Risk assessment related to biogenic amines occurrence in ready-to-eat baby foods

Accepted Manuscript Risk assessment related to biogenic amines occurrence in ready-to-eat baby foods Anna Czajkowska-Mysłek, Joanna Leszczyńska PII:

S0278-6915(17)30164-3

DOI:

10.1016/j.fct.2017.03.061

Reference:

FCT 8980

To appear in:

Food and Chemical Toxicology

Received Date: 15 February 2017 Revised Date:

24 March 2017

Accepted Date: 29 March 2017

Please cite this article as: Czajkowska-Mysłek, A., Leszczyńska, J., Risk assessment related to biogenic amines occurrence in ready-to-eat baby foods, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.03.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Risk assessment related to biogenic amines occurrence in ready-to-eat baby foods Anna Czajkowska-Mysłeka, Joanna Leszczyńskab a

Department of Food Quality, Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology

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36 Rakowiecka, 02-532 Warsaw, Poland

Faculty of Biotechnology and Food Sciences, Lodz University of Technology 4/10 Stefanowskiego, 90-924 Lodz, Poland

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corresponding author: tel. +48 42 674 64 14, fax +48 42 674 81 24

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E-mail: [email protected]

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Keywords: biogenic amines; risk assessment; baby food; fish; vegetable; fruit

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ACCEPTED MANUSCRIPT 1.

Introduction

Biogenic amines (BAs) are biologically active compounds formed through natural biochemical processes taking place at a cellular level (Lee et al., 2015). Endogenous BAs may

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be presented in non-fermented food (Önal, 2007). However, if their levels in food reach a critical threshold, amines may be dangerous to human health (EFSA, 2011). The food adverse reactions caused by amines are mainly classified as intolerance reactions, known as

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pseudoallergies (Tahmouzi et al., 2011). High concentration of BAs is observed in food with high protein content, particularly fermented and ripened (Saaid et al., 2009; Silla Santos,

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1996; Suzzi and Gardini, 2003). This phenomenon is attributed to the higher microbial activity of decarboxylases in bacteria being the contaminating microflora or microorganisms intentionally added (Alvarez and Moreno-Arribas, 2014). Consumption of food containing large amounts of tyramine (TYR) cause dietary-migraines in sensitive individuals (EFSA,

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2011). Putrescine (PUT) and cadaverine (CAD) being the diamines, and spermine (SPM) and spermidine (SPD), classified as polyamines, are accountable for the regulation of cell growth and division (Halász and Baráth, 2002) or neutral transmission (serotonin, dopamine,

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norepinephrine) (Önal, 2007). Nonetheless, di- and polyamines are also identified as potential precursors for the formation of carcinogenic N-nitroso compounds (Wei et al., 2009).

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Histamine (HIS) has concerned particular attention, as a most common course of food poisonings. Consumption of food containing high doses of histamine potentiated by other amines (and possibly by other substances) is associated with a wide variety of physiological syndromes (Becker et al., 2001; Mo Dugo et al., 2006). These will occur in healthy individuals when a dose of 50 mg/meal/person histamine NOAEL (no-observed-adverseeffect-level) is consumed (EFSA, 2011). The European Union based on NOAEL established the acceptable level of histamine at 200 mg/kg in fish products with high histidine content

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ACCEPTED MANUSCRIPT (EC, 2007). In light of amine-related risk, only few risk assessments with estimation of exposure regarding biogenic amines occurrence in food (mainly fishery products or fermented food) were performed (Latorre-Moratalla et al., 2017). Baby foods are products intended to fulfill the particular requirements of healthy infants

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while they are being weaned and of healthy young children as a supplement to their diet and/or for their progressive adaptation to regular food (EC, 2013). In the case of infants feeding, the World Health Organization (WHO) recommends exclusive breastfeeding for the

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first 6 months of life (WHO, 2002). However, the introduction of solid foods during infancy (from 4 months of age) is essential for properly development and growth of a young child.

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But studies on the composition and consequences of introducing solid foods are very scarce in comparison to breastfeeding (Wu and Chen, 2009). Complementary foods such as ready-toeat food products targeted for infants (from 4 months of age) and young children (1-3 years old) are widely used due to its commercial availability and diversity. In Poland, about 60% of

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parents declare using commercially prepared foods (Weker et al., 2011). Ready-to-eat baby foods are available in quite many different variants of ingredients origin. Baby foods are mainly accessible as jars, pouches or boxes containing single-ingredient food often puree,

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mixed dinners, soups or desserts. They are labeled according to a baby’s stage development. Commercial baby foods are preserves ready to eat after or without heating. In general, baby

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food products stay fresh after opened in the refrigerator, and should be used within 48 h. In opinion of Committee of Nutrition (CoN) European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) there is a special attention and collaboration needed in review and evaluation of baby foods, focusing also on longer-term safety outcomes (Hernell, 2012). The safety concern is particularly acute for baby foods because of the vulnerability of babies to health effects from consumption of contaminated food. There is no doubt, that the potential adverse reactions among infants and young children could appear after consumption

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ACCEPTED MANUSCRIPT of food containing small amounts of toxic amines which can also enhance histamine release from basophils (Kovacova-Hanuskova et al., 2015) and provoke allergy-like responses (Jarisch, 2004). However, foods for infants and young children have never been the object of biogenic amine profiling. As a result, no data on the profiles and concentrations of individual

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BAs in baby foods are available. The primary cause of this is due to the lack of proper analytical methods with appropriate sensitivity and the secondary, that the expected low level of these compounds in food is not being hazardous for healthy adults. But, the appropriate

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feeding in early childhood can have a huge impact on the child’s physical and psychological development, with effects that continue into adult life (Wu and Chen, 2009). Therefore, baby

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food products should be screened for the presence of BAs, particularly direct toxic amines (HIS, TYR) or with cancerogenic properties (SPD, SPM), and for the presence of amines (PUT, CAD) which enhance histamine toxicity by inhibiting histamine metabolizing enzymes. BAs profiling in baby foods is a challenge, primarily due to the complexity of

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matrix, the presence of free amino acids and compounds that could interfere with the analytes, the low concentration of BAs and in some cases significant differences in the concentrations of individual amines in the amine profile. But the availability of highly-sensitive analytical

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tools for the determination of amines arising from the connection of separation technique – high-performance liquid chromatography with mass spectrometry detection could overcome

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all these issues.

The main objective of this work was to quantify 6 biogenic amines (putrescine, histamine, cadaverine, tyramine, spermidine, and spermine) selected from obtained amine profile in 68 samples of commercial ready-to-eat food products intended for infants and young children, produced by 10 available in Poland manufacturers. Moreover, for the first time, we also present the amine-related risk assessment for baby foods in relation to ARfD (Acute Reference Dose) and BAI (Biogenic Amine Index).

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Materials and methods

2.1

Chemicals

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2.

LC-MS grade Acetonitrile (VWR, Radnor, PA, USA) was used, and water purified using a MilliQ Direct 8 system (Merck, Darmstadt, Germany). Perchloric acid 60%, L-proline

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for biochemistry, diethyl ether assay ≥99.7%, sodium carbonate anhydrous assay 99.9% and acetone for LC were acquired from Merck (Darmstadt, Germany). Ammonium formate

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≥99%, dansyl chloride assay ≥99%, and 1,7-diaminoheptane assay 98% were purchased from Sigma-Aldrich (St. Louis, MO, USA). All certified materials, including histamine dihydrochloride

assay

99.0%,

tyramine

hydrochloride

assay

99.5%,

cadaverine

dihydrochloride assay 99.6%, putrescine dihydrochloride assay 99.0%, spermidine

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trihydrochloride assay 99.0% and spermine tetrahydrochloride assay 99.0% were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Sodium hydroxide micropills assay

Sampling

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2.2

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≥98.8% was obtained from POCH (Gliwice, Poland).

