Reconstituted UHT-treated milk: Effects of raw milk, powder quality and storage conditions of UHT milk on its physico-hemical attributes and flavour

Int. Darr>, Journal 7 (1997) 129-140 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:

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Reconstituted UHT-treated Milk: Effects of Raw Milk, Powder Quality and Storage Conditions of UHT Milk on its Physico-Chemical Attributes and Flavour Ernani L. Celestino, Mani Iyer* and Hubert Roginski Gilbert Chandler College, Faculty @Agriculture,

(Received

Forestry and Horticulture, The University 3030, Victoria, Australia 24 September

1995; accepted

28 August

of Melbourne. Sneydes Road, Werribee

1996)

ABSTRACT Reconstituted, UHT-processed milk was produced from whole milk powders that were manufactured from fresh (control) or stored (4f 1°C 48f2 h) raw milk and stored for different periods at 25& 1°C. Lipolytic and proteolytic activities, physicoochemical and flavour changes and gelation in UHT milk during storage at 3f 1°C or 251!~1“C for up to 6 months were investigated in relation to the biochemical status of raw milk and whole milk powder. The green-red and blue-yellow components of colour and the non-protein nitrogen content of UHT milk increased, while pH and colour lightness decreased with storage; the rate of change being greater at higher storage temperature. Sediment increased with longer storage period, but was independent of storage temperature. With longer storage at both 3f 1’C and 25f 1°C greater sediment and lower pH were observed in UHT milk processed from older milk powders. Rates of enzymatic and oxidative reactions appeared greater in UHT milk stored at 25 f 1“C and in those processed from older powders and contributed to the development of off-flavours in UHT milk with a prolonged storage period. Gelation was observed only at 25+ 1°C. Lipases and proteinases were reactivated during storage and their activity was greater in UHT milk processed from powder manufactured from stored raw milk. The taste of reconstituted UHT milk was affected more by lipolysis than by proteolysis. 0 1997 Elsevier Science Ltd

the physical and chemical characteristics of reconstituted UHT milk. The type of heat treatment employed during processing, as well as subsequent storage of the UHT milk, may also adversely affect its properties. Age gelation, which is one of the main problems associated with the keeping quality of UHT milk (Wade, 1990) has been attributed to products of proteolysis by both indigenous and microbial enzymes (Snoeren et al., 1979) and to polymerisation initiated by Maillard-type reactions (Andrews, 1975). Despite extensive studies of this problem, the mechanisms of age gelation in UHT-sterilised milk during storage are still not completely understood (Gunnis, 1982; Singh et al., 1989). The present study constitutes the final part of the research on the quality of raw milk (Celestino et al., 1996a), its effect on milk powder (Celestino et al., 1997) and the effect of these raw materials on the keeping quality of reconstituted UHT milk. While there is some information on recombined UHT milk, virtually no information is available on the use of whole milk powder in UHT milk manufacture. This could be attributed to problems of flavour deterioration associated with oxidation of whole milk powder under normal storage conditions (Kieseker, 1981). According to Newstead (1990) the shelf-life of UHT

INTRODUCTION The quality of most dairy products is closely related to the microbial status of raw milk from which they are Depending on the temperature, manufactured. conditions and length of milk storage, various groups of microorganisms can undergo a period of intensive growth producing high concentrations of enzymes, particularly lipases and proteinases. Although the microorganisms are destroyed by sterilisation, the enzymes produced may remain active in sterilised products. Even low activity of lipases and/or proteinases may cause defects in products stored for a long time, such as UHT milk and even dried milk where the enzymes are preserved and may be reactivated after reconstitution (Kishonti, 1975). Rancidity development and age gelation appear to be two important defects caused by these heat-resistant enzymes (Cogan, 1980; Wade, 1990). A recent study (Celestino et al., 1997) has shown that whole milk powder manufactured from refrigerated (4~tl”C, 48f2 h) bulk raw milk had greater free fatty acids content than that from fresh (control) raw milk and that physical and chemical changes may occur during storage of the powder. The quality of the raw materials used may thus influence *Corresponding

author. 129

E. L. Cefestinoet al.

130

milk is 3-6 months at ambient temperatures (20-30°C). Many publications (reviewed by Cerf, 1981) consistently confirmed that refrigerated storage of UHT milk limits the changes which occur in the product. In the present study, UHT milk stored at 3f 1°C served as the control. The objectives of the present investigation were (1) to monitor physical and chemical changes that occur during storage of UHT milk made from reconstituted whole milk powder and determine the influence of raw material quality on these changes; (2) to relate the biochemical status of raw milk and whole milk powder to the activity of selected enzymes in the resultant UHT milk and (3) to study flavour and gelation in reconstituted UHT milk and relate them to the biochemical changes.

MATERIALS

sample of milk, tempered to 20-22°C was centrifuged in a 50-mL conical centrifuge tube at about 15Oxg for 10 min using an unheated Babcock centrifuge, and sediment measured in millimetres using dividers and a millimetre scale. Colour The colour of UHT milk (about 30 mL sample placed in a test tube) was determined using a Minolta Chroma Meter (CR-200/CR-231; Minolta, Japan) which measured ‘L’ (luminosity or lightness), ‘a’ (green-red component) and ‘b’ (blue-yellow component) values. Viscosity The viscosity of UHT milk was measured at 20-22°C using a Brooktield Synchro-lectric Viscometer (Model LVT; Brookfield Engineering Labs Inc., Stoughton, Massachusetts, USA).

