Probiotics and DCs

DCs are also called detector cells because of their receptors that bind with a specific part of the pathogens. DCs are found in the mucosa and gut-associated lymphoid tissue (GALT). These cells also catalyze signalling pathways, such as c-type lectin receptors and pattern recognition receptors (PRRs), such as TLRs, that modify their characteristics and release cytokines [27]. Probiotics contain MAMPs that bind with PRRs of DCs and stimulate the cells to protect the IEB [22]. After DCs start phagocytosis and get antigen from the mucus or lamina propria, they migrate to the lymph nodes. Finally, DCs can induce T cells and elicit the immune response [19]. Bacterial cell wall components take part in immunity regulations by the DCs. Bacterial antigen presentation by the gut CD11cþ DCs is dependent on MHC-II molecules, which are vital for Th17. In the case of immune response, there are associations found with CPS A, Bacteroides fragilis, and TLR2 of DCs. Where DCs have been demonstrated to prevent intestinal colitis and release IL-10. For instance, a direct association has been demonstrated between specific fragments of Bacteroides fragilis capsular polysaccharide A (PSA) and TLR2 on mouse plasmacytoid DCs. These plasmacytoid DCs have been shown to express proteins that protect the gut against colitis and to promote IL-10 secretion by CD4⁺ T cells following exposure to capsular PSA [19].

Probiotics and macrophages

Macrophages have three main functions: phagocytosis, microbiocidal activity, and cytokine production. These functionalities increase with the presence of probiotics [29]. Probiotics can influence immunity indirectly or directly through stimulation of cytokine elevation, such as ILs, IFNs, TGFs, TNFs, and chemokines by macrophages. L. casei CRL 431 was found to produce IL-10 by macrophages [16]. Macrophages can perform phagocytosis of pathogens and are found in the intestinal subepithelial part, where antigens pass through the epithelium. Macrophages contain PRRs (TLRs), similar to DCs, that bind to MAMPs on pathogens. Macrophages work as antigen-presenting cells (APCs) [17]. Macrophages are secondary APCs that represent antigens to the memory T cells and are typically found in either of two subclasses: 1. M1 macrophages are involved in immunogenic activation and proinflammatory responses derived from TNF, IL-1, IL-6, IL-8, and IL-12; and 2. M2 macrophages are associated with mucosal homeostasis and tolerance, and activated by anti-inflammatory or regulatory cytokines such as IL-10, TGF, and IL-1Rα. These subclasses are potential targets of probiotics, such that M1 macrophages are inhibited in inflammatory pathologies and M2 macrophages are inhibited in suppressor pathologies, such as in mucosal cancers [20].

LTA, the cell wall of gram-positive bacteria, can induce nitric oxide (NO) synthase in macrophages and kill the virus, while also increasing receptors such as FcγRIII (CD16) and TLRs for phagocytosis. These APCs also participate in T cell responses by secreting cytokines and differentiate into subtypes of CD4⁺ T cells such as Th1, Th2, or Th17. T cells binding with APCs is vital for the host’s adaptive immunity [19]. L. plantarum was found to increase IL-10 production and secretion in the macrophages which has been obtained from the colon after inflammation. On the other hand, L. rhamnosus GG increased IFN, IL-12, and IL-18 [21].

T cells and probiotics

T cells are part of the adaptive immune response, which shows interactions with probiotics [26]. T cells are also found in the lamina propria. T cells and related inflammation become activated after DCs with antigen reach the lymph nodes. T cells regulate the adaptive immune system of the intestine through IgA secretion. Research indicates that elevation of Treg function is probably linked to the positive effects of probiotics on conditions such as allergies. Previous study results showed probiotics can regulate immune modulator secretion by innate immune cells, such as DCs, and regulate T cells. For example, many types of pathogen-associated molecular pattern molecules (PAMPs) are expressed by the Lactobacilli strains, which can stimulate cytokine production, and later these bind to the PRR on APCs. These cytokines work as an important signal for T cells and induce cytokines, such as Th1, Th2, Th17, or T regulatory cell responses [19]. Overall, probiotics are capable of increasing Tregs, which are important for inflammation regulation [27].

