Action and immunomodulatory mechanisms, formulations, and safety concerns of probiotics

Abstract

The global probiotics market has been continuously growing, driven by consumer demand for immune-enhancing functional foods, dietary supplements, and natural therapeutics for gastrointestinal and gut function-mediated diseases. Probiotic microorganisms represent a diverse group of strains with complex but generalized mechanistic patterns. This review describes the various immunomodulatory mechanisms by which probiotics exert their effects, including the competitive exclusion of pathogenic microbes, production of antimicrobial substances, modulation of the immune system, and improvement of gut barrier function. In addition, the various formulations and methods of delivery of probiotics and the safety concerns associated with these products are also discussed.

INTRODUCTION TO PROBIOTICS

The concept of probiotics is rooted in the idea of promoting a balance of microbial communities in the human body, particularly in the gastrointestinal tract. The Food and Agriculture Organization/World Health Organization (FAO/WHO) defines probiotics as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [1,2,3]. These microbes can stay viable for extended periods of storage and survive under intestinal conditions [4]. The most common probiotic strains are bacteria and yeasts belonging to the genera Lactobacillus, Bifidobacterium, Enterococcus, and Saccharomyces [2, 3, 5, 6]. In the last few decades, the impact of these microorganisms on human wellness has become a focal point of research leading to increased interest in probiotics from both consumers and industry [7, 8]. Recent estimates indicate that the probiotic market was valued at 68.56 billion USD in 2022, with projections suggesting a significant increase to 133.92 billion USD by 2030 [9].

Probiotics can exert their effects through various mechanisms, such as competitive exclusion of pathogenic microbes, production of antimicrobial substances, modulation of the immune system, enhancement of the gut barrier function [10], and synthesis of nutrients (e.g., amino acids, enzymes, vitamins, and carbohydrates) [11]. Consequently, they have been associated with several health benefits, including the prevention and management of diarrhea, relief from symptoms of irritable bowel syndrome (IBS), modulation of the immune system, and potential roles in metabolic health [3, 12]. One of the most important sources of these microbes is fermented foods [13]. Probiotics are ideal for food manufacturing because of their pH and heat stability, as well as their favorable sensory characteristics, such as being colorless, tasteless, and odorless [14]. The best matrices for probiotic delivery are cultured dairy foods. Specifically, approximately 20% of fermented dairy products in Europe contained probiotics in the early 21st century, and their market has been rapidly expanding ever since. However, the selection of non-dairy-based probiotic products (e.g., fruit juices, kimchi, sauerkraut, and tempeh) has also expanded [15, 16], offering alternatives for individuals with dietary restrictions and preferences [17]. Additionally, probiotic diet supplements in the form of capsules, tablets, or powders are also available on the market [11].

Although there has been a substantial body of evidence to support the beneficial effects of probiotics, research in this area has faced challenges, such as strain-specific effects, variability in individual response, and the need for well-designed clinical trials. Furthermore, the effects of probiotics can vary widely depending on the specific strain, dosage, and individual factors. Thus, the present review aims to provide some perspectives on the mechanisms of action, formulation, and delivery of probiotics. While these products are generally accepted as safe, the concerns raised by growing numbers of consumers are also discussed.

ROLE OF PROBIOTIC MICROORGANISMS IN ENHANCING HUMAN WELL-BEING AND GUT HEALTH

Bacteria and yeasts as probiotics

Probiotic organisms are capable of enhancing human well-being by improving gut homeostasis and reducing various metabolic disorders [11, 18]. Most probiotics are lactic acid bacteria (LAB), including Lactobacillus, Streptococcus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Oenococcus, and Weissella strains [19]. These gram-positive, non-spore-forming microorganisms have been utilized widely in food production [20], especially for fermentation processes that generate lactic acid through homo- and heterofermentative pathways [19]. For example, Lactobacillus acidophilus, a well-known probiotic naturally found in the human intestinal tract, is often used in dairy foods and dietary supplements. Previous studies have suggested its potential benefits in supporting gastrointestinal health by increasing lactase activity and producing bioactive compounds to inhibit the growth of pathogenic bacteria and viruses [3]. Additionally, Lacticaseibacillus rhamnosus is one of the most thoroughly studied probiotic organisms and renowned for its effectiveness in both preventing and treating various gastrointestinal conditions [7]. Lacticaseibacillus casei and Lactiplantibacillus plantarum are versatile probiotic microorganisms found in fermented foods and dietary supplements. They have been investigated for their potential health benefits, including antioxidant, anti-inflammatory, immunomodulatory, and gut health promotion effects [21,22,23].

