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Article

Sustainable Use of By-Products (Okara and Linseed Cake) in Tempeh Fermentation: Effects on Isoflavones and Lignans

by
Maciej Kuligowski
1,
Elżbieta Radziejewska-Kubzdela
1,*,
Oliwia Górna
1,
Iwona Jasińska-Kuligowska
2 and
Marcin Kidoń
1
1
Department of Food Technology of Plant Origin, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Wojska Polskiego 31, 60-624 Poznań, Poland
2
Department of Food Quality and Safety, Poznań University of Economics and Business, Al. Niepodległości 10, 61-875 Poznań, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(22), 9936; https://doi.org/10.3390/su16229936
Submission received: 2 October 2024 / Revised: 25 October 2024 / Accepted: 12 November 2024 / Published: 14 November 2024
(This article belongs to the Special Issue Emerging and Modern Approaches for Sustainable Food Production and Waste Management)
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Abstract

:
Okara and linseed cake are nutrient-rich by-products containing phytoestrogens, which are underutilized in food production. The aim of this study was to optimize hydrothermal treatment and fermentation time using common starters for tempeh fermentation. Changes in isoflavones and secoisolariciresinol diglucoside (SDG) were analyzed using the HPLC-PDA method. Okara and linseed cake were steamed for 5 to 40 min, followed by fermentation with Rhizopus oligosporus at 30 °C for 48 h. The optimal steaming times were 10 min for okara and 20 min for linseed cake. Isoflavone analysis revealed a change in total levels depending on the microflora, with a significant increase in aglycones during fermentation. SDG levels did not show a significant reduction. In 24-h fermented tempeh, the isoflavone acetylglycosides content increased after frying. These results suggest that steam treatment is an effective hydrothermal method, offering energy and water savings. Additionally, the ability to modulate isoflavone composition through fermentation creates new opportunities for pro-healthy food development. This study concludes that okara and linseed cake can be successfully used for tempeh production, providing a sustainable alternative to conventional tempeh substrates. By utilizing these by-products, this research promotes the reduction of food waste and supports the development of environmentally friendly food production practices.

