31P NMR-based phospholipid fingerprinting of powdered infant formula

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31P NMR-based phospholipid fingerprinting of powdered infant formula Dan Zhu, Alan Hayman, Biniam Kebede, Ian Stewart, Gang Chen, and Russell D. Frew J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03902 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Agricultural and Food Chemistry

31P

NMR-based Phospholipid Fingerprinting of Powdered Infant Formula Dan Zhu†, Alan Hayman†, Biniam Kebede‡, Ian Stewart†, Gang Chen§, Russell Frew†*

†Department

of Chemistry, University of Otago, Dunedin, 9016, New Zealand

‡Department

of Food Science, University of Otago, Dunedin, 9016, New Zealand

§Key

Laboratory of Agro-Product Quality and Safety, Institute of Quality Standards and Testing Technology for

Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China

*Corresponding author E-mail address: [email protected] (Russell Frew) Tel: +64 3 479 7913

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ABSTRACT

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Infant formula (IF), regarded as the optimal substitute for human breast milk (HBM), is very

3

important for infant growth and development. Phospholipids (PLs) are ubiquitous components

4

of infant formula as they have good emulsifier properties in addition to their nutritional and

5

biological functions. In this study, the PL contents in four different commercial IF brands

6

(indicated as A, M, O and W) were characterized and quantified using optimized

7

spectroscopy.

8

phosphatidylethanolamine (PE) and sphingomyelin (SM) occurred at lower concentrations

9

(5.72 mg∙100 g-1 and 8.89 mg∙100 g-1, respectively) in IFs from brand O while phosphatidic

Nine

PLs

were

identified

and

quantified

and

31P-NMR

among

these,

10

acid (PA) was higher (2.83 mg∙100 g-1) in IFs from brand W. In summary,

31P-NMR

11

spectroscopy, combined with the multivariate data analysis, proved to be an effective analytical

12

toolbox for evaluating the PL contents in IF and the comparative differences between IF brands.

13 14

KEYWORDS: Infant formula; phospholipid; 31P nuclear magnetic resonance; multivariate data

15

analysis.

16 17 18 19 20 21 22 23 24 25 26

INTRODUCTION


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Human breast milk (HBM) is considered to be the optimal food for infants and is recommended

28

as the sole source of nutrition during the baby’s first stage (six months) of life by the World

29

Health Organization. However, in some circumstances, some infants could not be breastfed,

30

and in order to satisfy their growth requirements, commercial infant formulas (IFs) provide a

31

nutritional and safe substitute.1 Usually, IF is prepared on cow milk or soy base with added

32

vitamins, minerals and iron to resemble HBM composition and nutrient profile.2

33

The lipid fraction is a crucial part of the HMB, and it provides almost 50 % of the child’s

34

dietary calories and physiologically active molecules.3 There are several classes of lipids in

35

milk, such as the mono-, di-and triacylglycerides, free fatty acids, phospholipids (PLs),

36

glycolipid and sterols.4 Among these lipids, PLs only account for about 1 % of milk fat. PLs

37

are regarded as important suppliers of energy and long-chain polyunsaturated fatty acids (LC-

38

PUFAs) and play a key role in growth and brain development in new-born infants.5 Milk PLs

39

can be divided into two major classes called glycerophospholipids and sphingolipids according

40

to the type of alcohol backbone (glycerol or sphingosine). The main PLs contained in

41

mammalian milk and dairy products are phosphatidylcholine (PC), phosphatidylethanolamine

42

(PE), phosphatidylinositol (PI), ethanolamine plasmalogen (EPLAS), phosphatidylserine (PS),

43

phosphatidic acid (PA), and sphingomyelin (SM).5 The PL classes are defined by their different

44

polar head group as well as the various fatty acid distributions, including the length and degree

45

of saturation.6

46

Some previous studies on the identification and analysis of the PLs have reported the PLs

