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Lipids in cheese

Article  in  Lipid Technology · March 2015

DOI: 10.1002/lite.201500015

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Lipid Technology April 2015, Vol. 27, No. 4 83

DOI 10.1002/lite.201500015

Feature
Lipids in cheese*
Dr. Michael H. Tunick
Dr. Tunick is a research chemist in the Dairy and Functional Foods Research Unit, Eastern Regional Research Center, Agricultural Re-
search Service, U.S. Department of Agriculture, Wyndmoor, PA. E-mail: michael.tunick@ars.usda.gov

Summary
Lipids are present in cheese at levels above 20% and are analyzed by several techniques. Scanning electron microscopy (SEM) and confocal
laser scanning microscopy (CLSM) are used to examine the microstructure, gas chromatography is employed to look at fatty acid composi-
tion, and differential scanning calorimetry is utilized to examine the melting profile. Differences resulting from storage, organic feeding regi-
men, substitution of milk from a different species, and homogenization may be determined.

Milk and cheese Microstructure

Milk fat has the most complex structure and composition of all li-
pids found in food. Lipids in raw milk are in the form of emulsified
globules consisting of a triacylglycerol core that is covered with the
mammary cell membrane when it is secreted from the cell; the pro-
tective layer is the milk fat globule membrane (MFGM). The glo-
bules range from 0.1 to 10 μm in diameter in bovine milk and the
MFGM is a surface-active layer 8–10 nm thick. The MFGM is
composed of about 30% proteins and 60% lipids, including phos-
pholipids and cholesterol. It protects the globule from coalescence,
lipolysis, and oxidation, and is stripped off of the globules if the
milk is homogenized. A milliliter of raw cow milk contains ap-
proximately 1.5 × 1010 fat globules with a surface area around 2 m2
[1].
Commercially, cheese is the second most important dairy pro-
duct in the U.S., behind only fluid milk. The solid curd is separated
from the liquid whey in cheesemaking, concentrating the fat, pro-
tein, and minerals in milk by factor of approximately ten. Nearly all
cheese varieties contain more than 20% fat, and most of the volatile
compounds responsible for flavor are fat-soluble. The wide array of
flavors comes from the breakdown of protein and lipids by en-
zymes generated from the bacteria and coagulant that are added
during cheesemaking. The tasty and nutritious product that results
has been the subject of research for decades, some of it centering
on the lipids it contains.
Dietary experts have long encouraged consumers to use low-fat
dairy products, including lite cheese, to reduce saturated fat intake.
Mammary glands produce saturated and unsaturated fat, however,
and natural selection would presumably cause the latter to be pre-
dominant if the former were harmful [2]. Moreover, milk fat carries
bioactive compounds such as conjugated linoleic acid, phospholi-
pids, and vitamins, and, as noted above, is structured differently
than other lipids. Investigations on physical properties of fat and
other cheese components have been conducted at our laboratory for
many years, and this feature will summarize USDA research on li-
pids in cheese.
Fat globules in cheese may be viewed by scanning electron micro-
scopy (SEM) and confocal laser scanning microscopy (CLSM).
SEM provides information on the appearance of the cheese matrix
and CLSM allows for differential staining of fat and protein.
Fat is removed by solvent extraction during the sample prepara-
tion for SEM so that the areas vacated by the globules may be ob-
served. In our laboratory, samples are fixed in a 1% solution of
glutaraldehyde in 0.1 M sodium cacodylate and dehydrated in a
graded series of ethanol solutions before extraction of the lipids
with three changes of chloroform. After freeze-fracturing and criti-
cal point drying, the specimens are coated with a thin layer of gold
and examined by secondary electron imaging [3].
Figure 1 shows the microstructure of Mozzarella cheese. The
voids that had been occupied by the fat globules in Mozzarella
cheese show up as black cavities within a gray casein matrix. Most
of the voids are elongated because of the stretching operation dur-
ing Mozzarella manufacture. Streptococcus thermophilus from the
starter culture is visible in some of the cavities in the sample from
1 wk of refrigerated storage (Figure 1a). Bacteria in cheese tend to
congregate at the fat-protein interface. Figure 1b was obtained from
the same sample 5 wk later and shows that the fat has coalesced
after proteolytic breakdown of the casein released the globules
trapped inside. The number of S. thermophilus colonies decreased
during the storage period, and the release of proteolytic enzymes
from them apparently contributed to the partial breakdown of the
casein structure [3].
For CLSM, samples were obtained from the cheese, fixed, and
stored as with SEM. Two dyes were then applied: Nile Red, which
stains milk fat globules yellow, and FCF Fast Green, which stains
protein green. CLSM of Queso Blanco, a popular Hispanic variety,
showed that the protein matrix became less continuous and the dis-
tribution of fat droplets became more extensive during storage (Fig-
ure 2). These results were consistent with proteolytic breakdown of
the microstructure by the coagulant enzyme chymosin. As with the
Mozzarella, the fat globules are released from the matrix and coa-
lesce as the bonds between casein molecules are broken.

