50 Rober t G . Jensen et al.

Wiman, K. , Curman, B. , Tragardh, L. , and Peterson , P . A. (1979). Demonstration o f HLA -

DR-like antigens o n mil k fa t globul e membranes . Eur. J. Immunol. 9 , 190-195 .
Wooding, F. B. P. (1971a). The mechanis m o f secretion of th e milk fat globule . / Cell Sci. 9 ,
805-821.
Wooding, F. B. P. (1971b). The structure of the milk fat globule membrane./ Ultrastruct. Res.
37, 388-400 .
Wooding, F . B. P. (1974). Milk fat globul e membran e materia l i n skim milk . y. Dairy Res. 41 ,
331-337.
Wooding, F. B. P. (1977). Comparative mammar y fine structure. In "Comparative Aspects of
Lactation" (M . Peaker, ed.) , pp. 1-41 . Academi c Press , Ne w York .
Wooding, F. B. P., Peaker, M., and Linzell , J. L. (1970). Theories of milk secretion: Evidenc e
from th e electro n microscopi c examinatio n o f milk . Nature (London) 226 , 762-764 .
Zaczek, M., and Keenan , T. W. (1990). Morphological evidence for an endoplasmic reticulum
origin of milk fat globules obtained usin g lipid-selective staining procedures. Protoplasma
159, 179-182 .
Zikakis, J. P., Dougherty , T . M., and Biasotto , N . O. (1976) . The presenc e an d som e prop -
erties of xanthin e oxidas e i n huma n mil k an d colostrum./ . Food Sci. 41 , 1408-1412.

B. Particulat e Constituent s
in Huma n an d Bovin e Milk s

ROBERT G. JENSEN
BERNARD BLANC
STUART PATTO N

I. Introductio n

Milks ar e biological fluids o f exceptiona l complexit y containin g thousand s

of compounds . Thes e ar e locate d i n severa l compartments , directe d ther e
by th e biologica l an d physicochemica l force s actin g durin g mil k synthesis ,
secretion, an d thereafter . Compartmentatio n i s on e o f th e factor s whic h
control th e flow o f mil k component s an d thei r product s int o an d throug h
the digestiv e an d absorptiv e system s o f th e consumer . Th e compound s i n
milk provid e nutriture , structura l component s fo r cellula r membranes ,
and nonnutritiv e messages , e.g. , immunologica l system s fo r hos t defense .
The compartments i n bovine milk , which is produced an d consumed i n th e
largest quantity , ar e altere d b y processing . Mos t o f th e mil k i s processed ,
i.e., pumped , agitated , pooled , cooled , clarifie d (centrifugatio n t o remov e
cells, etc) , th e fa t conten t standardized , usuall y t o a lowe r amoun t b y
controlled separatio n (centrifugation) , pasteurized , an d homogenize d t o
reduce th e siz e o f th e fa t globules . Unfortunately , compartmentatio n i n

H A N D B O O K O F MIL K COMPOSITIO N
Copyright ® 199 5 b y Academi c Press , inc.
All righ u reserved . N o reproductio n withou t permission .
2. Th e Structur e o f Mil k 5 1