47

concentrations in HBM and IF.7, 8 In addition, reviews on the PLs in milk, including different

48

mammalian milk, dairy by-products or IFs, have been reported recently.5,9,10 The most

49

commonly used techniques are traditional TLC (thin layer chromatography),11 31P-NMR (31P

50

nuclear magnetic resonance),7, 12, 13 HPLC (high-pressure liquid chromatography),14-17 and MS

51

(mass spectrometry).18-20 Among these techniques, TLC is one of the earliest and has been

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employed for both the PL identification and quantification. However, TLC is not often used

53

today as it is time-consuming with complicated extraction procedures. Moreover, it is hard to

54

differentiate multiple PL classes in one run. HPLC, coupled with UV (ultraviolet) or ELSD

55

(evaporative light-scattering detector), have been used for PL separation and detection.

56

However, standards are required to identify the PL classes, and the calibration linearity only

57

worked for small concentration ranges.6 MS, coupled with HPLC, has developed into an

58

extensively used technique for PLs, including the PLs class identification and also the fatty

59

acids compositions of the PLs.19, 21 However, the ionization efficiency differs as a function of

60

the chemical structure, thus, the MS-based result may be influenced by the non-uniform

61

responses. NMR is a technique that is suitable for untargeted analysis with high specificity and

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separation ability. In addition, NMR detection is non-destructive, which is a distinct advantage

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for valuable samples.22

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According to previous studies, the total quantities of PLs in bovine milk and HBM were 0.2-

65

1.0 % and 0.4-1.4 %, respectively.7, 17, 23 Compared with bovine milk, the SM and EPLAS

66

contents in HBM were much higher (29.7~35.7 and 11.4 % of total PLs) while the PE content

67

was lower (18.3 % of total PLs) .7, 17, 24 In bovine milk, the SM, EPLAS and PE contents were

68

reported as 19.9, 4.5 and 31.4 % of total PLs, respectively.7,13 In order to improve the

69

sufficiency of the nutritional PLs, it was important to improve the resemblance between IF and

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HBM. The PL concentrations in bovine milk-based IF have been reported as follows: PE was

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2.2-75 mg/100g, PI was 1.4-46 mg/100g, PS was 0.6-28 mg/100g, PC was 3.8-84 mg/100g,

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and SM was 1.0-82 mg/100g.15, 18, 21 The PLs concentrations detected in these studies varied

73

widely, and it was unclear if the variation was due to the different detection techniques

74

employed, or to actual differences between the IF samples. Therefore, it is necessary to build

75

an efficient method to measure and compare the variations of the PL contents in different IFs.


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The price of IFs varies widely according to the place of origin or manufacturer. The price

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differential is a motivation for fraud and unsafe IF products that have been discovered in world

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markets.25,

79

manufacturers but also poses health issues for the infants who may consume the adulterated

80

product. To mitigate this, it is of vital importance to have efficient tools to verify the

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authenticity of the IF. Zhao et al. utilised 1H-NMR analysis of low-molecular-weight

82

metabolites, such as the contents of acetate, ascorbate, choline, and citrate, to differentiate

83

IFs.27 However, 31P-NMR has not been used to evaluate the PL values in IF samples and let

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alone, to find out the PL comparative differences among IFs. In this study, a PLs

85

characterization and quantification method was developed based on the 31P-NMR technique.

86

In addition, the PLs content in commercial IFs was determined and compared to investigate

87

the compositional differences among the IFs. The purpose of this study was to investigate the

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PL contents in IFs and understand the PL differences among the IFs from different brands that

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were relevant to health benefits, and additionally to assess the potential of utilising the

90

NMR technique for verification of the authenticity of IF.

26

Such fraudulent behaviour not only damages the reputation of the authentic

31P-

91 92

MATERIALS AND METHODS

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Materials. The organic solvents, chloroform, methanol, acetone, trimethylamine (Et3N) and

94

N, N-Dimethylformamide (DMF) were all HPLC grade and purchased from Fisher Scientific.

