Fatty acid profile and the sn-2 position distribution in triacylglycerols of

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Agricultural and Environmental Chemistry

Fatty acid profile and the sn-2 position distribution in triacylglycerols of breast milk during different lactation stage Ce Qi, Jin Sun, Yuan Xia, Renqiang Yu, wei wei, Jingying Xiang, Qingzhe Jin, Hang Xiao, and Xing-Guo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01085 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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Journal of Agricultural and Food Chemistry

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Fatty acid profile and the sn-2 position distribution in triacylglycerols

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of breast milk during different lactation stage

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Ce Qi 1*, Jin Sun 1, Yuan Xia 1, Renqiang Yu 2, Wei Wei 1, Jingying Xiang 2, Qingzhe

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Jin 1, Hang Xiao 3, Xingguo Wang 1*

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1

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Province, School of Food Science and Technology, Jiangnan University, Wuxi

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214122, Jiangsu, PR China

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Wuxi Maternity and Child Health Care Hospital, Wuxi 214000, PR China

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3

Department of Food Science and Technology, University of Massachusetts, Amherst

Collaborative innovation center of food safety and quality control in Jiangsu

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01003, Massachusetts, USA

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*

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Address:

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Tel: +86-18151559929

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E-mail address: [email protected] (Dr. C. Qi); [email protected] (Dr. X. Wang)

Corresponding author 1800 Lihu Avenue, Wuxi 214122, PR China

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ACS Paragon Plus Environment


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ABSTRACT

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Fatty acid (FA) is the major energy resource in breast milk, which is important

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for infant development. FAs profiles with sn-2 positional preference were important

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part of triacylglycerols due to their better availability. It was still not replicated in

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artificial formulas. This study quantified the FAs profile of total and sn-2 position in

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human breast milk samples from 103 healthy volunteers during colostrum,

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transitional and mature stages. Multicomponent analysis showed significant

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differences in FAs profiles of different lactation periods, due to that with relative

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percentage less than 1%. Linoleic acid (LA), mostly located at the sn-1,3 positions of

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TAGs, was more common in the milk of Chinese women than in western. The

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majority of the breast milk did not meet the standard for the ratio of LA/α-linolenic

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acid for infant formula. FAs related to brain development, mainly at sn-2 in TAGs,

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were enriched in colostrum. Capric and lauric acids were enriched in transitional and

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mature breast milk, and capric acid showed sn-1,3 selectivity in TAGs. This study

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will aid the development of infant formula containing TAGs more similar to human

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breast milk.

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Key words: Breast milk, colostrum, transitional milk, mature milk, total fatty acids,

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sn-2 fatty acids

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Introduction

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Breast milk is the best natural food for the neonate, and the World Health

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Organization (WHO) recommends exclusive breastfeeding for the first 6 months of

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life because the nutrients in breast milk influence the growth and health of infants 1.

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Triacylglycerols (TAGs), the major energy resource in breast milk, are important for

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infant development. The fatty acids (FAs) composition of the milk TAGs correlates

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with that in the plasma and tissues of the breast-fed infant 2. The positional

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distribution of the FAs in the TAGs of human breast milk is also important. Studies

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on the TAGs in the milk of the Western population revealed that palmitic acid (PA;

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C16:0) is mainly located at the sn-2 position of the TAGs, whereas unsaturated FAs

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(UFAs), especially oleic acid (C18:1 n-9), typically occupied the sn-1/sn-3 positions

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3

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∼21%) and oleic–palmitic–linoleic (OPL; ∼17%) 4. This unique FA distribution

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enables optimized uptake of calcium, fat, and energy in infants, and is even important

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for the development of the microbiome 5.

. The major TAG species in mature human milk are oleic–palmitic–oleic (OPO;

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The FAs composition and positional distribution in TAGs are key factors in

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determining the nutritional value of infant formula. Recently, we comprehensively

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evaluated the FAs compositions of commercial formulas on the Chinese market in a

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statistically meaningful sample size. The data revealed that, although commercial

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formulas had similar compositions of the major FAs (>1%) to human breast milk, the

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levels of some micro-FAs were lower 6. The positional distribution of the FAs in

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formulas is markedly different from that in human breast milk, even though some 3



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formulas are supplemented with OPO. Therefore, manufacturers should take greater

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care with the FAs profiles and stereochemical distributions of FAs in commercial

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formulas.