Sixty-eight samples of commercial ready-to-eat food preserves (125-250 g in jars, 50250 g in pouches or plastic boxes) intended for infants and young children were purchased from local shops and produced by ten (coded from A to J) leading manufacturers available in Poland. The information about recommended age and product composition was taken from general product descriptions. Of the 68 products, 35% were aimed at infants from 4 months of age. The samples were classified according to the ingredients used into 4 food groups:

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ACCEPTED MANUSCRIPT vegetable containing 8-12% of fish (23 products), vegetable with 8-10% of meat (15 products), vegetable (15 products), and fruit (15 products) products. The preserves were stored at room temperature prior to analysis.

HPLC-APCI-MS method

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2.3

Such challenging assignment as the determination of BAs in baby foods requires an

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analytical methodology with lower limits of detection (LODs), and greater chromatographic separation to be applicable for routine analysis in comparison to current analytic methods for

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quantification of BAs in food. Therefore, we develop (data not shown) a new method to evaluate the bioactive amines level in food products intended for infants and young children. The method uses derivatization with dansyl chloride followed by high-performance liquid chromatography coupled to a single quadrupole mass spectrometry by atmospheric pressure

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chemical ionization interface (HPLC-APCI-MS).

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2.3.1 Preparation of standard solutions and samples

Briefly, standard solutions of selected amines and ISTD (1,7-diaminoheptane, 50

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ng/mL) were diluted with 0.4 M HClO4. Stock solutions were stored at 4°C for 3 months and calibration solutions prepared daily before analysis. The calibration curves range was based on the concentration levels of individual BAs determined in the analyzed samples. Two baby food preserves with diverse date codes were combined (jars/pouches/plastic boxes), and homogenized (if not homogeneous) using a laboratory mixer. Then, five gram samples were weighed in a 50 mL Falcon centrifuge PP tube, homogenized at 16000 rpm (3 min) with

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ACCEPTED MANUSCRIPT 45 mL of 0.4 M HClO4 and centrifuged at 10000×g in 4°C for 15 min. The sample extracts could be stored at -18°C for approximately 6 months prior to further analysis.

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2.3.2 Derivatization and liquid-liquid extraction methodology

A 1 mL of supernatant or standard solution was transferred into a 10 mL PP tube and mixed with 20 µL of ISTD (1,4-diaminohaptane), 150 µL 2 M NaOH and 300 µL saturated

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aqueous solution of Na2CO3 (pH=11.0). The mixture was vortexed at 1600 rpm for 30 s and 1

again at 1600 rpm for 30 s.

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mL of dansyl chloride in acetone solution (10 mg/mL) added. The mixture was vortexed

The mixture was shaken and heated in a water bath in the dark for one hour at 50°C. After the reaction, the mixture was cooled and the dansylation reaction was interrupted using 200 µL of L-proline solution (100 mg/mL). The mixture was vortexed at 1600 rpm for a further 30 s and

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left for an additional 15 min in the dark. Next, the solution was extracted using diethyl ether (3×1 mL) assisted by centrifugation at 3000×g for 2 min. The combined organic ether phases were evaporated to dryness under N2 at 40°C and re-dissolved in 1 mL of acetonitrile, filtered

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through a 0.2 µm×17 mm Nylon filter, and 10 µL injected into an HPLC system, in triplicate.

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2.3.3 Apparatus

High-performance liquid chromatograph Prominence UFLC (Shimadzu, Kyoto, Japan) equipped with a binary system of LC-20AD pumps, a DGU-20A3 degasser unit, an SIL20ACHT autosampler, and a CTO-10ASVP thermostated column oven coupled to an LCMS2020 detector with an APCI (atmospheric pressure chemical ionization) interface, conducted in selected ion monitoring (SIM) in positive ion mode, all supervised via CMB-20A controller

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ACCEPTED MANUSCRIPT were used in this study. Data analysis was performed using LabSolution software (ver. 5.72 Shimadzu, Kyoto, Japan). The BAs were separated on a Gemini-NX C18 column (150×4.6 mm, 3 µm particle size, Phenomenex, Torrence, CA, USA), with a pre-column (4×3 mm) containing the same stationary phase, operated at 25°C with a flow rate of 0.8 mL/min. The

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mobile phase consisted of 10 mM ammonium formate (A) and acetonitrile (B) with the following gradient elution program: 0.01-16.00 min 60-90% B; 16.01-24.00 90% B; 24.0130.00 60% B (re-equilibration). MS acquisition was performed under the following

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conditions: APCI temperature 450°C, nebulizing gas (N2) flow rate 4 L/min, drying gas (N2) flow rate 10 L/min, heat block temperature 300°C, desolvation line temperature 200°C. To

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quantify the BAs, the SIM(+) mode was used in 4 segments with detailed parameters presented in Table 1. Mass spectrometer in scan mode was operated at the range of m/z 1001300, followed by SIM analysis of methylamine (METH, [M+H]+=265), ethylamine (ETH, [M+H]+=279), phenylethylamine (FEA, [M+H]+=355),

tryptamine (TRP, [M+H]+=394),

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putrescine (PUT, [M+H]+=555), cadaverine (CAD, [M+H]+=569), histamine (HIS, [M+H]+=578), agmatine (AGM, [M+H]+=597), tyramine (TYR, [M+H]+=604), serotonin (SER, [M+H]+=643), spermidine (SPD, [M+H]+=845), dopamine (DOP, [M+H]+=853),

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norepinephrine (NE, [M+H]+=869), and spermine (SPM, [M+H]+=1135).

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2.3.4 Method validation parameters

The method was validated on the most complex baby food sample – vegetable with fish. All the validation parameters were evaluated including linearity, matrix effect, precision, and accuracy. Briefly, linearity was obtained with R2 ranging from 0.9990-0.9999. The method recovery (sample spiked with high, intermediate and low levels of standard BAs solution) was in the range of 86.0% for spermine and 105.2% for spermidine with RSD≤17.2%. The relative

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ACCEPTED MANUSCRIPT standard deviation ranges were 1.7-7.5% for intra-day analysis (one day, n=3) and 1.4-9.7% for inter-day analysis (three consecutive days, n=9). The sensitivity of the method reflected by the limits of detection (LODs) values was in the range of 0.07-1.67 ng/mL. The limits of quantification (LOQs) were in the range from 2.0 ng/g for histamine, cadaverine, and

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tyramine, 25 ng/g for putrescine to 50 ng/g for spermidine and spermine, respectively. The evaluation of matrix effect indicated the presence of very low suppression, observed as a reduction in signal response, ranged from an estimated -2 for cadaverine to -7 for spermine.

Statistical analysis

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2.4

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Mild signal suppression, over 10%, was estimated for tyramine -14.

Statistical studies were performed using Statistica 10.0 (StatSoft, Cracow, Poland). One-way ANOVA analysis was performed with a post-hoc Tukey’s test. Differences between

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means with a 95% (P<0.05) confidence level were considered statistically significant. Principal components analysis (PCA) was also used to simplify the data and their interpretation.

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The results are given as a mean of 3 parallel determinations with standard deviation (SD). The

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mean values of amine concentrations were calculated without the results less than the LOQs.

3.

Results and discussion

3.1

Biogenic amine profile

The biogenic amine profile of commercial ready-to-eat baby foods embraces BAs commonly found in food (HPLC-APCI-MS in scan mode): methylamine, ethylamine,

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ACCEPTED MANUSCRIPT putrescine, cadaverine, histamine, agmatine, tyramine, serotonin, spermidine, and spermine. Apart from the above mentioned BAs, other psychoactive amines commonly found in food including dopamine and norepinephrine have been detected, but only in fruit-based baby foods. The presence of psychoactive amines such as serotonin, dopamine or norepinephrine in

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vegetable and/or fruit samples was not surprising. Their occurrence in vegetables and fruit was firstly confirmed by Udenfriend, Lovenberg, and Sjoderma in 1959 (Moret et al., 2005).

