Free Fatty Acids in Milk: Origin and Effects on Milk Quality

Alejandro G. Marangoni, Ph.D., FRSC, FAOCS, FRSC (U.K.)

Professor and Tier I Canada Research Chair

Food, Health and Aging

Lipid composition of bovine milk

Jensen, 2002, JDS 85:295

How are milk triglycerides made?

FFA’s are then resynthesized into TAGs and assembled into a MFG

Lipoprotein LipaseThe only endogenous lipase found in milk?

Deeth, H.C. 2006. Lipoprotein lipase and lipolysis in milk. Int. Dairy J. 16: 555-562Bengtsson-Olivecrona and Olivercrona, 1991. Meth. Enz. 197: 345

• Catalyzes the hydrolysis of triglycerides in chylomicrons (CM) and very low density lipoproteins (VLDL)

• Structurally related to pancreatic lipase and hepatic lipase

• Also hydrolyzes partial glycerides, phospholipidsand other esters in lipoproteins

• Has specificity for the sn-1,3 positions in TAGs, with enhanced activity towards sn-1. No fatty acid specificity.

• Normally associated with the casein micelle• No known biological function and found in very

high amounts in milk (1-2mg active enzyme/L)

Lipoprotein lipase cont’d• It is inhibited by fatty acids because LPL binds preferentially

to fatty acid micelles or fatty acids that accumulate at the MFG interface during hydrolysis.

• Bovine serum albumin (and calcium) binds fatty acids preferentially, thus removing them from the reaction. Hence, the reputation for being an activator. In vivo, fatty acids would be metabolized by living tissue and not accumulate in micelles

• LPL is a 100kDa homodimer glycoprotein with two N-linked oligosaccharides

• Can be released from the casein micelle by NaCl and heparin. Heparin has a high affinity for LPL and stabilizes it.

• Optimal reactions are 37oC and pH 9.0 and dentures rapidly at higher temperatures. HTST conditions (72oC for 15sec) inactivate the enzyme almost completely – heat labile.

• Typical activity in milk is ~100nmol min-1 mL-1, which would lead to complete milkfat hydrolysis in less than 10 min. Luckily, milk fat is protected within the milkfat globule.

Milk lipids are structured in the milkfat globule

0.2-10 µm diameter (small, normal, large)

Free fatty acids are a product of triglyceride hydrolysis

Units of measurement of hydrolysis:a true mess!

• meq/100g fat• mmol/L• mmol/100g fat or milk• µeq/mL• no units at all • % not recommended since:

g fatty acids/100g of X (%) = (moles base used)*(molecular wt. fatty acid) for 100g of milk or the fat in milk.

Need to know the free fatty acid composition of the milk!

Fatty acids in milkfat

88g/mol 88ppm1 0.22%1

mg/L g/g fat2

MW

282 g/mol 282ppm1 0.70%1

mg/L g/g fat2

1 for 1mmol/L FFA2 assuming 4% fat content in milk

Spontaneous and induced lipolysis

• Spontaneous lipolysis initiated by cooling milk below 10oC soon after milking

• Induced lipolysis arises due to physical damage to the milk fat globule membrane (agitation, pumping and homogenization)

• Both proceed during storage, with most occurring within the first 24 hours

Spontaneous Lipolysis

• Lack of integrity of MFGM

• Activating and inhibiting factors (most probably apolipoproteins (apoC-II and apoC-I activate, apoC-III inhibits). Heparin-like glycosaminoglycans in milk may activate LPL while proteose peptone fraction PP3 inhibits lipolysis. Monoolein removes apoC-III inhibition….complicated

• High LPL amounts

• MFGM-associated LPL! This is governed by the apoproteins, which may enhance binding to the MFGM

Spontaneous lipolysis

• Occurs at the farm only and 10mmol/L levels are possible!

• Enhanced due to late lactation, poor quality feed and mastitis

• For mastitis it is due to decreased TAG synthesis (elevated initial FFA levels) as well as lipolysis

• Any transfer of blood components to milk enhance spontaneous lipolysis (apoproteins, BSA).

• And now for a mistery…….”temperature activation”; can happen when mixing precooled milk from a previous milking with warm milk (method: heat to 25-35oC and re-cool to less than 10oC).

Induced lipolysis of milk

• Any process that will disrupt the MFGM and make the fat within more accessible to LPL. Sometimes the concept of “free fat” has been used, but not recommended.

• Agitation, pumping, air incorporation, homogenization, mixing raw and homogenized milk, freezing/thawing.

Effect of activation temperature on lipolysis of-precooled milk under constant agitation

Induced lipolysis of milk

FFA levels in milk are enhanced after 24 hours due to increased milking frequency. MFG size and integrity were not affected

Wiking et al. 2006

Another source of lipase – psychrotrophiclipolytic bacteria (e.g., Pseudomonas)

MFGM does not stop these lipases and they survive pasteurization temperatures (140oC for 4s)

Why should we care?

You are killing my cappuccino!

FFAs kill foams by displacing protein from the bubble surface.

Deeth and Smith, 1983Buchanan, 1965Huppertz, 2010

Why should we care?

• Hydrolytic rancidity. Short chain free fatty acids have a very strong and unacceptable “rancid/butyric/astringent/bitter” flavor at concentrations as low as 1.5mmol/L.

• Impaired creaming ability during separation

• Increased churning time in the manufacture of butter

• Increased fat loss in whey during cheese production

• Inhibitory effect on starter cultures used in making fermented dairy products

Why should we care?

FFA’s and milking technology

FFA – free fatty acidsSCC – somatic cell countTBC – total bacterial countPliBC – psychrotrophic lipolytic bacteria Mikulova, 2011

Analysis methods

• Extraction and titration with alkali (KOH or NaOH)

• Potentiometric methods

• Fourier transform mid-IR

• Gas-liquid chromatography

• Biosensor assays

Future work

• Optimization of rapid analytical method

• Mechanism of heat activation of LDL activity

• Making sure a separate endogenous milk lipase is not present. Why does everyone ignore the work of UC Davis (Fox, Patel, Tarassuk, 1968) and that of Jensen and Pitas (1976)?

• Crystal structure of bovine milk LDL

Acknowledgments

• Natural Sciences and Engineering Research Council of Canada

• Canada Research Chairs Program