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The effects of refrigerated storage of raw milk on the quality of whole milk powder stored for different periods
ht.
Dairy Journal 7 (1997) 119-127 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain PI
ELSEVIER
I:
SO958-6946(96)00041-6
0958.6946:97/$17.00+0.00
The Effects of Refrigerated Storage of Raw Milk on the Quality of Whole Milk Powder Stored for Different Periods Ernani L. Celestino, Mani Iyer* and Hubert Roginski Gilbert Chundler College. Faculty of Agriculture.
(Received
Forestry und Horticulture, The University 3030, Victoria, Australia 24 September
1995; accepted
28 August
qf Melbourne, Sneydes Road, Werribee
1996)
ABSTRACT Whole milk powders were produced from fresh (control) and stored (4f 1°C. 48f2 h) raw milk in three seasons (spring, summer and autumn), and stored for up to 8 months at 25fl”C. The suitability of the milk powders for reconstitution processes was assessed on the basis of selected microbiological, physical, chemical and biochemical quality parameters. Storage of raw milk did not significantly affect total bacterial count (TPC) in the resultant powder. Free fatty acid (FFA) content in powder manufactured from stored raw milk was greater than that from the control. TPC and free fat content decreased while insolubility index, FFA and peroxide value increased during storage of powder; lipase and proteinase activities did not change. All powders manufactured from control and stored raw milk, and stored for up to 8 months, were found suitable for reconstitution on the basis of TPC, moisture content and amount of scorched particles but not based on insolubility index. 0 1997 Elsevier Science Ltd.
for reconstitution processes in terms bacteriological, physical and compositional
INTRODUCTION The microbiological quality of whole milk powder is determined by the initial number and type of microorganisms present in the raw milk from which it is manufactured and the extent of post-processing contamination. The primary psychrotrophic microflora of milk are Gram-negative rods (Law, 1979). Although are killed by these psychrotrophs most of pasteurisation, many species produce heat-stable lipases and proteinases which retain activity following 1980) and spray drying pasteurisation (Cogan, (Shamsuzzaman et al., 1987; Ipsen & Hansen, 1990). The presence and activity of these enzymes in milk powder may result in physico-chemical changes, as well as defects in functional and sensory properties of the powder, consequently affecting its keeping quality. Whole milk powder has been reported to have a maximum shelf-life of about 6 months (Anon., 1989) at room temperature. However, it was found that whole milk powder can have a shelf-life in excess of 12 months if packed in cans under vacuum or an inert gas, such as nitrogen, to inhibit the development of offflavours (Kieseker & Aitken, 1993). Recent investigations (Celestino et al., 1996) have shown an increase in numbers of lipolytic and proteolytic bacteria and dominance of psychrotrophs after storage of raw milk at refrigeration temperature (41tl”C) for 2 days. The present study aims to relate the quality of raw milk with the physical, chemical, microbiological and biochemical properties of the resultant whole milk powder and assess its suitability
*Corresponding
MATERIALS Manufacture, powder
of selected parameters.
AND METHODS packaging and storage of whole milk
A 600 L sample of bulk raw milk was taken in each season (spring, September 1992; summer, December 1992; and autumn, March 1993) to account for variations in milk composition and quality throughout the 1992/93 milk supply period. In Victoria, milk for processing into products other than market milk is collected for approximately 10 months, from August to June. In the present investigation, each sample was taken from the same 86,000L factory silo, representing approximately 5,700 cows from 45 herds. Details of bulk milk sample collection are available (Celestino et al., 1996). Each milk sample was standardised to a fat:solids-not-fat ratio of 3.8:8.5, then divided into two equal batches. One batch was stored in the factory at 4fl”C for 48*2 h while the other served as the no storage control. Each batch of milk was manufactured into powder. During powder manufacture, every effort was made to ensure that all variations in thermal processing were kept to a minimum. The milk was given a heat treatment suitable for a medium heat powder (75°C for 3min), using a tubular heat exchanger and holding tubes. The milk was introduced into a double effect falling film evaporator at a temperature of 60°C with the first effect at 80°C and the second at 63°C producing a concentrate with 45%
author. II’)
120
E.L.Celestino et al.
total solids. The concentrate was homogenised in a two stage (17.23 MPa/3.45 MPa) Creamery Package Homogeniser (Model 3DDl; The Creamery Package Manufacturing Company, Chicago, Illinois, USA) at 50°C. The homogenised concentrate was dried on a pilot-scale, single stage Niro Production Minor spray drier (Niro Australia Pty. Ltd., Blackburn, Victoria). The concentrate was preheated to 65°C prior to centrifugal atomisation at 21500 rpm with distributor holes of 1.2 mm diameter. The inlet air was at 180°C and the feed flow rate was adjusted to give an outlet air temperature of 85°C. The powder was removed at 15 min intervals from the collection bucket, bagged and cooled to about 30°C in a cold storage (4°C) room. The powder produced from each batch of raw milk was divided into five lots (about 5.5 kg per lot). Each lot of powder was packed in three-ply multiwall bags with polyethylene barrier lining (thickness 60pm) and sealed with an elastic ring. The respective lots of powder were stored at 25fl”C for 0, 2, 4, 6, and 8 months. Sampling, microbiological and physical analyses Sampling of powder for all the tests was according to the method of the Standards Association of Australia (SAA) [AS 1166 (SAA, 1974a); AS 1629 (SAA, 1974b); AS 1095.2.8 (SAA, 1983)]. The following bacterial counts were determined: total plate [AS 1095.1.2 (SAA, 1971); AS 1095.2.1 (SAA, 1981)], psychrotroph [AS 1095.3.5 (SAA, 1976)], lipolytic [AS 1095.3.4 (SAA, 1976)], proteolytic [AS 1095.3.6 (SAA, 1975)] and mesophilic, thermophilic and psychrotrophic aerobic, and mesophilic and thermophilic anaerobic spores [AS 1095.3.7 (SAA, 1979a; APHA, 1984)]. Milk powder samples were analysed for bulk density, insolubility index and scorched particles in accordance with AS 1629 (SAA, 1974b). Chemical tests Compositional tests and pH
The procedures set by the SAA (SAA, 1988SAA, 1991) for the determination of total solids/moisture [AS 2300.1.1 (SAA, 1988)], fat [gravimetric method, AS 2300.1.3 (SAA, 1988)] and total nitrogen [AS 2300.1.2.1 (SAA, 1991)] were followed. For nonprotein nitrogen (NPN) content determination, milk powder was reconstituted to 12.3% total solids. The sample was then treated as in determining NPN in liquid milk [AS 2300.1.2.2 (SAA, 1988)]. NPN was calculated as for liquid milk except that the value was multiplied by the appropriate dilution factor. The free fat content of powder was determined based on the method described by Niro Atomizer (1978). The pH was determined at 20-22°C using a digital pH meter (TPS Pty. Ltd., Brisbane, Australia), as specified by the SAA (1989) for determining pH of dried milk (AS 2300.1.6). Pyruvic acid (pyruvate)
Whole milk powder was reconstituted to 12.3% total solids. The sample was then treated as for liquid milk (Boehringer Mannheim GmbH Biochemica, 1984). Absorbance was determined at 340 nm using a SequoiaTurner spectrophotometer (Model 340; Sequoia-Turner
Corporation, Mountain View, California, USA). The pyruvate content of the sample was expressed in mg 100 g-’ milk powder. Whey protein nitrogen index (WPNI) was determined following the procedure AS 1629 set by the SAA (1974b). Peroxide value
Determination of peroxide value was based on the ferric thiocyanate method described by Newstead & Headifen (1981). However, in addition to the reagent blank, a fat blank was also prepared consisting of the chloroform-methanol solvent, fat sample and ammonium thiocyanate. Sample absorbance readings were corrected for both reagent and fat blanks. Absorbance was measured at 500nm using a Sequoia-Turner spectrophotometer (Model 340). The peroxide value of the fat was expressed as meq oxygen kg- ’ . Biochemical assays Preparation of samples for biochemical analyses was accomplished by reconstituting whole milk powder to 12.3% total solids. Lipolysis
The acid degree value (ADV) of the prepared sample was determined according to the procedure outlined by Deeth & Fitz-Gerald (1976) for liquid milk. ADV was expressed in meq lOOg-’ fat. The free fatty acid (FFA) content of the prepared sample was analysed by extraction-titration method (Deeth & Fitz-Gerald, 1976) and expressed in meqkg-’ milk powder or meq lOOg-’ fat. Lipase activity was measured by means of a spectrofluorometric technique. The samples were prepared according to procedures described by Liodakis et al. (1991) Iverius & Ostlund-Lindqvist (1986) and Negre et al. (1988) as outlined by Celestino et al. (1996). Lipase activity was expressed as pmol pyrenyl butyric acid min-’ gg’ milk powder. Proteolysis
The procedures for fluorescamine method (Chism et 1979) and polyacrylamide gel electrophoresis $AGE) d escribed by Richardson & Newstead (1979) and NglKwai-Hang & Kroeker (1984), were followed with some modifications as outlined by Celestino et al. (1996). Statistical analysis Analysis of variance of the data for each parameter (except for aerobic psychrotrophic and anaerobic thermophilic counts, and results of PAGE and scorched particles test, which were interpreted descriptively) was a split plot in randomised complete block design. In this design, raw milk storage treatment was the mainplot, and storage period of powder was the subplot. Treatment means were compared using the Duncan’s Multiple Range Test (Steel & Torrie, 1980; Freund et al., 1986). Results for proteinase activity (fluorescamine method) were analysed with unequal number of observations.
