Tempeh and Fermentation—Innovative Substrates in a Classical Microbial Process

Abstract

The growing consumer awareness of functional foods has increased interest in fermented plant-based products with enhanced nutritional and health-promoting properties. This comprehensive narrative literature review examines the potential of diverse raw materials for tempeh production beyond traditional soybeans, analysing their nutritional composition, bioactive compounds, and functional properties. A structured literature search was conducted on peer-reviewed publications up to July 2025, focusing on tempeh fermentation technology, chemical composition, and bioactive compounds from various substrates using recognised analytical methods according to Association of Official Analytical Collaboration (AOAC) standards. The analysis of over 25 different substrates revealed significant opportunities for enhancing tempeh’s nutritional profile through alternative raw materials including legumes, cereals, algae, seeds, and agricultural by-products. Several substrates demonstrated superior nutritional characteristics compared with traditional soybean tempeh, notably tarwi (Lupinus mutabilis) with exceptional protein content ((32–53% dry matter (DM)) and mung bean (Vigna radiata) exhibiting remarkably high polyphenol concentrations (137.53 mg gallic acid equivalents (GAE)/g DM). Fermentation with Rhizopus oligosporus consistently achieved substantial reductions in anti-nutritional factors (64–67% decrease in trypsin inhibitors, up to 65% reduction in phytates) while maintaining consistent antioxidant activities (39–70% 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition) across most variants. The diversity of bioactive compounds across different substrates demonstrates potential for developing targeted functional foods with specific health-promoting properties, supporting sustainable food system development through protein source diversification.

1. Introduction

Interest in the consumption of functional foods that have a positive impact on health is growing every day due to increased consumer awareness [1]. Fermentation is one of the oldest and most versatile biotechnological processes used in food production. Its importance goes far beyond traditional preservation functions—fermentation influences the nutritional value, microbiological safety, sensory properties, and health-promoting potential of food. Modern research confirms that fermentation, especially the fermentation of legumes (e.g., tempeh), is the basis for the development of modern, functional, and sustainable plant foods [2,3,4].

The fermentation process leads to the breakdown of complex macronutrients into more easily digestible forms, increases protein digestibility, releases essential amino acids, and improves mineral bioavailability by breaking down anti-nutritional compounds [2,3,5]. Fermentation produces bioactive compounds (peptides, isoflavones, polyphenols, B vitamins), and fermented products show higher antioxidant and immunomodulatory activity [4,6,7]. Fermentation reduces the development of pathogens, improves sensory characteristics, and extends food shelf life [8].

Tempeh is a traditional ingredient in Indonesian cuisine made from fermented soybeans (Glycine max) [9]. The importance of fermentation in tempeh production is to improve protein digestibility; reduce anti-nutritional factors; increase the content of peptides, isoflavones, and polyphenols; and shape the unique sensory characteristics of the product. Soya has traditionally been used in the production of tempeh, but other raw materials are now increasingly used. The choice of raw material influences the nutritional value, amino acid profile, presence of bioactive compounds, and sensory attributes of the product.

The aim of this article is to provide a comprehensive literature review on the potential use of various plant raw materials for the production of tempeh (not only soybeans), with particular emphasis on their nutritional composition, bioactive compound content, and functional properties. The authors analyse how fermentation with Rhizopus oligosporus affects the nutritional value, antioxidant activity, and reduction of anti-nutritional compounds in tempeh based on over 25 different substrates (including legumes, cereals, algae, seeds, and agricultural processing by-products). The article also aims to highlight the potential of innovative, functional food products based on tempeh that support sustainable food systems by diversifying plant protein sources.

2. Methods

A structured narrative literature review was conducted without adherence to PRISMA guidelines, as the aim was to provide a comprehensive and integrative synthesis of existing knowledge on tempeh production from diverse raw materials rather than a narrowly focused quantitative analysis. This approach allowed for the inclusion of varied research designs, processing methods, and analytical techniques, which would not have been feasible through a conventional systematic review. The search encompassed peer-reviewed publications up to July 2025, focusing on the fermentation technology, nutritional composition, bioactive compounds, and functional properties of tempeh produced from both traditional and innovative substrates.

Specific keywords included “tempeh”, “Rhizopus oligosporus”, “solid-state fermentation”, “legume fermentation”, “alternative substrates”, “soybean alternatives”, “protein digestibility”, “isoflavones”, “polyphenols”, “antioxidant activity”, “anti-nutritional factors”, “phytates”, “trypsin inhibitors”, “functional foods”, “plant-based protein”, “fermented foods”, “bioactive compounds”, “nutritional composition”, and “processing technology.” The reference lists of all retrieved articles and prior reviews were manually screened to identify additional relevant studies. Only studies published in English were included due to practical limitations related to translation and consistency.

All data on chemical composition presented in this review were obtained from studies using recognised reference analytical methods in accordance with AOAC. This review did not include a formal risk-of-bias assessment or meta-analysis and should be interpreted as a qualitative synthesis of the current literature on tempeh fermentation from diverse raw materials.

3. Characteristics of the Raw Materials Used for Tempeh Fermentation

The classic raw material for making tempeh is soybeans [2,5,7,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28], but tempeh is produced from a wide range of alternative raw materials. Among these are the following:

• Legumes: broad beans (Vicia faba L.) [15,29,30,31,32], chickpeas (Cicer arietinum) [5,15,33,34], Jack bean (Canavalia ensiformis) [6,35,36,37,38,39,40], cowpea bean (Vigna unguiculata), Bambara groundnut (Vigna subterranea) [41], mung bean (Vigna radiata) [26,42], winged bean (Psophocarpus tetragonolobus) [23], common bean (Phaseolus vulgaris) [15,43,44,45,46,47], large pea (Lathyrus sativus) [48,49,50], lentils (Lens culinaris) [15], narrow-leaved lupin (Lupinus angustifolius) [51,52], tarwi (Lupinus mutabilis) [53], white mimosa (Leucaena leucocephala) [54];
• Cereals: oats (Avena sativa) [31,55], pearl barley (Hordeum vulgare) [56], basmati rice (Oryza sativa) [56], rice (Oryza sativa) [42], sorghum [26];
• Seeds and nuts: lotus seeds (Nelumbo nucifera) [57,58,59], Moringa oleifera seeds [60], rubber seeds (Hevea brasiliensis) [61];
• Algae: Porphyra sp. [62,63], Eucheuma spinosum [64];
• Plant additives: butterfly pea (Clitoria ternatea) flower petals [65];
• Other plants: wild turmeric (Curcuma aromatica) [66];
• By-products: brewer’s thresh [67], linseed pomace [50], rapeseed pomace [68].

