Solid-State Fermentation as a Biotechnological Tool to Reduce Antinutrients and Increase Nutritional Content in Legumes and Cereals for Animal Feed

Abstract

Antinutritional Factors (ANFs) are compounds produced by plants as defense mechanisms, and in high concentrations, they inhibit nutritional properties. Reducing these ANFs increases the presence of proteins, antioxidants, and vitamins, which is crucial for optimizing animal feed, particularly in developing countries where traditional methods may be costly. Solid-state fermentation (SSF) has the potential to improve the nutritional quality of animal feed derived from cereals and legumes cultivated and non-commercially cultivated by reducing antinutrients and enhancing nutrient availability. This review also considers the potential of non-native species, including those exhibiting invasive behavior and taxonomic similarity to cultivated varieties, as alternative substrates for SSF. Additionally, SSF highlights the biological properties of ANFs when extracted and utilized for technological and industrial advancements. Solid-state fermentation with lactic acid bacteria could be an effective and straightforward method for reducing these antinutritional factors while simultaneously enriching protein content. The aim is to present solid-state fermentation as a biotechnological tool to reduce antinutritional factors and enhance the nutritional content of legumes and cereals that are not cultivated for animal feed. This perspective contributes to expanding the range of raw materials considered for SSF by including taxonomically related but underutilized and ecologically problematic plant resources.

1. Introduction

Recently, food security has gained relevance due to factors such as food price inflation caused by extreme climatic conditions that have affected crop yields, making it difficult for low-income people to access nutritious food. The production of cereals and legumes consumed by people, such as wheat, corn, rice, soybeans, beans, lentils and chickpeas, sometimes decreases or remains uncertain due to factors such as soil health, water availability, pest and disease outbreaks, and market demand, ultimately affecting food security. Additionally, if the same cereals and legumes consumed by people are used to feed, it creates competition for food, increasing the price and affecting the availability of these [1].

Given that sustainability is now seen as an essential part of food security, global organizations such as the Food and Agriculture Organization of the United Nations (FAO), World Food Programme (WFP), World Health Organization (WHO), Codex Alimentarius Commission (CAC), World Bank, and World Trade Organization (WTO) work on programs to maintain food security and sustainability. These programs adopt multidisciplinary approaches involving consumption patterns, social norms, behaviors, and lifestyles [2]. By understanding all these factors, it is possible to create smoother, more acceptable transitions that minimize waste, reduce ecological footprints, and lower carbon emissions—ultimately fostering food systems that support sustainable urban growth [3].

Although challenges such as political instability, lack of funding, and resistance to change are faced with the programs implemented, initiatives such as the “Safe Food” campaign to reduce food loss and waste globally [4] and the “Purchase for Progress” program to help smallholder farmers access markets and improve their livelihoods have been successful [5].

The new strategies implemented by various governments worldwide focus on creating a sustainable food chain, prioritizing the production of new sources of food and feed [6]. This approach is particularly relevant in urban and rural populations of developing countries, where livestock farming is not only a crucial source of food but also a stable source of income and employment [7].

Soybean remains the most important and preferred source of high-quality plant-based protein for feed and food production; however, its cultivation and production are quite costly for developing countries. The possibility of using native plant species with equivalent high-quality protein as soybeans is an important alternative to avoid competition for food. The problem is that plants have the inherent ability to biosynthesize both primary and secondary metabolites, such as proteins, lipids, carbohydrates, vitamins, antioxidant nutrients, and antinutritional factors (ANFs) [8]. In this context, the possibility of using noncultivated and invasive species taxonomically related to conventional crops emerges as an important, sustainable, and low-cost alternative. These species often cause ecological problems due to the impossibility of avoiding their natural propagation. Proposing their use as substrates for SSF not only diversifies feed sources but also contributes to environmental management strategies.

The use of biotechnological tools can not only enhance food security and sustainability by optimizing resource use, increasing crop yields, and reducing environmental impact but also allows the use of different plant species as feed by increasing their nutritional quality and reducing antinutritional factors [9,10]. Biotechnological techniques, such as solid-state fermentation (SSF) with lactic acid bacteria, have been assessed to improve the nutritional quality of several plant species and agricultural waste [11,12,13,14,15,16,17,18,19], as well as selective breeding and genetic improvement for the development of animal breeds that are more resistant to diseases and have better feed conversion rates [20]. Additionally, supplemented feeds with probiotics, prebiotics, and other additives improve animal health [21]. Sensors to obtain data and their analysis optimize feed, reducing waste and increasing efficiency [22]. Alternative sources of proteins, such as insect production for feed, development of transgenic plants with higher nutritional value, pathogen resistance, and culture systems without soil to produce feed in reduced spaces with low quantities of resources, have also been used [23,24,25].

This review explores the potential of SSF to enhance the nutritional quality of cultured and noncultured plant species by reducing their antinutritional factors. Additionally, it proposes the possibility of using invasive species as raw material, offering a novel perspective within the framework of sustainable animal feed production.

2. Relevant Sections

2.1. Antinutrients in Legumes and Cereals

Antinutrients can be defined as natural or synthetic compounds found in various kinds of foods, mostly in many plants, which may inhibit the digestion or utilization of the nutrients required by the body. Forage legumes and grain legumes are used in animal nutrition. Alfalfa is used for ruminants, while fava beans, peas, and lupins are used especially for poultry and pigs and, to a lesser extent, for ruminants. Grain legumes are used for their high protein content (25–45 g/100 g dry matter), so much so that they are considered alternatives to soybean meals and other oilseeds. However, the presence of secondary metabolites, as well as their high fiber levels (non-starch polysaccharides), has restricted the use of grain legumes in the feeding of monogastric animals (poultry and pigs) and, even more so, in ruminants [26].

In plants, antinutritional factors are secondary compounds that defend them against herbivores, insects, and pathogens or survive under challenging growing conditions. When these plants are consumed by farm and domestic animals, these compounds can lead to adverse physiological effects [27]. The concentration and type of antinutritional factors vary among different plant species. Only the most relevant ones are mentioned here (

Table 1. Antinutrients classification from legumes and cereals [28].

Antinutrients Classification	Chemical structure	Glycosides	Saponins Cyanogens
		Proteins	Protease inhibitors: Trypsin Lectins
		Phenols	Tannins L-DOPA
	Functional significance	Anti-minerals	Phytates Oxalates

). The classification based on their chemical structure allows the identification and characterization of these compounds at the molecular level. On the other hand, the classification based on their functional significance focuses on the physiological and nutritional effects these compounds have on the organism, providing insight into their impact on health and nutrient absorption [28].

