THE INFLUENCE OF APPLIED HEAT TREATMENTS ON WHEY PROTEIN DENATURATION

Reconstituted skim milk with 8.01% DM was standardized with 3% skim milk powder and with 3% demineralized whey powder (DWP), respectively. Gained milk samples are named as 8%, 11% and 8%+3%DWP. All samples were heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min, respectively. Untreated milk was used as control. Milk samples were coagulated by glucono-δ-lactone (GDL) at the temperature of 45oC until pH 4.60 was reached. Milk nitrogen matter content decreased during heat treatments, but linear relationship between applied heat treatments and nitrogen matter decreasing was not found. Nitrogen matter content of sera gained from both untreated and heat treated milk increased with the increase of milk dry matter content and with the addition of DWP. The higher temperature of applied heat treatment, the smaller sera nitrogen matter content. Nitrogen matter content in sera obtained from untreated milk were 64.90 mg%, 96.80 mg% and 117.3 mg% for milk 8%, 11% and 8%+3.0% DWP, respectively. Sera samples obtained from milk 8% heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min had 38.70 mg%, 38.30 mg% and 37.20 mg% of nitrogen matter, respectively. Sera samples obtained from milk 11% heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min had 55.90 mg%, 52.80 mg% and 51.30 mg% of nitrogen matter, respectively. Sera samples obtained from milk 8% heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min had 69.50 mg%, 66.20 mg% and 66.00 mg% of nitrogen matter, respectively. 1 Safet Fetahagić, B.Sc., PKB IMLEK, Zrenjaninski put bb, 11213 Padinska Skela, FR Yugoslavia 2 Ognjen Maćej, PhD., Professor, Jelena Denin-Djurdjević, M.Sc., Research Associate, Snežana Jovanović, PhD., Assistant Professor, Department of Food Technology and Biochemistry, Faculty of Agriculture, 11081 BelgradeZemun, Nemanjina 6, FR Yugoslavia S. Fetahagić et al. ___________________________________________________________________________________________ 206 Distribution of nitrogen matter from untreated milk to milk sera were 12.01%, 11.14% and 17.69% for milk 8%, 11% and 8%+3.0% DWP, respectively. Distribution of nitrogen matter from milk 8% heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min to sera samples were 6.99%, 6.72% and 6.59%, respectively. Distribution of nitrogen matter from milk 11% heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min to sera samples, were 6.02%, 5.32% and 5.21%, respectively. Distribution of nitrogen matter from milk 8%+3%DWP heat treated at 85oC/10 min, 90oC/10 min and 95oC/10 min to sera samples were 9.64%, 8.66% and 8.67%, respectively. Whey protein denaturation increased with increasing of the temperature of the applied heat treatment. Denaturation was the most significant for milk sample 11%.

Whey protein denaturation increased with increasing of the temperature of the applied heat treatment.Denaturation was the most significant for milk sample 11%.

