Furosine in consumption milk and milk powders

Furosine in consumption milk and milk powders

ht. Dairy Journal 6 (1996) 371-382 Copyright 0 1996 Elsevier Science Limited Printed in Ireland. All rights reserved. 0958-6946/96/$15.00 ELSEVIER 09...

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ht. Dairy Journal 6 (1996) 371-382 Copyright 0 1996 Elsevier Science Limited Printed in Ireland. All rights reserved. 0958-6946/96/$15.00 ELSEVIER

0958-6946(95)00060-7

Furosine in Consumption Milk and Milk Powders

Roland Government

Van Renterghem*

& Jan De Block

Dairy Research Station, Brusselsesteenweg 370, B-9090 Melle, Belgium

(Received 20 March 1995; revised version accepted 15 September 1995)

ABSTRACT Furosine, a marker for the Maillard reaction, can be determined by a HPLC method. Concentrations of furosine in Belgian consumption milk samples were compared with contents of Italian and Spanish consumption milks reported in the literature. Further, the relation between acid-soluble /3-lactoglobulin and furosine content in different milk samples prepared by a pilot UHT-installation was verified. As expected, low concentrations of furosine were found in samples with high levels of soluble fi-lactoglobulin, but the relation was rather poor. In contrast, a good correlation was found between lactulose and furosine contents in UHT-milk and sterilized milk. Furosine determination on peroxidase-positive pasteurized milk can be used to demonstrate the addition of at Ieast 6% reconstituted milk to raw milk. Further, the furosine content of skim milk powders produced under different process conditions was studied. It was demonstrated that only under extreme conditions (holding for more than 60 s at preheating temperatures > 100°C) was the furosine formation influenced by the process conditions. Apart from these extreme conditions, furosine formation was affected mainly by the drying process and storage; the preheating conditions seemed to have little or no effect. Finally, the formation of furosine in milk powders due to storage conditions was studied. Storage temperature seemed to be the most important parameter for the formation of furosine, followed by the relative humidity. Copyright 0 1996 Elsevier Science Ltd

INTRODUCTION The thermal treatment of a sugar-protein system, such as a food, causes a nonenzymatic browning reaction, i.e. the Maillard reaction, between a reducing sugar, e.g. lactose, and a free amino group of proteins, mainly the E-amino group of lysine (O’Brien & Morrissey, 1989). The Maillard reaction, which occurs even at low temperatures, is relatively slow in high-moisture foods, but is the predo*To whom correspondence

should be addressed. 371

372

R. Van Renterghem & J. De Block

minant reaction at ambient temperatures in foods with a low moisture content, such as milk- or whey powders (Labuza & Saltmarch, 1981). During the early Maillard reaction, Schiffs bases are formed, resulting in the formation of glucosylamines and subsequently t-N-deoxylactulosyl-Llysine, the main stable Amadori compound during the early Maillard reaction. 6-N-Deoxylactulosyl-L-lysine can be partially converted by acid hydrolysis to 1972). the stable t-N-2-furoylmethyl-L-lysine, i.e. ‘furosine’ (Finot & Mauron, Furosine can be considered as an indicator of the extent of the early Maillard reaction related to the type and intensity of the food processing conditions, as well as to the storage conditions. Therefore, furosine can be used as a suitable indicator of the quality of dairy products. Brandt & Erbersdobler (1973) determined furosine in acid-hydrolysed milk products of different origin and demonstrated that it is a useful indicator of lysine damage in milk products. More recently, Resmini et al. (1990) proposed an ion-pair reversed phase HPLC method for the determination of furosine in acid-hydrolysed dairy products. The furosine index can be used as a quality parameter to identify the presence of reconstituted milk powder in raw or the presence of furosine in pasteurized milk (Resmini et al., 1992). Further, processed liquid milk samples either of commercial origin or processed under known conditions was investigated (Resmini & Pellegrino, 1992, 1994). Corzo et al. (1994a), who determined the ratio of lactulose to furosine in commercial UHT and non-fat dry milks and studied the changes in the concentrations of furosine and lactulose during storage of UHT-milk, demonstrated that the ratio of lactulose to furosine can be used as an indicator of the quality of commercial milks. In this paper, the concentrations of furosine, /I-lactoglobulin and lactulose in different milk samples obtained from the Belgian market were investigated. Further, furosine was determined in skim milk powders produced under different processing conditions and skim milk powders from different heat classes stored under different conditions in order to establish the extent to which these different heating and storage parameters influence the formation of furosine.

