Eur Food Res Technol (2008) 227:117–124 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 José Contreras-Calderón · Eduardo Guerra-Hernández · Belén García-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 [4–6]. 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-Calderón · E. Guerra-Hernández (&) · B. García-Villanova Departamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja, 18012 Granada, Spain e-mail:
[email protected] 123 one whey hydrolysate low in lactose (sample 14). Sweden). USA).2 ml/min. hexane. A sample of 24 mg protein was hydrolyzed and was made up to 5 ml with water.95 M HCl were hydrolyzed at 120 °C for 23 h.1). leucine aminopeptidase (86 U/mg. potassium ferrocyanide. trichloroacetic acid (TCA). 2-furaldehyde. 16]. MA. Duplicate samples were analyzed.4). Methods HMF and furfural determination Furosine was determined following the method described by Resmini et al.4.23. centrifuged Selecta Centrolit (Barcelona. because it closely reXects changes during storage and does not decrease with storage time. USA) and Hewlett Packard model 3394A integrator (Avondale.500g for 15 min. Spain) with 20 l injection loop. 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. PA. Available lysine determination Furanic compounds were determined following a method described elsewhere [15. Xuoro-2. Thymol. tri Xuoroacetic acid. Spain). The liquid chromatographic system consisted of Konic model 500A chromatograph (Barcelona. followed by high-pressure liquid chromatography (HPLC) analysis of 50 l.118 Eur Food Res Technol (2008) 227:117–124 amount of pyrraline was proposed as a suitable parameter for evaluating the extent of advanced MR in heat-treated or long-stored foods. 3. acetic acid glacial and chloride potassium were obtained from Panreac (Barcelona. Morales (Instituto del Rio. hexamethyldisilazane. CSIC. hydroxylamine hydrochloride. Duplicate samples were analyzed. USA) prewetted with 5 ml of methanol and 10 ml of deionized water and was then eluted with 3 ml of 3 M HCl. A 0.24. one sodium caseinate (sample 10).-2. Pyrraline was eluted with 1. threhalose. USA) and a Perkin-Elmer model 235 diode array detector (Norwalk. 125 l of Carrez I [15% potassium ferrocyanide (w/v)] and 125 l of Carrez II [30% zinc acetate (w/v)].1). Spain).000 U/mg.5 ml of methanolic solution (80:20 water:methanol). Twenty microlitres of Wltered solution were separated in a reversed-phase C18 column (250 mm £ 4. Sep-Pack cartridges (C18) were purchased from Waters Millipore (Milford. Kromasil. NE. CT. furfural. zinc acetate. 3. model 235 array diode detector and model 1020 computer-integrator. Pyrraline was donated by Dr. Germany). Spain). Bohus. Approximately 0.dinitrophenyllysine (DNP-L-Lysine) HCl. and one casein hydrolysate (sample 15). adjusted to pH 6.8 g of sample was added to 4 ml of deionized water and clariWed with The -NDP-lysine was determined by HPLC.2. Alltech. three calcium caseinates (samples 7 to 9).4. Madrid. Konic model 200 UV/VIS detector (Reno. USA). myoinositol. 123 . Furosine was separated using a C8 furosine-dedicated column (250 mm £ 4. following the method applied to infant cereals by Ramirez-Jimenez et al. one soy Xour (sample 13). France). IL. EC. 20 l of sample or standard was eluted using a gradient program at Xow rate of 1. prolidase (44 U/mg.4.13. 3. Waters) at room temperature. 3. magnesium sulphate. 5-(hydroxymethyl)-furfural. MA. [17]. The HPLC system consisted of a Perkin-Elmer model 250 pump (Norwalk. with a Waters 717 autosampler.4. USA).5 ml of hydrolysate was applied to a Sep-pack C18 cartridge (Millipore.6 mm. Samples Fifteen protein ingredients were analyzed (Table 1): six whey proteins with diVerent lactose contents (samples 1 to 6).9). EC. Methanol (HPLC grade) and hydrochloric acid. furosine and pyrraline. Duplicate samples were analyzed.4-dinitrobenzene (FDNB) were purchased from Sigma-Aldrich (Madrid. EC.5 mg protein/ ml of 7. all from Perkin-Elmer. Spain) at 9.6 mm. USA) with a Waters 717 autosampler (Milford. Data were collected with a 1020 software data system Perkin-Elmer. DeerWeld. ethanol and pyridine were purchased from Merck (Darmstadt. 