Eur Food Res Technol (2008) 227:117124 DOI 10.

1007/s00217-007-0700-2

ORIGINAL PAPER

Indicators of non-enzymatic browning in the evaluation of heat damage of ingredient proteins used in manufactured infant formulas
Jos Contreras-Caldern Eduardo Guerra-Hernndez Beln Garca-Villanova

Received: 23 February 2007 / Revised: 6 June 2007 / Accepted: 14 June 2007 / Published online: 18 July 2007 Springer-Verlag 2007

Abstract Twelve infant formula ingredients of animal origin (caseinates, whey proteins and hydrolysates of casein and of whey proteins) and three of vegetable origin (soybean proteins) were analyzed. Furosine, hydroxymethylfurfural (HMF) and pyrraline were studied as indicators of thermal damage and available lysine as nutritional indicator, determined by HPLC in phase reverse and UV detector. The objectives were to evaluate heat damage to ingredients used in commercial infant formulas by measuring nonenzymatic browning indicators and to determine the nutritional value of these ingredients by available lysine determination. Very high furosine values were detected in whey proteins, ranging from 354 to 1,435 mg/100 g of protein. Lower furosine values were found in the remaining ingredients, ranging from 1.36 mg/100 g in hydrolyzed casein to 60.5 mg/100 g in sodium caseinate. Available lysine content ranged from 1.85 g/100 g of protein in hydrolyzed casein to 7.91 g/100 g in calcium caseinate. HMF was detected in whey protein samples between 0.16 and 2.47 mg/100 g of protein. Pyrraline was only detected in one sample of whey proteins at 41 mg/100 g of protein. Similar ingredients from diVerent manufacturers show varied heat damage and nutritional values Keywords Infant formulas Non-enzymatic browning Ingredient proteins

Introduction The most widely used proteins in commercial infant formulas are caseins, caseinates, whey proteins, protein hydrolysates and soy proteins. The treatments applied to these ingredients can be drastic, e.g., evaporation, pasteurization and drying. One of the most important modiWcations induced by heating and long storage is the Maillard reaction (MR), which involves amino acids and reducing carbohydrates and can produce a loss in nutritional value [1]. Protein ingredients used in the preparation of infant formulas must have a chemical index (lowest of the ratios between the quantity of each essential amino acid of the test protein and the quantity of each corresponding amino acid of the reference protein) 80% that of human milk, i.e., a high nutritional quality [2]. Chemical indicators to assess the quality of heat-treated foods have proven useful to monitor treatment processes and optimize manufacturing conditions. Hydroxymethylfurfural (HMF) is a recognized indicator of the deterioration produced by excessive heating or storage in a wide range of carbohydrate-containing foods [3]. Furosine determination has also been used to study early stages of the MR during the heat treatment and storage of infant formulas [46]. Available lysine content is an indicator of both early and advanced MR phases [7], and several studies have been published on lysine loss due to the heat treatment and storage of infant formulas [5, 6, 8] and model systems [9]. Pyrraline is an advanced Maillard product formed by reaction between the -amino group of lysine and 3-deoxyglucosulose, which is a degradation product of reducing carbohydrates and aminoketoses [10]. Various authors have determined this compound after total enzymatic hydrolysis of the protein, using either amino acid analysis with photodiode array [11, 12] or an RP-HPLC approach [13, 14]. The

J. Contreras-Caldern E. Guerra-Hernndez (&) B. Garca-Villanova Departamento de Nutricin y Bromatologa, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja, 18012 Granada, Spain e-mail: ejguerra@ugr.es

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amount of pyrraline was proposed as a suitable parameter for evaluating the extent of advanced MR in heat-treated or long-stored foods, because it closely reXects changes during storage and does not decrease with storage time, unlike furosine [11]. The objectives of the present study were (1) to evaluate heat damage to ingredients used in commercial infant formulas by measuring the non-enzymatic browning indicators HMF, furfural, furosine and pyrraline, and (2) to determine the nutritional value of these ingredients by studying their available lysine content.