Occurrence level of biogenic amines

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3.2

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To evaluate the toxicity potential of ready-to-eat food products for infants and young children analyzed in this study, only 6 amines were selected from 10 BAs identified in their profile, due to their direct toxicity (HIS, TYR), their potential to enhance histamine toxicity (PUT, CAD) or their carcinogenic properties (SPD, SPM).

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The results of 6 BAs concentrations found in 68 samples of baby foods are shown in Table 2. The HPLC-APCI-MS method was fully adequate for quantification of all individual amines

LOQs (Table 2).

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present in the samples within a suitable concentration range, with only 10 results beyond the

Overall, the amine levels in baby food products vary a lot, from 2 ng/g (CAD, HIS or TYR) to

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53416 ng/g (PUT) (Table 3). The individual BAs were quantified as being in the range of 704-53416 ng/g for putrescine, 2-1263 ng/g for cadaverine, 2-2375 ng/g for histamine, 2-1668 ng/g for tyramine, 408-46680 ng/g for spermidine, and 34-5619 ng/g for spermine. The obtained amine quantities as mean concentrations in decreasing order were as follows (in ng/g): putrescine (5654 ± 8690)>spermidine (4907 ± 7689)>spermine (1278 ± 1111)>tyramine (300 ± 322)>histamine (186 ± 345)>cadaverine (90 ± 209) (Table 3). The amine profile of ready-to-eat baby foods consists mainly of putrescine and spermidine. These

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ACCEPTED MANUSCRIPT amines were the most abundant ones, representing 86% of the profile. Indeed, di- and polyamines are the predominant ones in plant cells (Santiago-Silva et al., 2011). Therefore, they could be found in all analyzed samples. Other amines, beginning from spermine, through tyramine, histamine to cadaverine covered only 14% of the amine profile.

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The maximum level of individual amines was obtained for histamine (2375 ± 6 ng/g) in vegetable sample with spinach, for tyramine (1668 ± 27 ng/g) in fruit product contain banana, for putrescine (53416 ± 1425 ng/g), cadaverine (1263 ± 22 ng/g) and spermine (5619 ± 364

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ng/g) in vegetable product with fish and green peas, and for spermidine (46680 ± 1435 ng/g) in vegetable sample with green peas. Distributions of particular amine concentrations found in

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analyzed 68 samples are presented in Fig. 1 as the frequency histograms.

The most aminogenic baby foods are those containing 43-70% of green peas (56239101423 ng/g) and 48-50% of banana (26958-36845 ng/g) (Table 2). The summary amine levels in analyzed baby food products were found in the range of 1283-101421 ng/g (Table 2).

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Fig. 2 shows the profile of BAs estimated for 10 manufacturers (A-J), among them an A, B, C are market leaders. In general, the amine contents were independent of the product manufacturers which may e.g. use lower quality raw materials or apply different production

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processes, rather by the use of the specific product ingredients. However, both the maximum summary BAs contents and the most variable levels of BAs were found in products purchased

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from B manufacturer and resulted from a high content of green peas (Fig. 3). Among all analyzed products, only 21% of the samples contained less than 5000 ng/g

of 6 BAs (Fig. 4). Nearly half of the 68 products contained amines at the concentration range of 5000-10000 ng/g. Next 21% of the samples ranged from 10000 to 20000 ng/g of amines, but in some particular samples, the amine level exceeded over 80000 ng/g up to 110000 ng/g. This indicates the need of unification of amine level designated for baby foods.

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ACCEPTED MANUSCRIPT Concerning the diamine (CAD+PUT) and polyamine (SPD+SPM) levels in all analyzed samples, a strong positive correlation (R=0.8188, P<0.0001) was found (Fig. 5). Consequently, only 4 out of 6 BAs have been taken under consideration during further aminerelated risk assessment.

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The statistical analysis results indicated a number of important, significant correlations between biogenic amine contents vs. sample components and between the individual amine concentrations found in the samples. Regarding individual amines of all baby foods, positive

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correlation (P<0.05) was found between putrescine and spermidine (R=0.8295), spermidine vs. spermine (R=0.6535), putrescine vs. cadaverine (R=0.5122), cadaverine vs. spermine

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(R=0.5022), putrescine vs. spermine (R=0.4828), cadaverine vs. spermidine (R=0.4535), and tyramine vs. putrescine (R=0.4317) levels.

Principal component analysis (PCA) was used to assess the similarities and differences between obtained BAs profiles. The selected 2 factors, as revealed by the PCA analysis,

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captured over 67% of the total variance (Fig. 6). The PCA analysis exposed the negative correlation between histamine and polyamine contents (SPD, SPM). The most variable level of amine concentrations was assumed for tyramine. The most correlated results were

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estimated for di- and polyamines, PUT and CAD vs. SPD and SPM, respectively. The significantly different amine profiles were found for samples with green peas (SPD, SPM, No.

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2, 22, 52), with spinach (HIS, No. 41, 46), with rice (PUT, No. 38), and with banana (TYR, No. 57, 68) (Fig. 6). Whereas most similar amine profiles were observed for samples with tuna (No. 39 vs. 46, No. 16 vs. 20), containing banana (No. 55 vs. 63, No. 57 vs. 68), and for vegetable samples (No. 33 vs. 53) (Fig. 6). Based on a one-way ANOVA and Tukey’s post-hoc tests of 4 food types organized by main ingredients we indicated the presence of individual components responsible for the variability of BAs levels within the food type. In general, the quantity and quality

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ACCEPTED MANUSCRIPT composition of particular food components in the samples was so different, even among selected food types that make their direct comparison to literature data’s almost impossible. The 23 vegetable samples with fish contained 8-12% of fish species such as salmon, cod, pangasius, tuna and other sea fishes. The results suggest that the BAs profiles were

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affected by the fish species present in the samples (Table 2). The results obtained for histamine (mean 519 ± 115 ng/g, 0.0002≤P≤0.0055) and tyramine (mean 567 ± 165 ng/g, 0.0009≤P≤0.0011) in 4 samples with tuna, and for tyramine (mean 546 ± 123 ng/g,

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0.0017≤P≤0.0052) in 3 products containing cod were significantly different (P<0.05) from levels found in the samples containing other fish species (Table 2). The fish species such as

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tuna contains high levels of free histidine (histamine amino acid precursor) (Lehane and Olley, 2000). Therefore, higher contents of histamine are to be expected. In case of tyramine, fresh fish contains little or no tyramine, but a large amount can be found in spoiled or fermented fish (Prester, 2011). The endogenous histamine and tyramine levels in tuna meals

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could be potentially toxic to sensitive babies. Indeed, it may be necessary to remove tuna as an ingredient of ready-to-eat baby foods, due to elevated level of the most toxic amines found in these meals. The higher mean levels of other amines including cadaverine (477 ± 682

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ng/g), putrescine (22574 ± 27145 ng/g) and spermidine (16590 ± 20981 ng/g) were observed in samples containing cod, and spermine (2817 ± 2722 ng/g) in samples with tuna.