AND METHODS Chemical tests

Processing, packaging and storage of reconstituted UHT milk

The whole milk powders manufactured from fresh (control) or stored (4f 1°C 48f2 h) farm raw milk in three replicates (spring, summer and autumn), as described in the previous study (Celestino et al., 1997) were reconstituted and processed into UHT milk. Details of composite milk sample collection and standardisation are available (Celestino et al., 1996a). Powders from five lots (stored at 25fl”C for 0, 2, 4, 6 and 8 months, respectively) were reconstituted to produce milk with 12.3% total solids. The powder was dispersed in water (3941°C) with mechanical agitation for lo-15 min until all the milk solids were dissolved. The reconstituted milk was heated to about 55°C and then homogenised in two stages at 14 MPa/3.5 MPa using a Creamery Package Homogeniser (Model 3DDl; The Creamery Package Manufacturing Company, Chicago, Illinois, USA). The homogenised milk was processed on an Alfa-Lava1 VTISjVTS indirect pilot UHT plant, located at the CSIRO experimental dairy factory, Highett, Victoria; preheating to 75°C was followed by UHT treatment at 138-140°C for 3 s, regeneration cooling to 85-88°C and cooling to about 24°C prior to tilling. The product was filled into 200- and 400-mL gamma-irradiated, screwcapped plastic containers under laminar flow of filtered sterile air. The UHT-treated milk produced from each lot of powder was divided equally into two sub-lots stored at 3 & 1°C or 25 f 1°C. Milk samples from each treatment were analysed and then sampled every month during 6 months’ storage.

The pH of UHT milk was determined at 20-22°C using a digital pH meter (TPS Pty. Ltd, Brisbane, Australia). The procedure set by the Standards Association of Australia (SAA; AS 2300.1.6-1989) was followed. Total solids, fat, total nitrogen and non-protein nitrogen (NPN) contents were determined as specifically described by the SAA for homogenised liquid milk (AS 2300-1988, 1991). For percentage NPN computation, the actual fat and protein contents of the sample were used in the calculation of the volume of precipitate. Free fat was determined as in testing raw milk (Te Whaiti & Fryer, 1975) with the following modifications: the sample was weighed into a skimmilk (O-0.5%) Babcock bottle; after the second centrifugation, the samples were refrigerated (7-13°C) for 18-20 h, instead of 13°C overnight, before proceeding to the next steps of the procedure; centrifugation and rewarming times of samples were doubled to allow the free fat, which was expected to be minimal, to rise and separate more efficiently. The test for homogenisation efficiency was carried out immediately after the manufacture of reconstituted UHT milk as per SAA (AS 2300.2.1-1980). Determination

of gelation

Reconstituted UHT milk samples were visually examined weekly for signs/symptoms of gelation. Complete coagulation was taken as a criterion in assessing the onset of gelation (Mittal et al., 1988). The viscosity of gelled UHT milk samples was measured using a Brookfield Synchro-lectric Viscometer (Model LVT) .

Physical tests

Biochemical

Fat separationlcreamline Duplicate samples of 400-mL UHT milk from each treatment were examined visually and the creamline thickness measured (Mittal et al., 1988).

Lipolysis The free fatty acid (FFA) content and lipase activity were determined as described in Celestino et al. (1997).

Sediment Sediment was measured according to the procedure of Zadow (1971), with slight modifications. A 40-mL

tests

Proteolysis The procedures for fluorescamine method and polyacrylamide gel electrophoresis (PAGE) were followed as described by Celestino et al. (1997).

Reconstituted UHT-treated milk Sensory evaluation Preparation cf samples Samples for the sensory evaluation were tempered to room temperature (18-22°C) and dispensed in 15-mL sample cups. A cup of water was provided as rinse. Selection ?~~judgtJsipanelists A triangle test (Gatchalian, 1981) was conducted three times to screen the panelist’s ability for evaluation of the reconstituted UHT milk samples. Out of the 18 panelists invited, live were chosen. Selection was based on the test results and on the interest and availability of the panelists. Evaluation qf’reconstituted UHT milk Samples were evaluated by five panelists for flavour and viscosity using the multi-sample test with the application of ordinal numerical scaling (Gatchalian, 1981). The judges evaluated the flavour of samples on a 7-point scale from ‘extremely unpleasant’ (Score= 1) ‘satisfactory’ (Score = 4) to ‘extremely through pleasant’ (Score = 7). Any off-flavour detected was further evaluated for its degree of intensity on a 5point scale, i.e., from ‘very slightly noticeable’ to ‘very pronounced’. After the flavour test, the same samples were evaluated for viscosity. The description used was ‘too thin’ (Score= 1) through ‘just right’ from (Score = 4) to ‘too thick’ (Score = 7). Statistical

analysis

Analysis of variance of the data (except for homogenisation efficiency which was not statistically analysed; and PAGE and gelation results which were evaluated descriptively) was applied according to split/ split plot in randomised complete block design, with unequal number of observations. In this design, the raw milk storage treatment, the storage period of powder, and the storage temperature/period of UHT milk were the mainplot, subplot, and sub-subplot, respectively. Treatment means were compared by the Duncan’s Multiple Range Test (Steel & Torrie, 1980; Freund et al., 1986).

