Chemical indicators of heat treatment in fortified and special milks

Carbohydrate and furosine contents in 12 commercial fortified and special milk samples (pasteurized goat's and ewe's milks; ultrahigh-temperature (UHT) goat's milk, UHT milks fortified with calcium, magnesium, fiber, or royal jelly and honey; and lactose-hydrolyzed milks) were analyzed. Except for lactose-hydrolyzed milks, furosine, lactose, lactulose, galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, and myo-inositol contents were similar to the previously reported values for UHT or pasteurized milk samples. In lactose-hydrolyzed milks, lactulose was not detectable and lactose was present in low amount; high levels of glucose, galactose, fructose, tagatose, and furosine were also detected in this type of milk. Results found in commercial milks were compared to those obtained in laboratory-prepared UHT milks with lactose hydrolyzed prior to heating. Hydrolysis of lactose before thermal treatments promoted elevated accumulation of reducing sugars (galactose and glucose) that could be partially converted to the corresponding isomers (tagatose and fructose) during heating. In addition, the reducing sugars could also react with the amino groups of proteins, giving rise to the corresponding Amadori compound. According to the obtained results, heating prior to hydrolysis of lactose is suggested to avoid a considerable loss of available lysine.     

Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids

To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.     

Kinetics of formation of three indicators of the maillard reaction in model cookies: influence of baking temperature and type of sugar

The Maillard reaction (MR), despite its impact on flavor, color, and texture of cereal products, must be controlled for possible deleterious effects on protein nutritional quality. The present study aims to simultaneously monitor three indicators of the MR reaction (acid-released lysine, furosine, and carboxymethyllysine (CML)) by GC/MS in model cookies and evaluate the effect of formulation and baking temperature. Whereas furosine followed a bell-shape kinetic, indicative of an intermediary compound, CML linearly accumulated, proving to be a good indicator of the advanced MR. Acid-released lysine continuously decreased during baking. A reference baking level was defined to compare differently processed cookies using fluorescence synchronous spectra, highly sensitive to the dough physicochemical properties. Furosine was maximal in glucose-containing cookies, but only accounted for 5-50% lysine blockage, depending on the sugar and baking temperature. High oven temperatures and the use of fructose as the sugar source were associated with lowest the lysine damage and CML formation.     

Formation of Maillard reaction products during heat treatment of carrots

As indicators of the early stage of the Maillard reaction in carrots, N-(furoylmethyl) amino acids (FMAAs) formed during acid hydrolysis of the corresponding Amadori products were analyzed using RP-HPLC with UV detection. N(ε)-FM-Lys (furosine), FM-Gly, FM-Ala, FM-Val, FM-Ile, FM-Leu, and FM-GABA were identified using synthesized standard material by means of mass spectrometry. Furthermore, N(ε)-carboxymethyllysine (CML) and pyrraline were analyzed as indicators for advanced stages of glycation. For commercial samples with high water content, the formation of Amadori compounds predominates, whereas the advanced stage of Maillard reaction plays only a minor part. Carrot juices, baby food, and tinned carrots showed quite low rates of amino acid modification up to 5%. For dehydrated carrots, significantly higher values for Amadori products were measured, corresponding to a lysine derivatization of up to 58% and nearly 100% derivatization of GABA. Drying experiments revealed great differences in reactivity between the amino acids studied. Whereas furosine reached constant values quite quickly, some FMAAs showed a continuous increase with heating time, indicating that selected FMAAs can be used as a hallmark for the early Maillard reaction to control processing conditions.     

Ratio of maltose to maltulose and furosine as quality parameters for infant formula

Nonenzymic browning reactions in commercial infant formulas were evaluated through their furosine content as well as the isomeric disaccharides formed during processing. Lactulose was observed only in samples containing appreciable amounts of lactose, whereas maltulose was present in all samples due to the isomerization of maltose. Because formation of maltulose depends on the initial amount of maltose present, the ratio maltose/maltulose was used for comparative purposes. The ratio maltose/maltulose varied within a wide range, 27-167; therefore, low values in maltose/maltulose ratio may indicate severe processing conditions during manufacture, whereas high values may indicate mild processing conditions. Variable amounts of furosine content in samples with similar maltose/maltulose ratios may be attributed to different conditions used during storage. Levels of furosine higher than those reported for milk powder were detected in most studied samples. Determination of both furosine and maltose/maltulose ratio would yield information retrospectively about the heat treatment applied during processing and the storage conditions of commercial infant formula.     