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Among 15 vegetable samples with meat classified by 7 kinds of meat (beef, chicken, lamb, veal, rabbit, turkey and pork) results analysis revealed no significant difference (P<0.05) between the type of meat and obtained BAs profile (Table 2). However, elevated levels of histamine (mean 380 ± 102 ng/g) were obtained for 5 samples containing beef, and for 2 out of 4 samples with chicken (mean 349 ± 85 ng/g). The highest tyramine content (605 ng/g) was estimated for sample with lamb, putrescine (mean 6559 ± 7747 ng/g) and spermidine (mean 4748 ± 4044 ng/g) in 2 products with veal, and spermine (mean 2520 ± 469

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predominant ingredients including spinach, carrot, pumpkin and other vegetables (Table 2). The obtained mean levels of histamine (1327 ± 909 ng/g, 0.0032≤P≤0.0045) and tyramine (501 ± 105 ng/g, 0.0009≤P≤0.0027) in 3 samples containing over 25.5% of spinach were

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significantly different (P<0.05) in comparison to the results found among other vegetable ingredients. The analyzed spinach baby foods have been produced by three different

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manufacturers. But elevated levels of tyramine (4-32 mg/kg in frozen spinach puree) and histamine (2-20 mg/kg in fresh spinach) were also obtained in commercial foods for adults (Kalač et al., 2002; Moret et al., 2005). Certainly, the high level of histamine reaching 2375 ng/g (sample No. 41) could not be allowed in baby foods, especially among hyper-sensitive

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infants from 4 months of age. Therefore, we recommend to remove or reduce (considering high nutritional value) the amount of spinach added to baby foods to e.g. 10% of the product. That amount of spinach has not resulted in increasing of histamine and tyramine contents and

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could be found in sample No. 11 (Table 2). The biogenic amine profile of vegetable baby foods consists mainly of di- and polyamines. The highest level of putrescine with spermidine

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(79083 ng/g) was found in a sample containing 70% of green peas (intended for 4 month infants). According to the literature, higher concentrations of spermidine in green vegetables are to be expected (Valero et al., 2002). Considering the higher concentration levels of di- and polyamines obtained in products with green peas as the main ingredient we recommend reducing the amount of peas to approximately 10-20% of the product, particularly in foods targeted for infants.

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ACCEPTED MANUSCRIPT In 15 samples of fruit products intended for babies only 2 main ingredients were specified: an apple and a banana (Table 2). Among baby desserts, the statistically significant differences (P<0.05) were found for histamine (mean 15 ± 5 ng/g, P=0.0003), tyramine (mean 1004 ± 419 ng/g, P=0.0030), putrescine (mean 14764 ± 8840 ng/g, P=0.0120), spermidine

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(mean 3572 ± 1448 ng/g, P=0.0021) and spermine (mean 309 ± 153 ng/g, P=0.0275) between 6 banana-based samples and the rest of desserts based on an apple. The assayed amine profile of fruit products targeted for the youngest consumers consists mainly of putrescine, the amine

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found in 2 samples at the highest concentration exceeding 20000 ng/g. The amine profile obtained for fruit-based baby products differs significantly among other baby food profiles,

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mainly according to the lowest polyamine levels (Fig. 7). Fruit are particularly rich in putrescine (Shalaby, 1996). Essentially, among other amines found in commercial banana fruits, putrescine is the predominant one (Baston et al., 2009; Tanasa et al., 2015). The higher putrescine level found in baby preserves with banana, as regards the aforementioned green

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peas is not directly toxic, but enhances histamine toxicity, strengthened by a higher dose of toxic tyramine. Furthermore, analyzed baby desserts are often eaten just after the main meal e.g. baby food containing meat or fish with an elevated level of dietary histamine. Taking into

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consideration the obtained BAs profiles of fruit-based baby foods we recommend the consumption of apple-based baby foods with a progressive introduction of products

3.3

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containing banana, especially among infants.

Amine-related risk assessment

The number of studies concerning a potential no-observed-adverse-effect-level (NOAEL) of histamine for adults are limited (EFSA, 2011), and in the case of healthy young children, there is an almost total lack of knowledge. Taking into account a potential acute

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ACCEPTED MANUSCRIPT reference dose (ARfD) of 5 mg/meal/70 kg person histamine (10-fold lower than the ARfD suggested by EFSA) for sensitive adult individuals (Karovičová and Kohajdová, 2005) calculated according to the WHO child weight-for-age standards for boys and girls (WHO, 2006) we assessed the percentage of ARfD ingested through consumption of 1 baby meal per

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day. The highest %ARfD of histamine was estimated for baby food products containing tuna (from 11% to 26%ARfD) and spinach (from 21% to 65%ARfD) (Fig. 8). This could explain why many so-called pseudoallergies appear after consumption of products containing tuna or

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spinach intended for young children, especially among infants. The calculated potential doses are associated with the consumption of one meal only. The WHO recommendation for infants

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between 6-8 months of age is to introduce complementary foods 2-3 times a day, increasing to 3-4 times daily between 9-24 months of age (WHO, 2016). Therefore, the estimated %ARfD of histamine will certainly increase by consumption of the following portion of baby food. An additional concern is associated with people suffering from histamine intolerance by diamine

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oxidase (DAO) deficiency, with health problems, or gastrointestinal disorders results in an escalation of the adverse effects (Jarisch, 2004; Latorre-Moratalla et al., 2017). In the case of children, especially infants at the beginning of the development of an efficient digestion this

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concern should not be neglected.

Although there is currently insufficient information related to establishing the NOAEL

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for tyramine in humans, this amine may be a causative agent of certain food-induced migraines, especially among individuals with lower genetic or physiological activity of monoamine oxidase (MAO) enzymes, and also act as a histamine potentiator (EFSA, 2011). Therefore, the appearance of tyramine should also be considered as an indicator of the potential toxicity of consumable baby food products. Especially taking into account recently results of the in vitro analysis, suggested that tyramine has a stronger and more rapid cytotoxic effect than histamine (Linares et al., 2016).

15

ACCEPTED MANUSCRIPT The assessment of potential toxicity of ready-to-eat baby foods based on the two most toxic amines (HIS, TYR) is not sufficient (Fig. 9). The obtained maximum total concentration of 55351 ng/g for 4 amines (PUT, CAD, HIS, TYR) would have exposed a 9-months infant to 7.2 mg of BAs in 130 g of consumable portion, or 157% of 40 mg/meal/person (Shalaby,

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1996), the value of BAs considered potentially toxic for healthy adults. This may support the application of common used Biogenic amine Index (BAI) for amine-related risk assessment of newly tested baby foods, as a safety parameter, according to the equation (Eq. 1). ng / g HIS + ng / g PUT + ng / g CAD + ng / g TYR 1000 ng / g

SC

BAI baby food =

(1)

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As seen in Fig. 10, there are 5 samples (containing green peas or banana) with the BAI in the range of 20-55. The composition of these products should be immediately re-evaluated to reduce the dietary amine intake. The most interesting is that among all samples, the bananabased baby foods seem to be the most potentially toxic ones (elevated tyramine content

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accompanied by the high amount of putrescine). In general, healthy adult tolerance levels are irrelevant to babies. The maximum level of 4 the most toxic BAs in baby foods should be established at 20 mg/kg (BAI=20) in view of higher vulnerability of babies. Our

EP

recommendations of BAI indexes are as follows: BAI<10 for baby food products safe for consumption by the youngest consumers, 10
AC C

foods, and BAI>20 for products being potentially harmful for infants and young children’s health due to adverse effects of exposure to BAs. The obtained BAI results undoubtedly indicate the need to pay special attention to properly components selection in foodstuffs intended for babies in order to protect them from the adverse effects of toxic amines intake.

4.

Conclusions

16

ACCEPTED MANUSCRIPT To our knowledge, this study, for the first time, gives a profile of biogenic amines and presents the occurrence of 6 the most toxic amines in commercially available ready-to-eat baby foods. We have pointed out that some ingredients present in baby foods are associated with the occurrence of specific compounds – bioactive amines with the most potential to

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provoke allergy-like responses and other adverse reactions at the level being hazardous for very young consumers. The biogenic amines presence in baby food is not the consequence of food-associated spoiled microorganisms’ activity, but the activity of endogenous

SC

decarboxylases and the presence of appropriate amino acids, as the substrates for amines production. This indicates that the biogenic amines level in baby foods is an equilibrium

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between the levels of amines present in the individual product components. In light of that most biogenic amines are thermostable compounds (besides polyamines), they will be present in baby foods even after pasteurization. Therefore, the only solution is to re-evaluate their composition taking into consideration the low amine level to be obtained.