RESULTS

homogenised milk, the membrane of the fat is altered (Mulder & Walstra, 1974). The study of van Boekel & Folkerts (1991) showed that homogenised caseincovered milk fat globules are subject to appreciable aggregation, especially after heating at temperatures above 135,-C. In the present study, the separated fat could have included additional proteins which were bound to the fat globule after heating. Aggregation of these protein-bound fat globules/clusters may explain the extensive phase separation, which was greater at a storage temperature of 25& l”C, possibly because at 3f 1°C. milk was more viscous. Furthermore, homogenisation of the concentrate prior to powder manufacture could have caused some losses of the fat globule membrane phospholipids and other emulsifying compounds, contributing to instability of fat globules after UHT treatment of reconstituted milk. Sediment Storage of raw milk did not significantly affect sediment formation in UHT milk (P>O.O5). However, a relationship between storage of powder and tendency to form sediment in UHT milk during storage was detected (P < 0.05). During storage at both 3* 1°C and 25f 1°C sedimentation was greater in UHT milk processed from older powders (P < 0.05; Figs 1 and 2). Very little sediment was found in any of the samples immediately after UHT milk manufacture, and the increase during storage was generally very slight. The increase was found to be independent of storage temperature. Presence of brown sediments which appeared like scorched particles was also observed in all samples one day after UHT milk manufacture. The low level of sedimentation may be due to the use of medium heat powder. Zadow & Hardham (1978) and Mittal et al. (1988) found that recombined UHT milk processed from such type of powder was more resistant to sediment formation than that prepared from low heat powder. Furthermore, sediment formation in directly heated milk was reported to be greater than after indirect heating (Perkin et al., 1973; Burton, 1972). Burton (1968) suggested that sediment in UHT 4,

Fat separutionjcreamline Storage of raw milk and of the respective powders did not significantly affect the formation of a creamline in reconstituted UHT-treated milk (P > 0.05). Very slight fat separation was observed after about 20 days in samples stored at 3 f 1“C and after 6 days in samples stored at 25f 1“C. However, the separated fat mixed well with the rest of the milk upon gentle mixing. The creamline increased to almost 7 mm and 10 mm after 6 months of storage at 3f 1°C and 25f l”C, respectively. Homogenisation tests showed an average value of 4% homogenisation index which indicated that the homogenisation was efficient. It has been suggested that the instability in the milk fat dispersion during storage of UHT milk is due to changes in the nature of the fat globule surface. In

I

3.5 -- - .

AND DISCUSSION

Physical properties

131

z s

. . _. . . . . . . .

3---

z .g 2.5 -- B *

2--1.5’

: 0

1 2 3 4 5 Storage Period of UHT Milk (Months) -0

-+2 -9-4 -36

Fig. 1. Sediment in reconstituted

6

-+8

UHT milk, processed from whole milk powders stored for 0, 2, 4, 6 and 8 months, and kept for up to 6 months at 3 f 1°C. Differences between treatments (powder storagexUHT milk storage temperature and period; n=6, except for fresh powders where n=4) were significant (P < 0.05).

E. L. Celestino et al.

132

-

3.5 -- . __ .__ .._ . . - - ._ 83.

vE! 3--_,

83. . _ _. _. _. _.

.g 2.5 -- . w

L 83.

2 _-. __ ____.____._____.____.____.~_ 1.5 1

83

/

f o

J 1 2 3 4 5 6 Storage Period of UHT Milk (Months)

I

...___....._..._._..._.__...._...................... _....._....... _.

7.8 t

+O

-2

+4

+3-6 -+8

Fig. 2. Sediment in reconstituted UHT milk, processed from whole milk powders stored for 0, 2, 4, 6 and 8 months, and kept for up to 6 months at 25* 1°C. Differences between treatments (powder storagexUHT milk storage temperature and period; n = 6, except for fresh powders where n = 4) were significant (P < 0.05).

milk is made of essentially the same material as that which can be deposited on heated surfaces.

r8tt __.__^ .-....-_. ..._... -.--a...-...

a

-3

Colour

Storage of raw milk did not affect the colour of UHT milk (P>O.O5). However, the colour values L, a and b of UHT milk significantly changed with storage temperature and time (P< 0.01). The L-value of samples stored at 3 f 1°C or 25% 1“C decreased with longer storage time and the a and b values increased with storage; in both cases, the rates were higher in samples stored at 25fl”C (Fig. 3). Storage of powder significantly affected the b-value of UHT milk (PC 0.01; Table 1). The b-value decreased when older powder was used. Storage of powder did not significantly affect the colour L and a values of UHT milk (P > 0.05). The change in colour during storage of UHT milk could be due to the browning reaction. During processing, and on prolonged storage, this effect is demonstrated by the amino-sugar or Maillard reaction which, as indicated by Renner (1988) proceeds to an appreciable extent only when the storage temperature is above 20°C. Various intermediates are formed during the reaction, such as 5-hydroxymethylfurfural which on Table 1. Colour L-, a- and b-value?