4 m. Pyrraline determination The enzymatic hydrolysis procedure for pyrraline evaluation followed the method of Morales and van Boekel [14]. and (2) to determine the nutritional value of these ingredients by studying their available lysine content. Pronase E (4. Furosine determination Materials and methods Chemicals All chemicals used were of analytical grade. CT. The analytical HPLC system consisted of a model 250 pump. unlike furosine [11].0–6. N.11. Furosine was obtained from Neosystem Laboratories (Strasbourg. Reversed-phase HPLC was performed in a Novapack C18 column (150 mm £ 4. Samples containing 6. two soy isolates (samples 11 and 12).4.6 mm. pepsin (10 FIP U/mg. MD. USA). The supernatant was clariWed in a prewetted Sep-Pack C18 cartridge with water. 25 § 0.7 5.53 § 0.3 4. 0.1 5.10 3.11 ND ND 60.03 0.51 n=2 ND not detected a Limit of detection.2 § 0.14 § 0.07 20. mg/100 g of protein HMFd mg/100 g of protein Ingredients Moisture (%) Whey protein 87.85 § 0.00 75.33 § 0.50 § 0.19 ND 5.4 3.75 § 0.02 ND ND ND ND ND ND 0.54 Sample 12 protein isolate 5.42 § 0.16 ND 11.15 § 0.67 § 0.33 § 0.20 – 4.83 £ 10¡4 g/100 g of protein c Limit of detection.3 § 2. 0.1 5. pyrraline.02 0.6 § 0.60 § 0.95 ND ND ND ND ND ND ND ND ND 1250 § 13 7.89 Sample 9 calcium caseinate 5.17 ND ND 22.6 § 1.7 7.61 § 0.90 Sample 5 4.06 § 0. 3.29 § 0.4 1.Table 1 Moisture.7 0.1 5.7 4.74 76.35 Soy Sample 11 protein isolate 5.01 ND ND Eur Food Res Technol (2008) 227:117–124 Sample 1 – Sample 2 6.31 ND ND ND 354 § 23 4.1 § 1.07 mg/100 g of protein b Limit of detection.8 § 0.16 1.22 § 0. sugars and HMF contents of ingredients used in manufactured infant formulas Protein (%) Galactose Glucose Lactose mg/kg Furosinea.19 0.3 § 0. protein.10 ND 1.04 0.06 41.36 § 0.58 § 0.91 § 0.7 § 2.16 § 0.87 § 0.58 § 0.57 ND ND 26.14 § 0.61 Sample 13 (Xour soy) 5.34 Sample 15 (casein) 5.0 § 0.01 2.8 76 64 30 16 13 100 90 90 85 90 90 40 82 90 1.4 6. available lysine.50 § 0.31 § 0.04 ND ND – 0.12 ND ND ND 1435 § 21 6.87 § 0.50 § 0.06 ND ND ND 464 § 7. g/100 g of protein Pyrralinec.42 40.02 ND ND ND ND ND ND 0. mg/100 g of protein Sugar content (%) Available lysineb.14 1.98 ND ND 39.54 Sample 3 4.46 Protein hydrolysates Sample 14 (whey) 7. 4.39 ND 33.45 Sample 10 sodium caseinate 6.06 ND ND ND ND ND ND ND ND 1.12 ND ND 644 § 18 5.21 § 0.8 § 1.9 mg/100 g of protein d Limit of detection.0 7.47 § 0.5 § 3.05 7.89 Sample 4 4.3 £ 10¡3 mg/100 g of protein 123 119 .73 § 0.85 § 1.30 ND ND ND 841 § 0.05 1.50 § 0.5 § 1.06 0.26 § 0.05 § 0. furosine.16 § 0.014 ND ND ND ND ND ND 60.50 Sample 6 – Caseins Sample 7 calcium caseinate – Sample 8 calcium caseinate 6. The integrator-computer used here was a 1020 Perkin-Elmer Nelson model. There was no need to purify the aqueous extract with organic solvent (trichloromethane) in this study.1% for furfural. The -DNP-lysine was determined by the external standard method. 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. Finally.diand trisaccharides were determined by the GC method of Troyano et al. The precision of the entire procedure. sample preparation and RPHPLC analysis. including acid hydrolysis. Calibration of the chromatographic system was realized by the external standard method. The detection and quantiWcation limits were realized on sample 8. 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.105 [19]. sample preparation and RP-HPLC analysis. 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 detection and quantiWcation limits were realized on the same commercial follow-up infant formula. [21] in their analysis of enteral formulas with similar ingredients. The quantiWcation limit (10£ detection limit) was 0.120 Eur Food Res Technol (2008) 227:117–124 [18]. The present results diVer from those obtained by RuWan-Henares et al. HMF values obtained in non-puriWed and in puriWed sample 3 were 0. and variation coeYcients were 2. was evaluated for a commercial follow-up infant formula (n = 8). 