Elucidation of mechanism of aminoreductone formation in the Maillard reaction of lactose

ORIGINALRESEARCH Elucidation of mechanism of aminoreductone formation in

the Maillard reaction of lactose

VU THU TRANG,1 TOMOKO SHIMAMURA,2 TAKEHIRO KASHIWAGI,2

HIROYUKI UKEDA2* and SHINYA KATSUNO3

1School of Biology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet, Hanoi, Vietnam,2Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan and 3Nippon Milk Community Co., Ltd, Saitama350-1165, Japan

*Author forcorrespondence. E-mail:[email protected]

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The aim of this study was to elucidate the formation mechanism of aminoreductone, an important indica-

tor for estimating the extent of Maillard reaction of lactose and amino compounds. Using the model sys-tem of lactose and butylamine, the model solution of lactose and milk proteins, and milks, it was

concluded that D-galactose was liberated at the same time as the generation of aminoreductone. It wasshown that the extent of the Maillard reaction and the lactose degradation during heating depended

closely on the concentration ratio of amino group ⁄ lactose in the sample solution.

Keywords Lactose, Galactose, Aminoreductone, Maillard reaction, Model solution and milk.

INTRODUCT ION

The Maillard reaction, a chemical reaction betweenamino groups and carbonyl groups, is a complexnetwork of chemical reactions which usually takeplace during food processing or storage (Boekel1998). Maillard reaction products are largelyresponsible for the colour, taste, flavour and thenutritional value of food products (Ramonaityt_eet al. 2009). Therefore, these products can be con-sidered as some of the most important influenceson food quality and acceptance (Rizzi 1994; Jaegeret al. 2010). In the case of milk, lactose reacts withfree amino groups of milk proteins to form anAmadori product, which is not available for diges-tion, leading to the decrease in the nutrient valuesof milk and dairy products (Finot et al. 1981; Henleet al. 1991). Therefore, the reactions of lactosehave been thoroughly investigated (Pischetsriederet al. 1998a). Many researchers have demonstratedthe chemical changes in milk during heating pro-cess (Olano and Calvo 1989). However, the evalu-ation of the extent of the Maillard reaction isdifficult because it is very complicated with manyparallel and consecutive reactions (Finot et al.1981; Morales and Jimenez-Perez 1998).Aminoreductone (AR: 1-[Ne-(Na-acetyllysinyl)]-

1,2-dehydro-1,4-deoxy-3-hexulose) was reportedthe first time by Pischetsrieder et al. (1998a) as a

product of the Maillard reaction in the heatingsolution of lactose and Na-acetyllysinyllysine. AsAR can be detected after short time heating (Pis-chetsrieder et al. 1998a), in the early stage of theMaillard reaction, it becomes a very important indi-cator for estimating the extent of Maillard reactionor the extent of heat treatment in milk. Moreover,AR was reported as an active component (Wuhret al. 2010). Many beneficial effects of AR such asan antioxidant activity (Pischetsrieder et al.1998b), a protective effect on riboflavin photo-deg-radation (Trang et al. 2008) and an antimicrobialactivity against Helicobacter pylori (Trang et al.2009) have been found. As the role and character-istics of AR were clarified, understanding themechanism of AR formation should be a veryinteresting topic for food scientists. Pischetsriederand Severin (1995), Pischetsrieder et al. (1998a)and Shimamura et al. (2002, 2004) implied thatD-galactose (Gal) might be released during theformation of AR. However, the lacking of a stoi-chiometric data on the generation of Gal in theMaillard reaction of lactose makes the mechanismof the AR formation to be not fully understood. Inaddition, one of the complications is that Gal wasalso reported as one of the main products of lactosedegradation during heating (Boekel and Berg1994; Belloque et al. 2001; Aider and Halleux2007). Although many researches investigated the

