Impact of Holder Pasteurization on Protein and eNAMPT/Visfatin Content in Human Breast Milk

doi:10.21203/rs.3.rs-4837352/v1

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Impact of Holder Pasteurization on Protein and eNAMPT/Visfatin Content in Human Breast Milk

https://doi.org/10.21203/rs.3.rs-4837352/v1

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Human milk proteins, a mixture of whey proteins including caseins, milk fat globule membrane (MFGM) proteins, various peptides, and their amino acids, play a crucial role in infant growth and development, as do non-nutritional bioactive components. The extracellular nicotinamide phosphoribosyltransferase (eNAMPT) or visfatin is a conserved cytokine/enzyme released by many mammalian cells, related to multiple metabolic and immune processes. Few investigations have been reported about detecting visfatin in skimmed milk and the hypothesis of its potential role in regulating infant adiposity through breast milk. Milk samples from a donated human milk bank were analyzed. After milk fractionation, skimmed milk and MFGM were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. An ELISA assay assessed visfatin content in skimmed milk, while MFGMs were analyzed by Western blot. The data showed a higher visfatin content in raw skimmed milk than in pasteurized samples. This is the first time visfatin has been identified associated with MFGM. Its levels appear to be higher in MFGM derived from pasteurized milk than in raw milk, an opposite result to that obtained by analyzing the skimmed milk samples. These results suggest the visfatin involvement in the features of MFGMs and their potential role for infants.

Biological sciences/Biochemistry/Proteins

Health sciences/Health care/Nutrition

human breast milk

holder pasteurization

visfatin

eNAMPT

ELISA

Western Blot

Maternal milk is considered the optimal source of nutrition for infants across species due to its comprehensive profile and health benefits. The World Health Organization (WHO) substantiates this assertion by recommending exclusive breastfeeding from birth through the first six months of life [1]. The composition of human milk is the result of millions of years of evolution: it comprises the perfect amount of water, proteins, lipids, carbohydrates, vitamins, and micronutrients, i.e., all the nutrients that infants need at every stage of their growth [2]. Indeed, human milk can be described as a dynamic and vital tissue in which the components vary in quantity according to the infant's needs between feedings and throughout the lactation period [3]. Human milk proteins are a mixture of whey, caseins, and various peptides: their amino acids play a crucial role in the growth and development of the child [4]. Some of these proteins, such as α-lactalbumin, lactoferrin, and lysozyme, are also important for infants as non-nutritional bioactive components [5]. Moreover, human milk contains cytokines, interleukins, interferon-γ, and transforming growth factor-β, providing immunomodulation and passive protection to the baby [6]. In addition, the protein extracellular nicotinamide phosphoribosyltransferase (eNAMPT), also known as visfatin [7, 8], is present in human milk [9]. Visfatin, or pre-B-cell colony-enhancing factor (PBEF) or NAMPT, is a 52 kDa protein secreted from the visceral adipose tissue, and it is correlated with several metabolic and immune processes [7, 8, 10–12]. The presence of visfatin in human milk may imply its potential role in regulating infant adiposity through maternal milk [9].

Since human breast milk is the recommended choice for feeding a newborn baby, it is essential for those infants who cannot be breastfed to be fed with donated human milk (DHM)[13]. DMH is collected in a Donated Human Milk Bank (DHMB), following local legislation, from healthy lactating women, and it is used to feed children hospitalized in neonatal intensive care. Immediately after donation, DHM undergoes the Holder pasteurization process to inactive pathogens, reduce bacterial contamination, and stabilize the milk [14]. While Holder pasteurization remains the recommended method according to international guidelines for establishing Human Milk Banks, ongoing discussion surrounds the impact of this pasteurization process on protein content [15–18].

In this work, we aim i) to evaluate the effect of Holder pasteurization on the protein content and quality of donated human milk, ii) to detect visfatin in both skimmed milk and MFGM-associated proteins and iii) to determine if there are differences in visfatin content between pasteurized and non-pasteurized human breast milk.

