Vitamin C Paste Alpha H
Abstract
Tempeh is a fermented food made of mainly soybeans and is a nutritious, affordable, and sustainable functional source of protein. Globally, tempeh is a widely accepted fermented product. Although there is a growing body of literature on tempeh, most research has focused on unfermented soybeans, thus the impact of tempeh fermentation on biological properties of soybeans has been largely left scattered. The objective of this review is to summarize the literature of tempeh fermentation over the past 60 years. A search of articles on tempeh published from 1960 to 2020 was performed using the Cochrane Library, Web of Science, EBSCOhost FSTA database, and Google Scholar. References from identified articles were reviewed for additional sources. In total, 321 papers were selected for this review, of which 64 papers were related to the health benefits of tempeh. This review concluded that sufficient evidence exists in the literature supporting tempeh fermentation as a low-cost, health-promoting, and sustainable food processing technology to produce protein-rich foods using various beans, legumes, and grains. This comprehensive review suggests further studies are needed on tempeh fermentation and its impact on human health; research and standardization of nonsoy tempeh; assessment of food safety-improving modification in tempeh production system; and initiatives supporting the sourcing of local ingredients in tempeh production.
1 INTRODUCTION
As meat consumption and production have been considered unsustainable in terms of public health and environment (Godfray et al., 2018), tempeh can be favorable food source due to its health benefits, affordability, and sustainability. Tempeh is an indigenous food from Indonesia, where it has been consumed as a staple source of protein for more than 300 years (Shurtleff & Aoyagi, 2020). Tempeh is usually made of soybeans fermented with Rhizopus spp., but it can be made using various nuts, grains, and beans (Karyadi & Lukito, 1996). Tempeh has been known as a source of significant amounts of protein, Vitamin B12, and bioactive compounds (Babu et al., 2009; Nout & Kiers, 2005). Compared to tofu and soy, tempeh has been less studied (Figure 1) but there is growing research interest of tempeh (Figure 2).
Number of publications found in search results for the keywords "soy," "tofu," "tempeh," and "tempe" on the Web of Science from 1960 to 2019 (overlapping titles in "tempe" keyword search results not included)
Number of publications found in search results for the keywords "tempeh" and "tempe" on the Web of Science from 1960 to 2019 (overlapping titles in "tempe" keyword search results not included)
Over the past few decades, studies have shown that fermentation is key to the increased protein amount and solubility of tempeh made from soybeans and other beans (Ashenafi & Busse, 1991d; Onoja et al., 2011; Stodolak & Starzynska-Janiszewska, 2008; Wronkowska et al., 2015). Furthermore, the fermentation process can decrease antinutrient and allergen contents, whereas it increased essential micronutrient content, for example, vitamin B12 and health-promoting bioactive compounds. The fermentation also promoted food sensory properties that are more versatile to be used as meats, providing a more sustainable and affordable way to produce such properties compared to that of conventional meats. The studies that supported these claims are discussed in Section 4.
Previous review papers have not been able to completely deduct the potential benefits of tempeh due to limitations in the number and scope of published research (Babu et al., 2009; Karyadi & Lukito, 1996; Nout & Kiers, 2005, Owens et al. 2015). The objective of this paper is to comprehensively and systematically review the health-promoting, affordability, sustainability, production, food safety, and processing aspects' evidence of tempeh and tempeh fermentation. This review aims to understand the potential of tempeh fermentation as a means of efficiently and effectively improving public health.
2 METHODS
A search of four large citation databases—the Web of Science, Cochrane Library, EBSCOhost Food Science and Technology Abstract (FSTA), and Google Scholar—was conducted for this review. Comprehensive mapping was conducted on all results using "tempeh" and "tempe" as the keywords for the search on all databases. A total of 715,747 documents on tempeh were identified. All 572 papers found on the Web of Science from 1960 to 2020 were first identified before being sorted based on the relevance to the subsections, access to full article, and language coverage, that is, English and Bahasa Indonesia. Searches on other platforms were conducted subsequently, prioritizing the relevance to the scope of this paper. Relevant references from the selected articles were also reviewed. In total, 524 papers were analyzed and 383 papers were cited and categorized into subsections. Sixty-four papers related to the health benefits of tempeh were analyzed and classified. The other 319 papers were assembled into the other subsections (Figure 3). Analyses of nutrition and price of tempeh were conducted based on the data mined from USDA FoodData Central, USDA Economic Research Service, and the Ministry of Trade Republic of Indonesia databases. Sixty-seven other papers were cited for complementing Sections 1 and 4 of this review.
Systematic literature screening and selection method used
3 RESULTS AND DISCUSSION
3.1 Tempeh
3.1.1 Definition
The official standard registered by the CODEX Alimentarius Commission (Food & Agriculture Organization–World Health Organization, 2017), coded as CODEX STAN 313R-2013, used the term "tempe," as it is spelled in Bahasa Indonesia, as the official label for the name of product. The term "tempeh" is used in this review to facilitate compliance with English language dictionaries. The CODEX standard described tempeh as a compact, white, cake-formed product, prepared from dehulled boiled soybeans through solid state fermentation with Rhizopus spp. The standard recognized only Rhizopus oligosporus, Rhizopus oryzae, and Rhizopus stolonifer inoculants. Tempeh can be mixed with cooked rice powder, rice bran powder, and/or wheat bran powder as inoculum. The texture of tempeh should be compact and not easily disintegrated upon cutting with knife. The color of tempeh should be white due to the growth of the mycelium of Rhizopus spp., limiting the degree of natural sporulation by the inoculant. The flavor of tempeh should be meaty, mushroom-like, and nutty. The odor of tempeh should be fresh and without any odor of ammonia. Tempeh should be typically free from food additives and from foreign matters such as other beans, husk, and small stones. The minimum composition of tempeh is a minimum protein content of 15% (w/w), a maximum moisture content of 65% (w/w), a minimum lipid content of 7% (w/w), and a maximum of 2.5% (w/w) of crude fiber (Food & Agriculture Organization-World Health Organization, 2017).
In this review, the term "tempeh" refers to soy tempeh and "tempeh fermentation" refers to fermentation with R. oligosporus, unless otherwise described. Soybean and R. oligosporus (Rhizopus microsporus var. oligosporus) were the most studied and commonly used combination (Shambuyi et al., 1992).
In Indonesia, tempeh has been standardized by the National Standardization Agency of Indonesia (Badan Standardisasi Nasional), coded as SNI 3144:2009, with more detailed specifications compared to that of CODEX (Table 1). The Indonesian standard requires a higher protein content by 1% (w/w) and a higher lipid content by 3% (w/w). The Indonesian standard also specifically limited the maximum content of metal, that is, Cd, Pb, Sn, and Hg, and microbial contaminants, that is, Coliform and Salmonella spp. Species of inoculant was not specified in the Indonesian standard. Both CODEX and Indonesian standards advised that tempeh production should be conducted hygienically in compliance with food production standards in terms of labeling and analysis.
| Category | Parameter | Unit | Indonesia SNI 3144:2009 | FAO-WHO CODEX STAN 313R-2013 |
|---|---|---|---|---|
| Condition | Aroma | NA | Normal, unique | NA |
| Color | NA | Normal | NA | |
| Taste | NA | Normal | NA | |
| Content | Moisture | % (w/w) | Maximum 65 | Maximum 65 |
| Ash | % (w/w) | Maximum 1.5 | NA | |
| Lipid | % (w/w) | Minimum 10 | Minimum 7 | |
| Protein | % (w/w) | Minimum 16 | Minimum 15 | |
| Fiber | % (w/w) | Maximum 2.5 | Minimum 2.5 | |
| Contamination | Cd | mg/kg | Maximum 0.2 | NA |
| Pb | mg/kg | Maximum 0.25 | NA | |
| Sn | mg/kg | Maximum 40 | NA | |
| Hg | mg/kg | Maximum 0.03 | NA | |
| As | mg/kg | Maximum0.25 | NA | |
| Coliform | MPN/g | Maximum 10 | NA | |
| Salmonella spp. | per 25 g | Negative | NA | |
| Inoculant(s) | Mold | NA | NA | Rhizopus spp. (R. oligosporus, R. oryzae, and/or R. stolonifer) |
3.1.2 Origin
The earliest reference of tempeh was found in Serat Centhini, a manuscript that was written in the 1600s and published in 1815 under the supervision of King Pakubuwono V of Surakarta Kingdom, Central Java the fact that tempeh was first mentioned in the Serat Centini was first discovered in 1984 by Shurtleff & Aoyagi (Shurtleff & Aoyagi 1984, in Shurtleff & Aoyagi 2020). Serat Centhini is a compilation of Javanese legends, traditions, and teachings, in which the word "tempe" and a tempeh dish called "sambal lethok" in Bayat, a subdistrict of Klaten Regency in Central Java, Indonesia were mentioned (Shurtleff & Aoyagi, 2020; Winarno et al., 2017; Astuti, 1999). The word "tempe" has been hypothesized to be derived from the word "tumpi," a white ancient Javanese food made of sago flour that tempeh resembled the appearance of (Purwadaria et al., 2016). The term "tempeh" was first introduced by Prinsen Geerligs in a Dutch article in 1896 and Van Veen and Schaefer in an English article in 1950. And the term "tempe" has been commonly used in Indonesia and has been registered in the regional standard of tempeh in FAO–WHO CODEX Alimentarius Commission (Food & Agriculture Organization–World Health Organization, 2017; Shurtleff & Aoyagi, 2020).
Within Indonesia, tempeh has historically been consumed as an affordable staple source of protein, especially on the islands of Java and Bali. It is consumed in many forms including fried, boiled, steamed, or grilled tempe benguk (made of tofu residue), tempe bongkrek (made of coconut oil or milk press cake), or tempe lamtoro (made of Leucaena leucocephala seeds) (Shurtleff & Aoyagi, 2020; Winarno et al., 1985; Winarno et al., 2017). Outside of Indonesia, tempeh has been introduced and consumed in the continents of Asia, Africa, North America, South America, Europe, and Australia (Shurtleff & Aoyagi, 2007). Shurtleff and Aoyagi (2020) reported that tempeh was introduced in Japan in 1912, in India in 1936, in Suriname in 1936, in the United States in 1958, and in Zambia in 1971.
In terms of production, tempeh was first made using soybeans wrapped in leaves, for example, banana, teak, or waru (Hibiscus spp.) leaves, suggesting that tempeh might originate from an accidental inoculation of soybean by Rhizopus spp. mold on leaf surface (Harahap et al., 2018; Winarno et al., 2017). The first tempeh inoculant identified was R. oryzae in 1895 followed by a study screening the use of various cultures and substrates in 1963 that identified R. oligosporus (NRRL 2710) as the best inoculant (Shurtleff & Aoyagi, 2020). The production of tempeh using plastic bags or tubes as containers was introduced in the United States in 1964 by Martinelli and Hesseltine. The use of various beans, grains, and legumes in tempeh production started in 1963 and gained popularity after 2005 due to concerns about soy in the popular culture (Shurtleff & Aoyagi, 2020).
In Indonesia, tempeh has been considered a "low-class protein" food commodity due to its low price, abundant supply, and accessibility for people across income brackets, including those who could not afford meat (Karyadi & Lukito, 1996). Recently, there is a global emergence of initiatives to rebrand tempeh as an affordable, sustainable, and healthy plant-based product, thus vegetarian- and vegan-friendly. These initiatives include the green marketing of tempeh internationally by the Indonesian Tempe Movement, which focused on the ecological aspect of tempeh (Ahnan-Winarno et al., 2019) and the proposal by the Rumah Tempe Indonesia (Indonesia Tempe House) for tempeh to be considered a UNESCO Intangible Heritage and their promotion of Good Manufacturing Practices in hygienic tempeh production (Astawan & Maskar, 2019). A study by Fibri and Frost (2020) reported that Indonesian millennials were proud of tempeh and preferred traditional over modern tempeh when product information was provided, for example, about the traditional usar inoculum, starter culture, and stainless steel factory used. The proudness scores significantly increased when subjects were provided the information that the tempeh samples consumed were of made of local ingredients and decreased when they were informed that the tempehs were made of imported soybeans. The proudness scores were not affected by knowing if the production method was traditional or modern (Fibri & Frost, 2020).
3.1.3 Production
Production of tempeh includes soaking, dehulling, washing, boiling, draining, cooling, inoculating, packaging, and incubating. Traditional tempeh production methods vary greatly (Owens et al., 2015). There are at least eight variations of how and in which order these main steps are conducted, including some repetitions of the same steps (Figure 4). Variations in tempeh production were found across different locations in Indonesia. The relatively simple method (green arrow in Figure 4) was found in Purwokerto and Pekalongan, Central Java, whereas the double-boiling method (black arrow in Figure 4) was found in Yogyakarta (Rahayu et al., 2015).
Variations of tempeh production flow (adapted from Rahayu et al., 2015)
The soaking step is usually conducted first, lasting for 6 to 24 hr. Soaking hydrates the soybeans and can make the hulls easier to peel. However, some production methods conduct dry dehulling with a machine (Nout & Kiers, 2005). In soybeans soaked in tap water for 24 to 36 hr at 20, 30, and 37 °C in Indonesia, Klebsiella pneumoniae, Klebsiella ozaenae, Enterobacter cloacae, Enterobacter agglomerans, Citrobacter diversus, Bacillus brevis, Pichia burtonii, Candida diddensiae, and Rhodotorula rubra were found, whereas Lactobacillus casei, Streptococcus faecium, Staphylococcus epidermidis, and Streptococcus dysgalactiae dominated (Mulyowidarso et al., 1989).
During the soaking step, natural acidification can happen (reaching pH 4.85), which can help inhibit or retard the growth of pathogens and/or spoilage-causing microorganisms (Nout et al., 1987; Tunçel & Göktan, 1990). Natural acidification did not happen if soybeans were boiled before soaking (Mulyowidarso et al., 1989). Various modifications have been added in the soaking step, including acidification and co-inoculation with Lactobacillus plantarum to improve the quality of tempeh produced as well as consistently inhibit the growth of unwanted microorganisms, including but not limited to Listeria monocytogenes, Bacillus cereus, Salmonella infantis, Staphylococcus aureus, and Escherichia coli (Ashenafi, 1991; Ashenafi & Busse, 1989, 1992; Nout et al., 1987). During the soaking step, some evidence indicated that the content of antinutrient phytates was reduced (Tawali et al., 1998). The levels of sucrose, stachyose, raffinose, propionic acid, formic acid, and acetic acid were decreased due to the activities of enzymes endogenous to beans, diffusion of sugars, and fermentation by microorganisms in the soaking water (Mulyowidarso et al., 1991a, 1991b).
Dehulling is an important step because the presence of soybean hulls in finished tempeh is considered contaminant according to CODEX (Food & Agriculture Organization–World Health Organization, 2017). Although the dehulling step was historically done by hands or feet (Fung & Crozier-Dodson, 2008), these methods have been eliminated in a hygienic tempeh production system and replaced with mechanical dehulling (Putri et al., 2018). The washing step is sometimes skipped in the production process as tempeh fermentation can be successful using soybeans dried directly from the boiled soak water (Babu et al., 2009; Nout & Kiers, 2005).
The boiling step, which usually lasts for 20 to 30 min, in tempeh production was critical because it removes the raw flavor through cooking as well as eliminates pathogens and spoilage organisms that can pose a food safety hazard and/or interfere with the fermentation process (Babu et al., 2009; Karyadi & Lukito, 1996; Nout & Kiers, 2005). The addition of 0.11 mol/L of lactic acid and 0.2 mol/L of sodium phosphate buffer can facilitate acidified boiling, resulting in pH values of 3.0 and 4.3 that can kill the spores of Bacillus stearothermophilus with a decimal reduction time of 2.8 min at pH 3.0 and 27 min at pH 4.3, respectively (Ruiz–Teran & Owens, 1996b). During the boiling step, the levels of flatulence-causing oligosaccharides in soybeans can also be reduced (Ferreira et al., 2011).
The draining step, which might also include a drying process, reduces the water content in tempeh as tempeh fermentation requires an optimum level of approximately 62% humidity and 0.99 to 1.00 water activity (a w) (Penaloza et al., 1992; Sarrette et al., 1992). Unless, the draining process was done rapidly, for example, using a centrifuge, it usually cools down the soybean to the desired range of 25 to 38 °C (Babu et al., 2009; Karyadi & Lukito, 1996; Matsumoto & Imai, 1990; Nout & Kiers, 2005). Sudarmadji and Markakis (1978) specifically reported that tempeh harvested at 30 hr after being fermented at 32 °C resulted the best organoleptic properties.
The inoculation step involves the dispersion of Rhizopus spp. sporangiospores, usually 104 CFU/g substrate, that grow into a dense mycelium biomass that can be harvested before it sporulates (Nout & Kiers, 2005; Penaloza et al., 1992). This would be accommodated by packing the soybeans into containers with limited air flow, for example, banana leaf or a perforated plastic bag (Bhowmik et al., 2013; Harahap et al., 2018).
The incubating step, usually at 25 to 38 °C for 18 to 72 hr (Babu et al., 2009; Karyadi & Lukito, 1996; Nout & Kiers, 2005), facilitates the growth of Rhizopus spp. that can increase the health-promoting potential of soybeans by enhancing nutrient bioavailability and eliminating antinutrients, for example, digesting protein into amino acids (Ashenafi & Busse, 1991d), digesting lipid into fatty acids (Ruiz-Terán & Owens, 1996a), transforming iron(II)-species into iron(III)-species (Tawali & Schwedt, 1998), breaking down isoflavone glycosides into aglycones (Kuligowski et al., 2017), reducing phytate content (Eklund-Jonsson et al., 2006), as well as producing Vitamin B12 through symbiosis (Liem et al., 1977, p. 12). The effects of tempeh fermentation on health-related components of soybeans are discussed in the next sections.
Harvested tempeh can be sold, cooked, and consumed fresh or after being pasteurized, it can be dried or frozen (Karyadi & Lukito, 1996; Nout & Kiers, 2005; Shurtleff & Aoyagi, 1979). The shelf life of fresh tempeh is approximately 3 days at ambient temperature (Moreno et al., 2002; Nout & Rombouts, 1990). Dried tempeh (a w = 0.48) can be stored for up to 30 weeks at a refrigeration temperature of 5 °C. Vacuum packaging can extend the shelf life of fresh tempeh by 2 days at 23 to 24 °C, 32 days at 4 to 6 °C, and 39 days at 0 to 4 °C (Astawan et al., 2016). High-pressure CO2 treatment for shelf life extension at 6.3 and 7.6 MPa for 5 to 20 min did not affect vitamins B1, B2, and B3, but decreased calcium, protein, fat, and water contents (Kustyawati et al., 2015).
The production scheme for producing tempeh using other legumes, grains, and nuts can be simplified as detailed in Figure 5. Although different substrates may require different conditions, the main principle for production would be similar to what was elaborated earlier. Techniques and effects of tempeh fermentation on various substrates are discussed in the next subsections.
