Impact of Variety and Agronomic Factors on Crude Protein and Total

Article pubs.acs.org/JAFC

Impact of Variety and Agronomic Factors on Crude Protein and Total Lysine in Chicory; Nε‑Carboxymethyl-lysine-Forming Potential during Drying and Roasting Grégory Loaec̈ ,† Céline Niquet-Léridon,† Nicolas Henry,§ Philippe Jacolot,† Céline Jouquand,† Myriam Janssens,# Philippe Hance,⊥ Thierry Cadalen,⊥ Jean-Louis Hilbert,⊥ Bruno Desprez,§ and Frédéric J. Tessier*,†,¶ †

EGEAL Unit, Institut Polytechnique LaSalle Beauvais, 19 rue Pierre Waguet, 60026 Beauvais, France Florimond-Desprez, 3 rue Florimond Desprez, 59242 Cappelle-en-Pévèle, France # Leroux SAS, 84 rue François Herbo, 59310 Orchies, France ⊥ Laboratoire Régional de Recherche en Agro-alimentaire et en Biotechnologie, Institut Charles Viollette, GIS GENOCHIC, Université Lille1 Sciences et Technologies, Bâtiment SN2, 59655 Villeneuve d’Ascq, France ¶ Université Lille, Inserm, CHU Lille, U995 - LIRIC- Lille Inflammation Research International Center, F-59000 Lille, France §

ABSTRACT: During the heat treatment of coffee and its substitutes some compounds potentially deleterious to health are synthesized by the Maillard reaction. Among these, Nε-carboxymethyl-lysine (CML) was detected at high levels in coffee substitutes. The objective of this study was to evaluate the impact of changes in agricultural practice on the lysine content present in chicory roots and try to limit CML formation during roasting. Of the 24 varieties analyzed, small variations in lysine content were observed, 213 ± 8 mg/100 g dry matter (DM). The formation of lysine tested in five commercial varieties was affected by the nitrogen treatment with mean levels of 176 ± 2 mg/100 g DM when no fertilizer was added and 217 ± 7 mg/100 g DM with a nitrogen supply of 120 kg/ha. The lysine content of fresh roots was significantly correlated to the concentration of CML formed in roasted roots (r = 0.51; p < 0.0001; n = 76). KEYWORDS: Nε-carboxymethyl-lysine, lysine, proteins, coffee substitute, chicory, agronomy

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INTRODUCTION

A great deal of research has been done recently to reduce the formation of another undesirable MRP in food, acrylamide, using the same strategy. A selection of raw plant materials with low contents of one or the two acrylamide precursors (i.e., asparagine and reducing sugars) was successfully used to mitigate acrylamide formation in cereal-based and potato-based products.11,12 Along with these products coffee substitutes made of roasted chicory are also known to be high in acrylamide and CML (474−4940 and 200−47000 μg/kg, respectively).13 We have recently presented evidence that a controlled reduction of the free asparagine content in chicory roots can mitigate the formation of acrylamide during the production of chicory-based coffee substitutes.14 Improved varieties accompanied by changes in agronomic practices were used for that purpose. The aim of the current study was to observe whether the means used to reduce acrylamide in roasted chicory could also be applied to reduce CML in the same food product. In the course of three years of cultivation, the contents of total lysine (i.e., free and protein bound lysine) and protein were compared among several cultivars with different levels of nitrogen fertilization and various harvesting dates. The chemical

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N -Carboxymethyl-lysine (CML) is a Maillard reaction product (MRP) formed between lysine and reducing sugars in foods during heating1 and in vivo on various proteins at 37 °C.2 Regardless of its origin, its potentially deleterious effects on human health have been widely discussed in the literature.3 Diets rich in CML and other MRPs seem to contribute to the development of degenerative diseases such as metabolic syndrome, diabetes, dementia,4 and Alzheimer’s disease.5 CML and other MRPs could also be a risk factor for hypertension, cardiovascular disease, and inflammation.6−9 We also showed that a diet with a large content of CML and other MRPs modulates biomarkers that suggest an increased risk of the development of type 2 diabetes and cardiovascular disease in healthy people.10 Reducing the exposure to foodderived CML is therefore an important topic of investigation if we want to prevent its adverse effects. Nguyen et al. concluded in a recent review of the literature on CML that it would be advisable to minimize the amount of it in food.3 Among different strategies to mitigate CML, we believe that a reduction of the levels of some precursors of CML in food matrices before heat treatment should be tested. To our knowledge, no study so far has focused on the reduction of the concentration of CML in processed foods through optimizing the composition of the raw food ingredients and, particularly, by reducing their lysine content. © 2015 American Chemical Society