Furosine: a suitable marker for assessing the freshness of royal jelly

Fifteen commercial samples of royal jelly, consisting of 10 imported samples, and 5 samples of known origin obtained freshly harvested from beekeepers, were analyzed for protein, lysine, and furosine content. In addition, a commercial sample of royal jelly, at the beginning of its commercial shelf life, was stored for 10 months both at 4 degrees C and at room temperature in order to assess the development of the Maillard reaction (furosine) and relative nutritional damage (blocked lysine). The commercial royal jelly products contained different amounts of furosine, ranging from 37.1 to 113.3 mg/100 g protein, evidence of different storage times and conditions. The average furosine content of the royal jelly samples of known origin and harvesting was significantly lower than that of the imported samples (41.7 versus 73.6 mg/100 g protein, respectively). With regard to shelf life, furosine content increased significantly from 72.0 mg/100 g protein to 500.8 mg/100 g protein after 10 months of storage at room temperature, while it increased to a much lower level (100.5 mg/100 g protein) when the royal jelly was stored at 4 degrees C. However, nutritional damage, expressed as blocked lysine (calculated indirectly from the furosine content), was minor or negligible, 11.9 and 2.3% of total lysine, in samples stored at room temperature and at 4 degrees C, respectively. Lysine was determined by an innovative procedure based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that furosine is a suitable index for assessing the quality and freshness of royal jelly.     

Maillard reaction and protein cross-linking in relation to the solubility of milk powders

Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.     

Storage stability assessment of freeze-dried royal jelly by furosine determination

The effect of freeze-drying and the assessment of the storage stability of freeze-dried royal jelly (RJ) were investigated by the determination of furosine and blocked lysine. The level of furosine in the RJ samples collected from cells at different times (1, 2, and 3 days after grafting) showed that the Maillard reaction had already occurred in the hive as indicated by the increase in furosine: from 9.6 to 20.8 mg/100 g of protein. Freeze-dried RJ was more prone to the early stage of the Maillard reaction than fresh RJ, as confirmed by the significantly higher furosine values found after 12 months, both at 4 degrees C (253.4 versus 54.9 mg/100 g of protein) and at room temperature (884.3 versus 332.5 mg/100 g of protein). After 18 months at room temperature, the lyophilized samples reached a furosine level of 1440.4 mg/100 g of protein, which corresponded to the blocked lysine levels, amounting to 24% of total lysine.     

Furosine as indicator of maillard reaction in jams and fruit-based infant foods

To investigate the presence of furosine in commercial samples of jams and fruit-based infant foods a simple method by ion-pair reversed-phase liquid chromatography is described. The yield of furosine during the hydrolysis with hydrochloric acid was optimized. The reproducibility in the repeatability and recovery of the method, expressed in relative standard deviation percentages, proved to be in the ranges of 4.1-8.3% and 1.0-4.4%, respectively. The recovery percentages of furosine varied between 86.7 and 95.3%. The obtained results support the suitability of the method. Furosine was detected in all studied samples. Although a high variability in the content of furosine was noticed, in general terms, the lowest levels of furosine were observed in samples of fruit-based infant foods and the highest were observed in jams of more than 60% sugar. These results could be due to different heat treatment, storage conditions, and/or differences in the values of water activity (a(w)) and amounts of sugar. The results obtained in the present paper point out the usefulness of furosine as an indicator of Maillard reaction for jams and fruit-based infant foods.     

Progress of browning reactions during storage of liquid infant milks.

Changes in furfural compounds and reactive lysine were monitorized in three commercial liquid infant milks during 9 months of storage at 20, 30, and 37 degrees C. Samples consisted of two ultrahigh temperature (UHT)-treated milks and one conventionally sterilized milk. Reactive lysine remained constant throughout storage at the three temperatures, whereas, in general an increase in furfural compounds was observed. The heat treatment used in the manufacture of milk is an important factor that influences the levels of furfural compounds although the composition of the milk is also a critical factor. Finally, a study was conducted to find the kinetic equations describing furfural compounds changes and allowing for the prediction of the influence of time and temperature of storage on those changes.     

Formation of early and advanced Maillard reaction products correlates to the ripening of cheese

The present study deals with the characterization of the ripening of cheese. A traditional German acid curd cheese was ripened under defined conditions at elevated temperature, and protein and amino acid modifications were investigated. Degree of proteolysis and analysis of early [Amadori compound furosine (6)] and advanced [N(ε)-carboxymethyllysine (4), N(ε)-carboxyethyllysine (5)] Maillard reaction products confirmed the maturation to proceed from the rind to the core of the cheese. Whereas 6 was decreased, 4 and 5 increased over time. Deeper insight into the Maillard reaction during the ripening of cheese was achieved by the determination of selected α-dicarbonyl compounds. Especially methylglyoxal (2) showed a characteristic behavior during storage of the acid curd cheese. Decrease of this reactive structure was directly correlated to the formation of 5. To extend the results of experimental ripening to commercial cheeses, different aged Gouda types were investigated. Maturation times of the samples ranged from 6 to 8 weeks (young) to more than 1 year (aged). Again, increase of 5 and decrease of 2 were able to describe the ripening of this rennet coagulated cheese. Therefore, both chemical parameters are potent markers to characterize the degree of maturation, independent of coagulation.     

