Smart and Functional Probiotic Microorganisms: Emerging Roles in Health-Oriented Fermentation

Abstract

The incorporation of probiotic microorganisms into fermented foods has long been recognized as a promising strategy to enhance gut health and overall well-being. Conventional probiotics, mainly from the bacterial genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus and yeast genus Saccharomyces, contribute to gastrointestinal homeostasis, immune modulation, and metabolic balance. Building on these foundations, recent advances in synthetic biology, systems microbiology, and genetic engineering have enabled the development of smart probiotics: engineered or selectively enhanced strains capable of sensing environmental cues and producing targeted bioactive compounds, such as neurotransmitters and anti-inflammatory peptides. These next-generation microorganisms offer precision functionality in food matrices and hold promise for applications in gastrointestinal health, immune support, and gut–brain axis modulation. However, their deployment also raises critical questions regarding biosafety, regulatory approval, and consumer acceptance. This review provides a comprehensive overview of the mechanisms of action, biotechnological strategies, and health-oriented fermentation applications of smart and functional probiotics, emphasizing their role in the future of personalized and evidence-based functional foods.

1. Introduction

The intersection of microbiology and food technology has driven substantial innovation in the development of functional fermented foods [1,2,3,4,5,6,7,8]. Central to this progress are probiotic microorganisms, traditionally defined as “live microorganisms that, when administered in adequate amounts, confer health benefits to the host” [1,2]. These include well-studied strains of bacterial genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus and yeast genus Saccharomyces, which are widely incorporated into fermented products such as yogurt, kefir, and fermented vegetables due to their ability to promote gut health, support the immune system, and enhance nutrient bioavailability [1,2,3,4,5,6,7,8].

Fermented foods, both artisanal and industrial, have long been associated with improved gastrointestinal function and overall well-being. However, the advent of health-oriented fermentation has extended their functionality by deliberately incorporating specific, well-characterized probiotic strains with targeted biological effects [9,10,11,12,13,14,15,16,17]. This shift has led to a clearer distinction between traditionally fermented foods that may contain beneficial microorganisms and functional fermented foods designed to deliver clinically validated health outcomes [1,3,4,5,6,7,8,9].

Building on this foundation, recent advances in synthetic biology, systems biology, and microbial engineering have led to the emergence of a new class of functional microorganisms called smart probiotics. These are next-generation probiotic strains that are either genetically engineered or selectively enhanced to perform highly specific, pre-designed functions in the host. By integrating biosensing and responsive genetic circuits, smart probiotics can detect defined physiological or pathological cues, such as pH fluctuations, inflammatory markers, or metabolite concentrations in the gastrointestinal tract and respond by activating tailored functional outputs [9,10,11,12,13,14,15,16,17]. These outputs may include targeted modulation of host immune responses, production of therapeutic bioactive compounds (e.g., neurotransmitters, antimicrobial peptides, short-chain fatty acids), competitive exclusion of pathogens, or restoration of metabolic balance.

Unlike conventional probiotics, whose effects are often broad and strain-dependent, smart probiotics enable personalized and programmable interventions, offering a level of precision and adaptability unprecedented in probiotic therapy. This opens new opportunities in both preventive and therapeutic contexts, ranging from targeted control of gastrointestinal infections and chronic inflammatory conditions to innovative applications in neuromodulation—such as mood regulation and cognitive enhancement via the gut–brain axis and metabolic health optimization. Furthermore, by coupling engineered safety features (e.g., kill-switch mechanisms, genetic stability safeguards) with regulatory-compliant design, smart probiotics are poised to become a strategic platform for next-generation live biotherapeutics that bridge nutrition, medicine, and precision health [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].

This review explores the emerging roles of both functional and smart probiotic microorganisms in fermentation processes aimed at improving human health. We discuss their mechanisms of action, technological applications, and future perspectives in the design and development of health-oriented fermented foods.

2. Probiotic Microorganisms in Functional Fermentation

2.1. Defining Probiotics and Their Role in Health-Oriented Foods

Probiotics are traditionally defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [1]. Most probiotic strains belong to bacterial genera such as Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus and yeast genus Saccharomyces, which have been extensively studied for their ability to improve gut health, modulate immune function, and enhance nutrient absorption [8,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32].

In the context of fermented foods, these microorganisms can be introduced intentionally to produce health-promoting metabolites or to survive gastrointestinal transit and colonize the gut. Their application in food not only adds probiotic value but also contributes to improving the technological and nutritional properties of the food and increasing its shelf life. By producing a diverse array of enzymes and metabolites, these microorganisms enhance the sensory qualities of food, improving texture, flavor, and aroma, while acting as natural biopreservatives that suppress pathogenic organisms and extend shelf life, reducing the need for chemical preservatives. They also improve nutritional value by increasing nutrient bioavailability, synthesizing essential vitamins, degrading harmful compounds such as phytic acid and mycotoxins, and generating bioactive molecules like peptides, fatty acids, and antioxidants with proven health benefits [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,33,34,35,36].

