Distribution of moniliformin in industrial maize milling and flaking process

doi:10.21203/rs.3.rs-3978577/v1

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Distribution of moniliformin in industrial maize milling and flaking process

https://doi.org/10.21203/rs.3.rs-3978577/v1

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Moniliformin (MON) is a widespread emerging mycotoxin often occurring in maize at not negligible levels. Few published studies investigated MON redistribution in maize derived products for human consumption; to better understand this issue, 5 maize lots with different level of MON contamination were processed following an industrial milling process to evaluate the redistribution of the mycotoxin in final products (grits), by-products destined to feed (bran and flour) and cleaning waste. A relevant MON reduction was obtained after sieve cleaning, scourer process and optical sorting, achieving a decrement of the concentration level close to 70%. The following other milling procedures showed a limited reduction from cleaned maize to small and large grits; considering the entire industrial process, the reduction percentage of MON contamination in the final products was 80.9 ± 9.3% and 81.0 ± 6.7% for small and large grits, respectively. The flaking process showed a very limited reduction of MON, close to 10%. Considering the widespread of MON occurrence in maize, the study highlights the importance of cleaning steps to achieve a low risk of exposure for the consumer.

moniliformin

maize

milling process

flaking process

Moniliformin (MON) is an emerging Fusarium mycotoxin occurring in cereals with high levels found in maize. MON is mainly produced by F. subglutinans, F. temperatum, F. verticilloides, and F. proliferatum (Logrieco et al, 2002; Scarpino et al., 2015; Scauflaire et al, 2012); it is a highly polar and acidic molecule (Fig. 1) and occurs as a water-soluble sodium or potassium salt (Springer et al, 1974). The Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA-CONTAM, 2018) indicated that cardiotoxicity and hepatotoxicity are its major adverse health effects, resulting in a shortage of energy, respiratory stress, and myocardial loss of functionality. MON can also increase oxidative stress, reducing the activity of glutathione peroxidase and glutathione reductase (Rossi, F.; et al, 2020); finally, its toxicity in combination with fumonisins was studied (Fremy et al, 2019). No regulatory limits have been fixed by EU Commission for MON, however EFSA recommended more data on its occurrence in cereals and derived products. Widespread contaminations were found in cereals (mainly maize) cultivated in both Northern and Southern Europe, showing that MON can be produced in different climate conditions (Scarpino et al, 2013; Bertuzzi et al 2019; Van Asselt et al, 2012; Herrera et al, 2017; Jajic et al, 2019; Janic et al, 2020). Although several studies on MON occurrence in maize have recently been published, few works investigated the distribution of this mycotoxin during the milling process. Pineda-Valdes reported a possible reduction of MON by heat processing and during alkaline cooking of maize (Pineda-Valdes 2002 and 2003); Tittlemier et al (2014) investigated the fate of MON during durum wheat milling, processing and cooking of spaghetti. Lately, Scarpino et al (2020) determined a relevant reduction of MON during maize large-scale dry milling process. In this work, distribution of MON during industrial dry milling process of different lots of maize intended for human consumption was evaluated. Five maize lots having different MON contaminations were processed and cleaning waste, by-products and final products were collected and analysed; finally, the influence of industrial flaking process for corn flakes production on MON contamination was also determined.

Maize milling process and sampling

Five different commercial lots of maize cultivated in 2023 in Northern Italy were processed in an industrial mill. Initially, maize was subjected to different cleaning steps; at first, a sieve cleaner was used to discard broken grains having a particle size inferior to 500 µm. Then, maize was passed to densimetric tables to separate defective grains and successively to a destoner to remove impurities. After a new passage to densimetric tables, maize was subjected to an intensive scourer with aspirator. A final cleaning step involved an optical sorting to remove grains undetectable by visual control. Cleaned maize was processed using a tempering degermination system consisting in adding warm water to achieve a moisture level of about 20%; in these conditions, germ becomes elastic and maize can be degerminated. After that, maize was sifted to obtain bran, flour, small and large size grits. Flour, bran, and a fraction of cleaning waste were processed for the production of animal feed flour. The sampled products represented a maize lot of about 40 t and were collected during the milling process according to European Commission Regulation EC 401/2006 (European Commission 2006). From the 5 maize lots considered, unprocessed maize, two cleaning waste (after sieve cleaner and after densimetric tables), maize before and after optical sorting, germ, bran, flour, small and large size grits were collected. For each product, the final sample (about 5 kg) was obtained by blending 30 incremental samples (150–200 g each) collected at regular intervals for 1 hour by means dynamic sampling procedure. The samples were kept at -20°C until the analysis. Moreover, two lots of cleaned maize were collected before and after an industrial flaking process (95°C for 35 min).