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Human milk has long been the leading reference in the development of

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commercial formulas. However, the optimal composition of TAGs in formula

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remains unknown because it is not constant and can be affected by various factors,

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such as maternal diet

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regional dietary characteristics in China should be considered when developing

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commercial formulas for China, where all babies generally receive formula. There

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have been several reports on the FAs composition of human milk in China

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However, the sample sizes in some of the studies were too small to facilitate

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statistically meaningful analysis, and the studies did not consider the stereochemical

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distribution of the FAs in the TAGs. The Yangtze Delta area is a fast-developing area

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in China, with typically healthy dietary patterns and great improvements in infant

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development. Wuxi, a city located in the heart of the Yangtze Delta, is a

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representative city for the study of ideal breast milk.

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and stage of lactation 8. Breast milk with representative,

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.

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We investigated the total and sn-2 FAs profiles of breast milk from women in

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Wuxi and compared the profiles during different lactation periods. Our aim was to

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elucidate the main characteristics of the FAs profile and sn-2 positional distribution

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of FAs in breast milk from women of the Yangtze Delta area, and to explain the

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differences to the breast milk from women in Western countries. Our results may

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prove helpful for the development of infant formula containing TAGs more similar to 4


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human breast milk.

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Materials and methods

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Materials

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Porcine pancreatic lipase (type II) and thin-layer chromatography (TLC) plates

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were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ammonia solution,

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ethanol, diethyl ether, petroleum ether, and potassium hydroxide were purchased

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from Sinopharm Chemical Reagent (Shanghai, China). Methanol (>95.0% purity)

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and hexane (>99.9% purity) were obtained from J&K Scientific (Beijing, China).

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The standard mixture of FA methyl esters (FAMEs) was purchased from

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Sigma–Aldrich (St. Louis, MO, USA). The other solvents and reagents were all of

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analytical grade (Sinopharm Chemical Reagent, Shanghai, China).

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Breast milk sample collection

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The breast milk samples used in this study were from voluntary donors. All

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participants received detailed information about the study and provided written

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informed consent; the study protocol was approved by the Ethics Committees of the

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respective participating institutions (the Medical Research Board of Jiangnan

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University and Wuxi Maternity and Child Health Care Hospital) (WXM201560). The

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study group comprised 103 healthy women volunteers from Wuxi, China. They were

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disease-free, based on medical history and physical examination, and had healthy

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infants. The times of gestation ranged from 37 to 39 weeks, with a mean of 38.5 5



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weeks. The mean age of the women was 27.86. The mean parity was 1.2. All

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participants received detailed information about the study and provided written

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informed consent; the study protocol was approved by the Ethics Committees of the

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respective participating institutions: the Medical Research Board of Jiangnan

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University and the Wuxi Maternity and Child Health Care Hospital.

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Breast milk was collected at approximately day 1–5 (colostrum), day 6–15

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(transitional milk), and more than 15 days (mature milk) after birth. Before sample

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collection, the volunteers were given oral and written instructions for standardized

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sample collection. A portion of whole breast milk was obtained at 10 AM by breast

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massage. The first drops of milk (approximately 500 µL) were discarded. The

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samples were kept frozen at −20 °C until delivery to the laboratory, then stored at

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−80 °C until further analysis. All samples were shipped to Jiangnan University for

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storage, processing, and lipid analysis.

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Total lipid extraction from breast milk

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The extraction of total lipids from breast milk was performed with the Mojonnier

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method (AOAC, 2000; method 995.19) as modified by Barbano et al. 10. Briefly, we

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added 1 mL ammonia water into 5 mL breast milk, then mixed thoroughly and

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incubated the mixture in a water bath at 65 °C ± 5 °C for 20 min. After the mixture

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cooled, we successively added 5 mL absolute ethanol, 12.5 mL ether, and 12.5 mL

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ligarine, and mixed to extract the lipids. After the samples were left to stand for at

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least 1 h, we collected the clear supernatants. The lipids were re-extracted as above,

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and the 2 fractions were pooled. The residual solvent was removed by nitrogen

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blowing.