Bacterial genera such as Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus, and yeast genus Saccharomyces are central to both traditional and modern fermentation, simultaneously contributing to preservation, nutritional enhancement, and the synthesis of bioactive compounds [1,2]. Upon ingestion, these beneficial microorganisms influence gut microbiota composition, promoting intestinal homeostasis and modulating immune responses [3,4]. Host benefits include enhanced digestive efficiency, improved barrier function, and potential psychobiotic effects, which have been increasingly recognized in smart probiotic applications [9,10,11,12,13,14,15,16,17]. This holistic interaction underscores the multifunctional potential of probiotics as both technological agents and bioactive health modulators [1,2,3,4,9,10,11,12,13,14,15,16,17].

2.2. Functional Properties of Conventional Probiotic Strains

Conventional probiotic strains, such as Lacticaseibacillus rhamnosus GG, Bifidobacterium longum, and Lactobacillus acidophilus, have been widely studied for their multifaceted functional roles in promoting host health [3,6,8]. This includes:

• Regulation and stabilization of the intestinal microbiota composition [3,6,8];
• Enhancement of epithelial barrier integrity and tight junction function [2,7];
• Synthesis of antimicrobial compounds such as bacteriocins, hydrogen peroxide, and organic acids, which inhibit pathogenic colonization [4,6];
• Modulation of host immunity through the stimulation of anti-inflammatory cytokine profiles and mucosal immune regulation [3,9,22].

Importantly, these health-promoting effects are strain-specific and influenced by multiple variables including cell viability, dosage, and the microorganism’s resistance to food processing, gastric acidity, and bile salts [1,2,19]. Advancements in fermentation biotechnology and smart delivery systems have significantly improved the incorporation, stabilization, and targeted release of these strains in both dairy and non-dairy fermented matrices. Functional food vehicles such as soy-based yogurts, kombucha, kefir, and kefir-enriched non-alcoholic beers are increasingly being explored for their ability to sustain probiotic viability and enhance delivery to the lower gastrointestinal tract [1,3,4,5,6,22].

Dairy matrices generally support strong survival and functionality of Lacticaseibacillus rhamnosus GG, Bifidobacterium longum, and Lactobacillus acidophilus, while non-dairy options such as soy yogurt and non-alcoholic beer are gaining relevance due to their technological and nutritional benefits, particularly for vegan and lactose-intolerant populations. Within this context, L. rhamnosus GG and L. acidophilus consistently exhibit high survivability across different food vehicles, whereas B. longum demonstrates robust functional activity, especially in lactic-based systems. Taken together, these findings emphasize the critical role of food matrix compatibility in shaping the performance of conventional probiotics and providing a clear transition toward more detailed comparative analyses of strain-specific outcomes in diverse fermentative systems [9,17].

2.3. The Interaction Between Probiotic Microorganisms and Prebiotic Compounds in Fermentation Processes

The concept of synbiotic refers to formulations that combine live microorganisms with specific substrates, typically non-digestible, fermentable dietary components such as inulin, oligofructose, and galactooligosaccharides, that are selectively utilized by beneficial microorganisms to confer health benefits to the host. Originally defined by Gibson and Roberfroid [37], the definition of synbiotic was revised during the 2019 International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus to: “a combination of live microorganisms and substrate(s) selectively utilized by host microorganisms that confer a health benefit on the host” [2].

ISAPP further categorizes synbiotic into two types: complementary, which includes independently established probiotics and prebiotics, and synergistic, which features a substrate specifically targeted to enhance the activity or survival of a co-administered microorganism, not necessarily classified as a conventional probiotic [2,9]. A classic example of a synergistic synbiotic is the pairing of Lactobacillus species with lactose, a disaccharide naturally present in milk and preferentially fermented by these strains [2,9].

To be considered effective, both categories of synbiotic must demonstrate measurable health effects in human hosts. In vitro studies suggest that the administration of synbiotics is more advantageous than the isolated use of probiotics or prebiotics, as the combination can lead to a more robust colonization and a more effective and long-lasting modulation of the gut microbiota. Documented benefits include modulation of gut microbiota composition, stimulation of immune responses, inhibition of pathogenic colonization, and supportive effects on hepatic and gastrointestinal health [3,4,22].

Although some fermented foods may inherently contain both probiotic and prebiotic-like compounds, not all qualify as synbiotic. This is due to processing conditions that may inactivate microorganisms or eliminate fermentable substrates. Therefore, accurate characterization of microbial load and substrate availability is essential to classify a food product as synbiotic [1,5].

Numerous studies have demonstrated the role of dairy and non-dairy fermented products enhanced with prebiotics, such as inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, and β-glucans and selected probiotic strains as effective synbiotic vehicles [1,2,3,4,5,6,7,8,9,36]. For example, yogurt formulations enriched with inulin and Levilactobacillus brevis exhibited resistance to gastric conditions and increased antimicrobial compound production, effectively inhibiting pathogens such as Salmonella typhimurium [38]. Likewise, fermented soy beverages supplemented with fructooligosaccharides (FOS) and Bifidobacterium animalis subsp. lactis showed improved intestinal colonization and short-chain fatty acid (SCFA) production [4,6]. GOS-enriched dairy drinks containing Lacticaseibacillus rhamnosus demonstrated enhanced adhesion to intestinal epithelial cells and immunomodulatory activity [7,8]. Products formulated with resistant starch and yeast Saccharomyces boulardii promoted butyrate production and antioxidant activity [31,33], while β-glucan-enriched kefir fermented with Lactiplantibacillus plantarum exhibited increased cholesterol-lowering potential and bioactive peptide formation [34,36]. Collectively, these findings highlight the versatility of synbiotic formulations and their potential to deliver both technological and health benefits to the consumer.