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The exposure to BAs through the consumption of baby foods appears to be higher than anticipated. Indeed, further research is required on amine-related risk assessment of newly tested baby food. A dietary exposure assessment is also needed to investigate the tolerable

EP

threshold of BAs in correlation with consumption data of food products intended for infants and young children.

AC C

Our amine-related nutritional recommendations concerning ready-to-eat baby foods are as follows: 1.

Tuna should be removed from the list of ingredients allowed in baby foods with regard to the elevated content of histamine and tyramine.

2.

Spinach should be removed from baby foods or reduced to the amount of 10% with regard to the very high level of toxic histamine.

17

ACCEPTED MANUSCRIPT 3.

Green peas content should be reduced to 10-20% of the product with regard to the high concentration of di- and polyamines.

4.

Beef usage in baby food should be reduced to babies beyond 12 moths with regard to the elevated level of histamine. Banana introduction to a baby’s diet needs a special attention, especially among infants

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5.

with regard to the high concentration of toxic tyramine and putrescine.

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Acknowledgements

The first author would like to thank SHIM-POL (A. M. Borzymowski, Poland) for providing

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the APCI-MS detector used in present work, and especially Dr. Paweł Stalica for his valuable support. Special thanks also to Dr. Arkadiusz Szterk for kindly providing the dansyl chloride used in this study.

This research was financially supported by the Polish Ministry of Science and Higher

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EP

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Education for the maintenance of research potential in 2014/2015.

18

ACCEPTED MANUSCRIPT References

Alvarez, M.A., Moreno-Arribas, M.V., 2014. The problem of biogenic amines in fermented

Trend Food Sci. Tech., 39, 146–155.

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foods and the use of potential biogenic amine-degrading microorganisms as a solution.

Baston, O., Moise, D., Barna, O., Pricop, E., 2009. Bioactive amines content in “Dwarf Cavendish Banana” stored at different temperatures. Lucr. Ştiinţ. seria Agron., 53, 603–

SC

606.

Becker, K., Southwick, K., Reardon, J., Berg, R., MacCormack, J.N., 2001. Histamine

M AN U

poisoning associated with eating tuna burgers. JAMA, 285, 1327–1330. EC, 2007. European Commission. Commission regulation 1441/2007. Amending Regulation (EC) No 2073/2005 of November 2005 on microbiological criteria for foodstuffs (05.12.07).

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EC, 2013. European Commission. European regulation (EU) 609/2013. On food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive 92/52/EEC,

EP

Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission

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Regulations (EC) No 41/2009 and (EC) No 953/2009 (12.07.13) EFSA Panel on biological Hazards (BIOHAZ). 2011. Scientific Opinion on risk based control of biogenic amine formation in fermented foods. EFSA J. 9 (10), 2393. Halász, A., Baráth, Á., 2002. Toxicity of biogenic amines – the present knowledge. In Food Science and technology COST 917 Biogenically active amines in food; EC Publication: Luxembourg, Vol. no. 6, pp. 131–141.

19

ACCEPTED MANUSCRIPT Hernell, O., 2012. Current safety standards in infant nutrition – a European perspective. Ann. Nut. Metab., 60, 188–191. Jarisch, R., 2004. Histamin-Intoleranz, Histamin und Seekrankheit (Histamine intolerance. Histamine and motion stickness). Stuttgart: Georg Thieme Verlag KG.

products. Food Chem., 77, 349–351.

RI PT

Kalač, P., Svecova, S., Pelikanova, T., 2002. Level of biogenic amines in typical vegetable

Karovičová, J.; Kohajdová, Z., 2005. Biogenic amines in food. Chem. Pap., 59, 70–79.

SC

Kovacova-Hanuskova, E., Buday, T., Gavliakova, S., Plevkova, J., 2015. Histamine, histamine intoxication and intolerance. Allergologia Immunopathol. 43, 498–506.

M AN U

Latorre-Moratalla, M.L., Comas-Baste, O., Bover-Cid, S., Vidal-Carou, M.C., 2017. Tyramine and histamine risk assessment related to consumption of dry fermented sausages by the Spanish population. Food Chem. Toxicol., 99, 78–85. Lee, S., Yoo, M., Shin, D., 2015. The identification and quantification of biogenic amines in

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Korean turbid rice wine, Makgeolli by HPLC with mass spectrometry detection. LWT – Food Sci. Technol., 62, 350–356.

1–37.

EP

Lehane, L., Olley, J., 2000, Histamine fish poisoning revisited. Intern. J. Food Microbiol., 58,

Linares, D.M., del Rio, B., Redruello, B., Ladero. V., Martin, M.C., Fernandez, M., Ruas-

AC C

Madiedo, P., Alvarez, M.A., 2016. Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine. Food Chem., 197, 658–663. Mo Dugo, G., Vilasi, F., La Torre, G.L., Pellicano, T.M., 2006. Reverse phase HPLC/DAD determination of biogenic amines as dansyl derivatives in experimental red wines. Food Chem., 95, 672–676. Moret, S., Smela, D., Populin, T., Conte, L.S., 2005. A survey on free biogenic amine content of fresh and preserved vegetables. Food Chem., 89, 355–361.

20

ACCEPTED MANUSCRIPT Önal, A., 2007. A review: Current analytical methods for the determination of biogenic amines in foods. Food Chem., 103, 1475–1486. Prester, L.. 2011. Biogenic amines in fish, fish products and shellfish: a review. Food Addit. Contam: Part A., 28, 1547–1560.

RI PT

Saaid, M., Saad, B., Ali, A.S., Saleh, M.I., Basheer, C., Lee, H.K., 2009. In situ derivatization hollow fibre liquid-phase microextraction for the determination of biogenic amines in food samples. J. Chromatogr. A., 1216, 5165–5170.

SC

Santiago-Silva, P., Labanca, R.A., Gloria, B.A., 2011. Functional potential of tropical fruits with respect to free bioactive amines. Food Res. Int. 44, 1264-1268.

Res. Int., 29, 676–690.

M AN U

Shalaby, A.R.,1996. Significance of biogenic amines to food safety and human health. Food

Silla Santos, M.H., 1996. Biogenic amines: their importance in foods. Int. J. Food Microbiol., 29, 213–231.

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Suzzi, G., Gardini, F., 2003. Biogenic amines in dry fermented sausages: a review. Int. J. Food Microbiol., 88, 41–54.

Tahmouzi, S., Khaksar, R., Ghasemlou, M., 2011. Development and validation of an HPLC-

EP

FLD method for rapid determination of histamine in skipjack tuna fish (Katsuwonus pelamis). Food Chem., 126, 756–761.

AC C

Tanasa, V., Moise, D., Stanca, M., 2015. Separation and quantification of biogenic amines in bananas by high performance liquid chromatography. Food Env. Safety, 14, 245–249. Valero, D., Martinez-Romero, D., Serrano, M., 2002. The role of polyamines in the improvement of the shelf life of fruit. Trends Food Sci. Tech., 13, 228–234. Wei, F., Xu, X., Zhou, G., Zhao, G., Li, C., Zhang, Y., Chen, L., Qi, J., 2009. Irradiated Chinese Rugao ham: Changes in volatile N-nitrosamine, biogenic amine and residual nitrite during ripening and post-ripening. Meat Sci., 81, 451–455.