* Mention of trade names or commercial products in this article

is solely for the purpose of providing specific information and Composition
does not imply recommendation or endorsement by the US De-
partment of Agriculture. USDA is an equal opportunity provider The composition of the lipids in milk is well known, and the tri-
and employer. acylglyceride and fatty acid profiles do not change when the milk

© Published 2015. This article is a U.S. Government work and is in the public domain in the USA. www.lipid-technology.com
Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
84 April 2015, Vol. 27, No. 4 Lipid Technology

Figure 1. Scanning electron micrographs of Mozzarella cheese after (a) 1 wk and (b) 6 wk of storage at 4°C. Arrows point to
Streptococcus thermophilus from the starter culture. Bar at lower right = 40 μm.

is converted to cheese. The triacylgylcerols contain 24–54 carbon Table 1. Major fatty acids and total fat in milk from the most common
atoms, and the arrangement of fatty acids on the glycerol backbone dairying species [5].
is not random owing to the specificities of the enzymes involved in Cow Buffalo Goat Sheep
biosynthesis [1]. Unique among lipids, the 4:0, 6:0, 8:0, and 10:0
g fat/100 g fat
fatty acids in milk fat are preferentially esterified at the sn-3 posi-
tion, and two-thirds of the fatty acids at sn-2 are 14:0 and 16:0 [1]. 4:0 3.3 3.6 2.6 4.0
Table 1 shows the fatty acid profiles of the milk from species most 6:0 1.6 1.6 2.9 2.8
commonly used for making cheese. Variations exist due to age, 8:0 1.3 1.1 2.7 2.7
breed, feed, health, season, stage of lactation, time since previous 10 : 0 3.0 1.9 8.4 9.0
milking, and other factors [4]. 12 : 0 3.1 2.0 3.3 5.4
The fatty acid profiles of milk fat are examined in our laboratory
14 : 0 9.5 8.7 10.3 11.8
by removing the fat from the milk by centrifugation, converting the
16 : 0 26.3 30.4 24.6 25.4
fatty acids to methyl esters, and obtaining a fatty acid profile by gas
16 : 1 2.3 3.4 2.2 3.4
chromatography. We recently completed a project comparing or-
18 : 0 14.6 10.1 12.5 9.0
ganic and conventional milk from adjacent farms, finding that the
compositions and proteins of the two types of milk are nearly iden- 18 : 1 29.8 28.7 28.5 20.0
tical. One notable difference is in the percentages of α-linoleic acid 18 : 2 2.4 2.5 2.2 2.1
(ALA) and conjugated linolenic acid (CLA) in the lipids. These 18 : 3 0.8 2.5 0.0 1.4
two fatty acids have been shown to confer numerous health bene- All 3.3–4.7 4.7–6.9 4.1–4.5 4.0–9.9
fits, and were found to be at significantly higher concentrations
during the grazing seasons. The organic herd did not consume spe-
cial feed intended to raise CLA and ALA levels, meaning that trations in organic milk, which is often a factor in consumer pur-
farmers can rely on pasture plants to elevate the amounts of these chases. These results would carry over to cheese made from organ-
fatty acids in milk. CLA and ALA are likely to be at higher concen- ic milk, which will be the subject of future studies.