95

N, N-Dimethylformamide-d7 (DMF-d7, ≥ 99.5 atom-% D), trimethyl phosphate (TMP),

96

Ethylenediaminetetraacetic acid tetrasodium salt hydrate (Na4-EDTA) and guanidinium

97

hydrochloride were purchased from Sigma-Aldrich. Milli-Q water (MQ, 18.2 MΩ cm−1 at

98

25 °C) was used for all aqueous solutions. The DMF/Et3N/GH+ solution was prepared by

99

adding 10 mL DMF, 3 mL Et3N, and 1 g GH+, respectively with 5 µL TMP, which was used

100

as an internal standard.



101

Samples. Commercially available IF powder of different brands (coded A, W, O and M)

102

were collected from retailers in New Zealand. For each brand, six samples from different

103

batches were chosen and used for the NMR analysis. All the IFs were produced using New

104

Zealand raw milk base but by different manufacturers. The selected IFs were sold in different

105

countries, including New Zealand, China and the United States. In addition, IFs were designed

106

for infants according to growing stages; stage I was designed for infants from 0 to 6 months,

107

and the follow-on formulas (stage 2~4) were prepared for infants older than 6 months. The IF

108

samples used in this study were from different stages. The origin and stage information of the

109

IF s were summarized in Table 1. Samples from brand A and M were combined and coded as

110

A&M in the following data analysis.

111

Sample Preparation. The PL extraction was based on previously published methods.12, 13

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First, the milk powder (2 g) was dissolved in 40 mL acetone, and the insoluble residue was

113

collected after centrifugation (3,000 rpm for 15 min). This process was repeated once. As only

114

apolar lipids dissolve in acetone, the purpose of this procedure was to remove the

115

triacylglycerols, which account for about 98 wt % of the milk lipids. Second, the residue was

116

extracted with 30 mL chloroform/ methanol 2:1 (v/v) and in order to avoid the presence of

117

metallic divalent cations, the extract was washed with the same volume of a 0.01M Na4-EDTA-

118

0.1M NaCl solution. It is reported that the presence of these cations would alter the chemicals

119

shifts of the phosphorous nucleus by interacting with anionic phosphates and forming

120

coordination complexes.12 Finally, the lower organic phase was evaporated under nitrogen after

121

centrifugation and stored at -20 °C until analysis. Each sample was prepared in triplicate.

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31P-NMR

Measurements. High-resolution 31P-NMR spectra were acquired on a Varian 400

123

operating at 161.97 MHz, using an inverse probe fitted with a gradient along the Z-axis.

124

Analytical samples were placed in standard 5 mm NMR sample tubes and measured at 25 °C

125

without spinning. The 1H-decoupled, one-dimensional

31P


spectra were obtained using the

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following conditions: spectral width 200 ppm, delay time (D1) 7 s, pulse width of 8.0 ms (90°

127

spin-flip angle), number of scans 3000, and number of data points 32 K. In order to acquire

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31P-NMR

129

was used as an external reference (2.50 ppm), as its resonance did not interfere with PLs peaks

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in the monophasic solvent system.

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The

spectra, the dried sample was dissolved in 0.5 mL DMF/Et3N/GH+ solution. TMP

31P-NMR

data was processed using Vnmrj 4.2 packaged with CRAFT (complete

132

reduction to amplitude frequency table) software, which converts time-domain data into

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frequency-amplitude data using Bayesian analysis. Compared with the peak height or peak area

134

extraction of the frequency domain data, quantitation using Bayesian time domain data

135

performs less sensitive to baseline and phase issues.28, 29 The quantification of the PLs was

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undertaken using the following equation provided that the peaks were well defined with little

137

or no overlap observed:

138 139

Molarity (PL) =

Amplitude (PL) × Molarity (TMP) Amplitude (TMP)

Where Molarity (TMP) was 3.28 mmol∙L-1.

