Clinical research review: usefulness of bovine lactoferrin in child health

Lactoferrin (LF) is abundant in human milk and plays an important role in the health of children. Bovine LF (bLF) has high homology with human LF and has been reported to have multiple biological functions. Several clinical studies have been conducted considering these properties, which reported the usefulness of bLF. This review was aimed to provide an overview of the clinical evidence in children. We searched clinical reports investigating the effects of bLF in children and identified 36 studies on the role of bLF in infections, iron metabolism, body growth, cerebral development, and fecal microbiome. Considering the accumulated evidence, bLF may contribute to the child health, particularly by suppressing or alleviating gastrointestinal and respiratory symptoms, and improving the iron status of children with anemia or those at high risk of anemia. The dose of bLF varies depending on the expected effect and target age, but may not necessarily have to be as high as human LF in human milk. Some of the beneficial effects of bLF have not been fully validated due to limited clinical evidence or being observed in the secondary analysis of some studies. Further clinical evidence would add significant value to the use of bLF in child health.


Introduction
Lactoferrin (LF) is an iron-binding glycoprotein and well-known for its multiple biological functions such as antimicrobial and immunomodulatory activities. LF is abundant in human breast milk and its concentration is maximum during early lactation, which rapidly declines thereafter and remains relatively constant from one month to two years of lactation (Rai et al. 2014). This means that LF is an important component for infants and children even after the age of one year.
Human and bovine LF (hLF and bLF, respectively) share 69% amino acid sequence identity based on the protein sequence alignment analysis (Pierce et al. 1991), and their bioactivities as assessed in vitro or in animal models are comparable (Steijns and van Hooijdonk 2000;Lönnerdal et al. 2011). bLF is manufactured on an industrial scale and used worldwide in a variety of food products such as powdered formula. From the 1980s to the present, multiple clinical studies have been conducted in children fed bLF. In this review, we focus on clinical studies that examined the efficacy of bLF in children and provide an overview of the potential contribution of bLF intake to child health.

Materials and methods
We searched clinical reports investigating the effects of bLF in children using PubMed, Google Scholar, and additional hand searching. In this review, we identified 36 studies and obtained full-length papers. The main effects discussed in these studies were protection from infection, modulation of iron metabolism, body growth, cerebral development, and regulation of the fecal microbiome.

