Macronutrient content and fatty acid composition and their positional distribution in human breast milk from Zhejiang Province, China in different lactation periods

Abstract Lactational changes in macronutrient content, lipid profile, fatty acid composition, and positional distribution of human breast milk were investigated in this study. A total of 378 milk samples of six different lactation periods, including 0‒5, 6‒14, 15‒30, 31‒90, 91‒180, and 181‒360 days, were collected cross‐sectionally from healthy lactating women in Zhejiang, China. As lactation progressed from 0‒5 to 15‒30 days, the lipid content and the percentages of C10:0, C12:0, C14:0, C18:2n‐6, and C18:3n‐3 increased significantly, while the protein concentration and the proportions of phospholipids, cholesterols, C16:0, C18:1n‐9, C24:1n‐9, C20:4n‐6, C22:4n‐6, C22:5n‐3, and C22:6n‐3 decreased notably. When lactation was further extended to 181‒360 days, the protein content continued to decrease, and the percentages of C12:0 and C14:0 continued to increase, whereas the levels of other tested nutrients remained stable. Although the triacylglycerol positional distributions of some fatty acids underwent significant lactational variations, C14:0, C16:0, C24:1n‐9, C22:4n‐6, C22:5n‐3, and C22:6n‐3 were located mainly at the sn‐2 position, while C18:1n‐9, C18:2n‐6, and C18:3n‐3 were primarily distributed at the sn‐1,3 positions. Compared with human breast milk reported in Western countries, samples in our study demonstrated higher percentages of C18:2n‐6, C18:3n‐3, C20:4n‐6, and C22:6n‐3, but lower proportions of C12:0, C14:0, and C18:1n‐9. The results from this study indicated a nutritional composition different from that of the Western countries and may provide useful data for the development of infant formulas closer to Chinese breast milk in terms of the fatty acid composition and its specified positional distribution on triglyceride structure.

To date, numerous studies on human breast milk composition have been carried out in western regions and have revealed that the macronutrient content, lipid profile, and fatty acid composition of human milk vary with lactation periods and geographical regions due to diverse dietary patterns and genetic backgrounds (Bravi et al., 2016;Gidrewicz & Fenton, 2014;Innis, 2014;Sosa-Castillo et al., 2017). To our knowledge, several studies in this field have been conducted in China, but only a few of these studies were involved in fatty acid positional distribution (Deng et al., 2018;Qi et al., 2018;Wu et al., 2019). In addition, most of the studies merely focused on milk samples within a lactation period of fewer than three months, which may not provide sufficient data support for the development of localized infant formulas at different stages (0-6 months and 6-12 months). Geographically representative breast milk from healthy, well-nourished mothers should be considered as the golden reference in the development of localized infant formulas. Zhejiang, one of the most developed provinces in China with typically healthy dietary patterns and good improvements in infant growth and development (weight, height, head circumference, nutritional status, etc.), is an ideal area for the study of Chinese human milk.
In the present study, we investigated the macronutrient content, lipid profile, fatty acid composition, and positional distribution of human breast milk in six different lactation periods (0-5, 6-14, 15-30, 31-90, 91-180, and 181-360 days) from healthy lactating women in Zhejiang, and compared with those of breast milk from Western countries. Our aim was to elucidate the characteristics of the lipid composition and structure of Chinese breast milk and to explain the differences with those of Western breast milk. The results provide a broad overview of the fatty acid composition events underlying the lactation periods changes and explore the valuable specified positional distribution of fatty acids on triglyceride structure of human milk that could be used to develop commercial infant formulas more suitable for Chinese babies.

| Subjects and milk sample collection
Three hundred and seventy-eight lactating women from Zhejiang Province of China who met the inclusion criteria were recruited for the study. The inclusion criteria were as follows: (1) Healthy breastfeeding mothers of full-term and singleton delivery babies; (2) no smoking and drinking; (3) no severe nutritional diseases (irondeficiency anemia, marasmus malnutrition, kwashiorkor, etc.) and malignant consumptive diseases (malignant tumor, pulmonary tuberculosis, etc.); (4) no chronic diseases of diabetes and hypertension; and (5) no nutritional supplements were taken recently. The basic characteristics of the participants, including childbirth age, gestational period, pregestation, and predelivery body mass indices (BMI), as well as birth weight and length of respective babies, were collected at the time of enrollment by a self-administered questionnaire. All participants received detailed information about the study and provided written informed consent. The study protocol was approved by the Ethics Committee of the College of Biosystem Engineering and Food Science, Zhejiang University.
Each participant was asked to provide one milk sample between 9:00 am and 12:00 am. Both sides of the mammary gland were evacuated completely with an electric breast pump, and the milk was carefully mixed. A portion of 20 ml of whole breast milk was taken and subpacked into four 5-ml frozen tubes, and then immediately stored in a low-temperature refrigerator at −80°C until analysis. The remaining milk was fed to the infants. Among the four frozen tubes, one is used for macronutrient content analysis, two were used for lipid and fatty acid composition determination, and the remaining one was used as a backup sample.