Reagents and standards

Chemicals and solvents used for the extraction and clean-up steps were ACS grade or equivalent (Carlo Erba, Milan, Italy). For LC-MS/MS analysis, water, methanol, acetonitrile and acetic acid were LC-MS grade (Merck, Darmstadt, Germany). MON and 1,2-diamino-4,5-dichlorobenzene (DDB) were purchased from Sigma (St. Louis, MO, USA). For MON, a stock solution of 10 mg l^− 1 was prepared in water-acetonitrile 1 + 9 v/v; working solutions of 10-2000 µg l^− 1 were prepared by diluting with water-acetonitrile 1 + 9 v/v.

LC-MS/MS analysis for MON determination

After milling of samples using a cyclone hammer mill (sieve 1 mm) and their homogenisation, MON was extracted from 25 g using 100 ml mixture CH₃CN:H₂O 1 + 1 (v/v) for 60 min. After filtration on folded filter paper and centrifugation (3000 g for 10 min), the extract was purified on a MycoSep® 240 Mon cleanup columns (Romer Labs®, Tulln, Austria); then, after filtration through 0.45 µm filter, the extract was injected (10 µl) into the LC-MS/MS system. The HPLC-MS/MS system consisted of a LC 1.4 Surveyor pump (Thermo Fisher Scientific, San Jose, CA, USA), a PAL 1.3.1 sampling system (CTC Analytics AG, Zwingen, Switzerland) and a Quantum Discovery Max triple quadrupole mass spectrometer; the system was controlled by an Excalibur 1.4 software (Thermo Fisher Scientific, San Jose, CA, USA). MON was separated on a HILIC zwitterionic column (BEH-Z-HILIC, 2.5 µm, 2.1 x 100 mm, Waters) using as mobile phase a mixture 25 mM ammonium formate (pH 3.2):CH₃CN 15 + 85 (v/v). The ionization was carried out with an ESI interface (Thermo-Fisher) in negative mode as reported by Bertuzzi et al (2019): spray capillary voltage was 3.5 kV, sheath and auxiliary gas 40 and 15 psi, respectively; skimmer 9 V, temperature of the heated capillary 350°C. The mass spectrometric analysis was performed in selected reaction monitoring (SRM). For fragmentation of the [M − H]⁻ ion (97 m/z), the argon collision pressure was set to 1.2 mTorr and the collision energy to 21 V. The detected and quantified fragment ion was 41 m/z. Five MON standard solutions among 0.005 and 0.4 mg l^− 1 were injected. The limit of detection (LOD) and of quantification (LOQ) were 7 and 20 µg kg^− 1, respectively. Three replicates were carried out for each sample.

For extraction and purification steps, quality control data are reported in our previous work (Bertuzzi et al, 2020); matrix effect was limited, below 6%.

Confirmatory LC-MS/MS method for MON determination

Considering the low molecular mass of MON and the production of one fragment ion, a derivatization with 1,2-diamino-4,5-dichlorobenzene (DDB) was carried out to increase the sensitivity and the accuracy of the analysis, in according to the study of Zollner et al (2003). A total of 20 samples were analysed using this method. Then, standard MON solutions and sample purified extracts (2 for each matrix) were derivatized as follows: 2 ml extracts were derivatized with 0.5 ml DDB solution (1mg/ml in HCl 1 M) at 60°C for 120 min. After evaporation under gentle flow of nitrogen, the extract was redissolved with 0.5 ml CH₃CN:H₂O 3 + 7 (v/v) and injected into the LC-MS/MS system. The HPLC-MS/MS system consisted of a Vanquish pump and autosampler, and a Fortis triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA); the system was controlled by an Excalibur 1.4 software (Thermo Fisher Scientific, San Jose, CA, USA). MON was separated on a Betasil RP-18 column (2.5 µm, 2.1 x 100 mm, Thermo Fisher) using a gradient elution H₂O (A):CH₃CN (B), both acidified with 0.2% formic acid, as follows: A from 70–15% in 5 min, isocratic for 4 min. The ionization was carried out in positive mode with an H-ESI interface: sheath and auxiliary gas 35 and 12 psi, respectively; sweep gas 2 psi, temperature of heated capillary and vaporization 270 and 200°C, respectively. The mass spectrometric analysis was performed in selected reaction monitoring (SRM). For fragmentation of the [M + H]⁺ ion (229 m/z), the dwell rime was 500 ms, the argon collision pressure was set to 1.5 mTorr and the collision energy to 30 V. The fragment ions were 124, 152 and 187 m/z. The retention time of MON was 8.4 min (Fig. 1). The limit of detection (LOD) and of quantification (LOQ) were 0.5 and 2 µg kg^− 1, respectively.