140

Multivariate Data Analysis. An unsupervised method, principal component analysis (PCA)

141

and a supervised method, partial least squares discriminant analysis (PLS-DA) were used to

142

build the multivariate model. The scores of variable importance in projection (VIP) were

143

calculated to measure the variable’s importance and select potential markers. Usually the rule

144

of “greater than one” was used for variable selection criteria, but the threshold of VIP really

145

depends on the data itself, such as the number of variables, the proportion of the number of

146

relevant predictors.30-32 In addition, Student’s t-test was used to test if there was a significant

147

difference in the selected variables between groups. Moreover, the fold change was also

148

calculated and combined with the VIP values from the multivariate data analysis, to help with

149

filtering the biomarkers. Statistical tests were performed using Microsoft Excel 2013®, and R

150

project (R-v3.5.3), which was an open-source freeware widely used for statistical analysis. 7 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Set-up of the

31P-NMR

Spectroscopy. The most important parameter for

31P-NMR

154

detection was to find an appropriate solvent system. There have been two main solvent systems

155

used for milk detection via

156

chloroform/methanol/water-EDTA, which was developed by Meneses and Glonek.33 The

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second class of solvent system was a monophasic solvent mixture, which was proposed and

158

optimized by Bosco et al.34 The monophasic solvent mixture, trimethylamine/dimethyl-

159

formamide/guanidinium chloride (Et3N/DMF-GH+) was used in this study as the chemical

160

shifts were more stable and reproducible using this system. In addition, the range of

161

chemical shifts was enlarged slightly, which helped to improve the resolution. Although it was

162

reported that the estimation of the PE content might be influenced due to the different adducts

163

formed between PE and guanidinium chloride, the possible PE adducts have been ascertained,

164

and the total PE content could be calculated accurately.7, 13

31P-NMR.

The first one was the biphasic solvent system,

31P

165

T1 relaxation is the process by which the net magnetization returns to its initial maximum

166

value. In order to prove the accuracy of the result in NMR measurement, T1 values of the

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targeted analytes were another key point for quantitative analysis. The T1 values of the principal

168

species of PLs detected in milk have been reported, and they were in the range of 0.85 ~ 1.2 s

169

in the DMF/Et3N-GH+ solvent system. The repetition time (TR, = at+D1) should ideally be as

170

long as 5T1 of the particular metabolites requires for accurate quantification.35, 36 Thus, 7s was

171

selected in the set-up 31P-NMR method, which has also been adopted in previous work.13

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The PLs detected by 31P-NMR spectra were extracted by the CRAFT software and quantified

173

with the internal standard, TMP. The CRAFT parameters were set as follows: line broadening

174

(1/at) (Hz) 0.26, CRAFT maximum linewidth (Hz) 20, CRAFT merge peaks within +/- (Hz)

175

0.26 and segment width (Hz) 3. In total, 13 ROIs (regions of interest) were selected in this


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study and the residual spectrum, the reconstructed spectrum and the extracted CRAFT models

177

were demonstrated in Figure 1a. The efficiency of the CRAFT model was validated visually

178

from the residual spectrum. The chemical shifts of PLs detected by 31P-NMR were identified

179

according to the previous study.7, 12, 13 The

180

formula samples iss presented in Figure 1 b.

31P-NMR

spectra acquired from different infant

181

The concentration of the PLs was calculated based on the internal standard, TMP and the

182

ratios of the moles (mol %) for each PL class were listed in Table 2. In addition, the main

183

classes of PLs detected previously in bovine milk and HBM were also summarized in Table 2.