Based on an analysis of the data in Table 1, the protein content in the tested materials shows a wide range of values, from a minimum of 0.3% in butterfly pea flowers (Clitoria ternatea) to a maximum of 52.0% in narrow-leaved lupin (Lupinus angustifolius). The classification of raw materials according to protein content allows three main categories to be distinguished: High-protein raw materials (≥35% DM), which include six raw materials, with a predominance of legumes. The highest protein concentration is found in lupin (Lupinus mutabilis, 44.7%) and narrow-leaved lupin (31.0–52.0%), confirming their potential as alternative protein sources to conventional raw materials. Soybeans (Glycine max, 39.0–40.0%) and rapeseed meal (38.1%) are excellent sources of plant protein used in both human nutrition and animal feed. Moderate-protein raw materials (20–34% DM) represent the most numerous group (15 materials), dominated by legumes such as field beans (Vicia faba, 26.0–33.0%), chickpeas (Cicer arietinum, 18.7–41.8%), and common beans (Phaseolus vulgaris, 20.0–27.0%). Low-protein raw materials (<20% DM) mainly include cereals and other plant raw materials, with the lowest protein content found in pea flowers (0.3%) and Eucheuma spinosum (6.0–7.3%).

Analysis of the data in Table 1 reveals a remarkable variation in fat content between the raw materials, ranging from trace amounts to values exceeding 50% of dry matter. The raw materials with the highest fat content include Brazilian rubber (Hevea brasiliensis), which by far leads the way in fat content with 42.5–54.2% of dry weight. Rubberwood seed oil is rich in unsaturated fatty acids such as 39.6–40.5% linoleic acid, 17–24.6% oleic acid, and 16.3–26% linolenic acid [69,70]. Moringa oleifera seeds rank second with 43.6% fat content.

Table 1. Characteristics of raw materials for tempeh production.