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doi: 10.1111/j.1471-0307.2010.00648.x

extent of the heat treatment in milk (Olano andCalvo 1989; Olano et al. 1989) by using thechange of Gal as an indicator, most studies did nottake into account that the noticeable amount of Galmight be formed by the Maillard reaction of lac-tose. In fact, Gal could be formed both in the Mail-lard reaction (Boekel 1998) and in the lactoseisomerisation ⁄degradation reactions (Olano andCalvo 1989; Olano et al. 1989). Thus, the reactionpathway of lactose and amino compounds duringheating can be outlined as shown in Figure 1. Asdescribed in Figure 1, in the system containingabundant amount of lactose, Gal could be formedby both the Maillard reaction and the lactose degra-dation, whereas AR formed only by the Maillardreaction. For this reason, the concentration of Gal ispredicted to be higher than that of AR. On the otherhand, in the system containing abundant amount ofamino compounds, redundant amino compoundscan also react with Gal just at the moment it wasformed leading to the formation of AR (Wuhr et al.2010). Thus, based on Figure 1, it can be postu-lated that the formation of AR and Gal closelydepends on the ratio of lactose and amino com-pound concentration in the solution before heating.As the determination of AR has been shown to

be useful in distinguishing the extent of the Mail-lard reaction in milk (Ukeda et al. 1995, 1996,1998; Shimamura et al. 2004), it is important todemonstrate the mechanism of AR formation andthe reaction network of lactose and amino com-pounds in the milk. Therefore, in this research, wefocused on the correlations between Gal and ARformation during heating by using the variousmodel solutions containing different ratios of lac-tose and butylamine concentration, model solutionof lactose and milk proteins and milks. The pur-pose of this study was to clarify the formationmechanism of AR during the Maillard reaction,and to unravel the reaction network of lactose

during the thermal treatment not only in the milkbut also in other dairy products.

MATER IALS AND METHODS

Experiment designIn this study, the model solutions consisting of lac-tose (127 mM) and various concentration of butyl-amine (0–635 mM) were used based on thehypothesis shown in Figure 1. The formations ofAR and Gal in the model solution were monitoredtogether with the increase in heating time and tem-perature. At the same heating temperature, the rela-tionships between those indices were examined inall samples. Besides investigating the mechanismof AR formation and the reaction network of lac-tose in the model solution of lactose and butyl-amine, the experiments were also applied for themodel solution of lactose and milk protein, andpractical milks (UHT milk and raw milk). All testswere performed at least in duplicate and the resultswere shown as average values.

Reagents, milk and model systemXTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrezolium-5-carboxanilide), a-lactalbumin andb-lactoglobulin were purchased from SigmaChemical Co. (St Louis, MO, USA). F-kit (Lac-tose ⁄D-Galactose) was purchased from Roche(Darmstadt, Germany). Lactose monohydrate andnutrose (Casein sodium) were obtained from Naca-lai Tesque Inc. (Kyoto, Japan). n-Butylamine wasobtained from Wako Pure Chemical Industries(Osaka, Japan). All other reagents were of the high-est grade commercially available.Milli-Qwater wasused in all procedures. The milks (raw and UHTmilk; 130�C, 2 s) were supplied by Nippon MilkCommunity Co. Ltd (Tokyo, Japan). Raw milk wastransported and stored at 4�C after collected fromhealthy cows. Soon after arriving at the laboratory(within 4 h), themilkwas used for the experiment.The solutions containing lactose and butylamine

were used as a model system of the Maillard reac-tion of lactose (model solution). The solutions wereprepared according to the previous report (Shi-mamura et al. 2004). Briefly, lactose monohydrate(127 mM) and butylamine (0–635 mM) were dis-solved in 1.28 M phosphate buffer (pH 7.0). Theconcentration of sugar was set to the value basedon that of lactose in milk (Ukeda et al. 1998).Moreover, the model solution containing lactosemonohydrate (127 mM) and butylamine (15 mM)was used as the model sample of milk system. Thebutylamine concentration was adjusted so as to be

Aminoreductone

Amino compounds

Aminoreductone

Amino compounds

Galactose

Galactose

Lactose

Isomerisation

Degradation

Epilatose

Saccharinic acid

Lactulose

Figure 1 Postulated reaction network of lactose and amino

compounds.

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almost the same amino group concentration as thatof the primary amino group of casein in milk (Uke-da et al. 1998). Besides that, milk proteins, b-lacto-globulin (0.32%), a-lactalbumin (0.12%) andcasein (2.6%) were dissolved in the solution of lac-tose monohydrate (127 mM) in 20 mM phosphatebuffer (pH 6.7) as a milk model system. The modelsolutions (1.2 mL) and milk samples were heatedunder the indicated condition. Immediately afterheating, the heated solutions were cooled on iceand used for the determination of Gal and AR.