1. Introduction

Okara, also known as biji (Korean), douzha (Chinese), tofukasu (Japanese), and ampas tahu (Indonesian), is a by-product of food processing derived from soybeans (Glycine max) [1,2]. It is a solid by-product of water-soluble soybean extraction, used for soy milk and tofu (soy curd) production. Most okara end up as animal feed or even waste [3,4], and only a small fraction (5%) is currently repurposed for human consumption [5]. The quantities of okara reported in the literature appear to be underestimated or outdated; for example, the amount of okara generated by the soybean curd manufacturing sector is reported as 2.8 million tons in China, 0.8 million tons in Japan, and 0.3 million tons in Korea [1], while Taiwan alone produced 0.5 million tons of okara [3]. Processing 1 kg of soybeans to produce tofu results in 1.2 kg of fresh okara [6]. These calculations do not take into account local production of soy beverages, which is an intermediate step in tofu production and is consumed in large quantities. Other evaluations place okara production in Asian countries at 4 million tons per year [7]. Okara has a high moisture content (approximately 80% by weight) and contains 50–63% total fibers, 25–43% proteins, and 10–16% lipids on a dry basis [8,9].
In a report presented by the United Nations, it was shown that the utilization of protein derived from plant or fungal sources is more sustainable for the planet [10]. In modern western societies, the trend to avoid meat consumption for health or ethical reasons is increasing [11]. As an alternative, soybean seeds are one of the richest sources of protein in the plant kingdom. Additionally, they are the main source of isoflavones in the human diet. These compounds, due to their similar structure to estrogen hormones, can bind to estrogen receptors and affect the human body.
In patients with a history of stroke, higher isoflavone intake has been linked to longer survival and a reduced risk of stroke recurrence [12]. Moreover, consumption of soy isoflavones has been linked to a lower incidence of prostate cancer in Asian populations [13]. Additionally, in women from Japan and Australia, menopausal hot flashes were significantly inversely associated with the consumption of soy products and isoflavone intake [14,15]. However, other authors found [16] that this effect was observed only in women with mild to moderate vasomotor symptoms in the early natural postmenopausal period.
Soy seeds contain twelve forms of isoflavones, including aglycones, glucosides, malonylglucosides, and acetylglucosides of the three compounds: genistein, daidzein, and glycitein [17,18]. The bioavailability of these isoflavones is largely determined by their chemical structure, which influences their absorption in the human body [19], affecting their physiological and health-related impacts.
Isoflavone aglycone treatments have been found to exert a moderate activity against estrogen-deficient bone loss in women, whereas studies with formulations containing isoflavone glycosides did not observe a protective effect against bone loss [20]. Differential effects were also observed between isoflavone aglycones, with genistein and daidzein showing differential activity in modulating the role of calcium in blood pressure regulation [21].
Some researchers suggested that isoflavones should be considered as the most prevalent xenoestrogens in the human diet, and they are also perceived as potential endocrine disruptors [22,23]. Also, some evidence suggests that the consumption of isoflavones may be associated with an increased risk of ulcerative colitis, particularly in females [24]. Genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of ulcerative colitis. Interestingly, these effects were not observed with daidzein [25].
The activity of isoflavones in humans is determined not only by their chemical structure and bonded form but also seems to depend on gender and age [16,26]. Moreover, it was found that isoflavones contribute to the reduction of prostate-specific antigens in the serum of Caucasian men, although these reductions were not observed in the serum of African American men [27], indicating that ethnic background also plays a role. Therefore, monitoring the changes in isoflavones during processing and culinary processing seems important to know the actual amount and form delivered with the meal. Providing food that meets individual needs depending on age, gender, ethnicity, and current health status, while enhancing well-being and optimal functioning, appears to align with the United Nations Sustainable Development Goal 3: good health and well-being [28].
Implementing sustainable development principles is a necessity, demanding a comprehensive approach to the relationship between humans and the environment. The intensification of livestock production and food waste significantly contributes to the ongoing degradation of ecosystems [10]. Ensuring sustainable consumption and production patterns is one of the European Sustainable Development Goals, aimed at improving resource efficiency and promoting a circular economy, decoupling economic growth from resource use and environmental degradation [29]. Simultaneously, the rapidly growing global population demands adequate food supplies. According to FAO forecasts, to meet the nutritional needs of the global population, food production will need to double by 2030 [30,31]. A significant challenge for increasing food production is the continuous decline in available agricultural land [32]. The use of non-meat protein sources, whose production can mitigate the negative environmental impact of industrial animal husbandry, is the subject of intensive research [11,32,33]. The utilization of okara, a nutrient-rich by-product, for food production appears to align with this trend. It may also help curb the growing competition for territorial resources, where the most important factors [32] are dietary choices, reducing post-harvest losses, and minimizing food waste, as well as contributing to the elimination of poverty and hunger while promoting good health and well-being.
Another by-product is linseed cake, which is produced during the extraction of oil from flax seeds. Linseed is primarily cultivated in Canada, Russia, China, India, and Kazakhstan, with the main producers in Europe being the United Kingdom and France [34]. In the European Union, the annual demand for linseed is approximately 0.6 million tons, of which the majority (approximately 75%) is used as ground linseed; 20% is used as animal feed, and approximately 5% is added to food products [35]. Global linseed production is estimated at 3.97 million tons in 2022, while linseed cake production is estimated at 1.25 million tons [34]. The cake produced as a by-product of cold-press oil extraction is mainly used in animal feed; however, it can serve as a valuable source of bioactive compounds in the human diet. Linseed cake, being a by-product, can be used directly without prior processing or can serve as a raw material for other products. It contains approximately 30–35% protein and 7–20% lipids (depending on the extraction method) [35]. Similar to okara, it also contains phytoestrogen compounds, namely lignans. Linseed is the richest known source of lignans, with concentrations ranging from 6.1 to 28.8 mg/g. The dominant lignan in linseed is secoisolariciresinol diglucoside (SDG) [36]. Lignans affect humans similarly to isoflavones, although their spectrum of effects is less known. We found no evidence of the adverse effects of lignans on humans. Despite some controversy surrounding the safety of foods containing phytoestrogens [22,23,25], a study by Katagiri et al. [37] found that a higher intake of fermented soy was associated with a lower risk of overall mortality, while this effect was not observed for total soy products. The fermentation process improves the bioavailability of soy isoflavones [26,38] through microbial enzymatic activity, which converts them to their aglycone forms [18]. An example of a fermented soy food is tempeh, which is an Indigenous product from Indonesia, fermented by Rhizopus spp. (mold), which may have originally been made from okara but is now mainly produced from soybeans. Bacteria are also often involved in fermentation. In a typical process, the soybeans are soaked in water, dehulled, and then cooked in boiling water. The fermentation time depends on the temperature and type of used starter. Mold growth develops into the entire mass, covered and bound by mycelium. This process changes the loose seeds into a stable, sliceable mass [33,39,40].
The trend to use plant-based meat alternatives, such as tempeh, is increasing, particularly in Europe and North America [11]. These products are composed of soybeans and contain isoflavones, so a deep understanding of their changes during technological and culinary processing is very important for consumer health.
The aim of this study was to examine the possibility of optimizing hydrothermal treatment and fermentation time during tempeh making from okara and linseed cake, as well as monitoring changes in phytoestrogens (secoisolariciresinol diglucoside (SDG) and isoflavones) during fermentation and culinary treatment.