125 l of Carrez I [15% potassium ferrocyanide (w/v)] and 125 l of Carrez II [30% zinc acetate (w/v)]. The liquid chromatographic system consisted of Konic model 500A chromatograph (Barcelona, Spain) with 20 l injection loop, Konic model 200 UV/VIS detector (Reno, NE, USA) and Hewlett Packard model 3394A integrator (Avondale, PA, USA). Twenty microlitres of Wltered solution were separated in a reversed-phase C18 column (250 mm 4.6 mm, Kromasil, Bohus, Sweden). Duplicate samples were analyzed. Furosine determination

Materials and methods Chemicals All chemicals used were of analytical grade. Methanol (HPLC grade) and hydrochloric acid, acetic acid glacial and chloride potassium were obtained from Panreac (Barcelona, Spain). Sep-Pack cartridges (C18) were purchased from Waters Millipore (Milford, MA, USA). Thymol, leucine aminopeptidase (86 U/mg, EC. 3.4.11.1), pepsin (10 FIP U/mg, EC. 3.4.23.1), prolidase (44 U/mg, 3.4.13.9), hydroxylamine hydrochloride, trichloroacetic acid (TCA), myoinositol, threhalose, hexamethyldisilazane, magnesium sulphate, N- -2,4- dinitrophenyllysine (DNP-L-Lysine) HCl, Xuoro-2,4-dinitrobenzene (FDNB) were purchased from Sigma-Aldrich (Madrid, Spain). Pronase E (4,000 U/mg, EC. 3.4.24.4), potassium ferrocyanide, zinc acetate, 5-(hydroxymethyl)-furfural, 2-furaldehyde, tri Xuoroacetic acid, hexane, ethanol and pyridine were purchased from Merck (Darmstadt, Germany). Pyrraline was donated by Dr. Morales (Instituto del Rio, CSIC, Madrid, Spain). Furosine was obtained from Neosystem Laboratories (Strasbourg, France). Samples Fifteen protein ingredients were analyzed (Table 1): six whey proteins with diVerent lactose contents (samples 1 to 6); three calcium caseinates (samples 7 to 9); one sodium caseinate (sample 10); two soy isolates (samples 11 and 12); one soy Xour (sample 13); one whey hydrolysate low in lactose (sample 14); and one casein hydrolysate (sample 15). Methods HMF and furfural determination

Furosine was determined following the method described by Resmini et al. [17]. Samples containing 6.5 mg protein/ ml of 7.95 M HCl were hydrolyzed at 120 C for 23 h. A 0.5 ml of hydrolysate was applied to a Sep-pack C18 cartridge (Millipore, MA, USA) prewetted with 5 ml of methanol and 10 ml of deionized water and was then eluted with 3 ml of 3 M HCl, followed by high-pressure liquid chromatography (HPLC) analysis of 50 l. The HPLC system consisted of a Perkin-Elmer model 250 pump (Norwalk, CT, USA) with a Waters 717 autosampler (Milford, MD, USA) and a Perkin-Elmer model 235 diode array detector (Norwalk, CT, USA). Data were collected with a 1020 software data system Perkin-Elmer. Furosine was separated using a C8 furosine-dedicated column (250 mm 4.6 mm, Alltech, DeerWeld, IL, USA). Duplicate samples were analyzed. Pyrraline determination The enzymatic hydrolysis procedure for pyrraline evaluation followed the method of Morales and van Boekel [14]. A sample of 24 mg protein was hydrolyzed and was made up to 5 ml with water, adjusted to pH 6.06.2, centrifuged Selecta Centrolit (Barcelona, Spain) at 9,500g for 15 min. The supernatant was clariWed in a prewetted Sep-Pack C18 cartridge with water. Pyrraline was eluted with 1.5 ml of methanolic solution (80:20 water:methanol). The analytical HPLC system consisted of a model 250 pump, model 235 array diode detector and model 1020 computer-integrator, all from Perkin-Elmer, with a Waters 717 autosampler. Reversed-phase HPLC was performed in a Novapack C18 column (150 mm 4.6 mm, 4 m, Waters) at room temperature; 20 l of sample or standard was eluted using a gradient program at Xow rate of 1.2 ml/min. Duplicate samples were analyzed. Available lysine determination

Furanic compounds were determined following a method described elsewhere [15, 16]. Approximately 0.8 g of sample was added to 4 ml of deionized water and clariWed with

The -NDP-lysine was determined by HPLC, following the method applied to infant cereals by Ramirez-Jimenez et al.