1 ml trichloroacetic acid (40%) was added to 1 ml of sample (2 g/ 25 ml bi-distilled water) and centrifuged at 4. because ATC forms a stable emulsion. 1 ml of the resultant mixture was added to 1 ml of internal standard (trehalose 0. 1. The curve was constructed in units of area against micrograms of pyrraline. The column used was a Chrompack CP-SIL 5 CB column (25 m £ 0. Fifty microlitres of Wltered solution were separated in a Novapack reversephase C18 HPLC column (150 mm £ 3.2% for HMF and 71. Hydrolysis of FDNB derivative was realized with HCl.5 l of the hexane layer was injected into a Perkin Elmer autosystem GC coupled to a Perkin-Elmer integrator.16 and 0.000g for 5 min.9 mm Waters) operating at room temperature. The detection limit (signal-tonoise ratio of two) was 3. Similar results were obtained when the other clarifying agent (trichloroacetic acid) was used. The detection and quantiWcation limits were realized on sample 4. 100 l hexane and 4 ml deionized water were added. The curve was constructed in units of area against milligrams/litre. Madrid. 920. The external standard method was used for the calibration. Precision of the entire available lysine procedure. The residue was dissolved in 2 ml hydroxylamine hydrochloride (5% w/v in anhydrous pyridine) and 0.03 mg/100 g of protein.05 [19]. Calibration was performed by adding increasing quantities of furosine standard.25 mm) (Sugelabor. was evaluated on a commercial follow-up infant formula (n = 8). respectively.42% for HMF and 1. Result and discussion HMF and furfural Performance of the methods Mean recovery values were 99. only Carrez can be used when a puriWcation step is included. However. within the expected concentration range. Duplicate samples were analyzed. The external standard method was used for the calibration. to a previously hydrolyzed raw milk sample. Total solids were determined by the gravimetric AOAC method no. A sample containing approximately 4 mg of protein was derivatized by addiction of FDNB.5% w/v and myoinositol 0. when an organic puriWcation step was necessary to avoid the interfering compounds generated during processing. BrieXy. The curve was constructed in units of area against micrograms of added furosine. and heating at 60 °C for 1 h. 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. Mono. The curve was constructed in units of area against milligrams/litre. 123 .15 mg/100 g of protein. 927. Recovery and detection limit of pyrraline were developed by our investigation group in enteral formulas [23] with similar protein ingredients to the present samples.23% for furfural [21]. detector 300 °C. Operating conditions were: injector 270 °C. Next. [20] with some modiWcations. and 1 ml of the supernatant was then mixed with 9 ml of ethanol–water (50:50) solution. Recovery of furosine was developed by our investigation group [22] in enteral formulas with similar protein ingredients to those in the present sample. Spain).25% w/v in methanol–water 60:40) and then evaporated under vacuum. A standard stock solution containing 744 mg/ml of furosine was used to prepare the working standard solution. After 24 h.5 g anhydrous magnesium sulphate. Additional determinations Protein was determined according to the Kjeldahl AOAC method no.3 £ 10¡3 mg/100 g of protein for both HMF and furfural. including acid hydrolysis. 1 HPLC-UV chromatogram of HMF in the sample 2 Fig. respectively). The correlation coeYcient was r2 = 1.85–33. Figure 2 shows a typical furosine chromatogram. Dogan et al. which had a similar protein content but diVerent sugar content. Analysis of the samples The furosine data (Table 1) provide an indirect measure of Amadori compounds. lactulosyllysine can be calculated as double the furosine under these study conditions [26]. The detection limit (signal-tonoise ratio of two) was 0.36–20.435 mg/100 g of protein.6 and 0. The equation for the curve was Y = 9 £ 106X ¡ 154830 (range. 