Front. Biosci.

(Elite Ed) 2024; 16(1): 3

https://doi.org/10.31083/j.fbe1601003

Review
Soy-Based Tempeh as a Functional Food: Evidence for Human Health
and Future Perspective
Gianluca Rizzo1, *
1 IndependentResearcher, 98121 Messina, Italy
*Correspondence: drgianlucarizzo@gmail.com (Gianluca Rizzo)
Academic Editor: Yiannis Kourkoutas
Submitted: 29 September 2023 Revised: 5 November 2023 Accepted: 24 November 2023 Published: 31 January 2024

Abstract
Tempeh is an Indonesian traditional food made from fermented soybeans, which offers wide culinary use in East Asian countries. Similar
to all fermented foods, its preparation offers the purpose of food preservation. However, preclinical studies have highlighted that microbial
action leads to a modification in the nutritional composition of the food’s matrix. Although there is a wide availability of data on the
beneficial effect of soy, tempeh remains relatively unexplored, perhaps due to its limited diffusion in the world, which limits its research
availability. However, available data suggest that tempeh may confer beneficial health effects due to the high bioavailability of nutrients
and phytochemicals, showing ameliorative action on oxidative stress, glycaemic control, and blood lipid levels. Furthermore, the high
biological value of tempeh means it can be used to optimize protein and caloric intake in athletes, vegetarians, and children. Moreover,
the microbial fermentation used in the production of tempeh, in addition to improving the bioavailability of minerals, proteins, fibre,
vitamins, and isoflavones, produces biopeptides whose biological effect is currently of great interest. Tempeh can be employed in
traditional preparations as well as second-generation foods, such as plant-based meat substitutes, to provide functional and nutritional
properties and a higher eco-friendly option compared to animal foods. This review aims to provide an overview of tempeh’s properties,
regarding human data and future research perspectives.

Keywords: tempeh; soy; functional foods; fermented soybeans; isoflavones; antioxidants; phytoestrogens; plant protein; gut microbiota;
paraprobiotic

1. Introduction The use of fermenting plant foods is widespread

Ever since man has stabilized sedentary habits, the throughout the world, with its roots in the culinary traditions
need for food conservation has become a fundamental role of various countries, such as sourdough, sauerkraut, miso,
in daily food availability. Food fermentation consists of tempeh, natto, kombucha, tamari, sufu, tofuyo, shoyu, kim-
processing staples using microorganisms, with the main chi, tape, doenjang, meju, douchi, cheonggukjang, kan-
purpose of protecting against the growth of pathogenic jang, thua-nao, kinema, hawaijar, tungrymbai, oncom, etc.
microbes that cause food to spoil and pose health risks [1,7,8]. Many fermented foods from Southeast Asian coun-
[1]. However, fermentation also allows the food matrix tries extensively employ soy as a starting staple [9–12].
to be modified, providing different aromas from the start- Tempeh or tempe, as per its original name [13,14], is
ing food, by altering the bioavailability of nutrients and a traditional Indonesian soybean food, which is obtained
phytochemicals, improving nutritional quality, and confer- following various processes of dehulling, soaking, boiling,
ring added functional properties [2–4]. Furthermore, the and fermentation [15–17]. During the last century, tempeh
microorganisms being used for transformations can pro- has spread to India, Japan, and the United States, more re-
duce new molecules, such as vitamins, and by-products cently to Zambia in the early 1970s [15].
derived from microbial metabolism, including biopeptides Multiple fermentation steps can be used during pro-
with nutraceutical activities derived from matrix polypep- duction, while treatments performed at different tempera-
tides, which have recently gained more interest for health tures can follow one another along the production process.
purposes [5,6]. The use of various microorganisms may also vary based
There are different types of fermentation, which can on the traditional use of environmental sources or through
be characterized by the presence of a selected starter, along- inoculations of specific isolated microorganisms. Even if
side the working conditions, and type of microorganisms there is a single decisive fermentation, other fermentation
being used. The most commonly employed microorgan- steps can occur to improve the product. Indeed, Fig. 1 dis-
isms are yeasts, moulds, lactobacilli, and streptococci, plays the main steps in the soy-based tempeh production
which limit pathogen growth through acidification of the process.
medium and microbial exclusion. Additionally, foods can In Indonesia, tempeh is considered a cheap and afford-
be transformed by a heterogeneous pool of environmental able staple-based food, which is rich in proteins and can
microorganisms. be consumed through a wide variety of cooking methods,

Copyright: © 2024 The Author(s). Published by IMR Press.