21

ACCEPTED MANUSCRIPT Weker, H., Barańska, M., Dyląg, H., Riahi, A., Więch, M., Strucińska, M., Kurpińska, P., Rowicka, G., Klemarczyk, W., 2011. Analysis of nutrition of children aged 13-36 months in Poland – a nationwide study. Med. Wieku Rozw., 15, 224–231. WHO, 2002. Butte, N.F, Lopez-Alarcon, M.G, Garza, C. Nutrient adequacy of exclusive for

the

term

infant

during

the

first

six

months

http://www.who.int/child_adolescent_health/documents/9241562110/en/ 29.01.17).

of

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breastfeeding

life.

(accessed

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WHO, 2006. Child Growth Standards. Length/height-for-age, weight-for-age, weight-forlength, weight-for-height and body mass index-for-age. Methods and development.

29.01.17) WHO,

2016.

Nutrition

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http://www.who.int/childgrowth/standards/Technical_report.pdf?ua=1/

topics.

Complementary

(accessed

feeding.

http://www.who.int/nutrition/topics/complementary_feeding/en/ (accessed 29.01.17)

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Wu, T.C., Chen, P.H., 2009. Health consequences of nutrition in childhood and early infancy.

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Pediatr. Neonatal., 50, 135–142.

22

ACCEPTED MANUSCRIPT FIGURE CAPTIONS Fig. 1. Distribution of putrescine, tyramine, histamine, cadaverine, spermidine, and spermine contents (ng/g) in 68 samples of ready-to-eat baby foods available in Poland. Fig. 2. Biogenic amine profile regarding product manufacturer (n=68).

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Fig. 3. Mean concentrations with standard deviations of biogenic amines in baby foods within manufacturers (n=68).

Fig. 4. Distribution of total amine concentrations found in tested 68 samples.

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Fig. 5. Correlation plot of diamines vs. polyamines revealed in baby foods (n=68).

Fig. 6. PCA scatterplot of biogenic amines with distribution of analyzed parameters (n=68).

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Fig. 7. Profile of total amine vs. polyamine contents in baby foods (n=68).

Fig. 8. The percentage of ARfD=5 mg/meal/sensitive adult histamine estimated for 68 baby food products.

Fig. 9. Occurrence of tyramine and histamine in baby foods (n=68).

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Fig. 10. BAI evaluated for 68 samples of ready-to-eat baby food products.

23

ACCEPTED MANUSCRIPT Table 1 SIM(+) conditions for the APCI-MS method. Voltage (kV)

Segment Time (min)

APCI

[M+H]+

DL Volt, Qarray

Detector DC, Qarray RF (V)

(m/z)

0.0-11.45

4.5

1.3

40, 30, 78

555.3

2

11.45-17.2

4.5

1.5

40, 30, 78

569.3, 578.3, 597.3, 604.2

3

17.2-19.0

4.5

1.3

40, 30, 104

4

19.0-24.0

5.0

1.5

40, 30, 130

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1

845.3

AC C

EP

TE D

M AN U

SC

1135.5

ACCEPTED MANUSCRIPT

Table 2 Biogenic amines in 68 samples of ready-to-eat baby foods (n=3).

2907.5 ± 113.4a 53416.1 ± 1425.2a 11430.5 ± 634.1a 3120.4 ± 186.3a 1814.2 ± 76.4a 2469.9 ± 58.4a 2938.9 ± 76.3a 1849.6 ± 4.7a 11993.6 ± 707.3a 2150.9 ± 22.4a 1102.7 ± 18.0a 1926.3 ± 10.7a 2543.4 ± 101.3a 2313.0 ± 14.0a 2714.7 ± 47.0a 4382.0 ± 50.6a 6174.2 ± 74.0a 2869.4 ± 90.5a 4063.7 ± 86.9a 4383.9 ± 98.0a 1441.0 ± 98.1a 26252.1 ± 1218.5a 1582.4 ± 19.1a

24 25 26 27 28 29 30 31 32 33 34 35

1-3yr 1-3yr 1-3yr 5mth 6mth 5mth 5mth 7mth 4mth 5mth 6mth 6mth

ABe ABe BCh CLa APo CVe ARa DCh DBe AVe ATr BBe

2806.1 ± 52.3a 2520.1 ± 26.9a 1322.8 ± 21.8a 2773.7 ± 40.6a 2332.9 ± 58.0a 1081.4 ± 16.5a 1729.1 ± 16.0a 950.9 ± 23.2a 3031.8 ± 37.7a 12037.4 ± 272.6a 1772.8 ± 83.1a 1713.0 ± 44.3a

3.9 2.7 5.5 6.0 4.2 2.4 2.6 0.3 5.9 1.0 1.6 0.6 4.0 0.6 1.7 1.2 1.2 3.2 2.1 2.2 6.8 4.6 1.2

57.3 ± 1.6a 1263.2 ± 21.9a 121.9 ± 3.7a 33.7 ± 2.0a 25.3 ± 0.7a 35.1 ± 0.5a 24.4 ± 0.4a 20.1 ± 0.2a 122.8 ± 4.4a 601.3 ± 3.2a 10.8 ± 0.3a 170.9 ± 2.6a 21.8 ± 1.3a 45.1 ± 2.2a 21.6 ± 0.5a 122.8 ± 1.8a 37.8 ± 1.8a 26.2 ± 0.8a 53.3 ± 3.1a 134.4 ± 1.5a 46.5 ± 2.9a 161.6 ± 11.9a 13.5 ± 0.1a

2.7 1.7 3.0 6.0 2.9 1.3 1.6 1.0 3.6 0.5 2.3 1.5 6.1 4.9 2.3 1.5 4.8 3.1 5.9 1.1 6.2 7.4 0.7

90.3 ± 1.9a 33.6 ± 1.1a 46.5 ± 2.2a 304.7 ± 5.4a 7.1 ± 0.3a 3.8 ± 0.2a 11.2 ± 0.3a 41.1 ± 2.6a 28.6 ± 0.7a 70.7 ± 1.4a 14.8 ± 1.5a 152.5 ± 6.7a

2.1 3.3 4.7 1.8 3.7 4.1 2.6 6.3 2.4 2.0 10.2 4.4

1.9 1.1 1.7 1.5 2.5 1.5 0.9 2.4 1.2 2.3 4.7 2.6

Spermidine ( ± SD)

RSD (%)

Spermine ( ± SD)

RSD (%)

Total amine content

4004.0 ± 69.0a 40452.4 ± 2156.4a 8992.4 ± 540.8a 3265.7 ± 187.3a 1693.7 ± 42.3a 2317.9 ± 11.4a 4984.6 ± 135.7a 5415.8 ± 109.9a 8286.3 ± 401.9a 2684.1 ± 53.1a 2628.2 ± 60.7a 2985.6 ± 83.7a 3490.6 ± 75.9a 1032.8 ± 25.3a 3829.8 ± 106.4a 5586.7 ± 239.7a 5965.5 ± 310.8a 3359.3 ± 145.5a 5163.1 ± 254.1a 4861.0 ± 157.7a 867.5 ± 13.5a 27532.1 ± 1914.4a 2988.5 ± 34.4a

1.7 5.3 6.0 5.7 2.5 0.5 2.7 2.0 4.9 2.0 2.3 2.8 2.2 2.4 2.8 4.3 5.2 4.3 4.9 3.2 1.6 7.0 1.2

1189.7 ± 21.2a 5619.2 ± 363.5a 2483.5 ± 232.1a 893.2 ± 46.2a 573.5 ± 28.2a 925.3 ± 35.4a 1533.7 ± 70.2a 1694.5 ± 94.8a 2650.5 ± 293.1a 1063.9 ± 37.8a 1442.9 ± 22.9a 1521.7 ± 52.2a 1342.8 ± 106.5a 182.2 ± 5.3a 962.4 ± 26.9a 2461.9 ± 72.2a 1619.7 ± 81.1a 1168.0 ± 36.3a 2183.4 ± 112.8a 2362.8 ± 193.3a 164.8 ± 9.9a 2212.1 ± 240.0a 1839.1 ± 16.7a