2.1.1. Glycosides

These compounds are formed from simple sugar molecules and other functional groups through the substitution of a hydroxyl group in the sugar molecule [29]. Glycosides are characterized by a distinct bitter taste in the plants where they are found [7]. The glycosides found in legumes and cereals include saponins and cyanogens.

Saponins are complex phytochemicals belonging to the triterpene or steroid families and are present in various crops [30]. They are characterized by their bitter taste and their toxicity at high concentrations [31]. Although saponins exhibit several biologically significant properties, such as antioxidant, antitumor, hypocholesterolemic, hypoglycemic, and anti-inflammatory activities, they also have adverse effects on animal nutrition [32]. Studies have reported that their inclusion in poultry diets can lead to reduced growth, decreased egg production, and lower feed intake [33].

Cyanoglycosides are plant compounds consisting of a glucose molecule linked to a cyanohydrin group and their enzymatic degradation releases cyanide. They spontaneously degrade, releasing hydrogen cyanide, which can be lethal [34]. This compound interferes with iodine organification, potentially leading to tropical ataxic neuropathy, goiter, cretinism, growth retardation, and cyanide poisoning, with symptoms including vomiting, nausea, dizziness, abdominal pain, weakness, headache, and diarrhea [35].

2.1.2. Protease Inhibitors

A protein is composed of a long sequence of amino acids linked by peptide bonds, which are formed when the carboxyl group of one amino acid reacts with the amino group of another, resulting in the release of a water molecule during the process [36]. Among the antinutrients with protein structures found in legumes and cereal are protease inhibitors and lectins.

Protease inhibitors (PIs) are low molecular-weight polypeptides that act as natural antagonists of proteolytic enzymes. They can form a stable enzyme-inhibitor complex and inhibit trypsin and chymotrypsin intestinal activity, causing physiological and productive alterations with endogenous loss of amino acids [26]. They play a crucial role in a range of biological functions, including cell growth, apoptosis, protein turnover, and cell migration in all live organisms, either directly or indirectly [37]. In addition to their general biological functions, these inhibitors offer specific benefits in medicine. For instance, protease inhibitors are used to block the activity of the HIV protease, a viral enzyme essential for virus replication. By inhibiting this enzyme, PIs prevent the formation of new viral particles, thereby limiting the ability of HIV to infect additional cells [38]. PIs from Vicia faba can cause pancreatic hypertrophy and have a limited effect on animal growth [26].

Lectins are proteins or glycoproteins found abundantly in the plant kingdom, characterized by their unique ability to bind with carbohydrate-containing molecules, exhibiting a high degree of specificity for sugar components. These proteins are notably resilient, not easily degraded, and resistant to stomach acid and digestive enzymes. Lectins can adhere to the intestinal wall, potentially damaging the intestinal lining. Since they are not broken down by digestive enzymes, they can affect intestinal permeability and enter the bloodstream. This interaction may lead to disruptions in intestinal function, which could be associated with conditions such as colitis, Crohn’s disease, celiac disease, irritable bowel syndrome (IBS), and increased intestinal permeability [27].

2.1.3. Phenols

Phenolic compounds can impair the digestibility of proteins and carbohydrates, as well as the bioavailability of vitamins and minerals. They inhibit the function of digestive enzymes, including amylase, trypsin, chymotrypsin, and lipase. Additionally, these compounds may harm the mucosal lining of the digestive tract and hinder the absorption of essential nutrients, such as vitamin B12 [27]. Tannins, L-DOPA, oxalate, and phytates or phytic acid are phenolic compounds.

Tannins are water-soluble phenolic substances that can form insoluble complexes when they interact with proteins or metal ions. These compounds are prevalent across numerous plant species and have diverse applications, including their use in tanning leather. Additionally, tannins are utilized in various pharmaceutical industries [39].

Although L-DOPA (L-3,4 dihydroxyphenylalanine) has medical applications in the treatment of Parkinson’s disease, in the context of animal nutrition, it has potential neurotoxic effects in rats, especially due to oxidative stress and interaction with monoamine oxidase B [40,41]. In pigs, L-DOPA in feed results in reduced intake and weight gain.

Oxalates are classified as antinutrients and are found in plants in two forms: a soluble form as oxalic acid and an insoluble form as calcium oxalate [42,43]. These compounds can bind to minerals, reducing their absorption and thus limiting the bioavailability of certain nutrients. Additionally, soluble oxalates, which can be toxic, may be transported to the kidneys, where they can crystallize into calcium oxalate, potentially leading to hyperoxaluria and the development of kidney stones, a condition known as nephrolithiasis or urolithiasis [44]. They can be found in spinach and rhubarb, among other foods.

Phytates are salts of phytic acid found in cotyledons and are the primary storage form of phosphorus and inositol in plant-based foods. It accounts for approximately 1–5% of the weight of oilseeds, legumes, and grains, which affects the nutritional value of these foods [45]. This acid can chelate divalent and trivalent metal cations, such as Fe²⁺, Fe³⁺, Ca²⁺, Mg²⁺, Zn²⁺, and Cu²⁺. The lack of phytase in the intestines of humans, birds, and other animals prevents them from properly absorbing phosphorus and other minerals from their diet [46].

In general, ANFs, such as tannins, trypsin inhibitors, saponins, phytates, and phytic acid, reduce nutrient absorption and affect livestock growth, necessitating costly processing or supplements to improve feed quality. Antinutrients such as L-DOPA, phenolic compounds, lectins, phytates, oxalates, tannins, and saponins, among others, are produced as defense mechanisms of plants to protect them from predators [47]. The consumption of these antinutritional compounds limits the bioavailability of essential nutrients and can have harmful effects on animal health, affecting their growth rate and productivity, causing hormonal changes, organ damage, reduced food intake, and weight loss [17]. However, in low doses, they prove to be beneficial to health because they show different bioactivities when consumed in certain amounts, such as antioxidants, hypolipidemic, and chemo-preventive agents [35].