MATERIALS

AND

METHODS

Determination of proteins The protein

content

was determined

using the Kjeldahl

method

(IDF,

1993a).

Determination of moisture content of milk powders The moisture content of the powders was determined the Belgian standard NBN-V21-008 (NBN, 1977). Determination

gravimetrically

according

to

of lactulose

Lactulose was determined using an enzymatic method (Geier & Klostermeyer, 1983). The Boehringer Mannheim Test-Combination D-Glucose/D-Fructose was used. This test-combination can also be used to determine lactulose if combined

Furosine in consumption milk and milk powders

with with P-galactosidase, triethanolamine catalase (Boehringer Mannheim, 1989). Determination

of j&lactoglobulin

/?-Lactoglobulin (1993b).

was determined

Determination

hydrochloride,

by reversed-phase

HPLC,

3-i-3

glucose

oxidase

according

and

to IDF

of furosine

of furosine, essentially the method described by Resmini et with the exception that 50 PL of hydrolysate were injected instead of 10 pL. A standard of pure furosine was used (Neosystem Laboratoire, Strassbourg, France). On applying the above-mentioned procedure to a milk sample containing about 165 mg furosine 100 g-r protein, a coefficient of variation (n = 10) of 3.3% was obtained. The detection limit, expressed as three times the chromatographic noise obtained for a blank determination, was about 2 mg furosine 100 g-i protein. For the determination

al. (1990) was followed,

Milk samples Ten raw milk samples were obtained from dairy farms. Samples (38) of consumption milks were obtained at production plants by the Belgian National Dairy Office. They included pasteurized milk (n = 7), direct UHT-treated milk (n = 7), indirect UHT-treated milk using a plate heat-exchange system (n = 5), indirect UHT-treated milk using a tubular heat exchange system (n = 6) and inbottle sterilized milk (n = 13). Samples (20) of direct UHT-treated milk were obtained from the pilot plant of the Dairy Research Station, Melle. Heating temperature varied between 105 and 150°C and holding time between 2.5 and 20 s. Powders Skim milk powders (36) were produced in the pilot plant of the Dairy Research Station, Melle. Milk was preheated in a plate system at temperatures between 65 and 115°C in 5°C increments and passed through holding tubes for holding times of 30, 60 and 180 s, before being evaporated to 50% solids and spray dried. To avoid adsorption of moisture, the milk powders were stored in closed polyethylene bags covered with five layers of brown paper. The milk powders were stored for one year at room temperature without control of the relative humidity. Three milk powders belonging to different heat classes, namely extra-low heat, low heat and medium heat, were obtained from a local dairy factory and were used for storage experiments. Samples of each powder were stored at three different temperatures and for each temperature the powders were held at three different relative humidities. For that purpose, a 0.5 cm layer of milk powder was placed in a petri dish (0 = 85 mm) and the dishes were stored in desiccators containing saturated solutions of KCl, NaN02 or K2C03, giving a relative

374

R. Van Renterghem & J. De Block

humidity of 86, 65 or 45%, respectively. The desiccators were stored in rooms at a constant temperature of 5, 14 or 37°C. The milk powders were sampled for determination of furosine after 2, 4 and 8 weeks.