Similar to LAB, bifidobacteria are naturally occurring commensal bacteria in the small and large intestines that play a protective role for the host by producing bacteriocins and engaging in competitive exclusion to fend off pathogens [24]. Bifidobacterium bifidum has been studied for its potential to improve gut health and support the immune system. In addition, Bifidobacterium breve, commonly used in probiotic supplements and infant formulas, has been investigated for its role in promoting infant gut health [25,26,27]. Moreover, promising probiotic strains of other bacteria (e.g., Bacillus spp., Clostridium butyricum, or Escherichia coli) have also been investigated [19, 28].

As for yeasts, they have been utilized in the food industry for their role in various fermentation processes, but little research has been done on their probiotic activities. The relatively larger size of yeast cells, in comparison to bacteria, limits their extensive use. However, they are generally resistant to antibiotics and do not contribute to the dissemination of antibiotic resistance genes. In addition, their translocation has not been previously reported [29]. Saccharomyces boulardii is commonly used to prevent and treat diarrhea resulting from antibiotic use or gastrointestinal infections, due to its resilience in antibiotic environments [30, 31]. This yeast is sold as a probiotic in many countries because it can survive transit through the intestinal tract, grows well at 37°C, and exhibits antagonistic activity against pathogens [29].

Mechanisms of action of probiotic microorganisms

Although allochthonous probiotic microbes may have beneficial effects on host health, the mechanisms through which they permanently colonize the gut are varied, complex, and frequently unique to each strain [12, 23]. Figure 1 illustrates the currently understood mechanisms of action of probiotics. These include interactions with the host’s microbiota, modulation of the immune system, and various direct or indirect effects on host physiology [32]. However, the existing resident microbial communities can shape the effectiveness of probiotic colonization by affecting resource availability and occupying ecological niches or through direct or indirect interactions with the probiotics, such as competition, facilitation, and predation among other processes [33].

Fig. 1.

Current understanding of the mechanisms of action exhibited by probiotic organisms.

GABA: gamma-aminobutyric acid, γ-aminobutyric acid.

Microbial competition and colonization

Probiotic organisms can contribute to a balanced and healthy gastrointestinal microbiota by preventing pathogen colonization of the epithelium through microbial competition and colonization. Nonetheless, the antagonistic effects of probiotic strains are recognized to be multifactorial. For instance, certain Lactobacillus and Bifidobacterium species can prevent the proliferation and overgrowth of pathogenic microorganisms through competition for essential nutrients and adhesion sites in the gastrointestinal tract [32, 34]. Various probiotic microbes have specific surface structures or adhesins enabling them to adhere to the intestinal epithelial cells lining the gut. By adhering to these cells, probiotics can physically block the attachment of pathogenic bacteria, limiting their ability to establish infections. The maintenance of intestinal epithelial barriers by probiotic organisms is crucial for normal physiological functions or mitigating the onset of specific intestinal conditions such as hyper-acidity and diseases like obesity, inflammatory bowel diseases, and irritable bowel syndrome [1, 35]. Previous studies have demonstrated that probiotic strains of L. plantarum, Lacticaseibacillus paracasei [36], S. boulardii, Saccharomyces cerevisiae, and Pichia kluyveri [37] can diminish the adhesion of various foodborne pathogens, such as enteropathogenic E. coli and Salmonella Enteritidis, under in vitro conditions. Additionally, pre-treatment with probiotics has been found to serve a prophylactic role in managing Helicobacter pylori infections [38]. Probiotics also protect against enteric pathogens by reinforcing the intestinal mucosa, enhancing intestinal motility, and boosting mucous production [39].

Antimicrobial metabolites

Probiotics can also prevent gastrointestinal infections by secreting antimicrobial metabolites, such as organic acids (mainly lactic and acetic acids), hydrogen peroxide, and bacteriocins [40, 41]. These compounds regulate the microbiota by fostering conditions that are hostile to pathogens, thereby impeding their growth and survival. For instance, the acidifying action of probiotic strains can produce conditions unsuitable for the proliferation of specific pathogenic bacteria that prefer a more neutral pH environment [1]. The intestinal protective effect of S. boulardii may be facilitated by a 54-kDa serine protease, which has been observed to cleave Clostridioides difficile toxin A and exhibit enzymatic activity against toxin B. In addition, it diminishes the ability of toxins A and B to bind to the human colonic brush border membrane, thereby inhibiting the detrimental effects of these toxins on colonic epithelial cells and the native human colonic mucosa [42]. Rao et al. [43] noted that the ability of Bacillus licheniformis to detoxify aflatoxin B1 could be linked to its extracellular proteins or enzymes.

