Lipase problems: Outline
Lipase problems in the dairy
industry
What is a lipase?
Importance for the dairy industry
The lipases of significance
 Milk
lipase
lipases
 Bacterial
Lipase action in milk
 Spontaneous
Hilton Deeth
 Induced
Effects on dairy products
NCDEA Webinar 20 March 2013
What is lipase
Lipase is an enzyme* which catalyses the
breakdown of fats (lipids)
[To many of us enzymes are a bit mysterious and
we are not sure how, or even if, they work.
Lipase in milk is a good example of an enzyme
whose presence we can demonstrate. More
later on this]
The breakdown is called lipolysis *.
Measuring lipase and lipolysis
Lipids and lipase action
The fat (lipids) in milk are mostly triglycerides
Lipase causes the breakdown of these triglycerides
(TG), i.e., lipolysis
Triglycerides have a glycerol backbone attached to
3 fatty acids
Lipase action produces free fatty
acids (FFA) and also diglycerides
(DG, 2 fatty acids) and
monoglycerides (MG, 1 fatty acid)
Lipase
TG FFA + DG
DG  FFA + MG
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Importance to dairy industry
 FFA, esp. short chain acids (i.e. butyric (C4),
caproic (C6), caprylic (C8) have strong
flavours and low flavour thresholds  flavour
defects - rancid, astringent, butyric, „bitter‟ but
also impart desirable flavours to some
cheeses like parmesan* and chocolate
The lipases of significance
Natural milk lipase
Bacterial lipases
 Partial glycerides (and FFAs) are surface
active* - cause steam foaming problems in
cappuccino coffee making
Milk lipase
 Originates from the blood
 In the body, the same enzyme is involved in
synthesis* of milk fat (triglycerides) but also
breakdown (hydrolysis) of fats or lipids i.e.,
lipolysis
 Present in all raw milk
 Role in milk unknown; is inactivated by acid in
stomach so of no use to newborn calf
 Inactivated by pasteurisation - FORTUNATELY!
 Therefore it causes no lipolysis in milk or dairy
products after pasteurisation*
Milk lipase in raw milk
 Raw milk contains enough lipase to breakdown all the
fat in milk (~ 1 mg can be isolated from 1 litre)
But it doesn‟t. Why not?
1. It cannot attack fat in intact milk fat globules (due
to protection of the milk fat globule membrane)
2. Lacks some activators: as a “lipoprotein lipase”, it
is activated by lipoproteins as found in the blood –
this can be demonstrated by adding some blood
serum to raw milk; lipolysis proceeds rapidly
3. Contains some substances which inhibit lipase
action [we’ll come back to this point]
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Lipolysis by milk lipase
There are two main ways this can happen:
by spontaneous lipolysis - at farm
Spontaneous lipolysis
Interesting phenomenon but not
completely understood
by induced lipolysis - at farm or
factory
Spontaneous lipolysis
 Initiated in milk of some cows just by cooling to <
100C
 In this type of milk (sometimes called
„spontaneously lipolytic‟ or just „spontaneous‟ milk)
after cooling, lipolysis occurs during refrigerated
storage and reaches a maximum after 12-16 hrs
 Occurs mostly in milk of :
 cows in late lactation
 cows on poor feed
 certain cows only
 FORTUNATELY, spontaneous lipolysis is greatly
reduced when „spontaneous‟ milk is mixed with
„normal‟ milk -
Cow facts
 Can identify „normal‟ cows and
„spontaneous‟ cows
 Some cows always „normal‟
 Some always „spontaneous‟
 Some „spontaneous‟ only at end of
lactation
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Patsy
Thelma
a ‘normal’ but celebrated cow
a nice cow but always ‘spontaneous’
Mixing spontaneous and normal
milk – an example
(Thelma plus Patsy)
Milks mixed (1:1) immediately after milking, then
milks cooled
Free fatty acids (FFA) measured in mixed milk and
individual milks after 16 h at 5ºC
FFA results (mmoles/litre):
Patsy: 0.5; Thelma 4.8; mixture 0.9 (if there was no
inhibition, the FFA would have been 2.65) *
Possible biochemical
explanations
Too much lipase
Weakness of the milk fat globule
membrane?