Raw Material	Protein (% DM)	Fat (% DM)	Carbohydrates (% DM)	Fibre (% DM)	Bioactive Compounds	Health-Promoting Properties	Anti-Nutritional Factors
Adlay (Coix lacryma-jobi)	20.0–31.7 [71]	1.0–8.2 [72]	56.0–75.0 [71]	2.5–17.0 [72]	Polyphenols, flavonoids, coixol, phytosterols, saponins [71]	Hypolipidaemic, antidiabetic, antioxidant, prebiotic [71]	Phytic acid, saponins, trypsin inhibitors [73]
Broad beans (Vicia faba L.)	26.0–33.0 [74]	<1.0 [74]	45.7–70.1 [75]	11.4–16.6 [74]	Polyphenols, flavonoids, L-DOPA, tannins, lectins [76]	Cholesterol lowering, heart support, antioxidant [77]	Phytic acid, trypsin inhibitors, saponins, vicine, convicine, lectins, tannins [74]
Chickpeas (Cicer arietinum)	18.7–41.8 [78]	2.7–6.5 cooked [79]	60.0–65.0 [79]	18.0–22.0 [80]	Polyphenols, sterols, carotenoids, tannins, isoflavones [80]	Cholesterol lowering, heart support, glycaemic control, antioxidant [81]	Phytates, lecithins, enzyme inhibitors, oligosaccharides [78]
Red algae (Porphyra sp.)	33.7–41.8 [82]	Up to 1.2 [83]	30.0–40.0 [84]	up to 48.0 [85]	Polysaccharides (porphyran), phycobiliproteins, polyphenols, flavonoids [84,86,87,88].	Antioxidant [86], immune support [84], cholesterol lowering, heart support [89], antidiabetic and anti-inflammatory [84], improved iron bioavailability [90]	Phytic acid, saponins [91]
Eucheuma spinosum	6.0–7.3 [92]	<0.1 [92]	69.0–70.0 [92]	15.0–20.0 [92]	Polyphenols, flavonoids, saponins, tannins, steroids, triterpenoids [93]	Antioxidant, antimicrobial, prebiotic, support for bone mineralisation [93]	Phytic acid, tannins [92]
Cowpea bean (Vigna unguiculata)	17.5–32.5 [94]	1.5 [95]	62.1 [95]	7.0–11.0 [96]	Polyphenols, flavonoids, saponins, tannins, phytosterols, L-DOPA [97]	Cholesterol lowering, glycaemic regulation, microbiome support, antioxidant [97]	Phytic acid, tannins, trypsin inhibitors [97]
Jack bean (Canavalia ensiformis)	25.2 [98]	5.2 [98]	58.4 [98]	7.1 [99]	Polyphenols, flavonoids, kaempferol glycosides, α-glucosidase inhibitors [100]	Antidiabetic [100], immunomodulatory [101]	Canavanine, trypsin inhibitors, lectins, oligosaccharides [99]
Bambara groundnut (Vigna subterranea)	15.0–37.0 [102]	1.3–7.4 [102]	45.0–64.0 [102]	3.7–6.4 [102]	Polyphenols, flavonoids, saponins, tannins, alkaloids, L-DOPA [103]	Cholesterol lowering, glycaemic regulation, satiety, antioxidant, anti-inflammatory [104]	Phytates, tannins, oxalates, trypsin inhibitors, lectins [104]
Mung bean (Vigna radiata)	14.6–32.6 [105]	1.2–1.9 [105]	61.0–67.1 [105]	5.5 [106]	Polyphenols, flavonoids, saponins, phenolic acids, alkaloids, bioactive peptides [107]	Cholesterol lowering, glycaemic regulation, antioxidant, anti-inflammatory, satiety [107]	Phytic acid, tannins, trypsin inhibitors, lectins [105]
Winged bean (Psophocarpus tetragonolobus)	27.2–45.0 [108]	15.2–23.4 [108]	14.2–35.7 [108]	1.6–26.2 [108]	Polyphenols, flavonoids, saponins, phytosterols, bioactive peptides [108]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant, anti-inflammatory [108]	Phytates, tannins, oxalates, trypsin inhibitors, haemagglutinins [109]
Common bean (Phaseolus vulgaris)	20.0–27.0 [110]	0.6–3.0 [111]	58.0–70.0 [110]	30.3–34.2 [112]	Polyphenols, anthocyanins, saponins, tannins, phytosterols [113]	Cholesterol lowering, heart support, glycaemic regulation, antioxidant [110]	Lectins, phytic acid, saponins, trypsin inhibitors, tannins [114]
Large peas (Lathyrus sativus)	17.7–25.6 [115] 20.0–24.9 [116] 18.0–34.0 [117]	1.7 [118]	48.0–52.3 [118]	1.1–1.7 [118] 4.0–7.0 [116]	Polyphenols, flavonoids, saponins, tannins, phytosterols, homoarginine, β-ODAP [118]	Antioxidant, hypolipidaemic, antidiabetic, anti-inflammatory [118]	β-ODAP, phytic acid, saponins, tannins [115,116]
Pearl barley (Hordeum vulgare)	13.6 [119]	2.8 [119]	63.9 [119]	4.7 [119]	β-Glucan, arabinoxylan, polyphenols, flavonoids, phytosterols [119]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant, anti-inflammatory [119].	Phytic acid, saponins, tannins [119]
Rubber (Hevea brasiliensis)	19.4–30.7 [120] 23.3 [121] 26.1 [122] 31.6 [123]	42.5–54.2 [120]	11.6–29.0 [120]	5.9 [121] 43.0 [122]	Polyphenols, flavonoids [120]	Antioxidant, anti-inflammatory, metabolic, requires detoxification [31,120]	Tannins, saponins [121,123]
Wild turmeric (Curcuma aromatica)	19.4 [124]	2.5 [124]	97.5 [124]	n.a.	Polyphenols (curcumin, demethoxycurcumin), flavonoids, terpenoids, essential oils [124,125]	Antioxidant, anti-inflammatory, anticancer, neuroprotective [124,126]	Saponins, tannins, alkaloids [124]
Flowers of butterfly pea (Clitoria ternatea)	0.3 [127]	2.5 [127]	2.2 [127]	2.1 [127]	Anthocyanins (300–500 mg/100 g), flavonoids (100–150 mg/100 g), phenolic acids, saponins, tannins, carotenoids, avenanthramides [127,128,129]	Potent antioxidant, anti-inflammatory, hypoglycaemic, neuroprotective, cardioprotective, anticancer, skin support [127,128]	Tannins, saponins, oxalates [127,128]
Lotus (Nelumbo nucifera)	16.0–21.0 [130]	2.4–3.0 [130]	61.0–62.0 [130]	2.8 [131]	Polysaccharides (porphyran), phycobiliproteins, polyphenols, flavonoids [132]	Antioxidant, immunomodulatory, prebiotic, antidiabetic [133]	Phytic acid, saponins [134,135]
Narrow-leaved lupin (Lupinus angustifolius)	31.6–34.6 [136] 31.0–52.0 [137]	6.0 [137]	<24.0 [138]	37.5–40.2 [137]	Polyphenols, flavonoids, bioactive peptides, saponins, phytosterols [137]	Cholesterol lowering, glycaemic improvement, satiety, antioxidant, anti-inflammatory [138]	Alkaloids, phytic acid, trypsin inhibitors [137]
White mimosa (Leucaena leucocephala)	26.6 [139] 31.1 [140]	5.6 [140] 31.8 [139]	18.6 [140] 15.3 [139]	13.2 [140] 15.5 [139]	Polyphenols, flavonoids, saponins, tannins, phytosterols [141]	Antioxidant, hypolipidaemic, immunomodulatory [141,142]	Mimosine, tannins, saponins [141]
Brewer’s malt	15.0–30.0 [143] 20.0 [144]	3.0–13.9 [144]	n.a.	up to 80.0 [143] 70.0 [144]	Phenolic acids (ferulic, p-coumaric), flavonoids, antioxidant peptides, fibre, melanoidins, healthy fatty acids, and minerals [145,146]	Cholesterol lowering, glycaemic regulation, prebiotic, antioxidant [143]	Phytic acid, tannins, trypsin inhibitors [144]
Moringa oleifera (seeds)	31.4 [147] 35.4 [148]	36.7 [147] 43.6 [148]	9.2 [148] 18.4 [147]	4.7 [148] 7.3 [147]	Polyphenols, flavonoids, saponins, phytosterols, bioactive peptides [149,150]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [151]	Phytates, glucosinolates, tannins, trypsin inhibitors [150]
Oats (Avena sativa)	10.0–17.2 [151] 13.7 [152]	2.1–10.3 [151] 7.6 [152]	47.9–74.3 [151] 62.7 [152]	2.1–15.4 [151] 10.1 [152]	β-Glucans, avenanthramides, polyphenols, flavonoids [152]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [152]	Phytic acid, saponins, β-glucan [152]
Rice (Oryza sativa)	16.8–24.1 [153] 6.0–7.8 [154] 5.5 [155]	1.6–2.8 [154] 0.8 [155]	82.7–84.5 [154]	2.1–2.7 [154]	Polyphenols, phytosterols, γ-oryzanol, tocopherols [156]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [156]	Phytic acid, tannins [156]
Basmati rice	7.6–9.1 [157]	1.6–2.4 [157] 3.0–3.5 [158]	77.4–79.4 [157]	1.0–1.8 [157]	γ-Oryzanol, tocopherols, phytosterols [158]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [158]	Phytic acid, tannins [157]
Lentils (Lens culinaris)	20.5–26.0 [159]	0.6–1.0 [159]	63.7–69.8 [159]	19.3–26.4 [159]	Polyphenols, flavonoids, saponins, phenolic acids, oligosaccharides [160]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [160]	Phytic acid, tannins, trypsin inhibitors, lectins [160]
Soybean (Glycine max)	39.0 [14] 40.0 [161]	17.0–20.0 [14] 20.0 [161]	18.0 [14] 31.1 [5]	5.1 [5]	Isoflavones, saponins, phytosterols, lecithins [161]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [161]	Phytates, trypsin inhibitors, saponins, lectins [161]
Sorghum (Sorghum bicolor)	8.0–12.0 [160]	1.5–3.5 [160]	65.0–75.0 [160]	6.0–10.0 [160]	Polyphenols, tannins, phytosterols, resistant starch [160]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [160]	Tannins, phytic acid, trypsin inhibitors [160]
Tarwi (Lupinus mutabilis)	44.7 [162]	15.4 [162]	n.a.	n.a.	Polyphenols, flavonoids, isoflavones, saponins [163]	Cholesterol lowering, glycaemic improvement, satiety, antioxidant, anti-inflammatory [163]	Quinolizidine alkaloids, phytic acid [163]
Linseed pomace	21.3 [164]	43.9 [164]	n.a.	6.2 [164]	Lignans, polyphenols, flavonoids, plant mucilages [164]	Cholesterol lowering, glycaemic regulation, prebiotic, antioxidant [50]	Cyanogenic glycosides, phytic acid [50]
Rapeseed pomace	38.1 [165]	33.5 [165]	n.a.	15.3 [165]	Polyphenols (mainly ferulic acid, hydroxycinnamic acids), flavonoids, glucosinolates, phytates, isothiocyanates, sinapic acid, tannins, and saponins [68]	Antioxidant and anti-inflammatory, support of lipid and carbohydrate metabolism, promotion of favourable gut microbiota [68]	Glucosinolates, phytates [68]

DM—dry matter, n.a.—not available.

Table 1. Characteristics of raw materials for tempeh production.