processed mil k has received litde attention. However , it is known that about
30% of th e casein is associated wit h the fa t phase i n homogenized mil k an d
that th e whe y protein s associat e wit h th e casei n an d eac h othe r a s mil k i s
heat-treated beyon d pasteurization .
If human o r bovine mil k is centrifuged i n a tube, milk components wil l
be separate d int o th e severa l compartment s (se e Chapte r 2A) . A t lo w
speeds, e.g., 300g, cell s and any tissue debris are sedimented a s a soft pelle t
and th e fa t globules ris e and for m a distinct cream layer . A s centrifugatio n
continues an d th e forc e i s increased , smalle r fa t globule s ente r th e crea m
layer whic h i s becomin g mor e compact . Casei n micelle s als o begi n t o
sediment. I n orde r t o attai n mor e o r les s complet e sedimentatio n o f th e
casein int o a pellet , abou t 100,000 g fo r I h r i s required . A so-calle d fluff
layer wil l b e layere d o n to p o f th e casei n pellet . Th e fluff laye r contain s
membrane fragments , smal l vesicles, sloughed microvilli , etc. (Stewart et ai,
1972). Thi s produce s a clea r infranatatan t compartmen t whic h contain s
the solubl e constituent s o f mil k includin g th e whe y proteins . Thus , i t i s
possible, usin g centrifugation, t o prepar e th e individua l compartment s fo r
further observatio n an d analysis . Becaus e huma n mil k contains only abou t
one-tenth a s muc h casei n a s bovin e milk , th e casei n pelle t obtaine d b y
centrifugation i s muc h smalle r fo r huma n compare d t o bovin e milk . I t i s
possible to isolate the fat globule compartments which contain nearly all the
fat, bu t no t al l th e smal l globules , b y a relativel y simpl e centrifugatio n
procedure (Patto n an d Huston , 1986) .
In dealin g wit h mil k fro m a n analytica l standpoint , i t should b e born e
in min d tha t i t is progressivel y changin g fro m th e amoun t o f secretio n b y
the lactating cell. I n the case of an animal that is milk fed, som e o f the mil k
is alread y "old " as a result o f accumulatio n i n th e gland . Th e change s ar e
mostly subtle , bu t larg e shift s fro m on e compartmen t t o another , an d
sometimes destructio n o f mino r o r trac e constituents , ca n occur . Simpl y
cooling an d holdin g mil k i n th e col d ca n caus e som e substance s t o redis -
tribute int o other compartments. I n particular, th e equilibria of substance s
absorbed o n fa t globule s an d casei n micelle s wil l change , membrane s wil l
fragment, an d membran e component s wil l ten d t o dissociate .
The compartment s ar e define d b y th e amount , size , an d solubilit y o f
milk constituent s therei n a s show n i n Tabl e I (Jense n et al., 1990) . Th e
compartments tha t w e wil l discus s ar e cell s an d membran e fragments ,
emulsified lipi d globules , an d casei n micelles . Informatio n abou t th e for -
mation and properties of the milk lipid globule membrane and the globule s
is give n i n Chapte r 2A .
52 Robert G . Jensen et al.

TABLE I
Compartments and Their Constituent s i n Mature Bovin e and Human Millcs^

Major constituents

Content (%)

Compartment Bovine (B ) Huma n (H ) Nam e Content (%)

description

Aqueous phase 87.3 87.6 1. Compounds o f Ca , Mg , 0.7 a s ash , B

True solutio n PO4, Na , K , CI , CO2, 0.2 a s ash , H
(1 nm) citrate, casei n
2. Whe y proteins : 0.6 B , H
Whey proteins a-lactalbumin.
(3 to 9 nm) lactoferrin, IgA ,
lysozyme, an d
serum albumi n B ,
20% o f tota l N;H , 70 %
3. Lactos e an d oligo - 4.9 B
saccharides: 4. 8 an d
0.1 B ; 7. 0 an d 1.0 % H 8.0 H
4. Nonprotei n nitroge n 30 m g N/d l B
compounds: glyco -
cyamme, urea , amm o
acids. B , 5 % of total N ;
H, 25 % 50 m g N/d l H
5. Miscellaneous : B
vitamins, ascrobi c aci d
Colloidal dispersio n 2.6 6. Caseins : B - and , a fo r 2.6 B
(11 to 55 nm, lO^^/dl) B, Ca , PO 4 0.3 H
Emulsion 3.7 4.0 7. Fa t globules : triacyl - 3.7 B
Fat globules glycerols, vitamin s 4.0 H
(4 fim; 1. 1 X lO^o/dl )
Fat globule membran e 8. Mil k fa t globul e
Absorbed layer membrane: protein s 2% o f total
Cells and fragment s phospholipids, lipid
(8 to 40 |im; lO' * t o cholesterol, enzymes ,
lOVdl) trace mineral s
9. Macrophages, neutro-
phils, lymphocytes, epithe-
lial cells, leukocytes,
cytoplasmic fragment s

''All figures are approximate. Adapte d fro m Jensen et al. (1990) .