B-Lymphocytes and probiotics

The GALT is a part of the mucosa-associated lymphoid tissue (MALT). GALT plays the most important part of the immune system. It presents a big source of B-cells and T-cells, which later move to the area where they show necessary immune responses. The intestine is responsible for containing around 75% B cells that produce IgA. Probiotics are responsible for the maturation of IgA-producing plasma cells (differentiated from B cells). Where IECs produce cytokines and chemokines and create a microenvironment in the intestinal lamina propria, where clonal expansion of B cells occurs and increases IgA production. IgAs move to the mucosa through the intestinal epithelial layer and later control bacterial adhesion to the host gut epithelium [27]. This is how B cells play an important role in adaptive immunity through secreting antibodies. B cells are found to prevent excessive immune response and control it by IL-10. A study found that, after Lactobacillus gasseri SBT2055 oral administration, the production of IgA was triggered and increased IgAþ cells in Peyer’s patches and lamina propria. In another human experiment with probiotics, Bifidobacterium animalis with xylooligosaccharide decreased CD19 expression in B cells, which gave a clear picture of the good outcomes of immune system modulations caused by the correctly chosen probiotics and/or prebiotics [19]. Moreover, in another study, after admission of L. rhamnosus GG to an infant with acute gastroenteritis elevated nonspecific adaptive immune response that increased IgG, IgA, and IgM. Bifidobacterium bifidum potentially improves antibody production. L. acidophilus, L. bulgaricus, Streptococcus (S.) thermophilus, B. bifidum, and Bifidobacterium infantis containing yogurts induced IgA response against the cholera toxin in mice [21].

Mechanisms of probiotic delivery

The microencapsulation technique is a widely used method of probiotic delivery. This method is used to avoid severe physicochemical stresses during processing, such as acidity, high temperatures, various digestive enzymes, microbiota, and passing through the gut. Because biological processes can decrease the viability of probiotics when they are consumed orally [30, 31]. Probiotic delivery mechanisms are crucial for ensuring the viability and effectiveness of probiotics in therapeutic applications. Various innovative strategies have been developed to enhance the stability and bioavailability of probiotics during their passage through the gastrointestinal tract (GIT). To introduce a new probiotic delivery, there are some barriers that need to be overcome by probiotic bacteria; these are given below.

Microencapsulation materials

The materials used to coat probiotics in encapsulated delivery systems must satisfy a number of criteria; they should be food-grade, be able to adequately protect probiotics during transit in the GIT while also releasing them at the targeted site, as well as be economical. In addition to the materials described in Table 2, many researchers have used combinations of different construction materials for probiotic encapsulation. Such combinations often utilize the most advantageous features of the individual materials, resulting in overall improved functionality.

Microencapsulation techniques

Encapsulation refers to the preparation of “carrier” capsules made of solid, liquid, or even gaseous components that are able to release their contents in a controlled manner. Nano- (< 0.2 μm), micro- (0.2–5,000 μm) encapsulation are the most common size-based types for ingestion [63]. Probiotics can be encapsulated either via physical (spray drying, freeze drying, spray chilling, spray cooling, extrusion, fluidized bed drying, electrospraying, and electrospinning) or chemical (coacervation, ionic gelation, and molecular inclusion) means [64].

Spray drying is mainly used to protect the heat-sensitive prebiotics and involves dissolution, emulsification, or dispersion of the probiotic in an aqueous or organic solution containing an encapsulating agent, followed by spraying the mixture in a drying chamber. The droplets from the spray dry instantly, resulting in encapsulated particles that are collected. L. plantarum and L. rhamnosus have been viably encapsulated with this technique [65, 66]. This technique is fast and has a low operational cost, and is therefore easily scalable. However, the simple particles produced might provide limited protection to probiotics.

Spray cooling is similar to spray drying, but instead of forming particles by evaporation, they are formed by cooling such that the matrix material solidifies around the probiotics [67]. Saccharomyces boulardii, L. acidophilus, and B. bifidum have been encapsulated using the spray chilling or cooling technique [68]. This technique is appropriate for heat-sensitive probiotics and is also easily scalable.

Freeze drying or lyophilization is suitable for thermally sensitive bioactives and involves rapid freezing followed by dehydration. Freeze drying has been used to stabilize L. rhamnosus, L. acidophilus, Lactococcus lactis, L. plantarum, and L. acidophilus [66, 69–71]. A study was started on a microencapsulated probiotic delivery method for L. plantarum, which would be cryoprotected by microcapsules of chitosan-inulin complex. It was freeze-dried so that probiotics can get maximum viability. It was assessed by using in vitro low pH intestinal tract conditions and stored at various temperatures. A Box-Behnken experimental design includes different concentrations of CaCl₂, chitosan, and inulin used as encapsulant components for developing L. plantarum microcapsules. Under optimum conditions, 68.33% cell viability was achievable, but the study showed that L. plantarum with small microbeads could elevate the probiotic viability to around 108 log CFU/mL. Efficacy of freeze-dried, microencapsulated L. plantarum was reported to be dependent on a number of factors, including cell viability during drying, gastrointestinal treatment, refrigerated storage conditions, and the physicochemical properties of the powders used [71, 72]. Lyophilization results in a long shelf life; however, the process is expensive and slow, which might limit scalability.

Fluidized bed coating is often combined with spray drying technology and involves depositing a coating agent (which could include aqueous solutions of gums, starch, cellulose, or proteins) on the surface of the bioactive [73]. Enterococcus faecium IFANo.045 and L. plantarum IFANo.278 have been coated with this technique using cellulose [74]. L. plantarum NCIMB 8826 has been coated using alginate-chitosan [75]. This technique results in a uniform coating and can be tuned to create multi-layer capsules; however, the equipment cost is high.