of storuge on quality @‘whole
E&V
RESULTS
AND DISCUSSION
Microbiological quality The total plate counts (TPC) obtained for all powders were in accordance with the recommended norm for the microbiological standard of whole milk powder for reconstitution processes which is 2 10,000 cfu gg ’ (Jensen, 1990). Sanderson (1979) recommended a maximum viable plate count of 50,000 cfu gg’ of whole milk powder for reconstitution. The differences in TPC, proteolytic (PC), lipolytic (LC) and psychrotrophic (PSC) bacterial counts between powders produced from control or stored raw milks were found to be insignificant (P> 0.05; Table 1). As expected, heat treatment employed during powder manufacture resulted in a decreased number of psychrotrophs. Most psychrotrophs are heat sensitive. Kwee et al. (1986) reported reduction of psychrotrophs to negligible levels, specifically during the preheating stage of powder manufacture. TPC decreased with storage of powders (P < 0.01; Table 2). LC decreased (PcO.01) at eighth month of storage while PC and PSC did not change (f’> 0.05) with storage of powder. The decrease in microbial population during storage could be attributed to the low water activity (a,) of milk powder. Most bacteria cannot grow at an a, below 0.9; generally, powders have an a, much below 0.6 (Early, 1992). Furthermore, the powders underwent oxidation during storage; thus, the products of chemical oxidation may have had an effect on the viability of the organisms. Several authors (Davis, 1955; Crossley, 1962 as cited by Lovell, 1990; Mair-Waldburg & Lubenau-Nestle, 1988) reported that the microbial 1974; Renner, population decreased during storage of milk powder. Table 1. Mean Bacterial Counts”
Raw Milk Treatment Control Stored
TPC’ (x 102)
in Whole
Milk Powders
h
PSCh (x 102)
;:102,
25.6 (5.4)d 1.9 (1.1) 45.7 (7.9) 3.1 (1.4)
2.4 (0.6) 5.2 (1.2)
121
Love11 (1990) stated that raw milk counts in excess of lo5 cfu mL_’ can result in counts of over 104cfu gg’ in the dried product. However, this was not observed in resent study. The powders contained TPC of ‘2”,0Ycfu ggr even when raw milks from which the powders were manufactured had TPC of l.2x105cfumLand l.lx106cfumL~ (Celestino et al., 1996). This may again suggest that it is not simply a question of bacterial numbers but also of types, and that thermoduric organisms, if present in the raw milk, together with heat-resistant sporeformers, may consequently occur in the dried milk product. No significant difference was found (P> 0.05; Table 1) in aerobic mesophilic and thermophilic, and anaerobic mesophilic spore counts, between powders manufactured from control and stored raw milk. Storage of powder did not affect the numbers of aerobic and anaerobic sporeformers (P > 0.05; Table 2). The number of aerobic and anaerobic mesophiles appeared greater than their thermophilic counterparts for all powder treatments (Tables 1 and 2). Aerobic psychrotrophic sporeformers were absent in powder. Physical properties Bulk densit?
As expected, bulk densities of powders manufactured from control or stored raw milk were not significantly different and did not change with storage of powder (P > 0.05; data not shown). Scorched purticles
Scorched that have evaporator
Manufactured (n= 15)
b
milk powder
Aerobic
particles usually originate from milk solids been held up somewhere within the and/or drier, and thereby have been
from Control
and Stored
Sporeformers
(411°C.
48f2 h) Raw Milk
Anaerobic
Sporeformers
(LxYO2) 4.2 (1.0) 6.3 (1.0)
Mesophiles’
Thermophilesb
Psychrotrophs”
Mesophilesb
Thermophiles’
25.0 (4.8) 44.0 (10.7)
7.3 (2.0) 13.0 (1.7)
nde nd
33.5 (8.1) 299.0 (150.3)
<2to2 ~2 to 6
a Means for powders (as cfu gg ‘) stored for all periods in all replicates. ’ No significant difference between raw milk treatments (P > 0.05). ‘Not subjected to ANOVA; result for anaerobic thermophiles is the range of 15 readings. d Figures in brackets are standard errors of the means. ‘nd = Not detected. Table 2. Mean Bacterial
Counts” h
Storage Period (Months)
TPC’ (x102)
0
54.3a (12.4)'
5.8a (3.7)
4.6~~(2.4)
2
50.&b
3.2~~(2.1)
3.8~1(1.6)
4
41.8b (8.1)
I.7a (1.1)
6
21.9c (10.5)
8
9.5d (4.3)
"
PSCh (x102)
(9.5)
in Whole Milk Powders
;:102,
b
Stored for Different
Aerobic
Periods
Sporeformers
at 2511°C
(n = 6)
Anaerobic
Sporeformers
:02, Mesophiles’
ThermophilesC
Psychrotrophsd
7.4a (1.8)
37.5 (14.0)
II.7 (4.6)
nd
15.8 (4.1)
<2to2
5.7~1(1.6)
35.0 (14.2)
9.2 (3.0)
nd
290.0 (264.1)
<2to5
3.8a (0.9)
6.8a (1.8)
34.2 (14.5)
9.2 (3.0)
nd
299.0 (121.0)
<2to5
I.la (0.6)
3.8a (1.5)
4.9a (1.2)
33.3 (14.8)
10.2 (3.1)
nd
56.5 (37.5)
<2to6
0.6a (0.5)
2.7a (1.6)
l.4b (0.7)
32.5 (14.9)
10.7 (2.8)
nd
170.0 (150.3)
<2to5
Means for powders (as cfu gg
‘) made from control and stored raw milk
b Compared using DMRT; means within a column followed by a common ‘No significant difference between storage periods of powder (P > 0.05). dnd = Not detected. ‘Not subjected to ANOVA and DMRT; result for anaerobic thermophiles ‘Figures in brackets are standard errors of the means.
in all replicates. letter are not significantly
is the range of 6 readings.
Mesophiles”
different
Thermophiles’
(P > 0.05).
122
E. L. Celestino et al.
overheated or burned. In the present study, occurrence of scorched particles gave a Disc A rating [or 7.5mg32.5g-’ powder; AS 1629P (SAA, 1979b)] for powders processed in spring and summer. This is within the recommended norm (maximum 7.5mg) for reconstitution processes for whole milk powder (Jensen, 1990). However, control and stored raw milk in autumn yielded powders with higher scorched particles (Disc B rating or 7.5-15.0 mg 32.5 g-’ powder; results not shown). These differences could be due to slight day-to-day variations in drier performance caused by changes in factors such as ambient air temperature and humidity. It is generally recognised that these factors are hard to control. Another possibility is that autumn raw milk is less stable because of its higher NPN (Ghatak et al., 1989) protein (Singh & Newstead, 1992) and, possibly, higher calcium content (Ghatak et al., 1989), resulting in greater burn-on tendency during evaporation. However, the autumn powders were still within the official standard limit for scorched particles content. Brady (1972) reported that milk is usually most stable in spring and early summer. For reconstitution, Sanderson (1979) recommended a Disc B rating or better for whole milk powder of less than 9 months age.
Table 3. Insolubility Index of Whole Milk Powders, Manufactured from Control or Stored (4&l”C, 48f2h) Raw Milk and Stored for Different Periods at 25fl”C
Insolubility index
PH Fat, % m/m Free fat, % m/m Crude protein, % m/m Non-protein nitrogen, % m/m True protein, % m/m Moisture, % m/m Whey protein nitrogen index, mg gPeroxide value, meq oxygen kg-’ fat Pyruvic acid, mg 100 gg ’
Insolubility indices of powders manufactured from stored raw milk were higher than those manufactured from control raw milk but the differences were not significant (P > 0.05; Table 3) and the values obtained were within the official standard specification of not greater than 0.5mL (Jensen, 1990). A maximum of 0. I mL was recommended for reconstitution processes (Sanderson, 1979; Jensen, 1990). Storage of powder at 25fl”C significantly increased the insolubility index of powder (P < 0.01; Table 3), with higher values obtained at 6 and 8 months. When all powders manufactured from the raw milk in different replicates were compared, insolubility index test results showed that freshly manufactured and 2- and 4-month-old powders produced from control or stored spring raw milk, and freshly manufactured powder from control summer raw milk, had insolubility index of 0.1 mL. An insolubility index of > 0.5 mL was measured at 8 and 6 months of storage in powders manufactured from control and stored autumn raw milk, respectively. Increases in the insolubility index of whole milk powder with storage were also reported by van Mil & Jans (1991). Kudo et al. (1990) cited several factors contributing to decreased solubility during storage, such as crystallisation of lactose, non-enzymatic browning, higher moisture content and elevated storage temperature of powder. However, the mechanism of the formation of insoluble material in milk powder when held at temperatures ranging from 20 to 100°C is still not known (Kudo et al., 1990). Chemical properties Composition and pH
The pH of powder was not affected by raw milk storage (P > 0.05; Table 4) and was found to gradually decrease with storage of powder (PC 0.01; Table 5). Freshly manufactured powder had a pH of 6.63 which decreased by only 0.03 after 8 months of storage. The slight change in pH may be due to the formation of
Insolubility Indexa (mL) Raw Milk Treatment
Storage Period of Powder (Months)
0 2 4 6 8
Control
Stored
0.13 0.17 0.14 0.30 0.37
0.20 0.21 0.18 0.43 0.43
(0.03) (0.04) (0.03) (0.10) (0.12)
(0.06) (0.05) (0.04) (0.13) (0.12)
aMeans for powders in all replicates. No significant difference between raw milk treatments (n= 15) (P>O.O5); significant differences between storage periods of powder (n= 6) (PC 0.01). Figures in brackets are standard errors of the means.
Table 4. Mean Value? for the Different Chemical Properties of Whole Milk Powders Manufactured from Control or Stored (4&1”C, 48f2 h) Raw Milk (n= 15) Chemical Attribute
Raw Milk Treatment Control
Stored
6.62 28.33 0.44 26.10 0.33 24.02 2.33 4.59
6.60 28.07 0.47 26.02 0.33 23.89 2.36 4.47
(0.01) (0.18) (0.03) (0.20) (0.02) (0.21) (0.05) (0.18)
(0.01) (0.15) (0.03) (0.22) (0.02) (0.21) (0.08) (0.18)
1.20 (0.20)
1.40 (0.24)
2.22 (0.12)
2.92 (0.14)
aMeans for powders stored for all periods in all replicates. No significant difference was found between raw milk treatments for any of the parameters (P > 0.05). Figures in brackets are standard errors of the means.
FFA as a result of oxidation as well as lipolytic activity during storage of the powder. Storage of raw milk did not significantly affect the composition of whole milk powder (P> 0.05; Table 4). occurred Some compositional changes, however, during powder storage (Table 5). Moisture content decreased slightly at the second month (PcO.05) but increased to approximately the initial level at the sixth and eighth months of storage. The values obtained at different periods of storage were below the maximum moisture content (3.0%) recommended by Sanderson (1979) for whole milk powder suitable for reconstitution. The change in moisture during storage of powder may be related to the change in the state of lactose, i.e. the shift from amorphous to the crystalline state. When moisture is absorbed by amorphous lactose, a part will be incorporated as water of crystallisation in the a-hydrate and the remainder will be released, since crystalline ahydrate is not hygroscopic (Supplee, 1926 as cited by Nickerson, 1974). The water of hydration of a-lactose is only partly removed by the standard oven-drying methods (Thomasow et al., 1972).