Milk Samples

In this study, we investigated human milk samples kindly provided by DHMB of Ospedale Sant’Andrea, Vercelli, Italy. Between January and December 2023, 10 milk samples were collected from 10 healthy lactating mothers. All pregnant mothers, except for donor 1 who had a cesarean section, delivered by vaginal delivery. All milk samples were mature and donated at least five weeks after delivery. Sample 3 was excluded from the subsequent analysis because, in the pasteurized version, the skimmed milk fraction could not be separated from the fat globule fraction. Its protein concentration and visfatin quantification range in the same interval as the other samples. Supplementary Table 1 reports the demographic characteristics of milk donors.

Protein quantification

Table 1 and Fig. 1 report the protein concentration of milk samples before (NP) and after Holder pasteurization (P). Four samples of skimmed milk show a statistically significant difference between the treated and raw samples, with a higher protein concentration in the NP ones (Fig. 1A). The MFGM-associated proteins exhibit an inverse trend. Except for samples 1 and 10, all samples subjected to the Holder pasteurization process demonstrate a higher protein concentration, as illustrated in Table 1 and Fig. 1B.

Table 1

Skimmed milk protein and MFGM-associated protein concentration for not pasteurized and pasteurized samples. The data are expressed as mg/ml and presented as the mean protein concentration, derived from three technical replicates, accompanied by the standard deviation. Additionally, corresponding p-values and t-values are reported to indicate statistical significance.
	Skimmed milk protein concentration				MFGM-associated protein concentration
Sample	Not pasteurized	Pasteurized	t-value	p-value	Not pasteurized	Pasteurized	t-value	p-value
1	4.351 ± 0.284	4.046 ± 0.362	-0.856	0.482	4.891 ± 0.058	4.222 ± 0.011	-17.739	0.003
2	6.314 ± 0.049	6.068 ± 0.068	-5.916	0.027	1.892 ± 0.011	3.352 ± 0.066	32.927	0.001
4	6.341 ± 0.317	6.169 ± 0.128	-1.392	0.298	2.970 ± 0.053	5.010 ± 0.010	86.477	0.000
5	4.776 ± 0.142	4.608 ± 0.086	-1.040	0.487	1.102 ± 0.015	2.827 ± 0.048	83.827	0.000
6	6.708 ± 0.164	6.312 ± 0.207	-7.844	0.016	2.570 ± 0.073	4.919 ± 0.031	40.090	0.001
7	7.032 ± 0.114	6.619 ± 0.106	-5.148	0.036	2.030 ± 0.076	2.567 ± 0.041	7.983	0.015
8	5.103 ± 0.226	5.072 ± 0.166	-0.553	0.636	2.153 ± 0.090	7.703 ± 0.682	16.201	0.004
9	6.428 ± 0.115	6.002 ± 0.222	-7.608	0.017	2.282 ± 0.094	3.233 ± 0.102	8.942	0.012
10	6.312 ± 0.107	6.064 ± 0.364	-1.335	0.313	1.402 ± 0.112	1.235 ± 0.077	-3.127	0.089

Protein profiling

The SDS-PAGE profile of pasteurized MFGM-associated proteins appears to have some more intense bands around 250, 150, and 75 kDa, in addition to those between 50 and 37 kDa and between 37 and 25 kDa (Supplementary Fig. 1).