Bacteriocins, which are peptides and peptide complexes synthesized by ribosomes, are recognized for their ability to inhibit (bacteriostatic) or kill (bactericidal) bacteria, usually those closely related to the producer organism (exhibiting a narrow spectrum of activity). However, there have been reports of bacteriocins that have a broad spectrum of activity and target a wider range of bacterial strains. These molecules may exert their effects at low concentrations, and due to their proteinaceous nature, they are degraded by proteolytic enzymes in the mammalian gastrointestinal tract. Consequently, pathogenic microorganisms are unable to develop resistance against them in the gut [44,45,46]. Most bacteriocins are primarily utilized as natural food preservatives; however, their application in the pharmaceutical industry as immune-modulating agents and clinical antimicrobials has also garnered attention [45, 47]. The production of bacteriocins can play a crucial role in the gut ecosystem by allowing the producing microbes to compete, providing the host defense against pathogens through their action as antimicrobial peptides and serving as signaling molecules [48]. Bu et al. [49] demonstrated that bacteriocin-producing L. plantarum inhibited the growth of Listeria monocytogenes, E. coli, S. Typhimurium, Shigella sonnei, and Staphylococcus aureus. Sharma et al. [50] evaluated the antibacterial properties of bacteriocins sourced from potential probiotic bacteria. In vitro tests revealed that crude, partially purified, and fully purified bacteriocins all exhibited antagonistic effects against Bacillus cereus and S. aureus, producing inhibition zones ranging from 10 to 22 mm and 10 to 21 mm, respectively. As a class I lantibiotic, nisin has shown significant effectiveness in treating a broad spectrum of infectious diseases [51]. Likewise, plantacyclin B21AG, synthesized by L. plantarum B21, is a food-grade circular bacteriocin with potential activity against food-borne pathogens and spoilage bacteria [45]. Pediocin GS4, produced by Pediococcus pentosaceus, has been effectively utilized to suppress S. aureus, L. monocytogenes, E. coli, and Pseudomonas aeruginosa [52]. The crude bacteriocin from Enterococcus hirae LD3, which possesses potential probiotic properties, has been found to reduce the growth of a wide array of food-borne pathogens, such as E. coli O157:H7, S. aureus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella Typhi, L. monocytogenes, Shigella flexneri, and Vibrio sp. [53].

Biofilm formation

Furthermore, probiotics can form biofilms, which not only help them withstand harsh environmental conditions but also allow them to adhere to gut surfaces, block pathogenic bacteria from attaching, and serve as media for exchanging beneficial molecules. A crucial aspect of these biofilms is their extracellular polysaccharide matrix, which safeguards the probiotic organisms against enzymatic and antibiotic actions [39, 54]. However, biofilm formation is influenced by a variety of factors and environmental stressors, such as microbial species, temperature, pH, and nutrient availability. Research has been conducted on both potential probiotic bacteria and yeasts for the development of protective biofilms [39, 55].

Immunomodulatory effect

To advance research in the field, Ashaolu and Fernandez-Tome [56] summarized the major action mechanisms of probiotics. These include 1) cellular and humoral immune modulation characterized by increased antibody production against pathogens [57]; 2) interplay with dendritic cells, regulation of phagocytic activities, and upregulation of cytokines against colonic inflammation [58]; 3) establishment of resistance against colonization by resident or invasive pathogens via tissue dominance [59]; 4) interaction with surface molecules such as pili, exopolysaccharides, mucin-binding proteins, and LPxTG-motif-binding proteins to enhance intestinal health [13]; 5) microbiota-mediated processes such as antagonism, support, or competitive exclusion regarding nutrients to maintain homeostasis [13, 60, 61]; and 6) production of organic acids through carbohydrate fermentation in the gut, which lowers the luminal pH and inhibits the growth of pathogens [41, 62, 63]. Drawing on Ashaolu’s research [1], Table 1 summarizes these immune-boosting actions of probiotics as examined in various studies.

Table 1. Immune-enhancement mechanisms of some reported probiotics [1].