| Macronutrient content analysis
Macronutrient content, including lipids, protein, and lactose, of human breast milk were determined by injecting an aliquot of a 2-ml milk sample into a mid-infrared human milk analyzer (HMA, Miris AB) according to the manufacturer's instructions (Zhu et al., 2017).
Before analysis, the milk samples were thawed in a water bath at 40°C and homogenized by an ultrasonic oscillatory mixing machine.
A daily calibration check and cleaning steps for every 10 analyses were performed using the calibration solution and cleaning solution, respectively. The accuracy (average recoveries, n = 6) and precision (relative standard deviations (RSD), n = 6) of the method were 88.46%-116.94% and 0.37%-1.86%, respectively.

| Total lipid extraction
Total lipids from the breast milk were extracted using the Association of Official Analytical Chemists (AOAC) official method 989.05 with some modification (Barbano et al., 1988). In brief, 5 ml of thawed and homogenized breast milk and 1 ml of ammonia water were placed in a 50-ml centrifuge tube, mixed thoroughly, and incubated in a water bath at 65°C for 20 min. After the mixture was cooled to room temperature, 5 ml of absolute ethanol was added and mixed for 1 min.
Then, 12.5 ml of anhydrous ether and 12.5 ml of petroleum ether were added and mixed for 3 min to extract the lipids. The mixture was centrifuged for 15 min at 1500g, and the clear supernatant was collected and dried with blowing nitrogen to obtain the milk lipid extract. Portions of 10, 20, and 30 mg of the lipid extract were taken to determine the lipid profile, total fatty acid composition, and sn-2 fatty acid composition, respectively.

| Lipid analysis
The lipid composition was analyzed using an Iatroscan thin-layer chromatography with flame ionization detector (TLC-FID) analyzer (Iatron Laboratories Inc.; Li et al., 2010). Briefly, 10 mg of human milk lipid extract, which had been dissolved in 1 ml of chloroform, was spotted on the starting point of the chromarods as 2 µl using an autospotter. After spotting, the rods were developed in developing tanks with a petroleum ether-ethyl ether-acetic acid (60:15:0.1, v/v/v) solvent system for 25 min and a petroleum ether-ethyl ether (56:4, v/v) solvent system for 30 min successively. Each time after development, the rods were placed in an oven (53°C) for 3 min to evaporate the residual solvents. Then, the chromarods were scanned with an Iatroscan MK-6s TLC-FID Analyzer. The air and hydrogen flow rates were 2000 ml/min and 160 ml/min, respectively, and the scan speed was set at 30 s/scan. The compositions of lipid classes were expressed in weight percentages (wt %) of the total lipids according to their peak areas, which were recorded and processed by Chromstar software. Glyceryl tripalmitate, lecithin, cholesteryl palmitate, cholesterol, glyceryl 1,2-dipalmitate, glyceryl 1,3-dipalmitate, and palmitic acid (Sigma-Aldrich) were employed as authentic standards for the quantitative analysis of TAGs, PLs, cholesterol esters (CEs), free cholesterols (FCHOLs), 1,2-DAGs, 1,3-DAGs, and FFAs. The accuracy (average recoveries, n = 6) and precision (RSD, n = 6) of the method were 92.67%-104.05% and 0.24-7.76%, respectively.

| Fatty acid methylation
Milk lipid extract (20 mg) was dissolved in 0.7 ml of potassium hydroxide-methanol solution (1 mol/L) and incubated in a water bath at 60°C for 3 min with shaking. Then, 1 ml of boron trifluoridemethanol solution (14%, w/v) was added and incubated in a water bath at 50°C for 15 min. After the mixture was cooled to room temperature, 2 ml of saturated NaCl solution and 1 ml of n-hexane were added and mixed thoroughly and then centrifuged at 2200g for 5 min. The supernatant containing fatty acid methyl esters (FAMEs) was collected and subjected to gas chromatographic (GC) analysis.

