The authors have declared that no competing interests exist.
Liège syrup is a Belgian traditional cooked fruit foodstuff, produced mainly from apples and pears. The process includes several hours of heating at high temperature during which complex chemical reactions occur, such as Maillard condensation between reducing sugars and amino acids. Aiming at understanding the modifications of the fruit juices during heating, different parameters were monitored throughout the process. It was shown that hydoxymethylfurfural was formed during the first step of concentration by heating. At the end of the process, hydroxymethylfurfural had totally disappeared and the deep brown color of the product suggested that this compound was transformed into melanoidins. A parallel increase in antioxidant capacity was also observed. To determine optimal conditions to reach high melanoidin content and high antioxidant capacity, different
Liège syrup (in french Sirop de Liège) is a Belgian traditional food produced from different fruits in the northeast of the province of Liège. It is not a jelly, nor jam, nor marmalade. Liège syrup is a bit like apple butter, gummy and super sweet: it is produced by reducing (boiling off the water from) a mixture of fruit juices. After several hours of long, slow cooking of apples and pears with water and sugar, the resulting product is a soft brown paste that is just barely translucent. It takes 400 g of fresh fruit to produce 100 g of Liège syrup. Besides apples and pears, dates or other fruit juices can be used as well.
This foodstuff is a mixture of compounds such as amino acids, carbohydrates, vitamins and minerals. Complex chemical reactions occur as a result of heat treatments. These include Maillard condensation between reducing sugars and amino acids, sugar browning, ascorbic acid browning and destruction of pigments
There is controversy concerning whether dietary Maillard reaction products (MRPs) represent potential harmful or beneficial effects. Some products of the Maillard reaction, such as heterocyclic aromatic amines and acrylamide are mutagenic/carcinogenic or neurotoxic. Also, certain melanoidins have negative effects on the structure of DNA and collagen, and could be involved in promoting Alzheimer disease, diabetes and cardiovascular diseases
Thermal treatments are used in the preservation of fruit products and in the manufacture of processed foods. The negative effects of these treatments include non-enzymatic browning, loss of nutrients and formation of undesirable products such as 5-hydroxymethylfurfural (HMF), an intermediate in the Maillard Reaction
HFM is formed not only from the Maillard Reaction, but also from hexoses degradation and caramelization for which the presence of amino groups is not needed. Moreover, it is one of the decomposition products of ascorbic acid. Although HMF is nearly absent in fresh and untreated foods, its concentration tends to rise during heating, so it is a useful marker of heat damage in foodstuffs.
HMF is a widespread heat-induced contaminant whose dietary intake is several orders of magnitude higher than that reported for other food toxicants such as acrylamide or furan
Based on data reported in literature, it is not clear whether human exposure to HMF represents a potential health risk
The objective of this study was to evaluate the antioxidant capacities and relationship with MRPs during the process of Liège syrup preparation. The content of HMF and antioxidant capacity of aqueous sugar (fructose and glucose) and different amino acid in
Various varieties of apples (Boskoop, Granny, Pinova, Elstar, Cox, Jonagold and Jonagored) were used for the production of Liège syrup by Siroperie Meurens (Aubel, Belgium). Sampling was done at various stages:
Homogenate: after the washing and the grinding of the apples (with skin, seeds…)
Juice: after the first cooking (3 h at 90°-95°C) and filtration (filter press) of the homogenate
Concentrate: after concentration of the juice with a concentrator (120°-140°C for 40 min)
Syrup: after mixing concentrates of various fruits at 65°C and heating at 105°C during 20 min. Sugar can be added during mixing.
The samples were diluted in water and centrifuged at 10000 g for 10 min before the various measurements.
In
Marmalades (lab preparations) were prepared by mixing and crushing fruits (10 g of apple or pear) with sucrose (6 g) and distilled water (6 mL). The mixture was heated in 50 ml disposable plastic tubes for 60 min in boiling water. In some experiments, the heating was extended to 120 min or the mixture was buffered to pH 5 or 9. In one experiment, sucrose was replaced by fructose (5 g instead of 6 g because the sweetness of fructose is higher than this of sucrose).
Total phenolics were determined according to the Folin-Ciocalteu method described by Mihalache Arion et al.
The antioxidant capacity was first determined by scavenging of the 2,2-diphenyl-1-picryhydrazyl (DPPH) radical as described by Sipel et al.
The antioxidant capacity was also determined by the ORAC assay as described by Kevers et al.
The results obtained with both assays are expressed in µmol Trolox equivalents (TE) per mL.