Probiotic organism	Functions	Mechanisms	Reference
Lactiplantibacillus plantarum MON03	Toxin detoxification	Binding via surface structures	Jebali et al. [156]
Lentilactobacillus kefiri KFLM3, Saccharomyces cerevisiae KFGY7, Acetobacter syzygii KFGM1	Toxin detoxification	Adsorption and biotransformation	Taheur et al. [157]
Lactobacillus helveticus ATCC 12046	Toxin detoxification	Binding via surface structures	Ismail et al. [158]
Bacillus licheniformis CFR1	Toxin detoxification	Enzymic degradation	Rao et al. [43]
Saccharomyces cerevisiae HR 125a	Toxin detoxification	Binding via surface structures	Ismail et al. [158]
Lactococcus lactis JF 3102, Lactiplantibacillus plantarum NRRL B-4496	Toxin detoxification	Binding via surface structures	Ismail et al. [158]
Streptomyces cacaoi subsp. asoensis K234, Streptomyces luteogriseus K144, Streptomyces rimosus K145	Toxin detoxification	Enzymic degradation	Harkai et al. [159]
Several other Lactobacillus strains	IgA secretion	Stimulation of dendritic cells to produce IL-6	Kikuchi et al. [69]
	IgA secretion	Stimulation of dendritic cells to produce TGF-β	Sakai et al. [160]
	Enhancement of natural killer (NK) cell activity	Stimulation of IL-12 secretion	Takeda et al. [68]
	Inhibition of Th2 activity	Stimulation of IL-12 secretion	Fujiwara et al. [161]
	Inhibition of Th2 activity	Stimulation of activated T-cell death	Kanzato et al. [162]
	Improvement of oral tolerance	Induction of Tregs	Aoki-Yoshida et al. [163]
	Reduced inflammation	Reduced pro-inflammatory cytokines and chemokines bydown-regulation of TLR-signals	Shimazu et al. [164]
	Toxin detoxification	Binding via surface structures	Chlebicz and Śliżewska [165]
Several other Bifidobacterium strains	Reduced inflammation	Induction of IFN-β	Kawashima et al. [166]
	IgA production	Upregulation of pIgR expression	Nakamura et al. [167]
	Modulation of anti-viral activity	Reduced A20 and improved IRF-3, IFN-β, MxA, and RNase L	Ishizuka et al. [168]
	Protection from enteropathogenic infection	Production of acetate and enhancement of intestinal defense with epithelial cells	Fukuda et al. [169]
	Allergy inhibition	Suppression of Th2 chemokines	Iwabuchi et al. [170]
	Reduced inflammation	Inhibition of IL-17	Miyauchi et al. [171]

Probiotics also modulate the host’s immune response by promoting the activation of immune cells involved in defense against pathogens, such as macrophages, dendritic cells, and lymphocytes [64]. In particular, probiotic organisms possess the ability to activate pattern recognition receptors (PRRs) found on both non-immune and immune cells [65]. The surface molecules of probiotics, including pili, flagella, surface layer proteins, lipoteichoic acid, capsular polysaccharide, and lipopolysaccharide, can specifically bind to PRRs, regulating various signaling pathways. These pathways include the mitogen-activated protein kinases, nuclear factor kappa B, and peroxisome proliferator-activated receptor gamma [66]. PRRs, including Toll-like receptors (TLR), nucleotide oligomerization domain-like receptors, retinoic-inducible gene-I-like receptors, and C-type lectin receptors, recognize pathogen-associated molecular patterns and trigger signaling pathways for the expression of various genes and production of immune mediators [67]. This immunomodulatory effect can contribute to the elimination of harmful bacteria in the gut. Takeda et al. [68] demonstrated that regular consumption of fermented milk containing L. casei Shirota enhanced natural killer cell activity and stimulated the secretion of interleukin (IL)-12. L. plantarum AYA was shown to increase IL-6 production in Peyer’s patch dendritic cells. Moreover, oral administration of this bacterium led to a rise in IgA production in both the small intestine and lungs of mice [69]. This contributes to the induction of immune tolerance and competence, as noted by Feleszko et al. [70]. Kitazawa et al. [71] reported that DNA motifs and cell wall components from immunobiotic LAB could facilitate the immunoactivation of gut-associated lymphoid tissues, particularly where TLR2 and TLR9 are abundantly expressed. Furthermore, probiotics can mitigate gut inflammation by modulating the production of inflammatory cytokines and promoting the synthesis of anti-inflammatory substances [56, 72]. Wang et al. [73] observed that a soluble factor produced by Bifidobacterium animalis subsp. lactis BB12 suppressed the TNF-α-induced production of IL-8 in Caco-2 cells, showcasing its anti-inflammatory potential.

Probiotics can also stimulate the production of mucin, a glycoprotein that forms a protective layer on the surface of intestinal epithelial cells and reinforces tight junctions among intestinal cells, thereby decreasing the permeability of the intestinal barrier [74, 75]. The resulting mucin layer thus acts as a physical shield, efficiently blocking the adhesion and invasion of pathogens. Additionally, certain probiotics can influence the host’s metabolism, such as the metabolism of dietary components and the synthesis of short-chain fatty acids. These activities may exert widespread effects on the body’s energy metabolism [76, 77]. Through fostering a favorable microbial community, probiotics contribute to maintaining a balanced and healthy gut microbiota, which is essential for digestive health and immune function. However, it is important to recognize the variability in probiotic mechanisms of action. For instance, specific strains have been identified to produce a range of neurochemicals, including dopamine, serotonin, γ-aminobutyric acid, and acetylcholine, among others. These compounds are known to enhance neurotransmission, stress management, and other neurological functions [77,78,79]. In a 12-week study, Lactiplantibacillus plantarum DR7 was shown to effectively reduce anxiety and stress symptoms in stressed adults. This probiotic’s administration also led to improvements in cognitive and memory functions. Notably, there was a decrease in plasma cortisol levels, alongside reductions in pro-inflammatory cytokines, such as interferon-γ and transforming growth factor-α. In contrast, the levels of the anti-inflammatory cytokine IL-10 increased. Additionally, DR7 supplementation boosted the serotonin pathway and stabilized the dopamine pathway [80, 81]. In research with mice reported by Varian et al., Limosilactobacillus reuteri lysate consumption increased plasma oxytocin levels and decreased corticosterone, the stress hormone [82]. Furthermore, Lactobacillus strains have been found to upregulate the expression of tight junction proteins, such as zonula occludens 1 and occludin, in human epithelial enteroids, enhancing intestinal barrier function [83].

