THE INFLUENCE OF GDL CONCENTRATION ON MILK pH CHANGE DURING ACID COAGULATION

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 85oC/10 min, 90oC/10 min and 95oC/10 min, respectively. Untreated milk was used as control. Acidification was carried out at 25oC, 35oC and 45oC during 240 min with GDL (glucono-δ-lactone), namely with the amount of 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0% and 3.0% of GDL, respectively. The results showed that all investigated factors, explicitly GDL concentration, acidification temperature and applied heat treatment of milk as well as added DWP influence the change of pH during acidification. Milk samples standardized with DWP had smaller buffer capacity and faster change of pH than samples standardized with skim milk powder. Only at acidification temperature of 25oC, added DWP did not influence the change of milk buffer capacity regardless of the change of casein:whey protein ratio. Under this acidification condition, both milk samples standardized with skim milk powder and DWP had similar final pH values.


I n t r o d u c t i o n
The fermentation of milk with lactic acid bacteria (production of fermented products: yogurt and acid-curd cheese varieties) and acidification with organic and inorganic acids (production of casein and coprecipitates) are regularly used for acidic coagulation of milk ( Since there are few information in available literature, the aim of this work was to investigate the influence of milk dry matter content and protein composition as well as applied heat treatments and acidification temperature on milk pH value change during acidification with GDL.

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 85ºC/10 min, 90ºC/10 min and 95ºC/10 min, respectively.Untreated milk was used as control.

Results and Discussion
Milk quality parameters are shown in table 1 The results shown in table 1. indicate that during heat treatment dry matter content of milk increased as a result of evaporation during heat treatment.On the other hand, nitrogen matter content of milk decreased during heat treatment, which agrees with the results of F e t a h a g i ć et al.The pH value of milk decreased faster when concentration of GDL was increased, as Figs. 1, 2 and 3 show.These results agree with those of D y b o w s k a and F u j i o , 1996, who concluded that higher concentration of GDL accelerated the onset time of gelation and the rate of network formation.The pH value of untreated milk samples decreased faster during acidification at 25ºC than pH of heat-treated samples.These results agree with the conclusion of H o r n e , 1999, who reported that heat treatment of milk alters the rate at which bonds between casein micelles are formed as well as the mechanism of micelles interactions during protein network formation.
The influence of acidification temperature and applied heat treatment on pH value after 60 min of acidification with GDL is shown in table 2 As expected, during the first 60 min, the reduction of pH value was greater at higher acidification temperature, since the rate at which GDL hydrolyzes increases at higher temperatures.At 25ºC, heat treated milk samples had higher pH value after 60 min than untreated milk samples, regardless of the content and composition of dry matter (as Figs. 1, 2 and 3 show), which indicates a greater buffer capacity of heat treated milk to GDL.According to K i m and K i n s e l l a , 1989, gelation of heat-treated samples starts at higher pH values, which shows that heat treatment increases the rate of gel formation.
The character of casein micelles influences forces that control gelation, therefore, changes on casein micelle depend on both temperature and pH value.Hydrophobic interactions, which play the most significant role in gelation of casein micelle, are favored at higher temperatures (G o d d a r d and A u g u s t i n , 1995).Hydrophobic interactions are weaker while repulsive forces are enhanced at lower temperature, so repulsive forces have to be reduced to a greater extent during acidification before intermicellar attractions can dominate.This could explain variations in pH value at the onset of gelation at different temperatures (B r i n g e and K i n s e l a , 1990).
The increase of acidification temperature from 25ºC to 35ºC had a greater influence on the rate of pH decrease than increase from 35ºC to 45ºC did.This agrees with the results of D e a n e and H a m m o n d , 1960, who reported that an increase of acidification temperature from 20ºC to 35ºC reduces coagulation time by 75-80%.K i m and K i n s e l l a , 1989, concluded that the increase of acidification temperature leads to the decrease of coagulation time, while the greatest reduction of coagulation time is noticed when temperature is increased from 45ºC to 50ºC.Gelation started after 180 min, 128 min, 68 min, 33 min and 16 min, respectively, at acidification temperatures of 35ºC, 40ºC, 45ºC, 50ºC and 55ºC, which indicates that higher temperatures increase the rate of GDL-induced acidic coagulation.According to B a n o n and H a r d y , 1991, the first signs of coagulation of samples coagulated at 15ºC and 20ºC occur at pH 5.0 and 5.1, respectively.D y b o w s k a and F u j i o , 1996, found that gelation started after 145 min, 110 min and 50 min (namely, at pH 4.90, 4.95 and 5.18), respectively, when coagulation temperature was increased from 30ºC to 35ºC and then to 40ºC.
Untreated milk samples A and C had similar pH values after 60 min of acidification regardless of the applied acidification temperature, which indicates that casein has the most important role in maintaining buffer capacity of raw milk.On the other hand, pH drop of heat-treated samples vary depending on GDL concentration, acidification temperature and applied heat treatment.
Heat-treated samples B and C show irregular drop of pH during acidification, which indicates a greater buffer capacity and exchanged pattern of coagulation, as Figs. 2 and 3 show.Milk samples B heat-treated at 95ºC/10 min had the most pronounced irregular decrease of pH during acidification, particularly at 25ºC.The increase of acidification temperature to 35ºC or 45ºC reduced irregularity and to a great extent improved the pattern of pH change.
The influence of applied acidification temperature and concentration of GDL on final pH value after 240 min is shown in table 3 Of the samples of untreated milk, samples B had the highest final pH value regardless of applied acidification temperature and used concentration of GDL, as table 3 shows.It again confirms our hypothesis that casein content present in milk controls milk buffer capacity.A greater amount of GDL has to be used to attain certain pH value when protein content of milk is increased without modification of casein:whey protein ratio.On the other hand, milk samples that had modified casein:whey protein ratio due to the addition of DWP (milk C) achieved lower pH values at 35ºC and 45ºC than milk A. At lower acidification temperature (25ºC) modified casein:whey protein ratio did not influence the change of pH value.
According to the above results, it could be concluded that casein has a greater buffer capacity than whey proteins.This means that standardization of milk dry matter intended for the production of fermented milk with DWP leads to the shortening of fermentation duration due to a lower buffer capacity, as the results of D e n i n D j u r d j e v i ć , 2001, and D e n i n D j u r d j e v i ć et al., 2002, showed.Naturally, this question needs further investigation since there are controversial interpretations in available literature.