Presence of activators?
Lack of inhibitors?
All of the above?
Note: most people can detect an off-flavour when the FFA reaches about 1.5-2
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Induced lipolysis (in raw milk)
 Most common cause is disruption of the milk fat
globule membrane; this allows lipase contact with fat
Induced lipolysis
An ongoing problem
 Can be caused by:
Agitation - with air*
Pumping – particularly with air intake
Freezing and thawing
Homogenisation – very effective
In commercial practice, homogenisation is always combined with
pasteurisation (~72°C/15 sec) which destroys milk lipase
Mixing homogenised (pasteurised) milk and raw milk. This is
an effective way of producing milk with a high FFA - A no no
in the dairy industry (a trap for young players!)*
Detecting milk fat globule damage
 Several methods have been proposed
Induced lipolysis – a case study
 When the damage occurs in raw milk, the FFA level is a good
 Raw milk was being sent by tanker from a regional
indication of the extent of damage
 Another way is to measure the amount of free fat, that is, fat
from which the milk fat globule membrane has been removed.
This works OK if:
the damage is not caused by homogenisation
a non-polar solvent such as hexane is used
the extract is done very carefully as to not increase the
damage to the MFGM
 Another method proposed is to measure the amount of lipolysis
produced when a lipase* is added that cannot attack the fat in
an in-tact fat globule; calibration of this method is tricky.
factory to a factory in a capital city
 On one occasion the milk, after pasteurisation, had an
off flavour
 The milk was analysed and found to have a FFA of 8.0
 Trace back showed that the regional factory had some
leftover pasteurised (homogenised) milk on the day
and decided to put it in the tanker with the raw milk
rather than wasting it – a reasonable thing to do
unless you know about lipase and lipolysis!
 This caused severe (induced) lipolysis
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How induced lipolysis occurs in
practice
 Can occur on-farm or in the factory
 On farms:
 due to excess air intake at teat cups and milk surging (and
foaming) in vertical pipes; warm milk is very susceptible
 Spontaneous milk is more susceptible than normal milk
 In factories:
 pumping, particularly if sucking air (faulty seals)
 excess pumping of milk through pipes, especially over long
distances;
 mixing raw and homogenised (pasteurised) milk
Bacterial lipases
Lipolysis by bacterial
lipases
a good reason for keeping bacterial
counts low
How do bacterial lipases differ from
milk lipase
 Lipases are produced by many bacteria
 Most important in milk and milk products are
lipases produced by psychrotrophic bacteria,
especially Pseudomonas species, that grow in
cold milk before pasteurisation
 Are produced when bacterial count is ≥ 106/mL,
in late log – early stationary phase
 Lipases and proteases are often produced by the
same bacterium - some are very heat-stable and
can survive even high temperature processing
such as UHT
1. Most are heat-resistant; are not inactivated by
pasteurisation
2. The milk fat globule membrane is no barrier to
bacterial lipases; hence they can attack fat in
intact fat globules
3. Not activated by blood serum or lipoproteins
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Bacterial lipolysis
 Usually affects long-shelf-life products only
 Amounts of lipase are always very small
 Small amounts of active lipase in products kept for
months, sometimes at room temperature (such as
UHT milk), can cause considerable lipolysis and
hence off flavours
Lipolysis in milk
Market milk:
In pasteurised milk, usually due to the action of
milk lipase before pasteurisation
In extended shelf life (ESL) milk, due to
bacterial lipase if it develops during storage
Noticeable at FFA > 2 mmol/L
UHT milk:
Rancidity may develop during storage
Due to traces of heat-resistant bacterial lipases
Lipase and lipolysis in
dairy products
most products can be affected
Lipolysis in powders
Rancidity is sometimes detected in powders containing fat
after storage, i.e. between production and end use
This can be awkward if the powder has been exported
and the end user is overseas
Due to bacterial lipases if the level of free fatty acids
immediately after manufacture is low
In powders containing no fat or containing very little fat, the
main problem is bacterial lipases which can affect the
quality of the final product.
