Learning from Tradition: Health-Promoting Potential of Traditional Lactic Acid Fermentation to Drive Innovation in Fermented Plant-Based Dairy Alternatives

Abstract

Food fermentation using lactic acid bacteria (LAB) is an ancient technique that has been deemed a simple and economical way to modify nutritional contents of plant-based foods. In many cultures, this practice shows a long history with a wide variety of fermented liquid and semi-liquid traditional foods being produced from cereals, legumes, and tubers. Nutritionally relevant benefits of the fermentation process are becoming increasingly evident and can be linked to the accumulation of bioactive compounds (exopolysaccharides, short-chain fatty acids, bioactive peptides), degradation of antinutritional factors, and improved bioavailability of essential nutrients (amino acids, minerals, vitamins). This manuscript discusses the current understanding on the impact of LAB fermentation on plant macro- and micronutrients in traditionally fermented foods and how this knowledge could aid to drive innovation in the emerging research and development (R&D) sector of plant-based dairy alternatives. Key-points include that the improved health-promoting properties and rich sensory appeal found in traditional foods results from a low and slow fermentation (prolonged fermentation time at suboptimal temperatures), which depends on the complex interplay of mixed microbial cultures found in such foods.

1. Introduction

Food fermentation has accompanied human civilisation through the centuries to produce nutritious and tasty plant-based foods. While fermented bovine dairy products play a central role, especially in Western diets, a recent shift in consumer’s demand towards more plant-based diets has driven new markets of fermented dairy alternative products to emerge that mimic dairy in flavour and texture. The role of fermentation in the making of fermented plant-based products has received renewed attention since it can be applied commercially at a low cost to improve a variety of organoleptic qualities at once [1,2]. Flavour and texture are tightly linked with consumer acceptance and typically serve as a focal point during product developments. In traditional foods, fermentation is, however, increasingly being recognised for its health-beneficial impact on nutrient bioavailability and the accumulation of bioactive metabolites, which likely contributed to the popularity and historic adoption of traditional fermented foods [3,4,5,6,7].

Plant-based yoghurt alternatives can be described as colloidal mixtures of plant material, acidified through lactic acid fermentation, and are based on a wide variety of starting materials, including legumes (soy, pea, faba bean), cereals (oat, rice), coconut, and oilseeds. The choice of raw ingredients for the development of such products brings nutritional challenges in the form of anti-nutritional factors (phytic acid, tannin, enzyme inhibitor), a high glycaemic index, and poor ileal digestibility of protein due to their complex internal structure and entrapment in cell wall structures [8,9]. A lack of certain nutritional benefits compared to conventional dairy has also been cited as a criticism against plant-based products. This criticism, combined with a rising consumer demand for health-beneficial sustainable foods, has created a need to explore new strategies in optimising the nutritional value of dairy alternative foods.

Fermentation is an attractive processing method to modify food material due to its ease of use, low cost, and limited environmental impact. In traditional fermented plant-based foods from Africa and Eastern Europe, the observed health- and improved nutritional benefits are the consequence of a complex interplay of naturally occurring microbial activities and demonstrate the possibilities that can be achieved using lactic acid fermentation [3,4]. Commercial endeavours of fermented plant-based dairy alternatives, in contrast, apply fermentation processes using defined starter cultures. Bacterial strains with optimal lactic acid production are employed, combined with the addition of sugars as growth-enhancing substrates, to achieve a fast acidification rate and ensure food safety by inhibiting pathogenic growth. Hence, LAB fermentation may be underutilised in producing a great diversity of compounds that generate flavour, texture, and bioactive properties in traditional fermented foods. This review aims to provide a broad summary on the potential impacts of lactic acid fermentation on macro- and micronutrients in plant-based materials and their relevance to human health. Information on popular LAB-fermented cereal beverages and gel-type foods (type of microbiota used, preparation methods, composition) was collated and contrasted to recent development efforts on plant-based yoghurt alternatives to highlight potential areas where we can “learn from tradition” to innovate further in this exciting R&D area.

2. A Renewed Interest in Plant-Based Dairy Alternative Foods

In the past decades an increasing trend and renewed interest in plant-based foods [10,11,12], especially in developed countries, has been seen. One of the drivers was the proliferation of plant-based dairy alternatives to meet consumer demands of “healthy foods”, ethics in the use of animal-based foods, and environmental concerns with animal farming [13,14,15,16].

In 2021, plant-based milk alternatives constituted a significant proportion (i.e., 90%, ~USD 18 billion) of the global retail value of the plant-based dairy industry, which currently sits at USD 20 billion [17]. One implication of this market data is that the current plant-based dairy alternative market consists largely of “plant milks” and that plant-based “cheese” and fermented plant-based “milk” are not as popular. This insight is corroborated by our findings in Figure 1, which covers the number of scientific publications on “plant-based dairy alternatives” and “plant-based yoghurt alternatives”. One key finding from this data is that, whereas plant-based dairy alternatives were being investigated in the scientific literature as far back as 1992 (about 16 publications), it took another 20 years to get the same number of research publications on fermented plant-based dairy alternatives (16 publications in 2012). In other words, although fermentation is an ancient technology, research on fermented plant-based milk alternatives is currently lagging that on plant-based milk alternatives.

Figure 2 shows the cluster analysis of author keywords for publications on fermented plant-based dairy alternatives. The author keywords are grouped into four clusters. The biggest cluster (coloured red in Figure 2) includes publications that describe the process, impact, and health benefits of fermented plant-based milk alternatives. Some keywords in this cluster are “fermentation”, “milk substitute”, “volatiles”, “metabolites”, functional food, and health benefits. “Lactic acid bacteria”, and Lp. plantarum, and S. thermophilus, which are two of the most widely used cultures in fermented plant-based milk alternatives are also mentioned in this cluster. The second most dominant cluster (shown in blue, Figure 2) features studies on the dietary and health implications of fermented plant-based milk alternatives. This cluster contains keywords, such as “cholesterol”, “calcium”, “clinical trials”, “humans”, “animal”, “lactose”, “lactose intolerance”, “nutrient”, and “vitamin”. The third cluster (shown in green, Figure 2) contains articles describing the physicochemical properties and textural or rheological behaviour of proteins in fermented plant-based milk alternatives. There is also a focus on sensory analysis and sustainability implications of this product category. “Gelation”, “heat treatment”, “particle size”, and “stability” are among the keywords in this cluster. The last cluster (shown in yellow, Figure 2) contains articles on consumer insights, compositional analysis, and examples of some established plant-based milk alternatives. There are keywords, such as “consumer study”, “marketing”, “protein content”, “fatty acid”, “carbohydrate”, “analysis”, “peanut milk”, and “allergen”. There are only four clusters of author keywords showing that the “fermented plant-based milk” research area is still in its infancy.

3. Fermentation in Traditional Foods

Fermentation counts among the oldest food-processing technologies and is applied historically in almost every culture to prepare a wide variety of staple foods that play an important role in human diets. Fermented beverages, such as Mahewu (South Africa), or cereal puddings, such as Ogi (Nigeria), are prepared from raw cereal grains and consumed throughout the day or used as infant food. Spontaneous fermentation takes place during the preparation as a result of undefined mixed cultures with plant- and environmental origin and can be defined as controlled microbial growth that affects major and minor food components through enzymatic conversion [18]. As a result, starchy raw materials are preserved and rendered more palatable through the production of organic acids and the pre-digestion of plant material. Popular non-alcoholic fermented products are found across the African, Eastern-European, and Asian continents and are summarised in

Table 1. Overview of popular traditionally fermented beverage and gel-type foods from various cultures worldwide including dominating microbiota, food type, and preparation methods.