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. Analysis of the samples Table 1 lists the results obtained. showed the highest furosine concentrations.221 mg/100 g of protein. The relative standard deviation was 3.8–60.1–0. The lowest concentrations were observed in protein hydrolysate (1.3 mg/kg respectively.5 mg/l). The furfural curve was not constructed because no peak was observed. 0. 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. where Y is the peak area and X is the HMF concentration (0.4% obtained on a sample with a mean furosine value of 1. with an increase after hydrolysis to 4. Sugar content was almost always higher in whey proteins than in the other ingredients (Table 1).3 and 2.551 g). and remaining samples a tenfold lower HMF content. There are few published data on the HMF content of protein ingredients. However. which may have resulted from the drying process during manufacture or inadequate storage conditions. hydrolyzed or protein isolate soy samples. r2 = 0. Furosine values ranged from 1. whey proteins hydrolysates and soy isolate proteins may be due to their low sugar content and high protein content (Table 1). hence more furosine might be generated by the same heat treatment without representing lower nutritional quality.8 mg/kg.05 to 2. explaining their greater reactivity. Nevertheless. The caseins also diVered in their furosine values. No HMF compound was found in casein. followed by soy isolates or Xour (5. The HMF could only be used as an indicator of heat damage in the whey protein samples.4–33.5 mg/100 g of protein). 5).36 to 1. Compared with sample 4. [25] found considerably higher HMF values of 12.8%.5 mg/100 g of protein.3 mg/100 g of protein).8% [22]. Furosine Performance of the methods The recovery range was 97.Eur Food Res Technol (2008) 227:117–124 121 Fig.02432–0. which were correlated with their available lysine content (Fig. which were the only ingredients to contain sugars. HMF concentrations ranged from 0. No furfural was detected in any ingredient. Figure 1 shows a representative HMF chromatogram. 123 . 2 HPLC-UV chromatogram of furosine in the sample 2 The linear regression equation used was Y = 1. which were higher with greater sugar content.0006. Thus. The absence of HMF in calcium caseinates.8 mg/100 g of protein) and caseins (22. and the highest concentrations were found in whey proteins (354 and 1.5 mg/kg in whey protein concentrates.3–99. and the mean value was 98. sample 6 was found to have greater available lysine content (Table 1). Whey proteins with the same available lysine content (samples 3 and 4) showed diVerent furosine values.4911X + 0. which might indicate a more drastic heat treatment for sample 6. respectively). sample 2 showed a fourfold lower HMF content.9984.07 mg/100 g of protein. The highest HMF value in whey protein samples was obtained in sample 4.435 mg/100 g of protein). The quantiWcation limit (10£ detection limit) was 0. Samples 1 and 2. The same behaviour was observed for the protein hydrolysates (samples 14 and 15).7 mg/100 g of protein. although both presented the highest available lysine contents. 9999. RuWan-Henares et al. Analysis of the samples Table 1 lists the available lysine content of the ingredients.1669 in whey protein. 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. with values of 1. RuWan-Henares et al.9 mg/100 g of protein [23]. Analysis of the samples Pyrraline. the correlation is almost perfect (r2 = 0. When absorbance at 420 nm was measured in the same aqueous solution as used for the HMF determination.61 to 7. 