The scientific evidence accumulated over the past decade consolidates synbiotic foods as a powerful and multifaceted dietary strategy for promoting health. Innovation in this sector has evolved from simple formulations to highly sophisticated combinations, integrating modern encapsulation technologies to improve probiotic delivery and stability.

Encapsulation techniques—such as spray drying, freeze-drying with protective agents, extrusion, emulsion-based encapsulation, coacervation, and innovative nanoencapsulation approaches—have been successfully applied to enhance the survival of probiotics during processing, storage, and gastrointestinal passage [38,39,40]. For instance, microencapsulation of Lacticaseibacillus paracasei using biopolymer-based matrices has been shown to significantly improve their viability under acidic and bile salt conditions. Incorporating prebiotic-rich carriers, such as flaxseed and chia seed powders, not only provides a suitable encapsulation matrix and substrate for the probiotic but also enriches the final product with dietary fiber, polyphenols, and omega-3 fatty acids, enhancing its overall functional value. Additionally, the inclusion of green banana flour (GBF) in fermented milk formulations has demonstrated synergistic effects: protecting probiotic cells, sustaining high viable counts, and improving the biochemical profile through increased production of organic acids and volatile flavor compounds. These strategies exemplify how state-of-the-art encapsulation methods, combined with sustainable and nutrient-dense prebiotic sources, can potentiate the technological performance and health benefits of synbiotic products [38,39,40]. In another study, researchers adopted a “dual enhancement” approach: they enriched the kefir microbiota with additional, well-documented probiotic strains (Lacti. acidophilus LA-5 and B. bifidum BB-11) and added inulin as a prebiotic substrate. The study concluded that the combination resulted in synergistic synbiotic properties and that the type of milk (cow vs. goat) significantly influenced the sensory and physicochemical characteristics of the final product [38,39,40]. In another innovative approach to produce kombucha, researchers replaced traditional sucrose with yacon extract, a natural source of fructooligosaccharides (FOS), as the substrate for fermentation by the SCOBY. The microbial consortium was able to metabolize the FOS, resulting in a low-sugar beverage with clear synbiotic potential. The functionality was demonstrated by successful fermentation and the physicochemical characteristics of the product, with implied benefits for the gut microbiota [38,39,40].

Other innovations include cream cheeses fortified with β-glucan and phytosterol, which support the survival of Lacticaseibacillus rhamnosus during refrigerated storage [41]. Conversely, some prebiotics such as soluble maize fiber and polydextrose, while contributing to texture and taste, may not significantly enhance probiotic viability in all matrices [41]. Moreover, cereals are a promising base for synbiotic food design due to their content of fermentable fibers, proteins, and micronutrients. When fermented with probiotic strains, cereal-based products have shown potential in reducing risks associated with cardiovascular and gastrointestinal diseases [42].

Recent studies on non-conventional food matrices, such as dark chocolate, have demonstrated that synbiotic formulations combining Bacillus indicus HU36 spores with dietary fibers can improve sensory attributes and microbial viability while maintaining overall product quality [43]. Moreover, the synergistic interaction between probiotic microorganisms (e.g., Lactobacillus spp., Bifidobacterium spp.) and fermentable prebiotics (such as inulin, fructooligosaccharides, galactooligosaccharides, and lactose) is particularly noteworthy [37,38,39,40,41,42,43]. During fermentation, probiotics metabolize these substrates, leading to the production of diverse bioactive metabolites, including short-chain fatty acids (SCFAs), exopolysaccharides, vitamins, and antimicrobial peptides. These compounds play a key role in the functional properties of fermented foods, contributing to gut health, immune modulation, and enhanced nutritional quality [37,38,39,40,41,42,43].

2.4. Consumption of Probiotic Fermented Foods and the Regulatory Benefits on the Gut–Brain Axis

The gut–brain axis (GBA) is a complex bidirectional communication network that links the enteric and central nervous systems through neural, endocrine, immune, and metabolic pathways. At the core of this dynamic interaction lies the gut microbiota, whose composition and functionality play a key role in regulating host physiology, emotional states, and cognitive performance [27,44,45,46,47,48,49,50,51,52,53,54,55]. One dietary strategy that has gained attention for modulating this axis is the regular intake of probiotic fermented foods [27,44,45,46,47,48,49,50,51,52].