Food *	Substrate	Dominating LAB Species **	Preparation	Type	Origin	Ref.
Cereals
Ben-saalga	Millet	L. delbrueckii subsp. bulgaricus, Lp. plantarum, Lm. fermentum, Weisella confusa, L. amylolyticus, L. helveticus	Soaked, wet-milled, sieved, fermented, then cooked	Fermented gruel used to complement diets of infants and young children	Burkina Faso	[19,20]
Boza	Maize, Millet, and Rye	Lp. plantarum, L. acidophilus, Lm. fermentum, L. coprophilus, Lv. brevis, Lcc. raffinolactis, Leuc. mesenteroides	Boiled flour mixed with old batch, fermented for 24 h	Thick sweet–sour beverage with pale white-to-yellow colour	BulgariaTurkey	[21]
Bushera, Obushera	Millet, Sorghum	Lv. brevis, Lm. fermentum, Lp. plantarum, Lc. paracasei subsp. paracasei, Streptococcus thermophilus, L. delbrueckii	Boiled flour mixed with germinated unboiled flour as inoculant, fermented for 5 days at ambient temperature	Moderately thick, sweet–sour social drink and weaning food with pale brown colour	Uganda	[22]
Ikii	Maize	Lm. fermentum, Lp. plantarum, Weissella confusa, Lacticaseibacillus rhamnosus, Pediococcus sp.	Cooked, mixed with old batch, fermented for 72 h at ambient temperature	Thick porridge, popular among children, breastfeeding mothers, and aged populations and given to a sick and recovering person	Kenya	[23]
Koozh	Millet, Rice	Lp. plantarum, Bacillus amyloliquefaciens, Leuconostoc sp., Weissella sp.	Millet slurry, fermented overnight, mixed with rice porridge and cooked, fermented again, mixed with water before consumption	Fermented porridge as breakfast or lunch	India	[24]
Kunu-zaki	Millet	S. lutetiensis, Lm. fermentum, L. delbrueckii, Clostridium perfringens, Weissella confusa	Soaked grains, wet-milled, sieved, one part cooked mixed with uncooked part, fermented for 8 h	Sweet breakfast food drink	Nigeria	[25,26]
Mahewu	Maize, Millet, Sorghum	Lp. plantarum, Lv. brevis, Lm. fermentum, Pediococcus pentosaceus, Weissella confusa	Germinated ground millet mixed with cooked maise porridge, fermented 16–48 h	Creamy and sour beverage used as infant food	South africa	[27]
Ogi	Maize, Sorghum	Lp. plantarum, Lp. paraplantarum, P. acidilacti, P. pentosaceus, L. helveticus, Lm. fermentum, W. confusa, L. amylolyticus, Lcc. lactis	Soaked and pre-fermented grains, wet-milled, sieved, fermented for three days, then cooked until creamy	Fermented cereal pudding used as infant food	Nigeria	[25,28]
Poto poto	Maize	Lp. plantarum, L. gasseri, Enterococcus sp., Escherichia coli, L. acidophilus, L. delbrueckii, Bacillus sp., Lm. reuteri and Lc. casei	Soaked, milled, fermented for 10 h, dried for storage, boiled before consumption	Gruel used as weaning food	Congo	[29]
Pozol	Maize	Lp. plantarum, Lc. casei, L. delbrueckii, Lm. fermentum, Bifidobacterium sp.	Maize dough fermented for 3 days, soaked before consumption	Refreshing beverage, consumed for its curative properties and at religious ceremonies	Mexico	[30]
Swazi	Maize, Sorghum	Lp. plantarum, Leuconostoc sp.	Boiled, fermented for 72 h to 6 d	Liquid brown coloured beverage	Eswatini	[31]
Tarhana	Wheat	S. thermophilus, Lm. fermentum, Pediococcus pentosaceus, Leuconostoc pseudomesenteroides, Weissella cibaria, Lp. plantarum, L. delbrueckii spp. bulgaricus, Leuconostoc citreum, Lp. paraplantarum and L. casei	Soaked, fermented for 2 days, dried	Dried ingredient used for soups and stocks	Turkey	[32]
Ting	Sorghum	Lm. reuteri, Lm. fermentum, L. harbinensis, Lp. plantarum, Ll. parabuchneri, Lc. casei and L. coryniformis.	Sorghum slurry fermented 1–3 days, cooked to soft porridge	Fermented sour porridge	Botswana	[33]
Togwa	Maize, Millet	Lp. plantarum, Lv. brevis, Lm. fermentum, W. confusa, P. pentosaceus	Germinated ground millet mixed with cooked maise porridge, fermented 15 h	Industrially produced opaque, sweet beverage	Tanzania	[34]
Uji	Maize, Sorghum, Millet	Leuc. mesenteroides, S. faecalis, Lp. plantarum, Lv. brevis, P. cerevisiae	Mixed flour slurry, fermented for 3 days then cooked	Fermented porridge	Kenya	[35]
Tubers
Fufu	Cassava	Lp. plantarum, Leu. mesenteroides, Lm. fermentum, Lv. brevis, L. coprophilus, Lcc. lactis, L. bulgaricus	Steeped, fermented 3–4 days, mashed, sieved, expelled water is consumed	Liquid drink	Ghana	[36]
Garri	Cassava	Lp. plantarum, Lm. fermentum, L. pentosus, L. acidophilus, Lc. casei, Lc. mesenteroides	Mashed roots, fermented then dehydrated, reconstituted in water before consumption	Gruel	Nigeria	[37]
Legumes
Siljo	Faba beans	L. acidophilus, Lp. plantarum, L. delbruekii, Micrococcus spp., Bacillus spp.		Slurry	Ethiopia	[38]

* Selection is limited to non-alcoholic liquid and semi-liquid foods, ** taxonomy of lactobacilli has been adjusted according to a recent reclassification using the following genera: Lactiplantibacillus (Lp.), Lacticaseibacillus (Lc.), Limosilactobacillus (Lm.), Levilactobacillus (Lv.), Lactococcus (Lcc.), Lentilactobacillus (Ll.), Leuconostoc (Leuc.) [39].

with associated LAB microbiota.

3.1. Undefined Mixed Cultures of Traditional Foods

The fermentation by lactic acid bacteria (LAB) belonging to the family Lactobacillaceae is predominant in liquid and semiliquid traditional foods [40]. The production of organic acids by metabolising carbohydrate material to lactic acid, acetic acid, and, to a lesser extent, propionic acid, succinate, and acetoin leads to a pH reduction and characteristic flavours. Additionally, the accumulation of exopolysaccharides (EPSs) is a desired effect and contributes to texture development by impacting viscosity and water retention [41]. Additional fermentative processes take place and have to be acknowledged, which also includes the activity of yeast (Saccharomyces sp., Candida sp.). In boza, a fermented Bulgarian beverage, the presence of LAB and yeast was reported at a ratio of 2.2–2.6 [21]. Synergistic behaviour exists between LAB and yeast, but their presence in non-alcoholic foods is generally undesired based on sensory studies [42]. Fungi, spore-forming bacteria (Bacillus sp.), and additional alkaline fermenting species (Micrococcus sp.) have been attributed to protein fermentation in legume-based condiments, such as natto (Japan) or kinema (India) [43,44], and in liquid foods rich in carbohydrates, their role is however limited by the antagonistic effects of LAB fermentation. Dominating LAB from boza and other cereal-based products have shown the capacity to produce a wide range of antimicrobial substances, including bacteriocin, hydrogen peroxide, fatty acids, and diacetyl, which have attracted interest in them as probiotics [45,46,47]. Microbiota of traditional fermented foods are established from dispersed naturally occurring species found on the grain and depend on the ecological fitness of the individual organism, fermentation conditions, and availability of nutrients.