Simplified production scheme of tempeh using various legumes, grains, and nuts
3.1.4 Nutritional content
To investigate the nutritional content of tempeh, nutrition facts panels of 13 commercial tempehs in the United States obtained from United States Department of Agriculture's (USDA) FoodData Central were evaluated. The samples (identities not disclosed) included raw soy tempeh (S.SR, S.RC.C, S.TI1, S.TI2, S.GL, S.TH, S.SH, S.S); cooked soy tempeh (S.RC.C); raw soy, barley, brown rice, and millet tempeh (SBrM.L); raw soy, white rice, and brown rice tempeh (SBrWr.L); raw black bean tempeh (Bb.SH); raw black eyed pea tempeh (Bep.SH); and raw flaxseed tempeh (F.L). The nutritional profiles were presented in an 84-g serving, as defined by the US Food and Drug Administration (FDA)'s Reference Amount Customarily Consumed (RACC) and then compared to the daily recommended values based on a 2,000-Calorie diet, according to the US Food and Drug Administration (FDA) and National Institute of Health (Food & Drug Administration, 2018). Nutritional claims, for example, "high in protein," were deducted based on FDA's Code of Federal Regulations Title 21 Part 101 "Food Labeling" (Food & Drug Administration, 2019).
In the United States, commercial tempehs in their RACC portion (84 g) were all high in protein (100%, 14.5 ± 2.4 g), mostly high in fiber (69%, 7 ± 4.6 g), mostly low in saturated fat (85%, 0.6 ± 0.7 g), mostly free of sugar (62%, 0.8 ± 1.3 g), almost all were very low in sodium (92%, 32 ± 91.5 mg), and all free of cholesterol as well as trans fatty acids (Table 2, 3). The samples also contained considerable amounts of calcium (64.4 ± 22.3 mg), potassium (153.8 ± 151.2 mg), monounsaturated fatty acids (0.8 ± 1.1 g), polyunsaturated fatty acids (1.2 ± 1.6 g), and a relatively low amount of carbohydrates (14.2 ± 6.3 g) per 84 g.
| Nutrient | Unit | Maximum | Minimum | Mean | SD | DRV | Applicable claim | Percent with applicable claim |
|---|---|---|---|---|---|---|---|---|
| Energy | cal | 177.2 | 128.5 | 152.5 | 15.9 | 2,000 | NA | NA |
| Protein | g | 17.7 | 10.9 | 14.5 | 2.4 | 50 | High in protein | 100% |
| Total lipid (fat) | g | 9.6 | 0.0 | 4.5 | 3.2 | Low in fat | 31% | |
| Saturated Fat | g | 2.1 | 0.0 | 0.6 | 0.7 | 20 | Low in saturated fat | 85% |
| Carbohydrate | g | 25.3 | 6.4 | 14.2 | 6.3 | 275 | NA | NA |
| Sugars | g | 3.7 | 0.0 | 0.8 | 1.3 | 50 | Free of sugar | 62% |
| Fiber | g | 15.6 | 0.0 | 7.0 | 4.6 | 28 | High in fiber | 69% |
| Calcium | mg | 93.2 | 15.1 | 64.4 | 22.3 | 1,300 | NA | NA |
| Iron | mg | 3.3 | 1.4 | 2.2 | 0.5 | 18 | Good source of iron | 8% |
| Potassium | mg | 346.1 | 0.0 | 153.8 | 151.2 | 4,700 | NA | NA |
| Sodium | mg | 336.0 | 0.0 | 32.0 | 91.5 | 2,300 | Very low in sodium | 92% |
| Vitamin B-12 | μg | 0.1 | 0.0 | 0.0 | 0.0 | 2.4 | NA | NA |
| Monounsaturated fatty acids | g | 2.9 | 0.0 | 0.8 | 1.1 | NA | NA | NA |
| Polyunsaturated fatty acids | g | 4.0 | 0.0 | 1.2 | 1.6 | NA | NA | NA |
| Trans fatty acids | g | 0.0 | 0.0 | 0.0 | 0.0 | NA | Free of trans fat | 100% |
| Cholesterol | mg | 0.0 | 0.0 | 0.0 | 0.0 | 300 | Free of cholesterol | 100% |
| Tempeh | Beef | |||
|---|---|---|---|---|
| Nutrition | Average | SD | Average | SD |
| Energy | 181.54 | 18.89 | 152.64 | 72.84 |
| Protein | 17.21 | 2.80 | 12.64 | 4.45 |
| Total fat | 5.38 | 3.76 | 10.12 | 9.05 |
| Saturated fat | 0.71 | 0.80 | 4.48 | 3.55 |
| Carbohydrate | 16.93 | 7.51 | 3.11 | 4.85 |
| Sugars | 1.18 | 1.63 | 1.14 | 2.36 |
| Fiber | 9.88 | 4.33 | 0.30 | 0.58 |
| Trans fat | 0.00 | 0.00 | 0.24 | 0.64 |
| Cholesterol | 0.00 | 0.00 | 44.08 | 19.02 |
| Ca | 76.69 | 26.55 | 11.17 | 15.64 |
| Fe | 2.62 | 0.62 | 2.11 | 1.25 |
| Sodium | 38.15 | 108.87 | 587.11 | 365.59 |
Vitamin B12 levels were not provided by most samples but reached up to approximately 0.1 μg per 84 g in two soy tempehs and the black-eyed pea tempeh (Figure 6). Compared to nonsoy tempehs, soy tempehs contained higher levels of protein (Figure 7). Black bean and black-eyed pea tempehs were free of fat and contained higher levels of carbohydrates and sugars. Black bean tempeh had the highest iron content (3.3 mg per 84 g) (Figure 8). Only one raw tempeh sample provided values of other nutrients in units per 84 g (data not presented), including a high content of copper (0.47 mg), manganese (1.09 mg), and vitamin B2 (0.3 mg), a good amount of magnesium (68.04 mg) and phosphorus (223.44 mg), as well as considerable amounts of zinc (0.96 mg), vitamin B1 (0.07 mg), vitamin B3 (2.22 mg), vitamin B5 (0.23 mg), vitamin B6 (0.18 mg), folate (20.16 mg), and vitamin B12 (0.07 mg). The absence of Vitamin B12 and potassium levels in Figure 6 and Table 2 was due to data not provided by some tempeh entries. The following sections of this paper discuss the discrepancy of nutritional composition between the data presented in this section and other reports.
Levels of notable nutrients in U.S. commercial tempehs (adapted from USDA, 2019)
Levels of macronutrients in U.S. commercial tempehs (adapted from USDA, 2019)
Levels of micronutrients in U.S. commercial tempeh samples (adapted from USDA, 2019)
Documentation of the presence of vitamin B12 in tempeh has been patchy due to B12 being produced mostly due to contamination by K. pneumoniae and Citrobacter freundii (Keuth & Bisping, 1994; Liem et al., 1977; Okada, 1989). In Indonesia, where tempeh has been produced mostly traditionally, for example, using river water or in an unsanitized facility, tempeh samples were found to contain 0.34 to 2.44 μg/84 g (Liem et al., 1977). Currently, although in situ fortification of vitamin B12 using Propionibacterium has been successfully conducted (Signorini et al., 2018; Wolkers–Rooijackers, Endika, & Smid, 2018), there is no industrial standard for producing tempeh with sustained levels of Vitamin B12.
Compared to the six beef entries on the USDA FoodData Central database, the U.S. commercial tempehs contained similar protein content, less total and saturated fat, more carbohydrates, similar or higher sugar level, and higher fiber content (Figure 9). Tempeh generally contained more calcium, slightly more iron, less sodium, and slightly more sugars than beef (Figure 10).
Levels of macronutrients in U.S. beefs compared to commercial tempehs (adapted from USDA, 2019)
Levels of micronutrients in US beefs compared to commercial tempehs (adapted from USDA, 2019)
Phases of tempeh fermentation (adapted from Matsumoto & Imai, 1990; Ruiz-Terán & Owens, 1996a; Sparringa & Owens, 1999c; Sudarmadji & Markakis, 1978)
Based on the nutritional analyses above, tempeh can be considered as a nutritious source of protein given the high protein content, high fiber content, low saturated fat content, mineral content, and vitamin content. Compared to beef, tempeh was observed not to be inferior and potentially more favorable in terms of protein, total fat, saturated fat, fiber, cholesterol, calcium, iron, and sodium content (Table 4). To further understand the nutritional qualities of tempeh, bioassays in animal and human studies, for example, on protein efficiency ratio (Babji et al., 2010), need to be conducted.
| Tempeh | Beef | |||
|---|---|---|---|---|
| Nutrition | Average | SD | Average | SD |
| Energy | 181.54 | 18.89 | 152.64 | 72.84 |
| Protein | 17.21 | 2.80 | 12.64 | 4.45 |
| Total fat | 5.38 | 3.76 | 10.12 | 9.05 |
| Saturated fat | 0.71 | 0.80 | 4.48 | 3.55 |
| Carbohydrate | 16.93 | 7.51 | 3.11 | 4.85 |
| Sugars | 1.18 | 1.63 | 1.14 | 2.36 |
| Fiber | 9.88 | 4.33 | 0.30 | 0.58 |
| Trans fat | 0.00 | 0.00 | 0.24 | 0.64 |
| Cholesterol | 0.00 | 0.00 | 44.08 | 19.02 |
| Ca | 76.69 | 26.55 | 11.17 | 15.64 |
| Fe | 2.62 | 0.62 | 2.11 | 1.25 |
| Sodium | 38.15 | 108.87 | 587.11 | 365.59 |
3.2 Tempeh fermentation
3.2.1 Stages of fermentation
The duration and phases of tempeh fermentation have not been standardized by the FAO/WHO CODEX Alimentarius Commission. Here, studies investigating the relationships between duration of tempeh fermentation and organoleptic, nutritional, and biomass characteristics were reviewed and summarized. Tempeh fermentation at 30 to 32 °C underwent an active growing phase in the first 30 to 32 hr, indicated with mycelial growth, activities of lipase and protease, and alkalization (Ruiz-Terán & Owens, 1996a; Sudarmadji & Markakis, 1978); maturing phase until 46 hr, indicated with alkalization, optimum tenderness, and highest organoleptic scores (Sparringa & Owens, 1999c; Sudarmadji & Markakis, 1978); and an aging phase until 72 hr, indicated by the start of mycelial senescence and retained or deteriorated organoleptic scores (Ruiz-Terán & Owens, 1996a; Sparringa & Owens, 1999c; Sudarmadji & Markakis, 1978) (Figure 11). Tempeh fermented at 27 °C underwent a stationary phase between 20 and 22 hr, indicated with no change of temperature and biomass (Matsumoto & Imai, 1990). The stages of tempeh fermentation can result in color change due to the death phase of Rhizopus spp. and oxidized unsaturated fatty acids (Muzdalifah et al., 2017). Over-fermentation of tempeh (more than 72 hr) can gradually promote the production of bitter-tasting amino acids and degradation of umami-tasting compounds (Utami et al., 2016).
Based on mold growth, free fatty acid content, bacterial count, and temperature, Sudarmadji and Markakis (1978) classified tempeh fermentation at 32 °C into three phases. In phase 1 (0 to 30 hr), rapid increase in free fatty acid content, bacterial count, temperature, and mold growth was observed. Tempeh produced after 30 hr scored best organoleptically. In phase 2 (30 to 72 hr), there was little or no change in those parameters, except for the declining temperature. Tempeh produced after 72 hr kept good organoleptic quality. In phase 3 (longer than 72 hr), recommencement in free fatty acid content and bacterial growth, as well as signs of deterioration, was observed (Sudarmadji & Markakis, 1978).
Based on loss of dry matter, Ruiz-Terán and Owens (1996a) classified tempeh fermentation using R. oligosporus NRRL 2710 at 30 °C into mycelial growth (0 to 32 hr) and senescence (60 to 180 hr) phases. During the mycelial growth phase, tempeh fermentation decreased the total dry matter by approximately 10% (w/w), where activities of lipase and protease were detected (Ruiz-Terán & Owens, 1996a). During the mycelial senescence phase, approximately 12% of the total dry matter was lost almost entirely due to decrease in crude lipid (Ruiz-Terán & Owens, 1996a). At 27 °C, Matsumoto and Imai (1990) identified a stationary phase of tempeh fermentation at 20 to 22 hr, where there was no change of temperature and mycelium weight (Matsumoto & Imai, 1990).
Based on pH change throughout tempeh fermentation with R. oligosporus NRRL 2710 at 30 °C, mentioned were the maturing phase (pH changed from 4.6 to 6.6 after the first 46 hr) (Sparringa & Owens, 1999a) and the aging phase (pH changed from 6.6 to 7.1 at 46 to 72 hr) (Sparringa & Owens, 1999c). Based on texture, 24 to 72 hr of fermentation at 30 °C produced tempehs with acceptable texture, whereas 48 hr of fermentation resulted in the highest level of tenderness, indicated by texture weakness, modulus of elasticity, and surrender values (Handoyo & Morita, 2006). Ammonia level can be a limiting factor that inhibits tempeh mold sporulation and growth at the later stage of fermentation (Sparringa & Owens, 1999b).
3.2.2 Monitoring
Different analytical methods to analyze the changes of tempeh qualities during fermentation are summarized here, including the monitoring of volatile compounds, glucosamine content, texture, visual, temperature, mycelium weight, and dielectric permittivity. Volatile compounds produced during tempeh fermentation can be detected and identified using electronic nose sensors coupled with chemometrics, producing 97.33% accuracy (Hidayat et al., 2018; Hidayat & Triyana, 2016), as well as gas chromatography (Fujio, 1997). Tempeh fermentation done on different substrates (malt extract agar, barley, and soybean) can produce similar volatile compounds, but mushroom odor compounds, that is, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh (Feng et al., 2007). Glucosamine, a component of mycelium, can be detected to monitor biomass change with a conversion factor of 1 g of glucosamine per 12 g of dry fungal biomass (Sparringa & Owens, 1999a).
Texture change during tempeh fermentation can be analyzed based on hardness, cohesiveness, springiness, gumminess, chewiness, and resilience using response surface methodology (Nayak & Panda, 2016) and on modulus elasticity, surrender value, and weakness using a rheometer (Handoyo & Morita, 2006; Manurukchinakorn & Fujio, 1997). In soy, barley, and cowpea (Vigna unguiculata) tempehs, visual analysis was conducted based on color, surface structure, visibility of grain, and disorganization of grain cell structure, which then can be translated using image-processing algorithms to detect the correlation with physiological parameters, for example, protein, lipid, starch, and ergosterol contents (Feng et al., 2007; Handoyo & Morita, 2006). Using a thermogram, temperature can be monitored to detect the stationary phase in tempeh fermentation, which can be combined with mycelium weight (Matsumoto & Imai, 1990). Dielectric permittivity could also be measured at radio frequencies to monitor biomass changes in real-time and indicate fermentation stages (Davey et al., 1991).
3.2.3 Inoculum
3.2.3.1 Rhizopus oligosporus
The most widely used inoculum in tempeh production, R. oligosporus or R. microsporus var. oligosporus, can be regarded as the nontoxic, less-sporulating, and more vitamin-producing relative of R. microsporus. Rhizopus microsporus can have a toxin-producing endosymbiont bacterium in its mycelium such as Burkholderia that produces rhizonin, a hepatotoxic cycloprotein (Partida-Martinez et al., 2007). Compared to Rhizopus azygosporus, R. oligosporus had a defect in the spore formation process, producing 10% to 31% of irregular spores (Jennessen et al., 2008). A potentially infectious species of Rhizopus, that is, R. azygosporus, was previously isolated from the peritoneal cavity, kidney, and liver of three premature Australian babies who died due to infection (Schipper et al., 1996). The use of the domesticated and safe R. oligosporus in tempeh production is of similar resemblance with the use of the domesticated cheese fungus Penicillium camemberti, which also has a "wild" relative Penicillium commune (Schipper et al., 1996).
Several strains of R. oligosporus that have been studied include R. oligosporus NRRL 2710 (Wang, 1986), one of the first strains introduced in the United States, and R. oligosporus NRRL 2549, which showed more rapid mycelium growth (Hachmeister & Fung, 1993). Rhizopus oligosporus can grow at 25 to 37 °C with a temperature of 37 °C providing the most luxuriant growth and sporulation on rice or cassava root (Shambuyi et al., 1992). Rhizopus oligosporus can form vitamin B2, nicotinic acid, nicotinamide, and vitamin B6 at significantly higher levels compared to Rhizopus arrhizus and R. stolonifer (Keuth & Bisping, 1993).
3.2.3.2 Rhizopus microsporus var. chinensis
Like R. oligosporus, R. microsporus var. chinensis did not produce toxin (Jennessen et al., 2005) despite its close relationship to R. microsporus, which can have a toxin-producing endosymbiont bacterium in its mycelium (Partida-Martinez et al., 2007). Rhizopus microsporus var. chinensis hydrolyzed sucrose and raffinose, whereas R. oligosporus did not (Schwertz et al., 1997). When combined with Aspergillus oryzae, fermentation using R. microsporus var. chinensis in grass pea seeds resulted in higher bioavailability of protein, protein hydrolysis, and levels of free amino acid (Starzyńska-Janiszewska, Stodolak, & Wikiera, 2015a).
3.2.3.3 Rhizopus oryzae
Rhizopus oryzae has been reported in one of the first studies about the functionality of tempeh fermentation. Molecular identification studies by Febriani et al. (2018) and Vebliza et al. (2018) reported that some Rhizopus species that were morphologically identified as R. oligosporus UICC 116, R. arrhizus UICC 36 and UICC 55, and R. oryzae UICC 85, UICC 119, UICC 120, and UICC 135 were genetically identified as R. oryzae CBS 112.07(T) (Febriani et al., 2018; Vebliza et al., 2018).
Rhizopus oryzae improved the digestibility of soybeans by decreasing the hemicellulose content by about 50%, converting more than 50% of the protein content into amino acids, and promoting vitamin B1 content (150 μg per 100 g) (van Veen & Sohaefer, 1950). Fermentation with R. oryzae in oats resulted in decreased phytate content (by 74%) as well as increased total phenolic content and antioxidant activities (Cai et al., 2014). Rhizopus oryzae is autotrophic to vitamin B2 and vitamin B3 (Roelofsen & Talens, 1964). Supplementation of tempeh fermented using R. oryzae decreased cecal Enterobacteriaceae content, increased cecal propionate and acetate contents in rats (Yang et al., 2018), as well as decreased free cholesterol level in the livers of rats fed with a high-fat diet (Kameda et al., 2018).
3.2.3.4 Rhizopus stolonifer
Rhizopus stolonifer increased daidzein and genistein levels more than R. oligosporus and R. oryzae by up to twofold (Kameda, Aoki, Yanaka, Kumrungsee, & Kato, 2018). In rats fed with a high-fat diet, only supplementation of tempeh fermented with R. stolonifer improved liver function by significantly suppressing serum aspartate transaminase, total bilirubin, and ammonium levels (Kameda et al., 2018). Supplementation of R. stolonifer tempeh also improved gut health in rats by increasing the amounts of Bifidobacterium, Lactobacillus, propionate, and acetate, as well as decreasing cecal Enterobacteriaceae and Akkermansia muciniphila levels (Yang et al., 2018). Rhizopus stolonifer can form vitamin B2, nicotinic acid, nicotinamide, and vitamin B6 (Keuth & Bisping, 1993).