Fermented foods such as kefir, yogurt, kombucha, and a wide variety of fermented vegetables (e.g., sauerkraut, kimchi, pickled carrots, fermented olives) are natural sources of probiotic microorganisms, including bacterial genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus, and yeast genus Saccharomyces. For example, Lactobacillus spp. are abundant in sauerkraut and kimchi; Bifidobacterium spp. have been detected in fermented soy-based products such as tempeh and miso; Lacticaseibacillus spp. are commonly found in pickled cucumbers and artisanal vegetable brines; Levilactobacillus spp. occur in fermented carrots and beetroot; Lactiplantibacillus spp. are prevalent in olive fermentations and cassava-based products; and Saccharomyces spp., including S. boulardii, are present in kombucha and certain fruit-based fermentations. These beneficial microorganisms help reshape the gut microbiome by increasing microbial diversity, stimulating the growth of commensal bacteria, and reducing the abundance of potential pathogens, thereby supporting gut health and host immunity [27,45,49].

Beyond microbial modulation, these foods also serve as functional vehicles for delivering bioactive compounds that influence host–microbiota–brain interactions. One of the principal mechanisms through which probiotic microorganisms exert regulatory effects on the GBA is via the production of neuroactive metabolites. These include short-chain fatty acids (SCFAs) such as butyrate and propionate, γ-aminobutyric acid (GABA), serotonin, and precursors of dopamine and other biogenic amines. These molecules can cross the intestinal barrier and signal to the brain either through direct vagal activation or by modulating immune and endocrine responses [27,44,46].

Notably, strains like Lacticaseibacillus rhamnosus JB-1 have been shown to alter central GABA receptor expression and reduce anxiety-like behaviors in animal models, an effect dependent on vagus nerve signaling [44]. Experimental and clinical studies further support the psychobiotic potential of fermented foods in improving mood, reducing cortisol levels, and enhancing cognitive flexibility [27,45,49].

Additionally, these probiotics exert anti-inflammatory effects by reducing systemic pro-inflammatory cytokines, reinforcing intestinal epithelial integrity, and preventing gut permeability—factors that otherwise contribute to neuroinflammation and disruption of brain homeostasis [27,44,52,55].

The relevance of the microbiota–gut–brain axis extends beyond general mental health. It is implicated in the pathophysiology of major depressive disorder, neurodevelopmental conditions such as autism spectrum disorders (ASD), and age-related cognitive decline. Alterations in gut microbiota have been documented in individuals with depression and ASD [47,51], while animal studies demonstrate that transplantation of dysbiotic microbiota can transfer behavioral abnormalities to germ-free hosts [49]. Probiotic interventions, often in conjunction with polyphenols or prebiotic substrates, have shown promise in reversing such alterations and improving neurobehavioral outcomes [44,50,52].

Furthermore, aging is associated with shifts in gut microbiota composition and increased vulnerability to neurodegenerative diseases. Fermented foods and dietary polyphenols may attenuate these changes by modulating the microbiota–gut–brain axis, reducing oxidative stress and neuroinflammation, and promoting synaptic plasticity [50,52].

It is important to highlight, however, that not all fermented foods qualify as psychobiotic or synbiotic. Their health-promoting effects depend on multiple factors such as the viability and functionality of specific microbial strains, the fermentation substrate, matrix composition, and dosage. Nonetheless, the inclusion of fermented foods in the daily diet, particularly those combining probiotics with prebiotic substrates (synbiotics), offers a promising strategy to support gut and mental health, enhance emotional resilience, and mitigate risks associated with neuroinflammatory and neurodevelopmental disorders [27,44,45,48].

Fermented foods such as kefir, yogurt, kombucha, and fermented vegetables deliver live probiotic microorganisms (e.g., bacterial genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus, and yeast genus Saccharomyces), which interact with the host microbiota, enhancing microbial diversity and producing neuroactive compounds like GABA, serotonin, and SCFAs. These metabolites influence the central nervous system via the vagus nerve and systemic immune modulation, contributing to improved mental health, reduced inflammation, and better cognitive and emotional resilience [27,44,45,46,47,48,49,50,51,52] (Figure 1).

3. Smart Probiotics: Concepts and Innovations in Fermentation

3.1. From Conventional to Smart Probiotics

Smart probiotics represent a next-generation class of functional microorganisms that extend beyond the traditional benefits of conventional probiotics [9,10,11,12,13,14,15,56,57,58,59,60,61,62]. While classical probiotics, such as those from the genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus, and Saccharomyces, promote gut health, modulate immunity, and improve nutrient bioavailability [63,64,65], smart probiotics are intentionally designed or engineered to perform precise, programmable actions within the host [9,14,15].

These engineered strains are capable of:

• Detecting and dynamically responding to specific environmental cues in the gastrointestinal tract [10,11,60];
• Synthesizing and delivering bioactive molecules such as anti-inflammatory agents, neurotransmitters, short-chain fatty acids, or therapeutic metabolites (e.g., 3-hydroxybutyrate or phenylalanine-degrading enzymes) [10,11,60];
• Modulating host pathways involved in inflammation, metabolism, mood, and immune responses through gut–microbiota–host crosstalk [9,15,59,61].

The development of such probiotic strains leverages advances in synthetic biology, metabolic engineering, genome editing (e.g., CRISPR), and systems microbiology. These tools enable precise control over cellular functions, including enhanced mucosal adhesion, resistance to gastric conditions, targeted molecule delivery, quorum-sensing modulation, and metabolic circuit reprogramming [12,13,56,58].