Received: Revised: Accepted: Published: 10295

June 10, 2015 September 23, 2015 November 8, 2015 November 8, 2015 DOI: 10.1021/acs.jafc.5b02853 J. Agric. Food Chem. 2015, 63, 10295−10302

Article

Journal of Agricultural and Food Chemistry

Figure 1. Mean levels of total lysine and crude protein in roots of 24 chicory cultivars grown in 2011 under the same agronomic conditions. The vertical bars show the standard deviation. At the chicory-processing plant the roots were dried on a Variable Circulation Lab Dryer (VCLD) (CPM Wolverine Proctor LLC, Glasgow, UK). Briefly, the roots were placed on perforated trays in the VCLD and dried for 125 min at 110−130 °C with an alternative upward and downward air flow. Representative samples were taken for analysis, and the remainders of the dried roots were roasted for 20 min at 180 °C with the same device. The roasting conditions were representative of those in the industry related to chicory. The light roasting obtained was estimated by reflectance measurement, and a value of 180 arbitrary units was the target for all samples. All dried and roasted samples were finely ground and stored at room temperature protected from light before analysis. Analysis of Total Lysine in Raw, Dried, and Roasted Chicory Samples. Total lysine was quantified in triplicate according to the method of Niquet-Léridon and Tessier.15 Briefly, samples equivalent to 10 mg of protein were incubated in 5 mL of 6 M HCl at 110 °C for 20 h. A total of 300 μL of each acid hydrolysate was dried in a SpeedVac concentrator (Thermo Fisher Scientific, Courtaboeuf, France). Each dried residue was reconstituted in 300 μL of (15N2)-lysine (in NFPA 20 mM) and filtered through a 0.45 μm membrane before LCMS/MS analysis. The analytical separation was performed on a Hypercarb column (100 × 2.1 mm, 5 μm; Thermo Fisher Scientific) at a flow rate of 200 μL/min using a gradient of a solution of 20 mM NFPA in water (A) and acetonitrile (B). The percentage of solvent B increased from 0 to 50% in 20 min. Ten microliters of each sample was injected on column. LC-MS/MS was performed on a Surveyor HPLC system coupled, by an electrospray ionization (ESI) interface, to a Finnigan LTQ iontrap mass spectrometer (Thermo Fisher Scientific). The LC-MS/MS system was controlled by Xcalibur software, version 2.0 (Thermo Fisher Scientific). The conditions of the ESI-positive interface were similar to those described by Niquet-Léridon and Tessier.15 Tandem MS analyses were performed in selected reaction monitoring mode (SRM). The specific transitions m/z 147.0 → 130.0 and 149.0 → 131.0 were used for the detection and quantification of lysine and (15N2)-lysine, respectively. Quantification of total lysine was achieved by measuring their peak area ratio to their corresponding internal standard and comparison with the standard curves. Each analysis was done in triplicate. Analysis of Crude Protein in Raw Chicory Roots. Total protein was determined using nitrogen analysis. The nitrogen content was determined by combustion method using a LECO FP528 nitrogen analyzer (LECO France, Garges les Gonesse, France) according to the Dumas method.16 A total of 100 mg of crushed sample was weighed in

degradation of lysine during the drying and roasting of the chicory relative to the formation of CML is also presented.