I!$&-t of’ storage on qua&v qf’whole milk powder Table 5. Mean Value? for Chemical Properties of Whole Milk Powders Chemical
Attribute
Storage 2
0 PH Fat, % m/m Free fat, % m/m Crude protein, % m/m Non-protein nitrogen. % m/m True protein, % m/m Moisture, % m/m Whey protein nitrogen index, rn? g -’ Peroxide value, meq oxygen kg- fat Pyruvic acid, mg 100 g- ’
Stored for Different
6.63a (0.01) 28.87a (0.29) O.SOab (0.03) 26.34a (0.31) 0.26b (0.04) 24.72a (0.23) 2.44ab (0.06) 4.72a (0.19) 0.55d (0. I 1) 2.27~ (0.28)
6.62b (0.01) 28.19b (0.19) 0.5la (0.03) 26.05ab (0.37) 0.32a (0.03) 24.02b (0.29) 2.2oc (0. I I) 4.38a (0.28) 0.93cd (0.16) 2.86a (0.22)
123 Periods
at 25f 1°C (n = 6)
Period (Months) 4
6.61bc (0.01) 28.25b (0.12) 0.50ab (0.07) 26.05ab (0.38) 0.33a (0.03) 23.9513 (0.34) 2.21 bc (0.09) 4.14a (0.34) 1.09bc (0.20) 2.78ab (0.21)
6 6.61bc (0.01) 27.98b (0.24) 0.39b (0.02) 25.90b (0.36) 0.37a (0.01) 23.55b (0.3 I) 2.37abc (0.14) 4.78a (0.25) l.5lb (0.30) 2.45~ (0.23)
8 6.6Oc (0.01) 27.72b (0.25) 0.39b (0.02) 25.96b (0.34) 0.38a (0.01) 23.53b (0.30) 2.50a (0.11) 4.64a (0.32) 2.40a (0.33) 2.50bc (0.29)
‘Means for powders made from control and stored milk in all replicates; compared using DMRT; means within a row followed common letter are not significantly different (P > 0.05). Figures in brackets are standard errors of the means.
Fat content slightly decreased (P < 0.05) at the second month of storage and then remained constant up to 8 months. Free fat content slightly decreased at the sixth month of storage. The decrease in fat and free fat contents could be due to their hydrolysis during storage which could be attributed to lipases which survived processing and were active during storage of the powder (Andersson, 1980). Furthermore, the possibility that the free fat enters into a fat-protein complex which is stable to solvent extraction has been reported (Litman & Ashworth, 1957; de Vilder et al., 1977). Pyruvic acid (pyruvate)
The pyruvate content of milk powder is influenced primarily by the number of bacteria in the raw material. Higher pyruvate content was observed in powder manufactured from stored raw milk than in powder from control milk, but the difference was not significant (P > 0.05; Table 4). Pyruvate content of powder appeared to change during storage, with variations from 2.27 to 2.86mg lOOg_’ of powder (Table 5). Pyruvate appeared highest in powder manufactured from stored raw milk in the autumn replicate (3.54mg lOOg_‘), which also had the highest bacterial numbers (Celestino et al., 1996). Sjollema (1986) suggested that in skim milk powder, the level of pyruvate can give an indication of the bacteriological quality of the milk from which the powder was made and that a value lower than 9 mg lOOg-’ of powder gives a reasonable guarantee of absence of heat-stable enzymes. All results obtained in the present study were below this level; however, proteolytic and lipolytic activity was detected in the powder during storage (Tables 6 and 7). Whey protein nitrogen index
WPNI measures the proportion of undenatured whey protein remaining after heat treatment. Electrophoretic patterns of the whey proteins in powders showed apparent change in intensity of the bands as a result of heat treatment applied during powder manufacture (results not shown). It appeared that a-lactalbumin was the most stable, followed by /Glactoglobulins A and B and serum albumin. WPNI has influence on several properties (Pisecky, 1990) including flavour, sediment and other physicochemical attributes, both in powder and in products
by a
manufactured from such powder. Results for WPNI showed that the medium heat powders manufactured from control or stored raw milk (Table 4) were within the heat classification limit for such types of powder. Medium heat powder should give a WPNI of 1.515.99 mg g-’ powder (American Dairy Products Institute, 1992). Throughout the year, some variations can be observed for WPNI due to variations in the composition of raw milk (Walstra & Jenness, 1984; Steen, 1977 as cited by van Mil & Jans, 1991). The content of milk varies whey protein nitrogen throughout the season, from area to area and also in its susceptibility to heat (Kieseker, 1982). In the present study, differences in WPNI between replicates may reflect variations in milk composition between seasons. Peroxide
value, ,free fat and free futty acids
The peroxide value is used as a measure of the extent of deterioration of milk fat due to oxidation (Munro et al.. 1992). In the present study, the peroxide value increased with storage of powders (PC 0.01; Table 5). The peroxide values obtained during the 8-month storage period of the powders were slightly higher than those obtained by van Mil & Jans (1991). Compared to their study, lower preheat treatment was employed in the present work, thus there was probably a lesser release of sulfhydryl groups from milk proteins. Also, the milk powders were packed in bags with a polyethylene barrier lining which could have allowed permeation of oxygen during storage, resulting in increased peroxide values. Catalytic oxidative processes can principally occur with free fat (Shipstead, 1952 as cited by Shipstead & Tarassuk, 1953; Kirst, 1986) as well as FFA (Kirst, 1986). The present study showed decreased free fat and increased FFA with storage of powder (Table 6). The possible reasons for the decrease in free fat during storage of powder were discussed above. Further, FFA appeared to result from both lipolysis (lipase activity was present during powder storage), and oxidation reactions (Munro et al., 1992). Sonntag (1979) reported that fats in an advanced state of oxidation develop acidity through the accumulation of acidic cleavage products. Thus, oxidation of powder may have contributed to the increase in FFA results during storage. The present results support the report of Kirst (1986) that
E. L. Celestino et al.
124 Table 6. Mean Valuesa for Biochemical
Raw Milk Treatment
Control Stored
Free Fatty Acidsb (meq kg- powder)
Properties
of Whole Milk Powders Raw Milk
Free Fatty Acidsb (meq 100 g-l fat)
9.07 (0.50)
3.18 (0.18)
10.20 (0.53)
3.64 (0.19)
Manufactured
Acid Degree Valueb (meq 1OOg ’ fat)
1.08 (0.04) 1.22 (0.05)
aMeans for powders stored for all periods in all replicates (n= 15, except for proteinase stored). Figires in brackets are standard errors of-the means. b Significant difference between raw milk treatments (PC:0.05). ‘No significant difference between raw milk treatments (P > 0.05).
oxidative changes in the milk fat are indirectly connected with the lipolytic changes. Since oxidation involves reaction between oxygen and the fatty acids, either as free (Deeth & Fitz-Gerald, 1976), or in the triglycerides (Munro et al., 1992), the greater the lipolysis, the greater would be the formation of FFA, and this would consequently provide for increased oxidation reactions (Deeth & Fitz-Gerald, 1976). It is known that FFA oxidise more readily than the corresponding glycerides (Sonntag, 1979). As oxidation reaction proceeds during storage, the fatty acids react with oxygen, by a radical chain mechanism, forming fatty acid hydroperoxides (Lingnert & Eriksson, 1981). The FFA content of stored raw milk was greater than that of control raw milk (Celestino et al., 1996). Consequently, higher FFA and ADV were observed in powder manufactured from the stored raw milk than in powder manufactured from the control. Ipsen (1989) showed correlation of lipolysis (expressed by the acidity of the milk fat) in raw milk with the oxidative changes in stored whole milk powder. Earlier, Ipsen & Hansen (1988) found that oxidative stability of whole milk powder was lower when made from raw milk with high TPC and FFA content. In the present study, peroxide value in powder manufactured from stored raw milk was higher than that from the control but the difference was not significant (P > 0.05; Table 4). There was no linear correlation between FFA in raw milk and peroxide value of powder; further, only a weak positive linear correlation (r=0.645, n = 12) of ADV with oxidation in milk powder was found. Further research is needed to conclusively define the correlation of lipolysis in raw milk with the oxidative stability of the powder. Table 7. Mean Values” for Biochemical
Properties
from Control or Stored (4&1”C, 48*2 h)
Lipase Activity’ (firno pyrenyl butyric acid
Proteinase Activity’ (nmol LIeucyl-L-
min-’ 9-l)
leucine min-’ g-‘)
0.73 (0.08)
74.8 (9.3)
0.88 (0.09)
93.8 (10.3)
activity, where n = 14 for control
and 13 for
Biochemical properties Lipolysis There was no significant difference in lipase activity between powders manufactured from control or stored raw milk (P>O.O5; Table 6), although lipase activity of stored raw milk was significantly higher than that of control (Celestino et al., 1996). During whole milk concentration by evaporation and drying, milk solids are concentrated approximately 8-fold. Lipase activity in powders (Table 6) was approximately 9 times higher than that in the corresponding raw milk (Celestino et al., 1996), which suggests that no loss of lipolytic activity occurred during powder production. Storage of raw milk prior to powder manufacture resulted in whole milk powder with higher ADV and concentration of FFA (PC 0.05; Table 6). Lipase activity in the stored raw milk was higher than that in the control raw milk and this resulted in greater FFA content of the stored raw milk (Celestino et al., 1996). Although the lipase activities in the powders manufactured from control or stored raw milk were not significantly different, the FFA in the powder manufactured from stored raw milk was significantly higher than that in the product manufactured from the control raw milk. Lipase activity was stable throughout the S-months storage of powder (P>O.O5; Table 7). Significant increases in FFA and ADV were observed during storage (PC 0.01; Table 7). Renner (1988) reported that, in milk powder, enzyme activity ceases at an a, of < 0.6 or powder with approximately 4% water content. On the other hand, Andersson (1980) found that at uW values of 0.54 and lower, iipolysis took place at a fairly constant level in full-cream milk powder. Acker (1969),
of Whole Milk Powders
Stored for Different
Periods
at 25fl”C
Storage Period (Months)
Free Fatt Acidsb Y (meq kg- powder)
Free Fatty Acidsb (meq 100 g-’ fat)
Acid Degree valueb (meq 100 g-’ fat)
Lipase ActivityC (pm01 pyrenyl butyric acid min-’ g-‘)
Proteinase Activity” (nmol Lleucyl-lleucinemin-’ g-‘)
0 2 4 6 8
7.5% (1.18) 9. lob (0.80) 10.19ab (0.38) 10.38ab (0.28) 10.96a (0.53)
2.63~ (0.43) 3.52b (0.29) 3.60ab (0.15) 3.7lab (0.10) 3.95a (0.18)
0.87~ (0.03) 1.12b (0.06) 1.23a (0.07) 1.25a (0.05) 1.28a (0.07)
0.67 (0.19) 0.64 (0.14) 1.02 (0.13) 0.90 (0.08) 0.8 1 (0.07)
48.4 69.2 90.2 108.6 95.8
’ Means for powders made from control and stored milk in all replicates (n = 6, except for proteinase storage, where n = 5). Figures in brackets are standard errors of the means. b Compared using DMRT; means within a column followed by a common letter are not significantly ‘No significant difference between storage periods of powder (P > 0.05).