Raw Material	Protein (% DM)	Fat (% DM)	Carbohydrates (% DM)	Fibre (% DM)	Bioactive Compounds	Health-Promoting Properties	Anti-Nutritional Factors
Adlay (Coix lacryma-jobi)	20.0–31.7 [71]	1.0–8.2 [72]	56.0–75.0 [71]	2.5–17.0 [72]	Polyphenols, flavonoids, coixol, phytosterols, saponins [71]	Hypolipidaemic, antidiabetic, antioxidant, prebiotic [71]	Phytic acid, saponins, trypsin inhibitors [73]
Broad beans (Vicia faba L.)	26.0–33.0 [74]	<1.0 [74]	45.7–70.1 [75]	11.4–16.6 [74]	Polyphenols, flavonoids, L-DOPA, tannins, lectins [76]	Cholesterol lowering, heart support, antioxidant [77]	Phytic acid, trypsin inhibitors, saponins, vicine, convicine, lectins, tannins [74]
Chickpeas (Cicer arietinum)	18.7–41.8 [78]	2.7–6.5 cooked [79]	60.0–65.0 [79]	18.0–22.0 [80]	Polyphenols, sterols, carotenoids, tannins, isoflavones [80]	Cholesterol lowering, heart support, glycaemic control, antioxidant [81]	Phytates, lecithins, enzyme inhibitors, oligosaccharides [78]
Red algae (Porphyra sp.)	33.7–41.8 [82]	Up to 1.2 [83]	30.0–40.0 [84]	up to 48.0 [85]	Polysaccharides (porphyran), phycobiliproteins, polyphenols, flavonoids [84,86,87,88].	Antioxidant [86], immune support [84], cholesterol lowering, heart support [89], antidiabetic and anti-inflammatory [84], improved iron bioavailability [90]	Phytic acid, saponins [91]
Eucheuma spinosum	6.0–7.3 [92]	<0.1 [92]	69.0–70.0 [92]	15.0–20.0 [92]	Polyphenols, flavonoids, saponins, tannins, steroids, triterpenoids [93]	Antioxidant, antimicrobial, prebiotic, support for bone mineralisation [93]	Phytic acid, tannins [92]
Cowpea bean (Vigna unguiculata)	17.5–32.5 [94]	1.5 [95]	62.1 [95]	7.0–11.0 [96]	Polyphenols, flavonoids, saponins, tannins, phytosterols, L-DOPA [97]	Cholesterol lowering, glycaemic regulation, microbiome support, antioxidant [97]	Phytic acid, tannins, trypsin inhibitors [97]
Jack bean (Canavalia ensiformis)	25.2 [98]	5.2 [98]	58.4 [98]	7.1 [99]	Polyphenols, flavonoids, kaempferol glycosides, α-glucosidase inhibitors [100]	Antidiabetic [100], immunomodulatory [101]	Canavanine, trypsin inhibitors, lectins, oligosaccharides [99]
Bambara groundnut (Vigna subterranea)	15.0–37.0 [102]	1.3–7.4 [102]	45.0–64.0 [102]	3.7–6.4 [102]	Polyphenols, flavonoids, saponins, tannins, alkaloids, L-DOPA [103]	Cholesterol lowering, glycaemic regulation, satiety, antioxidant, anti-inflammatory [104]	Phytates, tannins, oxalates, trypsin inhibitors, lectins [104]
Mung bean (Vigna radiata)	14.6–32.6 [105]	1.2–1.9 [105]	61.0–67.1 [105]	5.5 [106]	Polyphenols, flavonoids, saponins, phenolic acids, alkaloids, bioactive peptides [107]	Cholesterol lowering, glycaemic regulation, antioxidant, anti-inflammatory, satiety [107]	Phytic acid, tannins, trypsin inhibitors, lectins [105]
Winged bean (Psophocarpus tetragonolobus)	27.2–45.0 [108]	15.2–23.4 [108]	14.2–35.7 [108]	1.6–26.2 [108]	Polyphenols, flavonoids, saponins, phytosterols, bioactive peptides [108]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant, anti-inflammatory [108]	Phytates, tannins, oxalates, trypsin inhibitors, haemagglutinins [109]
Common bean (Phaseolus vulgaris)	20.0–27.0 [110]	0.6–3.0 [111]	58.0–70.0 [110]	30.3–34.2 [112]	Polyphenols, anthocyanins, saponins, tannins, phytosterols [113]	Cholesterol lowering, heart support, glycaemic regulation, antioxidant [110]	Lectins, phytic acid, saponins, trypsin inhibitors, tannins [114]
Large peas (Lathyrus sativus)	17.7–25.6 [115] 20.0–24.9 [116] 18.0–34.0 [117]	1.7 [118]	48.0–52.3 [118]	1.1–1.7 [118] 4.0–7.0 [116]	Polyphenols, flavonoids, saponins, tannins, phytosterols, homoarginine, β-ODAP [118]	Antioxidant, hypolipidaemic, antidiabetic, anti-inflammatory [118]	β-ODAP, phytic acid, saponins, tannins [115,116]
Pearl barley (Hordeum vulgare)	13.6 [119]	2.8 [119]	63.9 [119]	4.7 [119]	β-Glucan, arabinoxylan, polyphenols, flavonoids, phytosterols [119]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant, anti-inflammatory [119].	Phytic acid, saponins, tannins [119]
Rubber (Hevea brasiliensis)	19.4–30.7 [120] 23.3 [121] 26.1 [122] 31.6 [123]	42.5–54.2 [120]	11.6–29.0 [120]	5.9 [121] 43.0 [122]	Polyphenols, flavonoids [120]	Antioxidant, anti-inflammatory, metabolic, requires detoxification [31,120]	Tannins, saponins [121,123]
Wild turmeric (Curcuma aromatica)	19.4 [124]	2.5 [124]	97.5 [124]	n.a.	Polyphenols (curcumin, demethoxycurcumin), flavonoids, terpenoids, essential oils [124,125]	Antioxidant, anti-inflammatory, anticancer, neuroprotective [124,126]	Saponins, tannins, alkaloids [124]
Flowers of butterfly pea (Clitoria ternatea)	0.3 [127]	2.5 [127]	2.2 [127]	2.1 [127]	Anthocyanins (300–500 mg/100 g), flavonoids (100–150 mg/100 g), phenolic acids, saponins, tannins, carotenoids, avenanthramides [127,128,129]	Potent antioxidant, anti-inflammatory, hypoglycaemic, neuroprotective, cardioprotective, anticancer, skin support [127,128]	Tannins, saponins, oxalates [127,128]
Lotus (Nelumbo nucifera)	16.0–21.0 [130]	2.4–3.0 [130]	61.0–62.0 [130]	2.8 [131]	Polysaccharides (porphyran), phycobiliproteins, polyphenols, flavonoids [132]	Antioxidant, immunomodulatory, prebiotic, antidiabetic [133]	Phytic acid, saponins [134,135]
Narrow-leaved lupin (Lupinus angustifolius)	31.6–34.6 [136] 31.0–52.0 [137]	6.0 [137]	<24.0 [138]	37.5–40.2 [137]	Polyphenols, flavonoids, bioactive peptides, saponins, phytosterols [137]	Cholesterol lowering, glycaemic improvement, satiety, antioxidant, anti-inflammatory [138]	Alkaloids, phytic acid, trypsin inhibitors [137]
White mimosa (Leucaena leucocephala)	26.6 [139] 31.1 [140]	5.6 [140] 31.8 [139]	18.6 [140] 15.3 [139]	13.2 [140] 15.5 [139]	Polyphenols, flavonoids, saponins, tannins, phytosterols [141]	Antioxidant, hypolipidaemic, immunomodulatory [141,142]	Mimosine, tannins, saponins [141]
Brewer’s malt	15.0–30.0 [143] 20.0 [144]	3.0–13.9 [144]	n.a.	up to 80.0 [143] 70.0 [144]	Phenolic acids (ferulic, p-coumaric), flavonoids, antioxidant peptides, fibre, melanoidins, healthy fatty acids, and minerals [145,146]	Cholesterol lowering, glycaemic regulation, prebiotic, antioxidant [143]	Phytic acid, tannins, trypsin inhibitors [144]
Moringa oleifera (seeds)	31.4 [147] 35.4 [148]	36.7 [147] 43.6 [148]	9.2 [148] 18.4 [147]	4.7 [148] 7.3 [147]	Polyphenols, flavonoids, saponins, phytosterols, bioactive peptides [149,150]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [151]	Phytates, glucosinolates, tannins, trypsin inhibitors [150]
Oats (Avena sativa)	10.0–17.2 [151] 13.7 [152]	2.1–10.3 [151] 7.6 [152]	47.9–74.3 [151] 62.7 [152]	2.1–15.4 [151] 10.1 [152]	β-Glucans, avenanthramides, polyphenols, flavonoids [152]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [152]	Phytic acid, saponins, β-glucan [152]
Rice (Oryza sativa)	16.8–24.1 [153] 6.0–7.8 [154] 5.5 [155]	1.6–2.8 [154] 0.8 [155]	82.7–84.5 [154]	2.1–2.7 [154]	Polyphenols, phytosterols, γ-oryzanol, tocopherols [156]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [156]	Phytic acid, tannins [156]
Basmati rice	7.6–9.1 [157]	1.6–2.4 [157] 3.0–3.5 [158]	77.4–79.4 [157]	1.0–1.8 [157]	γ-Oryzanol, tocopherols, phytosterols [158]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [158]	Phytic acid, tannins [157]
Lentils (Lens culinaris)	20.5–26.0 [159]	0.6–1.0 [159]	63.7–69.8 [159]	19.3–26.4 [159]	Polyphenols, flavonoids, saponins, phenolic acids, oligosaccharides [160]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [160]	Phytic acid, tannins, trypsin inhibitors, lectins [160]
Soybean (Glycine max)	39.0 [14] 40.0 [161]	17.0–20.0 [14] 20.0 [161]	18.0 [14] 31.1 [5]	5.1 [5]	Isoflavones, saponins, phytosterols, lecithins [161]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [161]	Phytates, trypsin inhibitors, saponins, lectins [161]
Sorghum (Sorghum bicolor)	8.0–12.0 [160]	1.5–3.5 [160]	65.0–75.0 [160]	6.0–10.0 [160]	Polyphenols, tannins, phytosterols, resistant starch [160]	Cholesterol lowering, glycaemic regulation, immune support, antioxidant [160]	Tannins, phytic acid, trypsin inhibitors [160]
Tarwi (Lupinus mutabilis)	44.7 [162]	15.4 [162]	n.a.	n.a.	Polyphenols, flavonoids, isoflavones, saponins [163]	Cholesterol lowering, glycaemic improvement, satiety, antioxidant, anti-inflammatory [163]	Quinolizidine alkaloids, phytic acid [163]
Linseed pomace	21.3 [164]	43.9 [164]	n.a.	6.2 [164]	Lignans, polyphenols, flavonoids, plant mucilages [164]	Cholesterol lowering, glycaemic regulation, prebiotic, antioxidant [50]	Cyanogenic glycosides, phytic acid [50]
Rapeseed pomace	38.1 [165]	33.5 [165]	n.a.	15.3 [165]	Polyphenols (mainly ferulic acid, hydroxycinnamic acids), flavonoids, glucosinolates, phytates, isothiocyanates, sinapic acid, tannins, and saponins [68]	Antioxidant and anti-inflammatory, support of lipid and carbohydrate metabolism, promotion of favourable gut microbiota [68]	Glucosinolates, phytates [68]