| 2-Monoacylglycerol (2-MAG) and its FAME preparation
Human milk lipids were hydrolyzed to 2-MAG by means of the method described by Luddy et al. (1964) with some modification.
Lipid extract (30 mg), Tris-HCl buffer (1 mol/L, 2 ml), sodium cholate (1 g/L, 0.5 ml), and calcium chloride (2.2%, 0.2 ml) were added to a 10 ml test tube and incubated in a water bath at 40°C for 1 min with shaking. Porcine pancreatic lipase (30 mg, type II, Sigma-Aldrich) was then added, and the mixture was mixed well and incubated in a water bath at 40°C for another 3 min. After the mixture was cooled, the HCl solution (6 mol/L, 1 ml) and diethyl ether (2 ml) were added and the solution was subjected to centrifugation at 4000 r/min for 5 min. The obtained clear upper phase containing hydrolytic product was collected and evaporated to dryness under nitrogen gas.
The residual-dried hydrolytic product was redissolved in 1 ml of n-hexane, and the 2-MAG in the hydrolytic product was separated and purified by an NH 2 solid-phase extraction cartridge (500 mg, 6 ml; Anpel). Acetone (5 ml) and n-hexane (5 ml) were used to activate the cartridge. Then, the hydrolytic product was loaded on the cartridge and eluted by 5 ml of acetone/n-hexane (15:85, v/v) and 5 ml of acetone/n-hexane (50:50, v/v) successively. The eluate of acetone/n-hexane (50:50, v/v) containing 2-MAG was collected and dried by nitrogen gas blowing and then subjected to the same methylation procedure as above to produce FAMEs.

| GC analysis
The FAME was analyzed using an Agilent GC 7890A (Agilent Corporation) equipped with a FID and a 100 m × 0.25 mm × 0.20 μm capillary column (Supelco 2560, Sigma-Aldrich). The injector and detector temperatures were 240°C and 250°C, respectively. Nitrogen was used as the carrier gas with a flow rate of 1.0 ml/min, and the split ratio was 1:10. The column temperature program was as follows: 60°C held for 2 min, followed by an increase of 15°C/min to 100°C, and subsequently elevated to 230°C at the rate of 3°C/min and maintained for 18 min. Fatty acids were identified by the comparison of retention time with a FAME standard mixture (GLC-746, Nu-Chek Prep). The compositions of fatty acids were expressed as weight percentages (wt %) of total fatty acids according to their peak areas. The accuracy (average recoveries, n = 6) and precision (RSD, n = 6) of the method were 94.18%-109.37% and 1.28%-6.45%, respectively.

| Fatty acid positional distribution calculation
The fatty acid positional distribution was calculated as the relative percentage of a specific fatty acid at the sn-2 position compared with the total amount of this fatty acid using the following equation: sn-2/total, % = percentage in total sn-2 fatty acids/(percentage in total fatty acids × 3) × 100.

| Statistical analysis
All determinations were made in duplicate, and the results were reported as the mean ± standard deviations (SD). Before statistical analyses, data were checked for normal distribution and variance homogeneity using the Shapiro-Wilk and Levene tests, respectively. If the data were normally distributed, one-way analysis of variance (ANOVA) was adopted to compare the nutritional composition of human milk in different lactation periods, and the Tukey-Kramer multiple comparison test was applied to test for differences between means at the 5% significance level (p < .05).
Otherwise, a nonparametric Kruskal-Wallis test was used. Oneway analysis of covariance (ANCOVA) was used to compare the nutritional composition of human milk in different lactation periods with adjustment for childbirth age, gestational period, TA B L E 1 Basic characteristics of mothers and respective babies corresponding to 378 breast milk samples with different lactation periods

Mothers
Childbirth age (years) 28.6 ± 3.5 pregestation and predelivery BMI, and Bonferroni multiple comparison test was employed to test for differences between means at the 5% significance level (p < .05). All analyses were conducted using SPSS 20.0 (SPSS Inc.).

| Basic characteristics
A total of 378 milk samples in 6 different lactation periods, includ-

| Macronutrient content
The macronutrient content of human breast milk from different lactation periods is listed in Table 2. The lipid content of breast milk increased dramatically from 2.81 g/100 ml at lactation 0-5 days to 4.05 g/100 ml at lactation 31-90 days and then remained stable thereafter, while the protein content decreased significantly during the whole lactation period, from 2.19 g/100 ml at lactation 0-5 days to 1.07 g/100 ml at lactation 181-360 days. Lactose content (6.63-6.97 g/100 ml) was consistent among different lactation times.    Note: Results are presented as mean ± SD. Values within the same row not sharing a common letter are significantly different (p < .05).