184

All the data shown in Table 2 were the averages or the ranges of the values. The RSD (relative

185

standard derivation) of all the detected PLs was within 10 % for each sample and within 20 %

186

for each group, representing good repeatability of the method. For all the IFs, PC content was

187

the highest with a ratio of 46.90 ~ 52.13 %, and the content of SM was second highest with a

188

ratio of 14.69~19.90 % in comparison to the bovine milk and HBM samples. It was reported

189

that the PC content of PLs in bovine milk was usually the highest (24.0 ~ 28.7 %), followed by

190

the PE (23.5 ~ 31.8 %) and SM contents (19.9 ~ 26.8).6, 7 Apparently, there was much more

191

PC detected in infant formulas, and this was possibly from the soy lecithin, which was

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commonly used as the emulsifier. PC contained more saturated fatty acids compared with the

193

other PLs with choline as a head group, and it was found to be an important component of the

194

membrane.10 The ratio of SM was much higher than PC in HBM compared with that in bovine

195

milk, as shown in Table 2.7 Consequently, the requirements of increased SM should be covered

196

by the infant formulas as SM was proved to have lots of benefits for human health, such as

197

reducing the cholesterol absorption.23

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Compositional Differences among Infant Formulas. All the PLs detected were quantified

199

and compared among the different IF brands. Unsupervised PCA was used to explore the data

200

and investigate potential outliers and trends. As can be seen, the first principal component (PC1)



201

adequately described the majority of the variation (72.8 %) with PC2 and PC3 describing less

202

at 13.5 % and 4.9 %, respectively (Figure 2a). Hence, three PCs were selected for the PCA

203

model, explaining a total of 91.2 % of the variation within the data. As illustrated in the PCA-

204

3D scores plots (Figure 2b), the IFs could be separated partially by the first three PCs, which

205

indicated the PLs compositional difference among the detected samples. However, it was

206

clearly shown in Figure 2b that brand A and M were positioned close to each other, and away

207

from brand O and W. Although brand M and A were sold in the market under different

208

packages and brands, they were produced in the same factory with similar raw milk sourced

209

from the same region in New Zealand (Table 1). Since these two IFs seemed to have a

210

comparable PL profile, they were grouped together and labelled as A&M in the next stages of

211

the multivariate data analysis (Figure 2c). PCA modelling was also used to investigate the

212

effect of stages (Figure 2d). However, the samples could not be clearly separated according to

213

their labelled stages. This result indicated that the formula of PLs in the infant milk might stay

214

the same among the samples designed for different stages from the same producer.

215

In order to identify metabolites that discriminate the three clearly separated classes (A&M,

216

O and W), a PLS-DA model was performed. For the PLS-DA model, the metabolites were used

217

as X-variables, and the IF samples were used as a categorical Y-variables. The validated model

218

had three components, with R2X = 0.895, R2Y = 0.818 and Q2Y = 0.718 (Figure 3a). In line

219

with the observation from the PCA modelling, the samples were separated into three clear

220

groups (A&M, O and W). To identify discriminant markers driving this separation, the VIP

221

plot of the variables from the PLS-DA model was used (Figure 3b). A cutoff point of 0.9 was

222

chosen in this PLS-DA model considering the significant difference of Student’s t-test and

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calculated fold change. The contents of these three selected phospholipids were calculated

224

according to the internal standard (TMP) and summarized in Table 3. The formula weights of

225

PA, SM and PE were calculated as 727, 729 and 744 g mol-1 according to a previous report.13


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In addition, the p values and fold changes of the selected PLs between groups were also shown

227

in Table 3.

228

In order to have an overview of the variable difference among groups, box plots of the

229

selected discriminant PLs were illustrated in Figure 4. As illustrated in Figure 4 and Table 3,

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three PLs were selected as biomarkers to distinguish samples from different brands, PA, SM

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and PE. In general, all these three selected PLs showed an obvious variation between the IFs

232

from brand O and W. The main differences between the IFs from brand A&M and O were the

233

levels of SM and PE. In addition, IFs from brand A&M and W showed different PA contents.