(14.X) (11.4) (11.9) (20.3) (8.4)
activity at 0 and 2 months different
(P > 0.05).
of
ITf;ect of storuge on quality of ~zhnle milk powder
as cited by Andersson (1980) explained that at low u, values the amount of available water is limited, thus changing the water-lipid interface whereby the number of reaction sites may be reduced. The fact that substrates for lipases are water insoluble and that the water phase is not the only location for the reactions catalysed by lipases may explain why lipolysis occurs at low u, values. In the present study, accumulation of FFA in the powder may be attributed to the stable lipase activity observed during storage. However, the effect of oxidation should also be considered. During storage of powder, FFA values ranging from 2.63 to 3.9.5meq lOOg_’ fat were obtained (Table 7) which are much higher than the threshold values of 1.5 to 2.0meq lOOg_’ fat reported for rancid flavour (IDF. 1991). However, high levels of FFA are not always associated with the development of off-flavours (Deeth ct al., 1979). FFA usually arise from lipolysis, but can be formed also as by-products of oxidation (Munro et cd., 1992). Thus, in the present study, increases in FFA and ADV during storage of powder may have been a result of the lipolytic activity as well as oxidation reactions. Compared to ADV, results of extractiontitration of FFA (in meq lOOg_’ fat) showed higher extent of lipolysis in powder (Tables 6 and 7). This could probably be due to the fact that ADV determination does not recover the short-chain FFA (C4-C8) in the fat separation process and only partially recovers the medium-chain (ClO-C16) FFA (Duncan & Christen, 1991). The fatty acids implicated in rancid flavour are C4-Cl2 (Scanlan et uf., 1965) and these vary in water solubility/insolubility. Some of these fatty acids, including butyric, caproic and even caprylic, may be partially released into the aqueous phase of the milk (Duncan & Christen, 1991). Thus, the ADV method is measuring only those FFA which are fat-soluble and remain in the fat during the separation procedure. A high ADV would primarily reflect a change in concentration of the long-chain fat-soluble FFA but increase in indicate an necessarily does not concentration of the volatile short-chain FFA (Duncan cr [Il., 1990). Proteolvsis
powder was greater in Proteinase activity manufactured from stored raw milk than from control raw milk, but the difference was not significant (P > 0.05; Table 6). This was supported by the results of PAGE as well as NPN content of powder. As shown in Table 4, the NPN content of powder was not affected by raw milk storage. Further, no change in intensity of the protein bands was observed during storage of the powder manufactured from control or stored raw milk (results not shown). The detected activities of proteinases in the powders manufactured from control or stored raw milk indicate resistance of some proteinases to the heat treatment applied during powder manufacture. Ipsen & Hansen (1990) attributed proteolysis in dried whole milk to heat-stable proteolytic enzymes which remain active after spray drying. The results of the present study show that proteinases retained their activity during storage of powder. In milk powders where a, is very low, degradation of proteins through the action of proteinases would not be expected. However, proteinases, especially the bacterial
125
types, may not be inactivated by the heat treatment employed during spray drying, and may thus possibly remain active during the powder storage (Ipsen & Hansen, 1990). In the present study, this has been shown by the proteinase activity detected (Table 7), but no proteolysis was evident as supported by the results of PAGE (results not shown).
CONCLUSIONS Storage of raw milk (4&l”C, 48f2 h) resulted in whole milk powder with higher values for FFA and ADV. There was an approximately 9-fold increase in lipolytic activity of the powder compared to that of corresponding raw milk which matches the degree of concentration and therefore suggests no overall loss of lipolytic activity during evaporation and drying. The activities of lipases and proteinases remained stable during storage of powder at 2511°C. The production of FFA appeared to be a consequence of lipolysis and oxidation reactions. Some changes in chemical and physical properties were observed during storage of powder. Free fat content slightly decreased, possibly due to either its hydrolysis or combining with an unstable protein to form a complex. Insolubility index and peroxide value increased with storage of powder. Bacterial count, moisture level and amount of scorched particles in powders made from control and stored raw milks, and stored at 25fl”C for up to 8 months, suggest that all powders were suitable for reconstitution.
ACKNOWLEDGEMENTS The authors wish to thank Professor William Sawyer for his assistance with lipase assay, and Mr Phil Clarke (CSIRO, Highett, Victoria) for the manufacture of whole milk powder. Helpful discussions with Dr Mary Ann Augustin (CSIRO) are gratefully acknowledged. This research project was financially supported by the Australian Agency for International Development (AusAID).
REFERENCES Acker,
L.W. (1969). Cited by Andersson, R.E. (1980). Microbial hpolysis at low temperatures. Appl. Environ. Microhiol., 39, 3&40. American Dairy Products Institute (1992). Standards for Grades of Dry Milks Including Methods of Analysis, Bulletin 9 16 (Revised). American Dairy Products Institute, Chicago, II, USA. Andersson, R.E. (1980) Microbial lipolysis at low temperatures. Appl. Environ. Microhiol., 39, 3640. Anon. (1989). Dairy Handbook. Alfa-Lava], Food Engineering AB. P.O. Box 64 S-221 00, Lund. APHA (1984). Compendium qf‘Methods,/& the Microbiological Examination ofFoods, 2nd edn.> ed. M.L. Speck. American Public Health Association, Washington, DC, USA. Boehringer Mannheim GmbH Biochemica (1984). Pyruvic acid (pyruvate) UV-method for the determination of pyruvic acid in foodstuffs and other materials. In Methods in Enzvmutic Analysis Using Sing/e Reagents.
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Boehringer Mannheim GmbH, Manneheim, Germany, pp. 57-58. Brady, L.P. (1972). Manufacture of specialized milk powders. In Seminar on Recombined Dairy Products, 2-3 Aug. 1972, CSIRO, Melbourne, p. 19. Celestino, E.L., Iyer, M. and Roginski, H. (1996). The effects of refrigerated storage on the quality of raw milk. Aust. J. Dairy Technol., 51, 59-63.
Chism, G.W., Huang, A.E. and Marshall, J.A. (1979) Sensitive assay for proteases in sterile milk. J. Dairy Sci., 62, 179% 1800. Cogan T.M. (1980). Heat resistant lipases and proteinases and the quality of dairy products. MF Bull. Dot. 118, pp. 2632. Crossley, E.L. (1962). Cited by Lovell, H.R. (1990). The microbiology of dried milk powders. In Dairy Microbiology, Vol. 1, The Microbiology of Milk, 2nd edn., ed. R.K. Robinson. Elsevier Applied Science, New York, pp. 245269. Davis, J.G. (1955). A Dictionary ofDairying, 2nd edn. Leonard Hill Ltd., London. Deeth, H.C. and Fitz-Gerald, C.H. (1976) Lipolysis in dairy products: A review. Aust. J. Dairy Technol., 31, 53-64. Deeth, H.C., Fitz-Gerald, C.H. and Wood, A.F. (1979) Lipolysis and butter quality. Aust. J. Dairy Technol., 34, 146149.
de Vilder, J., Moermans, R. and Martens, R. (1979) The freefat content and other physical characteristics of whole milk powder. Milchwissenschaft, 32, 347-350. Duncan, SE. and Christen, G.L. (1991) Sensory detection and recovery by acid degree value of fatty acids added to milk. J. Dairy Sci., 74, 2855-2859.
Duncan, SE., Christen, G.L. and Penfield, M.P. (1990) Acid degree value - does it really predict rancid flavor in milk? Dairy, Food and Environmental Sanitation, 1012, 715-718.
Early, R. (1992). Milk powders. In The Technology of Dairy Products, ed. R. Early. Blackie and Son Ltd., New York, USA, pp. 167-196. Freund, R.J., Littell, R.C. and Spector, P.C. (1986). SAS System for Linear Models. SAS Institute Inc., Cary, NC, USA. Ghatak, P.K., Bandyopadhyay, A.K. and Gupta, M.P. (1989) The relation between the heat stability of milk and its chemical composition. Asian J. Dairy Res., .8, 165168.
IDF (1991). A practical guide to the control of lipolysis in the manufacture of dairy products. Int. Dairy Federation, Brussels, Belgium, Bull. Dot., 264, pp. 2628. Ipsen, R. (1989). Factors affecting the storage stability of whole milk powder. Scandinavian Dairy Industry, 2, 24-26, 45.
Ipsen, R. and Hansen, F.S. (1988) Factors affecting the storage stability of whole milk powder. Dairy Sci. Abstr., 517, 297. Ipsen, R. and Hansen, P.S. (1990) Enzymic activity in raw milk-storage stability of dried whole milk. Dairy Sci. Abstr., 531, 64.
Iverius, P.-H. and Ostlund-Lindqvist, A.M. (1986) Preparation, characterization and measurement of lipoprotein lipase. Methods in Enzymology, 129, 691-704. Jensen, G.K. (1990). Milk powders: Specifications in relation to the products to be manufactured. In Proceedings of Seminar on Recombination of Milk and Milk Products, 1216 Nov. 1988, IDF, Brussels, pp. 104125. Kieseker, F.G. (1982) Recombined dairy products. Aust. J. Dairy Technol., 37, 132-135. Kieseker, F.G. and Aitken, B. (1993) Recombined full-cream milk powder. Aust. J. Dairy Technol., 48, 33-37. Kirst, E. (1986) Lipolytic changes in the milk fat of raw milk and their effects on the quality of milk products. Food Microstructure, 5, 265-275.
Kudo, N., Hols, G. and van Mil, P.J.J.M. (1990) The insolubility index of moist skim milk powder: Influence of
the temperature
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Kwee, W.S., Dommett, T.W., Giles, J.E., Roberts, R. and Smith, R.A.D. (1986) Nlicrobiological parameters during powdered milk manufacture. 1. Variation between processes and stages. Aust. J. Dairy Technol., 41, 3-6. Law, B.A. (1979) Reviews of the progress of dairy science: enzymes of psychrotrophic bacteria and their effects on milk and milk products. J. Dairy Res., 46, 573-588. Lingnert, H. and Eriksson, C.E. (1981) Antioxidative effect of Maillard reaction products. Prog. Food Nutr. Sci., 5, 453466.
Liodakis, A., Drew, J., Chan, R.Y.S. and Sawyer, W.H. (1991) Spectrofluorometric determination of lipase activity. Biochemistry International, 23, 825-834.
Litman, 1.1. and Ashworth, U.S. (1957) Insoluble scum-like materials on reconstituted whole milk powders. J. Dairy Sci., 40, 403409.
Lovell, H.R. (1990). The microbiology of dried milk powders. In Dairy Microbiology, Vol. 1, The Microbiology of Milk, 2nd edn, ed. R.K. Robinson. Elsevier Science Publishers Ltd., London, UK, pp. 245-269. Mair-Waldburg, H. and Lubenau-Nestle, R. (1974). Factors affecting the results of colony count determination in dried milk. 19th Intern. Dairy Congress, Vol. lE, 2-6 Dec. 1974, India, pp. 553-554. Munro, D.S., Cant, P.A.E., Macgibbon, A.K.H., Illingworth, D., Kennett, A. and Main, A.J. (1992). Concentrated milkfat products. In The Technology of Dairy Products, ed. R. Early. Blackie and Son Ltd., New York, USA, pp. 117-145. Negre, A., Salvayre, R., Dousset, N., Rogalle, P., Dang, Q.Q. and Douste-Blazy, L. (1988) Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice. Biochemica et Biophysics Acta, 963, 340-348. Newstead, D.F. and Headifen, J.M. (1981) A reappraisal of the method for the estimation of the peroxide value of fat in whole milk powder. New Zealand J. Dairy Sci. Technol., 16, 13-18.