Hydroxymethylfurfural and furosine reaction kinetics in tomato products

The reaction kinetics of two heat damage indices, HMF and furosine, were examined in four tomato products with different dry matter contents (10.2, 25.5, 28.6, and 34.5%) over a temperature-time range of 80-120 degrees C and 0-255 min. The reactions followed pseudo-zero order kinetics. E(a) and z-value were, respectively, 139. 9 kJ/mol and 19.2 degrees C for HMF, and 93.9 kJ/mol and 28.4 degrees C for furosine. The analyses of both indices in several samples of commercial and industrial tomato products showed very low levels of HMF (from 1 to 42 ppm) and a lack of correlation between HMF and furosine mainly because of the different evolution of the two indices during storage. The HMF level of a tomato paste sample stored at 25 degrees C decreased from 609 to 17 ppm after 98 days, while furosine increased from 458 to 550 mg/100 g of protein.     

2-Furoylmethyl amino acids and hydroxymethylfurfural as indicators of honey quality

Determination of changes in 2-furoylmethyl amino acids and hydroxymethylfurfural during the storage of four honey samples at 25 and 35 degrees C during 12 months was achieved to assess the potential use of both parameters, singly or in combination, as quality indicators. 2-Furoylmethyl amino acids increased during storage at both temperatures, whereas hydroxymethylfurfural only presented slight variations during storage at 25 degrees C but increased noticeably at 35 degrees C. The study of 2-furoylmethyl amino acids in 49 commercial honeys revealed that 2-furoylmethyl lysine (furosine) was present in all samples, whereas 2-furoylmethyl derivatives of arginine, GABA, and proline were only present in seven samples. Hydroxymethylfurfural can be considered as a good indicator of heat treatments applied to honey samples, whereas 2-furoylmethyl amino acids can be used as suitable markers of the storage period. The use of both parameters can be useful to detect adulteration with invert syrups, excessive heat treatments, or prolonged storage of honey samples.     

Chemical and physical changes in milk protein concentrate (MPC80) powder during storage

The solubility and chemical changes due to the Maillard reaction were investigated in milk protein concentrate powder containing 80% protein (MPC80) during storage at temperatures and relative humidities in the ranges of 25-40 °C and 44-84%, respectively. The Maillard reaction was studied by measuring furosine (a product of lactosylated protein after digestion with acid) and free hydroxymethylfurfural (HMF) contents by HPLC and L*, a*, b* values with a color-meter. Furosine, free HMF, and browning in MPC80 increased during storage, whereas the solubility decreased. The correlation between the Maillard reaction and solubility loss was explored in modified MPC80 to which glucose was added to enhance the rate of the Maillard reaction. More furosine and brown pigments were observed in the glucose-containing MPC80 than in MPC80 with added lactose. The opposite trend occurred for solubility, suggesting that the Maillard reaction may be a cause of solubility loss in MPC powder.     

Total chemically available (free and intrachain) lysine and furosine in pea, bean, and lentil sprouts

The effect of the germination of peas, beans, and lentils under differing conditions of illumination for different times on parameters linked to the Maillard reaction (chemically available free and intrachain lysine, lysine availability, and furosine) was evaluated. The chemically available free lysine content in the raw seeds of the three legumes was quite small compared to the chemically available intrachain lysine content, and furosine was detectable only in the beans and the lentils. The effect of germination was to increase lysine availability compared with levels in the raw seeds in all of the germinated samples, the smallest increase taking place in the lentils. In addition, furosine became detectable in all of the germinated samples. Quantities varied depending on the germination conditions but in all cases were higher than the quantities observed in the raw seeds. Linear correlations were observed to exist between some of the parameters considered in the three legumes tested.     