An aliquot of each skimmed milk sample was subjected to MALDI-TOF analysis to identify low molecular weight proteins. Upon examination of the spectra (Supplementary Figs. 2 and 3), discernible distinctions between not pasteurized (NP) and pasteurized (P) samples are not evident. In both not pasteurized and pasteurized DHM samples analyzed with the linear positive method, some peaks are visible, around 24,000 m/z, 14,000 m/z, 12,000 m/z, 8,000 m/z, 7,000 m/z, and the 4,000–5,000 m/z range. Information about m/z values and peak intensity was submitted to the GeenaR online platform to investigate differences in the molecular weight distribution of skimmed milk DHM samples. The principal component analysis has confirmed the hypothesis since it has not identified a discriminant between the pasteurization (P) and the not pasteurized (NP) samples.

eNAMPT/visfatin detection

Anti-eNAMPT/visfatin immunoreactive bands showed higher intensity in pasteurized MFGM samples than in not pasteurized milk. The bands that reacted are just above 50 kDa (Fig. 2), consistent with the presence of eNAMPT/visfatin, whose molecular weight is around 52 kDa. Sample 2, which has small amounts of visfatin, also follows the same trend, showing a slight band of reactivity in the pasteurized version compared to the unpasteurized one.

To confirm the observations, an optical density analysis of these bands was performed (Supplementary Fig. 4). In addition, a comparison was made with a recombinant eNAMPT/visfatin band (Supplementary Fig. 5) that allowed us to estimate the average protein amount of visfatin present and associated with milk fat globules. In raw (not pasteurized) samples, an amount of eNAMPT/visfatin of about 0.023 µg per milligram of MFGM-associated protein could be found. Analyzing the samples after heat treatment revealed an average of 0.925 µg per milligram of MFGM-associated protein.

An ELISA assay quantified the eNAMPT/visfatin concentration within the skimmed milk samples. With only one exception (Table 2 and Fig. 3), the trend demonstrates decreased eNAMPT levels after the pasteurization process. The difference is statistically significant in four out of ten sample pairs analyzed.

Table 2

Visfatin concentration is expressed as ng/ml of pasteurized samples, not pasteurized ones. The data are presented as the mean protein concentration, derived from three technical replicates, accompanied by the standard deviation. Additionally, corresponding p-values and t-values are reported to indicate statistical significance.
Sample	Not pasteurized	Pasteurized	t-value	p-value
1	1847.404 ± 124.152	88.637 ± 8.742	-21.552	0.030
2	2499.063 ± 286.929	2287.273 ± 180.615	-0.641	0.637
4	1883.835 ± 165.667	1106.02 ± 185.691	-54.936	0.012
5	1002.972 ± 110.562	72.236 ± 8.467	-12.897	0.049
6	915.376 ± 111.183	302.665 ± 16.459	-9.148	0.069
7	1238.406 ± 84.099	594.440 ± 18.484	-13.880	0.046
8	252.668 ± 42.354	52.994 ± 13.617	-9.826	0.065
9	229.025 ± 2.343	137.836 ± 31.499	-4.423	0.142
10	73.651 ± 16.539	219.975 ± 45.040	3.360	0.184

The amount of eNAMPT found in each mg of skimmed milk protein was calculated to compare the amount of visfatin present in milk fat globules. In this case, not pasteurized samples have a visfatin amount of about 86.982 µg per mg of proteins, while pasteurized ones observe a decrease to about 41.073 µg per mg of proteins. These values are higher than those usually reported in blood for eNAMPT [9, 19].

Examining the samples as a cohort can identify unique and statistically significant trends for all four parameters analyzed (Table 3 and Fig. 4). In particular, it was possible to determine a substantial decrease in the protein concentration of the skimmed phase after Holder pasteurization treatment. The eNAMPT/visfatin protein also follows the same trend, decreasing in pasteurized samples. At the same time, the concentration of milk fat globule membrane-associated proteins increases, as does the eNAMPT/visfatin protein concentration.