| Fatty acid positional distribution
The

| Nutritional composition after adjustment for basic characteristics of lactating mothers
In order to eliminate the influence of the difference of basic characteristics of lactating mothers on the nutritional composition of corresponding breast milk, the macronutrient content, lipid profile, fatty acid, and sn-2 fatty acid composition of human breast milk from different lactation periods were adjusted for childbirth age, gestational period, pregestation, and predelivery BMI of sampled mothers (Table S1-S4). By comparison, the adjusted nutritional compositions and lactational changes of breast milk were consistent with those of unadjusted (Tables 1-4).

| Macronutrient content
Due to the importance of human milk composition in the estimation of infant nutritional requirements and the common use of a newly developed rapid analytic method (mid-infrared spectrometry) for the determination of human milk macronutrients (Fusch et al., 2014), the lipid, protein, and lactose content of human milk from different regions and lactation periods have been widely investigated in recent years (Gidrewicz & Fenton, 2014;Leghi et al., 2020). Among macronutrients, the lipid content was directly affected by the sampling mode of human milk. Foremilk was reported to have a much higher lipid content (>2-fold) than hindmilk (Mitoulas et al., 2002).

| Fatty acid composition
The fatty acid composition of human milk was evidenced to vary with region and was susceptible to maternal diet ( Yuhas et al., 2006).  (Gardner et al., 2017;Miliku et al., 2019;Much et al., 2013;Yuhas et al., 2006).  et al., 2006). These variations are attributed mainly to the different regional dietary habits. In Western countries, people tend to consume more animal food, which contains high amounts of SFAs but low levels of PUFAs, in their daily life Wu et al., 2019).
However, the Chinese daily diet is characterized by high intakes of vegetable oils, especially soybean oil and corn oil, which are rich in LA and ALA, the precursors of n-6 and n-3 PUFAs . The Mediterranean diet contains large quantities of olive oil, which is high in OA (López-López et al., 2002), while coastal residents eat more seafood rich in DHA (Fu et al., 2016;Yuhas et al., 2006). Apart from regional variations, lactational changes in the fatty acid composition of human milk have also been widely reported (Floris et al., 2020;Koletzko, 2016). From our study, most fatty acids in human milk underwent significant differences as the lactation pe-  Note: Results are presented as mean ± SD. Values within the same row not sharing a common letter are significantly different (p < .05).

| Fatty acid positional distribution
In addition to the fatty acid composition, the specified positional distribution of fatty acids on the triglyceride structure of human milk is another key point . From the present study, up to 75.6%-81.0% of PA (the most abundant SFA) was located at the sn-2 position, whereas the relative percentage of OA (the predomi-  of OA (Spain: 12.22%-14.10%, Italy: 9.83%-15.80%) were comparable to our data López-López et al., 2002).
Although the relative percentages of PA and OA at the sn-2 position of human milk varied with region, it was certain that PA existed primarily at the sn-2 position, and OA esterified mainly at the sn-1,3 positions. These typical positional preferences make OA-PA-OA (OPO) one of the most abundant TAGs in human milk Morera Pons et al., 2000) and have been widely proven to be positively related to better fatty acid and mineral absorption and less hard stool and constipation incidence in infants ( Bar-Yoseph et al., 2013;López-López et al., 2001).  C16:1n-7 C18:1n-9 (OA) C20:1n-9 C22:1n-9 C24:1n-9 F I G U R E 4 Positional distribution (sn-2/total, %) of selected individual n-6 PUFAs in human breast milk from different lactation periods. Results are presented as mean ± SD. Values in the bars of the same type not sharing a common letter are significantly different (p < .05  in Western human milk. Therefore, the OPL structure needs to be considered more than the OPO structure when developing commercial infant formulas suitable for Chinese babies. The sn-2 positional selectivity for DPA, DHA, and C22:4n-6 in human milk has been well evidenced to be positively correlated with improved oxidative stability (Wijesundera, 2008) and improved absorption and enrichment in brain tissues (Christensen et al., 1995(Christensen et al., , 1998 According to the present study and some previous studies

| CON CLUS IONS
The present study investigated the macronutrient content, lipid pro-

CO N FLI C T O F I NTE R E S T S
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENT
The study protocol was approved by the Ethics Committee of the