Extrusion involves mixing bioactives into a hydrocolloid solution, which is then extruded through a nozzle into a curing solution (e.g., CaCl₂, AlCl₃, or FeCl₂) such that the initial mixture turns into a gel and pellets are created [76]. Various polysaccharides (most commonly sodium alginate) can be used for extrusion encapsulation and determine the size, shape, and viability of the encapsulated material [77]. L. plantarum 15HN has been encapsulated with this technique using alginate, an alginate-psyllium blend, and an alginate-fenugreek blend [78] while L. acidophilus La-14 has been encapsulated using a calcium chloride solution [79]. Alginate combined with natural polysaccharides and fructo-oligosaccharides has been used to encapsulate strains of L. casei 01 and BGP 93 via extrusion [76]. This technique uses relatively simple equipment; however, the process is slow, which may lead to low scalability.

Ionic gelation utilizes the electrostatic interactions between opposite charges involving at least one polymer and is most often used to prepare alginate particles [80]. The use of chitosan as a coating material on alginate beads containing the bacterium Bifidobacterium longum has been shown to improve its survival in gastric fluid and high-temperature conditions [81]. Different probiotics have been encapsulated via the ionic gelation process, such as L. casei, L. rhamnosus, L. plantarum, Pediococcus pentosaceus, Lactobacillus fermentum, and L. acidophilus [82–89]. Positively charged chitosan and negatively charged alginate can be used to coat E. coli Nissle 1917 for protection [33, 90] while encapsulated L. acidophilus can be encapsulated with carboxymethyl cellulose and chitosan to enhance their vitality in the GIT [33, 91]. This technique is also relatively simple and low cost, and can be easily scaled.

Complex coacervation involves the continuous stirring of three chemically immiscible parts such that the base substance diffuses into the coating solution and an outer layer solidifies around the bioactive via chemical or physical crosslinking reactions [65, 92, 93]. This process utilizes polysaccharides such as gum arabic (GA), chitosan, mesquite gum, PEC, alginate, xanthan gum, carrageenan, and carboxymethyl cellulose, as well as proteins including gelatin, whey protein, casein, albumin, and plant-derived proteins such as pea protein. or soy protein [65]. L. rhamnosus GG has been encapsulated by whey protein isolate-high melting point fat shortening oil (SO) and GA through complex coacervation [94]. Other probiotics encapsulated via the complex coacervation process include L. plantarum, L. casei, Lactobacillus paracasei, Bifidobacterium lactis, L. acidophilus, and L. reuteri [92–97]. This technique can be used to create sophisticated assemblies that are optimized for controlled release of probiotics in target locations of the GIT. However, optimization can be time-consuming and may limit scalability.

Electrospraying and electrospinning. Electrodynamic (EHD) is a technique that produces nano or microscale fibers by electrospinning and electrospraying that can be used for various applications, such as good stabilization and controlled release of sensitive components like essential oils, peptides, proteins, etc. Electrospinning and electrospraying are versatile, flexible, mild, and straightforward techniques which have emerged as techniques for probiotic stabilization in the last few years [98]. From these techniques, nano- or microscale fibers and spheres are made. The mechanism works as polymer solution extrusion is performed using a syringe pump equipped with a needle. Upon droplet formation at the needle tip, a Taylor cone develops. Subsequently, jet ejection occurs from the apex of the cone, followed by the deposition of the resulting fragmented droplets onto the collector. It was found that the survival rate of probiotic strain increased to 89.26% after using electrospinning, and when L. rhamnosus 1.0320 was kept for 21 days of storage, the survival rate was 84.63% by using polyvinyl alcohol (PVA)/PEC as the wall material. In another study, it was mentioned that the survivability was 90.07% and 91.96% in simulated gastric and intestinal fluids with PVA/PEC in the wall material [64]. In another study, the encapsulation of L. plantarum in electrosprayed whey protein concentrate (WPC) microcapsules demonstrated enhanced viability when fresh cultures were used instead of freeze-dried cultures. Compared to free cells, WPC encapsulation significantly improved bacterial survival during storage at elevated relative humidity (53%). Additionally, suspending the bacteria in the surfactant Tween 20^® increased the production yield of electrosprayed capsules. The viability of encapsulated L. plantarum was further assessed under in vitro digestion conditions and compared to freeze-dried samples. Encapsulation provided notable protection, with the majority of viability losses occurring during the gastric phase (pH 3) due to acidic degradation. Minimal differences in viability between encapsulated and freeze-dried bacteria were observed after the intestinal phase, likely because the capsules were fully disrupted by this stage. The marginal disparity may also be attributed to the inherent acid resistance of this particular strain. To enhance the protection of L. acidophilus under moist heat conditions, multilayered microcapsules were fabricated using a combination of electrospraying and fluidized bed coating. The core material, composed of alginate and glycerol, was electrosprayed into a shell solution of egg albumen (EA) and stearic acid (SA), followed by fluidized bed coating with cassava starch granules during drying. The encapsulation efficiency exceeded 90%, and cell viability was further improved by increasing the SA concentration. Although encapsulated cells exhibited a viability loss of only 0.6 log CFU/g, long-term stability studies are required to fully assess the efficacy of these microcapsules in probiotic preservation. Electrospun and electrosprayed structures are known to exhibit favourable mucoadhesive properties (Moreno et al. [99], 2018), which can significantly prolong the gastrointestinal residence time of probiotics and enhance their interactions with the gut microbiota. In a recent study, mucoadhesive electrosprayed microcapsules composed of alginate-starch and chitosan-coated alginate, loaded with L. plantarum, were evaluated for gastrointestinal delivery. The researchers employed an in vitro fluorescence imaging-based flow-through test using ex vivo porcine gastric epithelial mucosa to assess mucoadhesion. The chitosan-coated alginate particles demonstrated strong retention on gastric mucosa, comparable to that of pure chitosan controls. In contrast, alginate-starch particles (used as a negative control) exhibited weak retention, attributed to the non-ionic nature of starch and its inherently poor mucoadhesive properties [100].