I n t r o d u c t i o n
Several investigations showed that during heating of milk at temperatures above 70ºC whey protein denatured.The mechanism of whey proteins denaturation involves unfolding of polypeptide chains and exposition of buried thiol groups.Newly formed thiol groups become reactive enough to react with aecasein and form complex, namely coaggregates of milk proteins via thiol/disulphide interaction and intermolecular disulphide bridges (D a l g l e i s h , 1990, J a n g and S w a i s g o o d , 1990, M a ć e j , 1983, 1989, O l d f i e l d et al., 1998, S m i t s and v a n B r o u w e r s h a v e n , 1980, T e s s i e r and R o s e , 1964 ).During heating of milk at 90ºC, one thiol group/mol polypeptide chain of ßlactoglobulin becomes exposed.However, at temperatures higher than 100ºC thiol groups become either masked due to self-aggregation or transformed in intramolecular disulphide bridges formed by oxidation processes (M a ć e j , 1983, 1989, O l d f i e l d et al., 1998).It is assumed that during heating of milk, the following complexes could be formed: complex between α-lactalbumin and ßlactoglobulin; α-lactalbumin and ae-casein; ß-lactoglobulin and ae-casein; as well as complex between α-lactalbumin, ß-lactoglobulin and ae-casein (E l f a g m and W h e e l o c k , 1977, 1978a, 1978b Calcium ions influence protein association through formation of calcium bridges with ionic protein groups, which results in decreasing of intermolecular repulsion and formation of intermolecular hydrophobic bonds (S m i t s and v a n B r o u w e r s h a v e n , 1980).Hydrophobic, noncovalent interactions have significant role at temperature of 70ºC and during first few minutes of heating (H a q u e and K i n s e l l a , 1988, H a v e a et al., 1998, J a n g and S w a i s g o o d , 1990, S m i t s and v a n B r o u w e r s h a v e n , 1980 ).
Earlier investigations showed that heat-induced complex of whey proteins exist either attached at micellar surface or inside of micelle.By comparing micellar surface in untreated and heat treated milk, it is noticeable that newes formed surface is ragged with numerous filaments that protrude to sera.According to D a v i e s et al., 1978, casein and whey proteins do not form complex if milk has been heated to 90ºC and then immediately cooled.The surface of micelle remains smooth and appears as surface of micelle in untreated milk (D a v i e s et al., 1978, K a l a b et al., 1983).The temperature and time required for exchanges to take place are identical to those required for whey protein denaturation.The filaments composed of denatured ß-lactoglobulin are formed on the surface of casein micelle after heating of milk at 95ºC during 10 min, or after autoclaving at 121.7ºC during 15 min (D a v i e s et al., 1978, S m i t s and v a n B r o u w e r s h a v e n , 1980 ).
The rate and degree of whey proteins denaturation and interaction with casein micelle depend on processing conditions and temperature of heat treatments (C o r r e d i g and D a l g l e i s h , 1996).An increase of temperature or a decrease of milk pH lead to faster interaction between casein and whey proteins.At the temperature between 70-90ºC, interaction kinetics of both ß-lactoglobulin and αlactalbumin with ae-casein are similar, which indicates that complex formed between ß-lactoglobulin and α-lactalbumin participates in the formation of coaggregates (C o r r e d i g and D a l g l e i s h , 1996).Degree of interaction of whey proteins and casein micelles increases with time and temperature in region between 75-90ºC.The amount of whey proteins that reacts with casein micelle increases to the defined, maximal value, while higher temperatures induce faster protein-protein interaction (C o r r e d i g and D a l g l e i s h , 1999).According to the results of O l d f i e l d et al., 1998, although the amount of whey protein associated with casein micelle increases during heating in region 70-140ºC, the maximal level of β-lactoglobulin association attains ∼ 55%.On the other hand, maximum of α-lactalbumin association with casein is ∼ 55% at the temperature below 90 o C and ∼ 40% in temperature range 95-130 o C.
The aim of this work was to investigate of the influence of milk dry matter content, added demineralized whey powder and applied heat treatments on nitrogen matter content of sera gained by acidification with GDL, distribution of nitrogen matter from milk to milk sera, as well as the degree of whey protein denaturation.

Material and Method
Skim milk powder was reconstituted to obtain milk A (with 8.01% TS).Milk A was standardized with 3% of skim milk powder and 3% of demineralized whey powder (DWP), respectively, to obtain milk B (with 11.15% TS) and milk C (with 11.10% TS).
Milk samples were heat treated at: a) 85ºC/10 min, b) 90ºC/10 min and c) 95ºC/10 min.Untreated milk was used as control.
Coagulation of milk was carried out by GDL (concentration of 1% w/w) at 45ºC until pH 4.60 was reached.After coagulation, gained coagulum was cut and separated from sera by self-pressing during 10 min.

Analyses and measurements
Dry matter content determination -AOAC method 16.032 Determination of total nitrogen content by Kjeldahl method -FIL/IDF 20B: 1993 Fat content by Gerber method -FIL/IDF 105:1981 pH value by pH -meter Sentron 1001 Degree of nitrogen matter distribution from milk in milk sera and coagula was calculated on the basis of mass balance Degree of whey protein denaturation was calculated according to loss of whey protein solubility at pH 4.60 (modified method, according to S a v e l l o and D a r g a n , 1997).