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 a white covering texture by the mycelium, which com-
pacts the tempeh structure and causes a mushroom-like
flavour [29,30]. Although soy-based tempeh is the most
widespread, the same transformation process can be applied
to various legumes and grains [31].
As with many other fermented foods, the production
process reduces the concentration of antinutrients in the
food matrix (such as protease inhibitors, tannins, and phytic
acid), thereby causing an increase in the nutritional value of
the product compared to the starting food [3].
Although the properties of tempeh have been de-
scribed in preclinical studies, human studies are still very
Fig. 1. Production process of tempeh. The step order can change fragmentary if we consider the large availability of studies
based on specific needs. on soy [32–39].
This review aims to describe the possible benefits of
soy-based tempeh for humans, collecting available data
including boiled, fried, grilled, or steamed [15]. Different
from previously published literature. This document pro-
types of rice, wheat, or brans are normally used as tradi-
vided a comprehensive update of the current knowledge
tional inoculums. Heterogeneous pools of microorganisms
on biotechnology and biochemical characteristics of soy-
can be found in traditional tempeh, with dominant strains
based tempeh, with particular attention on human outcomes
such as S. dysgalactiae, S. faecium, S. epidermidis, and L.
and benefits related to its use. Although previous reviews
casei identified in the soaking step. Likewise, B. brevis, C.
on the topic are available, the information proposed is fre-
diversus, E. agglomerans, E. cloacae, K. pneumoniae, K.
quently based on animal and in vitro studies, with marginal
ozaenae, P. burtonii, R. rubra, and C. diddensiae have also
parts regarding the benefits on humans. A relevant part
been detected [18]. The use of Lactiplantibacillus plantar-
of this document was dedicated to nutritional values, nu-
ium (formerly known as Lactobacillus plantarium and L.
traceutical properties, and current health concepts, such as
arabinosus) during the soaking step seems to favour the
postbiotic and paraprobiotic actions of secondary metabo-
acidification medium, which inhibits the growth of vari-
lites from fermented food. Moreover, well-known bioac-
ous pathogenic microbes [19–22]. In the industrial produc-
tive molecules with health benefits for metabolic diseases
tion of tempeh, the fermentation step is conducted by pack-
are also discussed.
ing soaked and inoculated soybeans into perforated plas-
tic bags. However, naturally available materials are used
2. Nutritional Aspects
during the traditional fermentation process. Fig. 2 shows a
finished traditional tempeh product, whereby Hibiscus tili- Tempeh is a food low in carbohydrates and rich in pro-
aceus leaves are usually used for packing inoculated soy- teins, vitamins, and bioactive compounds [30]. Fermenta-
beans before the fermentation step. tion allows the release of free fatty acids from lipid fractions
[40] and transforms soy glycosidic isoflavones into uncon-
A single species of filamentous fungus is used in the jugated aglycone [41–43]. According to the Codex Alimen-
fermentation step, with the common employment of Rhizo- tarius Commission, the protein component must represent
pus species, such as R. oligosporus, R. oryzae, R. stolonifer, at least 15% w/w with a lipid content of 7% and dietary
R. arrhizus, and R. formosaensis, alongside other microor- fibres representing at least 2.5% [29]. According to the
ganisms belonging to Fusarium spp [23,24]. Among these United States Department of Agriculture Standard Refer-
moulds, Rhizopus microsporus var. oligosporus is the most ence (USDA SR) Legacy Foods database, tempeh provides
used globally for producing tempeh [25]. However, other 192 kcal per 100 g, 20.3% protein, 10.8% fat, and 7.64%
microorganisms such as bacteria, moulds, and yeasts may carbohydrates [44]. Furthermore, 100 g of tempeh consists
be present in traditional co-inoculums, such as Absidia spp., of 111 mg of calcium, 2.7 mg of iron, and 24 mcg of folate.
Mucor spp., Rhizomucor spp., etc. [26]. In non-traditional Fermentation leads to an increase in iron bioavailability ow-
co-inoculations, probiotics such as L. plantarium have been ing to the conversion of iron (III) into iron (II) [45,46]. Fur-
added to the main fermentation step, which can greatly in- thermore, there are 4.3 g of polyunsaturated fatty acids per
hibit the growth of pathogens during this phase [22]. The 100 g, the majority of which is represented by linoleic acid.
co-inoculation of B. subtilis, a bacterium used in the pro- This polyunsaturated fatty acid is an essential molecule in
duction of Natto, can also confer additional functional prop- the omega-6 series that is particularly represented in soy and
erties to tempeh [27], while the use of A. elegans can re- its derivatives [47]. In the USDA-branded foods database, a
duce the presence of non-digestible oligosaccharides that very similar composition can be found, with frequent use of
produce flatulence [28]. white, brown, or wild rice among ingredients. Nutritional
Solid-state fermentation changes the consistency of composition among different batches and preparations of
the starting matrix, producing a cake-formed product with tempeh can vary according to the microbial species being

2
Fig. 2. Traditional tempeh. Hibiscus leaves are used to package inoculated soybeans.

Table 1. Nutritional composition of tempeh (data per 100 g).