of Reconstituted

..._.- __.. .__ ..__ ..__ ......_ ......-...__....-__- .._. . ..” I&-

Fig. 3. Colour L-, a- and b-values of reconstituted UHT milk stored for up to 6 months at 3~t 1°C or 25f 1°C. Differences between treatments (UHT milk storage temperature and period; n = 28) were significant (P < 0.01).

further degradation produce other organic components and brown pigments (Adhikari & Singhal, 1991). It was reported that as the Maillard reaction progresses, pigment concentration increases (Burton, 1954; Zadow, 1970). Reddy et al. (1991) further reported that opacity development as a result of increased viscosity may also contribute to a decrease in the lightness of UHT whole milk. The present study, however, showed only slight increases in viscosity during storage at either 3% 1°C

UHT Milk Processed From Whole Milk Powders Stored for Different Periods at 25f 1°C

Storage period of powder (months)

n

Colour L-valueb

Colour a-valueb

Colour b-value

0 2 4 6 8

52 78 78 78 78

83.34 83.36 83.32 83.30 83.31

-3.02 -2.96 -2.85 -2.92 -3.01

7.90a (0.03) 7.56b (0.03) 7.53bc (0.03) 7.47bc (0.03) 7.32~ (0.03)

(0.07) (0.05) (0.05) (0.05) (0.05)

(0.04) (0.02) (0.02) (0.02) (0.02)

“Means from all UHT milk, processed from both control and stored (4f 1°C 48f2 h) raw milk in all replicates, and kept at both temperatures for all periods; Compared using DMRT; Means within a column followed by a common letter are not significantly different (P > 0.05). Figures in brackets are standard errors of the means. bNo significant differences.

Rrcnnstituted UHT-trrufed milk and 25& 1“C (results not shown) and thus, contribution of viscosity to colour changes may not have been very significant. Compared to L-values obtained in studies on directly heated UHT recombined milk (Mittal et al., 1988, 1990) and for indirectly heated UHT whole milk (Reddy rt al., 1991) L-values for reconstituted UHT milk in the present study were lower. A more extensive browning reaction could have occurred as a result of heating during processing and/or storage of the whole milk powder and this led to a greater colour change. It has been shown that non-enzymatic browning reactions also occur during prolonged storage of dried milk products (Renner, 1988; Kneifel, 1989 as cited by Kneifel et al., 1992). This may also explain the lower bvalues obtained when older whole milk powders were used (Table 1). In addition to the contributory effect associated with powder manufacture and storage on the colour of UHT milk, the indirect method applied during UHT processing could have also contributed to the greater change in colour due to more extensive Maillard browning. In general, direct processes cause less browning than indirect heating (Zadow, 1969; Blanc, 1980 as cited by Blanc & Odet, 1981). Because of the longer come-up time in heat exchangers, greater heat dose is imparted to the milk when the indirect method of heat treatment is applied, resuiting in comparatively longer equivalent holding periods (Nangpal & Reuter, 1990). Viscosity Refrigerated storage of raw milk had no significant effect on viscosity of the resultant UHT milk (P > 0.05; means of 2.13 mPa s for control compared to 2.14 mPa s for UHT milk obtained from raw milk subjected to refrigerated storage). A slight change in viscosity during storage of UHT milk at 3& 1“C and 255 1“C was observed. UHT milk stored at refrigeration temperature had higher (PC 0.01) viscosity (mean of 2.18 mPa s for combined storage periods) than that stored at the higher temperature (mean of 2.12 mPa s). At 251t 1“C, the highest viscosity value was observed at the third month of storage (2.16 mPa s), while at 3% l”C, this was observed at the fifth month (2.26 mPa s). Studies on directly heated recombined UHT milk (Blanc, 1978 as cited by Renner & Schmidt, 1981; Mittal et al., 1988; Alkanhal et al., 1994) showed similar results with regard to the effect of storage temperature, i.e. viscosity was greater in samples kept at refrigeration temperatures (5 or 6°C) than those at a

133

higher temperature (30°C). Other authors (Ashton, 1966; Harwalkar & Vreeman, 1978; Mittal et al., 1990; Reddy et al., 1991) have reported increased viscosity in stored UHT milk while Sur & Joshi (1989) did not find much change in viscosity of UHT whole milk (ranging from 1.97 to 2.44 mPa s) during storage at 22 and 37°C for 5 months.

