biotechnology, 1–9.
Valdar, W. S. J., & Thornton, J. M. (2001). Protein–protein interfaces: analysis of amino acid
conservation in homodimers. Proteins: Structure, Function, and Bioinformatics, 42(1),
108–124.
Van de Voort, F. R. (1992). Fourier transform infrared spectroscopy applied to food analysis.
Food Research International, 25(5), 397–403.
Wycherley, T. P., Moran, L. J., Clifton, P. M., Noakes, M., & Brinkworth, G. D. (2012). Effects
of energy-restricted high-protein, low-fat compared with standard-protein, low-fat diets: a
meta-analysis of randomized controlled trials. The American Journal of Clinical Nutrition,
96(6), 1281–1298.
Yu, X., Zhao, M., Hu, J., Zeng, S., & Bai, X. (2012). Correspondence analysis of antioxidant
activity and UV–Vis absorbance of Maillard reaction products as related to reactants.
LWT-Food Science and Technology, 46(1), 1–9.
Zhang, W., Li, N., Feng, Y., Su, S., Li, T., & Liang, B. (2015). A unique quantitative method of
acid value of edible oils and studying the impact of heating on edible oils by UV–Vis
spectrometry. Food Chemistry, 185, 326–332.

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