Ng-Kwai-Hang, K.F. and Kroeker, E.M. (1984) Rapid separation and quantification of major caseins and whey proteins of bovine milk by polyacrylamide gel electrophoresis. J. Dairy Sci., 67, 3052-3056. Nickerson, T.A. (1974). Lactose. In Fundamentals of Dairy Chemistry, eds B.H. Webb, A.H. Johnson and J.A. Alford. The Avi Publishing Company, Inc., Westport, Connecticut, USA, p. 289. Niro Atomizer (1978). Determination of free fat on the surface of milk powder particles Method No. AlOa. In Analytical Methods for Dry Milk Products, 4th edn. A/S Niro Atomizer, Copenhagen, Denmark, pp. 4647. Pisecky, J. (1990). 20 years of instant whole milk powder. Scandinavian Dairy Information., 4, 7476,
78.
Renner, E. (1988) Storage stability and some nutritional aspects of milk powders and ultra high temperature products at high ambient temperatures. J. Dairy Res., 55, 125-142.
Richardson, B.C. and Newstead, D.F. (1979) Effect of heatstable proteases on the storage life of UHT milk. New Zealand J. Dairy Sci. Technol., 14, 273-279.
Sanderson, W.B. (1979). Dairy products. IDF Bull. DOC. 116, pp. 40-42. Scanlan, R.A., Sather, L.A. and Day, E.A. (1965) Contribution of free fatty acids to the flavor of rancid milk. J. Dairy Sci., 48, 1582-1584. Shamsuzzaman, K., Modler, W. and McKellar, R.C. (1987) Survival of lipase during manufacture of nonfat dry milk. J. Dairy Sci., 70, 746-751.
Shipstead H. (1952). Cited by Shipstead H. and Tarassuk, N.P. (1953). Chemical changes in dehydrated milk during storage. Agric. Food Chem., 1, 613-616. Shipstead, H. and Tarassuk, N.P. (1953) Chemical changes in
&y&et of’ storage on quality of whole milk powder dehydrated milk during storage. Agric. Food Chem., 1, 613616. Singh, H. and Newstead, D.F. (1992). Aspects of proteins in milk powder manufacture. In Advanced Dairy Chemistry 1. Proteins, ed. P.F. Fox. Elsevier Science Publishers Ltd., London, UK, pp. 735-165. Sjollema, A. (1986). Recombination of dairy ingredients into milk, cream, condensed and evaporated milk. In Milk, the rital,force - Proceedings qf’the 22nd Intern. Dairy Congress, 29 Sept.-3 Oct. 1986. D. Reidel Publishing Co., Dordrecht. Germany, pp. 251-257. Sonntag, N.O.V. (1979). Reactions of fats and fatty acids. In Bailey’s Industrial Oil and Fat Products, Vol. 1, 4th edn., ed. D. Swern. John Wiley and Sons, New York, USA, pp. 99- 176. SAA (1971). Australian Standard. AS 1095 ~ Microbiological methods for the dairy industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1974a). Australian Standard. AS 1166 ~ Methods for sampling milk and milk products. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (I 974b). Australian Standard. AS I629 - Methods for the analysis of dried milk and whey. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1975). Australian Standard. AS 1095 ~ Microbiological methods for the dairy industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1976). Australian Standard. AS 1095 - Microbiological methods for the dairy industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1979a). Australian Standard. AS 1095 ~ Microbiological methods for the dairy industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1979b). Australian Standard. AS l629P ~ Scorched particle standards for dry milks. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (198 1). Australian Standard. AS 1095 ~ Microbiological
127
methods for the dairy industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1983). Australian Standard. AS 1095.2.8 ~ Microbiological examination ofdried dairy products. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1988). Australian Standard. AS 2300 ~ Methods of chemical and physical testing for the dairying industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1989). Australian Standard. AS 2300 -- Methods of chemical and physical testing for the dairying industry. Standards Association of Australia, North Sydney, New South Wales, Australia. SAA (1991). Australian Standard. AS 2300 ~ Methods of chemical and physical testing for the dairying industry. Standards Association of Australia, North Sydney, New South Wales, Australia. Steel, R.G.B. and Torrie, J.H. (1980). Principles and Procedures q/‘Statistics. 2nd edn. McGraw-Hill Book Co., New York, USA. Steen K. (1977). Cited by van Mil, P.J.J. and Jans, J.A. (1991). Storage stability of whole milk powder: Effects of process and storage conditions on product properties. Neth. Milk Dairy, J., 45, 145--167. Supplee, G.C. (1926). Cited by Nickerson, T.A. (1974). Lactose. In Fundamentals of Dairy Chemistry, eds B.H. Webb, A.H. Johnson and J.A. Alford. The Avi Publishing Company, Inc., Westport, CT, USA, p. 289. Thomasow, V.J., Mrowetz, G. and Delfs, E.-M. (1972) Die Bestimmung des Wassergehaltes von getrockneten Milchprodukten mit Hilfe der Kart-Fischer-Titration. Mil~h~~issensc,h~~, 27, 76-8 I. van Mil. P.J.J.M. and Jans, J.A. (1991) Storage stabilityofwhole milk powder: Effects of process and storage conditions on product properties. Neth. Milk Dairy J., 45, 145-167. Walstra, P. and Jenness, R. (1984). Dairy Chemistry and Physics. John Wiley and Sons, Inc.. New York, USA.
Dairy Journal 7 (1997) 119-127 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain PI
ELSEVIER
I:
SO958-6946(96)00041-6
0958.6946:97/$17.00+0.00
The Effects of Refrigerated Storage of Raw Milk on the Quality of Whole Milk Powder Stored for Different Periods Ernani L. Celestino, Mani Iyer* and Hubert Roginski Gilbert Chundler College. Faculty of Agriculture.
(Received
Forestry und Horticulture, The University 3030, Victoria, Australia 24 September
1995; accepted
28 August
qf Melbourne, Sneydes Road, Werribee
1996)
ABSTRACT Whole milk powders were produced from fresh (control) and stored (4f 1°C. 48f2 h) raw milk in three seasons (spring, summer and autumn), and stored for up to 8 months at 25fl”C. The suitability of the milk powders for reconstitution processes was assessed on the basis of selected microbiological, physical, chemical and biochemical quality parameters. Storage of raw milk did not significantly affect total bacterial count (TPC) in the resultant powder. Free fatty acid (FFA) content in powder manufactured from stored raw milk was greater than that from the control. TPC and free fat content decreased while insolubility index, FFA and peroxide value increased during storage of powder; lipase and proteinase activities did not change. All powders manufactured from control and stored raw milk, and stored for up to 8 months, were found suitable for reconstitution on the basis of TPC, moisture content and amount of scorched particles but not based on insolubility index. 0 1997 Elsevier Science Ltd.
for reconstitution processes in terms bacteriological, physical and compositional
INTRODUCTION The microbiological quality of whole milk powder is determined by the initial number and type of microorganisms present in the raw milk from which it is manufactured and the extent of post-processing contamination. The primary psychrotrophic microflora of milk are Gram-negative rods (Law, 1979). Although are killed by these psychrotrophs most of pasteurisation, many species produce heat-stable lipases and proteinases which retain activity following 1980) and spray drying pasteurisation (Cogan, (Shamsuzzaman et al., 1987; Ipsen & Hansen, 1990). The presence and activity of these enzymes in milk powder may result in physico-chemical changes, as well as defects in functional and sensory properties of the powder, consequently affecting its keeping quality. Whole milk powder has been reported to have a maximum shelf-life of about 6 months (Anon., 1989) at room temperature. However, it was found that whole milk powder can have a shelf-life in excess of 12 months if packed in cans under vacuum or an inert gas, such as nitrogen, to inhibit the development of offflavours (Kieseker & Aitken, 1993). Recent investigations (Celestino et al., 1996) have shown an increase in numbers of lipolytic and proteolytic bacteria and dominance of psychrotrophs after storage of raw milk at refrigeration temperature (41tl”C) for 2 days. The present study aims to relate the quality of raw milk with the physical, chemical, microbiological and biochemical properties of the resultant whole milk powder and assess its suitability
*Corresponding
MATERIALS Manufacture, powder
of selected parameters.
AND METHODS packaging and storage of whole milk
A 600 L sample of bulk raw milk was taken in each season (spring, September 1992; summer, December 1992; and autumn, March 1993) to account for variations in milk composition and quality throughout the 1992/93 milk supply period. In Victoria, milk for processing into products other than market milk is collected for approximately 10 months, from August to June. In the present investigation, each sample was taken from the same 86,000L factory silo, representing approximately 5,700 cows from 45 herds. Details of bulk milk sample collection are available (Celestino et al., 1996). Each milk sample was standardised to a fat:solids-not-fat ratio of 3.8:8.5, then divided into two equal batches. One batch was stored in the factory at 4fl”C for 48*2 h while the other served as the no storage control. Each batch of milk was manufactured into powder. During powder manufacture, every effort was made to ensure that all variations in thermal processing were kept to a minimum. The milk was given a heat treatment suitable for a medium heat powder (75°C for 3min), using a tubular heat exchanger and holding tubes. The milk was introduced into a double effect falling film evaporator at a temperature of 60°C with the first effect at 80°C and the second at 63°C producing a concentrate with 45%
author. II’)
120
E.L.Celestino et al.
