The role of probiotics in children’s immunity and overall health has garnered significant attention in recent years. Probiotics, defined as live microorganisms that confer health benefits to the host when administered in adequate amounts, are known to play a crucial role in maintaining the balance of gut microbiota, which is essential for proper digestive function and immune response. The colonization of the gut microbiota begins at birth and continues to evolve during early childhood, with the first three years being crucial for its maturation (1). Recent studies indicate that the intrauterine environment is non-sterile, suggesting that fetal intestines may be colonized even prenatally. This underscores the importance of a healthy maternal environment for optimal microbiome development from the earliest stages of in utero development (2). The composition of the intestinal microbiome, which serves as a distinct fingerprint unique to each individual, is influenced by a variety of factors, such as the mode of delivery, diet, the environment in which the child grows, and the use of various medications, particularly antibiotics. Understanding the factors that contribute to dysbiosis can aid in developing strategies to promote a healthy microbiome in children, which is essential for preventing various diseases later in life (3). Altered function and composition of gut microbiota during early childhood can lead to a variety of health issues. The improper balance of gut microbiota may disrupt the maturation of the immune system. This increases the likelihood of an exaggerated Th2 immune response, characterized by elevated production of specific molecules such as interleukins (IL-4, IL-5, IL-13) and IgE antibodies, which are key players in the development of allergic diseases, including asthma, eczema, and food allergies (4). Additionally, early-life dysbiosis is associated with an increased risk of metabolic diseases such as diabetes and obesity, as well as a higher susceptibility to developing inflammatory bowel diseases during adolescence (3).

Thus, in children whose microbiota is still developing and maturing, the early establishment of a healthy gut microbiome is linked to long-term health outcomes and the introduction of probiotics may offer protective effects against various disorders. Research indicates that probiotics may help prevent and manage common pediatric health conditions such as diarrhea, constipation, and colic. Moreover, emerging evidence suggests that the positive effects of probiotics extend beyond gastrointestinal health, including enhanced immune function, reduced inflammatory processes, mitigation of allergic reactions, improved skin health, regulation of metabolic disorders such as obesity and type 2 diabetes, and even support for mental well-being through the gut-brain axis (5–7).

Probiotics exert their effects through various mechanisms. These mechanisms include modulation of the gut microbiota composition by promoting the growth of other beneficial bacteria and enhancing the production of their metabolic byproducts. They also compete with pathogenic microorganisms for adhesion sites and nutrients, helping to prevent their colonization. Additionally, probiotics strengthen the intestinal barrier by reducing permeability, which in turn lowers inflammation and the risk of allergic reactions (8). Probiotics achieve many of their beneficial effects through the production of bioactive metabolites, such as short-chain fatty acids (SCFAs), bacteriocins, exopolysaccharides, vitamins, and neuroactive substances like gamma-aminobutyric acid (GABA) which play critical roles in their functionality. SCFAs such as acetate, propionate, and butyrate help maintain the gut’s protective barrier, enhance immune function, and provide energy to intestinal cells. These processes are particularly important in infants and young children, whose immune and digestive systems are still developing (9). Additionally, probiotics play a significant role in the absorption of essential nutrients in children. A recently published study has shown that these beneficial microorganisms can increase serum concentrations of vital vitamins such as vitamin D and A, along with minerals like calcium and zinc, thereby improving nutritional status and potentially boosting immunity (10). Probiotics can enhance respiratory, digestive, and immune functions due to their ability to promote humoral immunity maturation, particularly by increasing IgA antibody production (4).

What is important to know is that the effectiveness of probiotics can vary significantly based on individual health conditions and the specific strains used (9). This variability highlights the importance of personalized approaches to probiotic supplementation; what benefits one individual may not necessarily extend to another due to differences in gut microbiota composition or underlying health issues (11).