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Preparation of FAMEs for chromatographic analysis

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Milk lipids (10 mg) were suspended in 700 µL n-hexane and 125 µL

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KOH-CH3OH (2 M). After mixing for 2 min, we added 25 µL sodium methoxide and

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incubated the blend for 5 min with shaking, then added sodium sulfate and mixed

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thoroughly. The supernatant was removed after standing and passed through a

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0.22-µm filter. The resulting FAME was used for analysis by gas chromatography

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(GC).

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Preparation of 2-monoacylglycerol and its methyl esters

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Milk lipids were also hydrolyzed to 2-monoacylglycerol (2-MAG) by means of 11

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the method described by Sahin et al.

. We added pancreatic lipase (porcine

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pancreatic lipase, 30 mg), Tris buffer (pH 8.0, 7 mL), bile salts (0.05%, 1.75 mL),

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and calcium chloride (2.2%, 0.7 mL) to a test tube containing 30 mg fat. The mixture

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was incubated in a water bath (37 °C) for 3 min with shaking. It was mixed by vortex

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for 30 s, then incubated at 37 °C for 3 min. It was vortex again and incubated for 2

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min. After the mixture cooled, we added diethyl ether (2 mL), then centrifuged for 3

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min at 2500 ×g rpm. We transferred the supernatant to a new tube and evaporated the

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diethyl ether to a volume of 500 µL with nitrogen gas. The hydrolytic product was

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separated on a silica gel G TLC plate with the developing solvents hexane/diethyl

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ether/acetic acid (50:50:1, v/v/v). The band corresponding to 2-MAG was isolated 7



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and extracted twice with diethyl ether (1 mL). The solvent was removed by nitrogen

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gas, and the residue was methylated and analyzed by GC.

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GC analysis

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The GC instrument was an Agilent 7820A (Agilent, Santa Clara, CA, USA)

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equipped with a hydrogen flame ionization detector and a TRACE™ TR-FAME

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capillary column (60 m × 0.25 mm × 0.25 µm, Thermo Fisher Scientific, Waltham,

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MA, USA). Both the injector and detector temperatures were 250 °C. We used

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nitrogen carrier gas at 1.2 mL/min, and the split ratio was 1:100. The oven

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temperature was held at 60 °C for 3 min, then raised to 175 °C at the rate of 5 °C/min

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and maintained for 15 min at this temperature, followed by an increase to 220 °C (at

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a rate of 2 °C/min), which was maintained for 10 min. The FAMEs were identified

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by comparison of the retention times of the sample peaks with those of a mixture of

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the FAME standards.

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The relative percentage of each FA at the sn-2 position was calculated as relative

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percentage = (M/T×3) × 100, where M is the percentage of FAs at the sn-2 position

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and T is the percentage of the FAs in the TAGs 12. The FA contents were expressed as

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the weight percentage (%, w/w) of total FAs detected with chain lengths of 4–22

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carbon atoms.

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Statistical analysis

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We performed a partial least squares discriminant analysis (PLS-DA) to

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determine the differences in the total FAs and sn-2 FAs profiles in the 3 types of 8


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breast milk using SIMCA-P software (version 13.5, Demo Umetrics, Umea, Sweden).

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The dataset was mean-centered and pareto-scaled in a column-wise manner for the

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multivariate modeling 13. To find the specific FA profile of the milk from each stage,

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we subjected the data, which we displayed using Java TreeView, to hierarchical

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clustering using Cluster 3.0. The FAs data were adjusted by subtracting the

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sample-wise median from the value for each sample, so that the median value of each

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sample was 0. The adjusted data reflect their variation from the median. Then, the

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data

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centered-correlation and the average linkage method.

were

clustered

using

the

hierarchical

clustering

algorithm

with

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We analyzed the differences in the FAs or sn-2 FAs profiles of the breast milk

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from different stages using SPSS 22. We tested the data for normality (assessed by

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Shapiro–Wilk test) and homoscedasticity (assessed by Levene's test). In cases where

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the data met the normal distribution criteria, we performed a one-way analysis of

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variance. If differences were statistically significant, we used a Kruskal–Wallis test

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for multiple comparisons.

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Results

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Total lipid and macroscopic differences in FA composition

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The total lipid levels in breast milk from women in Wuxi significantly increased

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over the period of lactation (P

Fatty acid profile and the sn-2 position distribution in triacylglycerols of

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