DM—dry matter, n.a.—not available.

Moringa seeds are rich in protein, fat, calcium, iron, phosphorus, and vitamin E and can be used to produce a precious oil [166]. Soybean (Glycine max) has a fat content of 17.0–20.0%. Soybean oil has a high content of fatty acids with a predominance of unsaturated acids (oleic, linoleic, linolenic), lecithin, and vitamin E [161]. Four raw materials are characterised by trace amounts of fat: broad beans (Vicia faba L.): <1%, lentils (Lens culinaris): 0.6–1.0, red algae (Porphyra sp.): up to 1.2, and Eucheuma spinosum: <0.1%.

Analysing the data in Table 1, the highest carbohydrate content was found in wild turmeric (Curcuma aromatica), with as much as 97.5% DM, while the lowest content was found in flowers of butterfly pea (Clitoria ternatea), with less than 5% DM.

The analysis of the data in Table 1 shows that brewers’ milling is the richest source of fibre in the raw materials studied (70.0–80.0% DM), while basmati rice contains the least fibre (1.0–1.8% DM). A high fibre content brings numerous health benefits: regulation of intestinal peristalsis, increased feeling of satiety and reduced snacking (body weight control), stabilisation of blood glucose levels (prevention of diabetes), protection of the cardiovascular system by lowering Low-Density Lipoprotein (LDL) levels, reducing the risk of hypertension and heart disease, and a reduction in the risk of colorectal cancer by increasing stool volume and shortening the contact time of carcinogens with the mucosa [167].

Analysing the data in Table 1, it was noted that the raw materials used for tempeh fermentation differ significantly in terms of their bioactive compound profile. Chickpeas (Cicer arietinum), soybeans (Glycine max), and large peas (Lathyrus sativus) are rich sources of isoflavones and tannins; legumes mainly provide polyphenols and flavonoids, while algae (Porphyra sp., Eucheuma spinosum) stand out for their unique polysaccharides (porphyran, phycobiliproteins) with a broad spectrum of immunomodulatory and antioxidant activity [168]. Polyphenols and flavonoids neutralise free radicals, protect cells from oxidative stress, and reduce inflammatory reactions in the body [169]. Curcumin from turmeric (Curcuma aromatica) exhibits potent anti-inflammatory and anticancer properties through modulation of NF-κB and COX-2 pathways [170]. Porphyrans and phycobiliproteins from the red alga Porphyra sp. have prebiotic effects, modulate the composition of the gut microbiota, and stimulate macrophages and lymphocytes [168,171,172]. Saponins from a number of legumes can enhance the immune response and exhibit antiparasitic activity [173]. α-Glucosidase inhibitors (e.g., in Jack beans) slow carbohydrate digestion and lower postprandial glycaemia [100]. Phytosterols from rice (Oryza sativa) and oats (Avena sativa) help lower LDL cholesterol and improve lipid profile [174,175]. Coixol from adlay (Coix lacryma-jobi) inhibits cancer cell proliferation and has hepatoprotective effects [176,177]. Porphyran exhibits anticancer activity by inducing apoptosis and inhibiting the PI3K/Akt pathway [178].