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MATERIALS AND METHODS

Chemicals. Nonafluoropentanoic acid (NFPA, 97%), hydrochloric acid (37%), sodium hydroxide, sodium borohydride, boric acid, and lysine (98%) were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Both CML (97%) and (D2)-CML (97%) were provided by PolyPeptide Laboratories France SAS (Strasbourg, France), whereas (15N2)-lysine (98%) was from CortecNet (Voisinsle-Bretonneux, France). Stock solutions of calibrators (standard solutions) were prepared in 20 mM NFPA in water. Acetonitrile of HPLC grade and water of HPLC grade were purchased from Sodipro (Echirolles, France). Varieties, Agronomic Conditions, and Processing of Chicory Samples. Field trials were conducted by Florimond Desprez (Cappelle-en-Pévèle, France) near Coutiches in 2011 and Nomain in 2012 and 2013. The residual nitrogen at the end of winter was measured by colorimetry assay and was 144.5, 116, and 90 kg/ha, respectively. The experimental designs were randomized blocks of 9.1 m2. Plot populations were adjusted to 155 000 plant/ha. The yield of the varieties was measured in the different agronomic conditions tested and was presented in a previous publication.14 In 2011 a study comparing 24 genotypes (two replications/entry) was realized under advised solid nitrogen input (90 kg/ha of ammonitrate 27). The response of five commercial varieties (Malachite, Silex, Chrysolite, Calcite, and Orchies, named A−E in the figures) grown under five levels of nitrogen supply (0, 30, 60, 90, and 120 kg/ha) was evaluated in a four-replicated two-factor design. In 2012 the effect of the period of growth was studied. The same set of commercial varieties was evaluated in four replication designs harvested at seven different harvesting dates, every other week, from September to December. In 2013 cross effects of parameters of the previous two years were studied. Three varieties were selected (Malachite, Calcite, and Orchies), and each was grown with three levels of nitrogen supply (0, 90, and 145 kg/ha). Each combination was harvested at three dates from September to November. Each year, chicory roots of the entire plots were harvested and washed. Ten kilograms of roots randomly selected from each plot was delivered to the chicory-processing plant (Leroux, Orchies, France) to provide dried and roasted samples, and a homogeneous fresh sample was immediately freeze-dried and stored in a desiccator until analysis. 10296

DOI: 10.1021/acs.jafc.5b02853 J. Agric. Food Chem. 2015, 63, 10295−10302

Article


Figure 2. Levels of total lysine and crude proteins in roots of chicory cultivars (A) Malachite, (B) Silex, (C) Chrysolite, (D) Calcite, and (E) Orchies depending on the amount of nitrogen fertilizer (residual N in the soil, 145 kg/ha). The vertical bars show the standard deviation. Plot F represents the mean of the five varieties. Means followed by the same letter are not significantly different by the Conover−Iman post hoc test. triplicate. The universal protein-nitrogen conversion factor of 6.25 was used for the calculation of the protein content expressed in grams per 100 g of sample. Analysis of CML in Dried and Roasted Chicory Samples. CML content was measured according to the same method described above for lysine quantification.15 The only difference was that a reduction step was added to the protocol to avoid overestimation of CML content in the sample. Briefly, samples equivalent to 10 mg of protein were incubated with 1.5 mL of 0.2 M borate buffer (pH 9.5) and 1 mL of 1 M sodium borohydride at room temperature for 4 h. Then, HCl was added to a final concentration of 6 M. Tandem MS analyses were performed in SRM mode. The specific transitions m/z 205.0 → 130.0 and 207.0 → 130.0 were used for the detection and quantification of CML and (D2)-CML, respectively. Quantification of CML was achieved by measuring its peak area ratio to its corresponding internal standard and comparison with the standard curves. Each measurement was done in triplicate and averaged. Statistical Analysis. Data were analyzed by analysis of variance (ANOVA) with the XLSTAT software (Addinsoft, Paris, France). Results were considered statistically different from the null hypothesis if p < 0.05. Separation of means was performed with Tukey’s HSD test with α = 0.05. If ANOVA preliminary conditions (residuals normality and variances equality) were not filled, the nonparametric Kruskal− Wallis test with the Conover−Iman post hoc test was performed to assess differences. Correlations between phenotypic traits were tested using the Pearson correlation coefficient.