2. Th e Structur e o f Mil k 5 3

II. Cell s an d Membran e Fragment s

A. Human Milk

The cell s whic h hav e bee n identifie d i n huma n colostru m an d mil k an d

approximate number s ar e listed in Table II . Durin g early lactation, macro -
phages predominat e wit h number s o f al l types , excep t epithelia l cells ,
decreasing markedl y a s lactatio n progresses . Th e number s ar e represen -
tative an d var y considerabl y amon g individual s (Brooker , 1980 ; Riieg g
and Blanc , 1982 ; Hayward , 1983 ; Ogra an d Ogra , 1988 ; Lawrence , 1989) .
Beneficial immunologica l function s fo r th e infant an d the mammar y glan d
have bee n attribute d t o som e o f thos e cells , e.g. , th e macrophage s an d
lymphocytes (Rueg g an d Blanc , 1982 ; Hayward , 1983 ; Ogr a an d Ogra ,
1988; Lawrence , 1989) . Th e function s o f leukocyte s wer e describe d b y
Mandyla an d Xantho u (1986) , Buesche r an d Pickerin g (1986) , an d th e
lOM (1991) .
Brooker (1980 ) observe d membrane-boun d cytoplasmi c remnant s i n
the sedimentatio n pelle t o f centrifuge d huma n milk . Ther e wer e mor e
fragments tha n cells at all times postpartum studied . Mos t of th e fragment s
came fro m secretor y cell s i n th e mammar y gland . The y containe d vesicle s
of roug h endoplasmi c reticulum , lipi d droplets , an d Golg i vesicle s con -
taining casei n micelles . Thes e membrane s whe n folde d o r spherica l ar e
probably th e particle s name d mil k microsome s (Riieg g an d Blanc , 1982) .
The membranes , whic h i n genera l ar e calle d mil k lipoproteins , rang e i n
size fro m 1 0 t o 40 0 n m (Riieg g an d Blanc , 1982) . Som e o f th e fragment s
were probabl y associate d wit h th e cytoplasmi c crescent s see n o n abou t 7 %
of human an d 1 % of bovine lipi d droplets (Husto n an d Patton , 1990) , with
the array of glycoprotein filaments see n o n huma n bu t not bovine globule s
(Buchheim et ai, 1986) , and wer e displace d fro m thei r original site s by th e
processes o f isolatio n (se e Chapte r 2 A fo r mor e information) . Relativel y
little displacemen t o f eve n loosel y boun d materia l woul d b e expecte d t o
occur durin g th e shor t perio d o f transi t fro m th e breas t t o th e breast-fe d
infant's stomach . Suckin g b y th e infan t i s likel y t o b e mor e vigorou s tha n
hand expressio n o f th e milk , bu t no t a s muc h a s vacuu m pumping , t o
obtain mil k samples .
Bacteria, usually innocuous ski n species, are present. Th e number s ar e
low, but extreriiel y variable , an d ar e ofte n attache d t o squamous epithelia l
cells (Brooker , 1980) . Neubaue r et ai (1995 ) foun d tha t th e number s o f
bacteria an d leukocyte s wer e relate d t o th e incidenc e an d severit y o f
mastitis. Wit h n o mastitis , leukocyte s wer e < 1 x 10 ^ and bacteri a < 1 x
10^/ml. Fo r noninfectiou s mastitis , th e figures wer e ^ 1 0 ^ an d < 10^/m l
and fo r infectiou s mastiti s > 10 ^ and > 10^/ml . Th e majo r specie s presen t
in milk s fro m wome n wit h n o mastiti s (52% , n = 89 ) wer e ski n types . A n
increased incidenc e o f Staphylococcus aureus was associated wit h mastitis . I n
developing countries , man y o f th e wome n wh o are breastfeeding wil l hav e
54 Rober t G . Jensen et al.

TABLE I I
Cell Types and Numbers i n Human Mil k (per ^1 ) before and during Lactation

Time Total Macrophages Neutrophils Lymphocytes Epithelial*

Antepartum 3430 2140 360 240 —

Postpartum
Days 0- 4 2840 1490 1375 250 About 1 X lO' *
Days 5- 8 450 320 100 27 throughout
lactation

Weeks 1 or 2 69 52 4
Weeks 2- 4 51 52 8
Months 1 or 2 17 4 3
Months 2- 4 16 3 2
Months 4- 6 10 1 1

''Adapted fro m Haywar d (1983).