Product Energy (kcal) Protein (g) Fat (g) Carbohydrate (g) Fibre (g) Calcium (mg) Iron (mg) Ingredients
USDA SR Legacy 192 20.3 10.8 7.64 NA 111 2.7
Database
Lightlife (a) 190 21.4 5.36 14.3 7.1 104 2.38 Cultured organic soybeans
(soybeans, lactic acid from plant
source), water, and organic
brown rice
Nature’s Promise 224 23.5 9.41 12.9 4.7 94 1.65 Cultured organic soybean and
water
Lightlife (b) 202 16.7 5.95 21.4 8.3 71 2.14 Cultured organic soybeans
(soybeans, lactic acid from plant
sources), water, organic wild
rice, and organic brown rice
Franklin Farms 227 22.7 9.33 13.3 5.3 93 1.73 Cultured organic soybean and
water
Westsoy 211 21 9.21 11.8 3.9 105 2.37 Cultured organic soybean, water,
and organic white rice

used and the fermentation process conditions, such as time similar to unfermented soybeans, tempeh offers better pro-
and temperature. Table 1 shows the nutritional composi- tein quality. Fermentation increases the protein digestibil-
tion of tempeh according to the USDA SR Legacy Foods ity corrected amino acid score (PDCAAS) compared to the
and Branded Foods database. starting pulse [48]. The PDCAAS is a parameter that is
widely used in the evaluation of protein quality since it con-
2.1 Protein siders both the chemical composition of amino acids and
the physiological absorption of proteins. Furthermore, mi-
A protein-energy deficiency persists in low-income crobial degradation of proteins by microorganisms releases
countries, caused by low accessibility to food in many parts free amino acids into the matrix, which has possible func-
of the world. Furthermore, plant foods may have limited tional implications [49].
protein value due to their low amino acid score and di-
gestibility. Soy is a well-known food source that is rich Tempeh is considered a rich source of branched-
in essential amino acids. However, the digestibility of soy- chain amino acids (leucine, isoleucine, and valine) due
beans is limited by the presence of antinutrient substances to the increase in their bioavailability after fermentation
commonly found in legumes, such as phytic acid, tannins, [50]. Moreover, the fermentation process can slightly in-
and protease inhibitors. There is a lowering of phytic acid crease essential amino acid bioavailability compared with
concentrations during microbial fermentation and soaking, the starting staple [51]. The high protein quality of tem-
[46], which improves the bioavailability of proteins owing peh can be useful for ensuring affordable vegetable protein
to an increase in digestibility caused by a reduction of antin- sources with high nutritional values. Furthermore, these
utrients. Although the overall amount of protein is very characteristics are often attractive to those who follow ei-

3
ther a vegetarian or vegan diet and are looking for alter- tempeh can increase five-fold compared to the starting food
native nutritional sources. Considering that a plant-based [66–68]. Tempeh has also been identified as a source of er-
diet can be adopted for ethical, health, and environmental gosterol, a provitamin isoform of calciferol called vitamin
reasons [52,53], tempeh can be highly accepted among veg- D2, which is biosynthesized by fungi, including microbial
etarians and vegans. [69,70].
2.3 Antinutrients
2.2 Vitamins
As already mentioned, the reduction of antinutrients
Vitamin B12 is an essential molecule for humans. It
through the fermentative transformation process is a crucial
takes part in mammalian metabolic pathways as an enzy-
step in increasing the soy nutrients bioavailability found in
matic co-factor in metabolising odd-chain fatty acids and
tempeh, in particular, the polyphenols, minerals, and pro-
for homocysteine detoxification [54,55]. It is considered a
teins [3]. Although the cooking and soaking steps con-
vitamin of animal origin since the concentrations in plant
tribute toward the reduction in phytic acid, fermentation ap-
foods are negligible. However, its biosynthesis is carried
pears to be the most effective step for this activity [3,30].
out by microorganisms, meaning fermented foods can con-
This is possible thanks to the action of thermostable phy-
tain vitamin B12 if competent microbes are present [56,57].
tases of R.oligosporus, which work at an optimum temper-
Based on the microbial consortium composition of tempeh
ature of 44 °C and pH of 3–5 [71,72]. R. oligosporus phy-
fermentation, significant quantities of vitamin B12 derived
tases reduce the phytic acid content by approximately 65%
from symbionts can be found [58,59]. The production of
[73]. Some lactic acid bacteria can also show phytase ac-
vitamin B12 in tempeh usually depends on the presence of
tivity [72,74]. Similarly, oxalate levels in tempeh appear
K. pneumoniae and C. freundii in the microbial consortium
to be lower than in other plant foods, including textured
[58,60,61]. It seems that the main mould used for fermen-
soy protein, which shows levels that are almost 28 times
tation does not contribute to vitamin B12 production. Gen-
higher [75]. The levels of trypsin inhibitors are also reduced
erally, the presence of this vitamin is dependent on con-
in tempeh following fermentation, which also leads to im-
taminating and causal events and it can cause up to 12.4
proved protein digestibility [45].
mcg of vitamin B12 per 100 g of traditional tempeh [58].
Stachyose, raffinose, and alpha-
In countries of traditional production, where natural water
galactooligosaccharides are all reduced by the fermentative
sources are used, environmental bacteria can synthetize vi-
action of the microbes found in tempeh, thereby reducing
tamin B12 [58]. However, a cross-feeding network appears
flatulence resulting from indigestible oligosaccharides
to exist between vitamin B12-producing species and Rhi-
[73,76–78]. Moreover, regarding allergenicity, the effect
zopus [61]. Moreover, Propionibacterium species can be
of fermentation allows allergens hydrolysis to occur, which
useful in increasing vitamin B12 concentrations in tempeh
also happens in other fermented foods [79,80]. This aspect
batches and they could be industrially adopted for this pur-
can be further enhanced by selected co-inoculations that
pose [62,63].
favour the protein hydrolysis from the matrix [28]. A
Since the availability of vitamin B12 in plant-based
further improvement in protein quality occurs through the
foods is negligible among vegetarians, the possibility of fre-
reduction in phytates and indigestible oligosaccharides
quently using tempeh in steady amounts could help to guar-
as well as an increase in antioxidant power, which was
antee dietary vitamin B12 requirements [57]. However,
obtained by germinating the soybeans used for tempeh
considering that concentrations are still not standardized by
production [81–83].
the various producers, and tempeh is a food option not of-
Tempeh, unlike soybeans and other legumes, is among
ten consumed daily in countries different from the places of
the foods listed by Monash University resources as low in
traditional production, it remains premature to confer this
fermentable oligosaccharides, disaccharides, monosaccha-
nutritional role to tempeh.
rides, and polyols (FODMAPs) [84,85].
Although vitamin B12 is of great interest due to its
limited availability in nature, the fermentative action of mi- 2.4 Minerals
croorganisms can promote the bioavailability of other vi- Fermentation increases soluble iron from 24% to 67%
tamins. Co-inoculation of tempeh with Aspergillus oryzae owing to the release from the vegetal ferritin soy deposits,
compared to R. oligosporus alone shows a considerable in- probably due to the phytase activity [13]. The increase in
crease in vitamin B1 and vitamin B2 levels, although it has iron bioavailability also depends on the conversion of iron
been detected in grass pea and not soybean [64]. The in- (III) into iron (II) [46,86].
crease in vitamin B3 both in the form of nicotinamide and It has been suggested that soy products may provide
nicotinic acid has been observed in batches of tempeh fer- a potentially beneficial effect on bone health due to their
mented with C. freundii and Lactobacillus spp., while vita- isoflavone content [87–89]. Furthermore, microbial fer-
min B1 can be produced by C. freudnii [65]. The ferment- mentation in tempeh also improves calcium bioavailabil-
ing microbes increased vitamin B8 content by more than ity, which may contribute to improved bone density [13].
2 times compared to the basic matrix [66]. Folates also Haron and colleagues [90] evaluated calcium absorption
seem to increase from fermentation, while the content in from tempeh in 20 post-menopausal Malaysian women and