Composition and pH Raw milk storage had no significant effect on pH of the resultant UHT milk (I’> 0.05). However, UHT milk storage temperature and time affected the product’s pH (PC 0.01; Table 2). The pH decreased with storage of UHT milk, the rate of decrease being greater at 25% 1°C than at 3f 1°C. The effect due to the interaction of age of powder and UHT milk storage temperature/time was significant (P < 0.05). Lowest pH was observed at 6 months storage of UHT milk when 6- and 8-month-old powders (6.52 and 6.53, respectively; initial pH of UHT milk from both powders was 6.64) were used. The reduction in pH during storage has been linked to the hydrolytic dephosphorylation of casein, changes in the calciumphosphate equilibrium and the interaction between lactose and milk proteins (Same1 et al., 1971; Hansen & Melo, 1977). Andrews rt al. (1977) specifically ascribed the drop in pH during the Maillard reaction to the loss of positive charge on the protein molecules caused by the loss of free s-NH2 group of lysine. Further, result in the extensive Maillard browning may formation of various organic acids causing a fall in pH (Adhikari & Singhal, 1991). The greater decrease in pH of UHT milk stored at 25f 1°C could be due to the more pronounced extent of the Maillard reaction at higher temperatures as evidenced by the lower L-values obtained in UHT milk stored at 25* 1°C (Fig. 3). Reddy et al. (1991) have attributed rapid reduction in pH at elevated temperatures to enhanced Maillard and enzymatic reactions. Higher initial pH (Burton, 1954; Gothwal & Bhavadasan, 1992a) and higher protein level (Gothwal & Bhavadasan, 1992b) were reported to cause greater browning in milk. Milk with higher pH has higher numbers of unprotonated amino groups. Whitelaw & Weaver (1988) reported that only unprotonated amine can combine with sugars. Pyne & McHenry (1955) have noted that an increase in protein concentration in milk caused an increase in heat-induced acidity and they ascribed this to the acidity derived by Maillard

Table 2. Values of pH” for UHT Milk, Processed From Whole Milk Powder Manufactured From Control 481t2 hours) Raw Milk, and Kept for Different Periods at 3 % I C and 251t 1°C Storage temperature of UHT milk (&lC)

Raw milk treatment

or Stored

(4fl”C,

Storage period of UHT milk (months) 0

I

2

3

4

5

6

3

Control Stored

6.65 6.65

6.67 6.66

6.66 6.65

6.65 6.64

6.64 6.62

6.63 6.62

6.60 6.60

25

Control Stored

6.65 6.65

6.61 6.60

6.59 6.58

6.56 6.54

6.54 6.53

6.52 6.50

6.50 6.48

“Means from all UHT milk samples processed from powders stored for all periods in all replicates; significant difference between raw milk treatments (P > 0.05; n = 182); significant differences between milk (P < 0.01; n = 28).

compared using DMRT; no storage temperature of UHT

134

E. L. Celestino et al.

reaction. The results of the present study showed that compared to UHT milk processed from spring or autumn raw milk, that from summer raw milk had the highest initial pH (6.68, 6.62 and 6.65 for summer, spring and autumn raw milk, respectively) and the lowest crude protein content (3.13, 3.23 and 3.40% for summer, spring and autumn raw milk, respectively). UHT milk processed from autumn raw milk had a higher pH and higher protein concentration compared to that from spring raw milk. A faster rate of pH decrease was observed during storage of the former UHT milk than the latter. At 6 months of storage, the pH decreased from an initial of 6.65 to 6.53 and 6.62 to 6.56 for UHT milk processed from autumn or spring milk, respectively. This suggests that the higher concentration of proteins, as well as possible contributory effect of unprotonated amino groups, could have provided for more lactose-protein interactions, which therefore resulted in a greater increase in acidity with storage and consequently lower PH. A decrease in pH of UHT recombined milk and/or UHT whole milk during storage at room temperature and/or elevated temperatures has been observed by several investigators (Zadow & Birtwistle, 1973; Andrews et al., 1977; Gijrner et al., 1977; Kocak & Zadow, 1985; Mittal et al., 1988; Reddy et al., 1991). Refrigerated storage of raw milk and age of powder did not affect the chemical composition of UHT milk (P > 0.05; results not shown). Composition generally remained unchanged during storage of UHT milk at 3fl”C or 25fl”C. A significant increase in NPN was found in UHT milk during storage (results not shown). Higher NPN values were obtained at the higher temperature. The increase in NPN during storage could be attributed to decomposition of the proteins by reactivated proteolytic enzymes (Hostettler et al., 1957 as cited by Harwalkar, 1982; Samuelsson & Holm, 1966) which may have been more active at 25% 1°C than at refrigeration temperature. These enzymes may be indigenous (Driessen, 1981) or of bacterial origin (Mottar, 1981).

may be more effectively inactivated (Snoeren et al., 1980a as cited by Kohlmann et al., 1988; Corradini & Pecchini, 1981). Alternatively, the action of proteinases may be retarded by the greater amount of denatured Blactoglobulin present in milk sterilised by the indirect method (Snoeren et al., 1980a as cited by Kohlmann et al., 1988; Snoeren & Both, 1981). The role of proteolysis in gelation was investigated in the present study. Gelation occurred only in UHT milk stored at 25f 1°C where a higher degree of proteolysis was observed as compared to samples stored at 3f 1°C. PAGE results showed proteolytic activity during storage of UHT milk, particularly at 25% 1°C. Of the gelled samples, a greater number (eight out of ten) was processed from stored raw milk (in which greater proteolytic activity was measured; Celestino et al., 1996a). It would be difficult to determine exactly the proteinase activity at which the samples gelled because, prior to gelation, some samples showed no proteolytic activity. In those samples where proteinase activity was detected prior to gelation, greater proteolytic activity was observed at 25fl”C (range 14-30 nmol L-leucyl-L-leucine min-’ mL-‘) as compared to their counterparts stored at 3f 1°C (range 8-14 nmol Lleucyl-L-leucine min-’ mL-‘). The possibility of a physico-chemical effect contributing to gelation may explain this observation. As proposed by some researchers (Same1 et al., 1971; Andrews, 1975; Harwalkar & Vreeman, 1978) reactions resulting in structural changes of the casein micelles, may play a more significant role in gelation phenomena as compared to proteolysis. Since the rate of physicochemical reactions is faster at higher storage temperatures, gelation was faster at 25f 1°C. That gelation of UHT-processed whole milk is a consequence of a combination of physico-chemical and enzymic processes has been suggested by some authors (Kocak & Zadow, 1985; Pande & Mathur, 1992). Gelation was specifically described as a twostage process where proteolysis is followed by nonenzymatic physico-chemical changes (Kocak & Zadow, 1985; Manji, 1987; Pande & Mathur, 1992).

