1 Lipase problems: Outline Lipase problems in the dairy industry Hilton Deeth NCDEA Webinar 20 March 2013 What is a lipase? Importance for the dairy industry The lipases of significance Milk lipase Bacterial lipases Lipase action in milk Spontaneous Induced Effects on dairy products Measuring lipase and lipolysis 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 *. 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 Lipase action produces free fatty acids (FFA) and also diglycerides (DG, 2 fatty acids) and monoglycerides (MG, 1 fatty acid) TG FFA + DG DG FFA + MG
2 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 Partial glycerides (and FFAs) are surface active* - cause steam foaming problems in cappuccino coffee making The lipases of significance Natural milk lipase Bacterial lipases 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]
3 Lipolysis by milk lipase There are two main ways this can happen: Spontaneous lipolysis by spontaneous lipolysis - at farm by induced lipolysis - at farm or factory Interesting phenomenon but not completely understood Spontaneous lipolysis Initiated in milk of some cows just by cooling to < 10 0 C 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
4 Patsy a normal but celebrated cow Thelma a nice cow but always spontaneous Mixing spontaneous and normal milk an example (Thelma plus Patsy) Possible biochemical explanations 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) * Note: most people can detect an off-flavour when the FFA reaches about 1.5-2 Too much lipase Weakness of the milk fat globule membrane? Presence of activators? Lack of inhibitors? All of the above?
5 Induced lipolysis (in raw milk) Induced lipolysis An ongoing problem Most common cause is disruption of the milk fat globule membrane; this allows lipase contact with fat 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 When the damage occurs in raw milk, the FFA level is a good 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. Induced lipolysis a case study Raw milk was being sent by tanker from a regional 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
6 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 Lipolysis by bacterial lipases a good reason for keeping bacterial counts low Bacterial lipases 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 10 6 /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 How do bacterial lipases differ from milk lipase 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
7 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 Lipase and lipolysis in dairy products most products can be affected 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 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
8 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: Not normally a problem except when additives have active lipase, e.g. passionfruit 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 Lipolysis in processed cheese - a case study Cheese developed high FFA during storage Suspected bacterial lipase But cheese milk had low bacterial count Cause Build-up of solid material in pumping equipment Harboured very lipolytic bacterial contaminants Lipase released into final cheese product Measuring lipase and lipolysis
9 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 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 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 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
10 Concluding remarks Lipolysis in milk and dairy products is an ongoing 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