Responsive coating strategies

Responsive coating strategies for probiotics aim to enhance their survival and targeted delivery within the human GIT by overcoming challenges like inactivation during processing, storage, and passage through the harsh acidic environment of the stomach and the presence of bile salts in the small intestine. pH-responsive coatings are designed to be stable in the acidic environment of the stomach but dissolve as the pH increases in the intestine, allowing for the release of the probiotics at the target site. Luo et al. [101] 2022 reported the oral delivery of probiotics based on a pH-sensitive trigger mechanism, based on mucosal adhesion, enzyme degradation, spore or biofilm formation, or a redox-responsive trigger mechanism. Enzyme-responsive coatings are designed to be degraded by specific enzymes present in the gut, ensuring the release of probiotics at the desired location. Altogether, a combination of factors must be taken into consideration to ensure protected travel through the digestive system and the controlled release of probiotics at the target location. These factors include the characteristics of the probiotics themselves (such as initial quantity, particle size, diffusivity, partition coefficient, and concentration gradient), the properties of the carrier matrix or encapsulation system [including original size, shape and structure, porosity, pore formation and closure, and the (non)flocculated state of emulsion droplets], as well as external factors such as the surrounding environment and conditions within the GIT (e.g., the oral cavity and transit of the bolus into the stomach), chime going to the small intestine, and indigestible ingredients going to the colon [102, 103].

Production of antimicrobial substances

Side effects and limitations of probiotics

Before a bacterial strain can be considered appropriate to use as a probiotic, it must be ensured that it is harmless to the host, non-toxic, non-pathogenic (including not being an opportunistic pathogen), be genetically stable and not have any genotoxic properties [129]. The bacteria should also be resistant to the adverse conditions in the GIT, must efficiently adhere to the gut lining and should have a short generation time. Despite best attempts, limitations remain in the use of probiotics, and it is difficult to find a single ideal probiotic. For example, LAB are known to produce different varieties of bacteriocins with excellent antimicrobial properties; however, these bacteria are sensitive to digestive enzymes and must be protected by artificial encapsulation techniques [130]. Furthermore, the bacteriocins produced by these LAB may become toxic to humans at high concentrations already been seen. This risk is further complicated by the possibility of horizontal gene transfer of bacteriocin-associated genes as well as other virulence factor genes within the gut microbiome. This risk is most likely to be developed via long-term colonizing probiotics and other potential risks include disruption of the microbiome (e.g., through displacing a microbe typically performing a necessary function) and breaching of the gut barrier to access systemic circulation. For example, an increase in Lactobacillus-associated bacteremia was reported among intensive care unit patients receiving probiotics in a Boston hospital [131]. A specific Bifidobacterium strain has been reported to modify SCFA production in the gut and may be involved in antibiotic-associated diarrhea [132]. Individuals who use venous catheters or have severe diseases have been reported to be affected by Saccharomyces fungemia. Immunocompromised individuals as well as individuals with gut dysbiosis and/or impaired intestinal barriers are at an especially higher risk of side-effects associated with probiotics [129]. Populations at risk include the pregnant, newborns and the elderly as well as those undergoing chemotherapy, those who have had an organ transplant and those with HIV/AIDS [129, 133]. Theoretically, possible side-effects include systemic infections, unwanted metabolic activities, overstimulation of the immune system, and gene transfer [133]. In 100 studies of probiotic use during pregnancy, 11 reported gastrointestinal problems, nausea, and headache that might be caused by the treatment, but no serious health concerns of mother or infant were reported [131]. SCFAs produced via probiotic metabolism could also affect various organs such as the brain and liver [132]. L. paracasei bacteremia was reported in and 8-month-old infant girl being treated with probiotics [16]. Immunocompromised individuals must be adequately assessed prior to being administered with probiotics, even for supposedly “safe” probiotics. Some individuals may also experience relatively minor and temporary symptoms such as bloating or flatulence, but consumer concern may create an unfavorable view of probiotic use for all [129]. Conversely, probiotics can also reduce the side effects of other treatments even in compromised individuals. For example, a meta-analysis of probiotic use during cancer treatment reported that oral probiotics significantly decreased the side effects associated with radiotherapy and chemotherapy compared to placebo groups [133]. Another study in children being treated with chemotherapy for acute leukemia reported improved gastrointestinal symptoms when taking probiotics [134]. Due to the high variability in the target population, there is mixed evidence of probiotic benefits as well as drawbacks; therefore, a comprehensive safety evaluation is necessary for the full understanding of the effects of probiotics [133].