*Brooker (1980).

mastitis (Prentic e et al, 1985) . The importanc e i s that sever e mastiti s alter s
the composition o f mil k (se e Chapter 3F ) and destroy s lactatin g tissue, thu s
reducing th e volum e o f mil k availabl e fo r futur e lactations . Whil e natura l
defenses ar e operative , improve d hygien e woul d b e helpfu l (Prentic e et a/.,
1983).
T h e relationshi p i n dair y catd e wit h masutis , lowe r mil k production ,
and hig h somati c cel l count s (leukocytes ) i n thei r mil k ha s apparentl y no t
been studie d i n human s (Se e below) .

B. Bovine

L Cells

Bovine mil k contain s abou t 10 ^ t o 10* ^ cells/ml (Lipki n et al,, 1993) ,

although individua l variatio n i s large . T h e number s ar e usuall y reporte d
as somati c cel l count s whic h ar e a mixtur e o f epithelia l cell s (2% ) an d
leukocytes (98%) . Severa l enzyme s ar e foun d i n leukocytes , e.g. , catalase ,
proteases, etc . Mos t o f th e nuclei c acid s i n mil k originat e fro m thes e cells .
These mil k cells have been utilize d a s a source of deoxyribonucleic aci d an d
as a substrat e fo r th e polymeras e chai n reactio n (Lipki n et al., 1993) .
Somatic cel l count s ar e routinel y determine d i n th e U.S . Dair y Her d
Improvement Associatio n Program s usin g electroni c cel l counter s (Heald ,
1985). Hig h count s ar e associate d wit h reduce d mil k yield s an d increase d
incidence o f mastitis . I n on e study , th e mil k fro m 8 1 % o f 139,42 1 cow s
contained 18,00 0 m l 565,00 0 cells/ml . T h e averag e mil k yield s wer e 2 6 t o
2. Th e Structur e o f Mil k 5 5

21.6 kg ; milk productio n droppe d 0.6 8 k g for th e average o f al l cows eac h

time th e somati c cel l coun t doubled . Somati c cel l count s o f 200,00 0 t o
400,000/ml wer e associate d wit h lowe r mil k yield s an d greate r mastiti c
infection rate s (Jone s et a/., 1984) . They mentione d tha t somatic cell count s
above 500,000/m l hav e bee n use d a s a n indicato r o f significan t incidenc e
of mastiti s i n a her d o r nonspecifi c mastiti s if pathogeni c microorganism s
had no t been detected. Guidr y (1985 ) note d tha t leukocyte counts in excess
of 200,000/ml i n an individual co w sample suggested mastiti s with the nee d
for diagnosi s b y a chemical metho d suc h a s determinatio n o f chloride . I n
the Unite d States , th e Federa l regulator y limi t ha s bee n 750,000/ml , bu t
will b e lowere d t o 500,000/m l i n 199 4 (Bennett , 1993) . Thi s wil l b e don e
to alig n th e Unite d State s with th e requirement s i n th e EEC . The regula -
tory limi t wil l dro p t o 400,00 0 m l i n 1998 .
We mentione d earlie r tha t somati c cell s carr y enzyme s int o th e milk .
Verdi an d Barban o (1991 ) foun d proteolyti c activit y i n somati c cell s iso -
lated fro m mil k by ultracentrifugation. Th e protease s hydrolyze d P-casein .
They coul d caus e proteolysi s i n age d cheese s if no t destroye d durin g
processing. Neithe r cell s no r enzymes , wit h th e possibl e exceptio n o f
bacterial proteases , ar e likel y t o surviv e processing . Whe n sample s o f ra w
milk ar e froze n fo r subsequen t analytica l o r researc h purposes , i t shoul d
be remembere d tha t freezin g an d thawin g wil l disrup t cells . Degradativ e
enzymes wil l b e released . Thi s ca n b e prevente d b y a preliminar y heatin g
to 60° C t o inactivat e th e enzymes .
Verdi et al. (1984 ) observe d tha t mil k wit h highe r somati c cel l count s
had lower casein contents tha n milk with lower cell counts. They attribute d
this to proteolysis o f casein. This grou p (Seny k et al., 1985 ) later found tha t
proteolysis increase d whe n somati c cel l count s increase d fro m 50,00 0 t o
1,000,000/ml. Som e protelysi s wa s detected i n pasteurize d milk s wit h hig h
cell counts. The protease s associated with the cells damage ra w milk quality
during storag e an d hav e a n advers e effec t o n pasteurize d fluid mil k an d
milk during cheese making. Continuing their work, this group (Verdi et al.,
1987) foun d tha t proteolysi s o f casein s increase d with incubatio n wit h
either hig h o r lo w cel l counts .
Milk producer s ar e require d t o an d d o attemp t t o exclud e bacteri a
from milk ; however , som e microorganism s gai n entry . I n th e Unite d
States, the bacterial count in Grade A raw milk may not exceed 300,000/ml ,
in th e EEC , 100,000/ml , an d i n Switzerland , 80,000/ml . Whe n th e co w ha s
mastitis, microorganism s associate d with infection s suc h a s S. aureics, Strep-
tococcus uteris, an d Streptococcus agalactiae ar e foun d (Jone s et al., 1984 ;
Guidry, 1985) . Mil k fro m cow s wit h mastiti s mus t b e exclude d fro m an y
commercial processin g fo r huma n consumption , bu t some subclinica l case s
may no t b e detected . Pasteurizatio n destroy s mos t o f th e microorganism s
in mil k an d al l o f th e pathogens . I n th e Unite d State s th e uppe r limi t o f
bacteria i n pasteurize d mil k i s 20,000/ml . I n Switzerland , th e limi t i s
20,000/ml.
56 Rober t G . Jensen et al.