1.8 6.5 9.3 5.2 4.9 3.8 4.6 5.6 11.1 3.5 1.6 3.4 7.9 2.9 2.8 2.9 5.0 3.1 5.2 8.2 6.0 10.8 0.9

8489 101423 23913 7584 4310 5888 9831 8998 23778 7002 5652 7263 7571 4128 7655 13964 13879 7762 12303 12949 2819 56239 6478

2947.7 ± 61.0a 3510.5 ± 43.4a 2454.6 ± 103.7a 3644.4 ± 69.0a 1772.8 ± 34.7a 1888.2 ± 70.2a 2762.3 ± 156.5a 1931.3 ± 73.1a 4713.6 ± 264.6a 7607.8 ± 803.6a 2606.0 ± 346.4a 2602.3 ± 194.5a

2.1 1.2 4.2 1.9 2.0 3.7 5.7 3.8 5.6 10.6 13.3 7.5

2223.7 ± 128.2a 1844.1 ± 41.0a 2804.5 ± 108.1a 2040.1 ± 107.4a 1996.5 ± 81.7a 2775.6 ± 113.7a 1825.1 ± 102.4a 3032.8 ± 66.7a 2180.8 ± 160.2a 2045.2 ± 215.8a 1236.8 ± 152.1a 1024.3 ± 103.2a

5.8 2.2 3.9 5.3 4.1 4.1 5.6 2.2 7.3 10.5 12.3 10.1

8901 8649 6956 9411 6177 5858 6483 6129 10377 21864 5989 6101

RI PT

CSa BCoGp BTu DSa AOf COf ASa CSa BCo CSa DOf DPa DPa BCo ASa BTu ASa ASa BTu BTu BPa BOfGp BSa

Biogenic amine content (ng/g) RSD Tyramine RSD ( ± SD) (%) (%)

Vegetable products with fish 107.5 ± 3.4a 3.2 222.9 ± 11.0a 2.5 ± 0.2a 6.0 669.8 ± 5.9b b 397.7 ± 43.1 10.8 486.8 ± 51.3b a 5.6 ± 0.2 3.6 265.6 ± 20.1a 51.3 ± 1.4a 2.8 151.8 ± 4.4a a 28.3 ± 0.8 2.9 111.1 ± 6.0a 213.3 ± 3.3a 1.6 135.7 ± 5.3a a 4.2 ± 0.2 3.7 13.5 ± 0.8a a 179.8 ± 12.4 6.9 544.6 ± 35.5b 213.1 ± 7.9a 3.7 288.6 ± 2.6a a 242.5 ± 3.9 1.6 224.5 ± 3.2a 234.3 ± 3.1a 1.3 424.6 ± 14.3a a 4.1 ± 0.2 3.7 168.7 ± 6.8a 131.2 ± 1.9a 1.4 424.0 ± 10.8b a 43.0 ± 0.8 1.8 83.8 ± 5.6a b 660.6 ± 20.3 3.1 749.9 ± 39.1b 15.8 ± 0.9a 5.9 66.1 ± 5.5a a 173.8 ± 8.2 4.7 165.6 ± 11.0a 459.2 ± 6.8b 1.5 380.8 ± 17.1b b 557.4 ± 12.0 2.1 649.1 ± 20.5b a 71.0 ± 4.4 6.2 228.5 ± 19.4a 19.3 ± 1.5a 7.7 61.8 ± 4.5a a 12.9 ± 0.1 0.8 41.1 ± 0.4a Vegetable products with meat 453.1 ± 39.5a 8.7 379.9 ± 3.7a a 490.8 ± 20.4 4.2 249.7 ± 4.7a 142.9 ± 1.9a 1.3 184.3 ± 4.8a a 42.6 ± 3.1 7.2 605.2 ± 39.8a a 22.3 ± 0.3 1.2 45.7 ± 2.1a 109.5 ± 6.4a 5.8 <2.0 11.8 ± 0.4a 3.4 143.0 ± 9.5a 141.4 ± 4.1a 2.9 32.0 ± 3.1a a 392.3 ± 27.9 7.1 29.6 ± 0.9a 86.5 ± 0.8a 0.9 17.0 ± 0.6a a 43.9 ± 4.2 9.6 314.2 ± 44.4a a 328.0 ± 22.0 6.7 280.8 ± 21.4a

SC

1-3yr 9mth 6mth 1yr 8mth 11mth 5mth 15mth 8mth 5mth 7mth 11mth 4mth 8mth 5mth 1-3yr 1-3yr 6mth 9mth 1-3yr 11mth 12mth 5mth

RSD (%)

Histamine ( ± SD)

M AN U

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

RSD (%)

Cadaverine ( ± SD)

TE D

Putrescine ( ± SD)

EP

Sample code1

AC C

Sample Age No. group

4.9 0.9 10.5 7.6 2.9 5.4 3.9 6.2 6.5 0.9 1.4 3.4 4.0 2.5 6.6 5.2 8.3 6.7 4.5 3.2 8.5 7.2 1.0 1.0 1.9 2.6 6.6 4.6

6.7 9.7 3.1 3.6 14.1 7.6

ACCEPTED MANUSCRIPT

0.7 7.0 1.7

619.3 ± 7.8a 15.3 ± 0.5a 862.4 ± 6.5a

1.3 3.0 0.8

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

4mth 4mth 4mth 4mth 4mth 5mth 4mth 4mth 4mth 4mth 4mth 4mth 4mth 4mth 4mth

DSp GPu BSp HPu HCa COv COv CSp COv ACa ACa CCa BCa BOvGp BOv

2322.4 ± 132.0a 5303.5 ± 58.5a 2073.1 ± 43.1a 7839.9 ± 90.4a 823.4 ± 50.8a 1691.8 ± 30.6a 5651.1 ± 119.5a 1804.2 ± 22.1a 4423.3 ± 349.2a 2815.2 ± 0.1a 3778.4 ± 153.9a 2329.5 ± 16.8a 2345.9 ± 114.8a 32403.1 ± 1190.5a 3368.4 ± 198.2a

5.7 1.1 2.1 1.2 6.2 1.8 2.1 1.2 7.9 0.0 4.1 0.7 4.9 3.7 5.9

18.7 ± 1.1a 10.3 ± 0.4a 6.6 ± 0.5a 23.1 ± 1.1a 4.0 ± 0.1a 17.9 ± 0.4a 26.3 ± 0.4a 28.0 ± 0.7a 22.0 ± 0.7a 6.8 ± 0.6a 4.1 ± 0.2a 27.7 ± 0.5a 2.9 ± 0.1a 55.3 ± 2.8a 4.4 ± 0.1a

5.9 3.9 6.9 4.9 3.5 2.0 1.5 2.5 3.2 9.4 5.2 1.8 3.4 5.0 3.2

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

8mth 1yr 4mth 4mth 5mth 4mth 5mth 4mth 6mth 8mth 5mth 6mth 4mth 4mth 4mth

AAp DBa AAp ABa EAp EBa BAp HAp IAp BBa JBa HAp JAp CAp CBa

881.2 ± 39.8a 11765.6 ± 188.8b 1049.4 ± 20.6a 20726.6 ± 216.1b 704.1 ± 18.3a 6061.1 ± 102.0b 2626.5 ± 61.9a 1682.5 ± 30.7a 765.3 ± 18.0a 8586.6 ± 44.7b 11740.7 ± 119.9b 1262.5 ± 13.9a 1913.4 ± 63.7a 2063.7 ± 88.6a 29702.4 ± 406.0b

