Regular Consumption of Fortified Growing-up Milk Attenuates Upper Respiratory Tract Infection among Young Children in Indonesia: A Retrospective Cohort Study

1. INTRODUCTION

A functional immune system is essential to protect children against multiple pathogens. The immune system, along with other body systems, must develop well from an early age to achieve that purpose [1]. The proper development and functionality of the immune system are profoundly influenced by the relationship between the immune system and gastrointestinal microbiota [2]. The proper development of gastrointestinal microbiota in early life has been shown to be associated with protection against various diseases in adulthood, including asthma, obesity, and intestinal inflammation [2], and that gastrointestinal microbes and their produced metabolites are important in activating various players of the immune system [3-6]. The ecosystem of gastrointestinal microbiota within a host develops from birth and is heavily influenced by maternal (e.g., mother's gastrointestinal microbiota and vaginal infections during pregnancy) and postnatal factors (e.g., diet and antibiotic consumption). The microbiota profile of children gradually resembles the configuration of adults with a peak at 3 years of age [7].

One postulated way to support the proper development of the gastrointestinal microbiota as well as the immune system is by providing a variety of healthy foods to infants and young children. Two healthy foods of interest are prebiotics and n-3 long-chain Polyunsaturated Fatty Acids (LCPUFAs) [8]. Prebiotics are substrates that are selectively utilized by host microorganisms conferring a health benefit [9]. Prebiotics could modulate the host's health status through indirect (via metabolites produced during bacterial fermentation) and direct mechanisms (via interaction between prebiotics and surface receptors expressed by the gut-associated epithelial and innate immune cells) [10]. For example, it has been suggested that prebiotic intake could protect against Upper Respiratory Tract Infection (URTI) because the prebiotic supplementation could provide an anti-inflammatory effect at the site of infection as well as within the circulation [11].

An example of prebiotics is non-digestible oligosaccharides, including galactans and fructans, because they could enrich Lactobacillus and/or Bifidobacterium species, resulting in health benefits [9]. The health benefits of prebiotic intake in infancy have been investigated, either via individual supplementation, such as inulin [12, 13], Galacto-oligosaccharides (GOS) [14], or Fructo-oligosaccharides (FOS) [15-17], or a combination, such as a mixture of short-chain GOS (scGOS) and long-chain FOS (lcFOS) [18-20]. Of note, a specific mixture of scGOS and lcFOS in a ratio of 9:1 has been demonstrated to be similar to the proportion found within the human milk oligosaccharides [21, 22]. Furthermore, this mixture at a dose of 0.4 – 0.8 g/100 mL could modulate the intestinal microbiota and subsequently support immune development [21, 23-25].

Similarly, LCPUFAs are important fatty acids for immune development, and a balanced profile of LCPUFAs could result in optimal immune regulation and activation [26-30]. It is known that an imbalanced ratio of n-6:n-3 LCPUFAs could induce an inflammatory condition [31]. Thus, an increased dietary intake of n-3 LCPUFAs could balance the LCPUFAs ratios and support proper immune development, particularly during infancy or young childhood. Some of the most important n-3 LCPUFAs in the diet are Alpha-linolenic Acid (ALA), Eicosapentaenoic Acid (EPA), and Docosahexaenoic Acid (DHA). ALA is the precursor of n-3 LCPUFA, which is converted into EPA and DHA. While ALA is found in linseeds, rapeseed oil, and walnuts, EPA and DHA are found in fatty fish [32].

It has been reported that an intake of Growing-up Milk (GUM) supplemented with scGOS/lcFOS/n-3 LCPUFAs among young children could reduce the risk of upper respiratory tract or gastrointestinal infection [33]. However, the risk reduction was of borderline statistical significance, indicating that similar studies need to be performed [33]. We, therefore, aimed to assess whether young children in Indonesia who consumed scGOS/lcFOS /n-3 LCPUFAs-fortified GUM routinely for a minimum of 6 months would have a better immune system, as indicated by the reduced incidence of URTI in the subsequent 3 months.

2. MATERIALS AND METHODS

The study was a retrospective cohort clinical study conducted across Indonesia, covering all provinces in Java Island and more than ten provinces on other islands, between September and December 2022.

3. RESULTS

There were 154 children recruited into this study, comprising 78 and 76 subjects in the exposed and unexposed groups, respectively. The recruitment flow is explained in Fig. (1). Table 1 displays the demographic characteristics of those subjects and their respective mothers. The overall characteristics of the subjects were similar between the two groups, except that in the exposed group, there were more children born by the caesarian section (45; 57.7%) and fewer children of low birth weight (6; 7.7%). These characteristics were not statistically different (P>0.05). In addition, information on the number of school-age siblings was collected, in which there were more children with school-age siblings in the exposed group (22 out of 30 respondents with sibling: 73.3%) than in the non-exposed group (18 out of 33 respondents with sibling: 54.5%), but the difference was not statistically different (P>0.05). There were more mothers with university degrees (58: 74.4%) and more working mothers in the exposed group (59: 75.6%) than in the non-exposed group (38: 50.0% and 44: 57.9%, respectively). These characteristics were statistically different (P=0.004 and P=0.019, respectively). Next, the clinical characteristics of the subjects are shown in Table 1. Information on nutritional and immunization statuses was obtained as they might determine the proper development of the immune system among young children. However, there was no statistical difference observed within the nutritional and immunization statuses between the exposed and unexposed groups (P>0.05).