4
compared it to cow milk in a cross-over clinical trial. Fol- dogenous oestradiol and which, together with lignans and
lowing the intake of approximately 200 g of tempeh, com- coumestans, is classified as a phytoestrogen due to these
pared to 114 grams of milk (containing the same amount characteristics [33,110]. Even if some previous preclinical
of calcium), no significant differences were noted in food- studies have presented a possible negative endocrine effect
dependent absorption, thereby indicating that tempeh may of phytoestrogens, presently, we know that isoflavones can
represent a bioavailable plant-based source of calcium, with exert a beneficial effect on the human hormonal network,
fractional absorption of calcium similar to milk. Further- stimulating oestrogen receptors when circulating endoge-
more, urinary calcium excretion was not significantly dif- nous oestrogens are insufficient, and concurrently, compet-
ferent, although calcium balance was higher for tempeh ing with circulating oestrogens when their concentrations
compared to milk. are high, thereby mitigating their overall effects [34]. For
this reason, isoflavones, including soy-derived phytoestro-
3. Functional Aspects gens, are used to mitigate menopause nuisances.
The antioxidant effect of tempeh has been veri- Although phytoestrogens are present in many foods,
fied in vitro and is mostly associated with the presence such as seeds, legumes, and other vegetables, the main nu-
of isoflavones, such as genistein and in particular agly- tritional sources of isoflavones are soy and soy-based foods.
conic forms, in addition to the potential effect of trans- Isoflavones such as genistein, daidzein, and glycitein are
cinnamic acid [91,92]. However, soy contains non- found in soybeans in glycosylated forms as malonyl and
isoflavone polyphenols, including lignans—also classified acetyl compounds conjugated to sugars. However, these
as phytoestrogens—which can contribute to the antioxidant isoforms appear to be poorly bioavailable due to limited dif-
effect of tempeh [93]. fusion through the intestinal mucosa [111]. Many soy foods
The use of L. plantarium as a co-inoculum in tempeh contain mostly isoflavones in a glycoside form. Some soy
production favours the reduction of biogenic amines, such processing methods allow isoflavones to be metabolised,
as putrescine and tyramine, which can be formed due to transforming them into aglycone, which diffuses more eas-
the growth of unwanted microorganisms [94]. The strains ily through lipid membranes thanks to their lower hy-
used in this production can be decisive in obtaining various drophilicity and molecular weight, compared to their re-
functional properties related to tempeh since the microor- spective glycosides [112–115]. Fermentation during tem-
ganisms specific proteolytic activity can release the biopep- peh production is widely documented as being able to
tides responsible for some antioxidant, anticancer, antidia- perform this transformation and, therefore, confer greater
betic, and antihypertensive properties by tempeh [95]. The benefits from isoflavones resulting from the increase in
action of isoflavones could also influence cholesterol lev- their bioavailability after deconjugation [41]. The per-
els [96,97]. It also appears that free fatty acids from tem- centage of aglycone in fermented foods varies from 40%
peh exert a cholesterol-lowering effect by inhibiting hepatic to 100% as a result of the microbial beta-glucosidase ac-
3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) re- tion. Furthermore, the isoflavone content in traditional
ductase [98]. Furthermore, the hypocholesterolemic effect foods, such as tofu and soy milk is on average higher than
could depend on the cholesterol intestinal sequestration ef- in second-generation soy products [112]. However, tem-
fect of enterohepatic circulation by dietary fibre, while re- peh appears to be the soy-derived food with the highest
ducing the glycaemic response by inhibiting the absorp- levels of isoflavones [116]. Various production steps can
tion of macromolecules [99–101]. Tempeh has shown anti- favour the deconjugation of isoflavone, while the fermen-
aging effects in pre- and post-menopausal women, as as- tation time can also affect the final isoflavone levels [117–
sessed by skin quality, bone density, and uterine tissue 120]. Even frying the tempeh (one of the most traditional
health [102,103]. These mechanisms appear to be related to cooking methods) appears to further improve the concentra-
the interaction between isoflavones and the female hormone tion of aglycone isoflavones compared to raw tempeh, al-
network [104]. Furthermore, the oestrogen-like activity of though it simultaneously reduces the total isoflavones con-
isoflavones has been proposed to provide anticancer effects tent [121,122]. Pre-germination of soybeans appears to fur-
[105,106]. ther increase the isoflavones bioavailability by releasing the
germinated isoflavone content [123].
3.1 Isoflavones
Soy isoflavones are widely studied for their potential Isoflavone Absorption
beneficial effects. They are polyphenolic phytochemicals Equol represents the most active form of daidzein be-
that provide strong antioxidant effects [32]. However, they cause its structure is very similar to endogenous 17-beta-
are much better known for their interaction with oestro- estradiol and its presence in circulating fluids is closely
gen receptors that produce differential activity between the linked to the individual ability of metabolising isoflavones
ER-alpha and ER-beta; therefore, promoting their tissue- [106]. In addition to the greater estrogenic effect, it also
specific and oestrogen receptor-modulating effects [107– shows a greater antioxidant effect compared to daidzein
109]. Isoflavones can interact with oestrogen receptors [124–127]. Although clinical trial data are conflicting as to
thanks to their molecular structure, which resembles en- which soy foods (tempeh, soymilk, or texturized soy pro-