Gelation

Proteolysis

A small proportion of reconstituted UHT milk (10 out of a total of 925 samples stored at 25f 1C) gelled; this phenomenon was observed at 4.5-6 months of storage at 25f 1°C with viscosities ranging from 50 to 60 mPa s. The increase in viscosity with time could be indicative of progressive denaturation and unfolding of proteins (Ferry, 1948) and was thought to be connected with gradual aggregation of the protein micelles leading to the formation of a coagulum (Samuelsson & Holm, 1966). Gelation of only a few samples may be attributed to the intense heat treatment applied during UHT processing. In a study by McKellar et al. (1984), age gelation was not observed in indirectly heated UHT milk, with viscosity remaining constant throughout 30 weeks of storage at 20°C while gelation was observed at a viscosity of 100 mPa s between 6 and 10 weeks in directly heated UHT milk. Due to the comparatively longer equivalent holding periods involved in the indirect heating method (Nangpal & Reuter, 1990) the proteinase in indirect UHT milk

Proteinases, both indigenous (Driessen & van der Waals, 1978) and of bacterial origin (reviewed by Suhren, 1983), have been shown to withstand high heat treatments. Results of the present study showed higher (PC 0.01) proteolytic activity in UHT milk processed from stored raw milk than that from the control (12.3 and 8.7 nmol L-leucyl-L-leucine min-’ mL-’ for UHT milk processed from stored and control raw milk, respectively). The activity of residual proteinase was also manifested in the significant increase in NPN during storage (P < 0.05). Relatively small amounts of the enzyme present in the raw milk may result in proteolysis in the final product, as some proteases can increase both NPN and non-casein nitrogen fractions in milk (see review by Rogelj, 1992). Storage of powder did not significantly affect proteinase activity in UHT milk (P > 0.05). Changes in proteinase activity, however, were observed during storage of UHT milk at either 3 f 1°C or 254 1°C. Proteinase appeared to be more active at the higher

Reconstituted

temperature (PcO.01; Fig. 4). The detection of proteinase activity in UHT milk at 3 + 1°C supports the finding of Stepaniak et al. (1982). Indigenous and/or bacterial proteinases have been reported to be responsible for proteolysis in UHT-treated milk (Snoeren et af., 1979; Driessen. 198 1; Snoeren & Both, 1981; Suhren, 1983). Comparing these findings with the PAGE results, only slight differences in casein band intensity were observed between UHT milk processed from control or stored raw milk. During storage, UHT products which were reconstituted and processed from older powders showed a decrease in the intensity of the casein bands at an earlier stage. UHT milk processed from 2- and 4-month-old powders manufactured from either control or stored raw milk showed a slight decrease in intensity of the rx- and p-casein bands starting at the third to fourth month at 3f 1°C and at the second month at 251lzl”C (results not shown). For UHT milk processed from 6- and &month-old powders of both types, a change was noticed at the first-second and first month of storage at 31t 1°C or UHT milks processed from 25 f 1°C. respectively. freshly manufactured powders showed fainter casein bands starting at the fourth-fifth month at refrigeration temperature, and at the third-fourth month at 25f 1°C. The k-casein bands were not detected in the electrophoretograms. Electrophoretic patterns of the gelled samples showed decreased intensity of cx- and p-casein bands, which suggests in UHT milk processed from either proteolysis control or stored raw milk, with more gelled UHT milk samples processed from stored raw milk. Similarly, proteinase activity was greater in UHT milk processed from stored raw milk. These findings suggest a contribution of heat-resistant proteinases to gelation, in agreement with the reports of other authors on UHT whole milk and/or skimmilk (Zadow & Chituta, 1975; Carini & Todesco, 1977; Law rt al., 1977; Driessen & van der Waals, 1978; Snoeren et al., 1979; Driessen, 1983; de Koning et al., 1985). Apart from proteolysis, it appears that physico-chemical reactions may have also played a significant part in