To counteract the negative effects of ANFs, developing countries must invest in methods to reduce or eliminate these factors. The processing methods improve the nutritional profile of alternative feed sources, making local resources more viable for animal production. This approach is essential in areas where traditional feeds are either scarce or economically unsustainable, as it allows for the use of non-conventional feed resources, thereby reducing feed costs and promoting local resource utilization. Overcoming ANFs through these techniques is crucial to enhancing feed efficiency, lowering costs, and supporting sustainable livestock production in regions with constrained budgets and resources [48]. The presence of ANFs in non-conventional feed resources limits their utility due to lower nutrient availability, leading to reduced animal performance and economic inefficiency in production systems [49].

Treatments such as soaking, cooking, roasting, and germination, among others, have proven effective in reducing various antinutrients [50,51,52]. However, the energy costs associated with these processes are significant, making treatments with excessive energy consumption unfeasible in terms of both sustainability and profitability [17]. Fermentation is a process in which a substrate undergoes chemical and physical changes due to the enzymatic action of microorganisms, leading to positive effects on the proximate composition (proteins, carbohydrates, antioxidants), digestibility, and the reduction of ANFs [53,54].

Different types of fermentation have been used to improve the nutritional quality of various plant species. Submerged Fermentation (SmF) is a process in which microorganisms carry out enzymatic reactions in an abundant liquid medium, where the substrate is fully immersed, allowing high nutrient availability and efficient contact with the enzymes produced [55]. This method is commonly used in the production of beverages, enzymes, and various bioactive compounds. SmF is particularly effective for generating a high concentration of microbial products, including enzymes, proteins, and amino acids. However, control over conditions such as temperature, pH, and oxygen levels can be precisely regulated, enhancing microbial growth and product yield. Even though substantial amounts of liquid media, specialized equipment, and energy are required to maintain optimal conditions, they can render SmF costly, especially at the farm level.

Electro-fermentation (EF) is an innovative process that involves electrochemically controlling microbial fermentative metabolism with electrodes. The application of an electric potential can influence microbial metabolic activity, enhancing the efficiency of beneficial metabolite production such as organic acids, vitamins, or enzymes and reducing antinutrients, resulting in more nutritious and functional food and feed [56]. EF involves a prolonged fermentation process designed to improve specific nutritional properties or induce metabolic changes in the substrate, often lasting longer than standard fermentation cycles. Despite the time and resources needed, due to longer durations, which can escalate labor, time, and space requirements, challenges for farmers with limited resources are present.

Surface Fermentation (SF) is a process in which microorganisms grow and perform enzymatic reactions on the surface of a substrate or solid medium; it offers advantages such as lower installation and energy costs, but it requires significant labor and is sensitive to changes in the composition of the media [56].

Solid-state fermentation (SSF) involves the growth of microbial cultures on a moist, solid, and insoluble organic material that serves as both support and nutrient source for microorganisms in the absence or near-absence of free-flowing water [57,58]. SSF is considered a rapidly emerging approach in food processing and is viable for producing fermented foods, feeds and other useful industrial products [57]. SSF takes place in the presence of solid substrates, typically utilizing agricultural by-products; it enhances the breakdown of complex plant materials, improving the digestibility and nutritional value of the feed. It significantly reduces antinutritional factors (like phytic acid, trypsin inhibitors, and tannins) that interfere with nutrient absorption in animals, and small farmers can use raw materials available in the region, which usually are waste products from agriculture. For SSF, it is important to choose the right microorganism to have an efficient process. Otherwise, it can lead to poor results and contamination.

Each fermentation technique has its strengths and weaknesses, and its successful implementation depends on factors such as available resources, scale, and the specific nutritional goals for feed. Both SmF and EF require meticulous aseptic conditions, which present significant biotechnological challenges, as well as high operational costs and the need for specialized equipment and labor [59]. In contrast, SF is a more economical process, but it carries a higher risk of contamination due to the substrate’s exposure to air, making it susceptible to invasion by unwanted microorganisms. SSF, on the other hand, is easily scalable and applicable in rural communities, making it a particularly relevant option for improving animal nutrition in these regions.

The need for sterilization to produce animal feed is a critical step for the resource-limited context of smallholder farming and may represent the major limitation to the scalability of the fermentation process. In this context, the SSF can be implemented, assuring that the inoculation process does not require complex sterilization steps or non-sterilization. Some work supports this fact, but it is necessary after the SSF process to include microbial testing to assess the presence of pathogens and mycotoxins. SSF-assisted lactic acid bacteria (LAB) are recognized for their ability to produce bacteriocins, which are bioactive peptides with antimicrobial properties [60]. These peptides can selectively inhibit the growth of pathogenic bacteria, including various strains of Salmonella and Clostridium.

In addition, the formation of lactic acid during fermentation processes lowers the pH of the substrate and, combined with the activity of bacteriocins, effectively suppresses unwanted microbial growth [61], thereby enhancing the safety of fermented foods. Thus, inoculating the substrate with beneficial probiotic strains can help outcompete and inhibit the growth of pathogenic microorganisms. In a similar manner, results after SSF using microbial preparations containing microbial consortia rather than single species of lactic acid bacteria (LAB) have shown that suppressing harmful strains like Clostridium spores or Salmonella, supporting that the SSF of agro-industrial residues is a safety process [62]. However, uncontrolled SSF temperatures may favor the growth of undesirable strains, such as molds, potentially leading to mycotoxin contamination.

In summary, SSF offers several advantages over other techniques for reducing antinutritional factors present in plants used as raw material, primarily due to its low cost, as big and complex reactors are not necessarily required for achieving the same value-added feed production results. These benefits are particularly evident when employing microbial strains with specific enzymatic activities that are able to transform or neutralize key ANFs.

To better understand the microbial mechanisms behind these effects, the next section explores recent research on the role of different microorganisms in reducing antinutritional compounds during SSF. These results highlight how specific strains contribute to improving the nutritional quality and safety of fermented feed materials.