Some powders receive low heat treatment. e.g.,
pasteurisation, so bacterial lipases will remain active in the
powder
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Lipolysis in butter, ice cream, yogurt
Butter:
If present when fresh, due to milk lipase but if it
occurs after storage, due to bacterial lipases
Can determine which by FFA profile; short chain fatty
acids are water-soluble and lost in buttermilk if lipolysis
occurs before butter is made
Ice cream:
Usually caused by milk lipase – before
manufacture. Has caused problems in the past
Yogurt:
Lipolysis in cheese
Cheese:
A high FFA level is normal for some cheese types, eg
blue vein, parmesan, feta
Produced by mould or added lipases or even
homogenisation of raw milk before cheese
manufacture
In other types such as cheddar, it causes rancidity,
soapiness. Can cause huge losses – and has done.
If high free fatty acids are present when cheese is fresh,
due to milk lipase but if they develop during storage, due
to bacterial lipases; bacterial lipase is usually the culprit
Not normally a problem except when additives
have active lipase, e.g. passionfruit
Lipolysis in processed cheese a case study
Cheese developed high FFA during storage
Suspected bacterial lipase
But cheese milk had low bacterial count
Measuring lipase and
lipolysis
Cause
Build-up of solid material in pumping equipment
Harboured very lipolytic bacterial contaminants
Lipase released into final cheese product
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Measuring lipase
 Hardly anyone in the dairy industry measures lipase activity in
milk or dairy products
 Why?
Because measuring the native milk lipase does not help in
any way. Remember there is more than enough in raw
milk to cause extensive lipolysis and is absent from
pasteurised milk
Measuring bacterial lipase would be much more beneficial.
This may tell us which products are going to lipolyse during
storage.
Unfortunately, this is very difficult as the lipase is present in
only trace amounts and suitable methods of measuring such
low levels are not readily available
 However, a sensitive method which takes several days was
developed at University of Melbourne and published in 2011
Measuring free fatty acids (Total)
 No simple method
 Traditionally by BDI method
demulsification of fat with added Triton X-100 & Calgon plus
heat, separation of fat, then titration of an aliquot of fat with
methanolic KOH
OK but tedious for large numbers
 Another method used is a colorimetric method based on
making copper salts of the fatty acids (the copper soap method)
 Better is a method using solvent extraction followed by titration
of the extract, e.g., extracting with isopropanol-hexanesulphuric acid and then titrating the extract (containing the fatty
acids) with methanolic KOH
Widely used in original and modified forms
Measuring lipolysis
 This is much more common
 Measuring free fatty acids is the most common way
 The total concentration is usually measured, not the
individual acids [which can be done by gas
chromatography]
 Milk free fatty acids range from water-soluble to fat
soluble, so methods to measure them seldom
measure all of them
 Another measure sometimes used for raw milk is
foaming capacity. Useful if foaming is important but
correlation between lipolysis and foaming capacity is
not perfect
But is a solvent extraction-titration
method safe?
Although it uses flammable
organic solvents, it is normally
very safe
However, it can be a fire hazard
- if solvent extracts are stored in
the fridge before titration
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Concluding remarks
 Lipolysis in milk and dairy products is an on-
going problem
 It is important to be able to work out whether
a lipase problem is caused by milk lipase or
bacterial lipase – the remedies are quite
different
 The dairy industry needs to continually
ensure personnel are aware of the causes
and potential effects of lipase action on
product quality – and profitability
Thank you for your
attention
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