Table 3

Paired t-test results between Not Pasteurized and Pasteurized samples. Abbreviation OD: Optical Density, WB: Western Blot, MFGM: milk fat globule membrane. The results represent the mean of the nine samples ± the standard deviation.
Component	Not pasteurized	Pasteurized	t-value	p-value
Skimmed milk (mg/ml)	5.935 ± 0.942	5.662 ± 0.874	5.801	0.0004
Visfatin in skimmed milk (ng/µl)	1,104.711 ± 845.784	540.230 ± 737.943	2.970	0.0179
MFGM-associated proteins (mg/ml)	2.346 ± 1.090	3.896 ± 1.858	2.562	0.0336
OD of eNAMPT/visfatin in WB of MFGM-associated proteins	88,025.444 ± 107,058.056	912,134.889 ± 574,661.715	4.4740	0.0021

The research proposed in this paper adds a pillar to the study of Holder pasteurization's effect on the protein concentration in human milk before and after treatment. Holder pasteurization carried out at 62.5°C for 30 minutes, is presently considered the gold standard for milk processing in human milk banks. It is endorsed by numerous guidelines as the best balance between maintaining milk quality and ensuring microbiological safety [20, 21]. Conflicting results are reported in the literature concerning the decrease or increase in protein caused by the pasteurization process [17, 22–25]. Specifically, this study examined two phases: skimmed milk and the fraction of protein associated with milk fat globule membranes (MFGMs). Proteins in skimmed milk decreased significantly after treatment in only four of the samples analyzed. Looking at the whole cohort, this decrease in protein appears to be significant, which is in agreement with the findings of Vieira and colleagues [25]. While the pasteurization treatment caused a reduction in protein in skimmed milk, the proteins associated with fat globule membranes appear to be increased after pasteurization. These findings may suggest an interaction and subsequent aggregation between proteins present in the skimmed milk stage and those associated with MFGMs, as proposed by Ma et colleagues [26]. Otherwise, the fat globule membrane may reorganize during pasteurization. This change most likely involves an increase in the volume of the fat globule, which is achieved by a physical transfer from the skimmed milk fraction. This hypothesis aligns with recent findings observed in studies on the pasteurization of cow’s milk [27]. The appearance of more intense bands in the SDS-PAGE profile of the pasteurized sample supports these two hypotheses. Specifically, more intense bands appear in the profile of pasteurized MFGM-associated proteins around 250, 150, and 75 kDa, in addition to those between 50 and 37 kDa and between 37 and 25 kDa. However, the profile does not show the appearance or disappearance of any new bands. Looking at the gel image of the skimmed milk proteins, the intensity of the bands is almost unchanged between the samples, with the highest intensity bands around 75, 25 kDa, and 10 kDa. The very intense 75 kDa band corresponds to lactoferrin, while the thinner band below corresponds to serum albumin and immunoglobulin heavy chain. Just above the 25 kDa band is β-casein, as reported in our previous work [28]. The analysis of the skimmed phase of the milk at lower molecular weight, using the MALDI-TOF profiling, shows no structural or qualitative change in the proteins: the only change in skimmed milk is, therefore, quantitative.

Our study's original contribution is to investigate the presence of the eNAMPT/visfatin in skimmed milk and MFGM-associated proteins and its potential quantitative change after the Holder pasteurization process. Indeed, although the presence of eNAMPT/visfatin has already been described in skimmed milk [9], to our current knowledge, the presence of eNAMPT among milk fat globule membrane-associated proteins has not been reported in the literature before. Furthermore, the effect of pasteurization on this protein is unknown. The search for any differences between raw and subsequently pasteurized samples was carried out using two methodologies according to the nature of the samples. The skimmed milk phase, i.e., an aqueous phase, was investigated by a specific ELISA assay. MFGM-associated proteins, on the other hand, were analyzed by Western Blot because the extraction and solubilization buffer for these is highly denaturing and, when used in ELISA assay, caused denaturation of the antibody-protein sandwich. The ELISA results suggest decreased eNAMPT/visfatin concentration in samples subjected to Holder pasteurization with a loss of nearly 50% of eNAMPT/visfatin amount after treatment. This could be explained by a co-participation of this protein in the reorganization of the fat globule or its increased affinity to the fat phase (and thus easier transferring of this during reorganization). The western blot of MFGM-associated proteins also confirms the trend. Here, very intense bands can be observed in the pasteurized samples and less intense bands in the raw samples, suggesting the transfer of the eNAMPT/visfatin protein within or in association with the MFGMs. Indeed, this increase is confirmed by statistical analysis of their optical density (OD) value, which allows us to see an approximately 39-fold increase in the quantity of eNAMPT/visfatin in the samples after pasteurization.