2. Materials and Methods

2.1. Materials

Soy seeds (Glycine max (L.)) of the Augusta variety were obtained from Agricultural Company Dłoń, Poland. Linseed cake was supplied by PPHU Vitacorn, Poznań, Poland. Standards for daidzein, daidzin, genistein, genistin, glycitin, glycitein, and secoisolariciresinol diglucoside were purchased from Sigma Chemical Co., St. Louis, MO, USA. Acetyldaidzin, acetylglycitin, acetylgenistin, malonyldaidzin, malonylglycitin, and malonylgenistin were obtained from Nacalai Tesque, Kyoto, Japan. The Ragi tempeh starter was sourced from Raprima (Bogor, Indonesia). Kujawski-brand rapeseed oil was purchased from a local grocery store in Poznań, Poland.

2.2. Methods

2.2.1. Okara Preparation

Okara was prepared according to the method described by [2], where soybean seeds were soaked overnight (±12 h), then the water was removed using a sieve. The seeds were ground using a blender with added water (1:6.5 ratio). The mixture was boiled for 5 min and then filtered, resulting in okara as the solid residue and soy milk as the filtrate.

2.2.2. Hydrothermal Treatment

Linseed cake and okara were boiled in water for 10–30 min [40] or sterilized at 121 °C in an autoclave for 15 min [9] or exposed to steam according to the procedure shown in Figure 1. Before hydrothermal treatment, linseed cake was ground using a laboratory grinder.

2.2.3. Tempeh Fermentation

After hydrothermal treatment, the materials were cooled and inoculated with 1 g of Ragi tempeh or spore suspension then placed in Petri dishes with a 15 cm diameter. Fermentation was conducted at 30 °C for 48 h. The inoculating suspension was prepared from Rhizopus oligosporus NRRL 2710 cultured on a PDA medium for 72 h [41]. To each batch of material, 5 mL of spores and mycelia suspension with a concentration of 104 cfu/mL was added. The Ragi tempeh starter was added according to the manufacturer’s suggestion in the proper proportion—0.5 g of powder per batch. Tempeh samples were collected after 24 h and immediately frozen.

2.2.4. Culinary Treatment

After defrosting, the okara tempeh samples were cut into 1 cm2 pieces and fried in rapeseed oil at 150–180 °C for 1 min on both sides.

2.2.5. Isoflavones Analysis

One gram of previously lyophilized material (Alpha 2–4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) was ground using a laboratory grinder (WZ-1, ZBPP, Bydgoszcz, Poland), suspended in 80% acetonitrile, stirred intensively for 2 h at room temperature, and centrifuged at 1310 g for 15 min. The supernatant was evaporated under vacuum (R-215, Büchi Labortechnik AG, Flawil, Switzerland), and the residue was reconstituted in methanol to a final volume of 2 mL. Isoflavone analysis was performed using a Waters 2695 HPLC system equipped with a Waters 2996 photodiode array detector (PDA) (Waters Corporation, Milford, MA, USA). Separation was achieved on an Alltech Alltima C18 reversed-phase column (4.6 × 250 mm, 5 μm, Alltech Co., Deerfield, IL, USA) following the method of Achouri et al. [42] with modification [41]. The elution was conducted at a flow rate of 1 mL/min with the following solvent system: A: acetonitrile; B: 0.2% acetic acid in deionized water. After injection of a 10 µL sample, the gradient started at 15% A for 5 min, increased to 30% over 10 min, and was held for 10 min. It then increased to 50% over 10 min and was held for an additional 5 min. After 40 min, the system was recycled back to 15%. The column temperature was maintained at 26 °C, and eluted isoflavones were detected at 254 nm.