Results and discussion
Suppressive and alleviative effects on symptoms of infections We identified 10 published clinical studies that investigated the effects of bLF on the symptoms of infections (Table 1).
A meta-analysis of clinical studies on adults suggested that bLF could increase the eradication rate and suppress the side effects of anti-H. pylori therapy (Zou et al. 2009). On the other hand, clinical studies on the effect of bLF on H. pylori infection in children are limited and have not yet been fully investigated (Okuda et al. 2005). As an overall trend, numerous clinical studies on H. pylori eradication by bLF were conducted in the early 2000s, but only a few studies have been conducted in recent years, probably owing to the improvement in drinking water and standard anti-H. pylori therapy.
A recent meta-analysis including studies on adults and children suggested that bLF could reduce the risk of respiratory tract infections (Ali et al. 2021) supporting the usefulness of bLF in the management of respiratory infections in children.
Based on basic research, the mechanisms underlying these protective effects of bLF can be attributed to its antibacterial, antiviral, immunomodulatory activities, and barrier functions.
bLF exhibits antibacterial activity against various bacteria mainly by sequestering iron, which is essential for bacterial growth (Arnold et al. 1977), or by directly binding with lipopolysaccharide (LPS) embedded in the gram-negative bacterial surface, leading to destabilization and permeability of the bacterial outer membrane (Drago-Serrano et al. 2012). bLF and lactoferricin, an antimicrobial peptide derived from pepsin digestion of bLF, have been demonstrated to exhibit antibacterial activity against H. pylori in vitro (Dial et al. 1998).
Antiviral properties of bLF have also been extensively studied (Oda et al. 2020a). In vitro studies have suggested that bLF exerts antiviral activity against viruses that infect the gastrointestinal tract through inhibition of viral attachment to their target cells and suppression of viral replication via interferons (IFNs) (Superti et al. 1997;Ishikawa et al. 2013;Shin et al. 2017;Oda et al. 2021).
bLF also exerts immunomodulatory effects. Intelectin, a receptor for LF, is expressed in the small intestine, particularly the epithelium overlying the Peyer's patches (Talukder et al. 2003) where immune cells are located, suggesting that it may mediate the interaction between bLF and immune cells. In particular, orally-administered bLF activates natural killer (NK) cells via type I IFN (Kuhara et al. 2006). Moreover, bLF activates CD4 + and CD8 + T cells and enhances the production of IgA from plasma cells (Arciniega-Martínez et al. 2016). Recently, bLF has been reported to activate human plasmacytoid dendritic cells in adults, which produce type I IFN, activate NK cells, CD4 + and CD8 + T cells, and B cells, and play an important role in systemic viral clearance by modulating innate and adaptive immune responses (Miyakawa et al. 2021a). In contrast, bLF suppresses the production of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α (Zimecki et al. 1999).
bLF induces the growth and differentiation of enterocytes, strengthens the tight junctions, and thus enhances the integrity of intestinal epithelia . bLF also induces the production of type I and type III IFNs, antiviral cytokines from enterocytes (Shin et al. 2017;Miyakawa et al. 2021b). Collectively, orally-administered bLF exerts local antibacterial and antiviral activities, local and systemic immunomodulatory activity, and strengthens the intestinal barrier, leading to suppression and alleviation of gastrointestinal and respiratory symptoms.
The dose of bLF varied in different studies, and ranged from 35.8 to 1000 mg per day. Some studies were conducted using a high dose of bLF, which is almost equivalent to the amount of hLF that infants could consume from breast milk; on the other hand, some studies were conducted using a relatively low dose of bLF as that contained in commercially available formula. In a study, dose-dependent efficacy was observed with 91.5 mg of bLF being more effective than 47.2 mg of bLF in suppressing gastrointestinal symptoms (Chen et al. 2021). While, 200 mg of bLF has been reported to prevent acute gastrointestinal and respiratory symptoms Miyakawa et al. 2021b) and shorten the duration of infectious diseases (Oda et al. 2020b) in adults. Considering body weight, the dose of bLF might not necessarily have to be as high as that found in human breast milk to exert beneficial effects in children.
Two studies (Yen et al. 2011;Björmsjö et al. 2020) showed no beneficial effects of bLF fortification; subject characteristics, public health environment, or disease burden might have affected these results. The quality of bLF such as iron saturation, contaminant, and degradation might vary depending on the suppliers, and it might also affect the results. Properties of bLF were compared among suppliers by several in vitro assays, but no consistent features were observed (Lönnerdal et al. 2021). It is desirable to develop good in vitro assay models for each biological functions to be evaluated in human, and assess the activities of bLF prior to conducting clinical studies.
Five studies (Egashira et al. 2007;Zuccotti et al. 2009;Tolone et al. 2012;Cheng et al. 2019;Li et al. 2019) showed the beneficial effects of bLF in combination with other components often contained in breast milk or formula such as bifidobacteria, probiotics, lactulose, lysozyme, or milk fat globule membrane (MFGM), although relative contribution of each component was not determined (Table 2). Combination with other functional components may produce a synergistic positive effect on the child health. Some clinical studies have investigated the effect of bLF on immune responses in children and demonstrated its immunomodulatory effect to some extent ( Table 3).
Modulation of T cells, phagocytes, monocytes, and Th1/Th2 balance has been observed in HIV-infected children after bLF intake (Zuccotti et al. 2006(Zuccotti et al. , 2007. However, it is uncertain whether these effects can be observed in healthy individuals. Combination of bLF with curcumin also modulated T cells and monocytes in children with recurrent respiratory tract infection (RRTI) (Zuccotti et al. 2009) ( Table 2). Chen et al. observed dose-dependent effects of bLF on fecal biochemical indices such as human beta defensin 2 (HBD-2), cathelicidin LL-37, secretory IgA (sIgA), butyrate, and calprotectin in children (Chen et al. 2021).
These immunomodulatory actions could be a mechanism underlying the suppression of diarrhea and the regulation of the microbiome. Nonetheless, there are some limitations, such as few studies, small sample size, study design, and subject characteristics, including children with illness; therefore, additional clinical studies are needed to clarify whether bLF modulates the immune responses in children.