Several successful examples of smart probiotic systems have already emerged. For instance, engineered E. coli and yeast strains have been developed to sense gut inflammation and release therapeutic compounds in situ [10,60,61]. Additionally, commensal microorganisms have been genetically modified to act as live diagnostics or to mediate colorectal cancer chemoprevention through diet–microorganism interactions [62].

Overall, smart probiotics offer innovative opportunities for disease prevention, targeted therapy, and personalized nutrition strategies. However, their application in food systems and clinical settings still requires careful consideration of biosafety, regulatory frameworks, ecological impact, and host–microorganism compatibility [9,14,59].

While conventional strains such as bacterial genera Lactobacillus, Bifidobacterium, and yeast genus Saccharomyces support gut health via general metabolic activity, smart probiotics are engineered to sense gastrointestinal signals, produce targeted bioactive compounds, and modulate specific host pathways. Their mechanisms of action include mucosal adhesion, quorum sensing modulation, and metabolic reprogramming. Biotechnological strategies such as synthetic biology, genome editing, and metabolic engineering enable the design of these next-generation strains with enhanced therapeutic functions [9,14,59].

Several smart probiotics have already been evaluated and, in some cases, are being used in humans, mainly in clinical trials or as part of functional food formulations. Examples include genetically engineered Escherichia coli Nissle 1917 designed to produce 3-hydroxybutyrate for colitis management [10], Lactococcus lactis Gh1 modified to deliver interleukin-10 in patients with inflammatory bowel disease [39], and recombinant Bifidobacterium longum engineered to express therapeutic peptides targeting metabolic disorders [14,15]. While some strains remain under experimental use, these examples demonstrate the translational potential of smart probiotics in human health applications.

Before smart probiotics can be approved for human use, they must undergo a rigorous multi-phase evaluation process to ensure safety, efficacy, and regulatory compliance. Initially, strains must be well-characterized at the genomic and phenotypic levels to confirm taxonomic identity, assess genetic stability, and exclude transferable antibiotic resistance genes or pathogenicity factors [9,14,15]. Preclinical studies include in vitro assays to evaluate adhesion to intestinal epithelial cells, antimicrobial activity, metabolite production, and immunomodulatory effects, followed by in vivo testing in suitable animal models to assess toxicity, biodistribution, and functional outcomes [14,23,59]. Regulatory agencies such as the European Food Safety Authority (EFSA) and the U.S. Food and Drug Administration (FDA) require compliance with Good Manufacturing Practices (GMP), documentation of the Qualified Presumption of Safety (QPS) or Generally Recognized as Safe (GRAS) status, and detailed reports on stability and shelf-life [2,19]. For genetically engineered strains, additional biosafety assessments are necessary, including evaluation of horizontal gene transfer potential and containment strategies in accordance with WHO and national biosafety guidelines [10,11,60,61,62]. Human trials proceed through sequential phases: Phase I to confirm safety and tolerability in healthy volunteers, Phase II to explore dose–response and preliminary efficacy in target populations, and Phase III to validate clinical effectiveness and monitor long-term effects [15,19,61]. Only after successful completion of these steps can smart probiotics be considered for commercialization and widespread application in human health [9,14,15,19].

3.2. Design and Engineering of Smart Probiotic Strains: Functional Outcomes and Health Applications

The development of smart probiotics integrates genetic engineering, synthetic biology, and microbial biotechnology to enhance probiotic performance and enable targeted therapeutic functions [10,11,59]. Several strategic approaches have been explored to construct these advanced strains:

• Gene insertion technologies, enabling the expression of functional proteins such as neurotransmitter-synthesizing enzymes (e.g., glutamate decarboxylase for GABA or tryptophan hydroxylase for serotonin production) [10,11,59];
• Synthetic biosensors, which detect host-derived signals (e.g., pH shifts, inflammatory cytokines, or metabolic by-products) and activate specific genetic circuits in response [9,61];
• Bioencapsulation and delivery systems, designed to protect engineered strains from gastric degradation and ensure precise release at target intestinal sites [9,14].

Commonly used microbial chassis include bacterial genera Lactobacillus, Bifidobacterium, Lacticaseibacillus, Levilactobacillus, Lactiplantibacillus, yeast genus Saccharomyces, and non-pathogenic strains of Escherichia coli such as JM109, selected for their genetic accessibility and established safety profiles [10,56,59]. The functional applications of smart probiotics are expanding rapidly, with key areas of interest including:

• Gut–brain axis modulation: Engineered strains have been developed to produce neuroactive molecules such as GABA and serotonin, showing anxiolytic and antidepressant potential in preclinical models [10,11,60];
• Management of inflammatory bowel diseases (IBD): Smart probiotics expressing anti-inflammatory peptides or immunomodulatory proteins have shown efficacy in reducing intestinal inflammation and reestablishing microbial balance [14,60];
• Enhancement of nutrient bioavailability and detoxification: Some strains express enzymes that degrade antinutritional factors or sequester toxins, contributing to better nutrient absorption and gut protection [14,58].