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PA had the highest VIP score and was detected at the amount of over two times higher in

235

brand W than that in the other two brands. This makes sense as the concentration of PA in

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brand W was at the highest level (2.83 mg∙100 g-1) while the contents in the other samples were

237

not significantly different based p< 0.05 (1.07~1.14 mg∙100 g-1). The ratio of PA detected in

238

infant formula in our study was 1.22~3.02 mol%, which was in accordance with previous

239

reports.37 PA was the simplest phospholipid with an active H as the head group and was

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enriched in eukaryotic cell membranes.9 PA had an important role in glycerolipid metabolism

241

and membrane biogenesis with its anionic head group. Furthermore, PA could also act as a

242

signalling lipid.38 PA in raw bovine milk was detected in Gallier et al.’s study,11 however, the

243

PA detected was regarded to be caused by the hydrolysis of the neutral and polar lipids of the

244

membrane during a few hours of cold storage. In addition to this, PA was not found in bovine

245

milk or HBM according to other reports.7, 39 However, PA was detected in the soy lecithin,

246

which was used as an emulsifier in IFs as labelled, at about 2 wt%.40, 41 Therefore, the difference

247

of the PA content observed in IFs was most likely from the added ingredients.

248

SM had the second-highest VIP score and compared with PA, and SM showed a different

249

trend of variation among the IFs from different brands. Significant variation of the SM content

250

was observed in IFs from brand O, which was nearly one time lower than that in the other two



251

groups. Specifically, the average contents of SM were 17.49 mg/100 g, 8.89 mg/100 g and

252

13.76 mg/100 g in brand A&M, O and W, respectively (Table 3). SM was a type of

253

sphingolipids with phosphorylcholine as the polar head group. Thus, SM, together with PC,

254

were the main source of choline, which was crucial for the rapid growth of the brain and other

255

organs.42 In addition, SM, as reported, was the most saturated phospholipid with about 73 %

256

saturated fatty acids content and the SM contained in HBM was mainly composed of the C16:0

257

(palmitic acid), C18:0 (stearic acid), C22:0 (docosanoic acid) and C24:1 (tetracosanoic acid)

258

fatty acids.5, 6, 11 It found that the predominant sphingoid base in breast milk was sphingosine

259

(d18:1), which accounted for 83.6%.19 Therefore, the formula weight of the SM used as a

260

conversion factor was 729, SM (d18:1/18:1). The SM contents and the compositions of SM

261

were reported to be related to hydrolytic activity and the gut microbiota establishment, which

262

were of vital importance for the infant’s growth.43 In bovine milk, SM was found to be altered

263

by breeds, seasons and the lactation stages of the cow.44 Specifically, SM in milk fat from

264

Holstein cow was at a higher concentration than that from Jersey cows due to the different fat

265

globule sizes in milk between the breeds. In addition, the SM was observed more concentrated

266

in bovine whole-milk collected in summer or from cows at their late lactation stage as a result

267

of the increased milk fat content.44 The SM content in HBM during the lactation period was

268

also studied; however, there were different results about the SM content varieties. It was

269

reported that the percentage of SM remained constant during the different lactation periods,

270

while Shoji (2006) found that the SM percentage in mature milk was significantly higher.19, 45,

271

46

272

consumption by breastfed infants should be from 50~150 mg/day based on animal

273

experiments.47 IFs used in the study were all based on bovine milk and as reported, the relative

274

proportion of SM in bovine milk was lower than that in HMB (Table 2). However, the SM

275

concentration detected in IFs was within the HBM range, in which the SM level was reported

Considering the importance of the SM to the infant’s health, it was suggested that the SM


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to be about 5.0 ~ 13.3 mg∙100 g-1.7 Thus, additional SM was added as an ingredient of IFs to

277

resemble the SM content of HBM. The variations observed in the IFs from different brands

278

were possibly caused by both the raw milk and added formula.