Improvement in iron metabolism
We identified 16 published clinical studies that investigated the effect of bLF on iron metabolism (Table 4).
Among the seven studies (Fairweather-Tait et al. 1987;Schulz-Lell et al. 1991;Chierici et al. 1992;Lönnerdal and Hernell 1994;Hernell and Lönnerdal 2002;King et al. 2007;Björmsjö et al. 2020) investigating the effect of bLF in healthy and near-term newborns, five (Fairweather-Tait et al. 1987;Schulz-Lell et al. 1991;Lönnerdal and Hernell 1994;Hernell and Lönnerdal 2002;Björmsjö et al. 2020) did not show any beneficial effect on iron absorption or iron status after bLF intake during the first six months. Healthy term infants with normal birth  Hb, serum iron, and ferritin levels between the groups after 1.5 and 3 months of treatment, with the highest values in the bLF + iron group followed by the iron group weight are born with iron stores sufficient for their growth during the first six months of life (Dallman et al. 1980). In some of the above studies (Lönnerdal and Hernell 1994;Hernell and Lönnerdal 2002;King et al. 2007;Björmsjö et al. 2020), after intervention, hematological parameters such as hemoglobin (Hb) or serum ferritin showed relatively high values in accordance with the WHO guidelines (World Health Organization 2001). Therefore, it was suggested that bLF was administered when iron demand was not high, and these conditions might have affected the results. During the second half of infancy, the requirement for exogenous iron rapidly increases as the infant grows. In a study of infants who were previously exclusively breastfed and assumed to be at a high risk of anemia, after 3 months of bLF intervention, the iron status, including Hb, serum ferritin, transferrin receptor-ferritin (TFR-F) index, and total body iron content (TBIC) was improved, leading to a lower prevalence of anemia, iron deficiency (ID), and iron deficiency anemia (IDA) (Chen et al. 2015a). In addition, several studies on anemic children have shown that bLF consistently improved the iron status (El-Khawaga and Abdelmaksoud 2019; Chen et al. 2020;Mikulic et al. 2020;Kamal et al. 2021;El-Asheer et al. 2021;Omar et al. 2021;Atia et al. 2021). A recent meta-analysis of studies on pregnant women suggested that bLF improved IDA (Abu Hashim et al. 2017), and these findings are consistent with the results obtained from studies on children.
The mechanisms underlying the improvement in anemia by bLF are likely related to its anti-inflammatory activity. It has been reported that a part of anemia is associated with high levels of IL-6 that upregulates hepcidin, a peptide hormone mainly synthesized by hepatocytes. Hepcidin binds to ferroportin, causes its degradation, and leads to a significant decrease in iron export from the cells into plasma, and consequently, IDA may be detected (Rosa et al. 2017). Several studies on pregnant women (Paesano et al. 2009(Paesano et al. , 2010(Paesano et al. , 2012(Paesano et al. , 2014Lepanto et al. 2018) suggest that bLF decreases IL-6 levels, restores iron delocalization, and improves hematological parameters. Atia et al. observed a decrease in serum IL-6 and hepcidin levels in anemic children after bLF ingestion (Atia et al. 2021), which suggests that bLF improves iron metabolism in children by a mechanism similar to that reported in adults. According to the secondary analysis of some studies, ingestion of bLF protected children from infections (Chen et al. 2016(Chen et al. , 2021. This might be attributed to the decrease in inflammatory cytokines, including IL-6. Furthermore, Mikulic et al. reported that apo-bLF might promote dietary iron absorption in anemic infants (Mikulic et al. 2020). bLF was reported to solubilize a > 70-fold molar equivalent of iron at neutral pH in vitro (Kawakami et al. 1993), suggesting that ingested bLF could solubilize iron in the intestine, promote iron absorption, and consequently, improve anemia. Furthermore, bLF may improve intestinal iron absorption in children by promoting the maturation of intestinal epithelia (Yang et al. 2014;Hu et al. 2019).