2.2. Reduction of ANFs

Regarding protease inhibitors as trypsin inhibitors, Ramachandra et al. [63] identified that Bacillus subtilis can degrade these compounds found in various cereals and legumes. The enzyme produced by the bacteria acts on these inhibitors, breaking them into three inactive fragments. This process occurs at 40 °C and a pH of 8.0. The enzyme is a metalloenzyme dependent on calcium concentration, with its activity increasing at Ca²⁺ concentrations of 2.5 mM. Additionally, it exhibits moderate thermostability, maintaining activity for 4 h at 55 °C. The authors suggest that this enzymatic hydrolysis method using the enzyme from B. subtilis could be a potential process in the food industry for the removal of trypsin inhibitors (TIs). Meanwhile, Caminero et al. [64] found that Lactobacillus degrades wheat TIs. Gao et al. [65] optimized solid-state fermentation using Lactobacillus brevis and Aspergillus oryzae for TI degradation in soybeans. The authors employed a response surface methodology with a Box–Behnken design to optimize the solid-state fermentation. Treatment with the microorganisms reduced TI content by 57% and 87%, respectively. The optimal conditions for L. brevis were a pH of 5.1, an inoculum size of 10%, a duration of 72 h, and a substrate-to-water ratio of 1.5, while for A. oryzae, the substrate-to-water ratio was 0.81, the inoculum size was 4%, and the duration was 120 h. These treatments also reduced phytic acid, crude fat, and urease activity while increasing crude protein and crude fiber. Hoffman et al. [66] conducted soybean fermentation with rumen microorganisms to inactivate and degrade TIs. This process was monitored under controlled conditions in an in vitro fermentation system. The results indicate that this treatment is more effective at inactivating than degrading the compound, but it can be used to inactivate ANFs in legumes.

Regarding phenols, Priyadarshini et al. [67] conducted a review on engineering processes for microbial biodegradation of phenols, highlighting them as sustainable and cost-effective techniques for reducing and/or eliminating phenols in contaminated water. The enzymes produced by bacteria act as biocatalysts, accelerating biochemical reactions without affecting reaction equilibrium or degrading other contaminants in the matrix. They note that while the industrial use of enzymes can be costly, improvements are seen when these enzymes are immobilized. The advantages of using immobilized enzymes include reduced sludge volume generation (in wastewater), short retention times, and high specificity. Additionally, immobilization enhances biocatalyst stability and efficiency, facilitating recovery and reuse. This article cites several enzymes derived from microorganisms that have been used to degrade phenols in water.

Regarding tannins, Sharma [68] conducted a review on phytopathogenic tannases for tannin degradation. The author notes that tannins are plant secondary metabolites with a defensive role, acting as a barrier against various phytopathogens that can invade and cause plant diseases. Tannase is an enzyme known for its ability to degrade plant tannins. In the chemical industry, enzyme immobilization is highlighted as a technique that can minimize solvent effects in industrial-scale biotransformations. Additionally, several tannases derived from microorganisms, such as Aspergillus awamori, Serratia fecaria, Lactobacillus plantarum, Aspergillus niger, Paecilomyces variotii, Bacillus sphaericus, Rhodotorula glutinis, and Aspergillus nomius, have been immobilized to enhance this solvent effect. In terms of food and nutrition, tannases improve the quality of sorghum and other cereals by degrading tannins, which act as ANFs. Tannases from Paecilomyces varioti, Azotobacter, and Aspergillus niger are specifically mentioned. Sigona et al. [69] evaluated the role of bacterivores in fungus-based systems for tannin degradation. They found that while several fungal species are effective in degrading these compounds, high microbial content can affect the process. This research aimed to provide useful information for stabilizing fungus-based systems in non-sterile conditions. Specifically, white rot fungi produce various extracellular enzymes, including laccases, lignin peroxidases, and manganese peroxidases, which can degrade tannins. Some Ascomycetes, such as Aspergillus sp. and Penicillium sp., are also effective in this process due to their production of tannase.

For L-DOPA degradation, a study was found in which Rhizopus oligosporus was used to degrade this compound and pyrimidine glycosides from fava beans. A reduction of 100%, 91%, and 98% was observed for L-DOPA, vicine, and convicine, respectively, on the sixth day of fermentation. The authors recommend optimizing fermentation conditions to further improve and control the process [70].

The most relevant results reported in the literature on cereals have shown that SSF decreases their antinutritional components. Espinosa-Páez et al. [71] reported a 50% decrease in tannin content in Avena sativa using Pleurotus ostreatus; the authors noted that the fungus can reduce or eliminate the antinutrient tannins under growth conditions of 336 h (14 days) at room temperature, mainly by the action of tannase present in the fungus, which destroys tannins. The results also highlight the increase in protein and different important nutrients of cereal. This trend was also reported by [72], evidenced a reduction of tannin content with the use of Penicillium charlesii (tannase-producing fungus) in samples of wheat straw; the decrease of tannins was 91%; the authors also emphasized the use of fungi that can metabolize lignocellulose to improve the nutritional value in cereals by delignification. The use of tannase in food increases the bioavailability of nutrients by hydrolyzing phenols, which act as antinutritional factors.

Some studies suggest that an increase in phenol content promotes considerable health potential by functioning as antioxidants; in some cereals, they are found in a conjugated form that may affect consumer health and bioaccessibility. Kupski et al. [73] performed SSF on Oryza sativa with Rhizopus oryzae for 120 h at 30 °C to extract and enhance antioxidant compounds from the fermented biomass; the authors report that phenolic compounds in biomass extracts present different electron-donating potentials. Coumaric and ferulic acid are compounds that are the most active antioxidants due to the double bond in the molecule, participating in the stability of the unpaired electron radical. In this study, there was also a significant increase in protein and vitamins from fermented cereal. FFS in various types of cereals with fungi generates an increase in bioactive compounds, as reported by Xu et al. [74], who used three types of fungi: Agaricus bisporus, Helvella lacunose, and Fomitiporia yanbeiensis. These were individually inoculated on wheat, rice, oats, maize, millet, quinoa, buckwheat, soybean, peas, and sorghum. The results obtained showed that the antioxidant properties in the fermented products were stronger, that is, by the uptake of 1,1-diphenyl-2-picrylhydrazyl radicals, reducing power and chelating ability of ferrous ions and superoxide anion radicals.

Within the research developed with the use of bacteria, Adeyinka et al. [75] carried out the optimization of the growth conditions of Lactobacillus fermentum bacteria in the degradation of tannins of Sorghum bicolor; the authors reported that a temperature of 34 °C for 24 h promotes the growth of bacteria in the substrate and a decrease of 98.71% of tannins. This reduction is associated with the effect of tannins on other molecules, the tendency to self-polymerize and decompose into smaller molecules due to the reorganization and the ability of bacteria to metabolize these undesirable compounds in cereals.

