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Effects of mechanical agitation of raw milk on the milk-fat globule in relation

to the level of induced lipolysis

Article in Journal of Dairy Research · October 1978

DOI: 10.1017/S0022029900016599

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Journal of Dairy Research (1978), 45, 373-380 373

Effects of mechanical agitation of raw milk on the milk-fat

globule in relation to the level of induced lipolysis
BY HILTON C. DEETH AND CAROLYN H. FITZGERALD
Otto Madsen Dairy Research Laboratory,
Hamilton, Queensland 4007, Australia

(Received 29 November 1977)

SUMMARY. The amount of damage caused to the milk-fat globule by mechanical

agitation of raw milk was assessed by several methods and related to the level of
induced lipolysis resulting from the agitation. The results obtained by measuring the
amount of 'free' fat formed, the percentage of fat in the skim-milk phase after
separation, and the increase in the proportion of alkaline phosphatase associated
with the skim-milk phase, showed little correlation with the level of lipolysis induced
by agitation over a range of conditions. However, the level of induced lipolysis showed
good correlation with the amount of lipolysis caused by addition of an exogenous
lipase (from Candida cylindraceae) to the agitated milk. It is concluded that classical
methods of assessing milk-fat globule damage are unsuitable for predicting the
amount of fat available for lipase action and the lipolytic potential of raw milk
subjected to mechanical agitation.

Activation of the lipase system in milk by mechanical means has been attributed
by many workers to the increased accessibility of the milk fat to the natural milk
lipase caused by rupture of the protective milk-fat globule membrane (MFGM).
This process offers a reasonable explanation for the lipolysis resulting from severe
treatments such as homogenization and is probably the major effect of agitating
raw milk. Agitation, especially with simultaneous foam formation, has recently been
shown to cause another marked change in the milk lipase system - a redistribution
of the enzyme between the cream and the skim-milk phases (Deeth & Fitz-Gerald,
1977). In order to determine the significance of changes in the fat globule to the level
of induced lipolysis a suitable method for measuring these changes was sought.
Several approaches have been made to assess the extent of milk-fat globule damage
caused by mechanical treatment of milk. The amount of fat released from the damaged
globules, the so-called free fat, is usually measured by a heating-centrifuging pro-
cedure based on the method of Webb & Hall (1935) (Rothwell, 1962; Te Whaiti &
Fryer, 1975; Dillier-Zulauf & Wirasekara, 1971) or by solvent extraction (Lagoni &
Peters, 1959; Aule & Worstorff, 1975). The amounts of fat and of certain enzymes
normally associated with the MFGM which remain in the skim-milk phase after
separation of activated milk have been used as indexes of globule disruption (Hlynka,
Hood & Gibson, 1945; Solms-Baruth, 1971; Aristova et al. 1974; Stannard, 1975).
Another procedure measures the amount of lipolysis caused by certain fungal lipases,
374 H. C. DEETH AND C. H. FITZ-GEKALD
which attack unprotected fat but not the intact milk-fat globules, when added to the
activated milk (Deeth & Fitz-Gerald, 1975). Other reported procedures include the
determination of free fat by passing milk through a column of silica gel and eluting
the adsorbed fat with chloroform (Aristova, 1974), the measurement of the fat
globule size distribution (Solms-Baruth, 1971) and the examination of the milk sur-
face by microscopy (King, 1951). In this investigation 5 of the above methods have
been used to assess the extent of fat globule damage in milks agitated under different
conditions, in an attempt to relate this to the amount of lipolysis induced in the milk.