Of particular importance, yet often overlooked and insufficiently explored, is the fact that probiotics may interact with other drugs, influencing their absorption, metabolism, and effectiveness. Additionally, probiotics may modulate the activity of drug-metabolizing enzymes and transporters, thereby altering drug disposition (12–15). Understanding these interactions is vital for optimizing therapeutic outcomes, as inappropriate combinations can lead to reduced drug efficacy or an increased risk of adverse effects. Therefore, in addition to probiotic benefits it is essential to carefully evaluate the use of probiotics alongside medications, underscoring the importance of personalized approaches to treatment.

Since the effectiveness of probiotics depends on various factors, including the selection of the appropriate probiotic strain, the correct dose, and the duration of use, parental education about probiotics is of utmost importance. Well-informed parents are more likely to understand the benefits and limitations of probiotic therapy, which may lead to its appropriate use and maximize its potential health benefits for their children. Several studies have shown that parents’ awareness and practices regarding probiotics vary widely across different countries, with significant gaps in understanding (16–18).

To the best of our knowledge, no studies on this topic have been conducted in Serbia thus far. The only existing study, our previous research, assessed the knowledge, attitudes, and practices of health science students regarding gut microbiota and probiotics, revealing significant knowledge gaps and deficiencies in their practical application (19). Therefore, the aim of this study was to assess the knowledge, attitudes, and practices of parents regarding the use of probiotics in preschool-aged children, as well as to identify factors associated with probiotic supplementation. Additionally, the goal was to identify key factors that influence parents’ knowledge and attitudes toward probiotics and to guide future educational efforts aimed at enhancing awareness and promoting proper probiotic use.

As insufficient parental awareness about probiotics may lead to the underuse of probiotics in appropriate clinical indications, improper strain selection, incorrect therapy duration, and neglect of potential drug interactions, all of which can compromise their efficacy and safety, this study aimed to examine the knowledge, attitudes, and previous experiences of parents regarding the use of probiotics in preschool-aged children in Serbia. To the best of our knowledge, this is the first study of this type conducted in Serbia. The high level of interest in this topic is reflected in the substantial response rate, with 1,625 respondents completing the questionnaire.

Since only 30.9% of respondents exhibited good knowledge about probiotics, the study highlights several crucial areas where parental understanding of probiotics could significantly influence their decisions regarding the use of these supplements. While parents generally understand basic concepts such as the definition of probiotics and their role in the immune system, their knowledge, attitudes, and practices remain limited in key areas, including strain-specific effects, appropriate duration of use, safety considerations, and potential interactions with medications.

An important aspect is the insufficient understanding of probiotic strain specificity, as clinical effects may vary depending on the target condition, which could lead to unrealistic expectations regarding their efficacy. While single-strain probiotics offer strain-specific and well-documented benefits, for example, Lactobacillus rhamnosus GG in preventing necrotizing enterocolitis, multi-strain formulations are often believed to provide broader health advantages due to potential synergistic interactions among different strains (21, 22). These synergistic effects may result in enhanced immune modulation, improved gut colonization, or broader antimicrobial action (23, 24). However, combining strains may also lead to antagonistic interactions, where one strain inhibits the growth or function of another, potentially reducing the overall efficacy or leading to unpredictable outcomes (25). Despite these theoretical risks, strong evidence of clinically relevant antagonism is currently limited. The decision to use single- or multi-strain probiotics should therefore be based on the specific health indication, available clinical evidence, and the safety profile of the selected strains.

One particularly concerning gap is the lack of knowledge regarding probiotic use during pregnancy, which suggests inadequate access to reliable information or the presence of conflicting opinions among healthcare professionals. It is essential to provide parents with clear guidelines on the safety and proper use of probiotics during this period. Studies have demonstrated that probiotic supplementation is safe during pregnancy and can significantly reduce nausea, vomiting, and constipation, thereby improving the quality of life of pregnant women (26). Additionally, probiotic use during pregnancy may have a protective role against conditions such as preeclampsia (27), vaginal infections, gestational diabetes (28), and even childhood diseases by influencing the newborn’s microbiome (29). The World Allergy Organization also supports probiotic supplementation for pregnant women at high risk of allergies (30).