RESULTS

AND

DISCUSSION

Milk The furosine content of the 10 raw farm milk samples 100 gg’ protein. These results were similar to those Pellegrino (1992), which ranged from 3 to 5 mg 100 g-’ The. concentrations of furosine in the consumption Belgian market are presented in Fig. 1. The difference c ._ a

was between 4 and 5 mg obtained by Resmini & protein. milk samples from the in processing conditions

400-

5 ti 350UJ 8 ’

300-

g ‘G 2 2

250-

F 200

-

150

100

50

I Or

7 4 PASTEURIZED MILK N=7

qj I

1

uiir

MILK

DIRECT N=l

1 UHT

MILK

t

I UHT

MILK

INDIRECT

INDIRECT

TUBULAR

PLATE

N-6

N=S

STERILIZED MILK N-13

Fig. 1. Furosine concentrations in pasteurized milk (n = 7) UHT-milk treated by a direct system (n = 7), UHT-milk treated by an indirect tubular system (n = 6) UHT-milk treated by an indirect plate system (n = 5) and in-bottle sterilized milk (n = 13). Maximum and minimum concentrations are indicated, as well as the mean concentration (dashed line).

Furosine in consumption miIk and milk powders

375

was clearly reflected in differences in furosine content. Italian legislation limits the furosine content of peroxidase-positive pasteurized milk to 8 mg 100 gg’ protein. The aim of this legislation is to prevent the use of materials other than raw milk for the production of pasteurized drinking milk. For pasteurized milk, furosine concentrations of 4-7 mg 100 g-’ protein were found, which were in good agreement with those obtained by Resmini & Pellegrino (1992). However, for the direct UHT-milk, the furosine concentrations found ranged from 35 to 109 mg 100 g-’ protein and were significantly lower than those reported by Resmini & Pellegrino (1992), i.e. 5&170 mg furosine 100 g-’ protein. Furosine concentrations in direct UHT-milks on the Spanish market ranged from 89 to 155 mg 100 g-l protein (Corzo et al., 1994a). Since these authors expressed their results in mg L-‘, we assumed a protein concentration of 32.5 g L-’ for the calculation and comparison with the other results. Distinction was made between tubular and plate indirect UHT-systems resulting in a mean value of 180 mg furosine 100 g-’ protein for the plate system, which was slightly higher than the mean value of 168 mg furosine 100 g-’ protein for the tubular system. These furosine concentrations for indirect UHT-milks were somewhat higher than those for milks on the Spanish market (range 54263 mg furosine 100 g-’ protein, with a mean value of 135 mg furosine 100 gg’ protein) (Corzo et al., 1994a). In contrast, the furosine concentration in the indirect UHTmilk samples from the Italian market were significantly higher (150-300 mg furosine 100 g-’ protein as determined by Resmini & Pellegrino, 1992). Finallyi our results for sterilized milks ranged from 220 to 372 mg furosine 100 g protein and were lower than those reported by Resmini & Pellegrino (1992), i.e. 25&450 mg furosine 100 g-’ protein. Differences between our results and those of Resmini & Pellegrino (1992) can be explained by the fact that, although the same HPLC-method was followed, furosine was used as external standard in our case. Before it was commercially available, 2-acetylfuran was generally used as external standard, instead of furosine, since it was supposed to exhibit the same response factor (Resmini & Pellegrino, 1992). However, Hartkopf & Erbersdobler (1993) proved that the use of 2acetylfuran as external standard for HPLC led to an overestimation of furosine content of about 20%. In general, a large variation in furosine content is observed in commercial milk samples. Except for pasteurized and direct UHT-treated milk, an overlap is observed between the furosine content of milk arranged according to increasing heat intensity of the process used. In Fig. 2, the relation between acid-soluble P-lactoglobulin content and furosine content is given for milk samples processed in the pilot UHT-plant of the Dairy Research Station. As expected, at low furosine concentrations, high Blactoglobulin concentrations were found and vice versa. However, the relation was very poor, probably due to considerable differences in the kinetics of filactoglobulin denaturation (Dannenberg & Kessler, 1988) and the formation of Amadori compounds (Berg & Van Boekel, 1994). When the lactulose content of the consumption milk samples was compared with the furosine content [Fig. 3(a)], a much better correlation was obtained: [lactulose

(mg L-l)] = 3.69 [furosine

R= = 0.968.