MATERIALS AND METHODS

Milk. Uncooled bulk whole milk was obtained from a local commercial Friesian
herd and experiments were performed within 1-5 h. Milk from this farm was con-
sistently found to have extremely low susceptibility to both spontaneous lipolysis
(Tarassuk & Frankel, 1957) and lipolysis induced by temperature manipulation
' (Claypool, 1965).
Activation equipment and procedure. Samples were activated using a Sorvall
Omnimixer (Dupont Instruments, Newton, Conn., USA) with 400-ml capacity
stainless steel mixing vessels or a 2-speed Waring Commercial Blender (Waring
Products, Winsted, Conn., USA) with 1-1 capacity glass mixing vessel.
Whole milk was adjusted to the required temperature as rapidly as possible by
immersion in ice-water or hot water. Samples were activated immediately following
temperature adjustment. Directly after activation, samples were cooled to 5 °C in
ice-water.
Milk separation. Milk was separated at 36 °C into skim-milk and cream fractions
with a manually operated milk- separator (Nugget (Alfa-Laval AB, Stockholm,
Sweden) 451/h). Samples of skim-milk for alkaline phosphatase and skim-milk fat-
content assays were obtained by twice centrifuging 10-ml amounts of whole (cooled)
milk at 2000 g for 20 min at 5 °C.
Measurement offree fatty acids. The free fatty acid (FFA) contents of activated milks
and unactivated controls were determined on 3-ml samples after 20-h storage at
5 °C by the method of Deeth, Fitz-Gerald & Wood (1975). Results are expressed as
/i-equiv. FFA/ml.
Susceptibility to lipolysis by Candida cylindraceae lipase. Samples of milk (3-ml) were
incubated at 5 °C for 20 h with Candida lipase (Sigma, St Louis, Mo., USA) (0-5 ml,
2 mg/ml). Total FFA released were measured as above. Net lipolysis by Candida
lipase was obtained by subtracting the FFA content of controls incubated with
0-5 ml water in place of Candida lipase.
Fat analyses. Fat analyses of whole and skim-milks were carried out with a
Milkotester Mk I I I (Foss Electric, Hillerad, Denmark).
Measurement of alkaline phosphatase activity. Alkaline phosphatase (E.C. 3 . 1 . 3 . 1 )
activities were measured against ^-nitrophenyl phosphate using the following pro-
cedure: 0-1 ml milk, 0-3 ml 001 M-Mg2+, 0-3 ml 0-05 M-p-nitrophenylphosphate and
1-5 ml 0-1 M-carbonate buffer pH 10-2 were incubated at 25 °C for 5 min. The
reaction was stopped with 0-8 ml 0-5 M-NaOH and the mixture centrifuged. The
absorbance of the clear supernatant was read at 410 nm. Activities of skim-milks
were calculated as a percentage of the activity of whole milk.
 Mechanical agitation of milk 375
Free fat. The free fat method of Te Whaiti & Fryer (1975) was used to estimate
churned fat content of whole milks.
Measurement of solvent extractable fat. Milk (17-5 ml) and hexane (22-5 ml) were
measured into 50-ml centrifuge tubes, warmed to 40 °C and shaken gently by
hand for 20 s, then centrifuged for 5 min at 1000 g; 17-5 ml samples of the super-
natant layer were evaporated to dryness and the percentage weight of solvent
extractable fat in the original milk sample calculated.

EXPERIMENTAL AND RESULTS

Influence of temperature of milk and speed and duration of agitation on substrate
activation and lipolysis
Samples of milk (100 ml) were activated in a Sorvall Omnimixer at 2 speeds,
4000 and 15000 rev/min, for 20 s at 5, 10, 15, 20, 25, 30, 35 and 40 °C. Samples
were also activated at the lower speed for 10, 40 and 80 s at 5, 20 and 40 °C.
On each activated sample and on an unactivated control milk the following
analyses were performed: (i) degree of activation of lipolysis (FFA released in 20 h
at 5 °C); (ii) susceptibility to lipolysis by Candida lipase; (iii) fat content of skim-
milk; (iv) alkaline phosphatase activity in skim-milk; (v) solvent extractable free
fat; (vi) free fat. The dependence of factors (i-v) on temperature and time of agitation
for the 2 agitation speeds is shown in Figs 1 and 2 respectively.
(i) Degree of activation. Typical type (a) and type (b) activation curves (Deeth &
Fitz-Gerald, 1977) were produced by agitation at 4000 and 15000 rev/min respec-
tively. The degree of activation of lipolysis increased (non-linearly) with time of
agitation at rates dependent on the temperature of agitation (Figs la, 2a).
(ii) Susceptibility to lipolysis by Candida lipase. Curves reflecting the above activation
curves were obtained for the relationship between susceptibility to lipolysis by
Candida lipase and temperature of agitation, i.e. a peak was obtained at 15 °C for
the low speed agitation. With milks agitated at the higher speed, lipolysis by the
added lipase increased with increasing agitation temperature to a greater extent
than did lipolysis by milk lipase (Fig. 1 b). With increasmg time of agitation, lipolysis
by the added lipase increased almost linearly whereas lipolysis by milk lipase tended
to level out (Fig. 2b).
(iii) Fat content of skim-milk. For either speed of agitation, little increase in the fat
content of the skim-milk phase occurred for temperatures below 15 °C. Above 15 °C
only slight increases with temperature were found in the lower-speed agitated samples
while a rapid, almost linear increase was observed for the samples agitated at the
higher speed (Fig. lc).
With increasing time of agitation, the skim-milk fat levels showed no increase
at 5 °C, a slight increase at 20 °C and an appreciable increase at 40 °C (Fig. 2c).
(iv) Alkaline phosphatase activity in skim-milk. The release of alkaline phosphatase
into the skim-milk phase during agitation decreased as the temperature was in-
creased from 5 to approximately 20-30 °C and increased again as the temperature
was raised to 40 °C (Fig. Id). Little correlation with the degree of activation was
observed. The amount of alkaline phosphatase released increased with increased
time of agitation (Fig. 2d).
376 H. C. DEETH AND C. H. FITZ-GERALD
80 r 1 (a)

60 (ii) 60

§"40 "$• 40
3.