4. Technology for the Production of Tempeh

Tempeh is mainly made with moulds of the genus Rhizopus, especially Rhizopus oligosporus. The production process is based on solid-state fermentation (SSF), carried out under aerobic conditions, which results in the formation of a compact soy “cake”, united by a mould mycelium, with a white colour and a characteristic mushroom and nut smell [179]. The finished tempeh can be consumed after heat treatment (frying, boiling, baking) or stored under refrigeration for up to 10 days, possibly pasteurised or frozen to extend shelf life [4]. Fermentation technology leads to significant biochemical changes in the raw material. Under the influence of mould proteases, soy proteins are broken down into peptides and free amino acids, which increases the digestibility and nutritional value of the product [180]. Phytases produced by Rhizopus degrade phytates (reduction of up to 65%), which improves the bioavailability of iron, zinc, and calcium [179]. β-Glucosidase enzymes convert isoflavones from glycosidic forms to aglycones (genistein, daidzein), which have higher bioavailability and biological activity [4]. Fermentation results in an increase in B vitamins, including vitamin B12 (synthesised by companion bacteria, e.g., Klebsiella pneumoniae, Citrobacter freundii). The reduction of stachyose and raffinose reduces the bloating properties of soya [181]. The basic steps of tempeh production are illustrated in Figure 1, which presents a schematic representation of the typical fermentation process using Rhizopus oligosporus. The tempeh production process begins with the selection of legume seeds. This stage includes seed quality control. Seeds intended for tempeh production must be free from mechanical damage, mould, and other microbiological and physical contaminants. Soaking is a key stage in the preparation of the substrate, during which the seeds are hydrated at a temperature of approximately 25 °C for 6–18 h. This process aims to increase the moisture content of the seeds to a level that allows the subsequent stages of the technological process to be carried out effectively. During soaking, water-soluble components are also partially extracted. In some technologies, acidifying substances such as vinegar or lactic acid are added to lower the pH to around 3.5, which promotes the selective elimination of undesirable bacterial microflora. Dehulling involves the mechanical removal of the seed coat from legumes. Dehulling can be carried out by rubbing the seeds in water while stirring, which causes the husks to be released and float to the surface. The removal of the seed coat is important for the textural properties of the finished product and facilitates the penetration of Rhizopus oligosporus during fermentation. Thermal treatment of seeds involves cooking at 100 °C for 25–30 min or steaming. This process facilitates the availability of nutrients for Rhizopus oligosporus. Thermal treatment also softens the structure of the seeds, which promotes subsequent penetration of the mycelium hyphae. After cooking, the seeds are drained and dried to the appropriate moisture content. Inoculation involves introducing Rhizopus oligosporus spores into the prepared substrate. The spores are evenly distributed throughout the seed mass at a temperature not exceeding 35 °C (maintaining < 35 °C to prevent growth of thermophilic pathogens while supporting desired fungal development). Fermentation is a key stage in the process, during which R. oligosporus mycelium develops, and the substrate is transformed. The process takes place under fermentation conditions in a solid medium for 24–48 h at a temperature of 30–35 °C with a relative air humidity of 85–90%. During fermentation, Rhizopus oligosporus produces a dense network of white hyphae (mycelium), which mechanically bind the seeds into a compact structure resembling dough. At the same time, biochemical processes take place, including partial proteolysis of soy proteins; hydrolysis of oligosaccharides (raffinose and stachyose); and synthesis of enzymes, including phytase, which increases the bioavailability of minerals. After about 12–15 h of fermentation, endogenous heat production begins through the developing mycelium, which requires temperature control of the process. Cooling the product to room temperature completes the fermentation process and stabilises the structure of the tempeh. Rapid cooling stops the further development of Rhizopus oligosporus and extends the shelf life of the product. The finished tempeh is characterised by a compact, plastic structure; a white surface covered with mycelium; and a characteristic aroma with nutty and mushroom notes. Packaging takes place under controlled sanitary conditions, often using vacuum packaging or in a modified atmosphere to extend shelf life. The product can also be pasteurised [9].

5. Chemical Composition of Tempeh

All the data on the chemical composition of tempeh collated in this article are from studies using recognised reference analytical methods in accordance with AOAC. For the determination of protein content, the Kjeldahl or Dumas method was used [2,5]. For the determination of fat levels, the Soxhlet extraction or the Bligh and Dyer method was used [2,64]. Differential calculation or the phenol–sulphur method was used to determine carbohydrate concentration [5,62]. Total polyphenol content (TPC) was determined using the Folin–Ciocalteu method [2,6], and antioxidant activity was determined in two ways: as free-radical-neutralisation activity relative to the DPPH radical [2,5,6].

Table 2. Chemical composition of tempeh from different raw materials.

Type of Tempeh	Protein [% DM]	Fat [% DM]	Carbohydrates [% DM]	Fibre [% DM]
Tempeh with adlay (Coix lacryma-jobi)	n.a.	n.a.	n.a.	n.a.
Tempeh from broad beans (Vicia faba L.)	17.8 [15] 38.3 [32]	n.a.	16.72 [15]	n.a.
Chickpea tempeh (Cicer arietinum)	26.5 [5] 31.1 [33] 47.1 [15]	6.26 [5] 9.88 [33]	16.57 [33] 26.31 [15] 62.66 [5]	2.95 [5] 4.40 [33]
Tempeh from Porphyra sp.	17.6–18.5 [62]	6.81–7.22 [62]	11.58–12.15 [62]	n.a.
Tempeh from Eucheuma spinosum	11.6–16.7 [64]	4.34–6.61 [64]	8.04–10.12 [64]	14.40–17.99 [64]
Tempeh of cowpea bean (Vigna unguiculata)	n.a.	n.a.	n.a.	n.a.
Jack bean tempeh (Canavalia ensiformis)	9.6 [35] 15.8 [37] 31.6 [36] 31.8 [40] 41.0 [39]	1.6 [35] 4.1–4.5 [40] 6.2 [36] 10.9 [37]	12.5 [37] 25.1–26.0 [35] 57-1–57.9 [40]	16.6 (total) [36]
Tempeh from Bambara groundnut (Vigna subterranea)	22.6–23.4 [41]	1.4 [41]	12.8–13.0 [41]	n.a.
Mung bean tempeh (Vigna radiata)	n.a.	n.a.	n.a.	n.a.
Tempeh of winged bean (Psophocarpus tetragonolobus)	15.3–17.4 [23]	7.7–9.4 [23]	n.a.	n.a.
Tempeh of common bean (Phaseolus vulgaris)	16.3 [15] 21.4 [43] 23.3 [46]	1.0 [43] 1.3 [46]	27.0 [15] 55.5 [46] 68.4 [43]	17.5 [46]
Tempeh of large peas (Lathyrus sativus)	29.7 [48] 41.1 [49]	19.4 [48]	n.a.	n.a.
Pearl barley (Hordeum vulgare) tempeh	7.8 [56]	n.a.	n.a.	n.a.
Tempeh rubber (Hevea brasiliensis)	20.6 [61]	n.a.	n.a.	n.a.
Wild turmeric tempeh (Curcuma aromatica)	n.a.	n.a.	n.a.	n.a.
Tempeh topped with butterfly pea flowers (Clitoria ternatea)	15.6–17.1 [65]	9.3–11.8 [65]	12.1–12.9 [65]	n.a.
Lotus tempeh (Nelumbo nucifera)	9.4 [59] 38.4 [58]	5.8 [59] 5.6–8.3 [58]	19.5 [59] 53.6–65.6 [58]	n.a.
Tempeh of the narrow-leafed lupin (Lupinus angustifolius)	n.a.	n.a.	n.a.	n.a.
Tempeh from white mimosa (Leucaena leucocephala)	n.a.	n.a.	n.a.	n.a.
Brewer’s mill tempeh	5.0–7.9 [67]	n.a.	n.a.	n.a.
Moringa oleifera tempeh (seeds)	7.6–20.6 [60]	n.a.	n.a.	26.0–28.0 [60]
Tempeh from oats (Avena sativa)	n.a.	n.a.	n.a.	n.a.
Tempeh from rice (Oryza sativa)	n.a.	n.a.	n.a.	n.a.
Basmati rice tempeh	7.6–9.5 [56]	n.a.	n.a.	n.a.
Lentil tempeh (Lens culinaris)	15.1–46.2 [15]	n.a.	16.1-35–7 [15]	n.a.
Tempeh from soya (Glycine max)	14.6–17.4 [24] 16.5 [23] 16.8–18.6 [19] 25.0 [15] 37.4 [14] 44.7 [5] 44.3–44.9 [11]	5.0–10.8 [23] 8.2–11.8 [24] 16.5–17.1 [11] 17.3 [14] 23.2 [5]	7.3–12.0 [24] 9.9–18.4 [23] 27.1 [5] 32.6–33.6 [11]	4.2 [5]
Tempeh of sorghum (Sorghum bicolor)	n.a.	n.a.	n.a.	n.a.
Tempeh from tarwi (Lupinus mutabilis)	2.8–32.5 [53]	n.a.	n.a.	n.a.
Tempeh with linseed pomace added	n.a.	n.a.	n.a.	n.a.
Tempeh from rapeseed pomace	36.5 [68]	14.6 [68]		12.0 [68]