Probiotics and antibiotics

The relationship between probiotics and antibiotics is complicated. Probiotics have been shown to bolster the efficacy of antibiotic therapies by preserving the balance of gut microbiota, which is frequently disrupted by antibiotic administration. This maintenance of microbial equilibrium helps mitigate the colonization of multidrug-resistant bacteria. Wieërs et al. [137] (2021) demonstrated that probiotic interventions reduced the prevalence of resistant pathogens such as Pseudomonas aeruginosa and AmpC-producing enterobacteria. Cell-free supernatants of some probiotic strains have been shown to have synergistic effects with antibiotics like ceftazidime and gentamicin against resistant bacteria like Staphylococcus aureus and E. coli. El Far et al. [138] demonstrated how probiotics can increase the effectiveness of antibiotics against resistant pathogens. Probiotics are used together with antibiotics to treat Clostridium difficile infections, although their specific functions and best ways to administer them are still being studied [139].

Probiotic synergism has some risk factors; one is the possible spread of antibiotic resistance genes, especially in Lactobacillus strains. It can spread these genes to harmful bacteria and endanger public health [140]. Concerns regarding the possibility of antibiotic resistance genes spreading up the food chain are raised by the discovery of these genes in probiotics used in veterinary settings, such as in broiler chickens [141]. It was reported that Lactobacillus species are susceptible to common antibiotics, for example, tetracycline, erythromycin, and chloramphenicol, by inhibition of protein synthesis. Also, it was found that they are susceptible to penicillin but highly resistant to cephalosporins. Intrinsic resistance to colistin was found in many different types of lactobacilli [142]. Lactobacillus species tend to show intrinsic resistance against ciprofloxacin, co-trimoxazole, and nalidixic acid, which are not transferable horizontally [143]. Numerous Lactobacillus strains exhibit intrinsic vancomycin resistance and are commonly employed as probiotics for the prevention of C. difficile infection [144]. Nawaz et al. [145] (2011) demonstrated that Lactobacillus strains, while lacking the acquired resistance gene van(B), exhibited intrinsic vancomycin resistance. The possible ways of getting antibiotic resistance in LAB are intrinsic, acquired, and/or mutational with transposons and conjugative plasmids. In LAB, the most common resistant genes are tet(M) and erm(B). In addition, erm(A), erm(C), erm(T) are also found in lactobacilli and S. thermophilus [146]. In one study, it was found that LAB was found resistance against vancomycin, streptomycin, gentamicin, teicoplanin, kanamycin, bacitracin, furantoin, norfloxacin, sulfadiazine, cefoxitin, metronidazole, and trimethoprim [147]. Imperial and Ibana [148] (2016) reported intrinsic resistance in the LAB against bacitracin, kanamycin, teicoplanin, vancomycin, and betalactams. Lactococcus spp., Leuconostoc spp., and Lactobacillus spp. are found to be highly resistant to cefoxitin [148]. In another article, Sharma et al. [149], 2014 mentioned many species of lactobacilli, except L. delbrueckii subsp. L. bulgaricus, L. acidophilus, L. johnsonii, and L. crispatus, are intrinsically resistant to glycopeptides [149]. Bacillus, Streptococcus, and Enterococcus species exhibit broad-spectrum antibiotic resistance, likely attributable to their transferable resistance gene. In contrast, Bifidobacterium and L. lactis display minimal resistance, potentially due to the absence of transferable genes. These findings highlight the distinct and complex antibiotic resistance profiles among probiotic species, which may be determined by their respective resistance gene repertoires [147]. Probiotics also influence the host immune system and engage with gut microbiota, potentially mediating antibiotic resistance. For this reason, their use should be carefully considered, especially in susceptible groups like infants [150]. Probiotic and antibiotic use in early life can drastically change the ecology of gut microbes, affecting long-term health outcomes. This emphasizes the significance of using probiotics and antibiotics sensibly to maximize benefits and minimize risks [151]. Studies have confirmed that probiotics are effective in the treatment of Helicobacter pylori. In a previous study it was checked the relation between probiotic and antibiotic combination and antibiotic single treatment for 0–4 weeks and 4–8 weeks, respectively. The data showed that there is an effect of antibiotic (amoxicillin + clarithromycin) with or without probiotic administration, which can significantly affect the eradication. Administration of probiotics together with antibiotics may reduce the beneficial effect of probiotics. The reason behind it may be that the antibiotic can affect the live cells of probiotics. It is recommended to maintain an interval of at least 2 h between taking probiotics and antibiotics, even though in this study they did not mention the most suitable time for probiotic administration. For example, with H. pylori, the co-administration of probiotics can help in the self-protection rather than reducing the H. pylori. So after the eradication of H. pylori, probiotic uptake can be more beneficial for health. Many researchers advised that the course for probiotic use should be 2 weeks [152].