Quality parameters of milk and sera
Milk and sera quality parameters are shown in Table 1 The influence of applied heat treatment on nitrogen matter content in milk is shown in Fig. 1.As can be seen from the results shown in Table 1., nitrogen matter content increases with increase of dry matter.When skim-milk powder was used for dry matter standardization, an increase of nitrogen matter content was greater than when demineralized whey powder was used.On the other hand, as can be seen from Table 2., nitrogen matter content in sera was greater when demineralized whey powder was used for standardization.Gained results agree with those of D e n i n -D j u r d j e v i ć , 2001, and F e t a h a g i ć et al., 2002.The results presented in Table 1. and According to the results shown in Table 2. and Fig. 3., it can be seen that nitrogen matter content in sera decreased when temperature of applied heat treatment increased.Gained results agree with the results of C o r r e d i g and D a l g l e i s h , 1999.According to these authors, the amount of whey protein present in milk influences the amount of formed complex.The degree of interaction, besides time of heat treatment, depends on mass fraction of particular protein.Maximum value of δ (δ = g β-lg /g κ-casein ) is 2.2 during heating at 85 o C/20 and 1.4 during heating at 99ºC/20 min (L o n g et al., 1963, M a ć e j , 1983, 1989).However, C o r r e d i g and D a l g l e i s h , 1999, concluded that addition of ßlactoglobulin (2 g/l) does not lead to increase of amount of formed complex between ß-lactoglobulin and ae-casein during heating at 90ºC/60 min.Addition of α-lactalbumin in milk in the amount of 2 g/l leads to balance of α-lactalbumin and ß-lactoglobulin concentration in milk, so both proteins show similar kinetics of reaction.Also, similar amount of both proteins was found in formed complex (C o r r e d i g and D a l g l e i s h , 1999).When 2 g/l of ß-lactoglobulin was added in milk, which is later heat treated at 80ºC, the amount of formed complex attained maximum value after few minutes of heating.However, the amount of formed complex did not differ considerably from milk without added ß-lactoglobulin.This indicates that casein micelle has only certain number of place for interaction with ß-lactoglobulin (C o r r e d i g and D a l g l e i s h , 1999).The amount of complex formed between ae-casein and α-lactalbumin linearly increased during the first 20 minutes of heating.The amount of α-lactalbumin that interacts with ae-casein increased at higher temperatures and extended time of heating.Since processing conditions, primarily heat transfer conditions, have the influence on the amount of formed complex, the amount of formed complex was greater during indirect UHT sterilization than during direct sterilization (DSI) (C o r r e d i g and D a l g l e i s h , 1996).According to all aforementioned, it could be concluded that the increase of temperature leads to the increase of amount of α-lactalbumin, and therefore amount of total whey proteins that reacts with casein micelle.It also explains smaller amount of nitrogen matter is sera gained from heat-treated milk.
According to the presented results, it could be concluded that heat treatment at 95ºC/10 min had the greatest influence on coaggregate formation, which agrees with the results of C o r r e d i g and D a l g l e i s h , 1996, 1999, D a l g l e i s h , 1990, F e t a h a g i ć et al., 2001, M a ć e j , 1983, M a ć e j et al., 2000, P a r n e l l -C l u n i e s et al., 1988.On the other hand, several investigations showed that extended time of heating at 85ºC had the same influence on coaggregate formation as heating at 90ºC or 95ºC/10 min (C o r r e d i g and D a l g l e i s h , 1996, 1999, D a l g l e i s h , 1990, D e n i n -D j u r d j e v i ć , 2001).