Furthermore, many parents lack sufficient knowledge about the interactions between probiotics and medications. Although parents are moderately informed about the basic principles of using probiotics with antibiotics, many are not well-informed about the correct time intervals between taking these supplements and possible interactions with other medications. Numerous studies have confirmed that probiotics can alter drug pharmacokinetics and pharmacodynamics, which can lead to improved or reduced therapeutic efficacy, depending on the type of probiotics and medications used (12, 15, 31–34). Notably, this lack of knowledge is not limited to parents. A recent study conducted among university students of medical sciences in Serbia, who represent future healthcare professionals, revealed similar gaps in understanding regarding probiotic interactions with medications (19). These findings indicate a broader educational challenge that requires improvements in curricula and educational programs, both for the general population and for future medical professionals.

Similarly, several studies conducted in different countries have demonstrated significant variation in parental knowledge of probiotics, particularly regarding their definition, safety, and effects. For instance, a study in Turkey found that only 20.2% of mothers were familiar with the term “probiotics,” while 33.1% recognized specific probiotic products (35). In contrast, a study in China reported that while 95.7% of parents had a basic understanding of probiotics, they lacked the ability to distinguish high-quality probiotic products (36). Meanwhile, a survey in Australia found that 52% of parents were familiar with the term “probiotics” (16). These discrepancies suggest that factors such as educational background, socioeconomic status, and regional health campaigns play a crucial role in shaping parental knowledge and attitudes toward probiotics.

The analysis of parents’ attitudes on this issue shows that the majority of parents have moderately positive attitudes toward probiotics, but also harbor certain doubts and divided opinions on some aspects of their use. Similarly, parents’ attitudes toward probiotic use are generally positive in other studies as well, particularly when they perceive benefits for their children’s health (17, 18). The majority of parents agree that the involvement of healthcare professionals in informing about probiotics is necessary, which could contribute to the proper and safe use of probiotics in children. Parents largely recognize the positive impact of probiotics on the immune system and believe their use is safe from birth. However, a moderate level of agreement with the claim that probiotics can have an effect in the prevention of atopic dermatitis and other skin problems indicates some skepticism or insufficient information about these indications. Supplementation with prebiotics and probiotics appears useful for the reduction in the severity of atopic dermatitis. A systematic review and network meta-analysis involving 21 studies indicated that certain probiotic strains, particularly Lactobacillus rhamnosus GG and Lactobacillus paracasei, likely reduce the risk of developing atopic dermatitis in children (37). Additionally, about half of the respondents express some doubt about the general effectiveness of probiotics, which may stem from various personal experiences or inconsistent recommendations in available literature and expert advice. Nearly half of the parents consider natural sources of bacteria, such as fermented foods, to be a sufficient alternative to probiotic supplements. However, it is important to emphasize that probiotic supplements contain specific strains in concentrated doses, while natural sources of beneficial bacteria typically contain a mixture of strains in smaller amounts, so their effects are not comparable to the targeted use of probiotic supplements for specific indications (38, 39). A large number of respondents share the opinion that children should not use probiotics for extended periods, which corresponds with other findings of the study, where the majority of parents administer probiotics to their children for less than a month. Some studies suggest that at least three months of continuous use is necessary to improve the immune system (40, 41). There is evidence that long-term use of probiotics in children may contribute to reducing the incidence of allergies in early childhood, lowering the risk of atopy, and establishing a foundation for a healthier immune response during the child’s growth and development (42). Furthermore, although specific recommendations for continued probiotic use after completing antibiotic therapy are not clearly defined, some studies suggest that continuing probiotic supplementation could be beneficial for restoring the gut microbiota and supporting the immune system. Consequently, probiotics are often prescribed for 1–3 weeks after the completion of antibiotic therapy (43). The observed patterns in parents’ attitudes reflect the significant role of healthcare professionals as key sources of information and highlight the need for continuous education to reduce uncertainties and ensure the proper use of probiotics in the pediatric population.