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.73 to 5.33 to 7. [23] investigated the pyrraline content of casein and whey protein model systems and only detected pyrraline in whey protein that showed furosine values >1. Fig. Morales and Jiménez-Pérez [27] studied four model systems of sodium caseínate–lactose heated at 110–150 °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.74393 £ 109X ¡ 4.69 g/100 g of protein.0112–0.91 g/100 g in casein proteins. Figure 4 shows a typical DNP-Lysine chromatogram.83 £ 10¡3 g/100 g of protein.15 g/100 g of protein in soy proteins.6% in a sample with mean available lysine of 3. Thus. The detection limit (signal-to-noise ratio of two) for adapted infant formula was 4. with a correlation coeYcient (r2) of 0.300 mg/100 g of protein for the commercial whey protein system. Figure 3 shows a typical pyrraline chromatogram. absorbance was only obtained in sample 4. The equation for the curve was Y = 3.83 £ 10¡4 g/100 g of protein. which showed furosine values of >1.50 g/100 g of protein in whey milk proteins and from 5. 3 HPLC-UV (DAD) chromatogram of pyrraline in the sample 1 Fig. 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. 123 . [28] studied furosine content in sugar–protein model systems and found the following pre-treatment baseline furosine values: 60 mg/100 g of protein for casein system. 0. If this value (sample 4) is excluded. an indicator of high heat damage (last stages of MR). The concentration range was 1–10 mg/l. r2 = 0.9990. 50 mg/100 g of protein for laboratory whey protein system and approximately 1.85 g/100 g of protein in casein hydrolysate and 4. from 4. The linear regression equation used was Y = 684331X .54610.87 g/100 g of protein in whey protein hydrolysate. available lysine content ranged from 3.45101 £ 106 (range. with values ranging from 1.9746).1119 g).000 mg/100 g of protein.85 g/100 g of protein (sample 15) to 7.87 g/100 g of protein (sample 10). Pyrraline Performance of the methods The relative standard deviation was 3. was only detected in sample 1 (Table 1).33% and the detection limit (signal-to-noise ratio >2) was 0.122 Eur Food Res Technol (2008) 227:117–124 The correlation (r2) between furosine and HMF was 0. indicating that the MR was advanced and that furosine might be decreasing (higher degradation than generation). The same protein types showed a high variability. The quantiWcation limit (10£ detection limit) was 4. 4 HPLC-UV chromatogram of DNP-lysine in the sample 2 Available lysine Performance of the methods The relative standard deviation was 4.000 mg/100 g of protein. because its preparation required virtually no heat treatment. although the percentage of lysine loss was sometimes large. García-Villanova B. The furosine content of samples was used to estimate the percentage of blocked lysine. Ferrer E. Axel AW. The percentages of blocked lysine obtained in samples 7 and 8 were approximately 1. especially in casein samples (r2 = 0. [29] and adapted by Evangelisti et al. 15th edn. Acknowledgments This work was supported by the Comisión Interministerial De Ciencia y Tecnología (Project AGL 2001 2977). Reineccius GA. and furosine can be used as an indicator of blocked lysine in these types of samples. [31] studied the early MR in a lactose– whey protein model system and reported an initial percentage of blocked lysine of approximately 4%. Morales FJ. following the formula published by Finot et al. intermediate (HMF) and advanced (pyrraline) Maillard indicators. Corzo N. Battelli G (1990) Ital J Food Sci 2:173–183 18. Romero C. Recommendations for the composition of and adapted formula. Evaluation of the nutritional value of ingredients and the heat damage they have suVered requires analysis of available lysine and of early (furosine). Romero F. Romero R (1999) Food Sci Technol Int 5:447–461 8. Farre R. Alegria A. Morales FJ. Abellan P. Hernández A (2002) J Food Sci 67:328–334 9. Montilla J (1993) J Agric Food Chem 41:1254–1255 17. Guerra-Hernández E (2004) Food Chem 85:239–244 19. Romera JM (2002b) Int J Dairy Technol 55:234–239 7. Association of OYcial Analytical Chemists. Fig. Ferrer E. In general. León C. García-Villanova B.099). Sanz MA. RuWán-Henares J. The percentages of blocked lysine obtained in samples 1 and 2 were approximately 17 and 39%. Castillo G. respectively. Arlington 