279

In addition, PE was selected as another marker to distinguish IF samples from different

280

retailers with a VIP score greater than 0.9. Similarly with the SM content in the IFs, PE content

281

in the IFs from brand O was also at the lowest level (5.72 mg∙100 g-1 ) while IFs from the other

282

two groups were at the similar higher level (12.26~14.41 mg∙100 g-1). PE was highly

283

unsaturated with the ethanolamine as a head group and was found in all eukaryotic cells, which

284

accounted for about 25 % in cells and 45 % in the brain of the total phospholipids.46 It was

285

reported that the PE species found in milk were composed by the fatty acids with C16:0/C18:2,

286

C16:0/C18:1 and C18:0/C18:2.6, 49 According to previous research, the PE content increased

287

significantly both from colostrum to transitional milk and transitional milk to mature milk.49,

288

50

289

during their first growth stage. Moreover, PE was not only proven to be crucial for the

290

development of the membrane and brain but also found to play an important role in heart

291

health.48 Like SM, the PE in bovine milk was also found to have the highest level at the late

292

lactation stage of the cow.51 However, this difference was within 1.65 times (0.23 ~ 0.38),

293

which was not adequately accounted for the difference observed between the IFs from different

294

brands (Table 3). As PE was also detected in the soy lecithin at an amount of about 13.8 wt%,40

295

PE difference between groups might be contributed by both the raw milk and added ingredients.

296

As the best substitute for HBM, IF was required to be safe and satisfy the infant’s nutritional

297

requirements. In addition, the composition of IF was strictly regulated with established

298

guidelines that the retailer should follow.52 Though there were different results on the PLs

299

changes during the lactation period in HBM,45, 49 the PL amounts, including the PE and SM

300

contents, were found to have increased significantly in the brains of the newborns in their first

The increasing amount of PE indicated the high requirement of PE for infants’ development



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301

year of life.53 Thus, it was important to understand the different contents of the PLs in various

302

IFs and then chose the optimal feeding amount according to the guidelines for the infants at

303

different growing stages.

304

In summary, a

31P-NMR

method was set up in this study based on the DMF/Et3N/GH+

305

monophasic system with stable chemical shifts and good separation of the PL classes. Nine

306

phospholipid species were detected in the IFs, and moreover, PL contents detected in different

307

IF brands were quantified and compared. The results demonstrated that the PLs variances

308

existed among different IFs according to their various formulations used in the processing, such

309

as the contents of SM, PA and PE. Therefore, the

310

multivariate data analysis, were shown to be an effective tool for better understanding the PL

311

contents and differences among the IFs. This result could not only help with the feeding

312

guidelines but also help to verify the authenticity of the IFs from different brands. However,

313

the fatty acid compositions of the selected PLs were not measured in this study. Therefore, the

314

differences of the fatty acid distributions in relation to PLs among different IF brands needed

315

to be further studied in the future with the aid of other techniques.

31P-NMR

spectra, combined with the

316 317

ABBREVIATIONS

318

IF, infant formula; PL, phospholipids; TMP, trimethylphosphate; PC, phosphatidylcholine; PE,

319

phosphatidylethanolamine;

320

lysophosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; EPLAS,

321

phosphatidylethanolamine plasmalogens; SM, sphingomyelin; PI, phosphatidylinositol; PG,

322

phosphatidylglycerol; PA, phosphatidic acid.

MMPE,

Monomethylphosphatidylethanolamine;

323 324

Funding

325

This work was supported by University of Otago PhD Scholarship to Dan Zhu.


LPC,

Page 15 of 27


326 327

Notes

328

The authors declare no competing financial interest.

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355



356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

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491 492 493 494 495 496 497 498 499 500 501 502 503


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504


Figure captions

505 506

Figure 1. (a) The residual, reconstructed and experimental spectrum acquired from CRAFT and the

507

extracted CRAFT models of the 31P-NMR spectrum. (b) 31P-NMR spectra of the PL extracts from IFs.