Our study showed that the concentration of both protein portions, i.e., those present in the skimmed milk phase and associated with the fat globule membranes, appears to be influenced by Holder pasteurization. In particular, during the treatment, the fat globules seem to subtract protein at the skimmed milk stage, resulting in a higher protein concentration. Qualitative, SDS-PAGE and MALDI-TOF profiling of the skimmed milk fraction showed that the differences between treated and untreated samples are nearly negligible.

Furthermore, for the first time, the presence of eNAMPT/visfatin was demonstrated not only in the skimmed milk phase, already reported in the literature, but also in the protein phase associated with milk fat globule membranes. Holder pasteurization also affects this protein: its concentration decreases in the skimmed phase of pasteurized milk and increases in the protein-associated phase of MFGM. This can be explained by assuming that this protein was plausibly associated with a higher affinity for fat globules during their reorganization caused by the heat treatment. The eNAMPT/visfatin association with MFGM could support the hypothesis of its involvement in fat metabolism and infant body weight regulation [9, 11]. We can further suggest a potential active role of eNAMPT/visfatin in the immunological status of newborns [29]. Further investigation of MFG entrapment of eNAMPT/visfatin, which is a soluble protein, could add information on the role of this released cytokine.

Human milk samples

In this study, we investigated human milk samples kindly provided by DHMB of Ospedale Sant’Andrea, Vercelli, Italy. The mothers gave informed and signed consent to the study. This project was approved by the Department of Science, Technology and Innovation of the University of Piemonte Orientale (PROT #0006564–23/12/2021). All the experiments were performed in accordance with relevant guidelines and regulations. In particular, the recommendations stated in [14] were followed for donated human milk samples. Between January and December 2023, 10 milk samples were collected from 10 healthy lactating mothers. Donor exclusion criteria are reported in [14]. DHMs were subjected to bacteriological controls to exclude samples with theoretical risk for bacteria > 10⁵ CFU/ml. For DHM contaminated by Staphylococcus aureus and Enterobacteriaceae, discharge was warranted if the contamination exceeded 10⁴ CFU/ml. Immediately after donation, an aliquot was separated from the bottle and frozen at -20°C. Another aliquot was pasteurized for 30 minutes at 62,5°C (Holder pasteurization process) and then frozen at -20°C. The samples were transported, guarantying the cold chain, to the biochemistry laboratory and were stored at -80°C until analysis.

Milk phase separation and protein quantification

A protease inhibitor cocktail (cOmplete, Mini; Roche) was added to the milk aliquots, and the samples were then centrifugated at 2,000 x g for 10 minutes at 10°C to allow the separation of skimmed milk from fat globules (MFG). MFGs were washed with 0.9% saline and centrifuged at 3,000 x g for 10 minutes at 10°C three times, as indicated in [30]. Proteins associated with MFG membranes (MFGM) were extracted with an estimated equal volume of 2D buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 100 mM DTT, IPG-buffer (pH 3–10)). The protein concentration of both phases, skimmed milk and MFGM, was quantified using an optimized Bradford microplate assay with BSA (bovine serum albumin) as standard [31].

Protein profiling

Samples were analyzed by SDS-PAGE. Proteins (10 µg) were mixed to an equal volume of Laemmli reducing sample buffer [32] and heated at 95°C for 5 minutes. Skimmed milk proteins were loaded onto 12.5% polyacrylamide gel, and MFGM-associated proteins were loaded onto 10% polyacrylamide gel. SDS-PAGE was performed at 100–120 V with a Mini Protean System (BioRad) using a molecular weight protein standard (Precision Plus Protein ™ Dual Color Standard, BioRad). Gel staining was carried out with Blue Silver Stain [33]. After distaining, the gel images were obtained with a GS-900 densitometer (BioRad).