While the physiological effects and mechanisms of probiotics have been well documented, the exact therapeutic or health-improving benefits are still largely under investigation. Clinical intervention studies have shown probiotics to be effective in mitigating various gastrointestinal disorders [84, 85]. Yet, despite considerable advancements in understanding the mechanisms of probiotics through animal studies, translating this knowledge into clinically proven effectiveness for specific health conditions, such as inflammatory and autoimmune diseases, remains a challenge.

FORMULATIONS AND DELIVERY OF PROBIOTICS

The efficacy and potential health impacts of probiotics can be largely affected by their formulation or method of delivery. The viability of these beneficial microbes is fundamental to their function, necessitating the inclusion of a specific count of live colony-forming units (CFU) in each probiotic dose. This is critical for ensuring that the products confer their intended health benefits [85,86,87].

Typically, an effective daily dosage of these organisms is approximately one billion viable cells. However, their vulnerability to the harsh conditions of the gastrointestinal tract and the challenges posed by food processing can complicate the creation of probiotic foods and beverages [85]. For instance, exposing probiotics to gastric fluids with a pH range from 1 to 3 for 5 minutes can result in a decrease in CFU by up to a million-fold [88].

Probiotic products are often formulated with prebiotics, which are substances known to selectively foster the growth or activity of beneficial microorganisms. When probiotics and prebiotics are combined, the resulting products are known as synbiotics, which are aimed at enhancing the viability and functionality of probiotics. Available in a variety of forms, including capsules, tablets, powders, and fermented foods, probiotic products can vary in their effectiveness based on the chosen delivery method. This variance is due to multiple factors, such as the specific strains used and their stability [87, 89,90,91,92].

Products formulated with probiotics often include additional ingredients, such as carriers, stabilizers, and excipients. These additives are crucial for improving the stability of the product and extending its shelf life. It is vital to pay attention to the complete composition and quality of probiotic formulations. Consumers are encouraged to meticulously examine product labels to grasp the particular strains, dosage, and extra components contained in these formulations. Additionally, the efficacy of probiotics can be affected by several factors, such as the conditions under which they are stored, the way they are handled, and the unique reactions of individuals. Therefore, it is wise to consult healthcare professionals before adding probiotics to one’s regimen [92, 93].

Current trends in formulation of probiotics

The growing interest in novel delivery systems for probiotics centers on improving the survival, stability, and targeted delivery of beneficial live microorganisms to specific areas within the gastrointestinal tract [90]. Over the past decade, numerous innovative methods have been developed to enhance the performance of probiotics.

Microencapsulation of probiotics

Microencapsulation stands out as a promising technique that encapsulates probiotic cells within protective coatings to safeguard them against harsh environmental factors, such as the acidity of the stomach and bile salts. This approach notably improves the survival of probiotics both during storage and transit through the digestive system [94]. For crafting these microcapsules, four technologies have been predominantly utilized: freeze-drying, emulsification, extrusion, and spray-drying [95].