3.2.3.5 Other inocula
Recent molecular studies reported that several strains that were previously morphologically identified as R. oligosporus (UICC 27, UICC 40, UICC 51, UICC 67) and R. arrhizus (UICC 26, UICC39, and UICC 121) were genetically identified as R. delemar CBS 120.12(T) through rDNA Internal Transcribed Spacer (ITS) sequencing (Khasanah et al., 2018; Vebliza et al., 2018). Rhizopus arrhizus can form vitamin B2, nicotinic acid, nicotinamide, and vitamin B6 (Keuth & Bisping, 1993).
3.2.4 Starter culture
According to Nout et al. (1992), tempeh inoculation was performed traditionally by having the cooked ingredients rubbed with usar, an Indonesian term for waru (Hibiscus spp.) leaves grown with the mycelium of Rhizopus spp. that was heavily sporulated. Although waru leaves can contain other organisms, mainly Cladosporium spp., it did not provide growth-inhibition selectivity (Nout et al., 1992). Semi-traditionally, small pieces of freshly made tempeh can be used to initiate new fermentation, but this technique can lead to some food safety concerns due to contamination risk as well as the deteriorating quality of the dehydrated mycelia throughout the preservation (Wang et al., 1975).
Modern tempeh starter is usually made of R. oligosporus grown and desiccated (to a w = 0.48) on rice or cassava root powders (Shambuyi et al., 1992). Cassava root flour-based starter culture can last for up to 7 months at 5 to 25 °C and would be best used in less than 2 weeks when stored at the lowest temperature (Shambuyi et al., 1992). Rice flour-based starter culture can last for 1 year at 4 °C or room temperature as well as have tolerance against contamination up to 108 counts per gram of cooked soybeans (Rusmin & Ko, 1974).
Throughout the storage period, the number of dormant spores that can germinate decreased (Thanh et al., 2007). Factors that affect the activation and germination of spores include glucose, phosphate, and amino acids; in which L-alanine, L-leucine, and L-isoleucine stimulate, whereas L-proline inhibited alanine uptake (Thanh et al., 2005).
3.2.5 Wrapping material
Leaves, especially banana leaves, were used to wrap tempeh traditionally (Owens et al., 2015), but have been replaced by perforated plastic bags (polyethylene) for convenience and access in nontropical countries. On tempeh composition, both materials facilitated increases in antioxidant activities as well as total phenolic, daidzein, and genistein levels at different timings. Tempeh in banana leaf reached the peak of total phenolic content earlier, on days 1 and 2, whereas tempeh in plastic bag reached the peak on day 4. These results are likely due to difference in oxygen permeability that affects Rhizopus spp. growth, which is easier to control if perforated plastic bag is used instead of leaf. Because tempeh was most commonly and optimally harvested after 30 hr of fermentation at 32 °C (Handoyo & Morita, 2006; Sudarmadji & Markakis, 1978), the antioxidant content of tempeh wrapped in banana leaves can be superior to that of tempeh wrapped in plastic.
In terms of aroma, wrapping material can determine the presence of volatile aromatic compounds. A-pinene, a terpenoid that promotes salty and beany aromas, was found only in tempeh wrapped in banana leaves, whereas sec-butyl nitrite (promoting cereal-like aroma), α-bisabolene (promoting "green" aroma), and piperazine (no aroma) were found only in tempeh wrapped in plastic (Harahap et al., 2018). To reduce waste from wrapping materials, tempeh fermentation can also be done in a tray (Martinelli et al., 1964). However, further research is needed to determine how tray fermentation affects the quality and taste of the final tempeh product.
3.2.6 Perforation
The use of plastic bag instead of leaf allowed more control over perforation intensity and oxygen permeability. Bhowmik et al. (2013) reported significantly higher mold population density and texture parameters, that is, firmness, springiness, resilience, gumminess, and chewiness in tempeh with nine perforations compared to zero, seven, and eight perforations in plastic petri dishes after 36 hr of fermentation. Four perforations were located along the side of the petri dish, with 6.9 cm distance, and three, four, or five holes were located at the bottom of petri dish, 4 cm apart (Bhowmik et al., 2013).
Fermentation without perforation (anaerobic) was adopted as an additional step by Yusof et al. (2013) and Watanabe et al. (2007) to increase the levels of free amino acid (including gamma-aminobutyric acid [GABA]), peptide, and antioxidant activity in the tempeh product. Anaerobic fermentation was performed at 30 °C for 30 hr after fermenting soybeans with Rhizopus spp. 5351 for 30 hr at 30 °C (Yusof et al., 2013), or for 5 hr after normal fermentation with R. microsporus for 20 hr (Watanabe et al., 2007).
3.2.7 Microbial community and co-inoculation
Microorganisms other than Rhizopus spp. can be found in the traditional tempeh-making process, including lactic acid bacteria, Zygomycota (e.g., Absidia spp.), mold (e.g., Mucor spp. and Rhizomucor spp.), and yeasts (Wikandari et al., 2012). The lactic acid bacteria included Enterococcus faecium, Leuconostoc lactis, Leuconostoc spp. delbrueckii, and Alicyclobacillus spp. (Pisol et al., 2015), whereas Lactobacillus agilis, L. fermentum, and Enterococcus were the Firmicutes that were found predominantly in the tempeh and the water it was soaked in (Radita, Suwanto, Kurosawa, Wahyudi, & Rusmana, 2017). Clostridium was also found in the starter culture, but did not dictate the final bacterial composition in tempeh (Radita et al., 2017). Lim and Tay (2011) reported finding the following yeasts in tempeh: Pichia guilliermondii, Candida tropicalis, Pichia norvegensis, Sporopachydermia lactativora, and Trichosporon asahii, the latter of which was a food safety concern due to being the most frequently isolated species that can cause mild cutaneous infections and resistance to several antibiotics (Lim & Tay, 2011).
Tempeh fermentation can also be done by pairing the main inoculum, that is, Rhizopus with other microorganisms. The first example is the co-inoculation with L. plantarum, which has been identified as a probiotic (Helmyati et al., 2016; Nout et al., 1987). Supplementation of tempeh co-inoculated with L. plantarum resulted in the amelioration of hyperglycemia, hyperlipidemia, and hyperinsulinemia in rats with Streptozotocin-induced type 2 diabetes mellitus by altering intestinal bacterial distribution and increasing intestinal short-chain fatty acid levels (Huang et al., 2018). During tempeh fermentation, the addition of approximately 106 CFU/g of L. plantarum completely inhibited the growth of Salmonella infantis, Enterobacter aerogenes, E. coli, S. aureus, L. monocytogenes, Streptococci, Lactobacilli, and Micrococci in soy, fava bean, pea, and chickpea tempehs (Ashenafi, 1991; Ashenafi & Busse, 1989, 1991a, 1991b, 1992, 1992).
Co-inoculation of R. oligosporus and Bacillus subtilis on soybeans can produce tempeh-natto that exhibited a higher health-promoting potential in vitro and in vivo (Chung et al., 2009). The tempeh-natto had high in vitro α,α-diphenyl-β-picryl hydrazyl (DPPH)-scavenging effects and angiotensin-converting enzyme (ACE) inhibitory activity with the IC50 values of 66.9 and 0.6 mg/mL, respectively, and increased in vivo antioxidant status and decreased lung ACE activity in hypertensive rats at doses between 0.4 and 0.8 g/kg (Chung et al., 2009).
In soybeans, co-inoculation of R. oligosporus and Aspergillus elegans produced tempeh with reduced levels of flatulence-inducing oligosaccharides and IgE immunoreactivity, as well as increased soluble protein and peptide levels (Huang et al., 2019). In barley, co-inoculation of R. oligosporus with yeasts (Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia anomala, and Kluyveromyces lactis) slightly increased vitamin B6 and niacinamide levels and slightly decreased vitamin B content (Feng et al., 2007). In grass peas, combination of R. microsporus var. chinensis and an equal or lower dose of Aspergillus oryzae resulted in tempeh with increased in vitro bioavailability of protein, protein hydrolysis activity, and amino acid content (Starzyńska-Janiszewska, Stodolak, & Wikiera, 2015a). Grass pea tempeh fermented with R. oligosporus and Aspergillus oryzae produced tempeh with 70% higher radical scavenging ability, threefold higher vitamin B1 level, and twofold vitamin B2 level compared to tempeh inoculated only with R. oligosporus (Starzyńska-Janiszewska et al., 2012).
Bacteria isolated from tempeh can increase the levels of nicotinic acid and nicotinamide (Lactobacillus spp. and C. freundii) and thiamine (C. freundii) and transform phenolic compounds (glycitein to 6,7,4′-trihydroxyisoflavone/factor 2 by Brevibacterium epidermidis and Micrococcus luteus as well as daidzein to factor 2 and glycitein) (Denter & Bisping, 1994; Klus et al., 1993). Inoculation of K. pneumoniae and Trichosporon beigelii increased the levels of biogenic amines, that is, tyramine and putrescine, which are toxicants, by 11%. Addition of L. plantarum reduced these amines by 50% (Nout et al., 1993).
3.2.8 Fortification
According to the WHO, food fortification is a deliberate practice of increasing the content of essential micronutrient for the purpose of improving the nutritional quality of food supply and providing a public health benefit with minimal risk to health (Allen, 2006). Food fortification has been considered to be highly effective for preventing micronutrient malnutrition (Miller & Welch, 2013).
Tempeh has been reported as a promising medium for iron fortification, one of the three most prevalent forms of micronutrient malnutrition (Allen, 2006). In tempeh production, fortification can be done by adding the nutrient before the fermentation step and after the drying step. The addition of 28 to 112 mg/kg of sodium ferric ethylenediaminetetraacetic acid (NaFeEDTA) before fermentation at 32 °C for 16 to 32 hr did not alter organoleptic properties and increased hemoglobin levels in female Wistar rats at 24 ppm more than supplementations of FeSO4 and regular tempeh (Sudargo et al., 2015). Similar results were observed in anemic male Sprague–Dawley (SD) rats (Kusuma & Ermamilia, 2018). In tempeh made of 30 g of soybeans, fortification with 0.166% (w/w) of NaFeEDTA promoted iron (Fe) levels of 12.54 mg before cooking and 8.40 mg after boiling—meeting the recommended daily allowance (RDA) of 8 to 15 and 7.74 mg after frying (Mahardika et al., 2020). Helmyati et al. (2016) fortified tempeh with 50 ppm of FeSO4 and synbiotics (L. plantarum Dad13 and fructo-oligosaccharides) resulting in significantly increased blood hemoglobin and body weight in anemic Wistar rats (Helmyati et al., 2016).
3.2.9 Enzymatic activity
Physical and chemical changes during tempeh fermentation were led by the penetration of mold mycelium into substrate (approximately 2 mm deep within 40 hr of fermentation) (Jurus & Sundberg, 1976; Varzakas et al., 1997), in which enzymes play an important role. Cellulase, pectinase, amylase, protease, and lipase have been identified throughout the tempeh fermentation process (Manurukchinakorn & Fujio, 1997; Ruiz-Terán & Owens, 1996a), with the first four having the strongest correlation with degree of maceration (Manurukchinakorn & Fujio, 1997). These enzymes promoted degradation of protein, crude lipid, triglycerides, and glycerol and production of free ammonia and free fatty acids (Ruiz-Terán & Owens, 1996a). Heskamp and Barz (1998) identified intracellular, extracellular, and cell wall-bound proteases in nine strains of Rhizopus spp., including R. oryzae, R. microsporus var. chinensis, R. stolonifer, R. oligosporus (isolates Sama, J16, MS5), and R. oligosporus strain NRRL 2710. The dominant protease activity was in the cell walls of fungal hyphae (Heskamp & Barz, 1998).
3.2.10 pH Level
Sparringa and Owens (1999a) observed that alkalization in tempeh was mainly promoted by the release of ammonia, which accounted for approximately 40% of alkalization in mature tempeh (46 hr of fermentation) and almost entirely in aging tempeh (46 to 72 hr of fermentation) at 30 °C. This fermentation increased the initial pH level of 4.6 to 6.6 in mature tempeh (46 hr of fermentation) and to 7.1 in aging tempeh (72 hr of fermentation) (Sparringa & Owens, 1999a). Ammonia production contributed more to alkalinization compared to consumption of lactic acid, which accounted for only 3% (Sparringa & Owens, 1999c). After 18 hr of fermentation, the pH level of tempeh rapidly increased (Matsumoto & Imai, 1990).
3.2.11 Moisture and water activity
The optimum water activity (a w) for tempeh fermentation with R. oligosporus (NRRL 5905) in soy ranged from 0.98 to 1.00, where 0.99 to 1.00 was optimum for mycelial growth, polygalacturonase activity, and xylanase activity, whereas 0.98 was optimum for endocellulase, although reduced mycelial growth (Sarrette et al., 1992). The optimum humidity for tempeh fermentation with 3.5 × 104 CFU/g substrate of R. oligosporus (UCW-FF8001) was 620 g/kg (Penaloza et al., 1992). For the germination phase at 40 °C, 0.995 a w was optimum, 0.5% v/v oxygen was tolerable, and 5% to 10% (v/v) CO2 could be inhibitory (Han & Nout, 2000).
3.2.12 Substrate: Beyond soy
Although not standardized like soybean tempeh, tempehs made of other ingredients, pure or mixed, exist. These nonsoy tempehs are referred by mentioning the substrate before the word "tempeh," for example, pigeon pea tempeh. Some of these other types of tempeh that have been studied are chickpeas, lentils, white beans, black beans broad beans (Erkan et al., 2020), black gram (Yadav & Khetarpaul, 1994), green grams (Lakshmy & Usha, 2010), yam-beans (Azeke et al., 2007), velvet beans (Pugalenthi et al., 2005), rice bran (Nurrahma et al., 2018), barley (Feng et al., 2005), peanuts (Matsuo, 2006b), sunflower seeds (Vaidehi et al., 1985), lupin beans (Jiménez-Martínez, Hernández-Sánchez, & Dávila-Ortiz, 2007), pigeon peas (Ali, 2008), quinoa (Matsuo, 2006a), oats (Cai et al., 2014), millet (Anandito et al., 2018), cowpeas (Lakshmy & Usha, 2010), koro benguk (Mucuna pruriens) (Winarni & Dharmawan, 2017), buckwheat kernels (Wronkowska et al., 2015), red sorghum (Hachmeister & Fung, 1993), wheat (Hachmeister & Fung, 1993), fava beans (Berghofer et al., 1998), peas (Ashenafi & Busse, 1991d), koro kratok bean (Phaseolus lunatus) (Pertiwi et al., 2020), jack bean (Canavalia ensiformis) (Puspitojati et al., 2019), okara (filtration residue of soymilk production) (Matsuo, 1996), finger millet (Eleusine coracana) (Mugula & Lyimo, 1999), cottonseed kernels, and corn grits (Matsuo, 2000).
3.2.13 Other uses
The principle mechanisms of tempeh fermentation that increased protein content and bioavailability, antinutrient content, and mycelium mass; produced enzymes; and decreased antinutrient have also been applied in the productions of animal feed, high-protein fungal mass, and enzyme. Tilapia feed produced by fermenting chickpeas with R. oligosporus NRRL 2710 increased protein content by 13.1%, apparent digestibility of dry matter by 23.2%, and apparent digestibility of protein by 41.9%, as well as decreased phytate content by 45% (González et al., 2018).
Water-soluble fibrinolytic enzymes with thrombolytic activity were detected in black soybean tempeh (Poernomo, 2017), leading to isolation of fibrinolytic enzyme-producing organisms from tempeh, that is, Bacillus licheniformis RO3, B. pumilus, Fusarium spp. BLB, and B. subtilis (Afifah et al., 2017; Kim et al., 2006; Sugimoto et al., 2007).
3.3 Effects of tempeh fermentation on health-promoting effects of food ingredients
3.3.1 On nutritional content
Different kinds of tempeh demonstrated different characteristics and nutritional change (Table 5). In general, tempeh fermentation can increase crude protein, soluble protein, amino acid, antioxidant, crude fiber, ash, and vitamin contents as well as decrease the levels of antinutrients and crude lipid.
| Substrate | Effect of tempeh fermentation | Source |
|---|---|---|
| Soybean (Glycine max) | Increased crude and soluble protein, mineral, antioxidant bioavailability and activity, crude fiber, and ash levels; added vitamin B12 content; decreased antinutrient levels (phytate, trypsin inhibitor, oxalate, and oligosaccharides) | Ahmad et al., 2015; Areekul et al., 1990; Ashenafi & Busse, 1991d; Berghofer et al., 1998; Borges et al., 2016; Chang et al., 2009; Esaki et al., 1994; Ferreira et al., 2011; Kuligowski et al., 2017; Liem et al., 1977; Paredes-López & Harry, 1989; Stodolak & Starzynska-Janiszewska, 2008; Sudarmadji & Markakis, 1977; van der Riet et al., 1987; Wang & Murphy, 1996; Xiao et al., 2016 |
| Chickpea (Cicer arietinum) | Increased crude and soluble protein, antioxidant activity, fiber content, and ash levels; decreased antinutrient (oligosaccharides) level | Ashenafi & Busse, 1991d; Reyes-Moreno et al., 2000; Sánchez-Magana et al., 2014; Tewari, 2002 |
| Black gram (Vigna mungo) | Increased protein digestibility; decreased antinutrient (phytate) and polyphenol levels | Yadav & Khetarpaul, 1994 |
| Yam-bean (Pachyrhizus erosus) | Decreased antinutrient (cyanogenic glycoside) content | Njoku et al., 1991 |
| African yam-bean (Sphenostylis stenocarpa) | Decreased antinutrient (cyanogenic glycoside) content | Azeke et al., 2007 |
| Velvet bean (Mucuna pruriens) | Increased soluble protein, amino acid, and L-dopa contents; decreased antinutrient content (phytate) | Ariani et al., 2016; Higasa et al., 1996; Pugalenthi et al., 2005 |
| Common bean (Phaseolus vulgaris) | Increased in vitro protein bioavailability, protein efficiency ratio, antioxidant activity and content; decreased antinutrient (oligosaccharides, trypsin inhibitor) content | Paredes-López & Harry, 1989; Rochín-Medina et al., 2015 |
| Barley (Hordeum vulgare) | Decreased antinutrient (phytate) content | Eklund-Jonsson et al., 2006 |
| Lupin (Lupinus spp.) | Increased amino acid lysine content; decreased antinutrient (oligosaccharides and quinolizidine alkaloids) content | Chango et al., 1993 |
| Peanut (Arachis hypogaea) | Increased amino acid and fatty acid contents | Bujang & Taib, 2014; Matsuo, 2006c |
| Quinoa (Chenopodium quinoa) | Increased the levels of protein, free amino acid, fiber, phenolic acids, in vitro antiradical activity and ex vivo antioxidant activities in the liver | Matsuo, 2006a; Starzynska-Janiszewska et al., 2017 |
| Buckwheat (Fagopyrum esculentum) groat | Increased protein content and digestibility | Wronkowska et al., 2015 |
| Bambara nut (Vigna subterranea) | Increased protein and fat levels; decreased carbohydrate content | Amadi et al., 1999 |
| Oat (Avena sativa) | Increased antioxidant activity and content; decreased antinutrient (phytate) content | Cai et al., 2014 |
| Wheat (Triticum spp.) | Increased vitamins B1, B2, and B3 | Wang & Hesseltine, 1966 |
| Fava bean (Vicia faba) | Increased the levels of crude and soluble protein, crude fiber, ash, and phenolics as well as antioxidant activity; decreased crude fat content | Ashenafi & Busse, 1991d; Berghofer et al., 1998; Polanowska et al., 2020 |
| Rapeseed (Brassica napus) | Increased levels of aromatic amino acids and ammonia, decreased antinutrient (alpha-galactosides) content | Bau et al., 1994 |
| Pea (Pisum sativum) | Increased or decreased crude protein content; increased soluble protein, crude fat, crude fiber, and ash levels | Ashenafi & Busse, 1991d; Reiss, 1993 |
| Grass pea (Lathyrus sativus) | Increased protein bioavailability; decreased antinutrient (phytate, trypsin inhibitor, and ODAP) content | Kebede et al., 1995; Stodolak & Starzynska-Janiszewska, 2008 |
| Koro kratok (Phaseolus lunatus) | Increased angiotensin converting enzyme (ACE) inhibitory activity | Pertiwi et al., 2020 |
| Jack bean (Canavalia ensiformis) | Increased angiotensin converting enzyme (ACE) inhibitory activity | Puspitojati et al., 2019 |
| Spelt wheat (Triticum aestivum) | Increased soluble protein, phenolic acid, and ash contents; decreased starch content | Starzynska-Janiszewska et al., 2019 |
3.3.1.1 Protein content and bioavailability
Tempeh fermentation increased the amounts of crude and soluble protein in tempeh made from soybeans (9.6% to 16% and 25% to 66.4%), chickpeas (6.2% and 62.7%), buckwheat kernels (13.3% and 87%), fava beans (4.6% and 60.7%), peas (12.1% and 62.3%), black beans (9.5% and 24.5%), and bambara nuts (38% and 73.1%) (Ashenafi & Busse, 1991d; Bavia et al., 2012; Paredes-López & Harry, 1989; Pugalenthi et al., 2005; Wronkowska et al., 2015). In soy, protein solubility slightly increased within the first 18 hr and significantly increased after 18 hr of tempeh fermentation, whereas most amino acid content significantly increased after 16 hr of tempeh fermentation at 27 °C—aspartic acid, cysteine, methionine, phenylalanine, and arginine remained constant throughout fermentation (Matsumoto & Imai, 1990). Rhizopus spp. produced intracellular, extracellular, and cell wall-bound proteases, with the latter exhibiting the main proteolytic activity (Heskamp & Barz, 1998).