Recombinant DNA technology is widely used in the construction of these smart probiotics. Typically, a gene of interest is cloned into a plasmid vector (e.g., pGEM-T, Promega) and introduced into supercompetent bacterial cells (e.g., E. coli JM109) via electroporation [31]. This heterologous gene can drive the production of therapeutic proteins or metabolites [56,57] (Figure 2).

In addition to electroporation, horizontal gene transfer methods such as conjugation and transduction offer alternative means to construct smart strains. In conjugation, a plasmid containing functional genes is transferred through a conjugative pilus, while in transduction, bacteriophages mediate gene delivery [12,62] (Figure 3).

Smart probiotics are being positioned as living diagnostics and therapeutics, particularly for gut-related disorders. For instance, engineered E. coli strains have been developed to produce 3-hydroxybutyrate for treating colitis [10] or to function as inflammation-responsive biosensors in the gut [61]. Engineered yeast systems have also been shown to tune therapeutic responses in inflammatory models [60].

Nonetheless, while results in preclinical models (e.g., murine colitis) are promising, translation to human systems remains complex. The gut microbiota of rodents significantly differs from that of humans, which may impact colonization, gene stability, and probiotic efficacy. Additionally, safety and regulatory frameworks surrounding genetically modified probiotics need to be clarified to facilitate clinical adoption [14,59].

Despite these challenges, smart probiotics represent a promising frontier in nutritional medicine, with the potential to provide personalized, efficient, and natural therapies for complex diseases such as inflammatory bowel diseases (IBD) [9,14,15,60].

3.3. Smart Probiotic Strains Applied to Fermentation Processes and the Preparation of Fermented Foods

The integration of smart probiotic strains into fermentation processes represents a novel approach to enhance the nutritional and functional properties of fermented foods. Unlike conventional probiotics, smart probiotics are engineered or selectively enhanced to exhibit specific responses to environmental stimuli, produce bioactive compounds, and modulate host pathways, features that extend their application from therapeutic tools to agents of precision fermentation [1,9,14].

Smart probiotics are being increasingly incorporated into food fermentation systems to optimize microbial performance, modulate metabolic outputs, and deliver health benefits. These strains can be programmed to produce target metabolites such as neurotransmitters, short-chain fatty acids (SCFAs), bioactive peptides, or antioxidants during fermentation. Their inclusion improves not only the microbial ecology and sensory profile of fermented products but also contributes to immune modulation and gut–brain axis interaction [2,3,4,5,45].

3.3.1. Applications in Fermentation Processes

In traditional and industrial fermentations, smart probiotics enhance microbial process control and product consistency. Recent studies have shown that strains of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus can be genetically modified to synthesize bioactive metabolites directly in the food matrix, such as GABA and serotonin precursors, with potential psychobiotic effects [5,17,28,30]. These psychobiotics can be used to develop next-generation fermented foods aimed at mental wellness, aligning with the growing demand for mood-enhancing functional foods [3,14,45].

In non-dairy matrices, smart strains have also been used to ferment substrates such as fruit pulps, cereal blends, and cocoa by-products, promoting the development of novel probiotic beverages and synbiotic foods with improved shelf life, flavor, and antioxidant activity [1,5,35,38]. Notably, microencapsulation technologies and co-fermentation with prebiotics have been employed to protect the viability and functionality of smart strains during fermentation and storage [1,39,40].

The fermentation industry faces several challenges that can limit product quality, safety, and functional efficacy, including variability in microbial performance due to raw material heterogeneity, contamination risks during large-scale production, loss of probiotic viability during processing and storage, and inconsistent functional outcomes in the final product [1,2,9,14]. Conventional starter cultures often lack the capacity to adapt rapidly to fluctuating processing conditions or to deliver targeted health benefits with high reproducibility. Smart probiotics offer a promising solution to overcome these gaps by serving as precision-designed inoculants for fermented foods. Through synthetic biology and advanced strain engineering, these next-generation cultures can be programmed to sense environmental cues during fermentation, such as pH changes, oxygen levels, or substrate availability, and modulate their metabolic activity accordingly, ensuring consistent flavor development, enhanced nutrient bioavailability, and production of bioactive compounds [9,10,11,12,13,14,15,16,17,59,60,61,62]. In addition, engineered stress tolerance mechanisms and modern encapsulation technologies can improve their resilience to industrial-scale processing and storage, reducing viability losses and extending shelf life. By integrating smart probiotics into starter culture systems, the fermentation industry can move toward more robust, predictable, and multifunctional production processes, bridging the gap between traditional fermentation practices and precision-driven functional food manufacturing.

3.3.2. Functional Food Development and Health-Promoting Potential

Smart probiotics embedded in fermented products can act as live biotherapeutic agents, delivering bioactives that impact host metabolism, immunity, and neuroendocrine balance [3,7,22]. Their functional outcomes include:

• Blood pressure regulation, as seen in fermented milk enriched with ACE-inhibitory peptides [6];
• Modulation of gut microbiota composition and immune function, improving intestinal barrier integrity [3,7,22];
• Cognitive and emotional support, through modulation of the gut–brain axis via GABA and SCFA production [17,27,33].
• Moreover, in situ biosynthesis of health-promoting molecules during fermentation reduces the need for external supplementation, making these foods cost-effective and sustainable delivery platforms [1,4,47].