5
tein) are more effective in raising genistein or daidzein lev- and texturized soy. Following the ingestion of tempeh,
els in the urine following intake [128,129], major equol there was a greater increase in serum daidzein and genistein
concentrations have been found following the consump- compared to the other soy foods. However, the blood con-
tion of tempeh [128]. However, data are still insuffi- centration of isoflavones peaked faster following the con-
cient to definitively clarify these aspects. Equol and O- sumption of soy milk, thereby highlighting the favourable
desmethylangolensin (O-DMA) represent the products of influence of a liquid matrix compared to the solid state. Al-
daidzein intestinal metabolism after its transformation into though the highest concentration of total isoflavones was
the aglyconic form [130]. Nevertheless, since this step is found in texturized soy proteins, tempeh produced the high-
performed by the intestinal microflora, there may be indi- est concentrations of genistein and daidzein in aglyconic
vidual variability in the ability to produce equol [131–133]. form.
Some individuals are defined as equol-competent, equol-
producer, or equol-excreter if they excrete higher con- 3.2 Effects on Cognition
centrations of equol following daidzein intake [130,134– With the increase in the average lifespan of the pop-
137]. It is estimated that in the general population, only ulation, the occurrence of cognitive disorders has also in-
30% of individuals are equol-producers [138–141]. This creased, which results in greater healthcare costs. Cur-
also implies that individuals who are unable to metabo- rently, the clinical approach to cognitive decline is pallia-
lize isoflavones due to a lack of adequate intestinal micro- tive, with the aim of slowing down the disorder. Lifestyle
bial populations may not fully benefit from the activities approaches such as physical activity and nutrition remain
of isoflavone [134,142]. In mammals, isoflavones follow the most promising preventive interventions. However,
the enterohepatic circulation, meaning they are excreted in meta-analyses have highlighted a positive effect of oestro-
a conjugated form to glucuronic acids by bile [143]. Subse- gen administration on cognitive test outcomes for up to 2–3
quently, the isoflavones can be deconjugated again by bac- months [150,151]. Furthermore, the effects on cognitive
terial beta-glucuronidases and sulfatases. Therefore, the gut functions from modifying the microbiota appear to involve
metabolism of isoflavones is a complex biochemical pro- various mechanisms [152–154].
cess, whereby both the host and microbial enzymes partic- In an observational study on 142 elderly individuals
ipate [144–146]. from rural Indonesia (56–97 years old), it emerged that tem-
In 20 post-menopausal Malayan women, tempeh con- peh consumption was correlated with better immediate re-
sumption resulted in greater urinary excretion of equol, call scores [155]. More recently, Handajani and colleagues
daidzein, and genistein compared to milk consumption [156] conducted an intervention study on 84 participants
[147]. In one of the first works on the bioavailability of phy- aged over 60 years with mild cognitive impairment (MCI),
toestrogens from fermented soy foods, Hutchins and col- who had been randomized to consume two different tempeh
leagues highlighted how fermentation during tempeh pro- preparations compared to a control group. After 6 months
duction reduces the content of total isoflavones, especially of consuming 100 g of tempeh per day, there was an in-
those in the glycosidic form (daidzin and genistin), yet it crease in global cognitive scores in participants who had
increases the recovery of isoflavones in urine following in- assumed tempeh, although an improvement in language do-
gestion due to its increased bioavailability [148]. However, main scores was only associated with one of the two prepa-
in a study by Xu and colleagues [149], on 10 healthy fer- rations. Interestingly, the effective preparation for the lan-
tile women, the consumption of various types of soy foods guage domain was the preparation with the lowest concen-
(tempeh, tofu, textured soy, or soybeans) caused no signifi- tration of microorganisms, indicating that the qualitative as-
cant differences in short-term urinary isoflavone excretion. pect rather than the quantitative one may be more important.
However, the limited statistical power of these clinical tri- A probiotic strain of L. reuteri fermentum isolated from
als makes it difficult to conclude an overall outcome, also tempeh was used for a 12-week clinical trial on 93 Indone-
considering the influence of interindividual variability on sian individuals over 60 years old with memory impairment
isoflavone metabolism. Subsequently, a clinical trial was [157]. From the 16S rRNA analysis, the selected strain
performed with 27 men and women (both pre and post- contained the gene encoding for gamma-aminobutyric acid
menopausal), which tested the urinary kinetics of acute (GABA). The intervention improved the values in the cog-
isoflavone intake using soy milk, tempeh, and textured soy nitive domains of memory, learning processes, and verbal
proteins [128]. Recovery of urinary genistein was greater and visuospatial fluency.
in tempeh for premenopausal women than by soy milk, al- This suggests that tempeh consumption may be useful
though this was not observed in men and postmenopausal for a safe intervention in cognitive preservation, although
women. While there were no appreciable differences in only during a still receptive age window.
daidzein excretion related to the food matrix, consumption
of tempeh showed greater excretion of equol compared to 3.3 Physical Performance and Nutritional Adequacy
other soy foods. Similarly, Cassidy and colleagues [129] A drink with added tempeh extract was used in a
enrolled 59 individuals to investigate the pharmacokinetics double-blind controlled clinical trial on 18 semi-trained
of isoflavones from various soy foods: tempeh, soy milk, men aged 18–24 years to evaluate parameters associated