Determination of GalMilk or milk model solution (400 lL) was pre-treated by the protein precipitation (Richardson1990) with 200 lL of Carrez I solution (3.6%potassium hexacyanoferrate) and 200 lL of CarrezII (7.2% zinc sulphate) followed by centrifugationat 2000 · g for 5 min (Tomy high speed microcentrifuge MC-150, Tomy Seiko Co. Ltd, Tokyo,Japan). The supernatant was used for the determi-nation of Gal. The concentration of Gal determinedusing an F-kit (see below) was multiplied by a fac-tor of 2 in the calculation as the milk sample wassubjected to twofold dilution in the pretreatmentprocess.As the presence of AR (absorption maximum at

320 nm) could affect the measure of NADH(absorption maximum at 340 nm) in F-kit, AR inthe 100-lL model solution of lactose and butyl-amine was oxidised completely by adding 100-lLCu2+ (5 mM) (Ukeda et al. 1998). After that, theconcentration of Gal in heated solution was identi-fied. By using Gal solution, it was also confirmedthat the addition of Cu2+ does not affect the follow-ing assay.The concentration of Gal was determined using

the F-kit (Lactose ⁄D-Galactose) according to theinstructions of the manufacturer. The principle ofthe F-kit analysis is as follows: Gal was oxidisedby NAD to D-galactonic acid in the presence ofgalactose dehydrogenase at pH 8.6. At the sametime, NAD was reduced to NADH. The concentra-tion of NADH formed was stoichiometrically equalto that of Gal. The increase in NADH was mea-sured at 340 nm (UV–Vis spectrophotometer UVmini 1240; Shimadzu, Kyoto, Japan) before andafter the addition of the galactose dehydrogenase.

XTTassay procedureThe formations of AR in the heated model solutionsand milks were determined using an XTT assay,performed in a 96-well microtitre plate according tothe method described by Ukeda et al. (1995, 1996,

1998) and Shimamura et al. (2000). Each well con-tained 60 lL of 0.5 mM XTT prepared with 0.2 Mpotassium phosphate buffer (pH 7.0) saturated withmenadione. Heated milk sample (40 lL) was addedto the well and, after mixing in a microplate shakerat a speed of 500 rpm for 15 s, the difference in theabsorbance between 492 nm and 600 nm was readon a microplate reader (MPR A4i; Tosoh, Tokyo,Japan) as the absorbance at 0 min. After 20 min atroom temperature, the difference in absorbancewas again read and the increase in the absorbancewas recorded as the ability of a sample to reduceXTT (XTT reducibility). The concentration of ARwas estimated using the following equation:y = 0.606x + 0.046, where x and y represent theconcentration of AR (mM) and the reducibility ofXTT, respectively (Trang et al. 2008).

RESULTS AND DISCUSS ION

The formation of AR and Gal in the modelsolution of lactose and butylamine duringheatingIn an attempt to elucidate the mechanism of theAR formation and the reaction network of lactoseduring heating, we tried to determine the changesin AR and Gal formations when the ratios of lac-tose ⁄amino group concentration in the model solu-tion before heating were altered. In this study, themodel solutions of lactose (127 mM) and butyl-amine in which the ratios of butylamine concentra-tion to lactose concentration were set up to theranges of 0–5 were used. While investigating thelactose reactions in milk in the range of 100–150�C, Boekel (1998) implied that the Maillardreaction was more noticeable at lower temperatures(100�C) than isomerisation ⁄degradation reactionand vice versa. On the basis of this assumption, inthis study, we tried to elucidate the mechanism ofAR formation during the Maillard reaction byusing the heating temperature at 100�C. Besidesthat, to understand the reaction network of lactoseduring heat treatment at high temperature, themodel solution of lactose and butylamine was alsoapplied to 120�C. As the AR could be detectedusing the XTT assay (Shimamura et al. 2004), theXTT reducibility and the Gal level were monitoredwhile heating the model solution at 100�C and120�C for 0–20 min.As shown in Figure 2, both the XTT reducibility

(Figure 2a), an indicator of AR formation, and Gal(Figure 2b) increased gradually in accordance withthe rise of the butylamine ⁄ lactose ratio and heatingtime. Similar observations about the increase in the