Activity of protease and production of free ammonia were detected in the first 32 hr of tempeh fermentation (Ruiz-Terán & Owens, 1996a). After 46 hr of tempeh fermentation at 30 °C, 25% of the initial protein content was hydrolyzed, in which 65% remained in tempeh as amino acids and peptides, 25% was assimilated into mold biomass, and 10% was oxidized (Sparringa & Owens, 1999c). Bioactive peptides, which have been considered important due to their antihypertensive, antidiabetic, antioxidative, and/or antitumor activities, were found in hygienic tempehs in higher amounts compared to nonhygienic tempehs (Tamam et al., 2019).
In fava bean, tempeh fermentation increased the levels of released GABA and total amino acids by 10-fold (Polanowska et al., 2020). In spelt wheat, tempeh fermentation increased protein content, reaching 12.7 g per 100 g dw (Starzynska-Janiszewska et al., 2019). In white and colored quinoa, tempeh fermentation increased the levels of protein by 15% to 20%, free amino acid by 5.5 to 9-fold, and fiber by 48% (Starzynska-Janiszewska et al., 2017).
3.3.1.2 Lipid content, free fatty acids, and phytosterols
Decrease in crude lipids was observed in soybean (27.6%), chickpea (38.9%), fava bean (60.8%), pea (37.5%), black bean (12.5% to 25%), and bambara nut (73.2%) tempehs (Ashenafi & Busse, 1991d; Paredes-López & Harry, 1989; Pugalenthi et al., 2005). In soy, production of free fatty acid and lipase activity were identified in the first 32 hr of fermentation at 30 °C, resulting in 30% loss of crude lipid content (Ruiz-Terán & Owens, 1996a). Palmitic acid content was increased and linoleic acid content was decreased in soy, whereas in chickpea linoleic acid was increased (Paredes-López et al., 1991; Wagenknecht et al., 1961). In fava bean, tempeh fermentation released phytosterols, that is, stigmasterol and campesterol (Polanowska et al., 2020). Inversion of linolenic acid and increase of gamma-linolenic acid content by 21% were also observed during tempeh fermentation (Hering et al., 1991).
3.3.1.3 Carbohydrate
Tempeh fermentation left very little soluble carbohydrate in soy (van Veen & Sohaefer, 1950), decreased carbohydrate content by 10.3% in the common bean (Paredes-López & Harry, 1989), decreased starch content in spelt wheat (Starzynska-Janiszewska et al., 2019), and degraded ethanol-soluble sugars (alpha-galactosides, flatulence generator included) in rapeseed (Bau et al., 1994). In soy, the proportion of reducing sugar to total sugar was increased between 8 and 20 hr of tempeh fermentation at 27 °C (Matsumoto & Imai, 1990). In bambara nut, tempeh fermentation decreased carbohydrate content by 50% (Amadi et al., 1999).
3.3.1.4 Ash and mineral content
After tempeh fermentation, increased ash content was found in soybean (21.6%), chickpea (26.2%), fava bean (15.2%), spelt wheat (1.9%), and pea (17.4%) tempehs (Ashenafi & Busse, 1991d; Starzynska-Janiszewska et al., 2019), and decreased ash content was found in bambara nut (44.8%) and black bean (4.3%) tempehs (Paredes-López & Harry, 1989; Pugalenthi et al., 2005). In buckwheat kernels, tempeh fermentation increased Fe (31.6% to 35.9%), Cu (82% to 86.8%), P (16.3% to 17.8%), Mg (25.6% to 31.8%), K (24.9% to 30.9%), and Zn (13.7% to 22.7%), and maintained or decreased Ca content by 11.8% (Wronkowska et al., 2015). In terms of iron, tempeh fermentation increased Fe content and bioavailability by increasing the amount of iron(II)-species as well as decreasing complexed iron and iron(III)-species (Tawali & Schwedt, 1998).
3.3.1.5 Vitamin B12
Vitamin B12 deficiency has been a severe problem in the Indian subcontinent, Mexico, Central and South America, selected areas in Africa, and among vegetarians in Asia (Stabler & Allen, 2004). Vitamin B12 deficiency can lead to hazardous health conditions, for example, pernicious anemia, megaloblastic anemia, and hyperhomocysteinemia (Stabler & Allen, 2004). One of the main reasons of such a prevalence has been the limited number of dietary sources of vitamin B12, especially in the plant-based category, which has been regarded as a more sustainable option compared to meat in terms of public health and environment (Godfray et al., 2018; Stabler & Allen, 2004). Having been regarded as the richest plant-based source of vitamin B12 (Shurtleff & Aoyagi, 1979), tempeh has the potential to be a solution to the need for plant-based source of protein containing vitamin B12.
Vitamin B12 in tempeh was naturally produced by bacteria, for example, K. pneumoniae and C. freundii (Areekul et al., 1990; Keuth & Bisping, 1993; Liem et al., 1977; Okada, 1989). The vitamin B12-producing K. pneumoniae strains in tempeh were not pathogenic given that they did not produce enterotoxin and had different genetic profiles compared to those pathogenic to humans (Keuth & Bisping, 1993; Yulandi et al., 2016). In Indonesia, harmless Klebsiella spp. that did not have virulence-associated genes, for example, rmpA, could be found in most tempehs (Cesrany, Yulandi, Rusmana, & Suwanto, 2017).
Vitamin B12 content in tempeh can be highly varied (0.07 to 12.4 μg/100 g tempeh) because the presence of vitamin B12-producing bacteria has been mostly coincidental or due to contamination (Liem et al., 1977; United States Department of Agriculture, 2019). Commercial tempehs were found to contain 0.34 to 2.44 μg/100 g in Indonesia, 5.29 μg/100 g in Canada, and 15 μg/100 g in the United States, whereas the USDA FoodData Central listed some tempehs to contain 1.26 μg/100 g of vitamin B12 (Liem et al., 1977; United States Department of Agriculture, 2019). In lupin tempeh, in situ co-inoculation of Propionibacterium freudenreichii with R. oryzae or R. oligosporus resulted in 0.97 and 103.32 μg/100 g vitamin B12, respectively (Signorini et al., 2018; Wolkers–Rooijackers, Endika, & Smid, 2018). Applicable technology to produce vitamin B12-containing tempeh consistently is needed because fulfilling the RDA of 1.8 μg is within reach.
3.3.1.6 Other vitamins
Tempeh fermentation can promote the content of B vitamins in soy, barley, and wheat tempeh due to the ability of Rhizopus spp. to biosynthesize riboflavin, niacin, nicotinamide, and vitamin B6 (Feng et al., 2007; Keuth & Bisping, 1993; Roelofsen & Talens, 1964; Wang & Hesseltine, 1966). In pure culture models, R. oligosporus produced riboflavin, nicotinic acid, nicotinamide, and vitamin B6 in greater quantities than R. arrhizus and R. stolonifer (Keuth & Bisping, 1993). In wheat, tempeh fermentation increased riboflavin and niacin, but decreased vitamin B1 levels (Wang & Hesseltine, 1966). Tempeh fermentation could also increase beta-carotene and ergosterol, and decrease free tocopherols (Denter et al., 1998).
In barley, production of vitamin B6 and niacin was increased by S. cerevisiae co-inoculation, although vitamin B1 and biotin were decreased (Feng et al., 2007). Nicotinic and nicotinamide contents could also be increased by the presence of Lactobacillus spp. and C. freundii, which the latter can also produce vitamin B1 (Denter & Bisping, 1994). In buckwheat groats, tempeh fermentation increased thiamine and riboflavin contents by 2.5- and 7.5-fold, respectively (Starzynska-Janiszewska et al., 2016).
Total folate was found to be four to five times higher after tempeh fermentation (Ginting & Arcot, 2004; Murata et al., 1970). This result was due to de novo formation of folate compounds, that is, N5-formyl-5,6,7,8-tetrahydropteroyl-glutamic acid, 5-formyl-tetrahydrofolate, 10-formyl tetrahydrofolate, and rhizopterin/N10-formylpteroic acid by R. oligosporus (Ginting & Arcot, 2004; Sanke et al., 1971). Tempeh fermentation also increased biotin content by 2.3-fold (Murata et al., 1970).
3.3.2 On bioactive compounds
3.3.2.1 In soybean
Most of the biological activities of tempeh related to cancer inhibition, cognitive function, lung health, cardiovascular health, liver health, type 2 diabetes mellitus, skeletal muscle recovery, and malnutrition were hypothesized to be due to its soy isoflavone content. Tempeh can be one of the most bioavailable sources of isoflavones in comparison to other soy foods. Whole soybean foods, for example, soymilk, tofu, and tempeh contained higher concentrations of isoflavones compared to the "second-generation soy foods," for example, soy-based hot dog, burger, or noodles (Baiano, 2010). Among whole soybean foods, isoflavones in fermented soy foods, for example, miso and tempeh, were found to be more bioavailable compared to unfermented soy products, for example, soy protein and soymilk derivatives, by being higher in unconjugated isoflavone aglycone levels and lower in conjugated isoflavone glycosides such as malonyl glycosides (Baiano, 2010). Compared to some other fermented soy foods, that is, tofu and bean curd sheet, tempeh contained significantly higher levels of isoflavones in both raw and cooked forms (Haron et al., 2016).
In yellow and black soybean, tempeh fermentation decreased the levels of conjugated isoflavones, for example, malonyl glycosides, and increased the levels of unconjugated isoflavones, for example, daidzein and genistein. Tempeh fermentation decreased malonyl-genistin content after soaking and cooking, generated acetyl-daidzin and acetyl-genistin during heat processing (Wang & Murphy, 1996), and increased daidzein and genistein concentrations after fermentation due to fungal enzymatic hydrolysis (Berghofer et al., 1998; Borges et al., 2016; Esaki et al., 1994; Kuligowski et al., 2017; Rochín-Medina et al., 2015; Sánchez-Magana et al., 2014; Wang & Murphy, 1996). After 48 hr of fermentation, the levels of daidzein and genistein increased by approximately fourfold and sixfold, respectively, with increased antioxidant activity by four- to sixfold; meanwhile 4 to 5 days of fermentation increased the levels of daidzein and genistein by up to sixfold and ninefold, respectively, with increased antioxidant activity by up to 12-fold in 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) tests (Kuligowski et al., 2017).
In DPPH tests, tempeh fermentation also increased free-radical and superoxide scavenging activities, reducing power, and inhibitory activity toward lipid peroxidation (Ahmad et al., 2015; Chang et al., 2009; Xiao et al., 2016). Although the studies mentioned mostly used chromatography techniques, a contradictive result was found in a study using enzyme-linked immunosorbent assay, reporting that tempeh only contained 18.07% of the original soybean isoflavones (Fernandez-Lopez et al., 2016). Phenolic compounds binding with protein could be another factor that may affect the detectability and bioavailability of phenolic compounds in tempeh (Bartolomé et al., 2000; Ushijima et al., 2001).
Bacteria present in tempeh fermentation can also modulate soy phenolic composition. Micrococcus spp. and Arthrobacter spp. can hydroxylate soy phenolics, that is, genistein to 5,6,7,4′-tetrahydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone, biochanin A to 4′-methoxy-5,7,8,-trihydroxyisoflavone, and biochanin A to 4′-methoxy-5,6,7-trihydroxyisoflavone (Klus & Barz, 1998). The same bacteria could also convert glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and 7,8,4′-trihydroxyisoflavone, daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone, as well as glycitein to factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone (Klus & Barz, 1995). Brevibacterium epidermidis and Micrococcus luteus can transform isoflavone glycitein to factor 2, whereas Microbacterium arborescens converted daidzein to factor 2 and glycitein (Klus et al., 1993).
3.3.2.2 In humans
In human studies, supplementation of tempeh resulted in higher recovery of daidzein and genistein in saliva and urine compared to solid soy foods (Table 6). Compared to soybean, tempeh supplementation promoted significantly higher levels of genistein and daidzein in saliva after 24 hr of treatment in males (20 to 40 years, N = 22) (Hutchins et al., 1995). Compared to texturized soy protein, tempeh supplementation in postmenopausal women stimulated higher urinary recovery levels of genistein and equol, as well as higher or similar levels of daidzein, whereas in premenopausal women it promoted similar or higher urinary recovery levels of genistein and daidzein as well as higher level of equol, and in men it promoted higher urinary levels of genistein and daidzein (Cassidy et al., 2006; Faughnan et al., 2004).
| Subject | Sample | Study | Amount of soy isoflavone | ||
|---|---|---|---|---|---|
| Genistein | Daidzein | Equol | |||
| Premenopausal women | Urine | Faughnan et al., 2004 | Soymilk > Tempeh, TVP | Tempeh = soymilk = TVP | Tempeh > soymilk > TVP |
| Urine | Cassidy et al., 2006 | Soymilk > Tempeh > TVP | Tempeh > Soymilk > TVP | NA | |
| Postmenopausal women | Urine | Faughnan et al., 2004 | Tempeh, Soymilk > TVP | Tempeh = Soymilk = TVP | Tempeh > soymilk > TVP |
| Urine | Cassidy et al., 2006 | Soymilk > Tempeh > TVP | Soymilk > Tempeh > TVP | NA | |
| Men | Urine | Cassidy et al., 2006 | Soymilk > Tempeh > TVP | Soymilk > Tempeh > TVP | NA |
| Urine | Faughnan et al., 2004 | Tempeh = soymilk = TVP | Tempeh = soymilk = TVP | Tempeh > soymilk > TVP | |
| Saliva | Hutchins et al., 1995 | Tempeh > soybean | Tempeh > soybean | NA | |
Compared to soymilk, tempeh supplementation increased urinary levels of equol in postmenopausal, premenopausal women, and men (Cassidy et al., 2006; Faughnan et al., 2004), and increased urinary level of daidzein in premenopausal women only (Cassidy et al., 2006). In general, soymilk supplementation promoted earlier and higher maximum concentration of isoflavones in urine compared to tempeh and texturized vegetable protein at equalized dose (0.44 mg isoflavone per kg bodyweight). Further research is needed to compare the effects across similar food forms, that is, soybean versus tempeh or soymilk versus tempeh milk.
3.3.2.3 In nonsoy substrates
In nonsoy substrates, tempeh fermentation also increased total phenolic content and antioxidant capacity. In chickpeas, tempeh fermentation increased total phenolic content by 2.78-fold and antioxidant activity by 1.80- to 1.94-fold (Sánchez-Magana et al., 2014). In grass peas (Lathyrus sativus), tempeh fermentation increased DPPH radical-scavenging activity (Starzyńska-Janiszewska et al., 2008). In fava beans and oats, tempeh fermentation released phenolic acids (Polanowska et al., 2020) and increased antioxidative potential using lard and sunflower oil oxidations tests (Berghofer et al., 1998). In spelt wheat, tempeh fermentation increased soluble phenolic acid and ferulic acid contents by 25% and 300%, respectively (Starzynska-Janiszewska et al., 2019). In buckwheat groats, tempeh fermentation increased antioxidative activity by up to 124% in ABTS assay (Starzynska-Janiszewska et al., 2016). In dark common bean, tempeh fermentation increased the levels of soluble phenols by 29%, condensed tannins by 140%, flavonoids to 0.35 g/kg, and antioxidative activity by 45% (Starzynska-Janiszewska et al., 2015). In quinoa, tempeh fermentation increased soluble phenol content (vanillic acid, protocatechuic acid, and rutin) and antiradical activity by 160% (Starzynska-Janiszewska et al., 2017; Starzynska-Janiszewska, Dulinski, Stodolak, Mickowska, & Wikiera, 2016).
3.3.3 On toxins and antinutrients
Tempeh fermentation has been shown to reduce the levels of antinutrients, including phytate, oxalate, 3-N-oxalyl-L-2,3-diaminopropionic acid (β-ODAP), trypsin inhibitor, flatulence-causing oligosaccharides, and antinutritive phenols. Compared to other soy foods, tempeh contained relatively low amounts of phytates (approximately 4.31 to 6.17 mg per serving), which have the potential to disrupt mineral absorption in the body and cause micronutrient malnutrition (Al-Wahsh et al., 2005; Amarakoon et al., 2012). The soaking, cooking, and fermentation steps in tempeh production reduced the phytate content, with fermentation showing the highest level of reduction (Abu-Salem et al., 2014; Tawali et al., 1998). Reduction in phytate content was also observed in common bean tempeh (Paredes-López & Harry, 1989). During tempeh fermentation, R. oligosporus produced intracellular, extracellular, and active phytases that were thermostable (some had the optimum temperature of 44 °C), active at pH 3.0 to 5.0, and partly inhibited by high concentrations of substrate (Azeke et al., 2011; Sutardi & Buckle, 1988).
