Longitudinal changes in the bioactive proteins in human milk of the Chinese population: A systematic review

Abstract This systematic review aimed at investigating longitudinal changes in human milk bioactive protein concentrations in Chinese population. Both English and Chinese databases were searched. The data were pooled into six defined lactation stages. Weighted means of protein concentrations in each stage and the statistical significance of means of different lactation stages were calculated. The data of 11 bioactive proteins were retrieved. Concentrations of sIgA, IgM, and IgG decreased sharply during the first 14 days of lactation. The levels of α‐lactalbumin, lactoferrin, and β‐casein also decreased throughout lactation. Conversely, lysozyme levels increased over lactation. The changing patterns of the serum albumin, osteopontin, and bile salt‐stimulated lipase (BSSL) were not conclusive. This study represents the most comprehensive summary of bioactive proteins in Chinese human milk. In the future, mass spectrometry‐based analysis of human milk proteomics may be used to investigate the longitudinal changes of many more bioactive proteins.


| INTRODUC TI ON
Human milk provides infants with advantages in cognitive development, defense against pathogens, digestion and absorption of nutrients, lower risk of chronic diseases in later life, etc. (Lönnerdal, 2016b). Accordingly, the World Health Organization recommends that infants should be exclusively breastfed for the first 6 months of their lives. Breastfeeding beyond 6 months-with the addition of appropriate complementary foods-is also recommended (WHO, 2003). The components in human milk contain a complex matrix of nutrients, including proteins and amino acids, lipids, lactose and oligosaccharides, vitamins, minerals, and other substances.
The proteins in human milk are beneficial to infants in two distinct ways. First, amino acids derived from proteins can be assimilated by infants as their tissue proteins, catabolized as metabolic fuels, or converted to intermediate metabolites such as ornithine and citrulline (Kalhan & Bier, 2008). Second, some proteins exhibit beneficial effects on infants as intact proteins or partially digested products (peptides). These proteins are commonly defined as bioactive proteins (Lönnerdal, 2013). Bioactive proteins offer a wide variety of functions such as facilitating nutrient digestion and absorption, modulating immune functions, and defense against pathogens. Table 1 lists bioactive proteins with known or proposed functions (Artym & Zimecki, 2013;Haschke et al., 2016;Lönnerdal, 2016a).
Furthermore, it should be noted that the advancement of analytical tools has allowed an increasing number of bioactive proteins to be identified and quantified in human milk.
Different bioactive proteins in human milk follow different changing patterns throughout lactation . It is possible that the changing patterns of bioactive proteins meet specific needs of the infants during different stages of growth and development. For example, lactoferrin is a bioactive protein that can inhibit bacterial growth and has immunomodulatory properties (Legrand, 2016;Yin et al., 2014). Its concentration is the highest in colostrum, which is consistent with the fact that infants are more vulnerable to foreign pathogens during the first week of life when compared with the rest of infancy (Levy, 2007). Therefore, understanding the changing patterns throughout lactation can shed light on the physiological functions of bioactive proteins.
Genetic and dietary factors may lead to differences in the composition of human milk among different countries (Stam et al., 2013).
China has a population of more than 1.4 billion. The bioactive proteins in human milk in Chinese population have been investigated and published in original research articles in both English and Chinese.
However, to our best knowledge, there is currently no systematic review that compiles the data of bioactive proteins in Chinese human milk. Compared with original research articles, systematic reviews can provide a more comprehensive summary.

| Literature screening
The PRISMA guidelines were followed (Shamseer et al., 2015). Duplicates and obviously irrelevant articles were removed after reading the titles and abstracts. The full texts of the remaining articles were screened using the inclusion and exclusion criteria listed in PICOS (Table 2). Furthermore, a quality assessment was performed for all the included articles (Table S1). The literature screening process was performed independently by two investigators (Q. R. and Y. Z.). Discrepancies were discussed in the presence of Y. X. and S. J. until consensuses were reached.

| Data extraction and analysis
The means and medians were extracted, and the medians were converted to means as previously described (Hozo et al., 2005;Luo et al., 2015). All units were converted to mg/100 ml. The density of the human milk was assumed to be 103.2 g/100 ml for unit conversion (Neville et al., 1988). Lactation was divided into six stages-1-7,

| RE SULTS
The literature screening process is shown in Figure 1. The included studies are listed in Table 3 (Affolter et al., 2016;Bruun et al., 2018;Cai et al., 2018;Chen, 2007;Chen et al., 1986;Dai & Guan, 1985;Dou et al., 1986;Elwakiel et al., 2019;Han et al., 2010;Hsu et al., 2014;Jackson et al., 2004;Jiang, 2017;Jiang et al., 1999;Liu et al., 1994Liu et al., , 2018 Min, 1989;Sha et al., 2019;Shan et al., 2011;Shi et al., 2011;Urwin et al., 2013;Wang et al., 2012;Wang, 1987Wang, , 2012Wang & Lin, 1997;Wei & Pan, 1991;Wu et al., 1995;Yang et al., 2018;Yuen et al., 2012). The included studies covered 23 out of 34 provinces in China. Furthermore, 20 of the included studies specified that milk from mothers who delivered full-term infants was investigated, whereas the remaining 12 did not indicate the gestation age at delivery. Three of the included studies specified that milk was collected from vaginal delivery mothers, whereas the rest of the studies did not mention the types of delivery. Two studies collected foremilk, four studies collected full expression from one side, and the rest did not specify the means of milk collection (Table 3).