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generation of AR (Shimamura et al.1998) and Gal(Olano and Calvo 1989) during the prolongation ofthe heating time were obtained in previous reports.Although the same amount of lactose (127 mM)was contained in all model solutions, the formationof Gal much rapidly proceeded in the model sys-tem containing butylamine compared with that inthe model solution containing only lactose. It couldbe seen that if the model solution had the higheramount of butylamine, the greater Gal formationwas also found. Concretely, only low level of Galwas detected at 100�C (0.86 ± 0.07 mM after 20-min heating) in the solution containing only lac-tose, whereas a wide range of the Gal levels (from1.57 ± 0.03 mM to 26.4 ± 0.99 mM) were formedat 100�C for 20 min in the presence of butylamineat the concentration from 12.70 to 635 mM. Theformation of AR also correspondingly increasedwith the formation of Gal. These results could sup-port the hypothesis that Gal was formed in theMaillard reaction at the formation step of AR.Moreover, although the formation of Gal from

lactose solution (127 mM) was found with thehighest amount after 20-min heating (0.86 ±0.07 mM), it was quite small amount comparedwith considerable concentrations (18.07 ± 0.22 and26.4 ± 0.99 mM) of the Gal generated in themodel solutions of lactose (127 mM) and butyl-amine (317.50 and 635 mM respectively). These

observations suggested a possibility that in themodel solution containing the abundant amount ofamino compounds comparing with lactose, Galwas mainly generated in the Maillard reaction oflactose.From the literature (Boekel 1998), the degrada-

tion of lactose to form Gal proceeded more rapidlyat 120�C than at 100�C. Similar results were alsoobtained in this study. While heating lactose solu-tion in the absence of butylamine, the formation ofGal at 120�C was more noticeable (19.64 ± 0.74mM), approximately 20 times higher than at 100�Cfor 20-min heating (0.86 ± 0.07 mM) (Figure 3).As shown in Figure 3, the reactions of lactose werealso effectively enhanced by the increase in heatingtemperature; a more rapid increase in the formationof both Gal and AR at 120�C compared with at100�C was noted. The maximum amount in theXTT reducibility values were almost obtained assoon after 10 min of the heating time (Figure 3a).After that, the decreases in the XTT reducibilityand Gal were observed during the prolongation ofthe heating time at 120�C. It might be referred tothe progress of the advanced reactions of AR thatcommonly take place in the complicated sequencesof the Maillard reaction during heating (Pischets-rieder et al. 1998a). In the solution containingabundant amount of butylamine (317.50 and635 mM), there was a possibility that Gal formed

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Figure 2 XTT reducibility (a) and Gal formation (b) in the model solution of lactose and butylamine during heating at 100�C.Lactose (127 mM) and butylamine (•: 12.7 mM; h: 15 mM; s: 31.75 mM; : 63.5 mM; : 127 mM; : 317.5 mM;

*: 635 mM; -: 0 mM) in 1.28 M phosphate buffer (pH 7.0).

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during heating could also be involved in the Mail-lard reaction with the butylamine excess leading tothe formation of C4-AR (Pischetsrieder et al.1998a; Wuhr et al. 2010). As a consequence, aconsiderable formation of AR was observed in themodel solution containing butylamine at 317.50and 635 mM (Figure 3a), whereas the Gal concen-trations in those samples were less than that inother model solutions during heating at 120�C(Figure 3b). The progress of the Maillard reactionin the model solution containing very high concen-tration of butylamine (317.50 and 635 mM) wasquicker than that in the model solution containinglower concentration of butylamine. In that case, at120�C, although the model solutions containedabundant amount of butylamine, the concentrationof AR and Gal in heated samples drasticallydecreased as soon after 10 min of heating. Thoseresults indicated that the significant part of AR andGal formed had already taken part in the followingsteps of the Maillard reaction. Except for the modelsolution containing the high concentration of butyl-amine (317.50 and 635 mM), the increases in theformation of Gal and AR were accompanied by theincrease in butylamine ⁄ lactose ratio. In an attemptto elucidate the mechanism of AR formation in theearly stage of the Maillard reaction of lactose, wefocused on discovering the relation of Gal and ARin the given range of heating time in which the val-ues of Gal and AR belonged to the progressivestage.