An inoculation step is often included in traditional preparations and uses small amounts of previous successful batches as a starter culture (Table 1). Remarkably, by using this so called “back-slopping” approach, undefined mixed cultures were found to remain stable, down to the strain level, over decades if substrates and fermentation conditions are maintained [48]. This phenomenon has been explored mainly in fermented dairy products so far. In such environments, mixed cultures have shown superior resilience against phage attack and contaminants when compared to defined single-strain starter cultures [49,50]. This increased stability is economically advantageous to the food producer as it reduces the risk of failed batches and limits the need to monitor phages during the fermentation. Additionally, the continuous sub-culturing process in fermented food produced using back-slopping has been shown to allow for metabolic adaptations to the food environment in dairy-associated species, such as L. delbrueckii [51]. Such adaptations can permit the emergence of cultures with superior performance without the need for gene manipulation. Model mixed cultures of cereal-fermented foods remain poorly explored to this date but continue to attract attention as a valuable source of LAB strains with beneficial technological and probiotic properties [46,47,52].

3.2. Metabolic Processes during Fermentation

Cereals, such as maize, millet and sorghum, but also tubers (cassava) or legumes (faba bean), are among plant materials that are widely used as substrates for fermentation. Typically, the type of fermentation ingredient will depend on the country or region and availability. These plant materials are often pre-processed through mashing, grinding, and soaking to promote access by microorganisms to the fermentable compounds. Across this diversity of raw materials, starches are the most abundant (i.e., 35–90%) fermentable macromolecule and serve as an important carbon source. Pre-processing steps, such as mashing, grinding, and soaking, result in the release and stimulation of endogenous enzymes (α-amylase, maltase, glucoamylase) in the early stages, allowing for starch hydrolysis into fermentable sugar (glucose, maltose, maltodextrin) [43]. Additionally, amylolytic LAB (ALAB) that show the expression of extracellular α-amylase have been isolated from fermented cereals [53]. ALAB are capable of metabolising starches directly and form synergies with other LAB species. In Nigerian Ogi, ALAB make up 14% of isolated bacteria and were found to be essential for its biodiversity [54]. Genomic markers for the responsible enzymes (α-amylase, α-glucosidase, glycosyltransferase) can be found across most LAB species; however, their expression is rare and specific to certain strains [55]. Lp. plantarum is recognised as the dominant ALAB, while few other examples exist for L. acidophilus, Lm. Fermentum, and Lm. reuteri [53]. B. amyloliquefaciens, as one of the most amylolytic species, can also be found in fermented foods [24,56], but a poor pH tolerance limits the activity of such spore-forming bacteria.

Species that are considered “nomadic”, Lp. plantarum, Lc. Casei, and Lv. brevis, dominate microbial communities in traditional fermented food and possess broad carbohydrate-metabolic capabilities, whereas host-adapted species (L. delbrueckii, Lm. reuteri) have lost their ability to metabolise diverse carbohydrates due to niche adaptation and often rely on the presence of glucose, lactose, and sucrose in the substrate [51]. The separation of LAB into two main groups based on evolutionary history is useful. Homofermentative LAB (L. delbrueckii, L. acidophilus) convert glucose to lactic acid following the Emden–Meyerhof–Parnaz Pathway and are used as starter cultures in modern food fermentation, while heterofermentative LAB (Lv. brevis, Lm. fermentum, Lm. reuteri) are abundant in traditional fermentation and convert glucose to lactic acid and CO₂ using variations of the phosphoketolase pathway. Ethanol can collect as a side product but normally only at low quantities due to a rate-limiting effect, which leads to the accumulation of flavour active alternatives, such as the fruity acetate [41]. Lp. Plantarum, Lc. Rhamnosus, and L. paracasei are examples of facultative heterofermenters that possess the ability to switch between the two metabolic pathways depending on environmental factors [48]. The diversity of carbohydrates present in cereals is beneficial for such organisms and allows for the formation of complex mixtures of metabolites [53]. Hexose (fructose, mannose), pentose (xylose, ribose), and organic acids (citrate, malate, or pyruvate) for instance were shown to support the growth of heterofermenters and are involved in the production of EPS [57].

Species-dependant auxotrophy for essential amino acids is a characteristic of LAB. To sustain growth, a variety of proteolytic systems (cell-wall bound protease, transporter, and intracellular peptidase) are expressed once free amino acids are depleted [58]. The ability to metabolise peptides is widely found among LAB, unlike protease-type enzymes, which are found on plasmids and can be lost during prolonged propagation [59,60]. Substrate specificity of the expressed enzymes differs between species and combined with the limited proteolytic activity of species, such as Lp. plantarum and Leuconostoc sp., encourages a symbiotic lifestyle of LAB, where required metabolites (amino acids, glucose, vitamins) are exchanged. As such, the effect on protein materials and the release of amino acids/peptides depends on the combined proteolytic capabilities and metabolic demand of the present mixed culture. In the context of food, the fermentative breakdown of protein has been shown to contribute to the flavour, generation of bioactivities, and improvement of digestive properties [59,61].

4. Impact of Lactic Acid Fermentation on Macronutrients and Relevance to Human Health

Fermentation has been adopted historically as a simple and economic method for food preservation and to improve the organoleptic qualities of poorly palatable plant materials. Health-promoting properties of fermented foods that extend beyond the benefits of the starting material are, however, becoming increasingly evident [40]. Mechanistic routes that can deliver a benefit to the consumer include the modification of nutrients to improve their bioavailability and the synthesis of bioactive compounds, which both relate to LAB metabolic activity during fermentation [62]. The probiotic effects of fermented food on gut health have received considerable attention and can take place if viable organisms with beneficial properties are present during consumption [63]. Traditional preparations often include a cooking step after fermentation to ensure food safety (Table 1). This reduces the presence of viable probiotics; however, emerging evidence suggests that non-viable probiotics and their released bioactive material are equally beneficial [64]. Para probiotics are defined as “inactivated microbial cells that confer a health benefit to the consumer” and include structural cell components (cell wall, fimbriae, pili), whereas the term postbiotics has been suggested for beneficial metabolites that are released upon cell death (peptides, vitamin, fatty acids) [64]. In contrast to probiotics, para- and postbiotics are more suitable for immunocompromised individuals, such as the elderly.

Therapeutic claims for fermented foods exist for non-communicable diseases (obesity, diabetes, cardiovascular disease, hypertension, metabolic syndrome), gut health (allergies, food intolerance, inflammatory diseases) and gastrointestinal tract (GIT) cancer [4]. Still, they remain debated due to a lack of conclusive evidence from clinical trials. Bioactive substances from indigenous African foods have also been linked to an observed high immunity of the population [7]. Using bacterial isolates of plant origin, a body of research has identified desirable metabolic effects that can take place during fermentation (

Table 2. Substrate-specific effects of lactic acid fermentation, using isolates or natural microbiota, on macronutrients (carbohydrates, proteins, lipids) and secondary metabolites in cereals and legumes.