2. Membrane Fragments
These component s ar e simila r t o thos e foun d i n huma n mil k excep t
that there wil l be less derived fro m lipi d globul e cytoplasmi c crescent s (se e
Section II , A) . Onl y 1 % of th e globule s i n bovin e mil k hav e th e crescent s
(Huston an d Patton , 1990) . However , bovin e mil k ha s well-characterize d
fluff fraction s i n the skim milk phas e (Stewar t et ai, 1972) . Bovine globule s
do no t hav e glycoprotei n filament s clustere d o n thei r surface s a s are see n
on th e huma n globule s (Buchhei m et al., 1988) . A s mentioned , th e mem -
brane fragment s hav e bee n terme d mil k lipoprotein s (Riieg g an d Blanc ,
1982). Thes e component s ma y b e irreversibl y denature d o r otherwis e
altered b y processin g althoug h thi s ha s apparend y no t bee n reported .

III. Lipi d Globul e Emulsio n

A. Introduction

Almost al l o f th e lipi d i n huma n an d bovin e milk s i s foun d i n disperse d

globules. Th e stabilit y o f th e emulsio n i s maintaine d b y th e amphiphili c
components i n th e globul e membrane , particularl y th e stron g negativ e
change carrie d b y som e o f th e glycolipid s an d proteins . Triacylglycerol s
make up 98% or more of th e lipid with polar compounds i n the membran e
and th e nonpola r component s i n th e core . Th e cor e i s almos t totall y
triacylglycerol, whil e al l o f th e phospholipid s an d mos t o f th e cholestero l
are i n th e membrane . Th e purpos e o f th e emulsio n appear s t o b e t o
provide a unit amoun t o f disperse d lipi d globule s wit h larg e tota l surfac e
area uniformly disperse d in a unit volume of milk. After ingestion , the milk
forms a ge l (curd ) i n th e stomac h an d th e globul e surface s ar e accessibl e
to enzymati c actio n b y gastri c lipas e an d othe r enzymes . Rapi d lipolysi s
occurs. Th e globule s ar e resistan t t o digestio n i n th e smal l intestin e b y
pancreatic lipas e an d th e bile-salt-stimulate d lipas e i n huma n mil k unles s
first conditioned b y exposure t o gastri c lipase i n the huma n o r probably t o
pregastric lipase in the calf (see Chapters 6A and 6B). I f the lipids were no t
dispersed a s globules , th e fa t woul d ris e an d merg e int o a layer . I t coul d
not b e secrete d no r digested .