The dependence of the AR ⁄Gal ratio on thelactose ⁄amino group concentration ratio in themodel solutions of lactose and butylamineThe changes in AR and Gal formation dependingon the change of butylamine ⁄ lactose ratio wereinvestigated. The relationships between the ARand Gal formation during heating in all sampleswere examined. The ratio of AR to Gal formation

in the heated sample could be obtained from theslope of the regression equation between those twoindices. As shown in Table 1, good linear relation-ships (r > 0.98) were found in all samples. In linewith our expectations, the slopes of the regressionequation (0.326 to 1.730 at 100�C; 0.137 to 0.764at 120�C) increased consistently with the increaseof butylamine ⁄ lactose ratio (0.1 to 5).In the model solution containing lactose and bu-

tylamine at 1:1 in concentration ratio, the amountof Gal formed was significantly higher (8.59 ±0.02 mM at 100�C, for 20 min) than the formationof Gal in the lactose model solution without thepresence of butylamine (0.86 ± 0.07 mM at 100�Cfor 20 min) (Figure 2). Moreover, the slope of theregression equation was 1.075. This result stronglysuggested that the formation of one AR moleculewas accompanied by the release of one Gal mole-cule in the Maillard reaction of lactose. That is tosay, the formation of Gal was stoichiometricallyequal to that of AR during the Maillard reaction.Although the formation of Gal by the Maillardreaction of lactose was suggested in several litera-tures (Boekel 1998; Pischetsrieder et al. 1998a,b),those studies had not elucidated its formationmechanism. Based on the obtained data, the pres-ent results clarified the generation pathway of ARand Gal in the Maillard reaction of lactose asfollows: Gal was generated at the same time withthe formation of AR in the Maillard reaction.On the basis of obtained results described above,

the reaction network in Figure 1 was clarified, andthe phenomenon about the changes of the AR ⁄Galratios depended on the butylamine ⁄ lactose ratios inthe model system was explained. In the modelsolutions containing less amount of butylamine(<127 mM) compared with lactose, the slopes ofthe regression equation were <1. It meant that theformation of Gal was higher than the formation ofAR in this model solution. This phenomenon

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Figure 3 XTT reducibility (a) and Gal formation (b) in the model solution of lactose and butylamine during heating at 120�C.Lactose (127 mM) and butylamine (•: 12.7 mM; h: 15 mM; s: 31.75 mM; : 63.5 mM; : 127 mM; : 317.5 mM;

*: 635 mM; -: 0 mM) in 1.28 M phosphate buffer (pH 7.0).

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results from the fact that excepting for forming bythe Maillard reaction pathway together with AR,Gal was also formed by the lactose isomerisa-tion ⁄degradation pathway. Gal probably reactswith butylamine in the Maillard reaction followedby the formation of C4-AR (Pischetsrieder et al.1998b; Wuhr et al. 2010). However, in the solutioncontaining a redundant amount of lactose, thequantity of this C4-AR could be negligible (Boekeland Berg 1994). On the other hand, more abundantamount of AR was found compared with Gal inthe model solution containing the higher concentra-tion of butylamine. This phenomenon could beexplained that in the model solution containing anabundant amount of butylamine (the value ofbutylamine ⁄ lactose ratio was more than 1), ARmight be produced by the Maillard reaction of lac-tose and it also could be formed by the reaction ofGal (Pischetsrieder et al. 1998b; Wuhr et al. 2010)and excess butylamine as soon after the formationof Gal. Those reasons resulted in an increase in ARformation and a decrease in Gal amount in theheated model solution, leading to an increase inthe AR ⁄Gal formation ratio. Thus, the slopes of theregression equations in the heated model system ofbutylamine ⁄ lactose at 2.5 and 5.0 in ratios of theconcentration values were 1.348 and 1.730 (morethan 1). Our results were in remarkable agreementwith the proposed reaction network in Figure 1.

Boekel (1998) attributed that the Maillard reac-tion is more noticeable at lower temperature(<100�C) than the degradation and vice versa. Inagreement with the hypothesis suggested by Boe-kel, the decrease in the slope of the regression equa-tion at 120�C is the result of a correspondingchange in the extent of reaction in which the forma-tion of Gal via the lactose degradation became moreimportant during heating at 120�C than at 100�C.Using the model systems of lactose and butyl-

amine, our study provided a new insight on themechanism of the Maillard reaction of lactose. Itwas clarified that the reaction network of the Mail-lard reaction and the lactose degradation duringheating strongly depended on the concentrationratios of amino group ⁄ lactose: the Maillard reac-tion remarkably took place in the solution contain-ing the high concentration of amino groups. Basedon our study, the researcher might predict the ten-dency of the reaction network of lactose accordingto the primary constituents.