Human milk collection location
Term/ preterm

Concentrations
The lysozyme levels were similar during the first 60 postnatal days-except for the 8-14 postnatal day stage-and increased gradually during the remainder of lactation (Figure 2). No clear pattern could be observed in the levels of serum albumin, whose concentrations were higher in the 8-14 and 15-30 postnatal day periods than during the preceding or subsequent stages. For bioactive proteins with data of less than four stages available, the levels of β-casein and κ-casein were higher during the first 7 postnatal days than in later stages. Conversely, the levels of osteopontin and BSSL in the first 7 postnatal days were lower than those in later lactation stages.
The percentages of each bioactive protein in total protein generally followed the trends in protein concentration in milk, except for α-lactalbumin and β-casein (Figure 2). The percentage of α-lactalbumin in total protein increased during lactation, which was contradictory to the change in its concentration in human milk. The percentage of β-casein in total protein during the first 7 postnatal days was lower than those in later stages, whereas its concentration in human milk was higher in the first 7 postnatal days when compared with later stages.

F I G U R E 2
The longitudinal changes in the protein concentrations and the percentages of the total proteins of the bioactive proteins in the human milk of the Chinese population

| D ISCUSS I ON
This study represents the most comprehensive overview of bioactive protein concentrations in the human milk of the Chinese population to date. We found that α-lactalbumin, lactoferrin, sIgA, IgM, and IgG decreased whereas lysozyme increased throughout lactation.
The trends of the rest of the included proteins are less conclusive due to a lack of data in the literature. Furthermore, we revealed that the concentrations of haptocorrin, MFGM proteins, and other bioactive proteins have never been reported in Chinese population.
The longitudinal changes in human milk bioactive proteins in Chinese population were compared with studies in other populations (Table S3). The trends we found are generally consistent with the findings in non-Chinese populations with three minor exceptions. First, Lonnerdal et al. found the concentrations of serum albumin increased in the first 60 days of lactation and decreased thereafter, whereas we did not observe such a trend in our study.
Although insufficient number of studies available may cause the observed discrepancies in human milk bioactive proteins in different populations, we cannot rule out the possibility that genetic or dietary factors may cause the discrepancies. For instance, serum albumin in the blood is an indicator of the nutritional and hydration status (Akpele & Bailey, 2004). Therefore, it is entirely possible that the serum albumin in human milk is also related to maternal nutritional and hydration status.
The longitudinal changes in the bioactive proteins may have implications in infant nutrition and health. There is a sharp decrease in immunoglobulin (sIgA, IgM, and IgG) concentrations after the first 14 postnatal days, indicating that during the first 14 days of life, infants require their mothers to produce antibodies for the protection against pathogens. In this way, mothers may endow their infants with adaptive immune responses via human milk and protect them from pathogens that both the mothers and infants are exposed to (Levy, 2007). After 2 weeks of life, infants may be able to produce sufficient antibodies on their own and become less dependent on human milk for immunoglobulins (Gao et al., 2012). Additionally, the increase in the lysozyme concentration at the beginning of the 8-14 postnatal day stage suggests an increasing involvement of lysozyme in the protection of the infants against pathogenic bacteria.
This study was only able to reveal the longitudinal changes in 11 bioactive proteins. Previously, Lonnerdal et al. retrieved 7 bioactive proteins in their systematic review .
Most of these proteins were quantified by immunoassays (Table 3), which were dependent on the development of specific antibodies.
Moreover, the specificity and sensitivity of the antibodies used could contribute to variations among different studies. It should be noticed that more than 1,600 proteins have been identified in human milk due to the advances in mass spectrometry (Beck et al., 2015). In the future, mass spectrometry-based proteomic analysis of human milk may be used to shed light on the longitudinal changes of many more bioactive proteins in human milk (Cao et al., 2017).
There are limitations in our study. First, the association between the means of delivery and the bioactive protein levels in human milk remains controversial (Affolter et al., 2016;Liu et al., 2019;Yang et al., 2018). Unfortunately, only three of the included studies specified the means of delivery, making it impossible for us to extract data and analyze the association between the means of delivery and the bioactive protein concentrations in human milk. Second, it is well-known that premature infants are more prone to infections and that human milk decreases the rate of infections (Patel & Kim, 2018). Therefore, it is possible that milk from mothers who delivered premature infants may contain higher concentrations of immunomodulatory proteins such as immunoglobulins and lactoferrin (Bernloehr et al., 2016). Nevertheless, 20 out of the 32 identified article reported milk from mothers who delivered full-term infants, whereas the rest did not specify the gestation age at delivery. Therefore, it is impossible for us to analyze differences in bioactive protein levels between milk from mothers delivered at different gestation ages.

| CON CLUS IONS
This systematic review aimed at investigating the longitudinal changes in bioactive proteins in the human milk of the Chinese population. Data from 20 and 12 publications that were originally published in Chinese and English, respectively, were combined.
The concentrations of α-lactalbumin, lactoferrin, β-casein, and three immunoglobulins (sIgA, IgM, and IgG) decrease during lactation. Particularly, sharp decreases are evident in the immunoglobulin concentrations during the first 14 postnatal days. Conversely, the lysozyme concentrations increase during lactation. The patterns of longitudinal changes in serum albumin, osteopontin, BSSL, and κ-casein are less conclusive, mainly due to the limited data available. This study represents the most comprehensive report on the bioactive proteins in the human milk of the Chinese population to date.
The findings in the Chinese population are similar to those in other populations. Furthermore, it is revealed that future studies should document factors such as means of delivery, gestational age at delivery, and the protocol of milk collection to examine the association between these factors and the bioactive protein concentrations in human milk. Additionally, mass spectrometry-based analysis of human milk proteomics may be used to investigate the longitudinal changes in many more bioactive proteins.

ACK N OWLED G M ENTS
This study was supported by "Bai-Qian-Wan Engineering and Technology Master Project" (Grant # 2019ZX07B01), which was funded by the Government of Heilongjiang Province of the People's Republic of China.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.