DM—dry matter, n.a.—not available.

shows a comparison of the chemical composition of tempeh obtained from different raw materials. Values in the table in the form of a range indicate differences due to different test methods or raw material varieties.

Based on the analysis of the data in Table 2, it can be concluded that the average protein content of the tempeh samples tested is about 23.3% DM, with a minimum value of 2.8% for tempeh from tarwi and a maximum value of 47.1% for tempeh from chickpea. The cowpea beans (Vigna unguiculata) and lentils (Lens culinaris) with 46.2% DM are also rich sources of protein in tempeh from innovative substrates. The level of protein in these substrates is comparable to that in tempeh from soya (Glycine max)—44.9% DM. The most protein-poor substrates are brewer’s milling, rice, and barley—less than 10.0% DM is protein.

An analysis of the data in Table 2 found that the average fat content of the tempeh samples tested was about 9.2% DM, with a minimum value of 1.0%—for tempeh from common bean (Phaseolus vulgaris)—and a maximum value of 23.24%—for tempeh from soybean (Glycine max). Tempeh from soybeans (average 15.7%) and tempeh from large peas (Lathyrus sativus) (19.4%) are products with a markedly higher fat content, which may affect the creamier texture and higher calorie content of the finished product. The fat content of legume tempeh varies over a wide range from 1.2% to 19.4%, indicating a strong influence of raw material species and fermentation conditions. The other tempehs show moderate levels of fat (average 8.5%), with rapeseed pomace tempeh as an exception with a fairly high fat content (14.6%). The lack of fat content data for the cereal category makes it difficult to assess the influence of cereals on the fat profile of tempeh.

Tempeh fermentation is a complex biochemical process in which Rhizopus oligosporus produces specific enzymes responsible for the transformation of nutrients. The main mechanisms include protein proteolysis, which increases their bioavailability; the production of phytase, which eliminates phytic acid and increases the absorption of minerals; and the biosynthesis and release of polyphenols with antioxidant properties. These biochemical transformations make tempeh a product with a significantly higher nutritional value compared with the raw material.

Based on the analysis of the data in Table 2, the average carbohydrate content of the analysed tempehs was found to be around 31.2% DM, with a minimum value of 7.3% for tempeh from soybean (Glycine max) and a maximum value of 73.0% for tempeh from lentil (Lens culinaris). The highest concentration of carbohydrates is found in tempeh from lentils (Lens culinaris) (average 44.6%) and large peas (Lathyrus sativus) (66.3%), making them an excellent source of energy. The lowest content is observed in tempeh from soybean (Glycine max) (average 21.4%), which is due to the higher proportion of protein and fat in this raw material. Tempeh from legumes shows a wide range of carbohydrate content (7.3–73.0%), reflecting the diversity of their composition and fermentation methods. Tempeh from algae (Porphyra, Eucheuma) has a relatively low range of carbohydrate content (8.0–12.2%) due to the specific carbohydrate profile of these raw materials. The lack of data for cereals suggests the need to complete studies on tempeh based on barley (Hordeum vulgare), rice (Oryza sativa), and oats (Avena sativa) to assess their impact on the carbohydrate profile of the product.

Analysing the data in Table 2, the highest fibre content was observed in tempeh from Moringa oleifera seeds (average 27.0%). High levels of fibre are shown in tempeh from Jack bean and Eucheuma spinosum—content of approximately 16.0–17.0%. Raw materials with a high fibre content can enrich tempeh to aid peristalsis and increase its health benefits.

6. Content of Polyphenols, Isoflavones, and Antioxidant Activity

Antioxidants are a desirable functional property of food products, known for their beneficial health-promoting properties [26]. Tempeh is a source of valuable polyphenols—mainly isoflavones, flavonoids, and phenolic acids, which are responsible for antioxidant, anti-inflammatory, and anticancer activity and promote hormonal regulation [44,182]. Polyphenol content and antioxidant activity are key indicators of the health-promoting potential of tempeh.

Table 3. Summary of polyphenol content and antioxidant activity of tempeh from different raw materials.