The distribution of nitrogen matter from milk to sera
The distribution of nitrogen matter from milk to sera gained after acidification with GDL and separation of coagula is shown in Fig. 3.The results shown in Fig. 3. indicate that increase of temperature from 85ºC to 95ºC had significant influence on whey protein denaturation and formation of coaggregates.Also, it could be concluded that increase of dry matter content had the smallest influence on nitrogen matter distribution from milk heat treated at 85ºC/10 min.Distribution of nitrogen matter decreased from 6.99% (sera gained from milk with 8.01% DM) to 6.02% (sera gained from milk with 11.15% DM).On the other hand, samples heat treated at 90ºC/10 min and 95ºC/10 min had more manifested decrease of nitrogen matter distribution from milk to sera.Nitrogen matter distribution from milk heat treated at 90ºC/10 min to sera decreased from 6.72% (sera gained from milk with 8.01% DM) to 5.32% (sera gained from milk with 11.15% DM), while nitrogen matter distribution from milk heat treated at 95ºC/10 min to sera decreased from 6.59% (sera gained from milk with 8.01% DM) to 5.21% (sera gained from milk with 11.15% DM).
Gained results agree with the results of M a ć e j , 1983, who concluded that nitrogen matter utilization was 93.26-95.34%during co-precipitate production, and the results of M a ć e j et al., 2000, who reported that nitrogen matter distribution from milk heat treated at 87ºC/10 min to sera gained by precipitation with HCl and lactic acid, respectively, was 6.93% and 7.28%.According to the results of D e n i n -D j u r d j e v i ć , 2001, nitrogen matter distribution from milk to sera decreased when dry matter increased.
The greatest distribution of nitrogen matter from milk to sera was observed for milk samples standardized with demineralized whey powder.Nitrogen matter distribution from milk heat treated at 85ºC/10 min, 90ºC/10 min and 95ºC/10 min, respectively, was 9.64%, 8.66% and 8.67%.Gained results indicate that although addition of DWP changes casein:whey protein ratio, heat treatment induces formation of the significant level of coaggregates.The greatest distribution of nitrogen matter in sera (9.64%) was from milk heat treated at 85ºC/10 min, which indicates that 90.36% of nitrogen matter remained in coagula.
Gained results agree with the results of D e n i n -D j u r d j e v i ć , 2001, and M a ć e j et al., 2001, so it can be concluded that DWP can be used for standardization of milk dry matter.As a result, gained product has high nutritive value due to high content of essential amino acids.

The degree of whey protein denaturation
Applied heat treatment, dry matter content and added DWP had significant influence on sera nitrogen content as well as on nitrogen matter distribution from milk to sera.For obtaining as good as possible rheological properties of acid casein gel, it is necessary to obtain appropriate degree of whey protein denaturation.According to M o t t a r et al., 1989, the degree of whey protein denaturation directly influences proteins water-binding capacity, their hydrophobic-hydrophilic properties at pH 4.6 and rheological properties of acid casein gel.In this part we calculated degree of whey protein denaturation as influenced by selected factors.
The degree of whey protein denaturation increases with both temperature and time of heat treatments.The degree of whey protein denaturation was calculated with modified method after S a v e l l o and D a r g a n , 1997, as described by D e n i n -D j u r d j e v i ć , 2001.The method is based on difference of soluble nitrogen matter in sera gained from untreated and heat treated milk at pH 4.60.The difference was shown as a percentage of soluble nitrogen of sera gained from untreated milk, while loss of solubility at pH 4.6 was expressed as relative index of whey protein denaturation (WPD%) (D e n i n -D j u r d j e v i ć , 2001).
The influence of investigated factors on the degree of whey protein denaturation is shown in Fig. 4.
According to gained results it can be concluded that applied heat treatments have significant influence on WPD, especially in combination with dry matter content.
Smaller degree of WPD at lower temperatures can be explained with information reported by C o r r e d i g and D a l g l e i s h , 1996, 1999, that a longer time is needed for interaction between ß-lactoglobulin and ae-casein at lower temperatures (80ºC) of heating, while higher temperatures induce faster proteinprotein interaction.
WPD were smaller in milk samples standardized with demineralized whey powder than in samples with the same dry matter content standardized with skimmilk powder.WPD was 40.77%, 43.59% and 43.72%, respectively, when heat treatment at 85ºC/10 min, 90ºC/10 min and 95ºC/10 min was used.According to the above results, it can be concluded that applied heat treatment (namely, temperature of heat treatment) had significant influence on WPD of milk samples standardized with demineralized whey powder (with exchanged casein: whey proteins ratio).Gained results agree with the results of S a v e l l o and D a r g a n , 1997, who concluded that the greatest WPD could be obtained with heating by VAT system.D e n i n -D j u r d j e v i ć , 2001, concluded that addition of demineralized whey powder up to 2% did not has influence WPD, which agrees with the results of our investigation.