In grass pea tempeh, traditional tempe gembus (tofu curd tempeh), soy, lamtoro (L. leucocephala), and common bean tempehs in Indonesia, phytate-degrading activities might come from lactic acid bacteria (Damayanti et al., 2017). Phytase-producing lactic acid bacteria have also been found in other fermented foods such as sourdough bread (Reale et al., 2007).
Tempeh contained relatively low amounts of oxalate, which can bind with calcium and promote the formation of kidney stones (Al-Wahsh, 2005; Massey et al., 2001). The level of oxalate in tempeh (23 mg/serving) was relatively low compared to other soy food products such as texturized vegetable protein (496 to 638 mg/serving), soy beverage (336 mg/serving), tofu (43 to 235 mg/serving), soy burger (58 mg/serving), and peanut butter (225 mg/serving) (Massey et al., 2001).
In grass peas, the main toxic compound is the nonprotein amino acid (β-ODAP), in which overconsumption of the neurotoxin can cause lathyrism in humans and animals (Yan et al., 2006). Tempeh fermentation can greatly reduce or diminish ODAP content in grass peas, where the processes prior to inoculation were more efficient in achieving this goal—the cooking step resulted in approximately 77% of reduction (Kebede et al., 1995; Stodolak & Starzynska-Janiszewska, 2008).
In soybeans and grass peas, tempeh fermentation can diminish trypsin inhibitors, which can directly interact with proteolytic enzymes secreted by the pancreas and reduce the digestibility of proteins in the diet (Hajos et al., 1995). In grass peas, tempeh fermentation reduced the level of trypsin inhibitor by 99%, with the cooking step contributing to the greatest level of reduction (Stodolak & Starzynska-Janiszewska, 2008). In soy, tempeh fermentation increased the antitryptic activity of the 85% ethanol extract, suggesting that tempeh fermentation released and increased the solubility of trypsin inhibitor compounds (Liu & Markakis, 1991).
In African yam bean (Sphenostylis stenocarpa), tempeh fermentation with 1% (v/v) citric acid added during soaking diminished cyanogenic glycoside content, which can be enzymically hydrolyzed to release cyanohydric acid that is toxic due to its ability to bind with metals, for example, Fe, Mn, and Cu (Azeke et al., 2007; Francisco & Pinotti, 2000). Tempeh fermentation is a more effective and less energy-intensive method to prepare the African yam bean for consumption compared to the traditional preparation, which involves boiling the yam for 4 hr (Azeke et al., 2007).
In lupin (Lupinus mutabilis and Lupinus campestris), tempeh fermentation diminished the quinolizidine alkaloid content, which is a toxic factor (Jiménez-Martínez, Hernández-Sánchez, & Dávila-Ortiz, 2007). In soy, chickpea, pea, faba bean, and lupin, tempeh fermentation can greatly reduce or diminish undigestible and flatulence-causing oligosaccharide content, including alpha-galactooligosaccharides, stachyose, raffinose, and verbascose (Nassar, Mubarak, & El-Beltagy, 2008; Ruiz-Teran & Owens, 1999; Tewari, 2002; van der Riet et al., 1987). In dark common bean, tempeh fermentation can decrease the levels of stachyose, raffinose, and verbascose by 57%, 67%, and 53%, respectively (Starzynska-Janiszewska et al., 2015). Tempeh fermentation was also reported to remove 70% of the total cyanide content in bitter apricot seeds (Tunçel et al., 1990).
3.3.4 On allergens
In general, tempeh had negligible or very low immunoreactivity similar to other hydrolyzed or fermented soy foods such as soy yogurt and miso, likely due to the cooking and fermentation process that breaks down allergenic proteins (Song et al., 2008). Tempeh contained relatively low antigenicity of protein P34, the immunodominant allergen in soybean, compared to other commercial soy ingredients, that is, soy flour, soy protein isolate, extracted soy protein, and soy protein concentrate (Wilson et al., 2008). Selection of soy cultivar with low levels of protein P34 can further reduce the level of P34 protein in soy tempeh (Wilson et al., 2008).
Tempeh fermentation with co-inoculation of Actinomucor elegans, Neurospora crassa, and R. oryzae can significantly reduce IgE immunoreactivity in vitro (Huang et al., 2019). Fermenting buckwheat into tempeh before processing soba noodles can also decrease allergenic protein levels significantly (Handoyo et al., 2006). Compared to hydrolyzed vegetable protein, acid-hydrolyzed soy sauce, and soybean sprouts, tempeh showed lower allergenicity in radioallergosorbent inhibition assays and most importantly did not contain the antigens commonly found in raw soybean (Herian et al., 1993).
3.4 Health benefits of tempeh
There is a limited number of in vitro, ex vivo, in vivo, clinical, and population studies on the health benefits of tempeh (Figure 12). The current literature body consists of evidence on the potential health benefits of tempeh on gut health, cancer, cognitive function, lung health, cardiovascular health, anemia, liver health, bone health, type 2 diabetes mellitus, obesity, skeletal muscle recovery, and malnutrition. Most of the health benefits were linked to the isoflavone, protein, mineral, as well as para- and probiotic contents in tempeh (Figure 13).
Number of citation per health benefit topic
Tempeh fermentation and its related health-promoting potential
3.4.1 Effects of tempeh on gut health
In a human study, supplementation of steamed tempeh (100 g/person per day) for 2 weeks in eight healthy males and females (20 to 23 years of age) enhanced the production of IgA and increased the fecal number of A. muciniphila (Stephanie et al., 2017). In an animal study, feeding 6-week-old female SD rats with tempeh (ad libitum, the dose was adjusted so tempeh increased the protein content of the basal diet by 10% per serving per rat) for 28 days resulted in increased amounts ileum IgA through gene expression stimulation (Soka et al., 2015). Such an effect has been linked to the presence of paraprobiotics, that is, heat-killed probiotics that can stimulate immune response (Taverniti & Guglielmetti, 2011). In an in vitro simulation of human digestive tract, raw soy tempeh (6 g in 60 mL water) stimulated most the growth of Bifidobacterium spp.; raw black bean tempeh stimulated most the growth of E. coli; fried soy tempeh stimulated an increase in Lactobacillus; and fried black beans stimulated the highest increase of Bifidobacterium and E. coli (Kuligowski et al., 2013).
Different tempeh fungi resulted in different gut health effects. In male SD rats fed a high-fat diet (mean body weight: 109 g), a 3-week supplementation of 20% tempeh fermented with R. stolonifer (9, 10, 12, 14, and 15 g/rat on days 1, 2 to 4, 5 to 7, 8 to 12, and 13 to 21, respectively) increased the cecal numbers of A. muciniphila, Bifidobacterium, and Lactobacillus, whereas that of tempeh fermented with R. microsporus increased the level of A. muciniphila and fecal mucin (Yang et al., 2018). Both forms of tempeh as well as the tempeh fermented with R. oryzae decreased the cecal numbers of Enterobacteriaceae and increased cecal propionate and acetate levels (Yang et al., 2018). In rats (eight animals per group), tempeh supplementation (9 g/rat for day 1; 10 g/rat for days 2 to 4; 12 g/rat for days 5 to 7; 14 g/rat for days 8 to 13; and 15 g/rat for days 14 to 21) also significantly decreased the level of lithocholic acid (a risk factor of colon cancer) as well as increased the levels of fecal mucins (indices of intestinal barrier function) and IgA (index of intestinal immune function) (Utama et al., 2013). The constipation-preventing activity of tempeh could be due to beyond its fiber content, because supplementation of okara tempeh in rats resulted in shorter gut transit time compared to that of cellulose (Matsuo, 1995b). In terms of probiotics, supplementation with L. plantarum I-UL4 isolated from tempeh showed suppressed growth of colonic Enterobacteriaceae in SD rats (Foo et al., 2003).
Tempeh supplementation alleviated diarrhea severity. In 6- to 24-month-old Indonesian children with acute diarrhea (N = 304), supplementation of formula food containing tempeh shortened the duration of diarrhea and improved body weight and nutritional status (Partawihardja, 1990). In a study in Kenya, supplementation of tempeh-yellow maize porridge (dose adjusted to provide 3.9 g of protein and 101 calories per day for each serving per subject) in malnourished children (N = 56) resulted in shorter diarrhea duration (15 days) compared to that of milk-yellow maize porridge (20 days) (Kalavi et al., 1996). In piglets, supplementation of a high-molecular-weight soluble fraction of tempeh (approximately 10 g of tempeh equivalent per 4 × 4 Latin-square design of piglet) significantly reduced fluid loss in E. coli-infected small intestine compared to saline control, but the effect was not significantly different than cooked soybean (Kiers et al., 2006; Kiers et al., 2007).
Antiadhesion activity against enterotoxigenic E. coli of tempeh (0.2 g per milliliter phosphate-buffered saline) has been observed in hemagglutinated hamster red blood cell, piglet small intestinal brush border membrane, and Caco-2 cells (Kiers et al., 2002; Mo et al., 2012; Roubos-van den Hil, Nout, van der Meulen, & Gruppen, 2010; Roubos-van den Hil, Schols, Nout, Zwietering, & Gruppen, 2010). Administration of black soybean tempeh (25% to 100% [w/w] of tempeh in basal diet) also improved serum composition in enteropathogenic E. coli-induced rats (Nurrahman & Mariyam, 2019).
3.4.2 Effects of tempeh on cancerous cell lines
Tempeh fermentation has displayed the capability to transform isoflavone glycosides into their more-bioavailable form, isoflavone aglycones (Ahmad et al., 2015; Berghofer et al., 1998; Borges et al., 2016; Chang et al., 2009; Cheng et al., 2010; Esaki et al., 1994; Ferreira et al., 2011; Kuligowski et al., 2017; Murakami et al., 1984; Rochín-Medina et al., 2015; Sánchez-Magana et al., 2014; Starzyńska-Janiszewska et al., 2008; Wang & Murphy, 1996; Xiao et al., 2016).
In T47D breast cancer cells, ethanol extract of tempeh containing 0.681% (w/w) genistein showed inhibitory activity with IC50 value of 196.066 ± 15.956 μg/mL (Yuliani et al., 2016). In Caco-2 human colon adenocarcinoma cells, water extract of tempeh showed inhibitory effects (Hsu et al., 2009). In other human carcinoma cell lines, Cheng et al. (2011) reported that the extract of black bean tempeh milk exhibited cytotoxic activity toward human carcinoma cells Hep 3B (IC50 = 150.2 mg/mL), but not in human carcinoma cells HeLa, Hep G2, CL-1, and normal human lung fibroblast cells MRC-5 (Cheng et al., 2011). In HuH-7 human hepatocellular carcinoma cells, an antioxidant isolated from tempeh, 3-hydroxyanthranilic acid (HAA), showed cytotoxic activity and induced apoptosis at 600 to 700 μM supplementation concentrations (Matsuo et al., 1997). In MCF-7 breast cancer cells, extracts of overripe tempeh fermented for 60 and 180 hr showed cytotoxic activities with IC50 values of 8.70 and 5.2 μg/mL, respectively (Athaillah et al., 2019; Muzdalifah et al., 2018).
Although showing some potential, the significance of tempeh fermentation in modulating chemopreventive potential of soybean is still largely unknown because no unfermented control was tested in most of the studies reviewed, except for one study by Kiriakidis et al. (1997). In mouse myeloma cells, tempeh glycolipid showed higher inhibitory activity (reaching 96% at 100 μg/mL of concentration) compared to soybean (Kiriakidis et al., 1997).
In chicken chorioallantois membrane assays, supplementation of tempeh genistein, daidzein, 3-hydroxygenistein, and 8-hydroxydaidzein inhibited in vivo angiogenesis by 75.09%, 48.98%, 67.96%, and 24.42%, respectively (Kiriakidis et al., 2005). All isoflavones also inhibited the expression of Ets 1, a blood vessel formation transcription factor (Kiriakidis et al., 2005). In male SD rats, a 12-week supplementation of 300 mg/kg body weight (BW) of soybean or 600 mg/kg BW of tempeh significantly reduced the number of aberrant crypt foci (ACF) in the colon of 1,2-dimethylhydrazine dihydrochloride-treated rats. In particular, daily intake of 600 mg tempeh/kg BW reduced the number of ACF that was composed by more than four crypts. Increase in superoxide dismutase (SOD) activity was only observed in rats fed with 300 mg soybean/kg BW (Hsu et al., 2009). Although soybean and tempeh supplementations have shown chemopreventive effects in animal studies, the efficacy in clinical studies and the mechanisms of action are still largely unknown.
3.4.3 Effects of tempeh on cognitive function
In Indonesian elders (N = 15), tempeh flour supplementation (35 g per day per person) resulted in increases in Mini-Mental State Examination (MMSE) and Hopkins Verbal Learning Test (HVLT) scores compared to that of casein (Kridawati et al., 2019). A population-based study (N = 142) by Hogervorst et al. (2011) showed that both tofu and tempeh consumptions were associated with better immediate memory recall in younger, but not in older, rural Indonesian elderly. The tempeh consumption ranged from zero to three times a day with the median consumption of seven plus/minus five times a week. In a previous study with a larger sample (N = 719) in the same region of Indonesia, Hogervorst et al. (2008) found that high tofu consumption was associated with lower memory test scores and high tempeh consumption was associated with better memory scores among elderly Indonesians. The relatively better benefits of tempeh in improving memory could be due to its higher folate and vitamin B12 levels, which are enhanced by the fermentation process (Mo et al., 2013).
In rats with scopolamine-induced cognitive dysfunction, Ahmad et al. (2014) reported that supplementation of isoflavone extract from tempeh at 40 mg/kg, by mouth or per os (p.o.) significantly improved memory, reversed the scopolamine effect, and reduced inflammation compared to that of unfermented soybeans. Similar results were observed at 10, 20, and 40 mg/kg, p.o. of tempeh isoflavone extracts, which significantly increased acetylcholine and reduced acetylcholinesterase levels compared to unfermented soybeans. Soybean isoflavone extract only showed significantly better improvements in cholinergic activities than those of tempeh at 10, 20, and 40 mg/kg isoflavone equivalent doses (Ahmad et al., 2014). In normal 12-month-old female rats, supplementation of tempeh flour (dose adjusted to tempeh providing 14% [w/w] of total protein) resulted in faster maize completion time compared to those of tofu, estradiol, and casein (Kridawati et al., 2013).
3.4.4 Effects of tempeh on lung cells and tissues
Soy consumption has been linked to better lung function in several population and meta-analysis studies (Seow et al., 2002; Smith et al., 2004; Yang et al., 2011). However, the association with tempeh has not been extensively studied. Matsuo et al. (1997) reported that HAA, an antioxidative intermediate metabolite of tryptophan that can be found in tempeh, inhibited the formation of a lipid oxidation product 12-hydroxyeicosatetraenoic acid at high concentration (1,000 μM) but not at low concentrations (0.1 to 100 μM) in ex vivo rat lung models. This result suggests that regular tempeh consumption might not provide any protective effect from oxidation in the lung if HAA is the sole responsible compound. Another in vitro study by Cheng et al. (2011) reported that extracts of black bean tempeh milk exhibited cytotoxic activity toward human carcinoma cells Hep 3B (IC50 = 150.2 mg/mL), but neither toward human carcinoma cells HeLa, Hep G2, CL-1, nor normal human lung fibroblast cells MRC-5. Phenolic compounds were hypothesized to be responsible in promoting anticarcinogenic activity (Cheng et al., 2011). In one in vivo study, a decrease in ACE activity in the lungs of spontaneously hypertensive rats was correlated with the supplementation of tempeh-natto, a product made by co-inoculating soybeans with R. oligosporus and B. subtilis (Chung et al., 2009).
3.4.5 Effects of tempeh on cardiovascular health
A quasi-experimental clinical study where women with hyperlipidemia (N = 41) were given 103 and 206 g/day of tempeh gembus, which is made of soymilk curd, reported decreases in the levels of low-density lipoprotein (LDL) (27.9% and 30.9%, respectively) and total cholesterols (17.7% and 19.8%, respectively) and an increase in high-density lipoprotein (HDL) (3.91% and 8.79%, respectively) and triglycerides (2.3% and 3.1%, respectively) levels (Afifah et al., 2020). Similar results were observed in a study using tempeh drink supplementation (96.46 g of fresh tempeh equivalent per day per subject, consumed in three doses per day), which resulted in decreased total cholesterol, LDL, and triglyceride levels in male and female subjects (N = 51) (Wirawanti et al., 2017). In subjects with hypertension and hypercholesterolemia (N = 30), supplementation of germinated tempeh drink (105 g per day per subject, consumed in three doses per day) decreased systolic blood pressure (Ansarullah et al., 2017). These studies suggest that the hypolipidemic effects of tempeh may be exhibited in hyperlipidemia, but not in normal subjects.
Tempeh fermentation has been reported to increase soy isoflavone content, which has been associated with the amelioration of hyperlipidemia (Chen et al., 2017; Chen et al., 2014; Eslami & Shidfar, 2019; Kohno, 2017), a risk factor for cardiovascular disease (Nelson, 2013; Nordestgaard et al., 2009; O'Keefe & Bell, 2007). In koro kratok bean (Phaseolus lunatus) and jack bean (Canavalia ensiformis), tempeh fermentation process promoted the release of peptides that inhibit ACE in in vitro gastrointestinal digestion process (dose adjusted to tempeh providing 5 mg/mL of protein), resulting in a capacity of 90.5% and 88.2% ACE inhibition, respectively (Pertiwi et al., 2020; Puspitojati et al., 2019).
Supplementation of tempeh co-inoculated with L. plantarum in high-fat diet-induced hyperglycemic rats (40 mg/kg body weight per day for 4 weeks) significantly reduced serum total cholesterol, triglycerides, free fatty acid, and LDL levels while increasing HDL content (Huang et al., 2018). In normal rats, supplementation of okara tempeh (50% [w/w] of tempeh in diet, ad libitum for 7 days) lowered liver cholesterol level more than that of okara (2.8 mg/g compared to 4.7 mg/g), as well as lowered plasma cholesterol (69 mg/100 mL compared to 92 mg/100 mL) and bile acid levels compared to that of casein–cellulose mix (Matsuo & Hitomi, 1993). In spontaneously hypertensive rats, although supplementation of tempeh (0.1% and 0.5% [w/w] of tempeh in diet, ad libitum for 8 weeks) enriched with GABA did not result in apparent effect on plasma cholesterol and triacylglycerol, it significantly lowered blood urea nitrogen levels (Aoki et al., 2003). Elevated blood urea nitrogen level can be a predictor of mortality in decompensated heart failure patients (Aronson et al., 2004).
3.4.6 Effects of tempeh on anemia
In a quasi-experimental study with pregnant Indonesian women in the third trimester of pregnancy (N = 32), 100 g/day tempeh milk + iron supplementation for 30 days increased serum hemoglobin level as well as protein and iron uptake, compared to iron-only supplementation (Novianti, Asmariyah, & Suriyati, 2019). In a randomized controlled clinical trial with undernourished Indonesian children (N = 30), supplementation of tempeh-based formula (containing 13.6 g of tempeh equivalent per day for 14 days) promoted levels of blood hemoglobin and iron similar to supplementation with the World Health's Organization's (WHO) F100, which is made of skim milk (Iva et al., 2012). F100 is a WHO-standardized ready-to-use therapeutic food for severely malnourished infants aged less than 6 months in the rehabilitation phase of severe acute malnutrition (SAM) (World Health Organization, 2005). Iva et al. (2012) provided supportive evidence for the prospect of tempeh as a food intervention for malnourished children.