A variety of encapsulating agents have been employed in the microencapsulation of probiotics, including soy and whey proteins, pea proteins, pectins, alginates, chitosan, and carrageenans, which are among the most commonly used materials [96]. Additionally, recent research has explored various synthetic and natural polymers as promising encapsulation matrices. These polymers are particularly valued for their ability to protect probiotics and enable their targeted release within the intestines [97]. Pupa et al. [98] developed a double-layer coating technique that utilizes alginate and chitosan and is specifically tailored for LAB strains. This novel alginate–chitosan encapsulation, applied through extrusion, emulsion, and spray-drying methods, markedly improved the survival of the studied bacteria over a 6-month storage period. Furthermore, these encapsulation strategies successfully preserved the antibacterial efficacy and the ability of the LAB strains to withstand acidic and bile conditions. Conversely, Jin et al. [99] observed that microencapsulation using alginate alone did not significantly enhance the survival of L. casei Shirota in simulated gastrointestinal environments. However, the introduction of additional protective layers, such as xanthan, carrageenan, or acacia gums, improved defense against adverse environmental conditions. Zaeim et al. [100] developed a double-stage procedure using Ca-alginate and chitosan through electrohydrodynamic processing, incorporating L. plantarum along with prebiotics such as inulin and resistant starch. The integration of these microcapsules, featuring a synbiotic blend of prebiotics and probiotics, into ice cream was shown to improve the viability of probiotics. Rossi et al. [101] employed a microencapsulation technique for producing yogurt, using a starter culture that included L. plantarum VP-3.3 and Streptococcus thermophilus. A 7% concentration of this culture yielded an optimal product, maintaining a total LAB count of 9.98 log₁₀ CFU/mL following 28 days of refrigerated storage. Additionally, Nami et al. [102] demonstrated that encapsulating Lactococcus lactis ABRIINW-N19 in herbal-based hydrogels, combined with prebiotics, significantly preserved the viability of probiotics in orange juice over 6 weeks of storage. Gyawali et al. [103] reported on the benefits of administering microencapsulated L. paracasei to mice, noting improvements in fecal moisture content and an increase in lactobacilli levels in the feces. Furthermore, encapsulating the probiotic in polyacrylate resin was linked to substantial enhancements in intestinal health, as evidenced by a higher villus height to crypt depth ratio in the small intestine, increased mRNA expression levels of tight junction proteins and intestinal MUC-2, secretory immunoglobulin A and mucin concentrations.

Formation of probiotic nanoparticles

Nanoparticles and nanogels have emerged as promising carriers for probiotic microorganisms, offering protection against harsh conditions and overcoming limitations associated with traditional encapsulation techniques, such as inconsistent particle size control [88]. These nanotechnological methods ensure controlled release and enhanced stability, thereby preserving the viability of probiotics during storage and passage through the gastrointestinal tract. Currently, there is a growing interest in employing nanoparticulate materials, including gold and selenium nanoparticles, as well as nanolayers, nanobeads, nanoemulsions, and nanofibers in combination with probiotics. This approach aims to create products with potential health benefits, such as anticancer, antimicrobial, antioxidant, and photo-reactive properties [104, 105]. Electrospinning is commonly used to produce nanofibers, a technique that preserves the properties of probiotics without necessitating toxic organic solvents. Various techniques, such as the nanoprecision and antisolvent coprecipitation methods, are used for forming nanoparticle-encapsulated probiotics, whereas nanocoating offers a strategy for encapsulating individual cells to impart exogenous traits [95]. Atraki et al. [106] demonstrated that the viability of probiotic lactobacilli and bifidobacteria under simulated gastrointestinal conditions significantly improved with the use of probiotic-loaded nanofiber mats made from corn starch and sodium alginate. Kiran et al. [107] and Han et al. [108] further validated the effectiveness of nanoparticle encapsulation in protecting probiotic lactobacilli, utilizing a pH cycling method and a one-step shell construction based on natural materials (e.g., eggshell membrane hydrolysates and coffee melanoids), respectively. Hashem et al. [109] observed positive effects on the antimicrobial activity of a synbiotic combination of S. cerevisiae and Moringa oleifera leaf extract through nanoencapsulation. Despite their benefits, certain challenges, like instability at high temperatures and susceptibility to flocculation, coalescence, agglomeration, and precipitation, have been noted with nanoliposome techniques [110].

Formation of probiotic biofilms

Probiotic biofilms, which involve growing probiotics on surfaces to form protective matrices, offer an innovative delivery strategy for coatings on food products or supplements, promoting extended release and stability. This technique represents a forward-looking approach to enhancing the durability and effectiveness of probiotics in various applications [97].

Genetically engineered probiotics

Rapid advancements in genetic engineering have significantly improved probiotic strains, enhancing their therapeutic efficacy. Engineered probiotics can now produce specific proteins or peptides, augmenting their therapeutic capabilities [111]. Furthermore, the scope of engineered probiotics has expanded to include the diagnosis, prevention, or treatment of various health conditions, such as inflammatory bowel disease, tumors, metabolic diseases, and bacterial infections [112]. These synthetic probiotics act as live therapeutic agents, facilitating the targeted release of various therapeutic molecules like peptides, DNA, enzymes, and cytokines, enriching their application in prophylactic and therapeutic contexts [113, 114].

A notable application by Steidler et al. [115] involved the use of genetically modified L. lactis to produce IL-10 in situ, which led to a significant reduction in chronic colitis in a mouse model through intragastric administration. Similarly, modifications in L. lactis to express anti-(m)TNF nanobodies and trefoil factors have shown promise in reducing inflammation in dextran sulfate sodium-induced colitis models in mice [116, 117]. Metabolic engineering has also been employed to enhance the production of valuable fermentation products and metabolites, such as tagatose, nisin, and exopolysaccharides, optimizing their production rates and yields [113].