The relationship between the formation of ARand Gal in the milk model solutions and milksamplesAlthough the reaction network of lactose andamino compounds was clarified in the modelsystems of lactose and butylamine, it was alsovery important to confirm this hypothesis by

Table 1 Relationship between Gal formation and AR formation in the model solution of lactose and butylamine during

heating

Heating

temperature (oC)

Butylamine

concentration (mM)

Ratio of amino

group ⁄ lactoseSlope of regression

equation

aCorrelation

coefficient (r)

100 635.00 5.00 1.730 0.994

317.50 2.50 1.348 0.999

127.00 1.00 1.075 0.997

63.50 0.50 0.869 0.998

31.75 0.25 0.726 0.998

15.00 0.12 0.436 0.997

12.70 0.10 0.326 0.991

0 0 0 n.d.

120 127.00 1.00 0.764 0.986

63.50 0.50 0.528 0.992

31.75 0.25 0.354 0.985

15.00 0.12 0.221 0.992

12.70 0.10 0.137 0.981

0 0 0 n.d.

Each value was obtained by establishing the regression equation of AR concentration plotted against Gal concentration

formed in the model solution at the same heating condition, in there AR and Gal concentration values represent the mean

of duplicate experiments.

n.d., not detectable; AR, aminoreductone; Gal, D-galactose.an = 3–5.

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investigating the reaction network in the practicalsamples. By considering the reaction network andthe Maillard reaction in milk, the model solution oflactose (127 mM) and butylamine (15 mM) wasused in which the concentrations of lactose and bu-tylamine were similar to the concentration of lac-tose and the concentration of the amino groups ofcasein in milk (Ukeda et al. 1995). As shown inTables 1 and 2, the slopes of the regression equa-tion in this model solution were 0.436 at 100�Cand 0.221 at 120�C. Those values indicated thatGal formed by lactose degradation pathways wasmore prominent than that formed by the Maillardreaction when the ratio of butylamine concentra-tion to lactose concentration was 0.12 (<1) in themodel solution. The results found in the currentstudy were in agreement with the data obtained byother previous studies; Gal mainly formed by thedegredation of the reducing moeity of lactose inmilk processing (Olano et al. 1989; Troyano et al.1996; Boekel 1998; Belloque et al. 2001). Adecrease in the slope value at 120�C also con-firmed that at temperature above 100�C, the degra-dation reaction was predominant than the Maillardreaction (Boekel 1998; Brands et al. 2002).The model solution of lactose and butylamine

was able to describe the course of the reaction net-work of lactose during heating. However, in foods,free amino acids occur only in lower quantities andmost of the free amino groups are present as theconstituent of proteins (Finot et al. 1981; Pischets-rieder et al. 1998a). Therefore, a model solution

of lactose and milk proteins including casein(2.6%), a-lactalbumin (0.12%) and b-lactoglobulin(0.32%) was expected to give more useful informa-tion than the simple model solution. Althoughthere were notable differences between model sys-tems and practical milk, model of milk systemswas of a great value in elucidating the mechanismsof reactions and also in suggesting the changes inmilk during heating (Olano et al. 1989). In linewith our expectation, independently of the kinds ofamino compounds, the slopes of the regressionequation between the concentration of AR and Gal(0.255, r = 0.980) (Table 2) in the milk modelsolution were not significantly different with thevalue obtained using the model solution of lactose(127 mM) and butylamine (15 mM) (0.221,r = 0.992) (Table 1) at the same heating condition(120�C).To emphasise our hypothesis, the practical milks

were used in this study. As shown in Table 2, thesimilarity in the slopes of the regression equationof AR and Gal in heated-milk model solution(0.255, r = 0.980) and heated-milks was found(0.310 in the raw milk and 0.295 in the UHT milk).The results indicated that the reaction network inmilk was also very similar to that in the model sys-tem. For this reason, the reaction network of practi-cal milk could be achieved by applying theexplanation about the reaction network of lactosein the model system. The small values in the slopeof the regression equation of both raw milk (0.310,r = 0.995) and UHT milk (0.295, r = 0.991) sup-ported the results of the previous studies that thedegradation of lactose via the Maillard reaction inheated milk is quantitatively of less importancethan the degradation route (Boekel and Berg1994). As the formation of Gal by the Maillardreaction pathway could be deduced from the for-mation of AR in heated milk, the slopes of theregression equation of both raw milk and UHTmilk (0.3 approximately in value) suggested apossibility that 30% of Gal formed in heated milkwas by the Maillard reaction pathway at 120�C.In view of the Maillard reaction, Shimamura

et al. (2004) hypothesised that Gal is formed dur-ing the formation of AR. In this study, we clarifiedthis mechanism of the lactose reaction network andprovided the stoichiometric data for the formationof Gal during the Maillard reaction by using themodel systems of lactose and butylamine. Design-ing the reaction network of lactose must be per-formed in a comprehensive way considering boththe Maillard reaction and the degradation reactions.The similar slope of the regression equation of AR