Skimmed milk proteins were also analyzed through the MALDI-TOF spectrometer. 1 µl of samples at 1 mg/ml protein concentration was mixed with 10 µl of matrix solution (10 mg/ml sinapinic acid dissolved in acetonitrile: 0.1% TFA, 30:70% v/v – TA30), and 1 µl of each mix was spotted on a ground steel MTP 384 target plate (Bruker Daltonics GmbH, Bremen, Germany) in triplicate and let dry at room temperature overnight. MALDI-TOF spectra were acquired by Ultraflextreme mass spectrometer (Bruker Daltonics GmbH, Bremen) equipped with a Smart-beam 2 Nd:YAG laser operating at 355 nm, using FlexControl software (version 3.3, Bruker Daltonics GmbH, Bremen, Germany). Skimmed milk proteins were analyzed in linear mode in positive ion acquisition in a mass-to-charge ratio range of 2,000–50,000 and reflectron mode in positive ion acquisition in a mass-to-charge ratio of 700–3,500. The mass spectra obtained are the sum of 6000 shots for each sample, and the random walk feature with partial sample mode was activated. Protein mass spectra were calibrated through “Protein Calibration Standard I”, hydrolysates using “Peptide Calibration Standard I” (Bruker Daltonics GmbH, Bremen) in FlexAnalysis software (version 3.3, Bruker Daltonics GmbH, Bremen).

To analyze the principal component of spectra these were subjected to GeenaR (https://proteomics.hsanmartino.it/geenar/) with the following parameters: trimming from 1,400 to 3,500 (for spectra analyzed with reflectron method) or 2,000 to 30,000 (for spectra analyzed with the linear method); variance stabilization: square root; smoothing: Savitzky–Golay with half window size of 10; baseline removal: TopHat, with half window size of 75; normalization: total ion current (TIC). Peak alignment was carried out using MAD (median absolute deviation) for noise estimation (half window size: 20, SNR: 2) and LOWESS (local weight scatterplot smoothing) for phase correction (mass tolerance: 0.002). Then, peaks were selected by applying the strict method for binning and a coverage (minimum frequency of peaks for their selection) of 0.5. Finally, principal component analysis was applied to the processed mass spectra [34].

Visfatin detection

A western blot was performed to detect the presence of eNAMPT/visfatin (UniProt P43490) in MFGM proteins. 20 µg of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4–20% Cat. #4568093) and transferred using Trans-Blot® TurboTM Transfer System to nitrocellulose membranes (all from Bio-Rad). A recombinant NAMPT (rec) form was prepared in-house [35] and used as a control. To detect NAMPT, an anti-NAMPT was used (A300-779A, Bethyl Laboratories, Montgomery, TX, USA) with a mouse anti-rabbit HRP-conjugated as the secondary antibody (sc-2357, Santa Cruz Biotechnology). Western blot chemiluminescence reactions were visualized with ECL (Cat. #170–5061) (Bio-Rad, Segrate, Milan, IT) using the Invitrogen iBright CL1500 Imaging System (Thermo Scientific). Densitometric analyses were performed using Image Lab 6.1 Software (Bio-Rad) or iBright Analysis Software 5.2.1 (Thermo Scientific).

Skimmed milk eNAMPT/visfatin concentrations in not pasteurized (NP) and pasteurized (P) milk were determined using a human NAMPT Enzyme-Linked Immunosorbent Assay (ELISA) kit (Adipogen, AG-45A-0006YEK-KI01). Samples were diluted 300-fold to the assay range (0.125-8 ng/ml) with assay diluent, and the assay was performed according to the manufacturer’s instructions [9].