Recombinant bioengineered probiotics offer pathogen- and toxin-specific activities, preventing colonization by target pathogens, regulating virulence, producing antimicrobial agents, and stimulating robust immune responses [114]. Notable achievements include reprogramming S. boulardii to express Vibrio cholerae toxin tcpA, aiming to create an oral vaccine against Vibrio cholerae [118], engineering lactic acid bacteria to detect autoinducer peptides from S. aureus [119], and using L. reuteri WXD171 to express antigens for protection against S. aureus infections [120]. Additionally, E. coli Nissle 1917 has been engineered to combat C. difficile infections through bile salt hydrolase expression, showcasing the potential of engineered probiotics to modulate intestinal signals and inhibit pathogen growth [121]. Furthermore, genetically engineered L. casei expressing a Clostridium perfringens α-toxin toxoid has been developed to elicit comprehensive immune responses against the toxin [122]. Despite these advancements, the development and application of genome editing tools for LAB remain constrained by stringent food legislation and consumer apprehensions regarding genetically modified organisms in the food industry [123]. This underscores the need for ongoing research, dialogue, and regulatory considerations to maximize the benefits of genetically engineered probiotics while addressing public concerns.

In summary, probiotics can be seamlessly integrated into a variety of food matrices, including dairy foods, cereals, and snack items. Utilizing food models serves a dual purpose: it not only safeguards the probiotics, ensuring their viability, but also offers a practical way for consumers to incorporate these beneficial microorganisms into their diets. These innovative delivery mechanisms are instrumental in advancing the production of probiotic products characterized by enhanced stability, superior survival rates, and precise targeting within the gastrointestinal tract, thereby amplifying their beneficial health impacts [105]. Consequently, continuous research in this domain is imperative for the discovery of new technologies and methods to refine the efficacy of probiotic delivery systems.

SAFETY CONCERNS ASSOCIATED WITH THE USE OF PROBIOTIC PRODUCTS

While numerous probiotics have been successfully integrated into foods, their approval and usage come with complexities [124]. The European Food Safety Authority (EFSA) and the US Food and Drug Administration (FDA) have generally recognized the use of probiotics as safe for treating infections [88]. However, it is crucial to consider individual safety concerns and variations in response to probiotics. What is safe for one individual might not be for another, especially regarding infection risks. This concern is heightened for those with weakened immune systems, such as chemotherapy patients or organ transplant recipients, who may be more susceptible to adverse effects of probiotic use. This increased risk is particularly significant for individuals with serious underlying health conditions [125].

Documented cases have shown probiotic consumption linked to fungemia and bacteremia, yet exact rates of opportunistic infections related to probiotics have not been well-established [126]. Lactobacillus bacteremia has been reported in vulnerable groups, including preterm neonates with growth restriction or short bowel syndrome, and patients with ischemic or ulcerative colitis, cancer, or immunocompromised states. The mortality rate for Lactobacillus bacteremia patients has been estimated at 30%, with underlying conditions often being the direct cause of death [127]. In compromised hosts, lactobacilli have been linked to a range of diseases, including pleuropulmonary infections, bacterial endocarditis, gastrointestinal abscesses, endometritis, conjunctivitis, dental caries, and urinary tract infection [128]. Meini et al. [129] documented a case of bacteremia induced by L. rhamnosus GG in a patient with severe active ulcerative colitis, where the bacteremia developed with concurrent candidemia while the patient received a probiotic formulation containing the same bacterial strain. Similarly, Kunz et al. [128] reported two cases of L. rhamnosus GG sepsis in patients under probiotic therapy. Sherid et al. [130] observed that the use of probiotic Lactobacillus species might result in bacteremia and liver abscesses in susceptible individuals. In Finland, a study by Rannikko et al. [131] across five university hospitals found 46 patients diagnosed with Saccharomyces fungemia, with a significant portion having used S. cerevisiae var. boulardii probiotics shortly before their diagnosis.

The safety of probiotics has been generally established through numerous clinical studies, showcasing their non-toxic nature across various demographics, including pregnant women, infants aged 0–2 years, both healthy and hospitalized children, healthy adult volunteers, and immunocompromised patients [132,133,134,135,136]. Nevertheless, critiques from respected medical and scientific publications have cast doubts on the safety of probiotics. Concerns have emerged regarding the scientific integrity and methodology of some probiotic clinical trials, particularly in relation to the determination of clinical outcomes [136,137,138].

Furthermore, the issue of low-quality and contaminated probiotic products exacerbates safety concerns. The assurance of the safety of probiotics is heavily dependent on the quality control standards enforced by manufacturers. Contamination of probiotic products with harmful microorganisms or other substances is possible, and the absence of standardized regulations may be worrisome. Although the FDA has established good manufacturing practices for dietary supplement production, it is common for manufacturers to demonstrate non-compliance with these standards. Instances of non-compliance for the production of high-quality and safe products, especially those marketed as dietary supplements, have been documented [126, 139].