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Table 1 Moisture, protein, furosine, available lysine, pyrraline, sugars and HMF contents of ingredients used in manufactured infant formulas Protein (%) Galactose Glucose Lactose mg/kg Furosinea, mg/100 g of protein Sugar content (%) Available lysineb, g/100 g of protein Pyrralinec, mg/100 g of protein HMFd mg/100 g of protein

Ingredients

Moisture (%)

Whey protein 87.8 76 64 30 16 13 100 90 90 85 90 90 40 82 90 1.36 0.4 1.85 0.07 20.3 0.7 4.87 0.57 ND ND 26.7 2.4 3.73 0.19 ND 5.85 1.1 5.15 0.39 ND 33.8 1.3 4.06 0.014 ND ND ND ND ND ND 60.5 1.7 7.87 0.98 ND ND 39.6 1.4 6.50 0.17 ND ND 22.8 0.1 5.61 0.11 ND ND 60.3 2.0 7.91 0.06 ND ND ND ND ND ND ND ND 1.14 0.12 ND ND 644 18 5.58 0.31 ND ND ND 354 23 4.33 0.30 ND ND ND 841 0.1 5.31 0.06 ND ND ND 464 7.7 5.14 0.12 ND ND ND 1435 21 6.33 0.10 ND 1.75 0.16 1.58 0.16 ND 11.67 0.42 40.53 0.00 75.1 1.74 76.5 3.95 ND ND ND ND ND ND ND ND ND 1250 13 7.50 0.06 41.2 0.7 0.25 0.05 1.50 0.10 3.50 0.20 4.6 0.14 1.0 0.05 7.42 0.19 0.26 0.04 0.29 0.02 ND ND ND ND ND ND 0.21 0.04 ND ND 0.60 0.02 0.16 0.01 2.47 0.06 0.16 0.03 0.22 0.02 ND ND ND ND ND ND 0.05 0.01 ND ND

Eur Food Res Technol (2008) 227:117124

Sample 1

Sample 2

6.54

Sample 3

4.89

Sample 4

4.90

Sample 5

4.50

Sample 6

Caseins

Sample 7 calcium caseinate

Sample 8 calcium caseinate

6.89

Sample 9 calcium caseinate

5.45

Sample 10 sodium caseinate

6.35

Soy

Sample 11 protein isolate

5.54

Sample 12 protein isolate

5.61

Sample 13 (Xour soy)

5.46

Protein hydrolysates

Sample 14 (whey)

7.34

Sample 15 (casein)

5.51

n=2 ND not detected a Limit of detection, 0.07 mg/100 g of protein b Limit of detection, 4.83 104 g/100 g of protein c Limit of detection, 0.9 mg/100 g of protein d Limit of detection, 3.3 103 mg/100 g of protein

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[18]. A sample containing approximately 4 mg of protein was derivatized by addiction of FDNB. Hydrolysis of FDNB derivative was realized with HCl. The HPLC study was performed in a Perkin-Elmer 250 model with a Waters 717 automatic injector and Perkin-Elmer 235 UV diode array detector. The integrator-computer used here was a 1020 Perkin-Elmer Nelson model. Fifty microlitres of Wltered solution were separated in a Novapack reversephase C18 HPLC column (150 mm 3.9 mm Waters) operating at room temperature. Duplicate samples were analyzed. Additional determinations Protein was determined according to the Kjeldahl AOAC method no. 920.105 [19]. Total solids were determined by the gravimetric AOAC method no. 927.05 [19]. Mono- diand trisaccharides were determined by the GC method of Troyano et al. [20] with some modiWcations. BrieXy, 1 ml trichloroacetic acid (40%) was added to 1 ml of sample (2 g/ 25 ml bi-distilled water) and centrifuged at 4,000g for 5 min, and 1 ml of the supernatant was then mixed with 9 ml of ethanolwater (50:50) solution; 1 ml of the resultant mixture was added to 1 ml of internal standard (trehalose 0.5% w/v and myoinositol 0.25% w/v in methanolwater 60:40) and then evaporated under vacuum. The residue was dissolved in 2 ml hydroxylamine hydrochloride (5% w/v in anhydrous pyridine) and 0.5 g anhydrous magnesium sulphate. After 24 h, trimethylsilyl oximes of the corresponding sugars were formed in 1 ml of the previous solution by adding 1 ml hexamethyldisilazane and 100 l triXuoroacetic acid, and heating at 60 C for 1 h. Next, 100 l hexane and 4 ml deionized water were added. Finally, 1.5 l of the hexane layer was injected into a Perkin Elmer autosystem GC coupled to a Perkin-Elmer integrator. The column used was a Chrompack CP-SIL 5 CB column (25 m 0.25 mm) (Sugelabor, Madrid, Spain). Operating conditions were: injector 270 C, detector 300 C, oven at 180 C heated at 2 C/min to 206 C to elute monosaccharides and then heated at 10 C/min to 300 C and held at 300 C for 30 min to elute disaccharides and trisaccharides. Validation of the methods Recovery and precision of HMF and furfural were developed by our investigation group [21] in enteral formulas with similar protein ingredients to those in the present sample. The detection and quantiWcation limits were realized on sample 4. The external standard method was used for the calibration. The curve was constructed in units of area against milligrams/litre. Recovery of furosine was developed by our investigation group [22] in enteral formulas with similar protein ingredients to those in the present sample. The precision of the