Macronutrient	Effect *	Function and Reported Improvement in Bioactive Properties	Reference
Carbohydrate	Reduction in fermentable oligo-, di-, monosaccharides and polyols (FODMAPS) a,i,j,k	Reduced risk for irritable bowel disease (IBD)	[65,66,67]
	Production of exopolysaccharides (EPS) b,e,c,g,h	Prebiotic effect, immunogenic, anti-inflammatory	[68,69,70]
	Reduction in raffinose family oligosaccharides (raffinose, verbascose, stachyose) a,b,c,d,e,k,j,i	Reduced risk of indigestion and flatulence	[66,71,72]
	Production of organic acids and acidification a,b,c,d,e,f,h,k,j	Flavour, food safety	[66,68,70,72,73,74,75,76]
	Improvement of glycaemic index by the reduction of soluble carbohydrates a,e,k,j	Reduced blood glucose spikes during digestion	[66,77]
	Generation of short-chain fatty acids from fibre fermentation a,i	Regulative effect on gut microbiota, reducing the risk for dysbiosis and associated disease	[78,79]
Protein	Improvement of in vitro protein digestibility a,b,c,d,e,k,h,j	Improved availability of essential amino acids	[66,68,70,72,80,81,82,83]
	Release of bioactive peptides a,c,d,f,i	Antihypertensive, antithrombotic, antimicrobial, angiotensin-converting enzyme (ACE)-inhibitory activity, dipeptidyl peptidase-IV (DPP-IV)-inhibitory, hypolipidemic	[83,84,85,86,87,88]
	Conversion of glutamate to γ-amino butyric acid (GABA) b,c,d,h,i,j,e	Antidiabetic via the induction of hypotension; relaxation effect can alleviate anxiety	[70,72,83,89,90]
Lipid	Conversion of linoleic acid to conjugated linoleic acid i	Antioxidant, anti-cancer, prevention of atherosclerosis, reduction of body fat	[89,91,92,93]
	Modification of polar lipid fraction a	Anti-inflammatory, anti-platelet properties	[94]
Secondary metabolites	Degradation of anti-nutritional factors (tannins, trypsin inhibitors, phytic acid) a,b,g,h,k,i,j	Antioxidant	[66,69,72,72,81,95,96]
	Generation of bacteriocins b,h,m,n	Food safety	[47,97,98]
	Increase in polyphenol availability and bioactive properties (through hydrolysis, production, or modification) a,e,f,i,h,l	Antioxidant	[75,81,91,92,99,100]
	Generation of vitamins (riboflavin and folate) f,i		[89,101]

* Used substrates include ^a rice, ^b sorghum, ^c wheat, ^d barley, ^e quinoa, ^f oat, ^g emmer, ^h millet, ⁱ soybean, ^j chickpea, ^k lentil, ^l cowpea, ^m maize, ⁿ rye.

). Specific impacts of fermentation on macronutrients (protein, carbohydrates, lipids), as well as minor secondary metabolites, are discussed as follows.

4.1. Carbohydrates

The glycaemic response of a food is governed by its carbohydrate composition and the speed at which it can be absorbed into the bloodstream as glucose after consumption. A link between the glycaemic response and satiety is also found and influences total food intake [102]. As such, food items with low glycaemic index (GI) are recommended by health-care professionals to combat common lifestyle-associated diseases, especially in Western countries. Fermentation is of interest in this context as it readily reduces soluble carbohydrates in starchy foods, thus improving GI and relative contents of dietary fibre and resistant starches [66,102]. Patients suffering from irritable bowel syndrome are generally instructed to avoid some cereals, as a high content of fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) can lead to complications. During natural fermentation, FODMAPs are reduced by the complementary carbohydrate metabolic activity of various organisms present, with reports ranging between 50 and 90% [4], allowing for the development of low-FODMAP foods [103]. Of particular interest is the degradation of raffinose-family oligosaccharides (RFOs), which include sucrose derivatives containing one (raffinose), two (stachyose), or three (verbascose) galactose units. RFOs are found abundantly in legumes and whole grain cereals and have been linked to the development of gastrointestinal discomfort, flatulence, and indigestion [4]. LAB fermentation has been shown to be effective in degrading RFOs via enzymatic processes (typically via the action of α-galactosidase), especially for species, such as Lp. plantarum [71], Lv. brevis [66], Lc. casei, Leuconostoc sp., Bifidobacterium sp., Weissella sp., and to a lesser extent using yoghurt starter culture bacteria [67]. In fermented maize products from Ghana, strains of Lp. plantarum and Leuc. mesenteroides demonstrated a decrease in raffinose by 79–85% and 46–50%, respectively [104], which improved the nutritional value and reduced potential digestive discomfort.

Short chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in varying amounts following fibre fermentation and, in addition to flavour development, are increasingly recognised for their role in gut health [80]. Studies on colonic fermentation have emphasised the role of SCFAs in gut microbiota regulation and the prevention of dysbiosis-associated diseases, such as IBD [105]. Multi-strain mixtures of B. longum, B. breve, Lc. Rhamnosus, and L. acidophilus have shown fibrolytic capabilities and allowed for production of SCFAs from fermented rice fibre fractions, with acetate being the most abundant product, followed by propionate and butyrate [79]. In plant-fermented foods, SCFAs are typically found in higher quantities compared to unfermented raw material [4]. Fermented soy and rice beverages using multi-strain mixtures (Lm. fermentum, Lp. plantarum, L. helveticus, B. bifidum, and B. longum) showed significant increases in acetate and butyrate, while undesired volatiles decreased [79]. The accumulated SCFA contents in fermented foods has been suggested to complement intestinally produced SCFAs and support gut homeostasis.

In addition to the metabolic breakdown, sugars (hexose, pentose, oligosaccharides) are involved in the assembly of significant bioactive metabolites. As the most abundant organic acid in fermented foods, lactic acid (produced up to 1%) can act as an antioxidant and may lower starch digestibility to further improve the GI by inhibiting amylolytic digestive enzymes [4]. Polysaccharides are produced in diverse forms by fermenting LAB, which is dependent on substrate availability, and are either secreted or tightly attached to the cell surface as part of capsule formation. Excreted EPSs are indigestible and have shown prebiotic potential by positively modulating the intestinal microbiota [106]. Homopolysaccharides, consisting of glucose and fructose only, can be utilised by intestinal bifidobacteria for growth. For dextran/oligodextran-type EPSs, a decrease in undesirable bacteria was reported [107]. Antibiofilm activity was found for EPSs from different LAB and has been suggested to inhibit the intestinal attachment of common pathogenic bacteria (E. faecalis, B. subtilis, S. aureus) by preventing initial auto-aggregation at the intestinal epithelial cell surface [108]. Capsular EPSs are a known bacterial stress-response, and for probiotic cells found in cereal fermented foods, their expression improves survival during gastrointestinal passage and adhesion to intestinal surfaces. As such, the production of capsular EPS is vital for a positive probiotic effect [109]. Heteropolysaccharides (HePSs) are more difficult to utilise by intestinal bacteria, owing to a more complex composition and highly branched nature, but have been attributed significant immunogenic properties [19]. EPSs produced by strains of Lp. paraplantarum, Lc. rhamnosus, and Lp. plantarum can, for instance, impact cytokine production in intestinal epithelial cells, with the reported increased production of the regulative IL-10 [110,111], TNF (tumour necrosis factor), IL-6, and IL-12 [112]. Anti-inflammatory, antiulcer, and antitumour activities of EPS have been suggested [106]. The presence of diverse EPSs in fermented cereal foods is clearly beneficial; however, in vivo studies aimed to confirm specific therapeutic effects are still limited and demand further detailed investigation.