Tempeh can be a good or excellent source of iron, containing about 10.6% to 28.69% of the RDA amount (8 to 18 mg for adults) in 85 g of food, RACC (Food & Drug Administration, 2019; National Institute of Health, 2019; United States Department of Agriculture, 2019). Iron in tempeh can also be in more bioavailable forms, given that tempeh fermentation can decrease iron(III)-species and complex iron as well as increase iron(II)-species (Tawali & Schwedt, 1998).
Iron-deficient rats had significantly higher liver iron concentrations and SOD activity with tempeh supplementation (18.9 g of tempeh mean weight per day for 11 days) compared to unfermented soybeans (Kasaoka et al., 1997). The levels of thiobarbituric acid-reactive substances (TBARS), a biomarker for lipid peroxidation, were higher in rats supplemented with unfermented soybean (18.3 g of unfermented soybean mean weight per day for 11 days), but no significant difference in TBARS was observed between tempeh- and casein-supplemented rats (Kasaoka et al., 1997).
In healthy rats, tempeh supplementation (ad libitum, diet containing 21% [w/w] tempeh for 42 days) reduced dialuric acid-induced hemolysis by about 20% compared to unfermented soybean supplementation, which showed no reduction (Murata et al., 1971). In an older study, a contradictive result was found in rats fed with a vitamin E-deficient diet supplemented with 6-hydroxydaidzein (1 mg/day/rat for 7 days), a tempeh antioxidant (Ikehata et al., 1968). However, this result needs more evaluation given recent studies that have reported that isoflavones in tempeh are mainly genistein and daidzein in terms of amount (Cassidy et al., 2006; Setchell et al., 2011).
3.4.7 Effects of tempeh on liver health
In vitro and in vivo studies on the benefits of tempeh on liver health demonstrate correlation between increased antioxidative activities and hepatoprotective effects. In human hepatocellular carcinoma HuH-7 cells, HAA, an antioxidant isolated from tempeh, showed cytotoxic, apoptotic, and cell growth inhibitory activities at 800 μM of feed concentration (Matsuo et al., 1997). In mice, alcohol-induced liver damage was reversed by significant reduction of aminotransferase, alanine aminotransferase, cholesterol, triglyceride, malondialdehyde, and nitric oxide levels after supplementation of lyophilized tempeh fermented normally and then anaerobically at 1,000 mg/kg feed concentration ad libitum (Yusof et al., 2013). In healthy rats, the supplementation of tempeh and unfermented soybean, both at 21% (w/w) of concentration in diet (ad libitum), significantly lowered the level of liver lipid peroxidation biomarker thiobarbituric acid (0.20 ± 0.05 and 0.65 ± 0.13 optical density (O.D.)/g, respectively) (Murata et al., 1971). The level of glutathione peroxide activity was also decreased in rats supplemented with okara tempeh at the concentration of 50% (w/w) in ad libitum diet for 36 days (Matsuo, 1995a). In spontaneously hypertensive rats, increase in liver DPPH scavenging activity and ACE inhibitory activity was observed after supplementation with 0.4 to 0.8 g/kg of tempeh-natto, which was co-inoculated with B. subtilis (Chung et al., 2009).
3.4.8 Effects of tempeh on bone health
The quantity of calcium intake and its bioavailability are critical factors in maintaining healthy bone mass and functionality (Cashman, 2002). Tempeh contained 81.6 to 94.35 mg of calcium, which can fulfill 6.80% to 7.86% of the RDA per 85 g RACC (Food & Drug Administration, 2019; National Institute of Health, 2020; United States Department of Agriculture, 2019).
In postmenopausal Malay women (N = 20), calcium absorption from tempeh supplementation (206 to 240 g) was slightly higher, although did not differ significantly, compared to whole milk supplementation (114 to 130 g) containing an equal amount of calcium (36.9% ± 10.6% vs. 34.3% ± 8.6% of urinal calcium level after 24 hr of intake, respectively) (Haron et al., 2010). In SD rats, tempeh supplementation (averaging 34.6 g/day per rat for 4 weeks) promoted a higher calcium absorption ratio, by up to 20% higher compared to unfermented soybeans (Watanabe et al., 2008).
3.4.9 Effects of tempeh on type 2 diabetes mellitus
Tempeh fermentation can increase the levels of isoflavones and probiotics as well as decrease the fat content, in which these effects have been associated with the amelioration of type 2 diabetes by decreasing blood glucose level, total cholesterol level, and body weight (Hsu et al., 2003; Huang et al., 2013; Jayagopal et al., 2002; Lee, 2006). In high-fat diet-induced hyperglycemic rats, supplementation of tempeh fermented with R. oligosporus and L. plantarum (40 mg/kg body weight per day for 4 weeks) improved serum glucose and lipid levels by inhibiting cholesterol synthesis and promoting lipolysis through the modulation of gut lactic acid bacteria content (Huang et al., 2018). In a cohort population study (N = 232) in West Java, Indonesia, homeostatic model assessment of insulin resistance was negatively correlated with tempeh consumption, but not tofu only or tofu and tempeh combined (Febrianti et al., 2019).
3.4.10 Effects of tempeh on obesity
To the best of our knowledge, there has been only one in vivo study by Harun et al. (2017) and another one in Astawan et al. (2018) that analyzed tempeh and its effect on obesity biomarkers, which in this case are serum lipid composition and malondialdehyde (MDA) for oxidative stress (Sankhla et al., 2012). Astuti (1997) reported that daily consumption of tempeh-based drink (dose undisclosed) for 3 months decreased total cholesterol in human subjects (N = 24) (by 8.6% in men and 10.25% in women), LDL (by 12% in men and 9.67% in women), and MDA levels (by 23% in men and 15% in women) (Astawan et al., 2018). In highly active rats, supplementation of 3 g tempeh per 200 g BW per day for 1 month improved serum MDA level (Harun et al., 2017). In an in vivo study in Astawan et al. (2018), high-GABA tempeh supplementation (32.2% [w/w] of tempeh in diet, ad libitum for 6 weeks) resulted in greater improvement of triacylglyceride, HDL, and LDL levels compared to unfermented soybean and casein in male Wistar rats. The anti-obesity effects discussed were hypothesized to be due to the enhanced antioxidative and glycolytic enzyme (amylase and glycosidase) inhibitory activities (Gibbs et al., 2004; McCue & Shetty, 2003).
3.4.11 Effects of tempeh on skeletal muscle recovery
The potential use of tempeh to expedite muscle recovery and improve strength is due to its branched-chain amino acid (BCAA) and antioxidative isoflavone contents. BCAA consumption has been associated with reduction of creatine kinase and muscle soreness as well as increase in muscle strength (Howatson et al., 2012; Jackman et al., 2010). Tempeh fermentation released more bioavailable soy isoflavones, which can inhibit inflammation (Kuligowski et al., 2017), a marker for muscle damage during postexercise recovery (Peake et al., 2016).
In active pulmonary tuberculosis patients in Indonesia (N = 128), daily consumption of 166.5 g of boiled tempeh for 2 months along with standard therapy improved body weight and physical function parameters, that is, handgrip strength and 6-min walk test scores (Setiawan, 2016). The improvements in body weight and physical function were not associated with additional protein and caloric intake provided by the tempeh consumption, suggesting that the high isoflavone contents detected in tempeh may be responsible for the recovery due to their antioxidative properties (Kuligowski et al., 2017; Setiawan, 2016). In Indonesian student athletes (N = 18), postexercise tempeh drink supplementation (containing 8.5% [w/v] of tempeh per 600 mL dose) resulted in significantly lower serum creatine kinase, an indicator for muscle damage, and maximal strength at 24 hr after exercising compared to supplementation with whey and a placebo (Jauhari et al., 2013). The tempeh drink contained 23 g of protein per portion with 4.16 g of BCAAs (Setiawan, 2016), which have been associated with reduction of creatine kinase and muscle soreness as well as increase in muscle strength (Howatson et al., 2012; Jackman et al., 2010).
3.4.12 Effects of tempeh on malnutrition
Several studies have examined addressing malnutrition with tempeh as the main source of nutrients and as an ingredient in formulated food or diet. In a randomized controlled clinical trial with undernourished Indonesian patients aged 1 to 10 years (N = 30), Iva et al. (2012) reported that the supplementation of a tempeh flour-based formulation (containing 13.6 g of tempeh per day for 14 days) improved serum iron and hemoglobin levels similarly to the World Health Organization's F100 milk-based formula (Iva et al., 2012). F100 has been widely regarded as the gold standard formula for addressing severe malnutrition in infants aged younger than 6 months (World Health Organization, 2005).
In a protein–energy malnutrition (PEM) intervention model with Indonesian toddlers (N = 70) in Malang, East Java, Java Island, Indonesia, increases in weight, height, and blood hemoglobin levels as well as a decrease in blood albumin levels were observed after supplementation of biscuits made from dates, modified cassava flour, and tempeh as the main source of protein in a 50 g serving, in which tempeh provided 4.81 g of protein per day per subject for 12 weeks (Fatmah, 2018). In a quasi-experimental study with underweight children (N = 19) in Padang, Sumatera Island, Indonesia, 100 g/day supplementation of a mixture of tempeh and jicama (Pachyrhizus erosus) significantly increased body weight and blood albumin levels compared to a biscuit control (ingredients) (Symond et al., 2016). Besides body weight, blood albumin levels can be an indicator for malnutrition (Cooper et al., 2004; Gitlin et al., 1958). In pregnant women with iron deficiency (N = 252), daily intake of a tempeh-dominant supplementary food (600 g of tempeh, 30 g of meat, 350 g of guava, 300 g of papaya, and 100 g of orange per week) during pregnancy resulted in small decrease in blood hemoglobin, ferritin, and body iron compared to no intervention (Wijaya-Erhardt et al., 2011). In underweight children younger than 5 years of age (N = 46), provision of tempeh nuggets (containing 276.53 calories, 8.6 g of protein, 28.41 g of carbohydrate, 13.28 g of lipid, and 44.38 g of fiber per 100 g) for 30 days significantly increased energy intake (P <0.001) compared to no provision (Permatasari et al., 2018). In rats (N = 18), supplementation of 20 g/day of tempeh-based enteral formula for 30 days resulted in higher weight gain and serum albumin level compared to commercial product for malnutrition (Khasanah et al., 2015).
These studies discussed prospective use of tempeh as a food to ameliorate malnutrition, especially PEM and iron deficiency. However, further research on tempeh as the sole source of nutrients is needed, as well as clinical trials on the use of tempeh to address malnutrition in children under 5 years.
3.5 Food application as functional ingredient
3.5.1 Meat alternative and extender
Due to its meat-like consistency and high protein content, tempeh has been eaten like meat in the Western diet, for example, as burger patties, sausages, nuggets, and in stews (Permatasari et al., 2018; Sihite et al., 2018; Wang, 1984). Thiébaud et al. (1995) reported that tempeh burger produced significantly lower carcinogenic heterocyclic amines during frying compared to beef burger and bacon. The amounts of heterocyclic amines in the smoke condensates were 0.11 ng/g from fried tempeh burger, 0.37 ng/g from fried beef burger, and 3 ng/g from fried bacon (Thiébaud et al., 1995). In a mutagenicity assay on Salmonella typhimurium strain TA98, bacon was 350 times more mutagenic than a tempeh burger (Thiébaud et al., 1995).
In Brazil, burger patty made of white bean tempeh was sensorially accepted as much as soybean burger by 82 untrained panelists. The white bean tempeh burger had a similar appearance and crispy consistency compared to the soy tempeh burger, although it had lower flavor scores (Vital et al., 2018). In Ethiopia, wot, a traditional Ethiopian hot-spiced stew, made of fava bean, pea, or chickpea tempehs, was sensorially favorable and comparable to those made of meat or egg yolk (Ashenafi & Busse, 1991d).
Tempeh has been used as meat extender for its low production costs and nutrient profile, that is, fiber, vitamin, and minerals (Kuo et al., 1989; Taylor et al., 2013). Taylor et al. (2013) reported beef patties mixed with 10% and 20% tempeh resulted in better color stability by retaining a lighter color throughout storage and reduction in redness, but with significantly lower protein content. Kuo et al. (1989) incorporated tempeh into ham, where hams made with 2% to 3.5% tempeh obtained lower sensory acceptance levels and had lower moisture levels.
3.5.2 Flavoring ingredient
Seasoning powder made of overripe tempeh had higher levels of compounds that promote sourness, umami flavor, bitterness, saltiness, and pungent aroma compared to fresh tempeh (Gunawan-Puteri et al., 2015). Oven-drying overripe and fresh tempeh powders resulted in higher glutamic acid content (14.5% and 15.9%, respectively) compared to freeze-drying (13.9% and 13.9%, respectively) and no drying (12.8% and 12.6%, respectively) (Gunawan-Puteri et al., 2015). Stock cube made of overripe tempeh resulted in the best sensory results by mixing 27.35% of overripe tempeh powder with 2.34% of xanthan gum, 20% of oil, 16.83% of caramel syrup, 20.7% of salt, 6.48% of garlic powder, and 6.3% of pepper (Setiadharmaa et al., 2010). Miso made of tempeh had higher sensorial properties and antioxidant activities compared to that of unfermented soybeans (Matsuo, 2006b).
Chickpea, corn, and common bean tempehs have been made into flours with higher nutritional values compared to unfermented flours of the same ingredients. Reyes-Moreno et al. (2004) showed that fermenting chickpeas into tempeh before being processed into flour increased its in vitro and in vivo protein digestibility by approximately 10% and 5%, respectively. Tempeh fermentation also increased the in vivo protein efficiency ratio (PER), net protein retention, protein digestibility corrected amino acid score (PDCAAS), as well as the levels of isoleucine, methionine, cysteine, phenylalanine, and tyrosine (Reyes-Moreno et al., 2004). Similar results were found in corn tempeh flour, in which tempeh fermentation increased corn PER from 1.78 to 2.10 and PDCAAS from 0.55 to 0.83 (Cuevas-Rodríguez et al., 2006). In common bean tempeh flour, tempeh fermentation increased antioxidant capacity and total phenolic content by up to 2.2-fold (Gamboa-Gómez et al., 2016; Reyes-Bastidas et al., 2010).
3.5.3 Pasta and noodles
To increase protein content and amino acid quality, tempeh has been incorporated into carbohydrate sources such as pasta and noodles. Soybean and mung bean tempehs were mixed with nixtamalized yellow corn to produce pasta that helped accommodate the delivery of enhanced folate content; the limiting amount to achieve favorable results was 33.3% tempeh in the pasta mixture (Susilowati et al., 2018). In noodles, incorporation of tempeh can increase protein content but has been limited by sensory properties, that is, texture and taste (Aini et al., 2012). Aini et al. (2012) reported that 20% of tempeh flour was the maximum dose in corn flour noodles to produce acceptable sensory scores. In buckwheat soba noodles, Handoyo et al. (2006) found that tempeh fermentation improved the protein digestibility by increasing the levels of amino acids by up to 50-fold, which included isoleucine, leucine, lysine, valine, glycine, histidine, tyrosine, and gamma-amino butyric acid. Tempeh fermentation also significantly decreased phytate and allergenic protein contents in buckwheat soba noodles (Handoyo & Morita, 2006).
3.5.4 Bakeries
In bread, incorporating 5% of freeze-dried tempeh resulted in sensorially accepted bread with increased protein content and polyunsaturated/saturated fatty acids ratio (Melo et al., 2020). In vegan cookies, incorporation of tempeh paste to partially substitute wheat flour (tempeh:wheat flour 1:7) resulted in higher sensory acceptance scores compared to regular cookies without tempeh (Budsabun et al., 2019). In Brazil, incorporation of lyophilized tempeh flour for substitution of soy flour in coconut cookies improved the antioxidant levels, that is, isoflavone aglycones, while retaining acceptable sensory scores in texture, aroma, and flavor (Leite et al., 2013). In cereal bars, addition of 15% of freeze-dried tempeh resulted in sensorially accepted products with increased protein content and polyunsaturated/saturated fatty acids ratio (Melo et al., 2020). In crackers, tempeh fortified with calcium was incorporated to produce sensorially acceptable products (Haron & Halim, 2019).
3.5.5 Dietary supplements
Syida et al. (2018) produced tempeh protein isolate by defatting tempeh flour with hexane immersion then treating it with alkali and acid before neutralizing it. Compared to tempeh flour, tempeh protein isolate had higher protein content (by 50.5%) and amounts of essential as well as nonessential amino acids and lower levels of crude fat, total carbohydrate, total ash, moisture, and crude fiber (Syida et al., 2018). Germinating soybean before being processed into tempeh protein isolate increased protein content by 5% to 7% and protein digestibility by 1.2%, while decreasing fat content by 1.3% to 1.5% (Astawan et al., 2019).
3.5.6 Beverages
In green coffee beans, Lee et al. (2016) found that tempeh fermentation modulated the levels of aroma precursors by increasing proline and aspartic acid content, which exhibited high Maillard reactivity, by 1.5-fold. Tempeh fermentation also degraded ferulic and caffeic acids, which led to a twofold increase in the levels of total volatile phenolic derivatives (36% of total volatiles were generated during fermentation) (Lee et al., 2016). In milk, extracts of black soybean tempeh milk exhibited antioxidant and cytotoxic activities in human hepatoma cells Hep3B, but not in normal human lung fibroblast cells MRC-5 (Cheng et al., 2011).
3.5.7 Emergency food
In a human study, a tempeh-based emergency food formulae made by Iva et al. (2012) matched the effects of promoting serum iron and hemoglobin levels compared to WHO's F100 formula, which is the international standard for rehabilitation of severe malnutrition among children younger than 6 months. The tempeh-based formula consisted of 6.8 g of tempeh flour, 3 g of granulated sugar, 5 g of coconut oil, and 2 g of electrolytes in 100 mL of water. In contrast, the WHO F100 formula consisted of 8.5 g of skim milk, 5 g of granulated sugar, 6 g of coconut oil, and 2 g of electrolytes in 100 mL of water (Iva et al., 2012). Aini et al. (2018) created another tempeh-based formula, using tempeh flour mixed with corn flour, whole milk powder, sugar, and oil to produce an emergency food containing 8.1 g of protein, 20.67 g of lipids, 20.58 g of carbohydrate, and 298.04 kcal of energy. According to the standard for emergency foods (Zoumas et al., 2002), the nutritional content fulfilled the 7.9 to 8.1 g of protein and 233 kcal of energy per piece requirements, but not the 9.1 to 11.7 g of lipid and 23 to 25 g of carbohydrate requirements (Aini et al., 2018).