entire procedure, including acid hydrolysis, sample preparation and RP-HPLC analysis, was evaluated for a commercial follow-up infant formula (n = 8). The detection and quantiWcation limits were realized on sample 8. Calibration of the chromatographic system was realized by the external standard method. A standard stock solution containing 744 mg/ml of furosine was used to prepare the working standard solution. Calibration was performed by adding increasing quantities of furosine standard, within the expected concentration range, to a previously hydrolyzed raw milk sample. The curve was constructed in units of area against micrograms of added furosine. Recovery and detection limit of pyrraline were developed by our investigation group in enteral formulas [23] with similar protein ingredients to the present samples. The external standard method was used for the calibration. The curve was constructed in units of area against micrograms of pyrraline. Precision of the entire available lysine procedure, including acid hydrolysis, sample preparation and RPHPLC analysis, was evaluated on a commercial follow-up infant formula (n = 8). The detection and quantiWcation limits were realized on the same commercial follow-up infant formula. The -DNP-lysine was determined by the external standard method. The curve was constructed in units of area against milligrams/litre.

Result and discussion HMF and furfural Performance of the methods Mean recovery values were 99.2% for HMF and 71.1% for furfural, and variation coeYcients were 2.42% for HMF and 1.23% for furfural [21]. The detection limit (signal-tonoise ratio of two) was 3.3 103 mg/100 g of protein for both HMF and furfural. The quantiWcation limit (10 detection limit) was 0.03 mg/100 g of protein. There was no need to purify the aqueous extract with organic solvent (trichloromethane) in this study. HMF values obtained in non-puriWed and in puriWed sample 3 were 0.16 and 0.15 mg/100 g of protein, respectively. The present results diVer from those obtained by RuWan-Henares et al. [21] in their analysis of enteral formulas with similar ingredients, when an organic puriWcation step was necessary to avoid the interfering compounds generated during processing. Similar results were obtained when the other clarifying agent (trichloroacetic acid) was used. However, only Carrez can be used when a puriWcation step is included, because ATC forms a stable emulsion.

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Fig. 1 HPLC-UV chromatogram of HMF in the sample 2

Fig. 2 HPLC-UV chromatogram of furosine in the sample 2

The linear regression equation used was Y = 1,4911X + 0.0006, where Y is the peak area and X is the HMF concentration (0.10.5 mg/l). The correlation coeYcient was r2 = 1. The furfural curve was not constructed because no peak was observed. Figure 1 shows a representative HMF chromatogram. Analysis of the samples Table 1 lists the results obtained. HMF concentrations ranged from 0.05 to 2.5 mg/100 g of protein. No HMF compound was found in casein, hydrolyzed or protein isolate soy samples. No furfural was detected in any ingredient. The highest HMF value in whey protein samples was obtained in sample 4, which may have resulted from the drying process during manufacture or inadequate storage conditions. Compared with sample 4, sample 2 showed a fourfold lower HMF content, and remaining samples a tenfold lower HMF content. The absence of HMF in calcium caseinates, whey proteins hydrolysates and soy isolate proteins may be due to their low sugar content and high protein content (Table 1). There are few published data on the HMF content of protein ingredients. Jayaprakasha and Yoon [24] determined HMF in whey protein concentrates containing 70 and 80% of protein and reported similar values to the present Wndings (1.6 and 0.8 mg/kg, respectively), with an increase after hydrolysis to 4.3 and 2.3 mg/kg respectively. However, Dogan et al. [25] found considerably higher HMF values of 12.433.5 mg/kg in whey protein concentrates. The HMF could only be used as an indicator of heat damage in the whey protein samples, which were the only ingredients to contain sugars. Furosine Performance of the methods The recovery range was 97.399.8%, and the mean value was 98.8% [22]. The relative standard deviation was 3.4%