Probiotics and chemotherapy

According to current research, probiotics can effectively reduce gastrointestinal toxicity caused by chemotherapy, especially mucositis and diarrhea. According to a systematic review and meta-analysis of 23 randomized controlled trials, probiotics can help prevent diarrhea brought on by chemotherapy and radiation therapy, particularly high-grade diarrhea [153, 154]. Probiotic supplementation has been shown to lessen the severity and duration of chemotherapy-induced diarrhea in experimental models, including rats given FOLFOX chemotherapy [155]. Inhibition of the TLR4-NF-κB signaling pathway, which lowers inflammation and shields the intestinal mucosa, is one of the mechanisms behind these effects [156]. In order to prevent diarrhea and other gastrointestinal issues, probiotics also improve the function of the mucosal barrier and heal damaged jejunal villus [130].

Since side effects are uncommon and probiotics are generally regarded as safe, they are a good choice for cancer patients looking to modify their gut microbiota [157]. Furthermore, they encourage the synthesis of SCFAs, which support gut health and have anti-inflammatory qualities [158]. Even though the evidence is in favor of their use, the results are still controversial, and more excellent randomized controlled trials are required to verify their safety and effectiveness in relation to different cancer types and chemotherapy regimens [159]. There is still much to learn about the best probiotic strains, dosages, and treatment durations [160].

Probiotics and antifungal agents

Probiotics and antifungal medications have shown promise for working together, especially when it comes to reducing fungal biofilms and enhancing therapeutic results. Probiotics that have demonstrated strong growth inhibition and antibiofilm activity against Candida albicans include L. rhamnosus and Pediococcus acidilactici. The presence of SCFAs in probiotic supernatants, such as lactate and acetic acid, is partially responsible for this effect [161]. Moreover, probiotics can increase the effectiveness of antifungal medications when combined with them. As an illustration of their potential in biotherapeutic products, synbiotic cultures of Lactobacillus crispatus and L. reuteri in combination with plant extracts have demonstrated potent inhibitory effects on Candida biofilms [162]. The mechanisms behind this synergy include damage to the cell wall and membrane. Here, antifungal medications and synthetic antimicrobial peptides cause structural damage to Candida species, increasing the effectiveness of the drug [163]. Increased oxidative stress is another mechanism, as antifungal treatments such as direct current in combination with antifungals cause biofilm structures to be disrupted, oxidative stress to rise, and intracellular drug concentration to rise, all of which intensify antifungal effects [164].

Chronic and acute infectious diseases

Probiotics can aid in the treatment of a number of infectious diseases, including respiratory conditions, diarrhea, and UTIs [165, 166]. Probiotics combat intestinal infections by increasing both specific and non-specific immune responses, gut acidification, competition for resources and binding sites, and chemical production [166]. Probiotic cures are an intriguing alternative because uropathogenic microorganisms and other bacteria may become resistant to antibiotics as a result of antibiotic treatment for UTIs [167]. Using Lactobacilli probiotics may help increase the urogenital flora and offer protection from UTIs because Lactobacilli predominate in the urogenital flora of healthy premenopausal women [18]. Probiotics can also be used to treat diseases like SIBO and chronic bacterial infections like H. pylori and C. difficile [168]. Lactobacilli, Bifidobacterium, Bacillus licheniformis, and Saccharomyces have been effective in resolving the digestive problems associated with H. pylori because they can inhibit the urease function of H. pylori and generate reactive oxygen species that damage the bacterial cell wall and membranes [169].