Statistics

A paired t-test was used to compare continuous variables. Unless otherwise indicated, data are represented as mean ± SD. Statistical analyses were performed on RStudio version 2023.12.0 + 369. * p < 0.05, ** p < 0.01, *** p < 0.001.

Competing interests

The authors declare no competing interests.

Author Contribution

A.G, V.A., and M.C. devised the manuscript; A.G., C.G., I.F., and A.M.T. studied the samples; C.G. M.B., E.U., and G.C. collected the samples; A.G, V.A., and M.C wrote and revised the manuscript; V.A. and M.C supervised the whole research. All authors revised the manuscript.

Acknowledgement

V.A. is supported by a grant from the Italian Association for Cancer Research (AIRC-MFAG #26004) that co-founded the PhD fellowships of I.F. and A.M.T.

Data Availability

The sequence of eNAMPT/visfatin analyzed during the current study is available in the UniProt repository (UniProt P43490). The human milk protein sequences cited in the discussion section are available in the UniProt repository with the following accession numbers: lactoferrin (P02788), serum albumin (P02768), immunoglobulin heavy chain (P01877), and beta-casein (P05814).

World Health Organization, WHO Recommendations on Postnatal Care of the Mother and Newborn, World Health Organization, Geneva, 2014.
N.J. Andreas, B. Kampmann, K. Mehring Le-Doare, Early Hum. Dev. 91 (2015) 629–635.
O. Ballard, A.L. Morrow, Pediatr. Clin. North Am. 60 (2013) 49–74.
D. Yi, S. Kim, Nutrients 13 (2021) 3094.
B. Lönnerdal, Adv. Exp. Med. Biol. 554 (2004) 11–25.
C.E. Grosvenor, M.F. Picciano, C.R. Baumrucker, Endocr. Rev. 14 (1993) 710–728.
E. Semerena, A. Nencioni, K. Masternak, Front. Immunol. 14 (2023) 1268756.
M. Gasparrini, V. Audrito, Int. J. Biochem. Cell Biol. 145 (2022) 106189.
J. Bienertová-Vašků, P. Bienert, F. Zlámal, J. Tomandl, M. Tomandlová, Z. Dostálová, A. Vašků, Diabetes Res. Clin. Pract. 96 (2012) 355–361.
E. Adeghate, Curr. Med. Chem. 15 (2008) 1851–1862.
N. Mól, M. Zasada, P. Tomasik, K. Klimasz, P. Kwinta, PLOS ONE 13 (2018) e0204835.
S.H. Jia, Y. Li, J. Parodo, A. Kapus, L. Fan, O.D. Rotstein, J.C. Marshall, J. Clin. Invest. 113 (2004) 1318–1327.
N. Haiden, E.E. Ziegler, Ann. Nutr. Metab. 69 (2016) 7–15.
G., De Nisi, A.M., Ambruzzi, S., Arslanoglu, E., Bertino, A., Biasini, L., Gagliardi, G., Moro, F., Mosca, C., Profeti, G., Salvatori, P., Tonetto, Raccomandazioni per La Costituzione e l’organizzazione Di Una Banca Del Latte Umano Donato, New Magazione Edizioni, 2021.
F.-Y. Chang, L.-J. Fang, C.-S. Chang, T.-Z. Wu, Breastfeed. Med. 15 (2020) 576–582.
B. Adhisivam, B. Vishnu Bhat, K. Rao, S.M. Kingsley, N. Plakkal, C. Palanivel, J. Matern. Fetal Neonatal Med. 32 (2019) 3016–3019.
C. Peila, A. Coscia, E. Bertino, M. Cavaletto, S. Spertino, S. Icardi, C. Tortone, G.H.A. Visser, D. Gazzolo, Ital. J. Pediatr. 42 (2016) 36.