Table 2 Relationship between Gal formation and AR

formation in the milk model solution and milk sample

during heating at 120�C for 0–20 min

Sample

Slope of

regression

equation

aCorrelation

coefficient (r)bLactose and milk protein 0.255 0.980

Raw milk 0.310 0.995

UHT milk 0.295 0.991

Each value was obtained by establishing the regression

equation of AR concentration plotted against Gal

concentration at the same heating condition, in there AR

and Gal concentration values represent the mean of

duplicate experiments.an = 5.bThe model solution of lactose (127 mM) and the

mixture of three kinds of milk protein: b-lactoglobulin

(0.32%), a-lactalbumin (0.12%) and casein (2.6%) in

20 mM phosphate buffer (pH 6.7).

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and Gal was found in lactose-butylamine modelsolution and lactose-milk proteins model solution(0.221 and 0.255, respectively). This demonstratedthat the types of amino compounds did not affectthe reaction network of lactose during heating. Ourstudy permitted to estimate the tendency of theMaillard reaction and the lactose degradation in thereaction network depending on the ratio of lac-tose ⁄amino groups in the sample. The same resultsobtained in the model system and practical milkmeant that we can apply this study to explain or topredict the reaction network in other kinds of dairyproducts.

CONCLUS ION

In this study, to elucidate the formation mechanismof AR, we focused on the generation of Gal andAR while heating the model solutions of lactoseand butylamine, the milk model solutions contain-ing lactose and milk proteins and milks. The modelsolutions revealed the mechanism of AR formationduring the Maillard reaction. It could be suggestedthat the formation of Gal was correlated with thatof AR. In this study, we also reported for the firsttime that the formation of AR and Gal closelydepended on the lactose ⁄amino group ratio becauseof the competition of the Maillard reaction and thelactose degradation. On the basis of our results, theformation of Gal via the degradation pathway wasquantitatively of more importance than via theMaillard reaction when the concentration of lactosewas higher than the concentration of amino groupin the sample and vice versa. Furthermore, ourresults might provide more insight in the discoveryof those reactions in the milk and other dairy prod-ucts because it suggested a useful way to predictthe tendency of the Maillard reaction and to controlthe Maillard reaction during food processing basedon the identification of initial constituents.

R E F E R E N C E S

Aider M and Halleux D D (2007) Isomerization of lactose

and lactulose production: Review. Trends in Food Science

and Technology 18 356–364.

Belloque J, Villamiel M, Lopez-Fandino R and Olano A

(2001) Release of galactose and N-acetylglucosamine dur-

ing the storage of UHT milk. Food Chemistry 72 407–

412.

Boekel M A J S V (1998) Effect of heating on Maillard reac-

tions in milk. Food Chemistry 62 403–414.

Boekel M A J S V and Berg H E (1994) Kinetics of the early

Maillard reaction during heating of milk. In Maillard

Reactions in Chemistry, Food, and Health, pp 170–175.

Labuza T, Reineccius G, Monnier V, O’Brien J and Bay-

nes J, eds. Cambridge: Royal Society of Chemistry.

Brands C M J, Wedzicha B L and Boekel M A J S V (2002)

Quantification of melanoidin concentration in sugar-casein

systems. Journal of Agriculture and Food Chemistry 50

1178–1183.

Finot P A, Deutsch R and Bujard E (1981) The extent of the

Maillard reaction during the processing of milk. Progress

in Food and Nutrition Science 5 345–355.

Henle T, Walter H and Klostermeyer H (1991) Evaluation of

the extent of the early Maillard-reaction in milk products

by direct measurement of the Amadori-product lactulose-

lysine. International Journal of Food Research and Tech-

nology 193 119–122.

Jaeger H, Janositz A and Knorr D (2010) The Maillard reac-

tion and its control during food processing. The potential

of emerging technologies. Pathologie Biologie 58 207–

213.

Morales F J and Jimenez-Perez S (1998) Study of

hydroxymethylfurfural formation from acid degradation

of the Amadori product in milk-resembling systems.

Journal of Agricultural and Food Chemistry 46 3885–

3890.