total solids. The concentrate was homogenised in a two stage (17.23 MPa/3.45 MPa) Creamery Package Homogeniser (Model 3DDl; The Creamery Package Manufacturing Company, Chicago, Illinois, USA) at 50°C. The homogenised concentrate was dried on a pilot-scale, single stage Niro Production Minor spray drier (Niro Australia Pty. Ltd., Blackburn, Victoria). The concentrate was preheated to 65°C prior to centrifugal atomisation at 21500 rpm with distributor holes of 1.2 mm diameter. The inlet air was at 180°C and the feed flow rate was adjusted to give an outlet air temperature of 85°C. The powder was removed at 15 min intervals from the collection bucket, bagged and cooled to about 30°C in a cold storage (4°C) room. The powder produced from each batch of raw milk was divided into five lots (about 5.5 kg per lot). Each lot of powder was packed in three-ply multiwall bags with polyethylene barrier lining (thickness 60pm) and sealed with an elastic ring. The respective lots of powder were stored at 25fl”C for 0, 2, 4, 6, and 8 months. Sampling, microbiological and physical analyses Sampling of powder for all the tests was according to the method of the Standards Association of Australia (SAA) [AS 1166 (SAA, 1974a); AS 1629 (SAA, 1974b); AS 1095.2.8 (SAA, 1983)]. The following bacterial counts were determined: total plate [AS 1095.1.2 (SAA, 1971); AS 1095.2.1 (SAA, 1981)], psychrotroph [AS 1095.3.5 (SAA, 1976)], lipolytic [AS 1095.3.4 (SAA, 1976)], proteolytic [AS 1095.3.6 (SAA, 1975)] and mesophilic, thermophilic and psychrotrophic aerobic, and mesophilic and thermophilic anaerobic spores [AS 1095.3.7 (SAA, 1979a; APHA, 1984)]. Milk powder samples were analysed for bulk density, insolubility index and scorched particles in accordance with AS 1629 (SAA, 1974b). Chemical tests Compositional tests and pH
The procedures set by the SAA (SAA, 1988SAA, 1991) for the determination of total solids/moisture [AS 2300.1.1 (SAA, 1988)], fat [gravimetric method, AS 2300.1.3 (SAA, 1988)] and total nitrogen [AS 2300.1.2.1 (SAA, 1991)] were followed. For nonprotein nitrogen (NPN) content determination, milk powder was reconstituted to 12.3% total solids. The sample was then treated as in determining NPN in liquid milk [AS 2300.1.2.2 (SAA, 1988)]. NPN was calculated as for liquid milk except that the value was multiplied by the appropriate dilution factor. The free fat content of powder was determined based on the method described by Niro Atomizer (1978). The pH was determined at 20-22°C using a digital pH meter (TPS Pty. Ltd., Brisbane, Australia), as specified by the SAA (1989) for determining pH of dried milk (AS 2300.1.6). Pyruvic acid (pyruvate)
Whole milk powder was reconstituted to 12.3% total solids. The sample was then treated as for liquid milk (Boehringer Mannheim GmbH Biochemica, 1984). Absorbance was determined at 340 nm using a SequoiaTurner spectrophotometer (Model 340; Sequoia-Turner
Corporation, Mountain View, California, USA). The pyruvate content of the sample was expressed in mg 100 g-’ milk powder. Whey protein nitrogen index (WPNI) was determined following the procedure AS 1629 set by the SAA (1974b). Peroxide value
Determination of peroxide value was based on the ferric thiocyanate method described by Newstead & Headifen (1981). However, in addition to the reagent blank, a fat blank was also prepared consisting of the chloroform-methanol solvent, fat sample and ammonium thiocyanate. Sample absorbance readings were corrected for both reagent and fat blanks. Absorbance was measured at 500nm using a Sequoia-Turner spectrophotometer (Model 340). The peroxide value of the fat was expressed as meq oxygen kg- ’ . Biochemical assays Preparation of samples for biochemical analyses was accomplished by reconstituting whole milk powder to 12.3% total solids. Lipolysis
The acid degree value (ADV) of the prepared sample was determined according to the procedure outlined by Deeth & Fitz-Gerald (1976) for liquid milk. ADV was expressed in meq lOOg-’ fat. The free fatty acid (FFA) content of the prepared sample was analysed by extraction-titration method (Deeth & Fitz-Gerald, 1976) and expressed in meqkg-’ milk powder or meq lOOg-’ fat. Lipase activity was measured by means of a spectrofluorometric technique. The samples were prepared according to procedures described by Liodakis et al. (1991) Iverius & Ostlund-Lindqvist (1986) and Negre et al. (1988) as outlined by Celestino et al. (1996). Lipase activity was expressed as pmol pyrenyl butyric acid min-’ gg’ milk powder. Proteolysis
The procedures for fluorescamine method (Chism et 1979) and polyacrylamide gel electrophoresis $AGE) d escribed by Richardson & Newstead (1979) and NglKwai-Hang & Kroeker (1984), were followed with some modifications as outlined by Celestino et al. (1996). Statistical analysis Analysis of variance of the data for each parameter (except for aerobic psychrotrophic and anaerobic thermophilic counts, and results of PAGE and scorched particles test, which were interpreted descriptively) was a split plot in randomised complete block design. In this design, raw milk storage treatment was the mainplot, and storage period of powder was the subplot. Treatment means were compared using the Duncan’s Multiple Range Test (Steel & Torrie, 1980; Freund et al., 1986). Results for proteinase activity (fluorescamine method) were analysed with unequal number of observations.
of storuge on quality @‘whole
E&V
RESULTS
AND DISCUSSION
Microbiological quality The total plate counts (TPC) obtained for all powders were in accordance with the recommended norm for the microbiological standard of whole milk powder for reconstitution processes which is 2 10,000 cfu gg ’ (Jensen, 1990). Sanderson (1979) recommended a maximum viable plate count of 50,000 cfu gg’ of whole milk powder for reconstitution. The differences in TPC, proteolytic (PC), lipolytic (LC) and psychrotrophic (PSC) bacterial counts between powders produced from control or stored raw milks were found to be insignificant (P> 0.05; Table 1). As expected, heat treatment employed during powder manufacture resulted in a decreased number of psychrotrophs. Most psychrotrophs are heat sensitive. Kwee et al. (1986) reported reduction of psychrotrophs to negligible levels, specifically during the preheating stage of powder manufacture. TPC decreased with storage of powders (P < 0.01; Table 2). LC decreased (PcO.01) at eighth month of storage while PC and PSC did not change (f’> 0.05) with storage of powder. The decrease in microbial population during storage could be attributed to the low water activity (a,) of milk powder. Most bacteria cannot grow at an a, below 0.9; generally, powders have an a, much below 0.6 (Early, 1992). Furthermore, the powders underwent oxidation during storage; thus, the products of chemical oxidation may have had an effect on the viability of the organisms. Several authors (Davis, 1955; Crossley, 1962 as cited by Lovell, 1990; Mair-Waldburg & Lubenau-Nestle, 1988) reported that the microbial 1974; Renner, population decreased during storage of milk powder. Table 1. Mean Bacterial Counts”
Raw Milk Treatment Control Stored
TPC’ (x 102)
in Whole
Milk Powders
h
PSCh (x 102)
;:102,
25.6 (5.4)d 1.9 (1.1) 45.7 (7.9) 3.1 (1.4)
2.4 (0.6) 5.2 (1.2)
121
Love11 (1990) stated that raw milk counts in excess of lo5 cfu mL_’ can result in counts of over 104cfu gg’ in the dried product. However, this was not observed in resent study. The powders contained TPC of ‘2”,0Ycfu ggr even when raw milks from which the powders were manufactured had TPC of l.2x105cfumLand l.lx106cfumL~ (Celestino et al., 1996). This may again suggest that it is not simply a question of bacterial numbers but also of types, and that thermoduric organisms, if present in the raw milk, together with heat-resistant sporeformers, may consequently occur in the dried milk product. No significant difference was found (P> 0.05; Table 1) in aerobic mesophilic and thermophilic, and anaerobic mesophilic spore counts, between powders manufactured from control and stored raw milk. Storage of powder did not affect the numbers of aerobic and anaerobic sporeformers (P > 0.05; Table 2). The number of aerobic and anaerobic mesophiles appeared greater than their thermophilic counterparts for all powder treatments (Tables 1 and 2). Aerobic psychrotrophic sporeformers were absent in powder. Physical properties Bulk densit?
As expected, bulk densities of powders manufactured from control or stored raw milk were not significantly different and did not change with storage of powder (P > 0.05; data not shown). Scorched purticles
Scorched that have evaporator
Manufactured (n= 15)
b
milk powder
Aerobic
particles usually originate from milk solids been held up somewhere within the and/or drier, and thereby have been
from Control
and Stored
Sporeformers
(411°C.
48f2 h) Raw Milk
Anaerobic
Sporeformers
(LxYO2) 4.2 (1.0) 6.3 (1.0)
Mesophiles’
Thermophilesb
Psychrotrophs”
Mesophilesb
Thermophiles’
25.0 (4.8) 44.0 (10.7)
7.3 (2.0) 13.0 (1.7)
nde nd
33.5 (8.1) 299.0 (150.3)
<2to2 ~2 to 6
a Means for powders (as cfu gg ‘) stored for all periods in all replicates. ’ No significant difference between raw milk treatments (P > 0.05). ‘Not subjected to ANOVA; result for anaerobic thermophiles is the range of 15 readings. d Figures in brackets are standard errors of the means. ‘nd = Not detected. Table 2. Mean Bacterial
Counts” h
Storage Period (Months)
TPC’ (x102)
0
54.3a (12.4)'
5.8a (3.7)
4.6~~(2.4)
2
50.&b
3.2~~(2.1)
3.8~1(1.6)
4
41.8b (8.1)
I.7a (1.1)
6
21.9c (10.5)
8
9.5d (4.3)
"
PSCh (x102)
(9.5)
in Whole Milk Powders
;:102,
b
Stored for Different
Aerobic
Periods
Sporeformers
at 2511°C
(n = 6)
Anaerobic
Sporeformers
:02, Mesophiles’
ThermophilesC
Psychrotrophsd
7.4a (1.8)
37.5 (14.0)
II.7 (4.6)
nd
15.8 (4.1)
<2to2
5.7~1(1.6)
35.0 (14.2)
9.2 (3.0)
nd
290.0 (264.1)
<2to5
3.8a (0.9)
6.8a (1.8)
34.2 (14.5)
9.2 (3.0)
nd
299.0 (121.0)
<2to5
I.la (0.6)
3.8a (1.5)
4.9a (1.2)
33.3 (14.8)
10.2 (3.1)
nd
56.5 (37.5)
<2to6
0.6a (0.5)
2.7a (1.6)
l.4b (0.7)
32.5 (14.9)
10.7 (2.8)
nd
170.0 (150.3)
<2to5
Means for powders (as cfu gg
‘) made from control and stored raw milk
b Compared using DMRT; means within a column followed by a common ‘No significant difference between storage periods of powder (P > 0.05). dnd = Not detected. ‘Not subjected to ANOVA and DMRT; result for anaerobic thermophiles ‘Figures in brackets are standard errors of the means.
in all replicates. letter are not significantly
is the range of 6 readings.
Mesophiles”
different
Thermophiles’
(P > 0.05).
122
E. L. Celestino et al.
overheated or burned. In the present study, occurrence of scorched particles gave a Disc A rating [or 7.5mg32.5g-’ powder; AS 1629P (SAA, 1979b)] for powders processed in spring and summer. This is within the recommended norm (maximum 7.5mg) for reconstitution processes for whole milk powder (Jensen, 1990). However, control and stored raw milk in autumn yielded powders with higher scorched particles (Disc B rating or 7.5-15.0 mg 32.5 g-’ powder; results not shown). These differences could be due to slight day-to-day variations in drier performance caused by changes in factors such as ambient air temperature and humidity. It is generally recognised that these factors are hard to control. Another possibility is that autumn raw milk is less stable because of its higher NPN (Ghatak et al., 1989) protein (Singh & Newstead, 1992) and, possibly, higher calcium content (Ghatak et al., 1989), resulting in greater burn-on tendency during evaporation. However, the autumn powders were still within the official standard limit for scorched particles content. Brady (1972) reported that milk is usually most stable in spring and early summer. For reconstitution, Sanderson (1979) recommended a Disc B rating or better for whole milk powder of less than 9 months age.