4.2. Proteins

The physiological functions of fermented foods have frequently been linked to the release of bioactive peptides (BPs), a group of low-molecular-weight protein fragments that have shown resistance to gastrointestinal digestion and are readily absorbed into the blood stream by diffusion, endocytosis, and lymphatic pathways [113]. The presence of BPs in food is beneficial to chronic disease prevention, in particular diabetes, cardiovascular disease, and cancer. Lcc. lactis fermentation of dairy protein counts among the most well understood systems for the generation of BPs [59]; however, reports exist for a wide range of protein material, including cereals and legumes with biological properties that relate to the regulation of blood pressure, clotting, lipid levels, and immune functions (Table 2). Using an in-silico approach, Lacroix and Li-Chan [114] detected the presence of a total of 2256 peptide fragments that were known to exhibit DPP-IV inhibitory activity in 34 proteins of both animal (casein, collagen) and plant origin (barley, canola, soy, and wheat) with Gly-Ala, Gly-Pro and Pro-Gly being among the most frequent examples. Additional studies have since shown that cereal storage proteins, such as glutenin, gliadin (wheat), globulin, avenin (oat, amaranth), B/C-hordein (barley), prolamin, and glutelin (rice) encode di- and tripeptides of interest and suggest their presence in most dietary proteins at varying frequencies [87]. ACE-inhibitory activity, being among the most common bioactive properties, was demonstrated for isolated peptides, such as Leu-Gln-Pro, Ile-Gln-Pro, Leu-Arg-Pro, Val-Tyr, Ile-Tyr, and Thr-Phe from wheat [115] and Ile-Lys-Pro and Leu-Glu-Pro from amaranth [116]. The regular intake of foods rich in BPs has been suggested to promote health by controlling chronic diseases.

The release of BPs during fermentation is strain-dependant and has been observed predominantly in fermented soy products, which was attributed to the proteolytic activity of LAB Bacilli sp., yeast, and fungi [83]. For example, Lc. paracasei (previously L. casei) was used to release peptide fractions during the fermentation of soy protein isolate (37 °C, 36 h) with significant ACE-inhibitory activity that was linked to the peptide Leu-Ile-Val-Thr-Gln [86]. Four oligopeptides with strong angiotensin I-converting enzyme activity were also isolated after the fermentation of soy with P. pediococcus. Peptide fractions from quinoa fermented with native strains of Lp. plantarum (30 °C, 48 h) demonstrated antioxidative properties when exposed to pre-stressed epidermal cells [82]. From this study, five peptides ranging from 5 to 9 amino acid residues were identified and resisted further digestion. In the fermentation of pea protein with Lc. rhamnosus (30 °C, 24 h), antioxidative properties were detected mainly in low-molecular-weight fractions (<kDa) [117]. Traditional cereal beverages, such as boza, have shown a significant increase in peptide fractions with bioactive properties during fermentation, but no attempts have been made at identifying individual sequences to confirm their behaviour [83].

The generation of BPs in food varies based on the expressed proteolytic system and molecular make-up of the present protein material. During late fermentation stages, cereal endogenous enzymes are stimulated by a reduction in pH, which contributes to protein breakdown [118]. Depending on the source material and substrate specificity of expressed microbial proteases, proteins are broken down into peptide chains with differing chain lengths and amino acid sequences. Released BPs are thought to resist further hydrolysis and remain in the food, while other peptides may be absorbed by LAB for nitrogen assimilation [59,83]. Further research is needed to identify peptides that are released during cereal fermentation and persist in the product.

Peptides can also be assembled by ribosomal activity and excreted as secondary metabolites, which includes bacteriocin, a prominent class of antibacterial compounds that can impact shelf-stability of the fermented product and protect against food poisoning. The significant production of bacteriocin in plant substrates has been reported for strains of Lp. plantarum and Lc. rhamnosus, which led to the development of specialised adjunct cultures [59].

In addition to the release of bioactives, LAB fermentation impacts the nutritional quality of plant protein materials. An increase in in vitro protein digestibility can be observed in traditional fermented beverages from sorghum (uji, ting), pearl millet (ben-saalga), and maize (mawe) [119]. The partial hydrolysis of protein during fermentation improves the access to digestive enzymes of otherwise tightly folded storage proteins, leading to the enhanced release of essential amino acids [8]. The in vitro protein digestibility (IVPD) of zein protein, for instance, is increased from 74.9% to 93.5% during the natural fermentation of maize (37 °C, 24 h) [120], whereas fermented mixtures of millet, amaranth, and pumpkin achieved an IVPD of 92.8% [81]. Sorghum, known for its low level of bioavailable proteins showed improved IVPD from 30.0% to 53.5% after natural fermentation [121]. Using naturally occurring LAB, a consortium of bacteria cultures consisting of Lp. plantarum, Lm. fermentum, Lv. Brevis, Lc. rhamnosus, Lm. reuteri, and Pediococcus acidilacti improved the protein digestibility of sorghum further to reach between 79 and 84%, which was suggested to alleviate the negative impacts of cooking the grain [81].

Anti-nutritional factors (ANFs) that impact protein bioavailability in most cereals (whole grain) and legumes may further be degraded during fermentation. Enzyme inhibitors, for instance, actively block the active site of digestive enzymes (i.e., trypsin, chymotrypsin) and suppress protein digestion, most notably in soy, pea, and other legumes [122]. Trypsin inhibitors in pea and faba bean are heat-labile but have also been reduced effectively using LAB (Lp. plantarum) fermentation in the absence of heating [123,124]. Tannins, a type of phenolic compound with adverse effects on nutrient bioavailability were also reduced, as well as during the natural fermentation of cereals and pseudocereals [82]. Tannins, found abundantly in cereal bran of sorghum, millet, and hulls of peas and faba bean [122], contribute to the formation of insoluble complexes with protein, carbohydrates, and minerals. Tannins and their complexes remain unaffected by heating but are degraded readily in the presence of microbial tannase activity [66,73,82]. Phytic acid, owing to its reactive phosphate groups, acts as a strong chelating agent with minerals (Ca, Mg, Fe, Zn) and amino acid groups, thus reducing bioavailability in a similar manner by making proteins (as well as micronutrients) less available to the action of digestive enzymes. Endogenous phytase, present alongside phytic acid in most cereals, is stimulated at a pH range of 4.5–6.0 during fermentation. In combination with microbial phytase, the removal of the phytic acid of up to 90% has been achieved [125]. Despite the improved protein digestibility, detrimental effects of LAB fermentation are also possible if limiting essential amino acids are metabolised. For example, a reduction in methionine and cysteine during the fermentation of pea protein can lead to a reduced protein quality [123]. This negative effect should be considered when selecting for highly proteolytic LAB.

4.3. Secondary Metabolites

Plant ingredients contain a wide variety of compounds that do not contribute to the nutritional value of the food but instead provide beneficial effects in the prevention of disease. For example, phenolics (i.e., ferulic-, gallic-, chlorogenic-, coumaric acid flavonoids) act as potent antioxidants but are severely limited in their activity by poor bioavailability due to entrapment in cell wall components. LAB fermentation has been shown to be effective at increasing the release of soluble phenolics, especially when combined with yeast cultures or germination [119,126,127]. The beneficial modification of phenolics can also occur and has been reported for phytoestrogens found in soybean, where the β-glucosidase activity of Lp. plantarum was shown to promote the conversion of isoflavone glycosides to their aglycone counterpart that stimulated an enhanced protective effect against oxidative stress in vitro [128]. A co-culture of Lp. plantarum and Lv. brevis was used to further enhance this effect [91], which demonstrates potential synergistic effects between two or more organisms found in a complex natural fermentation. In addition, a separate study demonstrated the conversion of antioxidant plant-derived phenolic acids to catechol using similar LAB species [129].