Olano A and Calvo M M (1989) Kinetics of lactulose, galac-

tose and epilactose formation during heat-treatment of

milk. Food Chemistry 34 239–248.

Olano A, Calvo M M and Corzo N (1989) Changes in the car-

bohydrate fraction of milk during heating processes. Food

Chemistry 31 259–265.

Pischetsrieder M and Severin T (1995) The Maillard reaction

of disaccharides. In Maillard Reactions in Chemistry,

Food, and Health, pp 37–42. Labuza T, Reineccius G,

Monnier V, O’Brien J and Baynes J, eds. Cambridge:

Royal Society of Chemistry.

Pischetsrieder M, Schoetter C and Severin T (1998a) Forma-

tion of an aminoreductone during the Maillard reaction of

lactose with Na-acetyllysine or proteins. Journal of Agri-

cultural and Food Chemistry 46 928–931.

Pischetsrieder M, Rinaldi F, Gross U and Severin T (1998b)

Assessment of the antioxidative and prooxidative

activities of to aminoreductone formed during the

Maillard reaction: Effects on the oxidation of b-carotene,Na-acetylhistidine, and cis-alkenes. Journal of Agricul-

tural and Food Chemistry 46 2945–2950.

Ramonaityt_e D T, Kersien_e M, Adams A and Tehrani K A

(2009) The interaction of metal ions with Maillard reac-

tion products in a lactose-glycine model system. Food

Research International 42 331–336.

Richardson G (1990) Dairy products. In Official Methods of

Analysis of the Association of Official Analytical Chem-

ists, p. 811. Helrich K, ed. Virginia: Association of Offi-

cial Analytical Chemists.

Rizzi G P (1994) The Maillard reaction in foods. In Maillard

Reactions in Chemistry, Food, and Health, pp 11–19. La-

buza T, Reineccius G, Monnier V, O’Brien J and Baynes

J, eds. Cambridge: Royal Society of Chemistry.

Vol 64, No 2 May 2011


Shimamura T, Ukeda H and Sawamura M (2000) Reduction

of tetrazolium XTT by aminoreductone formed during the

Maillard reaction of lactose. Journal of Agricultural and

Food Chemistry 48 6227–6229.

Shimamura T, Ukeda H and Sawamura M (2002) Analysis

of Maillard reaction products in food using tetrazolium

salt XTT. Current Topics in Analytical Chemistry 3 145–

155.

Shimamura T, Ukeda H and Sawamura M (2004) Relation-

ship between the XTT reducibility and aminoreductone

formed during the Maillard reaction of lactose: the detec-

tion of aminoreductone by HPLC. Food Science and

Technology Research 10 6–9.

Trang V T, Kurogi Y, Katsuno S, Shimamura T and Ukeda H

(2008) Protective effect of aminoreductone on photo-deg-

radation of riboflavin. International Dairy Journal 18

344–348.

Trang V T, Takeuchi H, Kudo H, Aoki A, Katsuno S, Shi-

mamura T, Sugiura T and Ukeda H (2009) Antimicrobial

activity of aminoreductone against Helicobacter pylori

(H. pylori). Journal of Agricultural and Food Chemistry

57 11343–11348.

Troyano E, Villamiel M, Sanz J, Olano A and Martinez Q

(1996) Monosaccharides and myo-inositol in commercial

milks. Journal of Agricultural and Food Chemistry 44

815–817.

Ukeda H, Goto Y, Sawamura M, Kusunose H, Kamikado H

and Kamei T (1995) Reduction of Tetrazolium salt XTT

with UHT- treated milk: its relationship with the extent of

heat-treatment and storage conditions. Food Science and

Technology International 1 52–57.

Ukeda H, Goto Y, Sawamura M, Kusunose H, Kamikado H

and Kamei T (1996) A spectrophotometric microtiter-

based assay of the ability of UHT-treated milk to reduced

XTT. Food Science and Technology International 2 48–50.

Ukeda H, Shimamura T, Hosakawa T, Goto Y and Sawamura

M (1998) Monitoring of the Maillard reaction based on

the reduction of tetrazolium salt XTT. Food Science and

Technology International 4 258–263.

Wuhr A, Deckert M and Pischetsrieder M (2010) Identifica-

tion of aminoreductone as active components in Maillard

reaction mixtures inducing nuclear NF-KB translocation in

macrophages. Molecular Nutrition and Food Research 54

1021–1030.

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Elucidation of mechanism of aminoreductone formation in the Maillard reaction of lactose

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