The findings suggest a significant association between parents’ knowledge and attitudes toward probiotics and their likelihood of administering probiotics to their children in the past year. Parents with greater knowledge and more positive attitudes were more inclined to use probiotics, confirming the hypothesis that education on probiotics influences the decision to use them. This result is consistent with the study by Bezek et al., which also found a positive correlation between parental knowledge levels and the inclusion of probiotic supplements in children’s diets (16). However, contrasting findings have been reported in other studies. A study in China revealed that lack of knowledge was one of the primary reasons for not using probiotics in children (18). On the other hand, a previous study conducted in Turkey indicated that many mothers continued to administer probiotics to their children despite having insufficient knowledge about their benefits and appropriate use (35). Similarly, another study conducted in China did not identify a significant correlation between knowledge and practice scores related to probiotics (36). These discrepancies highlight the complexity of factors influencing probiotic use and suggest that, beyond knowledge, cultural practices, healthcare recommendations, and personal beliefs may also play a role in parental decision-making.

The results show a weak negative correlation between probiotic use and the child’s age, suggesting that younger children were more frequently given probiotics. Similar results were obtained in a study conducted in Slovenia, where the highest prevalence of probiotic consumption was also observed among children aged between one and two years (16). This higher use of probiotics in younger children can be partially explained by the more frequent use of antibiotics in this age group (44), as probiotics are recommended as adjunct therapy to preserve the gut microbiota and reduce the side effects of antibiotic treatment. According to the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), specific strains such as Lactobacillus rhamnosus GG and Saccharomyces boulardii are recommended for the prevention of antibiotic-associated diarrhea in children (45). In line with this, the most common reason cited by parents for giving probiotics to their children was concurrent use with antibiotics. The association between probiotic use and health issues in the past year indicates that parents of children who used probiotics during this period most frequently reported gastrointestinal problems, respiratory infections, skin issues, and allergic reactions. It is important to note that, given the cross-sectional design of the study, the observed association between probiotic use and reported health problems likely reflects reverse causation. That is, parents may be more likely to administer probiotics to children who are prone to infections or gastrointestinal issues, rather than probiotics contributing to increased incidence of such problems.

Generally, the most common indication for probiotic use reported in the study is for treating gastrointestinal problems, while their use for strengthening immunity is also common, though to a lesser extent. Furthermore, respondents indicated that they used probiotics less frequently for skin problems, allergic reactions, and respiratory issues, reflecting their lower awareness of the potential benefits for these conditions. Similarly, in a study conducted by Bezek et al., the primary reasons for introducing probiotics were digestive tract disorders, with antibiotic treatment being the most frequent factor (16). In contrast, an Australian study found that the most common reason for probiotic use was the improvement of overall health (54%), with half (51%) of parents believing that probiotics enhanced their child’s general well-being (17). In a study conducted in Denmark, however, parents were generally skeptical about the preventive use of probiotics, often perceiving them as a medicine necessary only when the child is ill (46).

The results also indicate a correlation between the prevalence of medication use and probiotic use in the last year, suggesting that parents of children with more health issues may be more inclined to administer probiotics in an effort to improve their children’s health or to complement medications, particularly alongside antibiotics and for mitigating gastrointestinal side effects (47). In this regard, parents of children who did not use probiotics rated their children’s immunity as significantly higher than those whose children used probiotics. One possible explanation is that parents are more likely to turn to probiotics when they perceive their child to have a weaker immune system, due to the previously discussed role of probiotics in the immune response.

Interestingly, a significant portion of parents stated that they do not pay attention to the composition of the probiotics they give their children, which is consistent with reported lack of awareness about the different effects of various strains. A rapid review of clinical evidence found that while the concept of strain-specificity is widely accepted, there is insufficient evidence to support the claim that probiotic effects in children are strain-specific (48). This lack of evidence may contribute to parents’ confusion and lack of awareness about the importance of specific strains. Parents may choose probiotic products based on general health claims rather than targeting specific health needs, potentially missing opportunities to address particular health issues. For instance, a study conducted in Australia found that parents primarily used probiotics to promote general health, rather than for treating specific conditions (17). This approach may limit the effectiveness of probiotic use in children, as the benefits of probiotics are often strain-specific and may vary depending on the health issue being addressed.