The bivariate analysis was subsequently performed to assess the impact of routine consumption of scGOS/lcFOS/n-3 LCPUFAs-fortified GUM on contracting URTI within the past 3 months. Information on three aspects was collected, i.e., the incidence and duration of URTI as well as the number of medical visits during an episode of URTI (Table 2). It was observed that subjects within the exposed group had statistically better protection against URTI as compared to the ones in the non-exposed group (RR=0.62; 95% CI=0.41-0.92; P=0.015) with an absolute risk reduction of 19.2% (95% CI= 4.0-34.4%). The exposed group exhibited a shorter duration of URI and fewer visits to the physician during the illness as well, but the differences were not statistically significant (P>0.05).

The multivariate analysis was finally performed to assess variables that might modulate the risk of the exposed group towards contracting URTI. Two statistically significant characteristics (Table 1, i.e., the educational and working statuses of the mothers, were included in the analysis. The nutritional and immunization statuses of the subjects were included as well, despite the differences being not statistically different, because these statuses might influence the adequate maturity of the immune system among young children. Table 3 demonstrates that upon adjustment of the maternal, educational, and working statuses as well as the subjects’ nutritional and immunization statuses, the exposed group retained protection against URTI as compared to the non-exposed group (RR=0.42; 95% CI=0.21-0.85; P=0.017). This suggests that daily consumption of scGOS/lcFOS/n-3 LCPUFAs-fortified GUM for a minimum of 6 months could provide adequate protection against URTI up to 58% within the past 3 months.

4. DISCUSSION

We have hereby reported a retrospective cohort study assessing URTI (within the last 3 months from the time of interview) among Indonesian children aged 18-36 months who consumed a minimum of 500 mL per day of GUM supplemented with 0.6 g/100 mL of scGOS/lcFOS (9:1) and 0.136 mg/mL of n-3 LCPUFAs for at least 6 months. Our retrospective study design has provided a quick and inexpensive way to obtain data [36], in which we chose 6 months of GUM consumption for young children as the minimum period to start benefiting from its health benefits. In addition, the subsequent 3-month recall period was chosen to allow observation as accurately as possible.

We observed that young children within the exposed group, who routinely consumed scGOS/lcFOS/n-3 LCPUFAs-fortified GUM, had a lower relative risk of contracting URTI (RR=0.62; 95% CI=0.41-0.92; P<0.05) as compared to the ones within the non-exposed group. This protective effect was supported by the multivariate analysis upon adjustment of several variables (RR=0.42; 95% CI=0.21-0.85; P<0.05). Those variables have been shown to be associated with the incidence of URTI, including sub-optimum nutritional status [37], incomplete immunization status [38], working mothers [39], as well as low levels of maternal education [40]. Hence, it was interesting to observe that after adjusting those variables, the protective effect of consuming fortified GUM remained. Our findings were partly supported by a previous finding [41], which demonstrated that the intake of infant formula supplemented with 0.4 g/100 mL of scGOS/lcFOS with a ratio of 9:1, as compared to the intake of standard infant formula, resulted in a lower number of URTI episodes and lower antibiotic intake. Furthermore, our findings have reinforced the published result on young children who consumed GUM supplemented with 0.8 g/100 mL of scGOS/lcFOS as well as n-3 LCPUFAs [33], suggesting protection against URTI due to a regular consumption of milk supplemented with certain prebiotics and omega-3 fatty acids.

Prebiotics have been extensively demonstrated to modulate intestinal microbiota and provide health benefits to the host [42, 43]. Breast milk-derived human milk oligosaccharides, the first prebiotics consumed by many infants, could promote the dominance of Bifidobacterium species within the intestinal tract during early life, which supports infant health [44]. It has also been shown that human milk oligosaccharides exhibit antimicrobial activity against gram-positive bacteria, which could prevent skin infection of the mother’s breast and protect infants from respiratory pathogens [45]. Other prebiotics have been reported to be utilized in milk formula as well, including resistant starch, pectin, gums, and oligosaccharides (e.g., xylo-oligosaccharides, gluco-oligosaccharides, GOS, and FOS) [9]. A recent meta-analysis of 17 clinical trials indeed suggested oral prebiotic supplementation in infants and children to significantly reduce the risk of a subject contracting respiratory tract infection [8]. This evidence has enhanced the conclusion of our study. Similarly, De Cosmi et al., Bryan D et al., and Pastor N et al. have declared how prebiotics can enhance the role of inflammatory mediators against infection [46-48]. This could be the underlying biomedical process of our study.

Our study has several limitations. First, there might be a recall bias as the data were collected retrospectively. Second, the data were collected only through guided interviews via phone calls. No clinical examination/visit was conducted to confirm the reported symptoms. Third, the data were collected from children living in the urban setting of particular regional areas in Indonesia. Despite that the subjects have been recruited from the majority of provinces in Indonesia, the sample size calculation has not been representative of the Indonesian population, especially from rural settings.

CONCLUSION

We observed routine consumption of scGOS/lcFOS/n-3 LCPUFAs-fortified GUM among Indonesian children aged 18-36 months old to have a protective effect on URTI compared to those not routinely consuming it. This result suggests that GUM supplemented with certain prebiotics and n-3 LCPUFAs for young children may provide possible protection against respiratory tract infection.

AUTHORS' CONTRIBUTIONS

It is hereby acknowledged that all authors have accepted responsibility for the manuscript's content and consented to itssubmission. They have meticulously reviewed all results and unanimously approved the final version of the manuscript.

REFERENCES