UHT-treated

milk

135

the gelation process, more so because gelation was only observed in samples stored at 2511°C. Previous reports have shown degradation of CI-and pcaseins in UHT milk brought about by the action of native and/or bacterial proteinases (Adams et al., 1976; Law, 1979; Snoeren vt al., 1980b as cited by Suhren, 1983; Fairbairn & Law, 1986). However, electrophoretic patterns of proteins in the present study could suggest that the decrease in intensity of the casein bands may also be partly due to its interaction with the whey proteins. After UHT processing, bands corresponding to P-lactoglobulins A and B either lost sharpness/became diffused or completely disappeared (results not shown). Further, electrophoretograms of the caseins did not show k-casein bands. During storage, two to four slow-moving undefined bands, possibly polymers, appeared in the electrophoretograms of the whey proteins. Polymerisation and associated Maillard reactions could have led to alterations in electrophoretic mobility and formation of various protein artefacts, resulting in a loss of definition of the bands (Andrews, 1975). Polymer formation appeared to increase with storage period, but not with temperature. A reduced number of fainter polymer bands were observed in electrophoretic patterns of UHT milks stored at 2511°C as compared to those stored at 31t 1’C. This may have been due to the lower amount of sulphydryl groups in UHT milks stored at 25& 1“C. Liberation of sulphydryl groups, resulting from heating of P-lactoglobulin, facilitates the formation of P-lactoglobuhn/k-casein complex and such complex makes the access of proteinases to caseins more difficult, and so reduces the degree of proteolysis (Rollema & Poll, 1986). Availability of sulphydryl groups during storage of UHT milk would depend on its degree of oxidation. In the sensory evaluation, oxidation in UHT milks was perceived to be faster at a storage temperature of 25f 1°C. Consequently, less sulphydryl groups could have been available to induce polymerisation or complex formation at such temperature, as suggested by Calvo et ul. (1993). Complex formation, which was shown in the present study to be more distinct at 31 l”C, may have also contributed to possible protection of the caseins against proteinase attack, thereby partly delaying gelation. Lipolysis

Storage Period (Months) /+

3+1”c

-

25+1 “C

Fig. 4. Proteinase activity (nmol L-leucyl-L-leucine min -’ mL_‘) in reconstituted UHT milk stored for up to 6 months at 3!~ 1°C or 25zt 1°C. Differences between treatments (UHT milk storage temperature and period; n=28) were significant (PC 0.01).

The results of the present investigation suggest that some lipases in the UHT milk were heat resistant as evidenced by their activity during storage. The lipolytic activity was likely of microbial origin; complete inactivation of native milk lipase by pasteurisation has been reported (Cogan, 1980). Free fatty acid (FFA) content was significantly greater (PC 0.05) in UHT milk processed from stored raw milk (1.27 and 1.19 meq L-’ for UHT milk processed from stored and control raw milk, respectively). Lipolytic activity was shown by the increases in FFA concentration (P < 0.01; Figs 5 and 6). Increases in FFA content were detected at both 3&l “C and 25f 1“C, but greater values were obtained at the higher storage temperature. For instance, when 2-month-old milk powder was used in processing UHT milk, FFA concentration of 1.23 meq L-’ milk was measured only at the sixth month of

136

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1.4 1

1.6 ,

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L

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2 3 4 5 6 1 Storage Period of UHT Milk (Months) -.-0

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2 3 4 5 Storage Period of UHT Milk (Months)

6

+-8

Fig. 5. Free fatty acid concentration (meq L-‘) in reconstituted UHT milk, processed from whole milk powders stored for 0, 2, 4, 6 or 8 months, and kept for up to 6 months at 3~t 1°C. Differences between treatments (powder storagexUHT milk storage temperature and period; n = 6, except for fresh powders where n = 4) were significant (P < 0.01).

Fig. 6. Free fatty acid concentration (meq L-‘) in reconstituted UHT milk, processed from whole milk powders stored for 0, 2, 4, 6 or 8 months, and kept for up to 6 months at 25 f 1“C. Differences between treatments (powder storage x UHT milk storage temperature and period; n = 6, except for fresh powders where n = 4) were significant (P < 0.01).

storage at 3f 1°C while a similar concentration was detected in UHT milk stored at 255 1“C as early as the second or third month. Results of previous studies on UHT recombined milk and/or UHT whole milk have shown increases in FFA during storage at refrigeration, room and/or elevated temperatures (Schmidt & Renner, 1978; Christen et al., 1986; Mittal et al., 1988; Shahin & Fahmy, 1988; Reddy et al., 1991). Lipase activity in UHT milk was higher when stored raw milk was used (P < O.Ol), and when manufactured from 2- or 4-month-old powders (P < 0.05; Table 3). At both storage temperatures of UHT milk processed from powders which were stored for different periods, the peak value of lipolytic activity was measured at the third month of storage using 2-month-old powder. Microbial lipases were reported to be most active at a temperature of 3&4O”C (Lawrence, 1967). However, the marked increase in FFA at 3f 1“C gives an indication that lipase can be active even at such a low temperature. A study by Landaas & Solberg (1978) showed high activity of Pseudomonas jluorescens lipase

even at temperatures

near 0°C. Results of the present study therefore also suggest that the presence of more active microbial lipases may cause increases in FFA regardless of the temperature of storage.