Cystic fibrosis

Cystic fibrosis (CF) is a mutation in the fibrocyctic membrane regulator gene, a hereditary disease. It may start in the gastrointestinal epithelium line and respiratory system then can spread to other organs of the body. In one study conducted in 2010 with CF patients were given probiotics daily that contained (L. acidophilus, L. bulgaricus, B. bifidum, and S. thermophiles) and it was found that there was a beneficial effect. According to the results, probiotics may have an inhibitory effect on pulmonary deterioration in CF patients and reduce the rate of pulmonary exacerbations. They also mentioned that probiotics may help reduce gastric inflammation in CF patients and enhance intestinal health [170].

Immune system and gut health

Probiotics play a vital role in immune system boosting. When taken in conjunction with a balanced diet, probiotics can help boost the body’s defenses against inflammation and infections. Because they promote the growth of beneficial microorganisms in the gut microbiome, probiotics are essential for maintaining proper digestive function. Additionally, probiotic bacteria ferment carbohydrates to produce organic acids like lactic and acetic acids. These organic acids help maintain an acidic environment in the stomach, which encourages the growth of good bacteria and prevents the formation of harmful ones. There are many other ways in which probiotics can improve immunity and gut health. L. plantarum BMCM12, for example, secretes extracellular proteins that reduce pathogen adherence and protect the intestinal barrier. One of the vital benefits of probiotics is pathogen’s competitive exclusion, such as E. coli Nissle 1917 (EcN) can prevent enterohemorrhagic E. coli (EHEC) growth by releasing the DegP protein. Additionally, H. pylori can be prevented to bind with epithelial cells by probiotics that secrete antibacterial compounds [171, 172]. IBS is a prevalent brain-gut axis disorder with complex, multifactorial pathophysiology. The emerging evidence bestows prominent significance on gut microbiota in the modulation of subjective symptom presentation in patients with IBS. Meta-analysis of 15 clinical trials (n = 1,793 IBS patients) depicted notable relief in abdominal pain and global severity symptom scores following probiotic treatment. Similarly, IBD, like Crohn’s disease and ulcerative colitis, is due to a dynamic interplay between genetic predisposition, environmental triggers, immune dysregulation, intestinal barrier defect, and microbial dysbiosis. Therapeutic application of some microbial species has been established in preclinical research: B. infantis and B. bifidum inhibited inflammatory markers and clinical symptoms through IL-10-mediated immunomodulation [11].

Diabetes

Diabetes is defined as a condition where there are elevated blood sugar levels and the pancreas cannot produce the hormone insulin, which helps transport glucose from food to reach cells and give us energy. Both type 1 and type 2 diabetes could occur from a genetic background. Type 2 diabetes causes insulin shortage, insulin resistance, or high blood glucose, which is a metabolic disease [173, 174]. Probiotics have been found to be an alternative way to reduce glucose levels in diabetic mice and postpone the consequences and issues associated with diabetes [175–177]. In many researches, it has been proven that probiotics can decrease blood glucose levels in people [178–181]. For six weeks, Ejtahed et al. [178] used yogurt made with L. acidophilus and B. lactis in 3.98 × 10⁹ CFU to treat type 2 diabetes. According to the results, its antidiabetic effect may make it useful as a treatment [182]. In addition, 20 individuals with type 2 diabetes mellitus who consumed 200 milliliters of soy milk per day enriched with L. plantarum A7 (2 × 10⁷ CFU) have been observed that a decrease in fasting plasma glucose compared to control [180]. In another trial, 68 adults with type 2 diabetes mellitus who took probiotic supplements containing L. acidophilus, Lactobacillus lactis, Lactobacillus bifidum, Lactobacillus longum, and Lactobacillus at a dose of 3 × 10¹⁰ CFU in 250 mL water twice a day for 12 weeks showed a decrease in fasting plasma glucose, fasting plasma insulin, hemoglobin A1c, homeostatic model assessment of insulin resistance [181]. Therefore, it can be said that probiotics can be used as therapy for both type 1 and type 2 diabetes by decreasing inflammation and beta cell death, respectively.

Skin diseases (anti-aging)

Skin is the most important outer barrier of the human body. There are a lot of factors, including intrinsic (hormonal and genetic) and extrinsic (environmental: pollution, smoking, radiation, etc.), that cause skin aging rapidly. Due to the increased metalloproteinase activity, the capacity to inhibit ROS increases the pH of the skin, and it elevates skin aging. Research showed probiotics and their metabolites play a role in changing the pH of the skin. During metabolism, probiotics produce acidic molecules and can lower the pH of the skin and help in controlling enzymatic activity, limiting the harmful bacterial growth in the skin, thus maintaining a good skin condition [182–184]. The aging process, along with environmental stress, ROS, and cellular damage, may hamper the natural antioxidant defense system. It was observed by the researchers that probiotics can reestablish these factors and slow down the aging process effectively [183]. Also, probiotics showed a reduction in the negative effects of UV exposure. For instance, people were given L. johnsonii and carotenoids for ten weeks before being exposed to both simulated and natural sunlight. Results showed that experimental supplementation improved immune system homeostasis recovery following UV-ray [185]. exposure and decreased UV-induced “Langerhans cell density” when compared to a placebo. Human volunteers who consumed 10¹⁰ CFUs/day of L. plantarum HY7714 for 12 weeks saw improvements in skin elasticity, gloss, and moisture content, as well as a reduction in the depth of wrinkles [186].