Various tempeh-based biscuits have also been made for different purposes. Targeted for PEM, iron deficiency anemia, zinc deficiency, and vitamin A deficiency, Lubis (2018) formulated tempeh-based biscuits that could provide protein adequacy for children aged 1 to 3 and 4 to 6 years by 34 to 55.8% and 25.3 to 41.4%, respectively. The biscuits were also rich in iron, zinc, and beta-carotene (Lubis, 2018). Similar biscuits were also made with soy or quinoa tempeh flour mixed with fish and millet and could be fortified with iron (Anandito et al., 2018; Setyawati et al., 2018). In those biscuits, tempeh masked the fishy aroma and metallic flavor as well as increased iron and alpha-tocopherol concentrations (Anandito et al., 2018; Matsuo, 2006a; Setyawati et al., 2018). Matsuo et al. (2006a) reported that biscuits made of 20% quinoa tempeh powder contained more than 2.5-fold higher iron and alpha-tocopherol content and resulted in higher absorption of iron in rats. Due to its high fat content, peanut tempeh can also be considered as an ingredient for emergency food (Matsuo, 2006c).
3.5.8 Foods for infants and the elderly
In Nigeria, Osundahunsi and Aworh (2002) formulated a tempeh-based weaning food that consisted of 20% soy tempeh or 40% cowpeas (V. unguiculata), 50% to 80% of ogi (a traditional Nigerian weaning food made of fermented maize flour) or maize flour, and 10% of melon seed flour. The vanilla-flavored versions of both the ogi–cowpea tempeh and the ogi–soy tempeh formulations resulted in very good overall acceptability (8.2 in a 1 to 9 scale: 1 for dislike extremely and 9 for like extremely) (Osundahunsi & Aworh, 2002). Both formulations contained approximately 18.6% to 18.62% of protein, 8.27% to 8.83% of fat, 1.72% to 1.83% of ash, 70.72% to 71% of carbohydrate, 6.70% to 7.61% of moisture, and 435 to 437 kcal of energy per 100 g, and were cost-efficient given that they were eight to 10 times cheaper than commercial products available in the area (Osundahunsi & Aworh, 2002). Another tempeh-based weaning food that was formulated by mixing 27% of black bean tempeh with 73% of cooked rice resulted in 86% in vitro digestibility with low content of oligosaccharides that could be indigestible and cause flatulence (Rodriguez-Burger et al., 1998). In Tanzania, fried tempehs made of the combinations of sorghums, bambara nut, sesame, cowpea, pigeon pea, chickpea, mung bean, sesame seed, finger millet, common bean, soybean, and groundnut were found sensorially acceptable as weaning foods (Mugula & Lyimo, 1999, 2000).
In elderly subjects, a tempeh drink was formulated by Kridawati et al. (2019) as a source of folate and isoflavones to improve cognitive function. The supplementation of the tempeh drink (35 g of tempeh flour) resulted in better MMSE and HVLT scores compared to casein supplementation (17.5 g) (Kridawati et al., 2019). Nakajima et al. (2005) found that allowing soybeans to germ/hypocotyl in tempeh fermentation can enrich isoflavone content in tempeh. This isoflavone-enriched tempeh could be granulated to create a nutritious supplement suitable for elderly (Nakajima et al., 2005).
3.6 Food safety
3.6.1 Outbreaks and policy implications
The most published food-borne illness outbreaks related to tempeh consumption were the bongkrekic acid toxin outbreaks in Indonesia in 1895 to 2014, as well as the gastroenteritis outbreak in North Carolina, USA in 2012 (Garcia, 1999; Griese et al., 2013; Shurtleff & Aoyagi, 1979). Bongkrekic acid is a mitochondrial toxin produced by Burkholderia gladioli pathovar cocovenans (B. cocovenans) that can grow in incompletely fermented tempe bongkrek, a traditional kind of tempeh in Indonesia made with coconut oil and/or coconut milk press cake (Anwar et al., 2017). Bongkrekic acid inhibits mitochondrial adenine nucleotide translocase that disrupts adenosine triphosphate and adenosine diphosphate synthesis and exchange, causing a wide range of symptoms including malaise, dizziness, jaundice, and, in extreme cases, shock, coma, and death (1 to 1.5 mg can be fatal in humans) (Anwar et al., 2017; Deshpande, 2002).
In Indonesia, bongkrekic acid poisoning affected more than 9,000 people and killed more than 1,000 people between 1951 and 2013, in which the number of cases went down from 1,036 cases and 125 deaths in 1975 to four cases and one death in 2013 (Anwar et al., 2017). The public health policy implication included avoiding the production of tempe bongkrek and use of coconut oil or milk press cake, adjusting the soaking water pH to 4.5, as well as implementing hygienic food production standards in tempeh production (Anwar et al., 2017; Buckle, 1985).
Griese et al. (2013) reported the gastroenteritis outbreak in North Carolina in 2012 caused by the consumption of unpasteurized tempeh affected 87 residents (eight hospitalized) from five states with symptoms including diarrhea, abdominal cramps, fever, vomiting, and bloody diarrhea. The outbreak was the first case in which tempeh was being a food vehicle of Salmonella enterica Paratyphi B variant L(+) tartrate(+) (formerly Salmonella var. Java), which has mostly been associated with contaminated poultry or eggs (Griese et al., 2013). The investigation found that the contamination came from the starter cultures of Rhizopus spp. produced in Indonesia (Griese et al., 2013). The food safety policy implications included implementing pasteurization in tempeh production, which can kill pathogens such as Salmonella enterica Paratyphi B variant L(+) tartrate(+) (Silva & Gibbs, 2012), especially in ready-to-eat products, as well as controlling cross contamination from contact with bare hands, surfaces, and raw materials (Griese et al., 2013).
3.6.2 Contamination by closely related strain or fermenter
In tempeh production, it is critical to avoid food intoxication hazard from contamination by R. microsporus var. microsporus (R. microsporus), which has been reported to be infectious and is closely related to the most commonly used and researched tempeh fermenter, R. microsporus var. oligosporus (R. oligosporus) (Dolatabadi et al., 2016). Both organisms have been classified as R. microsporus based on ITS and large subunit ribosomal ribonucleic acid sequence analysis (Walther et al., 2013). Based on sporulation ability as well as the DNA sequences of ITS, ACT, and translation elongation factor 1-α regions, there was no difference found between R. microsporus and R. oligosporus (Dolatabadi et al., 2014).
Rhizopus microsporus has been mostly isolated from environmental and clinical samples such as soil, wood chips, saw mill dust, and human tissue, but it has also been isolated from tempeh in Indonesia (Dolatabadi et al., 2016; Jennessen et al., 2005). Certain lines of R. microsporus harbor Burkholderia rhizoxinica as an endosymbiont, which can produce rhizoxin toxin that is antimitotic (Dolatabadi et al., 2016; Jennessen et al., 2005). Out of 15 tempeh samples analyzed by Jennessen et al. (2005) and Dolatabadi et al. (2016), in which 14 of them were from Indonesia and one was from The Netherlands, R. microsporus was isolated from three of them and only one sample contained Burkholderia rhizoxinica, whereas only R. oligosporus was isolated from the rest (including from the tempeh from The Netherlands). In a laboratory experiment, R. microsporus with rhizoxin-producing B. rhizoxinica endosymbiont can ferment cooked soybeans into tempeh (Rohm et al., 2010). Once tempeh is contaminated with R. microsporus, it is technically impossible to prevent its growth or selectively promote the growth of R. oligosporus given that the optimum growth conditions for both were the same, that is, 40 °C and a w = 0.995; both R. microsporus and R. oligosporus can grow at low oxygen level (0.5% [v/v]) (Han & Nout, 2000).
3.6.3 Acidification and co-inoculation to prevent pathogens
Acidification with or without the addition of lactic acid bacteria during the soaking step has been recommended by many studies to inhibit the growth of pathogens in tempeh. Acidification reaching pH 4.85 can happen naturally during soybean production, but it does not always occur (Nout et al., 1987; Tunçel & Göktan, 1990). When natural acidification occurred (pH 4.85), it did not inhibit the growth of Bacillus, S. aureus, and K. pneumoniae in tempeh consistently in ex situ experiments (Nout et al., 1987; Tunçel & Göktan, 1990). When natural acidification does not occur, 105 CFU/g of B. cereus in the soaking water can grow to 108 CFU/g in tempeh, causing spoilage (Nout et al., 1987).
Acidification and co-inoculation are two separate control measures in improving the microbial quality of tempeh, because acidification alone does not inhibit the growth of pathogens such as L. monocytogenes, Salmonella infantis, E. coli, and E. aerogenes in tempeh (Table 7) (Ashenafi, 1991; Ashenafi & Busse, 1989, 1989, 1991c). Nout et al. (1987) observed that preventing B. cereus growth in tempeh through acidification of soak water was best done by adding lactic acid to reach pH ≤ 4.4, although this process might reduce the quality of tempeh (Nout et al., 1985) in comparison to the addition of acetic acid reaching pH levels ≤ 5.5 and inhibiting the growth of R. oligosporus. Co-inoculating R. oligosporus with L. plantarum, L. casei spp. alactosus, and L. fermentum produced tempeh of excellent quality, but did not prevent B. cereus growth and subsequent spoilage without acidification (Nout et al., 1987).
| Level of inhibition | ||||
|---|---|---|---|---|
| Tempeh substrate | Pathogen | Acidification | L. plantarum co-inoculation | Acidification + L. plantarum co-inoculation |
| Soy | Bacillus cereus | + | + | ++ |
| Listeria monocytogenes | – | ++ | ++ | |
| Salmonella infantis | – | +++ | +++ | |
| Escherichia coli | – | +++ | +++ | |
| Enterobacter aerogenes | – | +++ | +++ | |
| Staphylococcus aureus | + | ++ | NA | |
| Chickpea | Bacillus cereus | + | + | ++ |
| Listeria monocytogenes | – | ++ | ++ | |
| Salmonella infantis | – | +++ | +++ | |
| Escherichia coli | – | +++ | +++ | |
| Staphylococcus aureus | + | + | NA | |
| Pea | Bacillus cereus | + | + | ++ |
| Listeria monocytogenes | – | ++ | ++ | |
| Salmonella infantis | – | + | – | |
| Escherichia coli | – | ++ | +++ | |
| Staphylococcus aureus | + | ++ | NA | |
| Fava bean | Bacillus cereus | + | – | ++ |
| Listeria monocytogenes | – | + | + | |
| Salmonella infantis | – | + | – | |
| Escherichia coli | – | – | ++ | |
| Staphylococcus aureus | + | + | NA | |
- +, inhibition; ++, marked inhibition; +++, complete inhibition; –, no inhibition.
Similar results were observed in tempehs made from Ethiopian beans. Ashenafi and Busse (1991b) reported that B. cereus can grow reaching 106 to 107 CFU/g in unacidified soy, chickpea, and pea tempehs within 40 hr, and 108 CFU/g in unacidified fava bean tempeh to cause spoilage. In unacidified soy, chickpea, and pea tempehs, inoculation with L. plantarum decreased the final B. cereus count by 2 log units but not in fava bean tempeh (Ashenafi & Busse, 1991b). Combination of acidification (pH 5.5) and co-inoculation with L. plantarum completely inhibited B. cereus growth (Ashenafi & Busse, 1991b).
According to Ashenafi (1991) and Ashenafi and Busse (1992), L. monocytogenes and S. aureus can grow to the concentration of 106 CFU/g in soybean, chickpea, pea, and fava bean tempehs in an ex situ experiment, which can pose a significant food safety hazard. Acidification alone did not show significant inhibitory effects, whereas the co-inoculation with L. plantarum on unacidified or acidified beans significantly or completely inhibited the growth of L. monocytogenes and S. aureus (Ashenafi, 1991; Ashenafi & Busse, 1992).
Lactobacillus plantarum co-inoculation with or without acidification inhibited Salmonella infantis growth completely in soybean and chickpea tempehs, but only retarded the bacterial growth until approximately 24 hr in pea and fava bean tempehs (Ashenafi & Busse, 1991c). Similar results were observed on the growth of E. aerogenes in soy tempeh (Ashenafi & Busse, 1989). The growth of E. coli can be completely inhibited by L. plantarum co-inoculation with or without acidification in soybean, chickpea, and pea tempehs, but marked inhibition could only be achieved by co-inoculation and acidification in fava bean tempeh (Ashenafi & Busse, 1989, 1991c).
Besides the presence of acids, the inhibitory effects of L. plantarum co-inoculation paired with acidification could be due to the presence of other compounds produced by L. plantarum, possibly bacteriocin (Ashenafi, 1991; Ashenafi & Busse, 1989, 1991b, 1991c, 1992). Bacteriocins produced by L. plantarum, that is, plantaricins, have been identified as broad-range antimicrobial glycolipoproteins that can be heat stable (60 min at 100 °C and up to 10 min at 121°C) and be active in a pH range of 2.0 to 8.0. Plantaricin can be produced by L. plantarum in sorghum beer, green olive fermentation, and the Nigerian fermented food, ogi. Plantaricin exhibited inhibitory effects on the growth of B. cereus, Staphylococcus spp., Enterococcus faecalis, Listeria spp., E. coli, but not on Candida albicans and Klebsiella spp. (Diep et al., 1995, 1996; Jiménez-Díaz et al., 1993; Ogunbanwo et al., 2003; Reenen et al., 1998).
Compared to other tempeh substrates discussed, fava bean tempeh required the highest measure of food safety handling, for example, the combination of acidification and L. plantarum co-inoculation. Furthermore, the growth of Salmonella infantis that cannot be inhibited by acidification and co-inoculation is a food safety concern. A combination of acidification and L. plantarum co-inoculation is recommended on top of implementing safe food production standards.
3.7 Processing
Improvements in time, nutrition, and nutrient bioavailability in tempeh fermentation can be achieved through pre-germination of inoculants, germination of substrates, incorporation of hypocotyl, replacement of the boiling step with pressure steaming, and choices of cooking methods. Pre-germinating the spores of R. oligosporus on rice or in potato extract–yeast extract–glucose broth for 8 to 12 hr at 30 to 35 °C prior to inoculation can reduce the incubation time by up to 4 hr (Kronenberg, 1984). Germinating soybean for 12 to 24 hr on water-saturated filter paper at 25 °C resulted in tempeh with increased levels of crude protein and protein efficiency ratio (from 2.26 to 2.19), as well as reduced levels of phytates, fat, and oligosaccharides (i.e., sucrose, raffinose, and stachyose) (Suparmo & Markakis, 1987). Soaking soybean with 10% (v/v) trifluoroacetic acid resulted in higher peptide recovery (Rusdah et al., 2017). Germination also lowered phytic acid content and increased antiradical activity in soybean (Puteri et al., 2018). In a protein isolate form, tempeh made of germinated soybeans had significantly higher in vitro protein digestibility by 1.2% compared to tempeh made of nongerminated soybeans (Astawan et al., 2019).
Effects of modifications in standard tempeh-making steps are discussed. Incorporation of defatted soybean germ increases the levels of isoflavone aglycones and isoflavone glycosides (Nakajima et al., 2005). Replacing the boiling step with pressure steaming can result in higher nutrient retention and Rhizopus spp. growth due to minimized contact with and nutrient diffusion to water (Kusumah et al., 2018). Hygienic production may increase bioactive peptide content in tempeh (Tamam et al., 2019).
Although a variety of cooking methods are utilized in tempeh preparation, frying was the most popular cooking method in Indonesia (Karyadi & Lukito, 1996; Kristianto et al., 2015). Fried tempeh contained more isoflavone aglycones (approximately 35 mg of daidzein and 31 mg of genistein per 100 g) than raw tempeh (approximately 26 mg of daidzein and 28 mg of genistein per 100 g), but had less malonyl glycoside and total isoflavone contents (Ferreira et al., 2011; Haron et al., 2009). These studies suggest that the frying process might break down isoflavone glycosides into isoflavone aglycones, which can increase their bioavailability. Frying tempeh in coconut oil significantly reduced the levels of free fatty acids by releasing them into the frying oil, but the final glyceride composition was not affected (Sudarmadji & Markakis, 1978). Deep-fat frying also decreased the levels of amino acids after 5 min and moreover after 7 min, where lysine and cysteine were the most susceptible to heat destruction compared to other amino acids. In contrast, steaming did not affect amino acid content in tempeh (Stillings & Hackler, 1965). Boiling and frying can change the flavor profiles of tempeh by increasing aliphatic aldehydes and decreasing aliphatic esters and alcohols to different degrees (Apriyantono et al., 2001). Application of sous vide cooking method for 3 days at 45 °C resulted in a more gel-looking tempeh with strong sweet and umami tastes (Guixer et al., 2017).
3.8 Sensory properties and consumer acceptance
Tempeh has been introduced and consumed in Asia, Africa, North America, South America, Europe, and Australia continents since the early 20th century (Shurtleff & Aoyagi, 2007). However, our research found that scientific reports on the sensory properties and consumer acceptance of tempeh or tempeh-related foods were limited to certain countries in the continents of Asia, that is, Indonesia, India, Japan, Thailand, and Turkey; Africa, that is, Benin, Nigeria, Ethiopia, and Tanzania; Europe, that is, Germany and Poland; South America, that is, Chile and Brazil; and Australia, that is, New Zealand. In this section, sensory properties and consumer acceptance of tempeh in these countries are discussed. The size of panelist population, scale of hedonic scoring, and other details are described, unless access to full report was limited.
3.8.1 In Indonesia
In Indonesia, tempeh consumption contributed to approximately 10% of total protein consumed, higher than meat (3.15%) and chicken eggs (1.25%) (Karyadi & Lukito, 1996). The average tempeh consumption in Indonesia was 10.1 to 33.75 kg/capita/year, contributing to up to 60% of the total national soybean consumption (Astawan et al., 2018; Astawan et al., 2017; Karyadi & Lukito, 1996). This suggests generally high sensory acceptance of tempeh in Indonesia.
Based on a sensory study in Yogyakarta, Indonesia, Fibri and Frost (2020) reported that in a 15-scale hedonic test (1 = dislike extremely, 8 = neither like or dislike, 15 = like extremely), soybean tempeh had the highest score (10.62) compared to black bean (8.5), jack bean (Canavalia ensiformis) (6.5), mung bean (6.9), and velvet bean (Mucuna pruriens) (3.9) tempehs. All samples were served to untrained subjects (N = 165, aged 18 to 40 years) deep-fried at 170 °C for 3 min after being marinated in 7% NaCl brine solution for 5 min, which was the most common and well-known methods of preparation (Fibri & Frost, 2020). Based on the source of soybean and production method, tempehs made of local soybeans using traditional methods resulted in higher hedonic score (11.3), compared to tempehs made of local soybean using traditional methods (9.8) as well as tempehs made of imported soybeans using hygienic methods (10.67). Traditional production method was defined as using usar leaves as inoculant in a noncertified facility, whereas hygienic method used industrial starter culture in a certified facility.
The same study by Fibri and Frost (2020) also reported that the sensory evaluation scores changed when product information was provided, which included raw materials (bean type), origin (local or imported), and production methods. Providing product information significantly (P < 0.05) increased the hedonic scores of all samples, except for the group of tempeh made of local soybean hygienically and one out of four groups of tempeh made of imported soybean hygienically (Fibri & Frost, 2020).