I.J. Paulaviciene, A. Liubsys, A. Eidukaite, A. Molyte, L. Tamuliene, V. Usonis, Breastfeed. Med. 15 (2020) 583–588.
V. Audrito, E. Moiso, F. Ugolini, V.G. Messana, L. Brandimarte, I. Manfredonia, S. Bianchi, F. De Logu, R. Nassini, A. Szumera-Ciećkiewicz, D. Taverna, D. Massi, S. Deaglio, J. Transl. Med. 20 (2022) 118.
S. Arslanoglu, E. Bertino, P. Tonetto, G. De Nisi, A.M. Ambruzzi, A. Biasini, C. Profeti, M.R. Spreghini, G.E. Moro, J. Matern. Fetal Neonatal Med. 23 (2010) 1–20.
D.L. O’Connor, J.B. Ewaschuk, S. Unger, Curr. Opin. Clin. Nutr. Metab. Care 18 (2015) 269–275.
ESPGHAN Committee on Nutrition, S. Arslanoglu, W. Corpeleijn, G. Moro, C. Braegger, C. Campoy, V. Colomb, T. Decsi, M. Domellöf, M. Fewtrell, I. Hojsak, W. Mihatsch, C. Mølgaard, R. Shamir, D. Turck, J. Van Goudoever, J. Pediatr. Gastroenterol. Nutr. 57 (2013) 535–542.
D. Silvestre, M. Miranda, M. Muriach, I. Almansa, E. Jareño, F.J. Romero, Acta Paediatr. 97 (2008) 1070–1074.
K. Hamprecht, J. Maschmann, D. Müller, K. Dietz, I. Besenthal, R. Goelz, J.M. Middeldorp, C.P. Speer, G. Jahn, Pediatr. Res. 56 (2004) 529–535.
A.A. Vieira, F.V.M. Soares, H.P. Pimenta, A.D. Abranches, M.E.L. Moreira, Early Hum. Dev. 87 (2011) 577–580.
Y. Ma, L. Zhang, Y. Wu, P. Zhou, Food Chem. 279 (2019) 209–215.
Y. Huang, T. Wei, F. Chen, C. Tan, Z. Gong, F. Wang, Z. Deng, J. Li, Food Res. Int. 167 (2023) 112580.
C. Cattaneo, A. Caramaschi, E. Uga, M. Braghin, G. Cosi, C. Peila, M.C. Strozzi, M. Sabatini, D. Gazzolo, M. Manfredi, M. Cavaletto, J. Immunol. Res. 2019 (2019) 1–12.
M. Cavaletto, A. Givonetti, C. Cattaneo, Nutrients 14 (2022) 4574.
M. Cavaletto, M.G. Giuffrida, D. Fortunato, L. Gardano, G. Dellavalle, L. Napolitano, C. Giunta, E. Bertino, C. Fabris, A. Conti, PROTEOMICS 2 (2002) 850.
M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
U.K. Laemmli, Nature 227 (1970) 680–685.
G. Candiano, M. Bruschi, L. Musante, L. Santucci, G.M. Ghiggeri, B. Carnemolla, P. Orecchia, L. Zardi, P.G. Righetti, ELECTROPHORESIS 25 (2004) 1327–1333.
E. Del Prete, A. Facchiano, A. Profumo, C. Angelini, P. Romano, Front. Genet. 12 (2021) 635814.
M. Gasparrini, F. Mazzola, M. Cuccioloni, L. Sorci, V. Audrito, F. Zamporlini, C. Fortunato, A. Amici, M. Cianci, S. Deaglio, M. Angeletti, N. Raffaelli, J. Biol. Chem. 298 (2022) 101669.

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Impact of Holder Pasteurization on Protein and eNAMPT/Visfatin Content in Human Breast Milk

Status:

Version 1

Abstract

Figures

Introduction

Results

Milk Samples

Protein quantification

Protein profiling

eNAMPT/visfatin detection

Discussion

Methods

Human milk samples

Milk phase separation and protein quantification

Protein profiling

Visfatin detection

Statistics

Declarations

Competing interests

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1