508

The signals were assigned as follows: TMP, 2.50 ppm, used as an internal reference; PC, 0.00 ppm; PE

509

adduct, 0.19 ppm; MMPE, 0.40 ppm; LPC, 0.45 ppm; PS, 0.54 ppm; PE, 0.56 ppm; EPLAS, 0.58 ppm;

510

SM, 0.83 ppm; PI, 1.02 ppm; PG, 1.25 ppm and PA, 5.40 ppm.

511 512

Figure 2. (a) Variance principal components scree plot; Three-dimensional PCA scores plot based on

513

the 31P-NMR data: (b) PCA-3D plot of four types of IF samples coded A, M, O and W. (c) PCA-3D

514

plot of three types of IF samples, coded A&M, O and W. (d) PCA-3D plot of two stages of IF samples,

515

stage 1 and stage 2&3.

516 517

Figure 3. (a) PLS-DA biplot based on the

518

variables with a VIP value over 0.9 were shown in bold). (b) Bar plot of the VIP scores from the PLS-

519

DA model. Blue line- VIP score was 1.0; the red line- VIP score was 0.90.

31P-NMR

data (the open circles were variables and the

520 521

Figure 4. Boxplots and the structure of the selected PLs to distinguish IFs (a) PA; (b) SM and (c) PE

522



Page 20 of 27

Tables Table 1 Information on the collected infant formula samples Group A&M

New Zealand USA

Stage 1 2 2

O

China

3

3

0

W

New Zealand

NL*

NL

NL

O W *NL:

Formulation Stage Stage 2 Stage 3 2 2 3 1

Manufacturer Code A M

Origin

Not labelled.


Page 21 of 27


Table 2 Relative proportion of PL classes in different infant formulas PL classes PC MMPE LPC PS SM PI PG PA PE a Results

Relative proportion (mol% of total PLs)a A&M O W 47.77 ± 6.24 52.13 ± 7.58 46.90 ± 7.23 2.57 ± 0.37 2.80 ± 0.33 3.77 ± 0.55 2.57 ± 0.41 2.56 ± 0.37 2.43 ± 0.36 5.79 ± 0.27 4.66 ± 0.28 6.60 ± 1.45 19.90 ± 3.44 17.93 ± 3.88 14.69 ± 2.87 5.74 ± 0.98 5.78 ± 1.06 6.32 ± 0.91 0.77 ± 0.15 0.53 ± 0.11 1.19 ± 0.22 1.22 ± 0.22 2.30 ± 0.23 3.02 ± 0.39 13.66 ± 2.97 11.31 ± 2.26 15.08 ± 1.27

Bovine milkb 24.0-28.7 0 1.5-11.7 19.9-26.8 3.6-14.0 4.6 1.8 23.5-31.8

presented as mean ± SD

b

Data references 7, 11, 16, 37

c

Data references 7, 43


Human milkc 18.7-24.5 0 8.1-15.3 29.7-39.6 3.8-14.1 0 8.7-18.3


Page 22 of 27

Table 3 Contents of PA, SM and PE in different IF samples PLs

Concentration (mg∙100g-1)a

FC b

A&M

O

W

A&M/O

A&M/W

O/W

PA

1.07 ± 0.19

1.14 ± 0.11

2.83 ± 0.36

0.94

0.38***

0.40***

SM

17.49 ± 3.02

8.89 ± 1.92

13.76 ± 2.64

1.97***

1.27*

0.65***

PE

12.26 ± 2.66

5.72 ± 1.14

14.41 ± 1.21

2.14***

0.85**

0.40***

a Results bAsterisk

presented as mean ± SD rating system was used for quoting the P value: P < 0.05 *, P < 0.01, ** P < 0.001***


Page 23 of 27


Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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Page 27 of 27


TOC graph


31P NMR-based phospholipid fingerprinting of powdered infant formula

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