The most notable sensory variables in tempeh according to the panelists were nutty, compact, savory, umami, firm, and salty properties, whereas other sensory variables that were noticed include mushroom-like, grass, rancid, earthly, buttery, chewy, soapy, bitter, oily, sour smell (volatile acidity), and sour taste (Fibri & Frost, 2020). The salty and beany properties could be caused by the presence of a terpenoid compound, that is, alpha-pinene, which was only found in tempeh wrapped in banana leaf, identical with traditional production method (Harahap et al., 2018). The mushroom-like sensory perceptions could due to the presence of 3-octanone and 1-octen-3-ol, which were detected in soybean and soybean tempeh (Feng et al., 2007). The grass or "green" sensory perception could be stimulated by the presence of (Z)-alpha-bisabolene that was found in tempeh wrapped in plastic bag (Harahap et al., 2018).
3.8.2 In Asia and Australia
In Bangkok, Thailand, 14% replacement of wheat flour with tempeh flour in vegan cookies resulted in significantly (P< 0.05) higher overall acceptance score (7.27 ± 1.08 out of 9) compared to regular vegan cookies (6.84 ± 1.34 out of 9) according to 40 untrained panelists (N = 40) between 18 and 21 years of age (Budsabun et al., 2019). In Japan, biscuits made from quinoa tempeh flour substituting wheat flour resulted in higher sensory evaluation scores for brittleness, color, and taste compared to normal biscuits (Matsuo, 2006a). In Bangalore, India, a blind sensory evaluation with nine semi-trained panelists (N = 9) resulted in the highest preference for curry and fried chips made of soybean–sunflower tempeh, then soy–groundnut tempeh, and lastly soybean tempeh (Vaidehi et al., 1985). In the same area, ready-to-prepare dry soups and porridges made with green-gram tempeh resulted in the average of 4 out of 5 hedonic score (Vaidehi et al., 1996).
In Kerala, India, cowpea and green-gram tempehs were sensory acceptable (Lakshmy & Usha, 2010).
In Antalya, Turkey, 13 semi-trained panelists (N = 13) gave highest preference and taste scores for deep-fried tempehs made of chickpea, then broad bean, white bean, green lentil, black bean, soybean, and lastly red lentil (Erkan et al., 2020). The same study reported that red lentil and white bean tempehs were significantly harder than chickpea, soybean, broad bean, and green lentil tempehs; black bean tempeh was significantly less springy compared to other tempehs; red lentil tempeh was significantly more cohesive than the others; white bean tempeh was significantly less gummy than the others; and red lentil tempeh was significantly more gummy, chewy, and resilient than the others.
In Otago, New Zealand, 15 untrained panelists (N = 15) scored that substituting 10% (w/w) beef with tempeh in a burger patty resulted in significantly decreased overall acceptability, but not appearance and color.
3.8.3 In Africa
In West Africa, Egounlety (2001) reported that fried tempeh snacks made of soybean, cowpea, and bambara groundnut were sensory accepted with the average hedonic score of 6.10 out of 9 among 300 untrained panelists (N = 300). The tempehs served were blanched in 1.5% salt solution for 2 to 15 min and seasoned with small quantity of onion, garlic, and Syzygium racemosum. The panelists consisted of people from Benin (74.3%), Burkina-Faso (0.3%), Ivory Coast (1.7%), Cameroon (3.3%), Congo (0.7%), Gabon (0.7%), Guinea (0.3%), Mali (0.3%), Niger (0.7%), Nigeria (17.1%), Senegal (0.3%), Chad (0.7%), Togo (0.3%), and Zaire (0.3%) (Egounlety, 2001).
In Ibadan, Nigeria, weaning foods made of ogi (dehydrated fermented maize flour) fortified with soybean, cowpea, and ground bean tempehs were found as acceptable as commercial weaning food Cerelac by 10 nursing mothers and their 6- to 24-year-old children (N = 10) (Egounlety et al., 2002). In Port Harcourt, Nigeria, yam-bean tempeh as a pie filling resulted in significantly (P< 0.05) higher sensory acceptability scores compared to beef (Njoku et al., 1991).
In Addis Ababa, Ethiopia, Ashenafi and Busse (1991) reported that 100 panelists (N = 100) gave the hedonic scores for wot, a traditional Ethiopian hot-spiced stew, made of horse-bean tempeh higher (7.14 ± 1.53) than those made of pea tempeh (6.52 ± 1.81) and chickpea tempeh (6.72 ± 1.69). Wot made from chickpea tempeh was preferred by most panelists (71%) compared to those made of egg yolk (12%) and beans (17%), whereas wot made of horse-bean tempeh was preferred by slightly higher number of judges (7%) compared to that made of meat (5%), but lower than that made of egg yolk (88%) (Ashenafi & Busse, 1991d). In Morogoro, Tanzania, fried and salted tempehs made from the combinations of sorghum–cowpeas–sesame, sorghum–common bean–groundnut, sorghum–bambara groundnut–sesame, sorghum–pigeon pea–sesame, sorghum–soybean–groundnut, and sorghum–mung bean–groundnut were sensory accepted by 20 semi-trained panelists (N = 20) with the overall acceptance scores of 3.6, 3.8, 3.6, 3.5, 2.9, and 3.5 out of 5, respectively (Mugula & Lyimo, 2000).
3.8.4 In South America and Europe
In Osorno, Chile, lupin tempeh was sensory accepted by 25 untrained panelists (N = 25), resulting in the average overall acceptance score of 3.5 out of 5 (Agosin et al., 1989). In Gioania, Brazil, among 82 (N = 82) untrained panelists, soybean tempeh burger was sensory more acceptable than white bean tempeh burger with the overall scores of 6.4 out of 9 and 5.1 out of 9, respectively (Vital et al., 2018). In Londrina, Brazil, coconut cookies made of 50:50 tempeh:soy flour were sensory acceptable by 150 untrained panelists aged 17 to 60 years (N = 150), resulting in the average overall scores of 8.45 ± 1.22 for those made with lyophilized tempeh and 7.71 ± 1.89 for those with roasted tempeh (Leite et al., 2013).
In Olsztyn, Poland, tempeh made of buckwheat groat was found to be sensory acceptable among 50 untrained panelists (N = 50), scoring 5.32 ± 0.31 for raw and 5.67 ± 0.75 for roasted samples. In Germany, pea tempeh in soup or fried was sensory accepted with nutty as a notable flavor (Reiss, 1993).
3.9 Sustainability and positive contributions to climate change
3.9.1 Protein delivery efficiency per unit energy and per unit greenhouse gas emissions
Total energy consumption (MJ/kg) and emission (kg CO2 eq./kg) of soy tempeh were calculated by including the measures in soybean production by González et al. (2011) as well as those in wood- and kerosene-fueled tempeh productions in Indonesia by Supartono et al. (2014) (Table 8). Conversion rates between tempeh and fresh, dry, and cooked soybeans were obtained from Sparringa and Owens (1999a), Hurburgh et al. (2008), and the USDA (2019).
| Production stage | Description | Energy (MJ/kg) | Emission (kg CO2 eq./kg) | Reference |
|---|---|---|---|---|
| Substrate production | Soybean production | 3.06 | 0.46 | González et al., 2011 |
| Tempeh production | Traditional processing—firewood-fueled | 5.00 | 0.46 | Supartono et al., 2014 |
| Traditional processing—kerosene-fueled | 5.62 | 0.46 | Supartono, Widyasari, & Purwadi, 2014 | |
| Traditional processing with electric boiling and splitting | NA | 0.76 | Putri et al., 2018 | |
| Traditional—firewood-fueled | NA | 0.96 | Wiloso et al., 2019 | |
| Modern processing—fully electric | NA | 1.14 | Putri et al., 2018 | |
| Modern processing—hygienic | NA | 1.04 | Wiloso et al., 2019 | |
| Average (soybean production + average of tempeh production) | 8.37 | 0.92 | ||
- Note. Conversion rates between fresh, dry, and cooked soybeans obtained from Hurburgh (2008), Sparringa and Owens (1999c), and United States Department of Agriculture (2019).
The calculated energy consumption and emission values of tempeh were 8.37 MJ/kg and 0.92 kg CO2 eq./kg, respectively (Table 8). Because raw tempeh contained approximately 145 g of protein per 1,000 g (United States Department of Agriculture, 2019), the protein delivery efficiency energy and protein delivery efficiency greenhouse gases (GHG) scores of tempeh would be 17.3 g protein/MJ and 124.8 g protein/kg CO2 eq., respectively (González et al., 2011).
The protein delivery efficiency energy score of tempeh is highly efficient compared to the animal-based protein sources with a score that was 3.94-fold that of beef, 4.12-fold that of mutton and lamb, 2.37-fold that of pork, 2.47-fold that of chicken, 3.40-fold that of fish, 1.92-fold that of eggs, 1.57-fold that of milk, and 2.66-fold that of cheese (Figure 14) (González et al., 2011). The protein delivery efficiency GHG score of tempeh was 22.22-fold that of beef, 20.76-fold that of mutton and lamb, 6.31-fold that of pork, 4.05-fold that of chicken, 2.35-fold that of fish, 3.76-fold that of eggs, 5.09-fold that of milk, and 5.63-fold that of cheese (Figure 15) (González et al., 2011).
Protein delivery efficiency energy of tempeh compared to other common sources of protein (adapted from González et al., 2011 and Table 8)
Protein delivery efficiency greenhouse gases (GHG) of tempeh compared to other common sources of protein (adapted from González et al., 2011 and Table 8)
Traditional tempeh production methods resulted in lower or similar energy usage and GHG production compared to modern methods (Putri et al., 2018; Supartono et al., 2014; Wiloso et al., 2019). Traditional methods use fire wood, gasoline, and/or kerosene as fuels; semi-traditional methods include the use of electricity; and modern methods were usually fully electric (Putri et al., 2018; Supartono et al., 2014; Wiloso et al., 2019). Modern tempeh production had better food safety ratings through the implementation of good manufacturing practices and hazard analysis critical control points (Table 8) (Putri et al., 2018; Wiloso et al., 2019).
Based on a life-cycle assessment by Wiloso et al. (2019), the main contributor to land use and eutrophication indicator results was soybean cultivation. Although traditional and modern tempeh production systems differed slightly, the main contributor to human toxicity, eco-toxicity, stratospheric ozone depletion, climate change, photochemical oxidation, and acidification was the transportation stage (Wiloso et al., 2019). Thus, utilization of locally grown legumes, grains, and nuts could reduce the negative environmental impacts and therefore improve the sustainability of tempeh production and consumption in various regions in the world (Wiloso et al., 2019).
3.9.2 Utilization of food production by-products
Affordability of tempeh fermentation could be due to the fact that tempeh can be made from food production by-products. In Indonesia, traditional tempe gembus was made of tofu or soymilk residue and tempe bongkrek was made from coconut oil or milk press cake (Damanik et al., 2018; Takeda et al., 2016). The fermentation in tempe gembus increased monounsaturated fatty acid content by 0.2%, decreased polyunsaturated fatty acid content by 8.11%, increased saturated fatty acid content by 0.14%, and decreased amino acid content by 0.60% (Damanik et al., 2018).
New types of tempehs made of food production by-products have been reported with enhanced functionalities, for example, on rice bran and flaxseed oil press cake (Cempaka et al., 2018; Nurrahma et al., 2018). In SD rats fed with a fructose-supplemented high-fat diet, supplementation of 2,205 mg/kg BW/day of rice bran tempeh extract increased HDL level (by 151%) and lowered total cholesterol (by 46%), triglyceride (by 36%), and LDL (by 64%) levels compared to the control group (Nurrahma et al., 2018). Soybean tempeh made with up to 20% (w/w) rice bran was sensorially acceptable for human consumption (Cempaka et al., 2018). In flaxseed oil press cake, tempeh fermentation reduced phytate content by up to 48% and increased phenolic content by up to 85%, radical scavenging activity by up to 200%, and reducing power by up to 30% (Duliński et al., 2017). Beneficial results were also observed in the slightly increased protein content and significantly decreased lipid content (Duliński et al., 2017; Stodolak et al., 2017). Incorporation of flaxseed oil press cake into grass pea seed tempeh resulted in increased omega-3 linolenic fatty acid content by 10-fold, improved omega-6/omega-3 fatty acid ratios from 11/1 to 0.5–2.5/1, increased sulfur amino acids by 10% to 46%, and decreased lysine content by 6% to 12% (Stodolak et al., 2013).
3.9.3 Treatment and utilization of production waste
Tempeh production by-products can be utilized to produce animal feed, biogas, fertilizer, and single-cell proteins. Soybean hulls can be utilized for feeding lamb, steer, and laying hen as energy and fiber sources (Anderson et al., 1988; Esonu et al., 2005; Hartini et al., 2018). Microbial fuel cell (MFC) system with methylene blue as a redox mediator can be used to treat tempeh wastewater using its own Gram-positive and Gram-negative bacteria as well as the biofilm formed (Arbianti et al., 2018; Mariana, Elisabeth, Utami, Arbianti, & Hermansyah, 2017; Siagian et al., 2017; Zuhri et al., 2016). Gram-positive and Gram-negative bacteria from tempeh wastewater were grown on selective media and selected before being added to the MFC reactor by 1 and 5 mL, resulting in reduction in chemical oxygen demand and biochemical oxygen demand levels by up to 29.32% and 51.32%, respectively (Arbianti et al., 2018). TiO2-N/bentonite–alginate can also be used to decompose tempeh waste water for approximately 53.66% degradation (Nisaa et al., 2018).
For large-scale tempeh industries that include soybean harvesting, up to 6.8 mL of biohydrogen can be generated from each gram of soybean straw and sludge, which contain carbohydrates (cellulose, hemicellulose, and lignin) and methane that can be digested using microbial consortiums consisting of Clostridium butyricum and Clostridium roseum in an anaerobic digester (Rengga et al., 2017). Waste from small-scale tempeh industries can be mixed with household waste and digested using a biogas balloon digester to produce biogas and fertilizer (Puspawati et al., 2019). In producing single-cell proteins by Chlorella spp., sea water-based cultivation mediums containing 30% tempeh waste yielded 37.1 × 106 cell/mL biomass with 52% protein content (Putri et al., 2018).
3.10 Affordability
A comparative price analysis of tempeh compared to other common sources of protein in Indonesia and the United States was conducted. Commodity prices were sourced from the Republic of Indonesia's Ministry of Trade, the USDA Economic Research Service, as well as six online retail websites (amazon.com, target.com, walmart.com, kroger.com, tokopedia.com, and shopee.com; accessed on May 20, 2020). Nutritional content was obtained from the description of the products as well as the USDA FoodData Central (Ministry of Trade Republic of Indonesia, 2020; United States Department of Agriculture, 2020).
In Indonesia, for the same amount of protein content, traditional tempeh can be cheaper than beef (6.92 times), chicken (1.83 times), egg (2.29 times), and milk (10.56 times) (Figure 16). In the United States., tempeh can be 33% cheaper compared to beef, but can also be more expensive compared to beef (by 19%), pork (by 42%), chicken (by 70%), and egg (by 98%) (Figure 17). The relatively cheaper price of tempeh compared to other source of protein in Indonesia could be due to its production volume supported by the high demand, given the average tempeh consumption of 10.1 kg/person annually and the existence of 100,000 small household producers that can produce 10 kg to 4 metric tons of tempeh per day (Astawan et al., 2018; Astuti et al., 2000).
Price of tempeh (per g protein) compared to other common protein sources in Indonesia (adapted from Ministry of Trade Republic of Indonesia, 2020; Winarno et al., 1985)
Price of tempeh (per g protein) compared to other common sources of protein in the United States (adapted from USDA, 2019, 2020)
Price of tempeh (per kg food) compared to other common sources of protein in Indonesia (adapted from Ministry of Trade Republic of Indonesia, 2020)
In terms of price per kilogram of food, traditional tempeh was cheaper than beef (by 7.79 times), chicken (by 1.88 times), egg (by 1.58 times), and milk (by 1.85 times) in Indonesia, and hygienic tempeh was cheaper by 1.43 times compared to beef (Figure 18). In the United States, tempeh on average can be 10% cheaper than beef per kg of food (Figure 19).
Price of tempeh (per kg food) compared to other common sources of protein in the United States (adapted from USDA, 2019, 2020)
Although the main positioning for tempeh could be as a plant-based source of protein, tempeh contains other health-promoting aspects that other common sources of protein, especially animal-based ones, might not have such as fiber and isoflavones. Accurate valuation of food contribution might be difficult to assess; however, such work might provide a rationale for positioning and marketing tempeh as a functional food.
4 CONCLUSIONS
Based on the semicentennial literature body discussed, tempeh fermentation is a low-cost and sustainable food processing technology that can produce meat-like sources of protein from various beans, legumes, and grains from around the world with enhanced the health-promoting potentials. This review of tempeh provides new research on tempeh, its health-promoting benefits, food safety issues, fermentation kinetics, current and future applications, sustainability, as well as affordability. This review identifies areas for further research including the health-promoting potential of tempeh, especially at clinical and epidemiological levels. To disseminate access to this promising fermentation technology, the need is clear for the standardization of nonsoy tempehs by the international food regulation body, FAO–WHO CODEX Alimentarius Commission. To further improve the food safety aspect of tempeh production in the industry, the evaluation of acidification and lactic acid bacteria co-inoculation in the soaking process is critical. To establish more sustainable tempeh production systems, the localization of ingredient sourcing in tempeh production is essential. Altogether, tempeh and tempeh fermentation as plant-based protein source and technology shall be considered and further studied as key parts of feeding the world in a sustainable way environmentally, economically, and public health-wise.
ACKNOWLEDGEMENTS
This work was in part supported by United States Department of Agriculture (Hatch MAS00556 and NIFA grant #2019-67017-29249 and 2020-67017-30835). In accordance with Comprehensive Reviews in Food Science and Food Safety policy and our ethical obligation as researchers, we are reporting that Amadeus Driando Ahnan-Winarno received funding from the Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan [LPDP]) for his doctoral study and conducting this research, has affiliation with the nonprofit Indonesian Tempe Movement, and has ownership of share of Better Nature Ltd. Florentinus Gregorius Winarno is also affiliated to the nonprofit Indonesian Tempe Movement. These interests have been fully disclosed to Comprehensive Reviews in Food Science and Food Safety for management of any potential conflicts arising from the involvements mentioned.
AUTHOR CONTRIBUTIONS
Amadeus Driando Ahnan-Winarno designed the general framework, chose the initial idea and topic, conducted the literature research, analyzed the literature search results, visualized data, as well as assembled the data obtained into the topics, subtopics, and narratives of this review. Lorraine Cordeiro designed and evaluated the adaption of literature research methodologies to be systematic and comprehensive, improved the narrations and writing style, reviewed the papers cited, provided guidelines for data visualization, and provided details in the nutrition-related sections. Florentinus Gregorius Winarno provided scientific research papers and books related to tempeh, translated ancient scriptures about tempeh, and provided navigations about the historical and cultural aspects of tempeh. John Gibbons provided the references for the genomic, evolution, and domestication aspects of tempeh fungi and their impact in food safety, health, and well-being. Hang Xiao conceived this review article, and he reviewed, revised, and finalized the manuscript.
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