In a 96-well microplate, 50 µL of water, sample (appropriately diluted), or standard (HMF) were mixed with 125 µL of p-toluidine solution (10% in isopropanol). 25 µL of barbituric acid (0.5%) were added. The absorbance at 550 nm was measured with a microplate reader (Multiskan Ascent, ThermoLabsystems, Finland). Results are expressed in µg HMF per mL.
Even if this method is not specific (it can also detect the presence of aldehydes other than HMF), it allows evaluating the modifications of HMF in
Melanoidin formation was evaluated by measurement of OD at 405 nm of 150 µL samples (triplicates) in a 96-well microplate according to Echavarria et al.
The assay was done with the Thermo scientific Pierce BCA protein assay kit according to the manufacturing instructions. Albumin was used as standard.
All results presented are means (±SEM) of three independent experiments except for
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34.1 ± 2.6a | 38.9 ± 2.3a | 29.6 ± 2.4a,b | 22.9 ± 1.9b |
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571.4 ± 53.2a | 845.2 ± 36.8b | 857.1 ± 43.9b | 594.4 ± 29.2a |
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45.6 ± 2.4a | 55.1 ± 2.2b | 75.6 ± 4.9c | 45.6 ± 3.1a |
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0.448 ± 0.028a | 0.415 ± 0.014a | 0.078 ± 0.003b | 0.012 ± 0.001c |
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0a | 0a | 0.153 ± 0.011b | 0.009 ± 0.001c |
The results were reported per mL of apple homogenate. Values with different superscript letters among columns are significantly different at p<0.05 using Tukey HSD’s post test (n=5).
The data were compared by ANOVA to evaluate the significant differences between samples using Tukey HSD’s post test P<0.05.
During the process, the fruits were first washed and grinded to obtain a homogenate. Then this homogenate was cooked at 90-95°C for 3 h and then filtered. The juice so obtained was concentrated at around 130°C for 40 min. and finally, the Liège syrup was obtained after cooling and further heating to 105°C. Thus this process includes at least 4 hours of heating at temperatures between 90°C and 140°C.
The antioxidant capacity (ORAC) and the total phenolic content were higher in juice than in homogenate (
The proteins present in the homogenate drastically decreased during the concentrate preparation. Proteins are easily denatured at high temperature and partially hydrolysed in acidic conditions. The released amino acid can then be used in the Maillard reaction. Indeed, in parallel, HMF appeared in the concentrate while in syrup, the final product, the content of this compound was very low. HMF is a furanic compound which forms as an intermediate in the Maillard reaction
Aiming at understanding how antioxidant capacity was modified and how the content in HMF and melanoidins varied during the process, in vitro model systems were studied. Each consisted in mixing one amino acid with either glucose or sucrose in equimolar concentration (100 mM). The different mixtures were heated at 100°C during 180 min.
For many amino acids, heating in the presence of fructose led to higher DPPH radical scavenging activity than heating it in the presence of glucose (
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10.7 + 1.0 | 89.1 ± 1.5 |
231.7 ± 34.6 | 432.9 ± 5.3 |
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0 | 24.0 ± 1.4 |
113.7 ± 5.5 | 268.6 ± 5.6 |
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27.0 ± 4.2 | 159.0 ± 7.1 |
230.7 ± 6.9 | 291.0 ± 14.0 |
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459.2 ± 5.9 | 311.7 ± 18.8 |
4759.2 ± 128.6 | 4357.1 ± 59.6 |
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207.1 ± 2.9 | 277.7 ± 12.3 |
15799.4 ± 269.4 | 15650.6 ± 125.0 |
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0 | 41.2 ± 0.6 |
127.2 ± 2.4 | 268.5 ± 5.4 |
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0 | 27.7 ± 1.4 |
923.4 ± 87.0 | 831.7 ± 19.9 |
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117.0 ± 21.2 | 132.0 ± 10.2 | 1331.1 ± 380.1 | 1811.0 ± 154.4 |
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105.5 ± 1.9 | 122.9 ± 1.5 |
2030.9 ± 323.2 | 3678.1 ± 87.9 |
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64.3 ± 0.8 | 21.2 ± 1.8 |
597.6 ± 6.4 | 221.9 ± 7.3 |
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47.4 ± 0.5 | 33.9 ± 0.2 |
264.9 ± 2.3 | 371.4 ± 11.9 |
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910.0 ± 49.1 | 1110.0 ± 26.5 |
6354.7 ± 256.2 | 6621.5 ± 526.5 |
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0 | 28.8 ± 2.8 |
18792.4 ± 2320.7 | 21393.2 ± 794.7 |
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2815.0 ± 229.0 | 3302.5 ± 21.7 |
14281.3 ± 369.7 | 18435.0 ± 541.6 |
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1701.3 ± 5.3 | 2376.7 ± 91.4 |
425281 ± 14950 | 377195 ± 8050 |
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6293.3 ± 219.4 | 6893.3 ± 204.3 | 546364 ± 10624 | 521219 ± 2199 |
Values in the presence of fructose with are significantly different from these in the presence of glucose at p<0.05 using Tukey HSD’s post test (n=3).