2.2.6. Secoisolariciresinol Diglucoside (SDG) Analysis

Linseed cake tempeh samples were ground, and 1.6 g of the material was defatted by adding n-hexane to the sample at a 1:5 ratio. The mixture was shaken on a roller shaker (Roll Mixer) for 15 min, then centrifuged (MPW-223e centrifuge, MPW-223e, MPW, Warsaw, Poland) at 600× g for 5 min. After a second lipid extraction and separation of the supernatant, the samples were left to allow complete evaporation of the solvent. Next, 10 mL of 70% methanol was added to the samples, and they were shaken for 2 h at 60 °C. Afterward, the samples were placed in an ultrasonic bath for 15 min and then centrifuged at 500× g for 10 min. The supernatant was collected into a flask, and the methanol extraction was repeated on the remaining precipitate. The resulting extract was evaporated using a vacuum evaporator at 37 °C. Following lyophilization, 15 mL of 0.3 M NaOH was added to the sample, which was then gently stirred and neutralized with 2 M acetic acid until the pH reached 6. The product was transferred to a 25 mL volumetric flask, brought to volume with deionized water, filtered through a syringe filter, and injected into chromatography vials [43]. Chromatographic analysis was performed under the same conditions as for the isoflavones (Section 2.2.5).

2.2.7. Statistical Analysis

All experiments were conducted in triplicate. Statistical analysis was performed using Statistica 12 (StatSoft, Inc., Tulsa, OK, USA) software. Tukey’s multiple comparison test was applied to assess differences between the samples, with a significance level set at α < 0.05.

3. Results and Discussion

3.1. Technological Treatment

Several publications describe different methods of hydrothermal treatment prior to fermentation. Based on these studies, we tested two hydrothermal strategies: sterilization at 121 °C for 25 min in an autoclave [9] and boiling in water [40]. While both methods were suitable for okara, linseed cake became dough-like after treatment, resulting in poor fermentation and incomplete mold coverage. Therefore, we explored the potential of using steam treatment as an alternative. To optimize the process, the tested by-products were subjected to steam exposure for various durations, ranging from 5 to 40 min in 5-min increments. The best results, characterized by no infection and adequate mycelial growth, were observed after a minimum of 10 min of treatment for okara and 20 min for linseed cake (Figure 1). Heat treatment prior to fermentation with R. oligosporus is essential [39,40], primarily for sterilizing the product before adding the starter. The R. oligosporus mold, commonly used for tempeh fermentation, does not grow without hydrothermal treatment of the raw material. Steam was used instead of traditional longer cooking because the okara had already undergone thermal treatment. The linseed cake, derived from cold-pressed oil, had not been hydrothermally treated, necessitating an extended steaming step. Steaming is a more water- and energy-efficient process and saves space [44], exemplified by the “several-layer steamers” used in traditional Chinese cooking. Previous fermentation processes for okara and linseed cake involved energy-intensive sterilization [9,45]. There is a growing trend towards non-sterile fermentation, which offers greater potential for energy reduction per process [46], but it is challenging to apply to tempeh production and more suitable for specific bacterial fermentations. The short thermal exposure time in our study significantly simplifies the 45-min steaming of okara before fermentation used in Indonesian research [2].