γ-Aminobutyric acid (GABA), a non-protein amino acid, is generated in fermented food through microbial glutamate decarboxylase (GAD) by catalysing the α-decarboxylation of glutamate. GABA has been of interest in dairy and soy fermented beverages due to its physiological role in neurotransmission, blood pressure regulation, and insulin release [92]. GABA conversion by LAB is pH-dependent (with pH optimal at 4.5) and thus only occurs in the later stages of fermentation. GABA conversion has been demonstrated to take place during the fermentation of cereals depending on the presence of available glutamate [130,131,132]. The fermentation of oat, buckwheat, amaranth, millet, and quinoa using Lp. plantarum and Lcc. lactis, consistently increased GABA concentrations, reaching 185, 643, 654, 110, and 415 mg/kg, whereas minimal increases were observed for soy, wheat, and rice [133].

4.4. Lipids

The effects of enzymatic reactions on lipids are limited during the LAB fermentation of plant-based milk alternatives, while the lipolysis of dairy milk fats releases free fatty acids, glycerol, and acyl glycerides associated with flavour formation in cheese [134]. The generation of bioactive lipids in plant matrices is largely linked to fungi and alkali fermentation. Still, the antioxidative properties of LAB-fermented foods can originate from the formation of conjugated linoleic acids (CLAs) [4]. Cereals and legumes, except for oats and rice, contain linoleic acids, which are converted to CLA by strain-dependent linoleate isomerase activity [4]. CLA production has been demonstrated in soymilk, using various strains of Lp. plantarum and Lv. brevis [92,93], but a relatively high bioactive dose of one gram of CLA per day has been found to be necessary to achieve a therapeutic effect [135]. Glenn-Davi, Hurley [94] separated the lipid contents of LAB-fermented coconut, rice, and almond milk alternative products and showed a significant increase in anti-inflammatory and anti-platelet biofunctionality of the polar lipid fraction, while the total lipid content did not change consistently. A rational was provided that fermentation altered the degree of fatty acid saturation toward a more unsaturated polar lipid fraction with improved bioactive properties. Similar results can be found in the context of fermented dairy [4]; however, studies focused on the effects of lipolysis in LAB-fermented plant-based foods are still limited.

4.5. Vitamins

The overproduction of vitamins during LAB fermentation has become another focal point for current research. The enrichment of B group vitamins is of particular interest, since deficiencies in B group vitamins are prevalent in individuals that follow a plant-based diet and can lead to significant complications over time [131]. Vitamin B12 (cobalamin) is known to originate predominantly from microbial action in the rumen of cows. Its generation has been observed in vitro by propionic acid bacteria and can be achieved in natural fermented foods, although the necessary enzyme machinery is complex [136]. The production of vitamin B9 (folate) by LAB (Lm. fermentum, Lm. reuteri, and Lp. plantarum) has been demonstrated in recent studies, even though the genetic capabilities of these organisms were found to be incomplete to fulfil the task [137]. The presence of appropriate precursors (glutamate, para-aminobenzoic acid) was found to be necessary, and these are present in a variety of grains or can be produced by additional organisms.

5. Current Trends in LAB-Fermented Plant-Based Dairy Alternatives, as Compared to Traditional Fermented Products

Fermentation using established LAB starter cultures was found to increase applications in the category of plant-based dairy alternative products. Primarily, fermentation is applied with the goal of modifying the flavour and texture of starchy plant materials to generate “dairy”-like characteristics. In cereal-based beverages, EPS-producing cultures have been applied to increase viscosity and creaminess, whereas in legumes, major interest exists in reducing “beany”-flavoured volatiles [1]. Similarly, traditional foods typically use starches as a thickening agent combined with fermentation, which modifies the texture through EPS production and amylolytic activity and produces a complex flavour profile in the process.

Table 3. Experimental development of plant-based yoghurt alternatives reported in the past 10 years (2013–2023).

Year	Substrate	Fermentation Conditions	Culture Type	Organism(s)	Topic of Study	Reference
Legumes
2022	Mung bean protein isolate, quinoa, coconut oil (3% w/v)	43 °C, 8 h	Co-	L. bulgaricus, S. thermophilus, Kefir microflora, B. lactis, B. longum, and B. infantis	Effects of pre-treatments on protein gelation	[138]
2022	Soy protein isolate (5% w/w), coconut oil (4% w/w), pectin microgel particle (1% w/w)	43 °C, 8 h	Co-	L. delbrueckii subsp. bulgaricus and S. thermophilus	Effects of added fats and fibre on protein gelation	[139]
2021	Faba bean flour, (10.37% w/v) or chickpea flour (9.86% w/v)	43 °C, 10 h	Co-	S. thermophilus, L. delbrueckii subsp. bulgaricus, Lc. casei, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, Le. species, Lc. lactis subsp. lactis biovar. Diacetylactis	Sensory and nutritional assessment	[140]
2021	Lentil protein isolate (4.35% w/w), sucrose (0.75% w/v), sunflower oil (1.5% w/v)	42 °C, 6 h	Co-	Commercial culture (Yoflex® Acidifix™, Chr. Hansen, Denmark)	Texture and functionality compared to soy and dairy yoghurt	[141]
2021	Pea, mung bean protein isolate (3% w/v), sucrose (5% w/v), sunflower oil (3% v/v)	37 °C, 120–200 min	Co-	S. thermophilus, L. delbrueckii subsp. bulgaricus, Lp. plantarum, L. acidophilus (NCFM®), and Bifidobacterium lactis (HN019™)	Comparative texture analysis	[142]
2020	Rice (10% w/w), lentil (5% w/w), and chickpea (5% w/w)	30 °C, 16 h	Mono-	Lp. plantarum DSM33326, Lv. brevis DSM33325	Nutritional analysis and probiotic stability	[66]
2020	Faba bean flour or faba bean isolate, rapeseed oil (3% w/w)	37 °C, 6 h	Co-	Commercial culture (ABT-1, Chr. Hansen, Denmark)	Effects of amylase pre-treatment and fractionation on texture	[143]
2019	Pea protein (10% w/w), rapeseed oil (4%), oat fibre (3%)	43 °C, 18 h	Co-	L. delbrueckii subsp. bulgaricus, S. thermophilus	Mechanism of pea protein gelation	[144]
2018	Chickpea flour (4.80% total solids),	42 °C, 16 h	Co-	S. thermophilus, L. delbrueckii subsp. bulgaricus, L. acidophilus	Sensory comparison to dairy yoghurt	[76]
2016	Lupin protein isolate (5% w/v), glucose (2% w/v), coconut oil (4% v/v)	30 °C, 14–35 h	Mono-	Lp. plantarum TMW 1.460 a. 1.468, P. pentosaceus BGT B34, Lv. brevis BGT L150 (at 8 log10)	Texture promotion by EPS, effects of heat treatment	[145]
	cereals
2023	Oats (33% w/w)	42 °C, 6 h	Co-	S. thermophilus, L. delbrueckii ssp. bulgaricus,	Volatile analysis and comparison to dairy yoghurt	[146]
2023	Oats, almonds	30/37 °C, 48 h	Co-	L. delbrueckii ssp. bulgaricus, Lp. plantarum ATCC 8014, Lp. plantarum PK 1.1.	Volatile analysis	[147]
2022	Rice flour, coconut, guar gum (0.05% w/v), date palm syrup (10% w/v)	43 °C, 8–12 h	Co-	S. thermophilus, L. delbrueckii subsp. bulgaricus	Nutritional analysis and comparison to yoghurt	[74]
2022	Sorghum, finger millet	30 °C, 8 h	Co-	Lp. plantarum 299 v, W. confusa/cibaria C2	Effects of novel co-culture on nutritional benefits	[70]
2022	Quinoa flakes, soybean at differing ratios (14.5% w/w)	42 °C, 12 h	Co-	L. delbrueckii subsp. bulgaricus and S. thermophilus	Optimised formulation based on sensory acceptance and nutritional value	[148]
2022	One of cooked rice (20% w/w), shredded coconut (20% w/w), almond (10% w/w)	37 °C, 24 h	Co-	L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus	Bioactive analysis, anti-inflammatory and anti-platelet properties of lipid fractions after fermentation	[94]
2020	Oat flour (12% w/w), aquafaba (3% w/v), coconut oil (5% w/v), lactose (5% w/v)	43 °C, 6 h	Co-	B. lactis, L. acidophilus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and S. thermophilus	Use of aquafaba as gelling agent	[149]
2019	Oat protein isolate (87% purity, 15% w/v), lactose (10% w/v)	45 °C, 24 h	Co-	L. delbrueckii subsp. bulgaricus, S. thermophilus	Use of oat isolates as ingredient	[150]
2018	Quinoa flour (15% w/w), sucrose (10% w/v)	30 °C, 24 h	Mono-	Weissella cibaria MG1	Texture promotion by EPS, effects of proteolysis on protein digestibility	[68]
2015	Oat, barley, malt flour (15% w/w)	37 °C, 10 h	Co-	L. acidophilus NCIMB 8821, Lp. plantarum NCIMB 8826, and Lm. reuteri NCIMB 11951	Volatile analysis and acceptance based on used starter culture	[151]
2014	Oat flakes (25% w/w)	30 °C, 12 h	Mono-	Lp. plantarum LP09	Sensory analysis and optimised formulation	[75]