Figure 2. Confocal laser scanning micrographs of Queso Blanco after (a) 1 wk and (b) 12 wk of storage at 10°C. Green areas
correspond to protein and yellow areas to fat globules. Bars at lower right = 20 μm.

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Lipid Technology
Figure 3. Melting profiles of Mozzarella cheeses made from
(A) water buffalo milk and (B) cow milk, along with melting
profiles of (C) water buffalo milk fat and (D) cow milk fat.
Thermal characteristics
Cheese softens when removed from the refrigerator because a large
portion of the lipids melt, allowing the protein matrix to become
more flexible. (The volatile compounds also become more detect-
able by the human nose, which is why cheese is best eaten at room
temperature). The fat in cheese melts from –30 to +40°C, with un-
saturated fatty acids accounting for the lower portion of the range
and saturated fatty acids the upper part. The differences in composi-
tion due to species, as shown in table 1, result in differences in
melting profiles.
On occasion, a cheese manufacturer or importer is accused of
secretly substituting cow milk for milk of a different species, and
then selling the product as genuine and at a premium. U.S. Customs
once contacted our laboratory about some imported Mozzarella
cheese that they suspected of containing bovine milk instead of
water buffalo milk, from which Italian Mozzarella is traditionally
made. We developed a rapid (less than 90 min) method for detect-
ing the type of milk used by looking at the thermal characteristics
of the fat with a differential scanning calorimeter (DSC). Each sam-
ple was heated for 20 min in a 120°C oven to evaporate the water,
weighed into a sample pan, held in the DSC at 50°C for 5 min to
erase the thermal history, cooled to –50°C at 5°C/min to solidify
the fat, held for 15 min, and heated to 50°C at 5°C/min to obtain
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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April 2015, Vol. 27, No. 4 85
the melting profile [5]. Curves A and B in Figure 3 show that low-
medium- and high-temperature melting regions of the fat in cow
milk Mozzarella differed from those of the fat in water buffalo milk
Mozzarella. These melting profiles were similar to those of the cor-
responding pure fats (C and D in Figure 3). Mixtures of the two
types of fat were also analyzed to provide an indication of the
amount of substitution of one fat for another. A previous study
showed that the addition of calcium caseinate to milk, which is ille-
gal if the resulting cheese is not labeled as imitation, is also detect-
able by DSC. The caseinate acts as an emulsifier, preventing some
of the fat from solidifying in the refrigerator. DSC scans reflect the
decrease in the melting peak of Mozzarella cheese fat between 10
and 20°C [6].
Fat droplets become surrounded by casein when the MFGM is
removed by homogenization, causing the thermal properties of
cheese to change. A 2°C delay in melting was observed in tests on
Mozzarella along with a virtual elimination of free oil, which is
created when the casein matrix collapses upon heating and allows
the fat globules to coalesce and flow to the surface [7]. If the milk
is homogenized or if the curd is milled into small particles, as in
Queso Fresco manufacture, the cheese will soften when heated but
not collapse and flow [8]. The milling step is employed to disrupt
the casein matrix and make the cheese crumbly, and homogeniza-
tion ensures that the fat globules are trapped by the casein and can-
not combine. Although both processes are used by some cheese-
makers, either should be sufficient to produce a non-melting pro-
duct.
Conclusions
Lipids are important constituents in cheese since they provide a ve-
hicle for delivering bioactive compounds and flavor while acting as
a significant structural component. Key properties of cheese are ex-
amined by looking at the microstructure, composition, and thermal
properties of the lipids it contains.
The author thanks Peter H. Cooke providing the SEM images and
Joseph Uknalis for the CLSM images.
References
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pp. 313–325.
[2] German, J.B., Sci. Aliments 2008, 28, 176–186.
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