Browning indicators in bread

Bread is the most important food in the Spanish household and represents the largest proportion of products produced by commercial bakeries. The browning indicators furosine, hydroxymethylfurfural (HMF), and color were determined to evaluate heat effects induced during manufacture of these foods. The breads analyzed were common, special, sliced toasted, and snack breads. Identical sample preparation and HPLC conditions were used to determine HMF in all breads. The precision tested at high and low HMF concentration in breads was 2.60% and 1.57%, respectively. Recovery of HMF was 96.2%. The HMF values ranged from 2.2 to 68.8 mg/kg. Color index (100 - L) ranged from 17.0 to 38.2. The linear correlations (r(2)) between 100 - L/HMF were above 0.70 for common, special, and snack breads. Similar correlation was obtained between 100 - L/HMF in a dough baking at different times. The furosine content in common bread ranged between 125 and 208 mg/100 g of protein. No linear correlation was found between furosine and HMF. Moreover, HMF and furosine were also determined in crumb and crust. Levels of HMF had a wide range (0.9-1.76 mg/kg) and furosine was between 43 and 221 mg/100 g of protein.     

Generation of furosine and color in infant/enteral formula-resembling systems

The extent of the Maillard reaction was studied by measuring furosine and color formation in infant and enteral formula-resembling model systems prepared by mixing calcium caseinate, laboratory-obtained or commercial whey protein with lactose or dextrinomaltose (ingredients similar to those used in infant and enteral formula manufacture) and heating the mixture at 100, 120, or 140 degrees C for 0-30 min. The furosine determination was performed by HPLC and the color determination by measuring colorimetric parameters L, a, and b in a reflection photometer. The first steps of the Maillard reaction could be followed by furosine determination when initial ingredients had low thermal damage. Hence, furosine may be an indicator of low thermal damage in ingredients with <100 mg/100 g of protein. At the concentrations used in these model systems, similar to those in infant and enteral formulas, furosine values (indirect measure of lysine losses) were higher in lactose than in dextrinomaltose systems, in which only glucose, maltose, maltotriose, and maltotetraose among all of the sugars present showed reactivity with casein. Finally, the advanced steps could be followed by color determination when the initial ingredients had high thermal damage or the model systems were heated at high temperature or for a long time. Among the parameters assayed, b was the most sensitive.     

Characterization and functional properties of lactosyl caseinomacropeptide conjugates

Ovine caseinomacropeptide (CMP) was modified with lactose through Maillard reaction under 44% relative humidity and 40 degrees C for various periods (0-11 days). Different lactosylated CMP forms were separated by capillary electrophoresis and reversed phase high-performance liquid chromatography (RP-HPLC) and identified by RP-HPLC coupled with electrospray ionization mass spectrometry (ESI-MS). Around 55-60% of CMP was lactosylated under the conditions assayed, with the monolactosylated form being the most abundant one, followed by the di-, tri-, and tetralactosylated species. During the first days of incubation amino acid analyses showed a decrease in lysine and NH(2)-terminal methionine, which coincided with an increase in the furosine content. However, from the ninth day of incubation, further degradation of Amadori compounds prevailed over their formation. Solubility, heat stability, and emulsifying capacity of the native and modified CMP were investigated. Lactosylation improved the emulsifying activity, but it did not modify the great solubility and heat stability of native CMP.     

Effect of malondialdehyde on the determination of furosine in milk-based products

For nutritional purposes the recombination of milk or milk proteins with polyunsaturated fatty acids had long been considered in newly designed products, such as functional foods. Four milk-resembling model systems were prepared having malondialdehyde content ups to 1 mM. Systems were heated between 110 and 150 degrees C for up to 30 min. The effect of the presence of bifunctional aldehydes on the determination of furosine as a heat-induced marker was investigated. Levels of 0.01 mM MAD in the reaction mixture could affect significantly the determination of furosine in a milk-based system. Impairment of lysyl residues through the analysis of furosine could be underestimated in the presence of malondialdehyde.     

Nonenzymatic browning of lactose and caseinate during dry heating at different relative humidities

Dry mixtures of lactose and caseinate were heated at 60 degrees C for up to 96 h at different relative humidities (RHs) ranging from 29 to 95%. The resulting nonenzymatic browning was studied by determining lactulosyl lysine formation in the caseinate (as measured by the conversion to furosine), amount of reacted lactose, loss of lysine, color formation, and fluorescent intensity. For each measurement, the maximum reaction occurred at intermediate RHs. While there is general agreement between the results obtained by different methods, discrepancies are understandable given the complex nature of nonenzymatic browning. It was shown that the degradation of the Amadori product, lactulosyl lysine, increased with RH. Moreover, the Maillard reaction, as opposed to caramelization of lactose, was the major pathway at all RHs. Visible browning occurred when the destruction of Amadori product became dominant, and interactions between sugar fragments and caseinate were not the rate-limiting steps in the nonenzymatic browning.