obtained on a sample with a mean furosine value of 1,221 mg/100 g of protein. The detection limit (signal-tonoise ratio of two) was 0.07 mg/100 g of protein. The quantiWcation limit (10 detection limit) was 0.7 mg/100 g of protein. The equation for the curve was Y = 9 106X 154830 (range, 0.024320.551 g), r2 = 0.9984. Figure 2 shows a typical furosine chromatogram. Analysis of the samples The furosine data (Table 1) provide an indirect measure of Amadori compounds. Thus, lactulosyllysine can be calculated as double the furosine under these study conditions [26]. Furosine values ranged from 1.36 to 1,435 mg/100 g of protein. The lowest concentrations were observed in protein hydrolysate (1.3620.3 mg/100 g of protein), followed by soy isolates or Xour (5.8533.8 mg/100 g of protein) and caseins (22.860.5 mg/100 g of protein), and the highest concentrations were found in whey proteins (354 and 1,435 mg/100 g of protein). Sugar content was almost always higher in whey proteins than in the other ingredients (Table 1), explaining their greater reactivity. Whey protein ingredients with similar sugar and protein contents (samples 5 and 6) showed very diVerent furosine values (354 and 644 mg/100 g of protein, respectively), which might indicate a more drastic heat treatment for sample 6. Nevertheless, sample 6 was found to have greater available lysine content (Table 1); hence more furosine might be generated by the same heat treatment without representing lower nutritional quality. Whey proteins with the same available lysine content (samples 3 and 4) showed diVerent furosine values, which were higher with greater sugar content. Samples 1 and 2, which had a similar protein content but diVerent sugar content, showed the highest furosine concentrations, although both presented the highest available lysine contents. The caseins also diVered in their furosine values, which were correlated with their available lysine content (Fig. 5). The same behaviour was observed for the protein hydrolysates (samples 14 and 15).

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The correlation (r2) between furosine and HMF was 0.1669 in whey protein. When absorbance at 420 nm was measured in the same aqueous solution as used for the HMF determination, absorbance was only obtained in sample 4, indicating that the MR was advanced and that furosine might be decreasing (higher degradation than generation). If this value (sample 4) is excluded, the correlation is almost perfect (r2 = 0.9746). Furosine could not be tested as an indicator of heat damage in the present study because of the lack of information on the heat treatment of the ingredients and the diVerent sugar and lysine contents of the samples. Morales and Jimnez-Prez [27] studied four model systems of sodium casenatelactose heated at 110150 C for up to 30 min and reported similar values (30 mg/100 g of protein) to present Wndings in untreated ingredient mixtures at the beginning of their experiments. RuWan-Henares et al. [28] studied furosine content in sugarprotein model systems and found the following pre-treatment baseline furosine values: 60 mg/100 g of protein for casein system, 50 mg/100 g of protein for laboratory whey protein system and approximately 1,300 mg/100 g of protein for the commercial whey protein system. The Wndings for casein and commercial whey milk are in agreement with the present results but the furosine content of laboratory whey milk was much smaller, because its preparation required virtually no heat treatment. The measurement of HMF and A420 in whey protein may be useful to establish the stage of MR (initial or intermediate) and to determine whether furosine is increased or decreased. Pyrraline Performance of the methods The relative standard deviation was 3.33% and the detection limit (signal-to-noise ratio >2) was 0.9 mg/100 g of protein [23]. The equation for the curve was Y = 3.74393 109X 4.45101 106 (range, 0.01120.1119 g), r2 = 0.9990. Figure 3 shows a typical pyrraline chromatogram. Analysis of the samples Pyrraline, an indicator of high heat damage (last stages of MR), was only detected in sample 1 (Table 1), which showed furosine values of >1,000 mg/100 g of protein. RuWan-Henares et al. [23] investigated the pyrraline content of casein and whey protein model systems and only detected pyrraline in whey protein that showed furosine values >1,000 mg/100 g of protein.