Sanders et al. [140] summarized the results of several studies evaluating the microbiological parameters of retail probiotic products, revealing the presence of microbes not listed on the labels. The presence of these microbes suggests either a failure of quality control procedures or an intention to mislead consumers. Furthermore, inadequately labeled excipients used in probiotic product formulations can lead to additional health problems, including allergic reactions. Documented cases of allergic reactions to components found in probiotic supplements, such as dairy proteins, have occurred in the past. Consequently, individuals with known allergies should carefully scrutinize the ingredients of these products.

The interaction of probiotics with pre-existing conditions could be significant. Individuals with specific pre-existing health conditions, such as short bowel syndrome, may experience adverse effects from probiotic consumption. Therefore, these patients must consult with healthcare professionals before using probiotic supplements. Other unwanted side effects may also result from consuming probiotics: certain individuals may experience mild gastrointestinal symptoms, such as bloating or gas, upon initiating probiotic supplementation [141,142,143,144,145]. Although these symptoms are typically temporary, consumers experiencing persistent or severe symptoms should seek medical advice.

Merenstein et al. [146] delineated emerging concerns regarding probiotic safety, encompassing both acute and long-term risks. Acute risk assessment entails scrutinizing alterations in microbiota composition or function, alongside examining transcriptional and metabolic readouts. Profiling the microbial composition before and after probiotic ingestion is pivotal in comprehending the role of probiotic organisms in shaping the microbiome. These assessments are crucial for elucidating the capacity of probiotics to deliver intended benefits to consumers [138, 147, 148]. Additionally, the horizontal transfer of antibiotic resistance genes can pose short-term risks. In rare instances, probiotics may translocate from the gastrointestinal tract, potentially leading to invasive infections [144, 146]. When consumed concurrently with other medications, probiotics may influence drug function. The gut microbiota can exert both direct and indirect effects on drug metabolism, impacting efficacy and toxicity [149, 150]. Long-term risks include the potential for sustained colonization of the microbiome, necessitating ongoing assessment of microbiota composition or function alterations. Current evidence suggests that most probiotics do not establish long-term colonization, as indicated by the fecal recovery of the microbes [146, 151].

The assessment of probiotic safety presents more challenges than initially anticipated. Since 2002, the WHO has introduced guidelines for evaluating probiotics. These guidelines involve identifying the genus/species/strain of bacteria, conducting in vitro tests, performing in vivo studies using both animal and human models, and providing guidance on health claims and labeling [152]. Despite extensive research on probiotic safety, there remains a lack of sufficient data from randomized controlled trials [144]. Overall, probiotics are regulated under categories such as foods or fermented food products, dietary supplements, and drugs/pharmaceuticals. These categorizations are determined jointly by regulators/authorities and probiotic manufacturers [2, 153].

To address safety concerns associated with probiotic products, it is crucial to subject them to thorough evaluation before approving them for marketing [144]. Similarly, emphasis should be placed on the harmful properties of microorganisms with probiotic potentials, such as translocation, production of harmful metabolites, gene transfer, and immunomodulation [154].

Ensuring the accurate assessment of taxonomic identity for new probiotic candidates should be prioritized early in the evaluation process. Currently, inappropriate identification methods are considered a major contributor to the mislabeling of probiotic products. Inconsistencies in microbial identification for commercial products can significantly impact their safety record and potential efficiency [140]. Furthermore, additional technological characteristics (e.g., manufacturing processes and marketing regulations) of the utilized strains should be evaluated to prevent undesirable outcomes [136]. Consumers are advised to consult with healthcare professionals before incorporating probiotics into their routine, especially if they have underlying health conditions or concerns about potential interactions. Additionally, adherence to recommended dosages and product guidelines can contribute to the safe use of probiotics. Given the lack of studies focusing on vulnerable populations, conducting research with longer durations would be beneficial for reaching more conclusive findings [155].

CONCLUSIONS

Despite existing safety concerns, probiotics have consistently played a vital role in health and nutrition. The future promises an expansion in their use across various formats, including dietary supplements, therapeutic capsules, functional powders, gels, injectables, and novel products. This anticipated growth underscores the necessity for ongoing, sophisticated research to deepen our understanding of how these microbes work, improve how they are delivered, and confirm their safety. Engaging in such cutting-edge studies is essential to maximize the benefits of these important microorganisms.

AUTHOR CONTRIBUTION

Conceptualization – TJA; writing, review, editing, final draft – TJA, BG and LV.

DATA AVAILABILITY

No new data were generated in this manuscript.

FUNDING

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest associated with this paper and its publication.