4.5 1.6 2.0 1.0 2.6 1.7 2.4 1.8 2.3 0.5 1.0 1.1 3.3 4.3 1.4

3.2 ± 0.2a 28.9 ± 0.8a 2.5 ± 0.2a 45.0 ± 1.0a 4.5 ± 0.2a 23.8 ± 0.7a 24.5 ± 0.7a <2.0 2.1 ± 0.2a 74.3 ± 0.5a 7.1 ± 0.6a 2.1 ± 0.2a <2.0 4.4 ± 0.2a 10.7 ± 0.2a

7.3 2.9 6.0 2.3 5.2 2.8 2.8

EP

9.5 0.6 9.1 9.5

AC C

1

289.5 ± 2.1a 0.7 290.3 ± 9.1a a 235.3 ± 16.4 7.0 328.7 ± 18.1a 409.4 ± 6.3a 1.5 542.4 ± 5.3a Vegetable products 757.0 ± 34.7b 4.6 439.2 ± 7.4b 338.5 ± 6.0a 1.8 13.3 ± 0.9a b 2374.6 ± 5.6 0.2 442.7 ± 26.3b a 43.9 ± 2.8 6.3 11.1 ± 1.0a 6.4 ± 0.4a 5.6 2.4 ± 0.3a a 117.0 ± 1.8 1.5 242.5 ± 2.1a 23.9 ± 1.6a 6.8 223.4 ± 0.8a b 848.0 ± 18.5 2.2 622.5 ± 0.7b a 13.9 ± 1.4 10.2 335.5 ± 30.1a 9.4 ± 1.1a 11.3 80.1 ± 1.3a a 7.3 ± 0.8 11.6 14.1 ± 0.8a 14.5 ± 1.8a 12.2 60.3 ± 6.6a a 4.0 ± 0.2 4.3 <2.0 5.6 ± 0.0a 0.0 12.4 ± 0.0a a 2.9 ± 0.4 13.2 9.4 ± 0.8a Fruit products 2.1 ± 0.2a 12.5 36.4 ± 2.8a 17.3 ± 0.7b 3.9 933.9 ± 9.2b a 4.6 ± 0.2 4.6 21.4 ± 1.0a 20.0 ± 0.7b 3.5 1355.1 ± 15.6b <2.0 12.7 ± 1.1a 11.0 ± 0.6b 5.0 657.5 ± 6.4b a 5.1 ± 0.4 8.0 335.9 ± 4.1a <2.0 12.7 ± 1.3a 2.0 ± 0.1a 5.0 72.0 ± 4.1a b 17.2 ± 0.5 2.7 771.1 ± 6.7b 7.4 ± 0.3b 4.1 637.0 ± 6.2b <2.0 479.9 ± 16.0a <2.0 <2.0 <2.0 2.8 ± 0.3a b 18.1 ± 0.9 4.8 1667.9 ± 27.3b

5.3 2.0

3.1 5.5 1.0

5557.9 ± 400.8a 2423.5 ± 138.8a 5070 ± 266.8a

7.2 5.7 5.3

2136.6 ± 231.1a 1245.9 ± 30.7a 2108.1 ± 187.8a

10.8 2.5 8.9

14451 6426 15298

1.7 7.1 5.9 8.9 12.9 0.9 0.4 0.1 9.0 1.7 5.5 10.9

3486.9 ± 312.0a 2734.4 ± 143.2a 3722.7 ± 91.9a 1810.1 ± 38.0a 1969.9 ± 10.0a 1184.0 ± 19.7a 10229.6 ± 0.7a 2116.1 ± 30.5a 3118.2 ± 269.4a 3045.3 ± 18.2a 3250.3 ± 138a 2506.7 ± 10.2a 2734.2 ± 69.9a 46679.9 ± 1434.6a 9892.9 ± 412.9a

8.9 5.2 2.5 2.1 0.5 1.7 0.0 1.4 8.6 0.6 4.2 0.4 2.6 3.1 4.2

811.2 ± 60.1a 2618.9 ± 170.1a 560.8 ± 50.7a 1221.2 ± 114.8a 60.2 ± 2.0a 136.7 ± 12.7a 1405.5 ± 10.6a 374.3 ± 13.3a 359.3 ± 40.9a 258.4 ± 26.9a 212.5 ± 15.4a 838.1 ± 63.9a 119.6 ± 3.2a 4163.0 ± 111.5a 2474.9 ± 98.0a

7.4 6.5 9.0 9.4 3.3 9.3 0.8 3.6 11.4 10.4 7.3 7.6 2.6 2.7 4.0

7835 11019 9180 10949 2866 3390 17560 5793 8272 6215 7267 5777 5207 83319 15753

865.9 ± 99.1a 2880.6 ± 86.8b 574.4 ± 48.2a 4616.7 ± 54.8b 882.9 ± 62.4a 2199.0 ± 113.0b 1552.6 ± 68.4a 1292.2 ± 119.3 407.5 ± 42.5a 3729.6 ± 102.6b 2833.6 ± 35.1b 1327.3 ± 107.9a 1694.3 ± 72.4a 1622.8 ± 139.7 5172.0 ± 40.5b

11.4 3.0 8.4 1.2 7.1 5.1 4.4 10.0 10.4 2.8 1.2 8.1 4.3 8.6 0.8

38.7 ± 0.6a 287.6 ± 13.7b 57.4 ± 2.8a 194.8 ± 14.3b 114.0 ± 5.4a 136.2 ± 12.5b 77.2 ± 4.5a 91.9 ± 7.3a 34.3 ± 3.3a 561.3 ± 34.4b 401.9 ± 16.3b 95.6 ± 11.2a 201.0 ± 15.6a 87.6 ± 6.0a 273.5 ± 8.5b

1.6 4.7 4.9 7.3 4.7 9.2 5.8 7.9 9.5 6.1 4.1 11.7 7.8 6.8 3.1

1827 15914 1710 26958 1718 9089 4622 3079 1283 13740 15628 3167 3809 3781 36845

RI PT

5557.2 ± 36.6a 2177.4 ± 152.8a 6306.2 ± 108.6a

SC

ECh FBe JCh

M AN U

6mth 4mth 5mth

TE D

36 37 38

0.0 8.4 7.7 1.0 4.6 1.2 8.4 1.0 1.2 10.0 5.7 0.9 1.0 3.3 11.6 1.6

letters A, B, C, D, E, F, G, H, I to J correspond to different manufacturers. Samples (No. 1-23) are coded by fish species: salmon (Sa), cod (Co), pangasius (Pa), tuna (Tu) and other sea fishes (Of), by kind of meat (No. 24-38): beef (Be), chicken (Ch), lamb (La), veal (Ve), rabbit (Ra), turkey (Tr), pork (Po), by vegetable ingredients (No. 39-53): spinach (Sp), carrot (Ca), pumpkin (Pu), other vegetables (Ov), green peas (Gp), and by kind of fruit (No. 54-68): apple (Ap), banana (Ba) SD: standard deviation Mean values in row marked with the same letter within selected food group are not (P>0.05) significantly different.

ACCEPTED MANUSCRIPT Table 3 Overall contents of biogenic amines found in ready-to-eat baby foods (n=68).

putrescine

No. of
cadaverine

2

2

1263

26

90

histamine

5

2

2375

44

186

tyramine

3

2

1668

224

300

spermidine

-

408

46680

2967

4907

7689

spermine

-

34

5619

1179

1278

1111

Max

Median

Mean

SD

704

53416

(ng/g) 2585

5654

8690 209

RI PT

Min

345 322

AC C

EP

TE D

M AN U

SC

Biogenic amine

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT • Occurrence level of biogenic amines in commercial ready-to-eat baby foods. • Identification of amine-related pseudoallergies components in baby foods. • Potential toxicity assessment in relation to ARfD and the BAI.

AC C

EP

TE D

M AN U

SC

RI PT

• Amine-related nutritional recommendations for baby foods.