B. Size Distribution

The averag e diamete r o f globule s i n al l specie s examine d (cow , human ,

goat, ewe, sow ) range s fro m 3 to 5 ^m . These figures ar e no t precis e sinc e
there are problems with determination o f mea n diameter . These problem s
are primaril y becaus e population s o f smal l globule s ( < 1 ^m ) canno t b e
counted wit h the light microscope o r Coulter counter [se e Chapter 2 A an d
Ruegg an d Blan c (1981 ; 1982)] . A relativel y ne w instrument , th e Coulte r
2. Th e Structur e o f Mil k 5 7

LSI30 photo n correlatio n spectrometer , whic h use s lase r light , ca n deter -

mine particles with diameters ranging fro m 1 to 10,00 0 nm. Measurement s
are base d o n lase r diffractio n an d scatterin g an d polarizatio n intensif y
differential scattering . Diameter s o f particle s i n mil k rangin g fro m 0. 1 t o
900 |Li m have bee n determine d (Blanc , unpublishe d data) . A broa d distri -
bution belo w 0. 6 [i m wa s observe d whic h wa s probabl y du e t o casei n
micelles. Blan c note d tha t th e casei n micell e distributio n wa s probabl y no t
correctly measure d becaus e th e calculation s wer e base d o n th e refractiv e
index o f mil k fat . Cy r et al. (1989 ) use d th e instrumen t t o determin e th e
size distributio n o f fa t globule s i n intravenou s fa t emulsions . Th e mea n
globule diamete r wa s 0. 3 pim . Ther e ar e thre e overlappin g siz e distribu -
tions o f huma n (Riieg g an d Blanc , 1981 , 1982 ) an d bovin e mil k globule s
(Walstra an d Jenness , 1984) : smal l wit h diameter s belo w 1 jim , interme -
diate wit h 3 t o 5 [A m diameters, an d larg e wit h a diamete r rang e o f 8 t o
10 pim. The smal l globule s mak e u p abou t 7 0 t o 90 % of th e tota l number ,
but onl y a smal l portio n o f th e tota l fat . Th e intermediat e grou p ha s th e
largest amoun t o f fat , bu t onl y abou t 1 0 t o 30 % of th e globul e numbers .
The large r populatio n range s fro m 8 t o 1 2 ^im , bu t ha s onl y 0.01 % o f
the fat .
Some importan t parameter s o f th e globul e dispersion s i n huma n an d
bovine milk s are presente d i n Table III . I n huma n colostrum , ther e i s less
fat, but also more globules , s o th e surfac e are a of fa t is 3.3 m^/ g compare d
to 1. 4 m 2 i n matur e milk . Thi s ma y b e a n adaptatio n i n respons e t o th e
neonate's relativ e inabilit y t o diges t fats . Colostru m i s exclude d fro m
commercial bovin e milk . Mos t o f th e bovin e mil k i s homogenized , a t leas t
in th e Unite d States , wher e th e fa t conten t i s standardized t o abou t 3. 3 t o
3.4%. Excep t fo r th e associatio n o f casei n wit h globule s mentione d earlier ,
we hav e ver y littl e informatio n o n th e redistributio n o f component s int o
compartments a s a result of homogenizatio n an d non e o n th e digestio n o f
bovine mil k lipid s i n human s b y th e gastric—pancreati c lipas e syste m
(Jensen et al, 1990 , 1992) . The globul e parameter s influenc e man y factor s
in th e processin g o f milk , e.g. , creaming , separatio n formatio n o f butter ,
clustering o f globules , etc . Fo r mor e informatio n se e Mulde r an d Walstr a
(1974) an d Walstr a an d Jenness (1984 ) an d Chapte r 2A .
The globul e siz e distributio n i n huma n mil k i s affecte d b y gestationa l
age o f th e infant . Th e averag e diameter s i n preter m an d ter m milk s wer e
identical and increased 2. 2 t o 2.7 ^i m to 40 days postpartum (Simoni n et a/.,
1984). Th e numbe r o f globule s wit h diameter s o f 1 to 1. 5 an d 8 to 1 3 jxm,
respectively, decrease d a s gestationa l ag e increased . A simila r declin e wa s
observed i n ter m milk , bu t th e number s o f globule s wer e lower . Th e
numbers o f large r globule s levele d of f t o thos e i n ter m mil k 5 0 day s
postpartum. Th e fatt y acid s in th e diet influenc e thei r profiles i n the lipid s
of huma n an d bovin e milks . Th e profile s influenc e th e liquidit y o f th e
triacylglycerols i n th e globule s i n bovin e milk . Timme n an d Patto n (1988 )
observed tha t cows fed certai n ration s produced globule s i n skim milk wit h
smaller diameter s an d altere d fatt y aci d profile s compare d t o thos e i n
58 Rober t G . Jensen et al.