Legumes can develop different textures, flavors, odors, and acidity due to the role lactic acid bacteria (LAB) play during fermentation [76]. In addition to preserving food by inhibiting harmful bacteria, LAB also acidifies the food and reduces the content of antinutritional factors (ANFs), making them highly valuable in the food industry [77]. Solid-state fermentation with LAB typically involves the conversion of carbohydrates into lactic acid [78]. The chemical reactions that occur during this process can be summarized as follows:

Glycolysis: LAB breaks down carbohydrates, such as glucose or sucrose, through glycolysis. In this process, carbohydrates are converted into pyruvate, a three-carbon molecule.

Lactic acid production: Pyruvate molecules are then converted into lactic acid via homolactic fermentation. In this step, pyruvate is transformed into lactic acid by the enzyme lactate dehydrogenase, generating two ATP molecules (energy source) from the original glucose molecule.

Acidification: The production of lactic acid results in a decrease in pH, acidifying the medium. This acidification inhibits the growth of other microorganisms, allowing LAB to dominate the fermentation process.

Proteolysis and lipolysis: During solid-state fermentation, LAB can also carry out proteolysis and lipolysis, breaking down proteins and fats, respectively, to produce additional nutrients and flavor compounds.

These reactions are facilitated by enzymes produced by LAB, which catalyze the conversion of substrates into the desired products [79]. Similarly, different reactions occur in the degradation of ANFs, although each reaction may vary depending on the specific compound targeted [51,80]:

Hydrolysis: ANFs such as tannins can be hydrolyzed by enzymes produced by LAB, leading to the breakdown of the compound into simpler molecules.

Oxidation: Polyphenols, another class of ANFs, can be oxidized by LAB enzymes, resulting in the formation of less harmful compounds.

Esterification: Some ANFs, like trypsin inhibitors, can be esterified by LAB, rendering them inactive. These reactions can enhance the nutritional quality and digestibility of food ingredients, ultimately leading to improved growth and health in animals.

A description of the relevant results from some research related to the degradation of ANFs using SSF with lactic acid bacteria in legumes is shown in

Table 2. Relevant research results related to ANF degradation using solid-state fermentation with lactic acid bacteria.

Legume	Microorganism	Main Findings	Reference
Vigna sinnensis var	L. plantarum	↓ Phytates (85%), ↓ TIs (50%)	[11]
Glycine max	L. plantarum	↑ TPC (21%), ↓ Phytic acid (55%), ↓ Saponins (4%), ↓ TIs (95%)	[13]
Mucuna cochinchinensis	L. plantarum	↓ Phytates (32%), ↓ Tannins (72%)	[14]
Tamarindus indica L.	L. licheniformis	↓ TPC (50%), ↓ TIs (86%), ↓ Tannins (75%)	[15]
Phaseolus vulgaris Cicer arietinum Lathyrus satires Lens culinaris Pisum sativum	L. plantarum L. brevis	↑ TPC (20–70%), ↓ Tannins (18%)	[16]
Mucuna deeringiana	L. rhamnosus	↓ TPC (51%), ↓ L-DOPA (91%), ↓ Tannins (97%)	[17]
Glycine max	Bifidobacterium animalis 937, Lactobacillus casei Zhang and Lactobacillus plantarum P-8 mixed with Bacillus subtilis natto	↑ Free amino nitrogen (100%), ↑ peptide with molecular weight less than 1000 Da increased (30.7–81.3%)	[81]
Glycine max	Bacillus licheniformis YYC4, Geobacillus stearothermophilus A75, Bacillus subtilis 10160	↑ Protein (12–18%), ↓ TIs (38–74%)	[82]
Zea mays	Lactobacillus plantarum and Saccharomyces cerevisiae	↑ Protein (38–55%), ↓ Phytate (66%), ↓ Tannins (75%), ↓ Trypsin inhibitor (64%)	[83]
Vicia faba L.	Lactobacillus plantarum	↓ Tannin (68–77%), ↓ phytic acid (18–35%), ↑ Protein (10–11%)	[84]
Glycine max	Bacillus velezensis Lactobacillus plantarum	↑ Protein (47–52%), ↑ amino acid profile (5%), ↓ glycinin (79%), ↓ β-conglycinin (73%)	[85]
Glycine max	Bacillus sp.	↑ Protein (11–13%), ↓ glycinin (82%), ↓ β-conglycinin (88%), ↓ ITs (99%), ↓ phytic acid (72%)	[86]
Glycine max	Bacillus subtilis	↓ ITs (99%), ↓ phytic Effect of solid-state fermentation on proximate composition, acid (20–38%), ↑ crude protein (49–51%)	[87]
Phaseolus lunatus, Parkia biglobosa	Bacillus subtilis, B. polymyxa, Lactobacillus casei, Leuconostoc mesenteroides, Micrococcus rubens and Staphylococcus aureus	↑ Protein (4–11%), ↓ Phytates (40–47%), ↓ Tannins (48–56%)	[88]
Faba bean meal	Lactobacillus plantarum	↑ Protein (10%), ↑ in vitro protein digestibility (4%), ↓ Tannins (80%), ↓ phytic acid (18%)	[89]

TPC—Total Phenolic Compounds; TIs—Trypsin Inhibitors, ↓—Decrease; ↑—Increase.

.

In legumes, SSF shows significant results in increasing key nutritional factors (protein, antioxidants, crude fiber, minerals, and digestibility) with the use of lactic acid bacteria (Table 2). Several authors [27,39,41,42,89] have reported favorable results in the decrease of ANF levels in the plant species studied using Lactobacillus plantarum bacteria. Olaleye et al. [14] conducted a comparative study of proximate and antinutritional factors of fermented dehulled and unhulled Lyon bean (Mucuna cochinchinensis). Additionally, the effect of fermentation time and temperature on the proximal composition and antinutrient content of shelled and unshelled Mucuna were investigated. The grains were fermented at 30 °C and 45 °C for 24, 48, and 72 h, respectively, and the fermented seeds were processed into flour. The results showed that fermentation time is a factor that greatly impacts the proximal composition of the grain, and the antinutritional compounds of the seeds decreased (28.5–5.76%) significantly with increasing fermentation time and temperature. This study showed that fermentation at 45 °C for 72 h increased the nutrient content of beans more effectively, decreased the content of antinutrients and favored the growth of L. plantarum.