Table 3. Insolubility Index of Whole Milk Powders, Manufactured from Control or Stored (4&l”C, 48f2h) Raw Milk and Stored for Different Periods at 25fl”C
Insolubility index
PH Fat, % m/m Free fat, % m/m Crude protein, % m/m Non-protein nitrogen, % m/m True protein, % m/m Moisture, % m/m Whey protein nitrogen index, mg gPeroxide value, meq oxygen kg-’ fat Pyruvic acid, mg 100 gg ’
Insolubility indices of powders manufactured from stored raw milk were higher than those manufactured from control raw milk but the differences were not significant (P > 0.05; Table 3) and the values obtained were within the official standard specification of not greater than 0.5mL (Jensen, 1990). A maximum of 0. I mL was recommended for reconstitution processes (Sanderson, 1979; Jensen, 1990). Storage of powder at 25fl”C significantly increased the insolubility index of powder (P < 0.01; Table 3), with higher values obtained at 6 and 8 months. When all powders manufactured from the raw milk in different replicates were compared, insolubility index test results showed that freshly manufactured and 2- and 4-month-old powders produced from control or stored spring raw milk, and freshly manufactured powder from control summer raw milk, had insolubility index of 0.1 mL. An insolubility index of > 0.5 mL was measured at 8 and 6 months of storage in powders manufactured from control and stored autumn raw milk, respectively. Increases in the insolubility index of whole milk powder with storage were also reported by van Mil & Jans (1991). Kudo et al. (1990) cited several factors contributing to decreased solubility during storage, such as crystallisation of lactose, non-enzymatic browning, higher moisture content and elevated storage temperature of powder. However, the mechanism of the formation of insoluble material in milk powder when held at temperatures ranging from 20 to 100°C is still not known (Kudo et al., 1990). Chemical properties Composition and pH
The pH of powder was not affected by raw milk storage (P > 0.05; Table 4) and was found to gradually decrease with storage of powder (PC 0.01; Table 5). Freshly manufactured powder had a pH of 6.63 which decreased by only 0.03 after 8 months of storage. The slight change in pH may be due to the formation of
Insolubility Indexa (mL) Raw Milk Treatment
Storage Period of Powder (Months)
0 2 4 6 8
Control
Stored
0.13 0.17 0.14 0.30 0.37
0.20 0.21 0.18 0.43 0.43
(0.03) (0.04) (0.03) (0.10) (0.12)
(0.06) (0.05) (0.04) (0.13) (0.12)
aMeans for powders in all replicates. No significant difference between raw milk treatments (n= 15) (P>O.O5); significant differences between storage periods of powder (n= 6) (PC 0.01). Figures in brackets are standard errors of the means.
Table 4. Mean Value? for the Different Chemical Properties of Whole Milk Powders Manufactured from Control or Stored (4&1”C, 48f2 h) Raw Milk (n= 15) Chemical Attribute
Raw Milk Treatment Control
Stored
6.62 28.33 0.44 26.10 0.33 24.02 2.33 4.59
6.60 28.07 0.47 26.02 0.33 23.89 2.36 4.47
(0.01) (0.18) (0.03) (0.20) (0.02) (0.21) (0.05) (0.18)
(0.01) (0.15) (0.03) (0.22) (0.02) (0.21) (0.08) (0.18)
1.20 (0.20)
1.40 (0.24)
2.22 (0.12)
2.92 (0.14)
aMeans for powders stored for all periods in all replicates. No significant difference was found between raw milk treatments for any of the parameters (P > 0.05). Figures in brackets are standard errors of the means.
FFA as a result of oxidation as well as lipolytic activity during storage of the powder. Storage of raw milk did not significantly affect the composition of whole milk powder (P> 0.05; Table 4). occurred Some compositional changes, however, during powder storage (Table 5). Moisture content decreased slightly at the second month (PcO.05) but increased to approximately the initial level at the sixth and eighth months of storage. The values obtained at different periods of storage were below the maximum moisture content (3.0%) recommended by Sanderson (1979) for whole milk powder suitable for reconstitution. The change in moisture during storage of powder may be related to the change in the state of lactose, i.e. the shift from amorphous to the crystalline state. When moisture is absorbed by amorphous lactose, a part will be incorporated as water of crystallisation in the a-hydrate and the remainder will be released, since crystalline ahydrate is not hygroscopic (Supplee, 1926 as cited by Nickerson, 1974). The water of hydration of a-lactose is only partly removed by the standard oven-drying methods (Thomasow et al., 1972).
I!$&-t of’ storage on qua&v qf’whole milk powder Table 5. Mean Value? for Chemical Properties of Whole Milk Powders Chemical
Attribute
Storage 2
0 PH Fat, % m/m Free fat, % m/m Crude protein, % m/m Non-protein nitrogen. % m/m True protein, % m/m Moisture, % m/m Whey protein nitrogen index, rn? g -’ Peroxide value, meq oxygen kg- fat Pyruvic acid, mg 100 g- ’
Stored for Different
6.63a (0.01) 28.87a (0.29) O.SOab (0.03) 26.34a (0.31) 0.26b (0.04) 24.72a (0.23) 2.44ab (0.06) 4.72a (0.19) 0.55d (0. I 1) 2.27~ (0.28)
6.62b (0.01) 28.19b (0.19) 0.5la (0.03) 26.05ab (0.37) 0.32a (0.03) 24.02b (0.29) 2.2oc (0. I I) 4.38a (0.28) 0.93cd (0.16) 2.86a (0.22)
123 Periods
at 25f 1°C (n = 6)
Period (Months) 4
6.61bc (0.01) 28.25b (0.12) 0.50ab (0.07) 26.05ab (0.38) 0.33a (0.03) 23.9513 (0.34) 2.21 bc (0.09) 4.14a (0.34) 1.09bc (0.20) 2.78ab (0.21)
6 6.61bc (0.01) 27.98b (0.24) 0.39b (0.02) 25.90b (0.36) 0.37a (0.01) 23.55b (0.3 I) 2.37abc (0.14) 4.78a (0.25) l.5lb (0.30) 2.45~ (0.23)
8 6.6Oc (0.01) 27.72b (0.25) 0.39b (0.02) 25.96b (0.34) 0.38a (0.01) 23.53b (0.30) 2.50a (0.11) 4.64a (0.32) 2.40a (0.33) 2.50bc (0.29)
‘Means for powders made from control and stored milk in all replicates; compared using DMRT; means within a row followed common letter are not significantly different (P > 0.05). Figures in brackets are standard errors of the means.
Fat content slightly decreased (P < 0.05) at the second month of storage and then remained constant up to 8 months. Free fat content slightly decreased at the sixth month of storage. The decrease in fat and free fat contents could be due to their hydrolysis during storage which could be attributed to lipases which survived processing and were active during storage of the powder (Andersson, 1980). Furthermore, the possibility that the free fat enters into a fat-protein complex which is stable to solvent extraction has been reported (Litman & Ashworth, 1957; de Vilder et al., 1977). Pyruvic acid (pyruvate)
The pyruvate content of milk powder is influenced primarily by the number of bacteria in the raw material. Higher pyruvate content was observed in powder manufactured from stored raw milk than in powder from control milk, but the difference was not significant (P > 0.05; Table 4). Pyruvate content of powder appeared to change during storage, with variations from 2.27 to 2.86mg lOOg_’ of powder (Table 5). Pyruvate appeared highest in powder manufactured from stored raw milk in the autumn replicate (3.54mg lOOg_‘), which also had the highest bacterial numbers (Celestino et al., 1996). Sjollema (1986) suggested that in skim milk powder, the level of pyruvate can give an indication of the bacteriological quality of the milk from which the powder was made and that a value lower than 9 mg lOOg-’ of powder gives a reasonable guarantee of absence of heat-stable enzymes. All results obtained in the present study were below this level; however, proteolytic and lipolytic activity was detected in the powder during storage (Tables 6 and 7). Whey protein nitrogen index
WPNI measures the proportion of undenatured whey protein remaining after heat treatment. Electrophoretic patterns of the whey proteins in powders showed apparent change in intensity of the bands as a result of heat treatment applied during powder manufacture (results not shown). It appeared that a-lactalbumin was the most stable, followed by /Glactoglobulins A and B and serum albumin. WPNI has influence on several properties (Pisecky, 1990) including flavour, sediment and other physicochemical attributes, both in powder and in products
by a
manufactured from such powder. Results for WPNI showed that the medium heat powders manufactured from control or stored raw milk (Table 4) were within the heat classification limit for such types of powder. Medium heat powder should give a WPNI of 1.515.99 mg g-’ powder (American Dairy Products Institute, 1992). Throughout the year, some variations can be observed for WPNI due to variations in the composition of raw milk (Walstra & Jenness, 1984; Steen, 1977 as cited by van Mil & Jans, 1991). The content of milk varies whey protein nitrogen throughout the season, from area to area and also in its susceptibility to heat (Kieseker, 1982). In the present study, differences in WPNI between replicates may reflect variations in milk composition between seasons. Peroxide
value, ,free fat and free futty acids
The peroxide value is used as a measure of the extent of deterioration of milk fat due to oxidation (Munro et al.. 1992). In the present study, the peroxide value increased with storage of powders (PC 0.01; Table 5). The peroxide values obtained during the 8-month storage period of the powders were slightly higher than those obtained by van Mil & Jans (1991). Compared to their study, lower preheat treatment was employed in the present work, thus there was probably a lesser release of sulfhydryl groups from milk proteins. Also, the milk powders were packed in bags with a polyethylene barrier lining which could have allowed permeation of oxygen during storage, resulting in increased peroxide values. Catalytic oxidative processes can principally occur with free fat (Shipstead, 1952 as cited by Shipstead & Tarassuk, 1953; Kirst, 1986) as well as FFA (Kirst, 1986). The present study showed decreased free fat and increased FFA with storage of powder (Table 6). The possible reasons for the decrease in free fat during storage of powder were discussed above. Further, FFA appeared to result from both lipolysis (lipase activity was present during powder storage), and oxidation reactions (Munro et al., 1992). Sonntag (1979) reported that fats in an advanced state of oxidation develop acidity through the accumulation of acidic cleavage products. Thus, oxidation of powder may have contributed to the increase in FFA results during storage. The present results support the report of Kirst (1986) that
E. L. Celestino et al.
124 Table 6. Mean Valuesa for Biochemical
Raw Milk Treatment
Control Stored
Free Fatty Acidsb (meq kg- powder)
Properties
of Whole Milk Powders Raw Milk
Free Fatty Acidsb (meq 100 g-l fat)
9.07 (0.50)
3.18 (0.18)
10.20 (0.53)
3.64 (0.19)
Manufactured
Acid Degree Valueb (meq 1OOg ’ fat)
1.08 (0.04) 1.22 (0.05)
aMeans for powders stored for all periods in all replicates (n= 15, except for proteinase stored). Figires in brackets are standard errors of-the means. b Significant difference between raw milk treatments (PC:0.05). ‘No significant difference between raw milk treatments (P > 0.05).