(mg 100 g-’ protein)]-20.7

376

R. Van Renterghem & J. De Block

0

’ 0

10

I

20

I

30

I

40

I

50

I

60

mg

I

70

I

80

I

90

I

100110

I

f urosine /lOOg

I

120

I

r

130 140

protein

Fig. 2. Relation between the acid-soluble

j?-lactoglobulin content and furosine content of direct UHT-treated milk samples processed in a pilot plant. Heating temperature ranged from 105 to 150°C; holding times ranged from 2.5 to 20 s.

In Fig. 3(b), the ratio of lactulose to furosine in the consumption milk samples is presented as a function of the lactulose concentration. Also here, furosine concentration is expressed as mg 100 g-i protein since the formation of furosine, in contrast to lactulose, is highly dependent on protein concentration. Large differences were observed in pasteurized milk. The reason for this is the moderate precision of the lactulose determination, since for the pasteurized milk samples, the lactulose concentrations were of the same order as the detection limit of the enzymatic method, which is about 10 mg L-’ (De Block et al., in press). On the other hand, for the same samples less variation was observed for the furosine determinations (5-7 mg furosin 100 g-* protein; detection limit: 2 mg 100 g-’ protein). As a consequence, large differences can be detected for the ratio of lactulose to furosine in pasteurized milk samples. For this reason, the ratios for the pasteurized milk were omitted for the calcultation of the regression line. The ratio ranged from 2.8 : 1 to 4.2 : 1. With the exception of the pasteurized milk samples, this ratio was almost constant and independent of the lactulose concentration (and thus the heating conditions).

Furosine in consumption milk and milk powders

z

377

2000

0 a t; B

1500-

./-

I-

_/

4 \ E” lOOO-

500-

0

I 200

I 100

0

mg

I 300

furosine / 1OOg

protein

7-

65____________-------

_____________-------- ___..-0 noo

4-- ____________-i------AA A u A 0 A. AA A Cl 0 0 A 3AA _____________-------- ___--A _____________-------a___________-------2ll 0

I 500

0

pasteurized milk A UHT-milk (direct system) A UHT-milk (indirect system) q sterilized milk l

I

I

1000

1500 mg

/

L lactulose

Fig. 3. Lactulose and furosine content of consumption milk samples: (a) relation between the concentrations of lactulose and furosine in consumption milk samples; (b) ratio of lactulose to furosine in consumption milk samples as a function of their lactulose content. Lactulose is expressed as mg L-’ and furosine as mg 100 g-’ protein. For the calculation of the regression line, the pasteurized milk samples were omitted. The confidence limits at f2 o are represented by dashed lines.