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The variation in the content of total lysine in 24 noncommercial chicory cultivars grown and harvested in 2011 is shown in Figure 1. The concentration of total lysine in the 24 freeze-dried raw samples varied in a narrow range from 197.1 ± 8.8 to 230.0 ± 23.9 mg/100 g DM. A variance analysis of the data indicates that no significant difference existed between varieties in terms of lysine content (p > 0.05). By comparing five commercial varieties (Malachite, Silex, Chrysolite, Calcite, and Orchies) harvested during the same year, we also found that the concentration of total lysine was not significantly different among the five cultivars when the amounts were compared under similar agronomic conditions (Figure 2). This low variation (4%) in the content of lysine between cultivars indicates that a varietal selection is not likely to be an efficient means of reducing this CML precursor in chicory roots. The 24 chicory cultivars did not show significant different accumulation of crude proteins either (p > 0.05, Figure 1). Concentration of proteins between 4.5 ± 0.3 and 5.1 ± 0.2 g/ 100 g DM were found in the 24 freeze-dried raw samples, and the same low variation of protein content was observed among the five commercial varieties (3.8 ± 0.1 to 5.3 ± 0.2 g/100 g DM, Figure 2). In addition, it should be noted that there was no correlation between the amount of total lysine and the crude protein (r = −0.15, p < 0.490) in the trial of the 24 noncommercial cultivars. Although no apparent variation of protein and lysine contents was detected between these 24 cultivars, it suggests nevertheless that difference in the amino acid composition of the proteins may occur from one cultivar to another. Effect of Nitrogen Fertilization on Total Lysine and Crude Protein Contents in Raw Chicory Roots. Five commercial varieties were grown in 2011 with five different amounts of nitrogen fertilizer (from 0 to 120 kg/ha in addition to the initial nitrogen level in the soil, which was 144.5 kg/ha). The trial was made with four different plots of each “variety/

RESULTS AND DISCUSSION

Varietal Effect on Total Lysine and Crude Protein Concentrations of Raw Chicory Roots. Very few data are available on the content of total lysine, the precursor of CML, in fresh chicory roots of various cultivars. To our knowledge the only complete study on the chemical composition of chicory was presented by Pazola in a book published in 1987.17 Surprisingly, only trace amounts of lysine were then reported in three cultivars, whereas other amino acids were quantified from 110 to 1810 mg/100 g dry matter (DM). 10297


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Figure 3. Relationship between the harvesting date and total lysine content or crude protein content in raw chicory roots. The plots are the means of the five commercial cultivars. Means followed by the same letter are not significantly different by Tukey’s HSD test.

Figure 3 shows the variation of the lysine and protein contents with the maturity of the chicory root. Because no significant difference was found between varieties, the date of harvest was considered as a variety-independent variable. Therefore, data from the five cultivars tested were combined to form a single graph. A mean concentration of 226 ± 13 mg lysine/100 g DM was found in the raw chicory roots harvested at the earlier date, and a significantly different mean concentration of 277 ± 18 mg lysine/100 g DM was found in the roots harvested 3 months later (the later date). When the protein content was measured in chicory roots harvested at the two extreme dates of the study, a significant difference was observed with 4.1 ± 0.2 g/100 g DM on the first date and 5.3 ± 0.4 g/100 g DM on the last date of harvest. The same trend was noted for each of the five cultivars (data not shown). Despite a tendency toward an increase in both lysine and protein levels with the maturity of the chicory, the analysis of the roots collected at the fourth and sixth harvest dates indicates a nonlinear accumulation of lysine and proteins in the roots during growth (Figure 3). The drops in both lysine and protein contents at two of the seven dates of harvest had already been observed in the free asparagine content of the same chicory samples.14 However, no causal relationship could be found between the temporary changes in chemical composition of the roots and any sudden environmental changes. The overall positive association between the length of cultivation and the concentrations of lysine and protein in the chicory roots was not confirmed a year later in the “cross-effect” study presented below. Cross-Effects of Variety × Nitrogen Fertilization × Date of Harvest on Total Lysine and Crude Protein Concentrations of Raw Chicory Roots. In 2013 a third and final trial was conducted to confirm the effects found in 2011 and 2012. This last trial was designed to simultaneously investigate the influence of variety (Calcite, Malachite, and Orchies), the level of added N fertilizer (0, 90, and 145 kg/ha), and the date of harvest (from September 9, 2013 to November 26, 2013). The narrow variability of the lysine and protein contents among varieties was confirmed (data not shown). The