oxidative changes in the milk fat are indirectly connected with the lipolytic changes. Since oxidation involves reaction between oxygen and the fatty acids, either as free (Deeth & Fitz-Gerald, 1976), or in the triglycerides (Munro et al., 1992), the greater the lipolysis, the greater would be the formation of FFA, and this would consequently provide for increased oxidation reactions (Deeth & Fitz-Gerald, 1976). It is known that FFA oxidise more readily than the corresponding glycerides (Sonntag, 1979). As oxidation reaction proceeds during storage, the fatty acids react with oxygen, by a radical chain mechanism, forming fatty acid hydroperoxides (Lingnert & Eriksson, 1981). The FFA content of stored raw milk was greater than that of control raw milk (Celestino et al., 1996). Consequently, higher FFA and ADV were observed in powder manufactured from the stored raw milk than in powder manufactured from the control. Ipsen (1989) showed correlation of lipolysis (expressed by the acidity of the milk fat) in raw milk with the oxidative changes in stored whole milk powder. Earlier, Ipsen & Hansen (1988) found that oxidative stability of whole milk powder was lower when made from raw milk with high TPC and FFA content. In the present study, peroxide value in powder manufactured from stored raw milk was higher than that from the control but the difference was not significant (P > 0.05; Table 4). There was no linear correlation between FFA in raw milk and peroxide value of powder; further, only a weak positive linear correlation (r=0.645, n = 12) of ADV with oxidation in milk powder was found. Further research is needed to conclusively define the correlation of lipolysis in raw milk with the oxidative stability of the powder. Table 7. Mean Values” for Biochemical
Properties
from Control or Stored (4&1”C, 48*2 h)
Lipase Activity’ (firno pyrenyl butyric acid
Proteinase Activity’ (nmol LIeucyl-L-
min-’ 9-l)
leucine min-’ g-‘)
0.73 (0.08)
74.8 (9.3)
0.88 (0.09)
93.8 (10.3)
activity, where n = 14 for control
and 13 for
Biochemical properties Lipolysis There was no significant difference in lipase activity between powders manufactured from control or stored raw milk (P>O.O5; Table 6), although lipase activity of stored raw milk was significantly higher than that of control (Celestino et al., 1996). During whole milk concentration by evaporation and drying, milk solids are concentrated approximately 8-fold. Lipase activity in powders (Table 6) was approximately 9 times higher than that in the corresponding raw milk (Celestino et al., 1996), which suggests that no loss of lipolytic activity occurred during powder production. Storage of raw milk prior to powder manufacture resulted in whole milk powder with higher ADV and concentration of FFA (PC 0.05; Table 6). Lipase activity in the stored raw milk was higher than that in the control raw milk and this resulted in greater FFA content of the stored raw milk (Celestino et al., 1996). Although the lipase activities in the powders manufactured from control or stored raw milk were not significantly different, the FFA in the powder manufactured from stored raw milk was significantly higher than that in the product manufactured from the control raw milk. Lipase activity was stable throughout the S-months storage of powder (P>O.O5; Table 7). Significant increases in FFA and ADV were observed during storage (PC 0.01; Table 7). Renner (1988) reported that, in milk powder, enzyme activity ceases at an a, of < 0.6 or powder with approximately 4% water content. On the other hand, Andersson (1980) found that at uW values of 0.54 and lower, iipolysis took place at a fairly constant level in full-cream milk powder. Acker (1969),
of Whole Milk Powders
Stored for Different
Periods
at 25fl”C
Storage Period (Months)
Free Fatt Acidsb Y (meq kg- powder)
Free Fatty Acidsb (meq 100 g-’ fat)
Acid Degree valueb (meq 100 g-’ fat)
Lipase ActivityC (pm01 pyrenyl butyric acid min-’ g-‘)
Proteinase Activity” (nmol Lleucyl-lleucinemin-’ g-‘)
0 2 4 6 8
7.5% (1.18) 9. lob (0.80) 10.19ab (0.38) 10.38ab (0.28) 10.96a (0.53)
2.63~ (0.43) 3.52b (0.29) 3.60ab (0.15) 3.7lab (0.10) 3.95a (0.18)
0.87~ (0.03) 1.12b (0.06) 1.23a (0.07) 1.25a (0.05) 1.28a (0.07)
0.67 (0.19) 0.64 (0.14) 1.02 (0.13) 0.90 (0.08) 0.8 1 (0.07)
48.4 69.2 90.2 108.6 95.8
’ Means for powders made from control and stored milk in all replicates (n = 6, except for proteinase storage, where n = 5). Figures in brackets are standard errors of the means. b Compared using DMRT; means within a column followed by a common letter are not significantly ‘No significant difference between storage periods of powder (P > 0.05).
(14.X) (11.4) (11.9) (20.3) (8.4)
activity at 0 and 2 months different
(P > 0.05).
of
ITf;ect of storuge on quality of ~zhnle milk powder
as cited by Andersson (1980) explained that at low u, values the amount of available water is limited, thus changing the water-lipid interface whereby the number of reaction sites may be reduced. The fact that substrates for lipases are water insoluble and that the water phase is not the only location for the reactions catalysed by lipases may explain why lipolysis occurs at low u, values. In the present study, accumulation of FFA in the powder may be attributed to the stable lipase activity observed during storage. However, the effect of oxidation should also be considered. During storage of powder, FFA values ranging from 2.63 to 3.9.5meq lOOg_’ fat were obtained (Table 7) which are much higher than the threshold values of 1.5 to 2.0meq lOOg_’ fat reported for rancid flavour (IDF. 1991). However, high levels of FFA are not always associated with the development of off-flavours (Deeth ct al., 1979). FFA usually arise from lipolysis, but can be formed also as by-products of oxidation (Munro et cd., 1992). Thus, in the present study, increases in FFA and ADV during storage of powder may have been a result of the lipolytic activity as well as oxidation reactions. Compared to ADV, results of extractiontitration of FFA (in meq lOOg_’ fat) showed higher extent of lipolysis in powder (Tables 6 and 7). This could probably be due to the fact that ADV determination does not recover the short-chain FFA (C4-C8) in the fat separation process and only partially recovers the medium-chain (ClO-C16) FFA (Duncan & Christen, 1991). The fatty acids implicated in rancid flavour are C4-Cl2 (Scanlan et uf., 1965) and these vary in water solubility/insolubility. Some of these fatty acids, including butyric, caproic and even caprylic, may be partially released into the aqueous phase of the milk (Duncan & Christen, 1991). Thus, the ADV method is measuring only those FFA which are fat-soluble and remain in the fat during the separation procedure. A high ADV would primarily reflect a change in concentration of the long-chain fat-soluble FFA but increase in indicate an necessarily does not concentration of the volatile short-chain FFA (Duncan cr [Il., 1990). Proteolvsis
powder was greater in Proteinase activity manufactured from stored raw milk than from control raw milk, but the difference was not significant (P > 0.05; Table 6). This was supported by the results of PAGE as well as NPN content of powder. As shown in Table 4, the NPN content of powder was not affected by raw milk storage. Further, no change in intensity of the protein bands was observed during storage of the powder manufactured from control or stored raw milk (results not shown). The detected activities of proteinases in the powders manufactured from control or stored raw milk indicate resistance of some proteinases to the heat treatment applied during powder manufacture. Ipsen & Hansen (1990) attributed proteolysis in dried whole milk to heat-stable proteolytic enzymes which remain active after spray drying. The results of the present study show that proteinases retained their activity during storage of powder. In milk powders where a, is very low, degradation of proteins through the action of proteinases would not be expected. However, proteinases, especially the bacterial
125
types, may not be inactivated by the heat treatment employed during spray drying, and may thus possibly remain active during the powder storage (Ipsen & Hansen, 1990). In the present study, this has been shown by the proteinase activity detected (Table 7), but no proteolysis was evident as supported by the results of PAGE (results not shown).
CONCLUSIONS Storage of raw milk (4&l”C, 48f2 h) resulted in whole milk powder with higher values for FFA and ADV. There was an approximately 9-fold increase in lipolytic activity of the powder compared to that of corresponding raw milk which matches the degree of concentration and therefore suggests no overall loss of lipolytic activity during evaporation and drying. The activities of lipases and proteinases remained stable during storage of powder at 2511°C. The production of FFA appeared to be a consequence of lipolysis and oxidation reactions. Some changes in chemical and physical properties were observed during storage of powder. Free fat content slightly decreased, possibly due to either its hydrolysis or combining with an unstable protein to form a complex. Insolubility index and peroxide value increased with storage of powder. Bacterial count, moisture level and amount of scorched particles in powders made from control and stored raw milks, and stored at 25fl”C for up to 8 months, suggest that all powders were suitable for reconstitution.
ACKNOWLEDGEMENTS The authors wish to thank Professor William Sawyer for his assistance with lipase assay, and Mr Phil Clarke (CSIRO, Highett, Victoria) for the manufacture of whole milk powder. Helpful discussions with Dr Mary Ann Augustin (CSIRO) are gratefully acknowledged. This research project was financially supported by the Australian Agency for International Development (AusAID).
REFERENCES Acker,
L.W. (1969). Cited by Andersson, R.E. (1980). Microbial hpolysis at low temperatures. Appl. Environ. Microhiol., 39, 3&40. American Dairy Products Institute (1992). Standards for Grades of Dry Milks Including Methods of Analysis, Bulletin 9 16 (Revised). American Dairy Products Institute, Chicago, II, USA. Andersson, R.E. (1980) Microbial lipolysis at low temperatures. Appl. Environ. Microhiol., 39, 3640. Anon. (1989). Dairy Handbook. Alfa-Lava], Food Engineering AB. P.O. Box 64 S-221 00, Lund. APHA (1984). Compendium qf‘Methods,/& the Microbiological Examination ofFoods, 2nd edn.> ed. M.L. Speck. American Public Health Association, Washington, DC, USA. Boehringer Mannheim GmbH Biochemica (1984). Pyruvic acid (pyruvate) UV-method for the determination of pyruvic acid in foodstuffs and other materials. In Methods in Enzvmutic Analysis Using Sing/e Reagents.
126
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