378

R. Van Renterghem & J. De Block

These results are in good agreement with those of Corzo et al. (1994a). Assuming a protein concentration of 32.5 g L-’ for their milk, an average lactulose to furosine ratio of 3.23 : 1 for direct UHT-milk and 3.28 : 1 for indirect UHT-milk can be calculated. For sterilized milks with high lactulose concentrations, this ratio increased to an average of 3.72 : 1. Finally, it must be mentioned that conditions of time and temperature of storage will also affect the furosine and lactulose content of milk. Corzo et al. (1994b) demonstrated that for UHTtreated milk samples, a storage for 90 days at 20°C caused an increase in the concentration of furosine in the range of 8.348.6 mg 100 g-’ protein. At 30 and 40°C the increase ranged from 77 to 157 mg 100 g-’ protein and from 166 to 312 mg 100 g-’ protein, respectively. For lactulose, an increase of 20f4 mg L-’ and 95flO mg L-’ during 10 weeks was observed at 22 and 30°C respectively (De Block & Van Renterghem, 1995). Corzo et al. (1994a) suggested to use the lactulose to furosine ratio to detect the addition of reconstituted milk to UHT-milk. Since high furosine concentrations are formed during milk drying processes, Resmini & Pellegrino (1992) suggested that the addition of low levels of reconstituted milk to pasteurized milk could be detected using furosine determination. During a normal pasteurization process (peroxidase-positive/alkaline phosphatase-negative milk), an increase in furosine concentration of about l-2 mg 100 g-i protein can be expected (Resmini & Pellegrino, 1992). According to these authors and to our results, raw milk contains up to 5 mg furosine 100 g- protein. Consequently, normal pasteurized milk, without addition of reconstituted milk powder, can contain up to 7 mg furosine 100 g-’ protein (Resmini & Pellegrino, 1992; our results). This corresponds with the Italian legislation which limits the furosine content of the peroxidase-positive pasteurized milk to 8 mg furosine 100 g-’ protein. It is possible to calculate the minimum volume fraction of reconstituted milk that can be added to raw milk to obtain a pasteurized milk with a furosine concentration > 7 mg 100 gg’ protein. There are three sources of furosine in such adulterated milk: 1) 2)

3)

The furosine present in the raw milk; [Fur]nM: furosine concentration (mg/lOO g protein) in the raw milk. The furosine present in the reconstituted milk powder; [FurIMP: furosine concentration (mg/lOO g protein) of the milk powder, which is the same as the furosine concentration of the reconstituted milk powder. The furosine formed during heating in a normal pasteurization process; [FurlaT: furosine formed during pasteurization.

Adulteration can be detected when it results in a higher furosine concentration than that of a normal, unadulterated pasteurized milk; so we can use following relation: [Fur]aM(l

-X) + [Fur]MpX + [FurlaT



[Furl,,

(1)

X being the volume fraction of the reconstituted milk powder. We assume that the milk powder is reconstituted to the same protein concentration as the raw milk. In order to estimate the minimum fraction of reconstituted milk powder that could be detected before the 7 mg furosine 100 gg’ protein limit for normal pasteurized milk would be exceeded, the lowest possible furosine content for raw milk, the lowest

Furosine in consumption milk and milk powders

379

possible furosine content for reconstituted milk powder and the furosine formed during a mild pasteurization should be taken into account. The lowest furosine concentration found for raw milk is 3 mg 100 g-’ protein (Resmini & Pellegrino, 1992). At least 60 mg furosine 100 g-’ protein will be formed during the production of extra-low-heat skim milk powder (Resmini & Pellegrino, 1994) and during a very mild pasteurization process, no more than 1 mg furosine100 g-’ protein will be formed (Resmini & Pellegrino, 1994). Using these data for relation (l), the minimum fraction (X) of reconstited milk powder that could be detected will be: 3 mg 100 g-i protein (1 -X) + 60 mg 100 g-’ protein protein > 7 mg 100 g-’ protein or X > 0.053.

X + 1 mg 100 g-’

This means that a volume fraction 2 6% reconstituted milk can be detected. It must be mentioned that in most cases milk powders have a much higher furosine concentration (165338 mg 100 gg’ protein, Corzo et al., 1994a; 65 to > 500 mg 100 g-’ protein; Resmini et al., 1992). Consequentlyl in most cases much smaller concentrations of reconstituted milk in pasteurized milk can be detected. Milk powder The furosine content of the skim milk powders produced under different processing conditions using the pilot plant of the Dairy Research Station is shown in

0



I

95

I

70

I

75

I

90

I

95

preheating

Fig. 4. Furosine

I

90

I

95

temperature

I

100

I 105

I 110

I 115

of the condenser

content of skim milk powder produced in a pilot plant. The milk was evaporated to 50% solids using holding tubes of 30, 60 or 180 s and using preheating temperatures between 65 and 115°C in 5°C increments and subsequently dried. The milk powders were stored for one year at room temperature in a room without control of the relative humidity.