3.2. Fermentation

The quality of tempeh fermentation is often determined by the mycelial overgrowth of the fermented material [9], although this is a relatively subjective parameter. The results of mycelial growth on the surface of fermented material (Figure 2, Figure 3, Figure 4 and Figure 5) show more intense mycelial growth on okara fermented with pure R. oligosporus NRRL 2710 culture, while the growth with the Ragi starter was less spectacular. On flax cake, both starters showed similar growth after 24 h. Our research indicates that the optimal fermentation time for okara and cake with the tested starters at 30 °C is 24 h. Extending the fermentation time to 48 h leads to the aging phase [40]. We have not yet found a description of the mycelium formed on linseed cake [45]. Among the many proposed methods for using okara, fermentation does not raise as much controversy as the production of okara flour due to drying cost issues [1]. Our studies show that obtaining tempeh from okara and flax cake is not complicated. These products could be used as human food and as components of other products. Matsuo [47] suggested that okara tempeh was more usable than unfermented okara as a foodstuff and could be used as a substitute ingredient.
Unlike in Indonesia, tempeh made from okara in other regions does not have the reputation of being a product for lower social classes [2,39,40].

3.3. Isoflavone Analysis

The results of the isoflavone analysis of soy and okara products are shown in Table 1. The isoflavone content in soybeans depends on the variety, growing conditions, and extraction methods [4,48]. Typically, the isoflavone content in okara ranges between 12% and 40% of the original content in soybean extraction methods [4]. Other authors found that total isoflavone content in soybeans can decrease by 14% when cooked for 5 min [49] and by 45–65% when cooked for 1 h [50]. In our study, we observed a 78% increase in isoflavones (Table 1), possibly due to analyzing all twelve forms of isoflavones in okara. Recent reports have focused mainly on four forms: daidzin, genistin, daidzein, and genistein [8]. Chen et al. [3] found higher concentrations of aglycones than glycosides in okara, which contrasts with our study (Table 1) and other articles [8]. These authors [8] also found a predominance of daidzein over genistein in okara, which we observed in soybeans.
Muliterno [51] published an interesting study analyzing all 12 isoflavone forms. They found 116.36 µmol/100 g solids of total isoflavones in okara, with 66% β-glycosides (42.2% genistin, 12.1% glycitin, and 11.7% daidzin), 26.4% malonylglycosides (19.3% malonylgenistin and 7.1% malonyldaidzin), and 7.6% aglycones, with no acetylglycoside isoflavones detected. In our research, we found acetylgenistin in okara (2.79%), 47% malonylglycosides, 22% glycosides, 27% aglycones, and 331.7 µmol/100 g dry matter (Table 1). The differences could be due to soy variety, isoflavone extraction methods, and technological differences in okara acquisition [4,48]. Jankowiak et al. [4] also did not find acetyldaidzin and acetylglycitin in okara.