Curiel et al. [16] exploited the nutritional and functional characteristics of traditional Italian legumes by fermentation with L. plantarum and Lactobacillus brevis bacteria. In this study, fermentation was carried out on 19 Italian legumes traditional in the diet for their nutritional values. Once fermented with the bacteria at 30 °C for 24 h, the results showed that the levels of antinutritional factors decreased. Also, important fermentation products such as lactic acid and acetic acid were reported to be produced from the bacteria used.

Álvarez et al. [17] reported that the formation of lactic acid by L. rhamnosus can be used as an indicator of the degradation of antinutritional factors, specifically L-DOPA in Mucuna deeringiana. Other ANFs, such as tannins and total phenolic compounds (TPC), are also degraded. The protein content increases by 12%, and the antioxidant capacity reaches 97%. As a result, they obtained a product with favorable nutritional characteristics for developing feed supplements.

With advancements in SSF, this technology has been recognized as a promising method for producing protein biomass using fungi to create products with enhanced nutrition (reduction ANFs) and functional value [90] (

Table 3. Relevant research results related to ANFs degradation using solid-state fermentation with fungi.

Legume	Microorganism	Main Findings	Reference
Lathyrus sativus	R. microsporus var. Chinensis, A. oryzae	↑ Free amino acids (12%)	[91]
Lens culinaris	Pleurotus ostreatus	↓ Phytic acid (88%), ↓ TPC (33%)	[92]
Phaseolus vulgaris	Lactobacillus plantarum	↓ Tannins (20%), ↑ protein (18%)	[93]
Brassica napus	Aspergillus niger	↑ protein (81.7%), ↓ Phytic acid (37.5%), ↑ TPC (64.5%)	[94]
Tylosema esculentum	Aspergillus oryzae, Aspergillus sojae	↓ Phytic acid (99%), ↓ TIs (68%), ↑ TPC (9%)	[95]
Glycine max	Pleurotus ostreatus	↑ Protein (53%), ↑ Phosphorus (36%), ↓ Phytic acid (29%), ↑ TPC (12%)	[96]
Lens culinaris	Pleurotus ostreatus	↑ Protein (23%), ↑ TPC (52%)	[97]
Lupinus spp	Aspergillus sojae, Aspergillus ficuum	↑ TPC (21.2–37.3%), ↓ Phytic acid (53.3 to 73.2%)	[51]
Glycine max	Lactobacillus acidophilus	↓ TIs (90%)	[98]
Phaseolus vulgaris	Pleurotus ostreatus	↓ Tannins (34–66%)	[71]
Glycine max	Aspergillus oryzae	↓ TIs (89.2%), ↓ Phytic acid (34.8%)	[65]
Vicia Faba L.	Aspergillus oryzae and Rhizopus oligosporus	↑ Protein (8–20%), a reduction in most antinutrients, with the exception of trypsin inhibitors	[99]

TPC—Total Phenolic Compounds; ITs—Trypsin Inhibitors, ↓—Decrease; ↑—Increase.

).

In summary, each microbial strain interacts differently with its substrate, leading to different levels of efficacy in degrading antinutritional factors, enhancing the nutritional value, and potentially influencing the digestibility of the feed or food product. In comparing the use of filamentous fungi and lactic acid bacteria for SSF, each microorganism offers distinct advantages depending on the desired outcome. Filamentous fungi excel in mimicking their natural environment within SSF, effectively reducing ANFs such as tannins and phytates, and enhancing the nutritional value of cereals by increasing protein content and antioxidant activity. However, LAB, such as Lactobacillus species, have shown superior adaptability to low water activity conditions and have proven particularly effective in the fermentation of legumes. LAB not only reduces ANFs like phytic acid, tannins, and L-DOPA but also significantly increases beneficial compounds like (TPC, proteins, and antioxidants). The variability in outcomes underscores the need for careful selection and optimization of microbial strains based on substrate type and the specific antinutritional factors targeted for reduction. Thus, the composition of the raw materials used in SSF plays a pivotal role in determining the efficacy of ANF reduction. Different substrates possess varying levels of ANFs, and their inherent nutritional quality influences how microorganisms perform during fermentation.

On the other hand, microbial metabolism and the efficacy of enzymatic content can enhance microbial activity, resulting in the effective breakdown of ANFs. The influence of fermentation time is critical, as prolonged fermentation periods have been associated with higher efficacy in degrading ANFs. For instance, studies on spontaneous fermentation of legumes observed significant reductions in ANFs like phytic acid and tannins over 24 to 72 h [100]. Moreover, the interplay between temperature and pH levels during SSF has demonstrated a correlation with metabolite production that can lead to enhanced nutritional profiles of legumes, transforming them into potential health foods [101]. Furthermore, when considering the substrate composition, different legumes exhibit varied susceptibility to microbial enzymatic action, which can be influenced by the aforementioned environmental factors.

pH plays a critical role in microbial metabolism and the efficacy of enzymatic activities that lead to the breakdown of ANFs. A neutral pH range of 6–7 is generally optimal for the growth and enzyme production of many filamentous fungi, including Rhizopus and Aspergillus, which are commonly utilized in SSF due to their robust proteolytic and amylolytic capabilities. For example, the use of Rhizopus during the fermentation of soybean or chickpea resulted in a marked increase in total protein digestibility while concurrently decreasing the concentrations of tannins, which are known for their adverse effects on protein absorption and overall nutrient uptake [91]. However, lactic bacteria lead to a decrease in pH as fermentation progresses. The lower pH achieved through lactic acid production is instrumental in enhancing enzyme activity that hydrolyzes these compounds, thereby improving nutrient availability. Lactobacillus acidophilus is noted for its rapid acid production, contributing to lower pH levels sooner than Lactobacillus plantarum, although both strains achieve similar end pH values by the conclusion of fermentation [102]. Additionally, Lactobacillus rhamnosus has been highlighted for its effectiveness in enriching protein levels while concurrently minimizing ANF content, showcasing the diverse capabilities of LAB depending on both the substrate and fermentation conditions used [17].

Overall, the degradation of ANFs through solid-state fermentation not only mitigates their adverse effects but also triggers biochemical transformations mediated by microorganisms and the environmental conditions, such as pH, that improve the nutritional profile of the final product. These include increases in protein content, enhanced bioavailability of minerals, and elevated levels of bioactive compounds.

The next section explores how these microbial-driven modifications translate into measurable improvements in the nutritional quality of legumes and cereals, highlighting key outcomes such as increased protein, fiber, antioxidant capacity, and overall digestibility.