However, most parents rely on recommendations from physicians and pharmacists when selecting probiotics. Nevertheless, nearly a fifth of parents reported that they never consult a doctor before giving probiotics, while the majority do so occasionally or regularly. Similar findings were observed in a study conducted by Bezek et al., where, although many parents followed healthcare professionals’ recommendations, almost 20% administered probiotics without prior consultation (16, 35).

Most parents did not notice any side effects from probiotics in their children; however, a small percentage reported gastrointestinal issues such as constipation, bloating, and diarrhea. These side effects are known to occur even in healthy individuals and children, though they remain relatively rare. While probiotics are widely considered safe for the general population, potential risks, albeit uncommon, should not be overlooked (43, 49). Theoretical concerns include systemic infections, excessive immune stimulation, and horizontal gene transfer (50, 51). These risks become more relevant in specific high-risk groups, such as preterm infants, immunocompromised individuals, elderly persons, and patients with underlying chronic conditions, where cases of bacteremia, endocarditis, or fungemia have been reported (52).

Regarding the concurrent use of probiotics with other medications, most parents administer them separately, indicating some awareness of potential interactions. However, due to the general lack of information and understanding about the potential interactions between probiotics and medications, as previously discussed, additional education on this topic would be highly beneficial.

The binary logistic regression analysis revealed that parents with higher education levels and those from healthcare professions have significantly greater odds of demonstrating good knowledge about probiotics. Additionally, parents who administer probiotics to their children for more than 10 days and those who consider probiotic strain selection are more likely to have sufficient knowledge and positive attitude toward probiotics. In contrast, parental age and the number of children do not significantly influence probiotic knowledge. Similarly, a cross-sectional study conducted in Turkey found that higher education levels were associated with better knowledge of probiotics, along with other factors such as employment, income, and family structure (35). However, unlike our findings, a study in China reported that parents of older preschool children (ages 5–6) were more likely to have positive perceptions of probiotic supplements, suggesting that a child’s age may play a role in shaping parental attitudes in certain populations (18). These variations highlight the influence of sociocultural and demographic factors on parental knowledge and perceptions of probiotics.

The strength of this research lies in being the first known study in Serbia to examine the knowledge, attitudes, and practices of parents regarding probiotic use in preschool children. An additional strength of the study is the large sample size, which allows for more reliable conclusions and analyses. However, several limitations should be acknowledged. First, the cross-sectional design of the study precludes any causal inferences between the examined variables. Second, the data is based on self-reports from parents, which carries the risk of subjective responses, including recall bias and social desirability bias, potentially leading to over- or under-reporting of actual knowledge and practices. Third, although efforts were made to disseminate the questionnaire through multiple social media platforms across different regions of Serbia, there is a possibility that not all regions or population groups were equally represented, which may limit the overall generalizability of the findings. Despite these limitations, the obtained results can serve as a significant foundation for further, more detailed and broader research on this topic, as well as for the creation of educational interventions aimed at improving parental awareness of probiotic use in children.

The obtained results provide valuable insight into the knowledge, attitudes, and practices of parents regarding probiotic use in children, highlighting both areas of good understanding and those that require further education. The study revealed that the majority of parents of preschool children in Serbia possess fair knowledge and attitudes about probiotics, with significant uncertainties regarding their use, composition, effects, and interactions with other medications, where the lowest level of awareness was observed. This lack of awareness may lead to inappropriate use or missed opportunities for targeted health benefits. The observed correlation between knowledge, attitudes, and probiotic practice emphasizes the importance of education and underscores the need for increased involvement of healthcare professionals in providing accurate information and guidance. The obtained results suggest that healthcare professionals are key trusted sources of information and could serve as effective channels for educational interventions aimed at improving parental knowledge and use of probiotics. Specifically, there is a need for more attention to the preventive use of probiotics and strain-specific effects. This would help ensure the proper and safer application of probiotics in the pediatric population.