fertilizer” combination. Figure 2 shows a general rise in the total level of lysine in the chicory roots in response to increasing level of N fertilization. This significant influence of N fertilization was observed regardless of the variety tested. The lowest lysine concentration was found in the chicory roots grown without added nitrogen fertilizer (177 ± 9, 173 ± 6, 179 ± 5, 176 ± 11, 174 ± 10 mg/100 g DM for cultivars A−E, respectively). An increase in lysine content proportional to the increase in N nutrition was mainly observed from 0 to 30 kg/ ha. It reached a maximum at 30 kg/ha, and no significant difference in lysine levels in chicory roots was found between 30 and 120 kg/ha. At this last amount of N fertilizer the lysine contents were 225 ± 19, 207 ± 12, 218 ± 13, 214 ± 7, and 223 ± 6 mg/100 g DM for cultivars A−E, respectively. Figure 2F shows the mean values of the five varieties with a statistical analysis which confirmed that lysine concentration in the chicory roots was significantly affected by N supply from 0 to 30 kg/ha. The same significant and positive effect of N supply was observed on the crude protein content. An increased application of nitrogen fertilizer from 0 to 120 kg/ha caused an increase of crude protein content from 3.9 ± 0.1 to 5.1 ± 0.2 g/100 g DM respectively (Figure 2F, data pooled across varieties). The effect of an increase in the nitrogen fertilization on the content of free asparagine in chicory roots,14 rye grain,18 and other plants has been described previously. The results presented in the current study on the contents of lysine and protein in chicory roots are in line with these previous studies. Influence of Date of Harvest on Total Lysine and Crude Protein Concentrations of Raw Chicory Roots. The concentration of lysine and crude protein in chicory roots may also be influenced by the period of growth from sowing to harvest. To investigate this, the roots of the five commercial cultivars were harvested at seven stages of maturity, every other week from early September to early December 2012. The five cultivars were grown in uniform agronomic conditions including a supply of nitrogen of 90 kg/ha (residual N level = 116 kg/ha). Each plot was repeated four times. 10298


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Table 1. Influence of N Fertilization and Date of Harvest on the Total Lysine and Crude Protein Contents of Three Chicory Cultivarsa n

total lysine (mg/100 g DM)

crude protein (g/100 g DM)

mean of three cultivars and three harverst dates

25 27 27

180 ± 16 a 212 ± 14 b 222 ± 14 c

3.2 ± 0.4 a 4.3 ± 0.4 b 5.0 ± 0.5 c

mean of three cultivars and three amounts of N supply

25 27 27

207 ± 18 ab 194 ± 25 a 216 ± 20 b

4.0 ± 0.7 a 4.1 ± 0.9 a 4.5 ± 0.8 a

factor tested added nitrogen (kg/ha)

harvest date

0 90 145 Sept 9, 2013 Oct 30, 2013 Nov 26, 2013

Results are from a multifactor trial conducted in 2013. Values in each column represent the mean ± standard deviation. Different letters in the same column and per factor indicate significant differences (p < 0.05) by Tukey’s HSD test.

a

Table 2. Total Lysine and Crude Protein Contents in the Roots of Three Chicory Cultivars (A, Malachite; D, Calcite; and E, Orchies) Monitored from 2011 to 2013a cultivar

2011

2012

2013

total lysine (mg/100 g DM)

A D E

204 ± 8 a 208 ± 8 a 216 ± 8 a

199 ± 10 a 203 ± 14 a 199 ± 10 b

197 ± 10 a 200 ± 10 a 208 ± 6 ab

crude protein (g/100 g DM)

A D E

4.6 ± 0.3 a 4.7 ± 0.2 a 4.8 ± 0.3 a

3.7 ± 0.1 b 3.8 ± 0.2 b 4.1 ± 0.3 b

4.2 ± 0.3 a 4.1 ± 0.2 ab 4.4 ± 0.1 ab

Values in each column represent the mean ± standard deviation. Different letters in the same row and per factor indicate significant differences (p < 0.05) by the Conover−Iman post hoc test. Cultivars were grown under the same amount of N in the soil (residual and supply) and harvested after the same period of growth. a

Figure 4. Relationship between total lysine in (A) raw and dried, (B) dried and roasted, and (C) raw and roasted chicory roots. Results are from a multifactor trial conducted in 2013. Each plot shows the Pearson correlation with r, p value, and the linear equation.