Growth promotion
We identified 8 published clinical studies that reported the effects of bLF on anthropometric indices (Table 5).
Several studies have reported that bLF promotes body growth such as increase in height and weight (Hernell and Lönnerdal 2002;King et al. 2007;Ochoa et al. 2008;Chen et al. 2015a;Li et al. 2019).
We identified 3 published clinical studies that reported the effects of bLF with other components such as lysozyme and MFGM on anthropometric indices (Table 6). Two studies (Cheng et al. 2019;Li et al. 2019) showed the beneficial effects, although relative contribution of each component was not determined.
In a neonatal animal model, dietary bLF has been suggested to promote intestinal maturation such as the development of crypt-villus structures and mucosal barrier, and increased enzyme activity (Yang et al. 2014;Hu et al. 2019). Intestinal development is important for the digestion and absorption of nutrients; therefore, bLF might have promoted body growth by improving the absorption of nutrients. Furthermore, bLF has been reported to suppress fat accumulation and promote lipolysis (Ono et al. 2013). bLF may facilitate efficient utilization of milk fat as an energy source. However, the intake of solid food or subtle differences in the amount of total protein in the test food can also affect the infant's body growth. To evaluate the growth-promoting effect of bLF, further studies are needed under strict control of the test food

Cerebral development and fecal microbiome
Several animal studies have suggested the potential involvement of bLF in neuroprotection, neurodevelopment, and learning. bLF administration through lactation showed a protective effect on impaired cerebral development in intrauterine growth restricted rats (Somm et al. 2014) and neuroprotective effect against hypoxia/ischemia-induced and LPS-induced brain injury Ginet et al. 2016). bLF has also been reported to promote early cognitive function and learning in neonatal piglets (Chen et al. 2015b).
On the other hand, there are limited clinical reports on the effect of bLF or bLF with other components on cerebral development (Tables 7, 8). One of them reported that bLF improved sleep quality (Miyakawa et al. 2020), but it was a secondary analysis of the study. Another reported improved neurodevelopment when bLF was administered in combination with MFGM (Li et al. 2019), but MFGM itself was also reported to have neurodevelopmental effects (Timby et al. 2014) and the net contribution of bLF did not evaluated. Due to above limitations, further clinical evidence is required. Differences in the fecal microbiome of breastfed infants and formula-fed infants have been indicated in previous studies (Yoshioka et al. 1983;, and the association of the fecal microbiome with immune function and cerebral development has also been discussed. In vitro studies have reported that bLF exhibits antibacterial activity against a wide range of pathogenic bacteria (Jenssen and Hancock 2009), whereas bLF and its digestive peptide show bifidogenic activity, particularly against infant-representative species (Oda et al. 2013). Increased secretion of HBD-2, cathelicidin, sIgA, and butyrate by bLF may also contribute to the regulation of the microbiome (Chen et al. 2021). However, no consistent effect of bLF or bLF with other components on the fecal microbiome was observed (Tables 7, 8). Compared with other components of breast milk such as human milk oligosaccharides and sIgA, the effect of LF on the fecal microbiome may be limited.

Conclusions
bLF may contribute to the child health, particularly by suppressing or alleviating gastrointestinal and respiratory symptoms and improving the iron status of children with anemia or those at a high risk of anemia. The dose of bLF varies depending on the expected effect and target age, ranging from 35.8 to 1000 mg per day, and it may not necessarily have to be as high as that found in human breast milk. Subject characteristics, field conditions and quality of bLF may have led to inconsistent results. In addition, some beneficial effects of bLF were observed in the secondary analysis of the study. Further clinical evidence as the primary endpoint is needed to accurately describe the beneficial effects of bLF on the child health.  Funding This research received no external funding.

Data availability
The data presented in this review are openly available on PubMed, Google Scholar, and the journal website.

Declarations
Conflict of interest All authors are employed by Morinaga Milk Industry.
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