TABLE II I
Some Parameters of the Fa t Globule Dispersion i n Human Colostru m and Mature
Human and Bovine Milks^

Human average (-I - SD) Bovin e (range )

Parameter Colostru m Matur e Mil k Homogenize d

Fat content (g/10 0 g ) 2. 6 1. 0 3. 3 0. 6 3.7-4. 1 3.7-4. 1

Globules (appro x 6xl0» o (2xlO»o ) l.lxlO^ ^ (3x109 ) 1.5-10^ o 10^2_ioi 4
No./ml)
Surface are a of 1 g 3. 3 0. 5 1. 4 0. 1 1.4-2.9 10-3 0
fat i n milk (m^)
Volume/surface 1. 5 0. 3 4. 0 0. 3 2.5-4. 6 0.2-0. 7
average diamete r

''Adapted fro m Rueg g and Blan c (1982).

cream. The author s suggeste d tha t the mammar y glan d regulate s th e fatt y
acid composition i n the globules t o maintain liquidit y at body temperature s
and tha t this may affect th e diameters of th e small globules . This influenc e
may b e irrelevan t industrially , sinc e her d milk s ar e poole d unles s mos t o f
the producers in a region are feeding th e same diet to their cows. Althoug h
changes i n die t markedl y an d rapidl y influenc e th e fatt y aci d profile s i n
human mil k (Chapte r 6A) , ther e ar e n o report s o f effect s o n globul e size s
and distributions .

IV. Casei n Micelle s

4. Introduction

These particle s exis t a s complexe s o f protei n an d salts , i n a colloidal ,

making u p 2 0 t o 40 % o f th e protei n i n huma n mil k an d abou t 80 % i n
bovine mil k (Rueg g an d Blanc , 1982) . Classicall y an d o f importanc e i n
cheese making , casein s ar e precipitate d fro m bovin e mil k b y acidificatio n
to p H 4. 6 a t 20°C (Eige l et al., 1984) . The casein s i n huma n mil k ar e mor e
difficult t o isolat e requirin g acidificatio n t o p H 4. 3 an d additio n o f CaCl 2
(Kunz and Lonnerdal , 1989a,b) . The amount s of the caseins in human an d
bovine milk s ar e show n i n Tabl e IV .
The casei n micella r system s i n th e milk s diffe r considerably . Th e
micelles i n huma n mil k ar e abou t 4 3 n m i n diamete r (Carrol l et ai, 1985) .
The averag e diameter s o f casei n micelle s i n bovin e mil k ar e abou t 8 3 n m
(Donnelly et al, 1984 ) or 120-18 0 n m (Farrell , 1990) . I n diameter, the y ar e
about 1/5 0 tha t o f a fa t globule .
2. Th e Structur e o f Mil k 5 9

TABLE I V

Amounts of Caseins in Mature Huma n and Bovine Mi lies

Human" (g/liter ) Bovine* (g/liter )
Protein
9.0 36.0
Total
2.7 29.5
Total casein Not presen t 11.9
a-Sl Not presen t 3.1
a-S2 2.3^ 9.8
P
p 1.2
H 0.4 3.5

''From Swaisgood, Chapte r 4B , Table 1 .

^Adapted fro m Kun z and Lonnerda l (1989a) .
'^Not precisely determined. Calculated by the authors based on x-casein being 15 % or less of
total caseins.
'V-Casein is a product o f th e proteolysi s of th e C-termina l o f P-casein.

B. Structure and Size Distribution

1. Human Milk

The structur e o f casein micelle s i n human mil k appears t o be based o n

the associatio n o f highl y phosphorylate d p-casei n whic h bind s calcium , a
low phosphorylate d form , an d glycosylate d x-casei n (Chapte r 5 A an d 7A) .
The interio r o f th e micell e contain s calciu m phosphate . I n huma n mil k
only 15 % of th e calcium i s bound t o casein an d i n bovine mil k th e amoun t
is abou t 65 % (Nevill e et ai, 1994) . Submicelle s ma y b e groupe d int o
spherical particles . Informatio n o n th e micelle s i s give n i n Tabl e V . Not e
that althoug h ther e i s les s casei n i n huma n tha n i n bovin e milk , th e
numbers o f micelle s ar e abou t th e same .