The HMF content was monitored during heating of one amino acid in the presence of glucose or fructose. Different amino acids showed different behaviours. Phenylalanine yielded similar high HMF amounts in the presence of glucose or fructose (
The antioxidant capacity (DPPH assay) of the reaction mixture increased with the heating time (as illustrated for glycine, isoleucine, lysine and phenylalanine in
Increasing the pH induced an increase in the HMF content and of the absorbance at 405 nm (
Many studies have reported beneficial effects associated with MRPs, including antioxidative properties
Development of antioxidant activity due to MRPs can be influenced by the characteristics of the food matrix. Therefore we also analysed the antioxidant capacity (DPPH and ORAC), the HMF content and absorbance at 405 nm during apple or pear marmalade cooking. According to the previous results, the cooking conditions used were: 100°C during 60 and 120 min in non buffered condition, or during 60 min at pH 5 or pH 9, or during 60 min with fructose added instead of sucrose).
Cooking increased both the DPPH antioxidant capacity (
When the mixture was buffered to pH 5, the antioxidant capacity after 60 min of heating was similar to that observed in non buffered conditions (
The replacement of sucrose by fructose induced an increase of the antioxidant capacity in apple marmalade. Reducing sugars in the fruit puree, mainly glucose and fructose, participate directly in the non enzymatic browning reactions while some disaccharides, such as sucrose are less reactive because they must be first hydrolyzed during thermal treatment, leading to the formation of glucose and fructose
The results of this study may have some useful practical implications at both the food technology and nutritional levels. Melanoidins are produced during the processing and storage of foods. The antioxidant properties of melanoidins can inhibit the oxidation of unsaturated lipids and functional food ingredients, such as vitamins, polyphenols and flavonoids. Moreover, their antimicrobial activity can inhibit the growth of microorganisms
Considering that melanoidins may preserve the quality and safety of foods
the cooking times and temperature,
the pH of the preparation,
the relative proportion of fruits or the addition of amino acids and reducing sugars responsible of the formation of the MRPs (reducing sugars are more rapidly transformed in MRPs than sucrose),
the addition of plant extract containing polyphenolics. The composition in polyphenols themselves responsible of antioxidant capacity can be an important factor that affects MRP formation
Concerning the cooking time and temperature, we have also to take in account that the degradation of ascorbic acid in food is one of the major sources of furan compounds
An increase of health properties of cooked fruits is associated with a higher antioxidant capacity, a decrease in HMF and an increase of melanoidins. Some phenolic compounds and plant extracts containing phenolic compounds could be used to prevent the formation of some MRPs before thermal process applications or during storage
The process of Liège syrup production includes several hours of heating at high temperature during which complex chemical reactions occur such as Maillard condensation between amino acids and reducing sugars present in the mixture. At the end of the process, the HMF formed have totally disappeared and the deep brown color of the product is probably due to the formation of melanoidins. These compounds could also contribute to the antioxidant capacity. In apple and pear marmalade’s, an increase of the antioxidant capacity, and of HMF and melanoidins contents was observed with time at high temperature. An increased of the pH also induced an increase in antioxidant capacity in the two marmalade’s while the HMF and melanoidins contents were increased by a shift of the pH from 5 to 9 in apple but not in pear marmalade’s. However, it is known that Maillard reaction has both desirable and undesirable effects on food products. Some parameters of the processing can be modified to improve the antioxidant properties and composition in HMF and melanoidins of cooked fruits such as cooking time, temperature, pH, addition of reducing sugars or amino acids,…
We thank ‘Siroperie Meurens’ for their cooperation and M. Chenut (student at the University of Lille 1) for his technical assistance. FB gratefully acknowledges the “Ministère des technologies nouvelles” (First subvention granted to « Haute Ecole de la Province de Liège »). The skillful assistance of the APE personnel (provided to CEDEVIT by the regional government of Wallonia) was greatly appreciated.