2-0 < 20

10 20 30 40 20 40 60 80

300 r

250 r 1

200

"f-15-0
5

< 100
u.

50

10 20 30 40 20 40 60 80

0-41- 1 (c)

0-3 0-31- 2 (c)

55 0-2

0-1

10 20 30 40 20 40 60

Fig. 1 (o-c) and Fig. 2 (a^c). For legend see opposite.

 Mechanical agitation of milk 377
90r- 2 (d)

801- 1 (d)

. 70
ID

60
Q.
ID

II 50

I I
40
10 20 30 40

10-0-r- 1 (e)

80

60

CD
CD
it 40

20

I I
10 20 30 40 20 40 60
Agitation temp., °C Agitation temp., °C

Fig. 1. Effect of agitation temperature on (a) degree of activation of lipolysis; (6), susceptibility
to lipolysis by Candida lipase; (c), fat content of skim-milk; (d) percent total alkaline phos-
phatase activity in skim-milk; (e), solvent extractable ('free') fat. Agitation conditions: Sorvall
Omnimixer, 20 s, 100 ml milk; • , 4000rev/min; A, 15000 rev/min. Unactivated controls (a)
O-5/t-equiv./ml; (6) 0-4/i-equiv./ml; (c) 0'04%; (d) 46-7%; (e) 2-2%. (i), Type (a) curve; (ii),
type (6) curve (Deeth & Fitz-Gerald, 1977).
Tig. 2. Effect of agitation time on (a), degree of activation of lipolysia; (6), susceptibility to
lipolysis by Candida lipase; (c), fat content of skim-milk; (d), percent total alkaline phos-
phatase activity in skim-milk; (e) solvent extractable ('free') fat. Agitation conditions: Sorvall
Omnimixer, 4000 rev/min, 100 ml milk; • , 5 °C; A, 20 °C; • , 40 °C.
378 H. C. DEBTH AND C. H. FITZ-GERALD
(v) Solvent extractable free fat. With high speed agitation the amount of free fat
extracted by hexane showed a maximum at intermediate temperatures of activation
and was much greater at all activation temperatures with high speed than with low
speed agitation (Fig. le). Differences in the degree of lipolysis initiated by the 2
treatments were much less pronounced (Fig. 1 a) than the differences in the amount of
solvent extractable fat (Fig. le).
In a separate experiment, commercially homogenized milk showed a negligible
amount of solvent extractable fat.
(vi) Free fat. For the milk used in this investigation (fresh, uncooled, Friesian
bulk milk). the measurement of free fat by the warming-centrifuging procedure
(Te Whaiti & Fryer, 1975) was most unsatisfactory. For short agitation times very
little free fat was obtained and no reliable indication of the effect of temperature of
agitation on the amount of fat released could be obtained. With increasing time
of agitation the amount of free fat usually showed an increase.