Raw Material of Tempeh	Polyphenols [mg GAE/g]	DPPH [% Inhibition] or [ppm]
Adlay (Coix lacryma-jobi)	58.2 [26]	647.0 ppm [26]
Broad bean (Vicia faba L.)	4.8 [15]	n.a.
Chickpea (Cicer arietinum)	4.3 [15]	6.4–14.7% [34]
Seaweed (Porphyra sp.)	n.a.	85.0% [63]
Seaweed (Eucheuma spinosum)	n.a.	n.a.
Cowpea bean (Vigna unguiculata)	n.a.	n.a.
Jack bean (Canavalia ensiformis)	4.0–10.7 [6] 12.0 [36]	2.5–3.6 [35] 69.7% [37] 457.0–3436.6 ppm [6]
Tempeh from Bambara groundnut (Vigna subterranea)	n.a.	n.a.
Mung bean (Vigna radiata)	137.5 [26]	128.0 ppm [26]
Winged bean (Psophocarpus tetragonolobus)	n.a.	39.1–51.8% [23]
Common bean (Phaseolus vulgaris)	2.1–3.6 [15]	43.0 [43]
Large peas (Lathyrus sativus)	2.1 [50]	n.a.
Pearl barley (Hordeum vulgare)	n.a.	n.a.
Rubber (Hevea brasiliensis)	n.a.	n.a.
Wild turmeric (Curcuma aromatica)	99.7 [66]	n.a.
With butterfly pea flowers (Clitoria ternatea)	n.a.	91.5% [65]
With lotus flowers (Nelumbo nucifera)	n.a.	72.6% [57]
Narrow-leafed lupin (Lupinus angustifolius)	n.a.	n.a.
White mimosa (Leucaena leucocephala)	n.a.	n.a.
Brewers’ spent grains	n.a.	n.a.
Seeds (Moringa oleifera)	n.a.	n.a.
Oats (Avena sativa)	0.8–1.8 [55]	n.a.
Rice (Oryza sativa)	n.a.	n.a.
Basmati rice (Oryza sativa)	n.a.	n.a.
Lentils (Lens culinaris)	2.7–4.3 [15]	n.a.
Soya beans (Glycine max)	6.1 [15]	45.1 [23] 50.0–84.0 [7]
Sorghum (Sorghum bicolor)	64.2 [26]	457.0 ppm [26]
Tarwi (Lupinus mutabilis)	n.a.	n.a.
Linseed oil-press cake	3.9 [50]	n.a.
Rapeseed oil-press cake	n.a.	n.a.

n.a.—not available.

summarises the polyphenol content and free-radical-neutralising activity against the DPPH radical of different tempeh types.

Based on the analysis of the data in Table 3, it was found that the average polyphenol content among the tempeh samples tested was typically in the range of 2.0 to 137.5 mg GAE/g DM.

The highest polyphenol levels (>50.0 mg GAE/g DM) were recorded in mung bean (Vigna radiata) tempeh—as high as 137.5 mg GAE/g DM; sorghum (Sorghum bicolor) tempeh—64.2 mg GAE/g DM; and adlay (Coix lacryma-jobi) tempeh—58.2 mg GAE/g DM. The lowest values (<5 mg GAE/g DM) are shown by tempeh from lentils (Lens culinaris), large peas (Lathyrus sativus), oats (Avena sativa), broad beans (Vicia faba L.), common beans (Phaseolus vulgaris), and chickpeas (Cicer arietinum).

Analysing the data in Table 3, it was noted that the vast majority of tempeh showed moderate-to-high DPPH-radical-neutralising activity: 39.0–70.0% inhibition. The highest antioxidant activities were recorded for soybean (Glycine max) tempeh—50.0–84.0% and Jack bean (Canavalia ensiformis) tempeh—up to 69.7%.

Fermentation of tempeh almost always increases polyphenol levels and DPPH radical scavenging efficiency relative to unfermented seeds. The differences between the tempeh samples analysed are mainly due to type and variety of raw material, duration and course of fermentation, and possible functional additives (e.g., spices [182], tea extracts [183]).

Reduction of Anti-Nutritional Factors Through Fermentation

Fermentation of tempeh results in a significant reduction in the anti-nutritional factors present in the raw legumes. The content of trypsin inhibitors decreases by 64–67% after fermentation, and additional frying of the product further reduces their levels [184,185,186,187]. In each of the variants analysed, there is also a marked decrease in the concentration of phytates and tannins, which are responsible for limiting the bioavailability of minerals [53]. The process of protein hydrolysis, which occurs during fermentation, leads to an increase in free amino acids and peptides, which directly translates into an improvement in the digestibility and bioavailability of the protein in the finished product [8,184,186,187].

7. Conclusions

This comprehensive review demonstrates that tempeh fermentation with Rhizopus oligosporus represents a versatile and sustainable approach for developing innovative plant-based protein foods from diverse raw materials [2,3,5,6]. The analysis of over 25 different substrates reveals significant opportunities for enhancing nutritional and functional properties beyond traditional soybean-based formulations. The fermentation process consistently achieves substantial reductions in anti-nutritional factors, with a 64.0–67.0% decrease in trypsin inhibitors and up to 65.0% reduction in phytates, while simultaneously enhancing protein digestibility and bioactive compound availability across all substrate categories.

Several alternative substrates demonstrate superior nutritional profiles compared with traditional soybean tempeh, notably tarwi (Lupinus mutabilis) with exceptional protein content (32.0–53.0% DM) and mung bean (Vigna radiata) exhibiting remarkably high polyphenol concentrations (137.5 mg GAE/g DM). The diversity of bioactive compounds identified across different substrates, from curcumin in wild turmeric tempeh to phycobiliproteins in algae-based variants, demonstrates potential for developing targeted functional foods with specific health-promoting properties. Consistent antioxidant activities (39.0–70.0% DPPH inhibition) across most tempeh variants further support their potential as functional food ingredients.

Tempeh technology enables the effective utilisation of underutilised raw materials including legumes, cereals, algae, seeds, and agricultural by-products, contributing to sustainable food system development through protein source diversification. The results indicate substantial potential for developing functional plant foods with enhanced nutritional profiles and diverse bioactive ingredient compositions, supporting both human nutrition and environmental sustainability objectives.

8. Remarks on Future Directions

Systematic studies are needed to optimise the fermentation parameters (temperature, humidity, duration, inoculum concentration) for each substrate category. The current literature reveals significant gaps in standardised processing protocols, particularly for cereal and algae-based substrates. Future research should prioritise the complete nutritional profiling of promising substrates, as numerous gaps exist in the current data (particularly with reference to fibre content, amino acid profiles, and mineral bioavailability). Advanced analytical techniques should be employed to characterise the bioactive peptides formed during fermentation.

Limited sensory evaluation data represents a critical research gap. Comprehensive consumer acceptance studies, texture analysis, and flavour profiling are essential for commercial viability, particularly for substrates with distinct organoleptic properties.

Life cycle analyses comparing the environmental impacts of different substrates should be conducted to support sustainable food system development. This includes evaluation of water usage, carbon footprint, and land use efficiency.

Research on large-scale production challenges, including substrate preparation, fermentation control, and quality standardisation, is crucial for commercial implementation. Development of standardised quality parameters and shelf-life studies for innovative tempeh variants are particularly needed.

Establishment of regulatory guidelines for novel tempeh substrates, particularly those containing algae or unconventional plant materials, will be essential for market acceptance and food safety assurance.

Human clinical trials investigating the health effects of specific tempeh variants, particularly those with enhanced bioactive compound profiles, should be prioritised to substantiate functional food claims.

Future research should explore synergistic combinations of different substrates to optimise nutritional profiles, potentially creating tempeh products with enhanced protein quality, improved amino acid balance, and complementary bioactive compound portfolios.

The evidence presented in this review strongly supports the potential for tempeh technology to contribute significantly to global food security and sustainable nutrition through the diversification of protein sources and the enhancement of plant food functionality. However, realising this potential requires coordinated research efforts addressing the identified knowledge gaps and technological challenges.