Fig. 3 HPLC-UV (DAD) chromatogram of pyrraline in the sample 1

Fig. 4 HPLC-UV chromatogram of DNP-lysine in the sample 2

Available lysine Performance of the methods The relative standard deviation was 4.6% in a sample with mean available lysine of 3.69 g/100 g of protein. The detection limit (signal-to-noise ratio of two) for adapted infant formula was 4.83 104 g/100 g of protein. The quantiWcation limit (10 detection limit) was 4.83 103 g/100 g of protein. The linear regression equation used was Y = 684331X - 54610. The concentration range was 110 mg/l, with a correlation coeYcient (r2) of 0.9999. Figure 4 shows a typical DNP-Lysine chromatogram. Analysis of the samples Table 1 lists the available lysine content of the ingredients, with values ranging from 1.85 g/100 g of protein (sample 15) to 7.87 g/100 g of protein (sample 10). The same protein types showed a high variability. Thus, available lysine content ranged from 3.73 to 5.15 g/100 g of protein in soy proteins, from 4.33 to 7.50 g/100 g of protein in whey milk proteins and from 5.61 to 7.91 g/100 g in casein proteins, with values of 1.85 g/100 g of protein in casein hydrolysate and 4.87 g/100 g of protein in whey protein hydrolysate.

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highest furosine and HMF values were obtained in whey protein samples. Ingredients with large available lysine content (high nutritional value) can show a large percentage of lysine blockage (high furosine values). Evaluation of the nutritional value of ingredients and the heat damage they have suVered requires analysis of available lysine and of early (furosine), intermediate (HMF) and advanced (pyrraline) Maillard indicators.
Acknowledgments This work was supported by the Comisin Interministerial De Ciencia y Tecnologa (Project AGL 2001 2977). The authors would like to thank Richard Davies for assisting with the English version.

Fig. 5 Correlation between available lysine and furosine

Figure 5 shows the correlations obtained between available lysine and furosine values for individual ingredients and globally. Overall, no correlation was found between available lysine and furosine (r2 = 0.099). In general, a larger amount of available lysine was associated with a larger amount of furosine, especially in casein samples (r2 = 0.996). A lower correlation was obtained in whey protein and soy protein. Samples with high furosine levels (samples 1 and 2) also showed high nutritional value (high available lysine content), although the percentage of lysine loss was sometimes large. The furosine content of samples was used to estimate the percentage of blocked lysine, following the formula published by Finot et al. [29] and adapted by Evangelisti et al. [30] for similar samples to those in the present study (% blockage = (3.1 furosine 100) 0.8/(chromatographed lysine + 1.86 furosine). The percentages of blocked lysine obtained in samples 1 and 2 were approximately 17 and 39%, respectively. This approach is not useful in samples with high furosine levels when the MR is advanced (presence of pyrraline), because there would be more blocked lysine than that estimated by the formula. The percentages of blocked lysine obtained in samples 7 and 8 were approximately 1.9 and 1%, respectively, and furosine can be used as an indicator of blocked lysine in these types of samples. Morgan et al. [31] studied the early MR in a lactose whey protein model system and reported an initial percentage of blocked lysine of approximately 4%. RuWan-Henares et al. [32] described similar available lysine contents to the present Wndings in model systems prepared with casein, laboratory whey protein and commercial whey protein.

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Conclusions Similar ingredients from diVerent manufacturers show varied heat damage (measured as furosine and HMF) and nutritional values (measured as available lysine). The

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124 23. RuWn-Henares JA, Guerra-Hernndez E, Garca-Villanova B (2004b) Eur Food Res Technol 219:4247 24. Jayaprakasha HM, Yonn YC (2005) Milchwissenschaft 60:305 309 25. Dogan M, Sienkewicz T, Oral RA (2005) Milchwissenschaft 60:309316 26. Krause R, Knoll K, Henle T (2003) Eur Food Res Technol 216:277283 27. Morales FJ, Jimnez-Prez S (2000) J Agric Food Chem 48:680 684

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