DISCUSSION
Agitation of whole milk can cause either aggregation (clumping, churning) or
disruption (splitting, dispersion) of the milk-fat globules (Mulder & Walstra, 1974).
The temperature of the milk, and hence the physical state of the milk-fat, and the
severity of agitation determine which of these effects predominates. In both cases
the fat loses its natural protection and becomes free and thus susceptible to lipase
attack. The extent of this susceptibility influences the extent of induced lipolysis.
The methods which have been used to assess the amount of fat globule disruption
caused by agitation of whole milk measure several different aspects of the process.
The most widely used method, the so-called free fat method, measures the amount of
churned-out fat obtainable by a warming and centrifuging procedure (Webb & Hall,
1935; Te Whaiti & Fryer, 1975). Using this method, Te Whaiti & Fryer (1975) found
that the optimal temperature for churning in milk was in the range 25-35 °C. In the
present investigation, this method was too insensitive and did not yield meaningful
results. The amount of free fat obtained was quite small and hence difficult to
measure accurately. Although the method has been successfully used in applications
where an estimate of the amount of churned-out fat was specifically required (Te
Whaiti & Fryer, 1975), it proved the least useful of the methods tested here for
assessing the extent of fat globule disruption as it relates to the degree of activation
of the lipase system. The milk used in this investigation was from a Friesian herd and
it is known that such milk is less susceptible to formation of free fat when agitated
than that from some other breeds (Te Whaiti & Fryer, 1976). Furthermore, it had
not been subjected to cooling or ageing, processes which are known to weaken the
protective MFGM and facilitate churning (Anderson et al. 1972; Anderson & Cheese-
man, 1975).
Free fat has also been estimated by a solvent extraction procedure (Aule &
Worstoff, 1975). This method calls for extreme care in manipulation and in stan-
dardizing of shaking conditions to achieve acceptable reproducibility. The method
gives a reasonable estimate of the amount of churned-out fat, but does not measure
finely dispersed or homogenized fat. The relationship of solvent-extractable fat to
 Mechanical agitation of milk 379
agitation temperature for the higher agitation speed (Fig. 1 e) showed a similar pattern
to that obtained by Te Whaiti & Fryer (1975) for free fat by the Webb & Hall (1935)
method. The amount of solvent extractable fat did not reflect the degree of activation
of lipolysis, particularly at the higher temperatures and with the lower speed of
agitation. In the latter case, appreciable lipolysis occurred for a release of free fat
considerably smaller than that found with the more vigorous agitation.
Measurement of the amount of fat in the skim-milk separated from agitated whole
milk under standard conditions provides an estimate of the extent of dispersion or
homogenization of milk-fat globules (Hlynka et al. 1945; Aristova et al. 1974; Aule &
Worstorff, 1975). Fig. 1 (c) shows that, under the agitation conditions used, dispersion
was negligible at temperatures below c. 15 °C and could not account for the lipolysis
observed at these agitation temperatures. A comparison of the relative amounts of
lipolysis and dispersed fat (Figs 1 a, c) for the 2 speeds of agitation at higher tem-
peratures shows that activation of lipolysis bears little relationship to the extent
of dispersion of the milk-fat globules. This is contrary to the results of Aule &
Worstorff (1975) who reported a high correlation (r = +0-996) between increase in
skim-milk fat content and FFA. Their results were obtained from agitation experi-
ments at only 2 temperatures (5 and 32 °C) and hence the lack of correlation observed
in the present investigation over a range of temperatures was not apparent.
Stannard (1975) suggested that the release of alkaline phosphatase (and xanthine
oxidase) from the MFGM could be used as an index of the extent of churning in
milk. He found that the level of alkaline phosphatase in the skim-milk increased
with the amount of agitation at a given temperature. The present work supports
this conclusion (Fig. 2d). However, the curve showing the amount of enzyme
released during agitation at different temperatures (Fig. Id) is unlike all other curves
shown in Fig. 1 and does not correlate well with the observed lipolysis pattern. In
fact, it is the inverse of the relationship between churning and agitation temperature.
At low temperatures (5 °C) considerable release of membrane material occurred but
only a minimal amount of lipolysis was initiated. Previous work (Deeth & Fitz-
Gerald, 1977) has shown that low temperature agitation results in considerable
transfer of lipase to the remaining MFGM. This attachment of the lipase to the fat
globules may be facilitated by the removal of some of the outside membrane material.
Fig. 1 (d) shows a minimal release of membrane enzyme in the temperature region
where considerable churning occurs (c. 20 °C) (Te Whaiti & Fryer, 1975).
Lipolysis by added Candida lipase showed a close parallel to the amount of
lipolysis by endogenous milk lipase, although it did not show a tapering-off with
increased agitation temperature (Fig. 16) or with increased agitation time (Fig. 26)
as did lipolysis by milk lipase (Figs la, 2a). Such differences could be explained if
the milk lipase showed greater substrate saturation and/or product inhibition effects
than did the Candida lipase at the level used in these experiments. Since this method
gives a measure of the surface area of accessible fat it does not distinguish between
churned fat and homogenized fat as do the free fat and fat-in-skim methods discussed
above.
The methods used here to estimate the extent of fat globule damage gave widely
differing results. Since each method measures a different consequence of the agitation
treatment and each of these varies in a different manner with temperature and
380 H. C. DEETH AND C. H. FITZ-GEEALD
severity of agitation, it appears that no single index can be used adequately to
describe the effects of agitation on the milk-fat globule as it relates to the extent of
lipolysis by milk lipase. Conversely, the FFA level developed in milk after agitation
cannot be taken as an accurate index of milk-fat globule damage. The results obtained
with the added Candida lipase showed greatest promise and gave the only indication
that the 15 °C peak observed in activation curves of type (a) (Deeth & Fitz-Gerald,
1977) (Fig. la) can be explained in terms of substrate activation alone. This method
is difficult to quantify and hence may be of only limited value in assessing fat
globule damage. Furthermore, preliminary results indicate that lipolysis by the
added lipase may not be as closely related to lipolysis by the milk lipase in stored
milks as in fresh milks.

This work was supported by grants from the Australian Dairying Research
Committee. Capable technical assistance by Messrs A. F. Wood and D. Mclntosh is
gratefully acknowledged.
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