Table 1. The content of isoflavones in soy products.
Table 1. The content of isoflavones in soy products.
Raw SoybeanOkaraR.o. NRRL 2710 24 hR.o. NRRL 2710 48 hRAGI 24 hRAGI 48 h
Daidzin8.73 ± 0.12 b28.49 ± 0.19 a2.77 ± 0.13 de8.56 ± 1.74 b5.47 ± 1.26 c3.17 ± 0.26 d
Glycitin3.55 ± 0.29 b5.09 ± 0.16 a2.29 ± 0.09 de3.08 ± 0.13 bc2.43 ± 0.71 cd2.19 ± 0.17 de
Malonyldaidzin53.42 ± 1.03 a48.93 ± 0.66 a21.04 ± 2.3 b20.95 ± 2.83 b15.66 ± 3.93 bc11.19 ± 0.78 cd
Malonylglycitin0 ± 0 e8.38 ± 0.06 a5.81 ± 0.18 b5.16 ± 0.41 b5.16 ± 0.78 b0 ± 0 e
Genistin13.26 ± 0.23 b40.82 ± 0.13 a3.97 ± 0.15 bcd12.96 ± 8 bc9.52 ± 3.06 bcd5.28 ± 0.17 bcd
Acetyldaidzin0 ± 0 d0 ± 0 d0 ± 0 d0 ± 0 d0 ± 0 d0 ± 0 d
Acetylglycitin7.65 ± 0.15 a0 ± 0 c4.63 ± 0.35 b0 ± 0 c0 ± 0 c0 ± 0 c
Malonylgenistin87.03 ± 2.55 b100.62 ± 0.3 a33.44 ± 1.5 c40.37 ± 4.39 c31.65 ± 8.33 c15.72 ± 3.4 d
Acetylgenistin4.55 ± 0.15 cd9.24 ± 0.65 a3.91 ± 0.05 d6.83 ± 0.69 b6.43 ± 0.51 bc5.27 ± 0.63 bcd
Daidzein4.52 ± 0.12 d8.53 ± 0.51 d18.49 ± 1.42 c17.92 ± 4.2 c27.73 ± 2.13 b50.03 ± 2.95 a
Glycitein0 ± 0 f1.04 ± 0.19 e1.92 ± 0.12 cd2.08 ± 0.46 c3.66 ± 0.12 b5.96 ± 0.37 a
Genistein2.88 ± 0.02 g80.52 ± 0.36 def128.99 ± 5.75 cd179.96 ± 46.83 c341.46 ± 16.13 b575.41 ± 10.95 a
Total185.6 ± 3.3 d331.7 ± 0.9 bc227.2 ± 12.9 c297.9 ± 43.3 bc449.2 ± 28.1 b674.2 ± 14 a
R.o. NRRL 2710—Rhizopus oligosporus NRRL 2710; RAGI—starter Ragi tempeh; 24 h and 48 h—fermentation time in hours. Averages of the same isoflavone marked with different letters differ significantly (α < 0.05, Tukey test) in Table 1 and Table 2. Values are the mean ± standard deviation of three replicates.
Table 2. The content of isoflavones in fried soy products.
Table 2. The content of isoflavones in fried soy products.
Fried R.o. NRRL 2710 24 hFried R.o. NRRL 2710 48 hFried RAGI 24 hFried RAGI 48 h
Daidzin2.67 ± 0.31 def0.38 ± 0.01 ef1.94 ± 0.11 def0 ± 0 f
Glycitin1.47 ± 0.07 ef0.91 ± 0 fg1.23 ± 0 f0 ± 0 g
Malonyldaidzin7.61 ± 0.17 de4.53 ± 0.09 de9.17 ± 0.51 cde2.2 ± 3.11 e
Malonylglycitin2.36 ± 0.05 cd1.53 ± 0.09 d2.82 ± 0.07 c0 ± 0 e
Genistin4.29 ± 0.75 bcd0.23 ± 0 d3.5 ± 0.34 cd0.16 ± 0 d
Acetyldaidzin7.14 ± 0.58 a2.02 ± 0.02 c3.73 ± 0.82 b0 ± 0 d
Acetylglycitin4.43 ± 0.13 b3.9 ± 0.04 b4.64 ± 0.04 b0 ± 0 c
Malonylgenistin8.79 ± 0.67 de1.77 ± 0.2 e13.58 ± 0.36 de1.62 ± 0.02 e
Acetylgenistin9.16 ± 0.84 a3.36 ± 1.16 de5.61 ± 0.12 bcd1.25 ± 1.76 e
Daidzein5.79 ± 0.39 d2.86 ± 0.3 d9.45 ± 1.04 d4.55 ± 0.07 d
Glycitein0.83 ± 0.04 ef0 ± 0 f1.27 ± 0.16 de0.8 ± 0.02 ef
Genistein36.37 ± 5.04 efg14.08 ± 3.73 fg91.39 ± 17.02 de30.17 ± 0.11 efg
Total90.9 ± 8.9 e35.6 ± 4.6 f148.3 ± 16.7 g40.8 ± 4.9 g
R.o. NRRL 2710—Rhizopus oligosporus NRRL 2710; RAGI—starter Ragi tempeh; 24 h and 48 h—fermentation time in hours. Averages of the same isoflavone marked with different letters differ significantly (α < 0.05, Tukey test) in Table 1 and Table 2. Values are the mean ± standard deviation of three replicates.