The integration of smart probiotic strains into fermentation processes enables the production of bioactive compounds during food processing, enhancing sensory quality, microbial safety, and health benefits. Functional metabolites such as GABA, SCFAs, and antioxidant peptides contribute to gut health, immune modulation, and gut–brain axis regulation [17,27,33].

Despite these advances, the implementation of smart probiotics in food systems faces challenges related to strain stability, regulatory approval, and consumer perception. Concerns regarding the use of genetically engineered microorganisms (GEMs) in food require clear safety assessments, strain traceability, and transparent labeling practices [9,14,19].

Nonetheless, with advances in synthetic biology, omics technologies, and bioencapsulation, smart probiotics are poised to transform fermented food systems into platforms for personalized nutrition and preventive health. Their ability to act at the interface between food, microbiota, and host physiology underscores their potential as cornerstones of the next generation of functional fermented foods [2,3,14,19,45].

4. Perspectives and Future Directions

4.1. Toward Personalized and Precision Fermentation

As research on smart probiotics evolves, the concept of precision fermentation is gaining traction. This approach integrates individual health profiles, gut microbiota composition, and nutritional needs to develop customized fermented foods with targeted health effects. The use of next-generation probiotics in personalized dietary strategies offers the potential to transform fermented foods into tools for preventive and therapeutic nutrition [1,14,15,17].

Technological advances such as multi-omics platforms (e.g., metagenomics, metabolomics, proteomics) and AI-driven bioinformatics enable prediction of host–microorganism and microorganism–microorganism interactions [2,9,14]; optimization of fermentation parameters for maximal functional metabolite production [1,3,47]; and personalization of microbial consortia to address specific clinical or physiological needs [14,19,28].

Such precision tools pave the way for microbiome-responsive foods tailored to modulate the gut–brain axis, immune system, or metabolic pathways, in line with emerging concepts of psychobiotic and nutribiotic nutrition [17,27,44,45].

4.2. Research Gaps and Clinical Validation Needs

Despite strong preclinical evidence, several research gaps hinder the clinical translation of smart probiotics, including lack of standardized safety and efficacy protocols for genetically modified or engineered strains [10,11,14]; scarcity of strain-specific clinical trials across diverse populations and life stages [18,22]; and limited regulatory frameworks to evaluate food-grade genetically engineered microorganisms (GEMs) [9,19].

To move forward, the field urgently requires longitudinal and multicenter clinical studies to assess functional outcomes, host compatibility, and long-term safety [14,19]; harmonized labeling regulations that clearly inform consumers while avoiding stigmatization of biotechnological innovations [6,19]; and ethical assessments on the use of synthetic biology in food applications, especially regarding environmental release and horizontal gene transfer [12,59].

4.3. Industrial and Regulatory Challenges

From an industrial standpoint, smart probiotics must overcome several production and commercialization barriers, including maintaining cell viability and functional expression during food processing and shelf-life [1,3]; addressing consumer skepticism related to genetically modified organisms (GMOs) and engineered strains in foods [19,22]; and scaling up cost-effective fermentation platforms that meet safety, traceability, and regulatory demands [6,9,14].

Innovative solutions include:

• The use of non-GMO strategies, such as adaptive laboratory evolution or CRISPR-based self-limiting expression systems, which circumvent regulatory hurdles [16,59];
• Bioreactor-based precision fermentation systems enabling scalable, reproducible production of probiotic-rich matrices enriched with health-promoting compounds [1,6,14].

These tools are critical to translating lab-scale benefits into market-ready functional foods that meet global health needs.

Furthermore, cold sterilization strategies are increasingly important in the production of probiotic and synbiotic foods, as they ensure microbial safety without exposing products to high temperatures that compromise probiotic viability. Unlike thermal pasteurization, these methods preserve the cell integrity and functional activity of heat-sensitive strains. Common cold sterilization techniques include microfiltration and ultrafiltration, which physically remove contaminating microorganisms while retaining the probiotics of interest; high hydrostatic pressure (HHP), which inactivates spoilage and pathogenic microorganisms at ambient or low temperatures; ultraviolet-C (UV-C) irradiation, effective for surface decontamination and transparent liquids; and pulsed electric fields (PEFs), which disrupt microbial membranes without significant heating [1,36,59]. In the context of smart probiotics, cold sterilization can be strategically applied to selectively control background microbiota in the food matrix before inoculation, thereby ensuring the dominance and functional expression of engineered strains during fermentation and storage. Such approaches enhance both the safety and the functional performance of probiotic-containing foods while aligning with clean-label and minimally processed product trends.

4.4. Biosafety, Regulatory Approval, and Consumer Acceptance of Smart Probiotics

The introduction of smart probiotic, engineered microbial strains with targeted functional or therapeutic properties demands careful evaluation beyond efficacy, encompassing biosafety, regulatory compliance, and societal acceptance.