Cardiovascular disease

A series of abnormal events that arise in the blood circulatory system involving veins, capillaries, and arteries is known as cardiovascular disease. If it happens, it is treated with medication, lifestyle changes, and sometimes surgery. Intestinal microflora can act as a fat-reducing factor, and with probiotic strains, blood lipid levels, such as low density lipoprotein cholesterol (LDL-C), can be controlled, which is a primary risk factor [187–189]. The endothelium, which creates the inner layer of blood vessels, helps in regulating healthy blood pressure in the human body with the production of sufficient NO, but the is some circumstances low level of NO or a lack of NO production leads to endothelial dysfunction, thus leading to cardiovascular disease [190].

Probiotics were previously found to be useful in reducing cholesterol in a group of people who consumed fermented yogurt [191]. Furthermore, the gut microbiome modulates cardiovascular health by metabolizing dietary components into bioactive compounds, such as SCFAs and trimethylamine N-oxide (TMAO). These metabolites influence critical physiological processes, including cholesterol metabolism, blood pressure regulation, and endothelial function [192]. Probiotics have been shown in studies to lower cholesterol levels. For instance, live L. fermentum 11976 significantly decreased the levels of LDL-C, triglycerides, and total cholesterol in hypercholesterolemic hamsters [193]. Together with other beneficial food ingredients, these bacteria alter metabolic pathways by reducing the synthesis of endogenous cholesterol [194] and digesting dietary fiber to produce SCFAs. Reducing and managing the degree of inflammation in inflammatory markers [195] is another consequence. In one study, rats were supplied with a high-cholesterol diet and also in addition, supplemented with lyophilized L. plantarum MA2 showed comparable outcomes to those fed a high-cholesterol diet alone, with a significant reduction in total cholesterol, LDL-C, and triglyceride levels [196]. Recent research has connected improved lipid profile levels to the mechanism of reducing inflammation. Emerging evidence indicates that specific gut microbiota compositions are linked to elevated blood pressure and reduced responsiveness to antihypertensive therapies, whereas other microbial profiles may confer protection against cardiovascular risk factors [31].

Respiratory disease

There is clinical emerging evidence supporting the therapeutic use of probiotics for allergic rhinitis (AR) and other respiratory diseases. In a randomized controlled trial, L. paracasei LP-33 was found to significantly improve nasal symptom scores in AR subjects. Probiotics have been found to have general efficacy against viral respiratory infections, including influenza and COVID-19, through multiple mechanisms: 1. Antimicrobial production: Bacillus subtilis 3 synthesizes aminocoumarin, a crucial antibiotic that repels respiratory pathogens and enhances host immunity. 2. Barrier reinforcement: L. rhamnosus GG reinforces epithelial strength in intestinal and pulmonary tissues, as confirmed in human trials. 3. Immunomodulation: Probiotic strains increase regulatory T cell counts and reduce pro-inflammatory cytokines (IL-6, TNF-α) in systemic infections. These properties make probiotics suitable adjuvants to SARS-CoV-2 and ventilator-associated pneumonia (VAP). Through the exclusion of pathogens by competitive exclusion and maintenance of microbiota homeostasis, probiotics can reduce VAP incidence without adding to antibiotic resistance [185].

Cancer

Probiotics are very promising adjuvant drugs in cancer treatment due to their ability to modulate gut microbiota and locally and systemically enhance immune responses. The therapeutic effectiveness of probiotics, as demonstrated by preclinical studies, in inhibiting tumor development, growth, and metastasis in various cancer models, including chemically-induced and transplantable tumors is remarkable. The anti-carcinogenic effect is exerted through multiple mechanisms whereby metabolites from the probiotics, like bacteriocins, immunomodulatory peptides, and SCFAs, are key. These substances regulate major oncogenic pathways by competing exclusion of procarcinogenic pathogens, butyrate production augmentation, carcinogenic bile acid inhibition, and binding of diet-derived mutagens. At the molecular level, probiotics inhibit NF-κB-dependent proliferative markers (cyclin D1, COX-2) and anti-apoptotic proteins (Bcl-xL, Bcl-2), promote pro-apoptotic TRAIL signaling and mTOR/4EBP1-mediated cell cycle arrest. They also inhibit tumor angiogenesis and aberrant crypt foci formation. These mechanisms, being multifaceted, provide the foundation for the use of probiotics as non-invasive adjuvants to conventional cancer therapy, particularly in microbiota-associated cancers [31].