three commercial varieties cultivated in 2013 accumulated similar concentrations of lysine in the roots (p > 0.05), indicating that a variety selection will, in all likelihood, not mitigate the lysine level in raw chicory roots, nor, therefore, will it affect the CML level in roasted chicory. The influence of the date of harvest observed in 2012 on the lysine and protein contents in raw chicory roots was not confirmed by this multifactor trial (Table 1). When the first and last dates of harvest were compared (78 day lag time), the lysine and protein contents were found to be not significantly different (207 ± 18 mg/100 g DM and 4.0 ± 0.7 g/100 g DM, respectively, at the lower maturity level and 216 ± 20 mg/100 g DM and 4.5 ± 0.8 g/100 g DM, respectively, at the higher maturity level). However, the analysis of the intermediate date of harvest confirmed the nonprogressive accumulation of lysine found in 2012. As shown in Table 1, the concentrations of lysine and proteins in chicory grown at 90 kg added N/ha were

significantly higher than those in chicory grown without added N. This variety- and maturity-independent impact of the N supply is in line with our first results in the 2011 trial. However, in the 2013 multifactor trial the N supply was found to have a significant effect up to the dose of 235 kg/ha (residual and added N = 90 + 145 kg/ha), whereas the effect reached a statistical maximum at the dose of 174.5 kg/ha (residual and added N = 144.5 + 30 kg/ha) in 2011. Growing Year Effect on Crude Protein and Total Lysine Concentrations of Raw Chicory Roots. It was shown by our previous study conducted over two years in succession that the year of cultivation had an effect on the free asparagine and protein contents in chicory roots.14 For the current study, data from three consecutive years (2011−2013) were available. Only three varieties grown under the same total N environment (residual and supply) and harvested after the same period of growth were used for the comparison of the three growing years. Table 2 shows that the crude protein 10299


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Figure 5. Multifactor trial conducted in 2013: (A) evolution trend of the CML contents in 76 chicory samples from dried to roasted; (B) correlation of total lysine contents in 76 raw chicory roots of different cultivars, nitrogen supply, and harvest date, with the resulting CML contents in the same chicory samples after roasting. Plot B shows the Pearson correlation with r, p value, and the slope.

content was significantly influenced by the growing year when 2011 and 2012 were compared (p < 0.05). Thus, in 2012 the protein contents were the lowest of all three years of study whatever the cultivar. Compared to 2011 the amount of protein decreased by 20, 19, and 15% in Malachite, Calcite, and Orchies in 2012, respectively. In contrast with the influence of the growing year on the protein content, no significant variation of the amount of lysine was noted over the three years of study (p > 0.05) (Table 2). This last observation suggests that the protein content in chicory roots is not fully correlated with the lysine content. The same conclusion was drawn with the trial of the 24 noncommercial cultivars in 2011. Effect of Drying and Roasting on the Total Lysine Content of Chicory. Because the last trial (2013) simultaneously combined three potential factors (variety, N supply, and date of harvest), the chicory roots from this trial were the ones chosen for drying and roasting. The content of lysine in dried chicory roots was significantly correlated with its content in raw chicory roots (Figure 4A, r = 0.68, p > 0.0001) but an average 20% loss of lysine was found after drying (slope = 0.802). The roasting step promoted an additional degradation with an average 83% loss of lysine between the dried and the roasted chicory samples (slope = 0.166) (Figure 4B). The content of lysine in roasted chicory roots was also significantly correlated with its content in dried chicory roots (r = 0.54, p > 0.0001). Figure 4C shows that the two-step process led to an overall degradation of 87% of the lysine originally found in the raw chicory roots: 20% + [(100% − 20%) × 83%], (slope = 0.133). Looking at the lysine content as a whole (not as a percentage of degradation), we observed that the more lysine that was present originally in the raw chicory roots, the more chemically modified lysine was also present in roasted chicory. This became clear from the chicory roots grown without any N fertilizer, the most significant factor tested, which contained not only the lowest amount of lysine in raw chicory roots but also the lowest amount of lysine after roasting (Figure 4C) and, of course, the lowest amount of modified lysine (not directly measured). The analysis of CML presented in the next paragraph showed that the formation of this Maillard reaction product cannot account on its own for the total degradation of the lysine. Many other MRPs derived from lysine were most likely formed during drying and roasting steps. Formation of CML during Drying and Roasting. Correlation with Lysine Content. The CML content was measured in the dried and roasted chicory roots, which came