• Biosafety remains a primary consideration, as genetic modifications may alter microbial metabolism, host interactions, and ecological persistence. Potential risks include unintended metabolic pathways, horizontal gene transfer, and disruption of native gut microbiota [12]. Comprehensive risk assessment should be integrated in in vitro and in vivo studies, focusing on genomic stability, metabolic profiling, and long-term host safety [9]. Advances in synthetic biology and metabolic engineering have enabled the development of controlled genetic circuits and self-limiting systems to minimize environmental and host-related risks [14,15,59].
• Regulatory approval pathways for smart probiotics are evolving and vary across jurisdictions. In the United States, the Food and Drug Administration (FDA) typically regulates these products under the live biotherapeutic products (LBP) framework, requiring pre-market evidence of safety, stability, and efficacy [16]. In the European Union, the European Food Safety Authority (EFSA) applies the Qualified Presumption of Safety (QPS) approach in conjunction with novel food regulations [23]. Products intended for medical applications face stricter clinical validation requirements, as demonstrated by engineered yeast probiotics for inflammatory bowel disease undergoing rigorous preclinical and clinical testing [60].
• Consumer acceptance is equally critical for market adoption. Public perception may be influenced by awareness of genetic engineering, trust in regulatory oversight, and a clear understanding of health benefits versus perceived risks [19]. Transparent labeling, accessible scientific communication, and educational outreach can improve acceptance, particularly when innovation aligns with sensory quality and cultural preferences [1,3]. Integrating consumer feedback early in the development process can guide both product formulation and dissemination strategies.
• Addressing biosafety, regulatory, and consumer dimensions from the earliest stages of research ensures not only scientific advancement but also ethical responsibility and societal readiness for the integration of smart probiotics into health-oriented foods and therapeutics.

4.5. The Future of Smart Probiotics

Smart and functional probiotic microorganisms represent a paradigm shift in health-oriented fermentation. Their engineered capacity to synthesize bioactive compounds, modulate host metabolic and immunological pathways, and sense and respond to environmental signals extends their application well beyond traditional nutritional supplementation. These microorganisms are increasingly recognized as potential live biotherapeutic agents that can be incorporated into diverse food matrices, thereby uniting food technology with medical innovation [9,14,17,66,67,68,69,70].

To unlock their full potential, the development and integration of smart probiotics require interdisciplinary collaboration across food science, microbiology, systems biology, synthetic biology, and regulatory science. Advances in omics technologies and computational modeling are providing new tools to optimize strain design, predict host–microbe interactions, and ensure safety and efficacy. Moreover, scalable fermentation platforms and novel encapsulation or delivery systems are crucial to maintain cell viability and functionality from production to consumption [66,67,68,69,70].

As clinical validation progresses and evidence of safety and efficacy accumulates, smart probiotics are expected to move closer to regulatory approval and consumer acceptance. These innovations hold the promise of personalized nutrition solutions, tailored to individual microbiome profiles and health conditions, while also addressing global challenges such as antibiotic resistance, metabolic disorders, and neuroimmune diseases [66,67,68,69,70].

Ultimately, engineered strains have the potential to transform the future of fermented foods, establishing a bridge between traditional dietary practices and next-generation precision health strategies. This convergence of nutrition, biotechnology, and microbial engineering sets the stage for a new era of functional foods that not only support general well-being but also deliver targeted therapeutic benefits [1,2,5,14,17,19,66,67,68,69,70].

5. Conclusions and Limitations of This Study

The integration of functional and smart probiotic microorganisms into fermentation processes marks a new frontier in the development of health-oriented foods. While conventional probiotics have long been recognized for their benefits in gut health and food preservation, smart probiotics offer the potential for precision interventions, enabled by engineered responsiveness to host and environmental signals. These next-generation strains can produce targeted bioactive metabolites, dynamically modulate host physiology, and pave the way for personalized nutrition and live biotherapeutic applications.

Their incorporation into fermented food systems can be further optimized by adopting cold sterilization strategies, such as microfiltration, high hydrostatic pressure, pulsed electric fields, and ultraviolet-C irradiation, to ensure product safety without compromising probiotic viability. Such approaches are particularly relevant for “Smart Probiotics”, where maintaining cell integrity and engineered functionality is critical for achieving the intended health benefits.

Nonetheless, the successful deployment of smart probiotics depends on interdisciplinary collaboration to address challenges related to biosafety, regulatory compliance, technological scalability, and consumer acceptance. Limitations of the present study include the lack of experimental validation for specific encapsulation and cold sterilization methods with smart probiotic strains and the need for in vivo trials to confirm strain-specific health outcomes. Future research should also investigate large-scale production feasibility and cost-effectiveness to facilitate industrial adoption.

By uniting advances in synthetic biology, systems microbiology, and food innovation, smart probiotics have the potential to transform functional foods from generalized wellness products into targeted, evidence-based solutions for promoting human health and resilience. This integrative potential, bridging microbiome science, food innovation, and biotechnology is illustrated in Figure 4, which summarizes their evolutionary progression, functional applications in precision nutrition, and the associated scientific and regulatory challenges.