Analysis of data

The quantification of MON distribution in the different sub-products of maize was calculated considering the following ratio:

MON ratio = MON concentration in the product/MON concentration in unprocessed maize

The maize lots showed different levels of MON contamination, from about 150 to 1100 µg kg^− 1, close to findings of surveys on maize cultivated in Northern Italy (Bertuzzi et al, 2020; Scarpino et al, 2015). The Table 1 shows contamination levels of MON (µg kg^− 1) found in the collected samples and the ratio of distribution respect the unprocessed maize calculated for each lot.

Table 1

MON contamination (µg/kg) in maize fractions collected during milling process.
	Maize 1	Ratio	Maize 2	Ratio	Maize 3	Ratio	Maize 4	Ratio	Maize 5	Ratio
Unprocessed maize	574.3		482.1		153.2		1090		755.4
Waste of sieve cleaner	5099	8.88	2059	4.27	645.9	4.22	9167	8.78	3602	4.90
Maize with particle size > 500 µm	325.5	0.61	420.7	0.87	118.5	0.77	421.5	0.39	524.4	0.69
Waste of densimetric tables	475.5	0.83	203.4	0.42	515.6	3.37	188.1	0.17	185.8	0.25
Cleaned maize after optical sorting	131.8	0.23	119.6	0.25	59.5	0.39	256.8	0.24	253.0	0.33
Small grits	32.3	0.006	92.5	0.019	33.2	0.22	192.4	0.18	237.9	0.31
Large grits	53.7	0.009	82.9	0.027	37.8	0.25	195.5	0.18	195.8	0.26
Germ	21.4	0.004	40.0	0.008	26.1	0.017	60.1	0.006	160.9	0.21
Bran	930.2	1.62	304.5	0.63	177.8	1.16	1205	1.11	1119	1.48
Flour	263.6	0.46	107.7	0.22	91.7	0.60	291.4	0.27	343.2	0.45

The samples (n = 20) analysed using the confirmatory LC-MS/MS method showed levels of contamination similar (+/-7%) to those obtained with the conventional method; using this method, lower LOD and LOQ were obtained (Fig. 2).

The results confirmed the efficacy of cleaning steps, mainly the use of sieve cleaner that removed high levels of MON with an average concentration in the waste about 6 times higher than in unprocessed maize (6.21 ± 2.40); the concentration in maize after this cleaning step was one third lower than in raw maize (mean: 0.67 ± 0.18). The waste of densimetric tables resulted less contaminated, at a concentration close to that found in the initial maize (mean: 1.01 ± 1.34). After the scourer process and the optical sorting, MON concentration in cleaned maize was about one third of that in the unprocessed maize (average ratio: 0.29 ± 0.07), achieving a global reduction of 71.3 ± 7.1%, remarkably higher that the values between 36–59% reported by Scarpino et al (2020). The use of the optical sorter confirmed to achieve a high reduction of MON contamination.

The following steps of the milling process showed high levels of contamination in bran fraction, reporting concentrations always higher in comparison with cleaned maize (average ratio: 3.93 ± 1.97). Germ was the fraction less contaminated, the average concentration ratio between germ and cleaned maize was 0.36 ± 0.19; higher ratios, but always inferior to 1, were calculated for both small and large size grits, showing very similar values, 0.65 ± 0.27 and 0.65 ± 0.15, respectively. Considering the entire process, MON concentration in both grits was one fifth than that found in unprocessed maize (0.19 ± 0.06 and 0.19 ± 0.09); then, the reduction percentage in the final product respect to the unprocessed maize was 80.9 ± 9.3% and 81.±6.7% for small and large grits, respectively, resulting very similar to 80% and 64% reported by Scarpino et al (2020) for flaking and medium hominy grits. However, in our study no significant difference in concentration of MON was found between large and small grits.