C o n c l u s i o n
According to all aforementioned, it could be concluded: Nitrogen matter content of milk increases with increase of dry matter.On the other hand, milk nitrogen matter content decreased during heat treatments.Nitrogen matter content in sera decreased when temperature of applied heat treatment increased.Nitrogen matter content of sera gained from untreated milk increased with the increase of milk dry matter content and with the addition of DWP.Sera samples obtained from milk 8% heat treated at 85ºC/10 min, 90ºC/10 min and 95ºC/10 min had 69.50 mg%, 66.20 mg% and 66.00 mg% of nitrogen matter, respectively.
Applied heat treatment had significant influence on the distribution of nitrogen matter from milk to milk sera.The distribution of nitrogen matter from milk with 8% DM and 11% DM to sera decreased when temperature of applied heat treatment increased.On the other hand, distribution of nitrogen matter from milk 8%+3%DWP heat treated at 85ºC/10 min, 90ºC/10 min and 95ºC/10 min to sera samples, were 9.64%, 8.66% and 8.67%, respectively.
Whey protein denaturation increased with increasing of the temperature of the applied heat treatment.The denaturation was the most significant for milk sample 11%.
Tokom termičkih tretmana dolazi do smanjenja sadržaja azotnih materija mleka, ali nije ustanovljena linearna korelacija sa povećanjem temperature termičkog tretmana.Sadržaj azotnih materija seruma se povećavao sa povećanjem sadržaja suve materije mleka i sa dodatkom demineralizovane surutke u prahu, kako kod netretiranih, tako i kod termički tretiranih mleka.Sa povećanjem temperature termičkog tretmana, smanjenje sadržaja azotnih materija seruma je više izraženo.Kod termički netretiranih mleka sadržaj azota u serumu je u proseku iznosio 64.90 mg%, 96.80 mg% i 117.3 mg% za mleka 8%, 11% i 8%+3.0%DSUP, respektivno.Kod uzoraka mlečnog seruma dobijenih od mleka , H a r t m a n and S w a n s o n , 1965, H a v e a et al., 1998, L y s t e r , 1970, M a ć e j , 1983, 1989, M a ć e j and J o v a n o v i ć , 2000, M e l o and H a n s e , 1978, Z i t t l e et al., 1962).Interaction occurs as a result of thiol-disulphide interchange between free thiol group of ß-lactoglobulin and disulphide bridges of α-lactalbumin (E l f a g m and W h e e l o c k , 1977, 1978a, 1978b, H a v e a et al., 1998, L y s t e r , 1970, M a ć e j and J o v a n o v i ć , 2000, M e l o and H a n s e , 1978, P u r k a y s t h a et al., 1967, S m i t s and v a n B r o u w e r s h a v e n , 1980), but thiol groups of ß-lactoglobulin have higher reactivity than disulphide bridges of α-lactalbumin (M e l o and H a n s e , 1978).It could be concluded that all proteins that contain cystein disulphide bridges may participate in interaction (C o r r e d i g and D a l g l e i s h , 1996, J a n g and S w a i s g o o d , 1990, L y s t e r , 1970).Except of thiol/disulphide interaction, in the formation of coaggregates protein-protein interaction participates via calcium bridges and hydrophobic reactions (H a q u e and K i n s e l l a , 1988, J a n g and S w a i s g o o d , 1990, O l d f i e l d et al., 1998, P a r r y , 1974, P a u l s s o n and D e j m e k , 1990, S m i t s and v a n B r o u w e r s h a v e n , 1980) as well as rearrangement of polypeptide chains structure (M c K e n z i e et al., 1971).

Fig. 1 .
show that during heat treatment nitrogen matter content of milk decreased, which agrees with the results of D e n i n -D j u r d j e v i ć , 2001, F e t a h a g i ć et al., 2001, J o v a n o v i ć , 2001, M a ć e j , 1983, 1989, and M a ć e j and J o v a n o v i ć , 1998.
The influence of dry matter content and applied heat treatment on nitrogen content of milkThe influence of applied heat treatment on nitrogen matter content in sera is shown in Fig.2.
Fig. 1. -Fig. 2. -The influence of dry matter content and applied heat treatment on sera nitrogen content