outlines current experimentally developed fermented plant-based products with a yoghurt-like characteristic using cereal and legume ingredients.

Nutritional benefits of commercial plant yoghurts depend on the selection of raw materials. Ingredients are selected from cereals and legumes based on consumer preference and fortified using minerals (e.g., Ca, Mg, Fe, and Zn), vitamins (e.g., B₁₂, B₂, D, and E) and amino acids to overcome the nutritional limitations of the starting material [152,153]. The use of plant protein isolates as an ingredient in yoghurt formulations has also gained traction, with recent studies that have focused mainly on the gelation properties of plant protein during fermentation as a novel texturising approach (Table 3). Isolates, produced using alkaline extraction, show reduced contents of ANFs and undesired volatiles [154,155]. However, a high energy and water consumption by the required extraction methods questions their sustainability.

Cereal-based raw materials remain interesting due to widespread availability, low cost of production, and popular organoleptic qualities, which have made them a common choice in traditional fermented food preparations. Sorghum, millet, and maize are among the most widely used (Table 1) and serve as a major source of dietary proteins in low socio-economic regions of Africa and Eastern Europe. Boosting their nutritional quality through fermentation plays an important role in combating nutritional insecurities in such countries [156]. Culturally, the advantage of fermenting cereals to improve flavour and nutrition are well established and has contributed to the continued popularity of traditional fermented foods. In the face of growing demands for sustainably produced foods that can satisfy both nutritional and organoleptic consumer requirements, fermentation technology is brought back to the forefront of food processing technology. Differences, however, exist when comparing the conditions, microbial species, and safety implications of traditional and “modern” fermentation methods, which suggests that further innovation is possible.

5.1. Fermentation Conditions

Most notably, the fermentation process used in most commercial and experimental plant yoghurts has been adapted from dairy yoghurt production. Established starter cultures (S. thermophilus, L. delbrueckii) and optimized growth conditions (37–43 °C, 6–10 h; see Table 3) are applied to achieve rapid acidification. This improves food safety by limiting the growth of potential contaminants. It also reduces production time. In traditional fermented cereals, however, fermentation typically occurs over several days (up to 5 days for bushera) at ambient temperatures depending on the regional climate [22]. Prolonged fermentation times are beneficial as they increase the production of several bioactive metabolites, such as EPSs, where increased yields in traditional fermented foods have been reported consistently [157,158,159,160]. The optimum pH for EPS production is found at pH 6.0 (with strain specific variations) [161], suggesting higher EPS yields if acidification progresses slowly. Moreover, suboptimal temperatures reduce LAB growth and equally promote higher EPS yields [108,162], which can be observed particularly for LAB that lack proteolytic activity, such as S. thermophilus [163]. Physiological stress from reduced temperatures has been suggested to promote EPS production as a stress response. In addition, the hydrolytic degradation of EPSs was found to be reduced at ambient temperatures [159]. Interestingly, ropy colony characteristics (attributed to EPS production) can be reduced or even lost entirely when LAB are cultured at elevated temperatures [157]. The loss of this characteristic may give a rise to mutant strains with reduced technological value.

Proteolysis during LAB fermentation is a beneficial process as it modifies protein bioavailability and leads to the release of BP. LAB metabolism of protein is typically low; however, new findings that linked the catabolism of amino acids (glutamine, glutamate, and arginine) to LAB acid-stress responses suggest that a metabolic shift takes place once a reduced pH of 3.5–4 is reached in the late fermentation stages [61,164]. In Lp. plantarum fermented barley, protein hydrolysis was first detected at 6 h, followed by a continuous increase in soluble peptide, amino acid, and phenolic content, together with antioxidative properties, whereas acidity and LAB counts peaked at 12 h and then slowly declined [161]. Studies that generated antioxidative, anticancer/inflammatory, antihypertensive, and antidiabetic properties due to the release of BP require a minimum of 10 h of fermentation time and include reports of peak bioactivity after 48–72 h [165,166].

Optimal temperatures for the production of bioactives, such as nisin by L. lactis, can also differ from optimal growth conditions (30 and 37 °C respectively) [167]. For Leuconostoc sp. and L. curvatus, similar findings exist, with an optimal production of bacteriocin at pH 5.5 and 25 °C but maximal biomass production at pH 6.0 and 30 °C [168]. Biosynthetic genes that regulate the release of metabolites, such as bacteriocin, are sensitive to environmental changes and may be activated at sub-optimal conditions as part of a cellular stress response. Furthermore, one study on the fermentation of pea, found temperatures of 22 °C and 30 °C to be beneficial for the release of peptides, whereas at 37 °C, peptide contents progressively decreased, likely due to their utilisation for cellular growth [169]. These findings suggest that sub-optimal growth conditions at ambient temperatures and long fermentation times, typically applied in traditional fermentation, can be advantageous to generate bioactives, such as EPSs, bacteriocin, BPs, and their associated functional properties.