380

R. Van Renterghem & J. De Block RELATIVE

HUMIDITY

45 % -_--________ I35 % TEMPERATURE

- _.-.........-._... “. 36%

c ._ al

3!

EXTRA

LOW

HEAT

S’C

14QC

37 oc

A

B

C

2000-

P

0, g

1500-

\ E ‘si

loo0

500-

_

o’,

, LOW

HEAT

,

,

___---_ . ........ .‘.“.“’

./.,,,_,.~.~‘: .s.c”‘_-

‘-.-.“m,7.n.~z”

(‘,

,

D

(,

,

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8’

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,...” _....

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2000

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/)..~’ :,‘:’i /

,.n’

;

0

I

I

I

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10

20

30

40

50

days Fig. 5. Furosine content of extra-low heat powder (A, B and C), low heat powder (D, E and F) and medium heat powder (G, H and I) stored for 8 weeks at different relative humidities and 5°C (A, D and G), 14°C (B, E and H) or 37°C (C, F and I).

Furosine in consumption

milk and milk powders

381

Fig. 4. For preheating temperatures below 105°C the furosine content of the powders ranged between 170 and 300 mg 100 g-’ protein and no significant effect of preheating was observed. For these preheating conditions, furosine formation is due mainly to the drying process and storage. This agrees with the findings of Resmini & Pellegrino (1994) who demonstrated that the amount of furosine found is strongly affected by the drying process and very little by preheating. This can be explained by the fact that during drying, the water activity passes through values optimal for the Maillard reaction (Renner, 1988). In Fig. 4, it is demonstrated that under extreme conditions of preheating (temperature > 105°C and the use of holding tubes of 60 and 180 s), the furosine level is also considerably affected by the preheating conditions. However, apart from these extreme preheating conditions, preheating seemed to have little or no effect on the furosine formation. The furosine content of the three milk powders from the different heat classes and stored under different, but controlled temperature and humidity conditions, are shown in Fig. 5. The freshly prepared powders had a furosine content of lo& 120 mg furosine 100 g-i protein. Comparable results were obtained for the furosine content of the three types of powder independent of their heat class. The influence of relative humidity on the furosine content was small, but significant. The furosine content of the different powders was higher when stored at 65% relative humidity than at 45 or 85% relative humidity. This agrees with data from Loncin et al. (1965), which showed that browning and loss of lysine were highest between 55 and 75% relative humidity. The temperature at which the milk powders were stored had the greatest influence on the furosine content of the milk powders. At 45% relative humidity and at temperatures below 14°C the furosine content of the powders remained practically constant. A small but significant increase in furosine content was observed at 14°C at relative humidities of 65 and 86%. However, the furosine content of the milk powders stored at 37°C was 10 to 20 times higher than that of the milk powders stored at a lower temperature. In contrast to this, Corzo et al. (1994b) observed an increase in furosine concentration from about 6&150 to 30&380 mg 100 g-’ protein for different batches of UHTmilk during storage at 40°C for 90 days. These results demonstrate that milk powders are far more susceptible to furosine formation than UHT-treated milks.

ACKNOWLEDGEMENTS We would like to thank the Belgian National Dairy Office for the collection of the milk samples from the market. Many thanks are due to Hendrik De Ruyck for the preparation of the milk powders. This work was part of an E.U. coresponsibility project (contract no. 1116/92-3.2).

REFERENCES Berg, M. E. & Van Boekel, M. A. J. S. (1994). Degradation of lactose during heating of milk. 1 Reaction pathways. Neth. Milk Dairy J., 48, 157-175. Boehringer Mannheim (1989). Lactose/D-glucose UV-method. In Methods of Biochemical Analysis and Food Analysis using Test-combinations. Boehringer Mannheim GmbH, Mannheim pp. 84-86.