20. suppl. Villamiel M (2002) Food Chem 79:513– 516 4. The Royal Society of Chemistry. Baynes JW (eds) Maillard reactions in chemistry. a larger amount of available lysine was associated with a larger amount of furosine. Serrano M. Kato H (1980) Agric Biol Chem 44:1201– 1202 11. Ingredients with large available lysine content (high nutritional value) can show a large percentage of lysine blockage (high furosine values). García-Villanova B. Morgan et al. García-Villanova B. 5 Correlation between available lysine and furosine Figure 5 shows the correlations obtained between available lysine and furosine values for individual ingredients and globally. Jiménez-Pérez S (1996) Food Chem 57:423–428 10. Acta Paediatr Scand. Mendoza MR. respectively. 262 3. Overall. Lowry KR. Pellegrino L (1994) Cereal Chem 71:254–262 14. Guerra-Hernández E. Romera J M (2002a) J Sci Food Agric 82:587–592 6. Guerra-Hernández E (2002) Food Res Int 35:527–533 Conclusions Similar ingredients from diVerent manufacturers show varied heat damage (measured as furosine and HMF) and nutritional values (measured as available lysine). [32] described similar available lysine contents to the present Wndings in model systems prepared with casein. RuWán-Henares J. Olano E (1992) Food Chem 45:41– 43 21. Pellegrino L. Hayase F. Klostermeyer HE (1993) Z Lebensm Unters Forsch 196:1–4 12. Monnier VM. Committee on Nutrition. Henle T. Klostermeyer HE (1994) In: Labuza TP. García-Villanova B. Henle T. [30] for similar samples to those in the present study (% blockage = (3. Guerra-Hernández E (2001) J Liq Chromatogr Relat Technol 24:3049–3061 22. Nakayama T. Samples with high furosine levels (samples 1 and 2) also showed high nutritional value (high available lysine content). Resmini P. Resmini P. Alegria A. Baker DH (1989) J Food Sci 54:1024–1030 2. Olano A. León C. O’Brien J. pp 195–200 13. García-Villanova B. Martínez Gómez ME. Montilla Gómez J (1992) J Liquid Chrom 15:2551–2559 16. EPSGAN (1977) European Society for Paedriatic Gastroenterology and Nutrition. Clemente G (2003) J Sci Food Agric 83:465–472 5.8/(chromatographed lysine + 1. García-Villanova B. Ramírez-Jiménez A.9 and 1%. This approach is not useful in samples with high furosine levels when the MR is advanced (presence of pyrraline). Corzo N.1 £ furosine £ 100) £ 0.996). Guerra-Hernández E. no correlation was found between available lysine and furosine (r2 = 0. laboratory whey protein and commercial whey protein.Eur Food Res Technol (2008) 227:117–124 123 highest furosine and HMF values were obtained in whey protein samples. because there would be more blocked lysine than that estimated by the formula. The 123 . Van Boeckel MAJS (1996) Neth Milk Dairy J 50:347–370 15. Troyano E. Guerra-Hernández E. A lower correlation was obtained in whey protein and soy protein.86 £ furosine). food and health. Martínez-Castro I. Abellan P. London. The authors would like to thank Richard Davies for assisting with the English version. References 1. Farre R. 1st edn. Guerra-Hernández E. AOAC (1990) OYcial methods of analysis. RuWan-Henares et al. RuWán-Henares JA. Krause R. Morales FJ. Yonn YC (2005) Milchwissenschaft 60:305– 309 25. Oral RA (2005) Milchwissenschaft 60:309–316 26. Appolonia-Nouzille C. Dogan M. Calcagno C. Jiménez-Pérez S (2000) J Agric Food Chem 48:680– 684 Eur Food Res Technol (2008) 227:117–124 28. Baechler R. Evangelista F. Oxford 5:345–355 30. García-Villanova B (2004b) Eur Food Res Technol 219:42–47 24. García-Villanova B. RuWán-Henares J. Prog Fd Nutr Sci. Knoll K. Pergamon Press.124 23. Raemy A (2005) Lait 85:315–323 32. Guerra-Hernández E (2006) Food Chem 98:685–692 123 . Guerra-Hernández E ( 2004a) J Agric Food Chem 52:5354–5358 29. García-Villanova B. RuWán-Henares J. Finot PA. Nardo S. Zunin P (1999) J Dairy Res 66:237–243 31. Jayaprakasha HM. Sienkewicz T. Bujard E (1981) In: Eriksson C (Eds). Deutsch R. Henle T (2003) Eur Food Res Technol 216:277–283 27. Guerra-Hernández E. Vautaz G. Morgan F.