2.3. Increase in Nutritional Quality

The reduction of antinutritional factors during solid-state fermentation (SSF) not only eliminates compounds that interfere with nutrient absorption but also promotes favorable biochemical transformations in cereals and legumes. These modifications result in increased protein content, improved digestibility, higher levels of vitamins and antioxidants, and enhanced bioavailability of minerals.

Furthermore, SSF modifies the nutritional qualities of plant-based feed materials by improving their palatability, organoleptic properties, and digestibility. Cereals and legumes, already rich in proteins, fats, vitamins and minerals [75], become even more valuable nutritionally after fermentation. This is particularly important for small farmers, who can utilize locally available agricultural by-products as substrates for SSF, transforming them into low-cost, high-quality animal feed.

The selection of microorganisms is critical for SSF as it directly affects the productivity and quality of the fermented product. Filamentous fungi are particularly well-suited for this technique, as it closely mimics their natural environment. Additionally, certain bacteria, such as Lactobacillus, can adapt to low water activity conditions [90]. The incorporation of bacterial probiotics during stressful periods for the intestinal microbiota improves dietary changes in animals. Lactic acid bacteria such as Lactobacillus, Bifidobacterium, and Enterococcus are the most used microorganisms because they are found in the gastrointestinal tract of humans and animals.

Zhang et al. [81] conducted a study on the SSF of soybeans using Lactobacilli and observed that after 48 h of fermentation, there was a rapid increase in the amino acid content of the final product, from 99.7 to 529.1 μmol/g due to the multiplication of microorganisms and the effect of the enzymatic system. Similarly, peptides with molecular weights lower than 1000 Da increased from 30.7% to 81.3%, indicating that this type of fermentation in soybeans can provide different probiotics and nutritious products. However, no other topic related to silage management has received as much attention from researchers and farmers as bacterial inoculants [57].

Many commercial feed products for ruminants with varying effectiveness are available. To achieve the intended effectiveness, each product must adhere to the recommended dosage and follow the described application method. Vanbelle et al. and Chaucheyras, Durand [103,104] suggest that many bacteria and yeasts can be used beneficially to maintain a healthy and balanced digestive flora in animals. The most used microorganisms include Lactobacillus sp., Streptococcus faecium, Bacillus subtilis, B. cereus, B. licheniformis, B. stearothermophilus, and Saccharomyces cerevisiae [21]. The Lactobacillus bacteria, which grow rapidly in the small intestine, are perhaps the most well-known for their ability to transform lactose into lactic acid. Since Lactobacillus prevalently appears in fermented foods, has a low infection rate, and are part of the normal microbiota of mucous membranes, they have low pathogenic potential, which is why they are considered GRAS (Generally Recognized As Safe) organisms [105]. These bacteria in the small intestine prevent intestinal colonization by pathogens by competing with them for essential nutrients and adhesion sites and by producing organic acids and other substances that make the intestinal environment unfavorable for pathogens [106].

The by-products formed after enzymatic action are used as prebiotics by probiotics, thereby improving the digestion of cellulose-rich foods. The incorporation of probiotics during stressful periods for the intestinal microbiota facilitates dietary changes in animals [107]. The main probiotics include lactic acid bacteria such as Lactobacillus, Bifidobacterium, and Enterococcus, which are inherent members of the gastrointestinal tract of humans and animals [108]. These organisms should be able to withstand the hostile environment, including the low pH and bile toxicity prevalent in the upper digestive tract [109].

In general, SSF not only enhances the nutritional profile of legumes and cereals by increasing protein levels, digestibility, and beneficial compounds such as vitamins and antioxidants but also creates conditions that influence other important aspects of feed quality, such as taste, aroma, texture, and color, which are significantly affected during fermentation. The following section explores how SSF contributes to improving the organoleptic properties of fermented products, which is crucial for feed palatability and consumer acceptance.

2.4. Improving Sensory Attributes

SSF can improve the palatability and digestibility of lignocellulosic materials, which are often underutilized in animal nutrition. Fermentation can lead to the production of various organic acids and flavor compounds that enhance the palatability of feeds. However, some authors argued that the reduction in AFN as tannins can improve the bitterness of some cereals; in some cases, as in lupin seed, the bitterness is associated with the overall alkaloid content in lupin seed more than with tannin or saponin levels [110]. Similarly, there is a general belief that tannins in sorghum confer objectionable sensory attributes. Still, the bitterness, astringency, and other sensory characteristics of tannin sorghum are similar to tannin-free sorghum [111]. In this case, the reduction of AFN does not contribute to sensory attributes. SSF enhances the development of desirable flavors through the production of volatile compounds, which helps increase the overall acceptability of the feed but is not always associated with the reduction of AFN; mixed lactic acid bacteria (LAB) strains have shown significant potential in minimizing undesirable odors, thus improving the palatability of the feed [112]. In this sense, Zhang et al. [113] reported a significant improvement in the overall sensory quality of solid-state fermented brown rice, with sensory scores rising significantly thanks to the development of distinctive flavors and aromas due to fermentation. According to the Cluster analysis (CA) and Principal Component Analysis (PCA) results, solid-state-fermented brown rice presents nutritional content and sensory characteristics that are different from unfermented rice. However, a relationship between the sensory properties and decreasing antinutritional factors was not established.

The textural properties of fermented feeds can also improve due to the breakdown of complex carbohydrates and proteins during fermentation. This alteration in texture can contribute to the acceptability of feeds among animals, thereby potentially increasing feed intake [112]. Breakdown of fiber and proteins during fermentation using Rhizopus oligosporus can create a softer, more palatable texture, improving the eating experience and, as a consequence, enhancing the digestibility and palatability of the feed [94].

As can be concluded, SSF can positively influence the sensory attributes of fermented legumes and cereals, such as flavor, aroma, texture, and palatability. These improvements contribute to increased feed intake and acceptance among animals. However, the benefits of SSF go beyond enhancing nutritional and sensory properties, as it also enables the production of valuable bioproducts such as organic acids, bioactive peptides, enzymes, and antimicrobial compounds that can contribute to animal health and performance.

The following section explores the types of bioproducts obtained through SSF and their applications in animal nutrition.

2.5. Bioproducts Obtained from SSF Applied in Animal Feed

Bioproducts used in animal feed are typically produced through SSF (

Table 4. Bioproducts obtained from SSF applied in animal feed.