6
with muscle tissue damage and recovery [158]. The tem- sidered the hunger hormone, and it is produced by gastric
peh drink significantly reduced creatine kinase levels and cells during fasting, while it is reduced following the in-
increased maximal muscle strength at 24 hours compared gestion of a meal [170,171]. Similarly, insulin is released
to the placebo. These effects showed no significant differ- after a meal by pancreatic cells to stimulate satiety [172].
ences compared to the whey protein control group. Creatine Furthermore, arginine is an amino acid that is released dur-
kinase levels were linked to muscle damage and indirectly ing protein digestion, which could act as a signal of meal
to muscle fatigue, while also reducing muscle strength and protein sufficiency [173]. This suggests that tempeh could
recovery [159]. Therefore, tempeh with its antioxidant improve the ability to regulate appetite, which may be ben-
properties could mitigate the effects of reactive oxygen eficial in individuals with metabolic syndrome.
species (ROS) produced by excessive training, which can
damage muscle cell membranes [160,161]. 3.4 Probiotic, Prebiotic, and Paraprobiotic Effect
In addition to these sport-related benefits, the nutri- Tempeh may have antimicrobial properties that can
tional characteristics of tempeh could be applied to improve positively influence the balance of intestinal microbiota
calorie-energy adequacy, especially in children. In a recent following ingestion. A liquid suspension of tempeh was
study, tempeh was provided as nuggets to 46 Indonesian able to inhibit the growth of B. subtilis and B. stearother-
children aged 24–59 months [162]. After 30 days, tem- mophilus in vitro [174,175]. R. oligosporus can inhibit the
peh consumption had improved energy intake compared growth of Bacillus spp, Staphylococcus spp and Streptococ-
to the control. Similarly, 30 g/day of tempeh nuggets cus spp. through heat-resistant proteins with antimicrobial
were offered to 30 children aged 36–60 months in a quasi- activity being released into the growth medium [176].
experimental study and showed a significant weight in- This aspect is particularly relevant since tempeh is
crease after 4 weeks [163]. In contrast, the control showed consumed after cooking, meaning a probiotic effect seems
a non-significant weight gain compared to baseline. unlikely. However, the substances released following fer-
Tempeh, given the effect of fermentation, can be a mentation, if resistant to cooking, can exert a paraprobiotic
highly digestible food for children [1,164]. Moreover, it effect, which modulates the intestinal microbiota ecology
does not result in an excessive intake of fibre, which in [35].
the case of a vegetarian or vegan diet could represent a An in vitro study that simulated human gastrointesti-
limit in the absorption of nutrients by children [165]. The nal digestion highlighted how tempeh can stimulate the
use of tempeh as an adequate nutritional source in chil- levels of Bifidobacterium spp. and Lactobacillus spp.
dren is a promising frontier, which would allow suitable [175]. Akkermasia muciniphila together with Bifidobac-
economic solutions for low-income countries to address terium spp. is more represented in the gut of lean individ-
calorie-energy insufficiency. At the same time, as a mini- uals rather than in subjects with metabolic syndrome [177–
mally processed source of legumes, it would help the objec- 179]. A. muciniphila colonizes the cecum and degrades the
tive of the FAO to increase the consumption of legumes, as glycoproteins that make up the mucin, thereby stimulating
an eco-friendly food security strategy, while also limiting the immune system and cell proliferation in the host mu-
food excesses in high-income countries [166,167]. Given cosa [180]. In an open-label study on 16 participants, con-
the high environmental impact of the animal food produc- suming 100 g of steamed tempeh for 16 days increased A.
tion chain (water use, carbon footprint, and greenhouse gas muciniphila numbers and immunoglobulin A (IgA) levels
emission), the use of plant-based foods with high nutritional in the stool compared to the baseline [181]. IgA acts as
value can encourage a transition toward a more plant-based a first defence against pathogens and toxins in the intesti-
diet, guaranteeing greater access to food and reducing the nal tract and promotes anti-inflammatory mechanisms that
ecological cost of production. shape the intestinal barrier through the microbial balance of
In a prospective, open-label intervention study on 35 the microbiota [182].
Indonesian patients with type 2 diabetes, daily consumption Subsequently, Stephanie and colleagues confirmed an
of 2 grams of tempeh for 3 months improved HbA1C and increase in A. muciniphila in the faeces after 28 days, also
triglyceride levels [168]. However, since this trial used a showing an increase in Bifidobacterium spp. compared to
freeze-dried sample of tempeh grounded into a powder, it is the baseline [183].
not possible to transfer the results to cases where fresh tem- The effectiveness of tempeh in this clinical trial con-
peh was consumed, which is normally cooked prior to con- firms the paraprobiotic effect since cooking before con-
sumption. In a randomized, double-blind controlled, cross- sumption inevitably destroyed the present microorganisms,
over trial, 13 Indonesian women aged 18–20 with a BMI which cannot act through a probiotic mechanism. However,
of 25–30 used tempeh or unfermented soy for an acute test the molecular motifs remaining from the microorganisms
[169]. A 30% reduction was observed in serum acyl-ghrelin present in fermented food can be recognized by the host’s
30 minutes after ingesting tempeh, yet not following unfer- immune system and they activate a response following the
mented soy consumption. Furthermore, the fermented food antigenic interaction [184]. Furthermore, the presence of
showed a greater insulin response and an increase in serum fermentable fibres in tempeh could favour the microbial
arginine at 120 min compared to the control. Ghrelin is con- production of short-chain fatty acids (SCFAs: acetate, bu-