5.2. Fermenting Organisms

The value of synergistic effects between two or more LAB species has been well recognised and forms the basis of established starter cultures (S. thermophilus, L. delbrueckii subsp. bulgaricus) in yoghurt production, where one proteolytic strain ensures the supply of amino acids and is in return promoted by growth-enhancing metabolites (CO₂, vitamin) from the other strain(s). The complexity of microbiota in traditional foods suggests the presence of many such interactions, which continues to spark interest. The fermentation of a peanut-based milk alternative, via a co-culture of Lp. plantarum and L. acidophilus, for instance, improved protein quality when compared to respective monocultures [95]. LAB naturally found in plant foods belonging to the family Lactobacillaceae have the capacity to hydrolyse protein but contain a narrow set of genes encoding proteolytic enzymes [60]. As such, mixed cultures are consistently superior at generating BPs in fermented food when compared to monocultures [83].

Similar effects can be observed for the degradation of ANFs and the production of vitamins in cowpea, where combined cultures of L. acidophilus and Lp. plantarum are found to be more effective at reducing phytic acid and trypsin inhibitors [96]. A co-culture with yeasts increased mineral availability via phytic acid degradation (up to 6-fold) in soymilk further, when compared to mono-cultures [170]. Co-culturing two strains (Lp. plantarum, P. freudenreichii) capable of producing vitamins B₁₂ and B₉ in fermented food increased their overall yield significantly and reached levels above those with any reported mono-culture [171]. Mixed culture studies were generally more successful in producing vitamins; however, a reduction can occur since vitamins are also a growth requirement in LAB fermentation [1].

Bacterial synergism can impact EPS expression and their related effect on texture and health-promoting food properties. For instance, co-culturing L. kefiranofaciens with non-EPS producing strains has shown effects on both EPS structure and yield [109]. ALAB provide a carbon source for EPS assembly through starch degradation and their importance as symbiotic organisms has been established in studies on ogi and pozol [53,54,134]. Weissella confusa, known for its high EPS yield, is the most abundant non-amylolytic LAB species found in pozol and was shown in a recent study to benefit from co-culturing with amylolytic Lp. plantarum A6 [172]. Similarly, Vila-Real, Pimenta-Martins [70] applied a co-culture of W. confusa 2LABPT05 and Lp. plantarum 299v in the development of a novel finger-millet/sorghum-based yoghurt alternative product and reported an increased overall nutritional value based on protein bioavailability, GABA, and EPS contents compared to the raw material. The identification of beneficial synergies and isolation of participating organisms from traditional foods is, however, still in the early stages and will be an essential strategy for the development of future LAB-fermented products with improved health-promoting properties.

5.3. Safety Implications

Food safety concerns have been a strong driving factor for the departure from spontaneous fermentation and use of defined starter cultures in modern industrial fermentation. While longer fermentation times can be beneficial in accumulating bioactive metabolites, the potential introduction of microbial-associated hazards needs to be addressed. As shown by Bartula and Begley [173], Listeria sp. and other common pathogenic species readily proliferate at relatively low temperatures in the range of 8 and 20 °C in popular plant-based beverages if left uncontrolled. The sporadic presence of enteric pathogens has also been reported for a variety of indigenous fermented foods [174,175]. Fast acidification rates, using high performing starter cultures at growth-optimised conditions, reliably inhibits the growth of randomly occurring pathogenic organisms, which are difficult to avoid when working with non-sterile food materials. The adoption of defined starter cultures accompanied by good manufacture practices (GMPs) reduces the risk for contamination and has been shown to improve the microbial quality of traditional fermented foods [176].

In addition to the production of organic acids, antagonistic effects of fermenting organisms against common pathogens can contribute to assuring food safety and have been observed frequently in traditional fermented foods. This includes both bactericidal and bacteriostatic activities against fungal (Aspergillus, Candida, Fusarium) and bacterial species (S. aureus, L. monocytogenes, Clostridium, Bacillus, Salmonella, Klebsiella, E. coli) and was linked to the production of hydrogen peroxide, EPS, and bacteriocins [109,146,177,178]. Bacteriocins describe a variety of short-chain peptides that result in the inhibition of pathogens mainly by directly targeting cell membrane components [59]. Nisin, known to be produced by Lcc. lactis, has been used as a preservative and shows the remarkable narrow inhibition of Gram-positive pathogens, which include Listeria monocytogenes, Clostridium difficile, and Staphylococcus aureus [167]. In the fermented foods poto poto and boza, strains of Lp. plantarum, Lm. fermentum, and Leuconostoc sp. showed the broad-spectrum inhibition of pathogens through the combined production of several bacteriocins that adhere to the surface of the producer cell [29,47,97]. EPSs with antiadhesive properties are produced by similar LAB species and can complement the effect of bacteriocin by preventing pathogens from generating protective biofilms [178].

Another risk factor during uncontrolled fermentation is the production of biogenic amines, which are basic nitrogenous compounds with recognised toxicity that can occur in fermented foods because of amino acid decarboxylase activity. For fermented cereal products, such as Boza, reports of biogenic amines at 25 to 65 ppm exist [179]. The enzymatic degradation of biogenic amines has been identified as a promising strategy for their reduction and can be achieved by food-indigenous bacteria if amine-degrading enzymes are expressed [180,181,182]. Complex starter cultures can thus ensure food safety by combining several strategies rather than rapid acidification alone. For a safe application, the absence of pathogenic species in the applied inoculum must be ensured, by either screening unknown mixed cultures or through the selection of individual strains as a defined culture.

6. Future Outlook

The potential of LAB fermentation in improving the nutritional and nutraceutical properties of fermented plant-based dairy alternatives has been demonstrated in various traditional fermented foods. Foods that undergo natural fermentation are a rich source of microbial populations. These microorganisms modify food components through synergistic and sequential enzymatic conversion and improve the nutritional composition of foods and the (over)production of bioactive metabolites (BP, EPS, B-vitamin, CLA). Partial enzymatic hydrolysis also leads to the pre-digestion of food materials, which improves the bioavailability of nutritionally relevant compounds, such as essential amino acids and phenolic compounds.

Advances in genomic-based research and the emergence of multi-omics techniques allow for a more comprehensive understanding of microbial interactions and their contribution to the health-related properties of traditional LAB-fermented plant foods. Selected traditional foods should be studied as models to drive the innovation of LAB fermentation methods to be used in the emerging sector of plant-based dairy alternatives. Purified isolates of fermented food origin are a useful starter culture that can be further optimised using tools of genetic manipulation (rDNA, CRISPR). However, the value of controlled mixed cultures should not be underestimated and may be more suitable depending on the desired outcome.

Aside from the importance of selecting a suitable starter culture, this review has highlighted the role of fermentation conditions. In the development of fermented plant-based milk alternatives, applying the correct fermentation conditions (temperature, time, oxygen content) can make the difference between increasing or decreasing nutritional benefits of the fermented food. The optimisation of growth condition can achieve a fast acidification rate but can impact a starter organisms’ capability to produce beneficial and health-promoting metabolites. Prolonged fermentation at sub-optimal temperatures, typically applied in traditional fermentation, has shown links to high yields of EPSs, BPs, B-vitamins, and bacteriocin and the degradation of ANFs. Optimised fermentation conditions depend on the starting material, pre-processing treatments, fermenting organism, and the desired outcome. These prerequisites need to be considered when developing nutritionally improved fermented foods. Microbial resources from traditional fermented food combined with knowledge on environmentally controlled enzyme expression will be necessary to update current industrial fermentation methods and permit the development of new yoghurt-like products from cereal and legume plant materials that satisfy a growing demand for sustainable healthy foods.