Application	Substrate	Microorganism	Improvement in Animal Feed	Reference
Pig feed	Soybean meal	L. plantarum, B. subtilis and S. cerevisiae	Reduction of trypsin inhibitor levels (97%) and enhanced crude protein levels (13%).	[114]
Fish feed	Soybean meal	Saccharomyces cerevisiae	Elevated levels of crude protein (13.7%) and amino acids (16.3%), with a decrease in phytic acid (93%) and trypsin inhibitor concentrations (8.5%).	[115]
Poultry feed	Faba beans, wheat bran, potato pulp	Lactic acid bacteria from a commercial product	Improved protein (13–16%) and phosphorus solubility (10–17%).	[116]
Ducklings	Flaxseed cake	Aspergillus niger, Candida utilis	Enhanced crude protein content (15,8%) and reduced hydrocyanic acid levels (73%), leading to increased nutrient bioavailability.	[117]
Broiler chickens	Wheat bran	Pleurotus eryngii	Boost in lignocellulolytic enzyme activity and antioxidant molecule expression following broiler consumption.	[118]
Chickens	Soybean meal	Bacillus subtilis ED-3-7	Crude protein and acid-soluble protein contents increased by 12% and 343%, and the trypsin inhibitor content was lower than the range specified in the detection kit.	[119]
Ovine feed	Groundnut meal	Saccharomyces cerevisiae	Increase in total protein (11–27%) and amino acids content (44%) with a reduction in phytic acid levels (69–72%).	[120]
Ruminants feed	Olive cake	Rhizodiscina cf. lignyota, Aspergillus niger	Rise in protein levels (94%) and decrease in phenolic compounds (43%), flavonoids (70%), and condensed tannins (42%).	[121]

), often utilizing a single substrate. This approach is favored because it simplifies process control and ensures the quality of the fermented product before it is incorporated as an additive in the final product formulation.

2.6. Applications and Future Perspectives

The expansion of the sustainable agro-food industry faces several challenges in the industrial application of SSF [122]. To overcome these challenges, future research should focus on integrating SSF with emerging technologies, thereby enhancing its feasibility in an industrial context. A search of the Science Direct database (www.sciencedirect.com (accessed on 1 May 2025)) for articles published between 2020 and 2024, which included the terms “solid,” “state,” and “fermentation” in the title, abstract, or keywords, was conducted to identify studies on SSF processes applied to agriculture and agricultural products. A total of 6020 articles were found (Figure 1).

Figure 1 indicates a growing interest in research on the subject, with a steady increase in the number of publications over the years. The most significant increase occurred between 2022 and 2023, while the growth between 2023 and 2025 was more moderate but still positive. This suggests a continuous expansion in the field of study, with a possible rise in interest or resources dedicated to research in this area.

Several patents related to animal feed incorporating probiotics and proteins have been developed, such as Hermetia illucens (CN110037165-A), Lactobacillus bulgaricus and Streptococcus thermophilus (CN110037189-A), and Bacillus licheniformis (CN1103841-A). These innovations enhance nutrient absorption, promote growth, and increase animal productivity. Additionally, fermented products for pig feed have also demonstrated significant benefits for animal health, including the preparation of a feed that stimulates intestinal peristalsis [112].

Solid-State Fermentation (SSF) has demonstrated significant potential for enhancing the nutritional quality of plant-based animal feed by reducing antinutrients and improving overall nutrient availability. The SSF process effectively lowers the levels of harmful antinutritional factors (ANFs) that can adversely affect animal health and productivity. By utilizing various microorganisms, including filamentous fungi and lactic acid bacteria, SSF not only diminishes these ANFs but also promotes beneficial compounds such as proteins, antioxidants, and vitamins. The research underscores the effectiveness of SSF in both cereals and legumes, showing improvements in nutrient profiles and digestibility. These advancements are crucial for optimizing animal feed, particularly in developing countries where the cost of traditional processing methods can be prohibitive. Future research should continue to explore the integration of local resources and the optimization of SSF processes.

The economic viability of solid-state fermentation (SSF) for smallholder farmers in developing countries can be assessed through various lenses, including the utilization of local agricultural by-products, production cost efficiencies, and other potential benefits. SSF benefits from the use of inexpensive raw materials, such as agricultural by-products, that are abundantly available in rural settings. Studies have shown that by using such materials, not only can production costs be significantly reduced, but also environmental sustainability is enhanced [123]. For instance, the ability of agro-industrial residues to serve as substrates in SSF is highly favorable among smallholders who often have limited access to financial resources. In regions where conventional nutrient sources are scarce or costly, the deployment of SSF could significantly lower the reliance on expensive processed feeds while enhancing the livestock’s uptake of nutrients from fermented agro-residues [124].

3. Conclusions

Solid-state fermentation (SSF) appears to be one of the most promising solutions for smallholder farmers regarding practical farm applications such as providing more nutritious, affordable, and sustainable animal feed. SSF allows farmers to actively reduce antinutritional factors (ANFs) in many locally available feed resources, such as crop residues, cereals, and legumes. Such practical improvements can make these feed materials more digestible and nutritionally valuable to livestock. For example, integration of SSF, on a small to medium scale, might include fermenting readily available agro-industrial by-products (soybean meal and cassava peel, for example) using microorganisms isolated locally. This would provide an economical means of improving feed quality while reducing reliance on expensive commercial feed.

Furthermore, exploring the application of SSF to non-cultivated and even invasive plant species such as Mucuna deeringiana offers a promising direction for expanding the diversity of feed sources. This approach could transform ecological challenges into valuable nutritional resources, particularly in the case of invasive species that represent an ecological problem and exhibit uncontrolled reproductive behavior. Future research should evaluate the broader potential of such species in SSF-based feed strategies.

4. Future Directions

In outlining the trends and new developments, further research activities focus on unlocking SSF methodological improvements, including the isolation of certain microbial strains that target ANFs or modulating protein and amino acid bioavailability. Other prospective innovations that might boost the efficiency and reliability of SSF include establishing newer techniques of microbial engineering and biotechnology (such as CRISPR-based modifications of microbial strains). Other persistent challenges include building more versatile fermentation facilities to accommodate variations in conditions on the farm and training farmers to adopt the SSF methodology effectively. Such lessons must be learned to ensure that SSF remains a more feasible and universally acceptable technique at the farm level toward sustainable livestock production.