from the last multifactor trial. Figure 5A shows that some CML was formed already during the drying step of the process. This indicated that this MRP is formed easily in drying conditions that were applied for the current study (110−130 °C for 125 min). We also noted that no acrylamide was formed during this process (data not shown). Our results are in line with the description of the CML formation at temperatures as low as physiological ones (37 °C)19 and the formation of acrylamide mainly above 120 °C.20 The CML concentration varied from 246 and 626 μg/100 g DM in the dried chicory samples. The amounts of CML in the same samples were in a narrower range of concentration after roasting and remained under 650 μg/100 g (393 to 642 μg/100 g DM). If the stability of CML during the course of the process is assumed, our data suggest that approximately 75% of CML was already formed during the drying step. If the assumption is incorrect, the apparent and relative stability of CML would be explained by a synthesis and a degradation, which would occur at the same time during roasting. Because the main goal of the study was to observe whether the mitigation of the lysine content in the chicory root could reduce the formation of CML during processing ,we investigated the correlation between CML and its precursor among the 76 samples of the 2013 trial. Figure 5B shows that the lysine content in raw chicory was moderately but significantly correlated with the CML formation (r = 0.51, p < 0.0001, n = 76). It appears clearly that chicory roots grown without the N supply contained the lowest amounts of lysine and had consequently the lowest amount of CML after roasting. A similar weakly positive correlation was found before between the free asparagine concentration in the chicory and the acrylamide formation, indicating that other genetic, environmental, and process factors must be investigated.14 The data from the last trial also indicate that, on average, 274 and 938 μmol of lysine/100 g DM were degraded during drying and roasting, respectively, whereas only 2 and 0.6 μmol of CML/100 g were formed, respectively. This suggests that other Maillard reaction products derived from lysine were formed as well, especially during roasting. It is known that the yields for the formation of advanced MRPs such as CML are usually very low compared to those for the formation of Heyns and Amadori compounds.21 An investigation of the quantity of these compounds, undoubtedly also formed in the process, was not part of this study. Conclusion. It has been shown previously that the formation of CML and other MRPs could be reduced in 10300



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various model foods by the addition of phenolic compounds with antioxidant properties.22,23 It has also proved possible to reduce the CML level in cakes made of several raw ingredients by a selection of ingredients with low glycation activities and the addition of potential inhibitors of glycation.24 In a previous study we found that the formation of another MRP, acrylamide, could be reduced in coffee substitutes by mitigating the accumulation of free asparagine, an acrylamide precursor, in the chicory root, its source material. Different agronomic conditions were then found to limit the formation of free asparagine in chicory.14 In the current study the level of fertilization was found to have the greater influence on the amount of lysine and proteins in the chicory root and consequently the greater influence on the extent of CML formation during roasting. The significant but weak positive correlation established between the lysine content of the raw chicory and the CML formation in roasted chicory indicates that other factors such as the amount of reducing sugars such as fructose formed during the drying and roasting processes have to be evaluated. It was, for instance, observed that the carbohydrate composition (e.g., the proportions of fructan and fructose) was influenced not only by the roasting process25 but also by the cultivar selection and the agronomic conditions.26 The length of the cultivation and the year of harvest seem to have uncertain impacts on the level of lysine in chicory roots. Our data also indicate that the accumulation of lysine in the raw chicory and the formation of CML in the roasted ones were not influenced by genetic factors because no significant difference was found between cultivars. Although the data presented here have to be taken as preliminary, it can be concluded that foods such as coffee substitutes, which are not consumed as a major source of protein and lysine, should be prepared from raw materials low in these nutrients to lower CML levels and limit their possible adverse health effects. When the protein and lysine contents are reduced in the chicory roots, there is probably an impact on the taste, and therefore the palatability, of the end product, but this remains to be properly evaluated.

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AUTHOR INFORMATION

Corresponding Author

*(F.J.T.) Phone: 33 3 20 62 35 61. Fax: 33 3 20 62 69 93. Email: [email protected]. Funding

This work is part of the Glycachic project (GIS Genochic) and was financially supported by the European Regional Development Fund, 1.2.32422, and the Regional Councils of Picardie and Nord Pas de Calais (France), 1012010944-10945. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the competitiveness clusters IAR and NSL for their support. ABBREVIATIONS USED CML, Nε-carboxymethyl-lysine; DM, dry matter; ESI, electrospray ionization; HPLC, high-pressure liquid chromatography; LC-MS/MS, liquid chromatography−tandem mass spectrometry; MRP, Maillard reaction product; NFPA, nonafluoropentanoic acid; SRM, selected reaction monitoring mode 10301


Article

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Impact of Variety and Agronomic Factors on Crude Protein and Total

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