3.3.1. Fermentation Effect

The results clearly show that fermented okara contains a statistically significantly higher amount of isoflavone aglycones compared to unfermented okara. Their total content increased with longer fermentation times. The percentage of malonyl derivatives in the total isoflavone content decreased after the first day of fermentation by 21% and 35% for R. oligosporus and Ragi, respectively. Hu et al. [9] determined a 3.9-fold reduction in genistein content and an 8.8-fold increase in genistein content in the extract after a two-day fermentation of okara by Rhizopus azygosporus. In our study of tempeh from okara fermented by R. oligosporus NRRL 2710, there was a 3.1-fold reduction and a 2.2-fold increase, and while using the commercial Ragi starter, there was a 7.7-fold reduction in genistin content and a 7.1-fold increase in genistein content (Table 1). The results suggest the possibility of modulating the quality composition of tempeh products through the use of appropriate microflora (the Ragi starter contained fungal and bacterial strains). Although the direction of changes caused by microflora appears to be similar, it is possible to manage the intensity of transformations [41].

3.3.2. Impact of Culinary Treatment

The decrease in daidzein, glycitein, and genistein in fried okara ranged from 56% to 100%, depending on the starter used and fermentation time (Table 2). Chien et al. [52] (2005) found similar changes during the heating of acetylgenistin at 150 °C. Another study [53] showed stable levels of aglycones at the same temperature for one hour. We observed a surprisingly stable daidzin and genistin content in okara fermented for 24 h by R. oligosporus NRRL 2710 (Table 1). A similar effect was observed in the study by Lee et al. [54], which explained this as the decomposition of malonyl forms to glycosides during heating. The appearance of acetyl forms in fried products suggests not only the possibility of degradation of malonyl forms but also their acetylation. So far, an increase in the acetyl forms of isoflavones in soybeans has been observed after 90 to 120 min of dry heating [55]. The variation in the stability of individual isoflavones during the frying process indicates the role of fermentation in affecting the properties of the fried product. This is especially observed in the Ragi starter and 48-h fermentation. The reduction of isoflavone levels in the product during cooking may be caused by their extraction. There have been reports on the presence of isoflavones in the medium in which they have been heated [48,56]. However, this concerned water and not fats, which have not yet been characterized. There are also opinions suggesting that traditional and domestic processing methods significantly reduce the isoflavone content in soy-based foods [57]. However, these claims appear to be primarily based on studies of isoflavone glycosides and aglycones, without considering the malonyl and acetyl forms. Furthermore, they do not account for the potential formation of specific isoflavone forms during heating (Table 2, [55]).

3.4. Secoisolariciresinol Diglycoside (SDG) Level

The fermentation process did not result in a statistically significant reduction in the SDG concentration in the linseed cake (Figure 6). With increasing fermentation time, the SDG concentration increased, especially with the use of R. oligosporus NRRL 2710. We did not find other research on the influence of tempeh fermentation on the SDG level. Feng et al. [36] described significant changes in linseed SDG concentration induced by lactic acid bacteria after 8 days of fermentation. It was found that SDG in linseed is stable, even when heated to 250 °C for 3.5 min [58]. For this reason, we decided not to analyze the SDG in fried linseed cake tempeh.
Our study is characterized by the limitations of a few hydrothermal treatments applied prior to fermentation. Using a combination of these could yield good results with an additional reduction in energy consumption. Increasing the number of soybean seed varieties and diversifying linseed cakes would allow a deeper understanding of the mechanisms involved in the effects of tempeh fermentation and cooking treatments on isoflavones and lignans. Further research should also include nutrient changes and sensory evaluation of the tempeh products manufactured.

4. Conclusions

The method presented for producing tempeh from okara is less energy-consuming than the method used traditionally in Indonesia. Also, the method for producing linseed cake tempeh is less energy-intensive than those previously described in the scientific literature. Tempeh fermentation can be used to create desired changes in the isoflavone content in okara tempeh by modulating the time and using specific strains of microorganisms. Frying significantly reduces the isoflavone content in okara tempeh. The secoisolariciresinol diglucoside in linseed cake does not appear to be affected by the changes caused by the microorganisms applied for tempeh fermentation within the typical process time. Using by-products such as okara and linseed cake contributes to the reduction of food waste and promotes environmentally sustainable food production practices. Importantly, the ability to modulate isoflavone content through the selection of appropriate microbial starters and fermentation times can influence the health effects of tempeh products on consumers. A thorough understanding of the role of isoflavones in human health could further enhance consumer well-being.