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R. Van Renterghem & J. De Block

Brandt, A. & Erbersdobler, H. (1973). Zur Bestimmung von Furosin in Nahrungsund Futtermitteln Landwirtschaft. Fors., 25, 115-l 19. Corzo, N., Delgado, T., Troyano, E. & Olano, (1994a). A. Ratio of lactulose to furosine as indicator of quality of commercial milks. J. Fd. Protect., 57, 737-739. Corzo, N., Lopez-Fandifio, R., Delgado, T., Ramos, M. & Olano, A. (1994b). Changes in furosine and proteins of UHT-treated milks stored at high ambient temperatures. Z. Lebensm. Unters. Forsch., 198, 302-306. Dannenberg, F. & Kessler, H.-G. (1988). Reaction kinetics of the denaturation of whey proteins in milk. J. Food Sci., 53, 258-263. De Block, J., Merchiers, M., Van Renterghem, R. & Moermans, R. Evaluation of two methods for the determination of lactulose in milk. Znt. Dairy J. (in press). De Block, J. & Van Renterghem, R. (1995). Onderzoek naar een nieuwe methode voor hitteklassiticatie van melk en melkpoeder met behulp van capillaire elektroforese. Report of the E.C. research program contract nr. 1116/2, Melle, Belgium, pp. 145-150. Finot, P. A. & Mauron, J. (1972). Le blockage de la lysine par la reaction de Maillard. II. Propriete chimiques des derives N-(desoxy-1-D-fructosyl-1) et N-(dCsoxy-l-D-lactulo~~1-1) de la lysine. Helv. Chirn. Acta, 55, 1153-l 164. Geier, H. Klostermeyer, H. (1983). Formation of lactulose during heat treatment of milk. Milchwissenschaft, 38, 475477. Hartkopf, J. & Erbersdobler, H. F. (1993). Stability of furosine during ion-exchange chromatography in comparison with reversed-phase high-performance liquid chromatography. J. Chromatogr., 635, 151-154. IDF (1993a). Determination of Nitrogen Content. IDF standard 20B, International Dairy Federation, Brussels. IDF (1993b). Determination of Acid Soluble /3-Lactoglobulin in Milk (Draft). IDF Questionnaire 3293. International Dairy Federation, Brussels. Labuza, T. P. & Saltmarch, M. (1981). Kinetics of browning and protein quality loss in whey powders during the steady state and nonsteady state storage conditions. J. Dairy Sci., 47, 92-l 13. Loncin, M., Jacqmain, D., Tutundjian-Provost, A. M., Lenges, J.P. & Bimbenet, J.J. (1965). Influence de l’eau sur les reactions de Maillard. C. R. Acad. Sci. Paris, 260,32083211. NBN (1977). Gravimetrische Bepaling van het Watergehalte van Melkpoeder. NBN-V21008, Belg. Best. voor Normal., Brussels. O’Brien, J. & Morrissey, P. A. (1989). Nutritional and toxicological aspects of the Maillard browning reaction in foods. Crit. Rev. Fd Sci. Nutr., 28, 21 l-250. Resmini, P. & Pellegrino, L. (1994). HPLC of furosine for evaluating Maillard reaction damage in skimmilk powders during processing and storage. Bulletin 298, International Dairy Federation, Brussels, pp. 31-36. Resmini, P. & Pellegrino, L. (1992). Analysis of food heat damage by direct HPLC of furosine. Znt. Chromat. Lab., 6, 7-11. Resmini, P., Pellegrino, L. & Battelli, G. (1990). Accurate quantification of furosine in milk and dairy products by a direct HPLC method. Ital. J. Fd. Sci., 3, 173-183. Resmini, P., Pellegrino, L., Masotti, F., Tirelli, A. & Prati, F. (1992). Determinazione de1 latte in polvere ricostituito nel latte crude ed in quell0 pastorizzato, mediante HPLC della furosina. Scienza e Tecnica Lattiero-Casearia, 43, 169-186.