C o n c l u s i o n
According to all aforementioned, it could be concluded: The pH value of untreated milk samples decreased faster than pH value of heat treated samples at acidification temperature of 25ºC.
During the first 60 min of acidification, pH value was faster decreased when acidification temperature was increased.After 60 min of acidification, heattreated milk samples had higher pH value than untreated samples, regardless of dry matter content.
Between 60 and 120 min of acidification, pH value of heat-treated samples decreased faster than pH of untreated samples, which indicates a smaller buffer capacity of heat-treated milk under these acidification conditions (between 60 and 120 min).
The increase of acidification temperature from 25ºC to 35ºC had a greater influence on the rate of pH decrease than increase from 35ºC to 45ºC did.
Untreated milk samples A and C had similar pH values after 60 min of acidification in spite of the applied acidification temperature.However, decreases of pH value in heat-treated milk depend on GDL concentration, acidification temperature and applied heat treatment.
Milk samples B heat-treated at 95ºC/10 min had irregular decrease of pH during acidification, particularly at 25ºC.The increase of acidification temperature to 35ºC or 45ºC improved the pattern of pH reduction.
Untreated milk samples B had the highest final pH value in spite of the applied acidification temperature and used GDL concentration.
A milk sample standardized with demineralized whey powder (milk C) that coagulated at 35ºC or 45ºC had a lower final pH value than milk samples A. At the acidification temperature of 25ºC modified casein:whey protein ratio did not influence final pH value in investigated samples.
D e n i n D j u r d j e v i ć , 2001, G u i n e e et al., 1993, J o v a n o v i ć , 2001, M a ć e j , 1983, M a ć e j et al., 2001).During the last 20 years, milk acidification with glucono-δ-lactone has been commonly used for investigating changes that occur on casein micelles during gel formation.The structure of casein micelle undergoes intensive change during acidic coagulation of milk as discussed elsewhere (M a ć e j et al., 2001, D e n i n D j u r d j e v i ć , 2001, R o e f s , 1986, B r i n g e and K i n s e l a , 1990, H e e r t j e et al., 1985, D a l g l e i s h and L a w , 1988).The most important changes that occur in casein micelle are: 1) Physicochemical changes of casein micelle (G u i n e e et al., 1993, H e e r t j e et al., 1985, R o e f s , 1986); 2) Dissociation of colloidal calcium phosphate (CCP) (v a n H o o y d o n k et al., 1986, R o e f s , 1986, D a l g l e i s h and L a w , 1989, B r i n g e and K i n s e l a , 1990, H o l t and H o r n e , 1996, P a r n e l l -C l u n i e s et al., 1988, W a l s t r a , 1990); 3) Dissociation of casein and factors that influence dissociation such as hydrophobic effect, solubilization of calcium and phosphate from casein micelle, izoelectric precipitation of casein (G u i n e e et al., 1993, v a n H o o y d o n k et al., 1986, H e e r t j e et al., 1985, B r i n g e and K i n s e l a , 1990, R o e f s , 1986, D a l g l e i s h and L a w , 1988, S i n g h et al., 1996); 4) Voluminosity changes during milk pH value reduction (v a n H o o y d o n k et al., 1986, G u i n e e et al., 1993, H e e r t j e et al., 1985, R o e f s , 1986); 5) Reduction of zeta-potential as influenced by milk pH value (v a n H o o y d o n k et al., 1986, G u i n e e et al., 1993, H e e r t j e et al., 1985, D a r l i n g and D i c k s o n , 1979); 6) Changes of casein micelle hydration (G u i n e e et al., 1993, P a r n e l l -C l u n i e s et al., 1988, D j o r d j e v i ć , 1987, T o r a d o d e l a F u e n t e and A l a i s , 1975); 7) Changes of casein micelle size (R o e f s , 1986, H o l t and H o r n e , 1996, G o d d a r d and A u g u s t i n , 1995); 8) Structural changes of casein micelle during acidification and gel formation (H e e r t j e et al., 1985, P a r n e l l -C l u n i e s et al., 1988, d e K r u i f and R o e f s , 1996, P a r k e r and D a l g l e i s h , 1977).
, 2001, D e n i n D j u r d j e v i ć , 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, 2000.Nevertheless, we did not find linear relationship between the temperature of applied heat treatment and decrease of nitrogen matter content.A milk sample that was standardized with DWP had the lowest nitrogen matter content in dry matter (DM), which agrees well with the results of D e n i n D j u r d j e v i ć , 2001.The influence of GDL concentration on pH change during acidification The influence of applied heat treatment, acidification temperature and GDL concentration on the change of pH value of milks A, B and C, respectively, is shown in Figs. 1, 2 and 3.