Flavour Refrigerated UHT milk samples were generally rated ‘satisfactory’ throughout the storage period of 6 months, while at 25f l”C, the samples obtained ‘satisfactory’ ratings up to 2-3 months of storage. UHT milk processed from stored raw milk obtained lower (PcO.05) flavour scores (3.85 and 4.01 for UHT

milk produced from powders manufactured from stored and control raw milk, respectively). A slight sweet taste which appeared acceptable to some panelists was noted after l-2 months at both storage temperatures. Hansen (1987) reported that a sweet taste in UHT milk was noted frequently by panelists, and appeared after the cooked flavour dissipated. Cooked/burnt flavour was detected in all

Table 3. Mean Lipase Activity in Reconstituted UHT Milk, Processed From Whole Milk Powders Manufactured and Stored (4f l”C, 48f2 h) Raw Milk, and Kept for Different Periods at 25f 1°C Storage period of powder (months)

0 2 4 6 8 Raw milk treatment meant

Lipase activity” (pm01 pyrenyl butyric acid min-’ mL_‘) Raw milk treatment Control

Stored

0.102 0.127 0.113 0.086 0.085 0.103 (0.004)

0.119 0.144 0.130 0.103 0.095 0.118 (0.004)

From Control Powder storage meanb

0.11 labc (0.008) 0.136a (0.007) 0.122ab (0.006) 0.094bc (0.004) 0.09oc (0.003)

aMeans from all UHT milk samples kept at both temperatures for all periods in all replicates. bCompared using DMRT; Powder storage means within the column followed by a common letter are not significantly different (P > 0.05; n = 78, except for fresh powder where n = 52). Figures in brackets are standard errors of the means. ‘Significant difference between raw milk treatments (P < 0.01; n = 182).

Reconstituted UHT-treated milk freshly manufactured UHT milk and dissipated more quickly in samples stored at 25* 1“C. A significant interaction effect of UHT milk storage temperature and time with raw milk treatment (I’< 0.05) and powder storage (P
131

Some panelists detected very slight rancidity at the fifth to sixth month and slight rancidity at the second month of storage of samples at 3 * 1“C and 25~t l”C, respectively. The degree of rancidity increased with storage period at 25f 1“C and was perceived as more pronounced in UHT milk processed from powder manufactured from stored raw milk. An astringent flavour, described as powdery/chalky, was detected at a ‘very slightly noticeable’ level at 4-6 months at 2511°C and at 6 months of refrigerated storage. A slightly bitter taste was also perceived in some samples at six months’ storage at 251t 1°C. Some panelists detected an astringent/powdery flavour, but without accompanying bitter taste, after the first month of storage of UHT milk at both temperatures. Astringency in highly heated milk has been attributed to (1) an interaction product involving whey proteins, calcium phosphate and caseins (Josephson et al., 1967) and (2) the production of polypeptides by the action of proteases that survived UHT treatment (Harwalkar, 1972: Harwalkar ct al., 1989). Although the present study has shown increases in proteinase activity at certain times of storage at 25+ 1°C (Fig. 4), such a phenomenon was only detected sensorically at six months, as indicated by the appearance of bitter flavour. Viscosity No difference in viscosity scores was observed in UHT milk samples stored at different temperatures (311°C or 25f- 1YZ) for the same period. However, viscosity measured instrumentally was greater in stored at refrigeration temperature (see samples Physical properties section).

CONCLUSIONS The main effects of storage temperature and/or time on the physical and chemical properties of UHT milk made from reconstituted whole milk powder were changes in colour, NPN, pH, sediment and creamline. Storage of raw milk (4rt 1°C. 48&2 h) did not affect the physical and chemical properties of UHT milk (P > 0.05). An effect due to the interaction between powder storage and UHT milk storage temperature and time was found for some parameters (PC 0.05). When older milk powders were used, colour b-values of UHT milk decreased. With longer storage at either 3% 1 C or 25f 1“C, greater sediment and lower pH were observed in UHT milk processed from older milk powders. Lipases and proteinases were active during storage of UHT milk, with the activities being higher in UHT milk processed from powder manufactured from stored raw milk. Enzymatic and oxidation reactions appeared to increase at higher storage temperature (25~t 1“C) of UHT milk, resulting in the development of off-flavours during prolonged storage. In contrast to previous reports on UHT milk (Mottar, 1981; Pande & Mathur, 1992), the present study showed that lipolysis appeared to affect the taste of reconstituted UHT milk more adversely than proteolysis because, lipolytic rancidity, apart from being more pronounced than bitter flavour, was perceived earlier during storage. Lipase activity in UHT milk was significantly affected by powder storage

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(PC 0.05). Proteinase activity in UHT milk remained constant regardless of storage period of powder. The interaction between powder storage and UHT milk storage temperature/period was found to affect lipase activity and FFA content in UHT milk. A high concentration of FFA in the powder could have influenced the flavour acceptance of the reconstituted UHT milk. Further, the fat in UHT milk may have played a possible protective role for protein against proteinase attack, as suggested by Lopez-Fanditio et al. (1993). The intense heat imparted during indirect UHT processing has probably inactivated a high proportion of proteinases, resulting in the occurrence of only a small number of gelled samples. Moreover, gelation, which was observed only at 25f 1°C could be attributed not only to proteolysis but also to physicochemical reactions. The strong effect of lipases may also indicate either the presence of the enzymes in large concentration resulting from high numbers of lipolytic bacteria in the raw milk (Celestino et al., 1996a), or the presence of more heat-resistant and/or more active bacterial lipases.

ACKNOWLEDGEMENTS The authors wish to thank Prof. William Sawyer for his assistance with lipase assay, and Messrs John Hardham and Andrew Lawrence (CSIRO, Highett, Victoria, Australia) for their assistance in UHT milk processing. Helpful discussions with Drs M. A. Augustin and J. G. Zadow are gratefully acknowledged. This research project was financially supported by the Australian Agency for International Development (AusAID).

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