7
tyrate, propionate), which show a beneficial effect in the the microbial component by limiting transformations such
gut by exerting a prebiotic effect [185]. Moreover, SC- as deep cooking and its transformation into flour [210].
FAs can exert anti-inflammatory and hypocholesterolemic
actions on the host by inhibiting the hepatic synthesis of 4. Food Technologies
HMG-CoA reductase [99,101,186]. The production of SC-
The consistency of tempeh and its nutritional proper-
FAs has also been linked to gut–brain axis (GBA) functions
ties make it ideal for use in second-generation foods, such
[187,188].
as meat substitutes, which are highly requested in West-
Depending on the fermentation conditions, GABA
ern countries [162,211,212]. Furthermore, tempeh-based
was found in tempeh [189]. GABA is a neurotransmitter
preparations can retain functional properties compared to
implicated in cognitive functions, of particular interest in
meat-based foods [213]. Tempeh can also be used as a
GBA communication mechanisms [190–192].
meat extender to improve the nutritional properties of meat-
3.5 Antioxidants based preparations and at the same time limit production
costs [214,215]. Similarly, the use of tempeh in mixtures
The antioxidant effect of tempeh components seems for pasta production allows for an increase in the content
to be related to fermentation times with greater ROS scav- of folates and proteins, while also improving the taste and
enging activity following prolonged fermentation [193– texture [216,217].
195]. In vitro studies have shown that tempeh has a greater
Acceptability and increase in polyunsaturated fatty
antioxidant effect than unfermented soy, showing greater
acid content were found when 5% dehydrated tempeh was
scavenging abilities on free radicals and superoxide an-
used in bread doughs and cereal-based bars [218]. The an-
ions [194]. In a recent in vitro study, microglial cells
tioxidant capacity is preserved even when employed for the
showed reduced ROS levels following tempeh adminis-
production of biscuits as a replacement for soy flour com-
tration after lipopolysaccharide (LPS)-induced oxidative
ponents [219]. This use does not alter the product accept-
stress [196]. This may depend on the content of polyphe-
ability. Tempeh has also been used as a fortifier to obtain
nols and their bioavailability [41,193,197]. The presence
an emergency food formula [220].
of greater isoflavone concentrations in the aglycone form
seems to explain the properties of tempeh compared to The use of tempeh in doughs has been successfully
other non-fermented soy foods and it identifies genistein used for the production of vegan biscuits to increase accept-
and daidzein in the aglycone isoforms as having greater an- ability [221]. Furthermore, good acceptability of beef pat-
tioxidant effects compared to their glycosides, as well as a ties containing 10% tempeh was observed compared to the
greater bioavailability [198–200]. Furthermore, microbial 100% beef control, with better sensory properties regarding
proteolytic activity allows proteins to be hydrolysed and re- the perception of tenderness and juiciness [215]. This out-
lease amino acids and biopeptides, which are more bioavail- come coincides with the participants’ desire to choose more
able and exert antioxidant activities [201,202]. balanced and antioxidant-rich foods. However, it should
be noted that food acceptability is also important if it is
The increase in antioxidant activity by tempeh may
to be used routinely. While acceptability in populations
also depend on the increase in phytochemicals as is the
that traditionally use tempeh may be high, in other coun-
case for beta-tocopherol, which shows a 200% increase in
tries where it is a little-known food, the aspect of sensorial
activity compared to unfermented soybean [203]. While
perception should not be underestimated. Neophobia can
beta-, gamma-, and delta-tocopherol concentrations seem
be a decisive element in the acceptability of culturally new
to increase during tempeh production, the concentration of
foods, such as tempeh, in some countries [222]. The accep-
alpha-tocopherol is not affected by the fermentation pro-
tance of tempeh-like products in the Danish market could
cess.
depend on the consumers’ predisposition to new foods, with
Another powerful antioxidant identified in tempeh is
a greater propensity by those who were already oriented
3-hydroxyanthanilic acid [204]. This showed an improve-
towards plant-based alternatives, avoiding mimicking the
ment in stabilizing soy products through the scavenging ef-
meat-based foods characteristics [223]. Consequently, al-
fect of NO [204,205]. The antioxidant power can be further
though it may be more difficult to include tempeh in the
increased by including an anaerobic fermentation step in
diet of populations with very different culinary traditions,
tempeh production [206]. The action of oxidative stress ap-
the search for sustainable food options could lead to greater
pears to be related to numerous chronic pathologies, such as
acceptance.
diabetes, inflammatory diseases, tumours, and cardiovascu-
lar [16,207–209].
The bioactive molecules contained in tempeh, in par- 5. Sustainability
ticular biopeptides, can exert a beneficial effect on cancer It has been proposed that tempeh may have a higher
prevention through a protective antioxidant effect on nu- protein delivery efficiency energy score compared to many
cleic acids. The same beneficial effect can be advantageous foods of animal origin and this represents an advantageous
in cancer therapy. However, to obtain the maximum ben- environmental aspect [224]. The most burdensome compo-
efit from consuming tempeh it may be useful to preserve nent on the environmental impact of tempeh appears to be