Author Contributions

Conceptualization, M.K. (Maciej Kuligowski) and M.K. (Marcin Kidoń); methodology, M.K. (Maciej Kuligowski), O.G. and I.J.-K.; software, I.J.-K. and E.R.-K.; validation, E.R.-K., I.J.-K. and M.K. (Maciej Kuligowski); formal analysis, M.K. (Maciej Kuligowski) and I.J.-K.; investigation, M.K. (Maciej Kuligowski) and O.G.; resources, M.K. (Marcin Kidoń); data curation, E.R.-K.; writing—original draft preparation, M.K. (Maciej Kuligowski); writing—review and editing, E.R.-K., I.J.-K. and M.K. (Marcin Kidoń); visualization, I.J.-K. and O.G.; supervision, M.K. (Maciej Kuligowski); project administration, E.R.-K.; funding acquisition, I.J.-K. and M.K. (Maciej Kuligowski). All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets presented in this article are not readily available due to technical and time limitations. The data presented in this study are available on request from the corresponding author.

Acknowledgments

We would like to thank Jerzy Nawracała, Department of Genetics and Plant Breeding, Faculty of Agronomy, Horticulture, and Bioengineering, Poznań University of Life Sciences, for providing the soybeans used in this research. We also thank the company Vitacorn for generously supplying the linseed cake free of charge. Special thanks go to Marcelus Arnold for delivering the Ragi Tempeh starter from Indonesia.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. The flow diagram of the process of tempeh production from by-products (okara and linseed cake).
Figure 1. The flow diagram of the process of tempeh production from by-products (okara and linseed cake).
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Figure 2. Fermentation stages of tempeh from okara inoculated with R. oligosporus NRRL 2710. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
Figure 2. Fermentation stages of tempeh from okara inoculated with R. oligosporus NRRL 2710. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
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Figure 3. Fermentation stages of tempeh from okara inoculated with Ragi starter. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
Figure 3. Fermentation stages of tempeh from okara inoculated with Ragi starter. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
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Figure 4. Fermentation stages of tempeh from linseed cake inoculated with R. oligosporus NRRL 2710. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
Figure 4. Fermentation stages of tempeh from linseed cake inoculated with R. oligosporus NRRL 2710. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
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Figure 5. Fermentation stages of tempeh from linseed cake inoculated with Ragi starter. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
Figure 5. Fermentation stages of tempeh from linseed cake inoculated with Ragi starter. (a)—before fermentation; (b)—24 h fermentation; (c)—48 h fermentation.
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Figure 6. The content of secoisolariciresinol diglycoside in the linseed products. R.o. NRRL 2710—Rhizopus oligosporus NRRL 2710. RAGI—starter Ragi tempeh. 24 h and 48 h—fermentation time in hours. Averages marked with different letters differ significantly (p < 0.05, Tukey test).
Figure 6. The content of secoisolariciresinol diglycoside in the linseed products. R.o. NRRL 2710—Rhizopus oligosporus NRRL 2710. RAGI—starter Ragi tempeh. 24 h and 48 h—fermentation time in hours. Averages marked with different letters differ significantly (p < 0.05, Tukey test).
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Kuligowski, M.; Radziejewska-Kubzdela, E.; Górna, O.; Jasińska-Kuligowska, I.; Kidoń, M. Sustainable Use of By-Products (Okara and Linseed Cake) in Tempeh Fermentation: Effects on Isoflavones and Lignans. Sustainability 2024, 16, 9936. https://doi.org/10.3390/su16229936

AMA Style

Kuligowski M, Radziejewska-Kubzdela E, Górna O, Jasińska-Kuligowska I, Kidoń M. Sustainable Use of By-Products (Okara and Linseed Cake) in Tempeh Fermentation: Effects on Isoflavones and Lignans. Sustainability. 2024; 16(22):9936. https://doi.org/10.3390/su16229936

Chicago/Turabian Style

Kuligowski, Maciej, Elżbieta Radziejewska-Kubzdela, Oliwia Górna, Iwona Jasińska-Kuligowska, and Marcin Kidoń. 2024. "Sustainable Use of By-Products (Okara and Linseed Cake) in Tempeh Fermentation: Effects on Isoflavones and Lignans" Sustainability 16, no. 22: 9936. https://doi.org/10.3390/su16229936

APA Style

Kuligowski, M., Radziejewska-Kubzdela, E., Górna, O., Jasińska-Kuligowska, I., & Kidoń, M. (2024). Sustainable Use of By-Products (Okara and Linseed Cake) in Tempeh Fermentation: Effects on Isoflavones and Lignans. Sustainability, 16(22), 9936. https://doi.org/10.3390/su16229936

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