Considering all the five lots of maize, the average yield of the cleaning fractions was 3% for both sieve cleaner and densimetric table waste; generally, cleaned maize after optical sorting was 92% of the unprocessed maize. For the degermination and milling process, the average yields respect to the cleaned maize were: 2% for bran, 5% for germ, 20% for flour, 55 and 10% for large and small size grits, respectively. Table 2 shows the amount of MON found in unprocessed maize and in the collected fractions.

Table 2

MON presence (µg) in maize fractions collected during milling process.
	Maize 1	Maize 2	Maize 3	Maize 4	Maize 5
Unprocessed maize	57425	48216	15324	108979	75547
Waste of sieve cleaner	18782	6176	1938	46983	13636
Maize with particle size > 500 µm	34189	40812	11500	40466	50871
Waste of densimetric tables	1427	610	1547	376	558
Cleaned maize after optical sorting	12127	11004	5483	23622	23275
Small grits	323	926	332	1924	2380
Large grits	2955	4563	2081	10756	10772
Germ	107	200	131	301	805
Bran	1860	609	356	2410	1206
Flour	5271	2155	1835	5828	6865

The percentage of MON content found in siever cleaner and densimetric tables waste respect to unprocessed maize were different among the lots; the average values, with high standard deviations, were 23.9 ± 13.5% and 3.0 ± 4.1%, respectively. After the entire cleaning process, the average percentage of MON remained in the cleaned maize was 26.4 ± 6.5%; these data confirmed that intensive scourer and optical sorting were very effective steps to remove the mycotoxin. Regarding degerminating and milling process, the sum of MON in bran, germ, flour and grits achieved 86.9 ± 6.5% respect to the MON in cleaned maize; this minor content found in the fractions can be due to the complexity of the sampling of several matrices. If compared to MON content in cleaned maize, MON distribution was: 39.1 ± 8.9% in large size grits, 7.1 ± 2.9% in small size grits, 30.1 ± 9.1% in flour, 8.5 ± 4.3% in bran, 2.0 ± 1.0% in germ. These results showed that, after the milling process, a relevant amount, about 50%, of MON remained in final products (grits), highlighting that the cleaning steps were more efficacious to reduce the MON presence.

The contamination levels of MON before and after the flaking process are reported in Table 3. It is possible to observe that MON reduction was very low, (mean of 2 process was 10.1%), showing that this mycotoxin is stable at temperature close to 100°C.

Table 3

MON contamination (µg/kg) before and after flaking process.
	Maize grits	Maize flakes	Reduction (%)
Lot 1	169.1	149.2	11.7%
Lot 2	41.8	38.2	8.6%

These results were similar to findings of Pineda-Valdes et al (2003), which observed a slightly higher reduction of MON, close to 20%, in corn grits extruded at temperature among 140 and 180°C.

It is well known that fumonisins (FBs) and MON often co-occur in maize; FBs redistribution was largely investigated in the milling fractions showing high reduction both in cleaning and in milling process; unlike MON was rarely studied. This study underlines that the cleaning steps are very efficient in reducing the content of MON in maize, as for FBs, and these operations are essential to reduce the risk of contamination of these Fusarium toxins. Moreover, the moderate reduction of MON during the following steps of milling might lead to a not negligible risk of exposure for the consumers, if a not accurate cleaning was carried out. The co-occurrence of MON and FBs was widely studied in maize samples, however there are few data regarding maize derived products intended for human consumption. The exposure to these mycotoxins could be relevant for consumers in the countries where maize is a staple food, for the baby food supply chain and for the celiac population. The confirmatory method for MON determination developed in this study could confirm its occurrence at trace levels obtaining a more accurate evaluation of risk.

Author Contribution

T.B. and P.G. wrote the manuscript; T.B.and A.A. carried out the chemical analysis. P.G. prepared tables and discussion. All authors reviewed the manuscript.

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No competing interests reported.

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Distribution of moniliformin in industrial maize milling and flaking process

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Abstract

Figures

Introduction

Materials and methods

Maize milling process and sampling

Reagents and standards

LC-MS/MS analysis for MON determination

Confirmatory LC-MS/MS method for MON determination

Analysis of data

Results and discussion

Declarations

Author Contribution

References

Additional Declarations

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