8
land use and eutrophication derived from soy cultivation. such as tempeh would guarantee greater access to food and
Tempeh exerts a reduction in greenhouse gas (GHG) pro- a lower ecological impact compared to the currently over-
duction compared to pork, beef, chicken, fish, eggs, and exploited animal sources.
milk, evaluated in grams of proteins per kg of CO2 eq.
based on life-cycle assessment (LCA) [225]. This implies Author Contributions
that to optimize the environmental impact of tempeh pro-
GR designed the research study, analyzed the infor-
duction it would be useful to use autochthonous legumes
mation, and wrote and edited the manuscript. GR con-
and grains that are adapted to a specific ecological niche.
tributed to editorial changes in the manuscript. GR read
Affordability is also part of the sustainability components
and approved the final manuscript. GR has participated suf-
together with the impact on health and environment. Al-
ficiently in the work and agreed to be accountable for all
though the costs of tempeh vary greatly based on produc-
aspects of the work.
tion factors, it has been highlighted that the price per kg of
tempeh is lower than the price of beef, with only traditional
Ethics Approval and Consent to Participate
products being cheaper than chicken, eggs, and milk [45].
Furthermore, transformation by fermentation can be Not applicable.
an optimal system for recovering by-products from other
food chains, which is the case for tempeh gembus since Acknowledgment
it is obtained from the okara of tofu or soy milk produc- The author wishes to thank Sandra De Dominici for
tion. Tempeh gembus has shown beneficial effects on total, language revision assistance.
low-density lipoprotein (LDL) and high-density lipopro-
tein (HDL) cholesterol fractions in women with hyper- Funding
lipidaemia [226]. The effect could be mediated by the
high quantities of fibres and several in vitro studies have This research received no external funding.
suggested further beneficial properties of tempeh gembus
[227–230]. Conflict of Interest
The author declares no conflict of interest.
6. Conclusions and Future Remarks
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