2. Bovine Milk

We mentione d earlie r tha t th e casei n micelle s i n bovin e mil k occu r a s

colloidal complexe s o f protein s an d salts , primaril y calciu m (Farrel l et ai,
1990). Whe n calciu m i s remove d submicelle s ar e produce d whic h contai n
four proteins : a-sl, a-s2 , P , and, x-caseins i n ratios of abou t 4:1:4:1. Thes e
compounds hav e a n averag e molecula r weigh t o f abou t 23,90 0 an d ar e
phosphorylated t o variou s degrees . Farrel l et ai, (1990 , 1993 ) hav e pos -
tulated tha t thes e hydrophobicall y stabilize d submicelle s ar e incorporate d
into the micelle . Accordin g t o Rollem a (1992) , the mode l fo r bovin e casei n
micelles whic h bes t fits experimenta l dat a i s th e associatio n o f severa l
subunits t o for m a larg e spherica l micelle . Th e implicatio n i s tha t bond s
between submicelle s i n th e calciu m phosphat e phas e an d hydrophobi c
60 Robert G . Jensen et al.

TABLE V
Some Parameters of the Colloidal Casein Dispersion in Human an d Bovine Milks^

Casein parameter Unit Human Bovine

Concentration g/dl 0.2-0.5 2.2-2.8

Types (a-sl:P:K ) Ratio ca. 0:7: 3 ca. 1:0.8:0. 3
Micellar Ca:P Ratio 0.2:0.6 2.2:2.8
Number of micelle s Per milliliter ca. 7x10^ 5 ca. 7xl0» 5
Average diamete r
of micelle s
dn nm 8-14 (43.0) * 21-24
d. nm 10-26 (44.9 ) 44-50
dy> nm 11-55(46.9) 90-100
^vm nm 16-88 (49.9 ) 104-140
Average diamete r
of submicelle s
du nm 6-8 10-11
dy nm 7-9 11-12
dyi nm 8-10 12-13
**vni nm 9-12 13-14

.^Adapted from Rtieg g and Blan c (1982).

^Carroll et al. (1989).

interactions betwee n submicelle s ar e responsibl e fo r th e integrit y o f th e

casein micelles . Th e highl y glycosylate d x-casei n als o ha s a structure -
stabilizing role. The fact that casein micelles carry a strong negative charge,
as indicate d b y thei r isoelectri c poin t o f p H 4.6 , make s the m strongl y
self-repeHing a t th e norma l p H o f milk , 6.6-6.7 . Tabl e V contain s th e
information o n bovine casei n micelles .

V Summar y

Again, th e extraordinar y complexit y o f th e physica l organizatio n o f th e

components i n huma n an d bovin e milk s i s obvious . Compartmentatio n
influences th e availability of th e components a s nutrient and as nonnutri-
tive messages . Th e effec t o n bovin e mil k processin g i s als o important .
Obviously, mor e informatio n i s needed.

Acknowledgments

The preparatio n of th e manuscrip t was supported i n par t by an NIH contrac t and by

federal fund s mad e availabl e throug h provisio n o f th e Hatc h Act , Scientifi c Contribution ,
2. Th e Structur e o f Mil k 6 1

Storrs Agricultura l Experimen t Station , Storrs , Connecticut . W e appreciat e th e advic e o f Dr .

H. M . Farrell , Jr. o n bovin e casei n micelles .

References

Bennett, R . (1993) . T he cas e fo r improvin g mil k qualit y regulations : Mil k somatic cell counts .
Dairy Food Environ. Sanit. 13 , 278-282 .
Brooker, B . E. (1980) . T he epithelia l cell s and cel l fragment s i n huma n milk . Cell. Tissue. Res.
210, 321-332 .
Buchheim, W. , Welsch , U. , Huston , G . E. , an d Patton , S . (1988) . Glycoprotei n filament
removal fro m huma n mil k fa t globule s b y hea t treatment . Pediatrics 81, 141-146 .
Buescher, E . S., an d Pickering , L . K. (1986) . Polymorphonuclea r leukocyte s i n huma n colos -
trum an d milk . In "Huma n Mil k i n Infan t Nutritio n an d Health " (R . P. Howell , F . H .
Morriss, Jr., an d L . K. Pickering , eds.) , p . 160-173 . Thomas , Springfield , IL .
Carroll, R . J., Basch , J . J., Phillips , J. G. , an d Farrell . H . M. . Jr. (1985) . Ultrastructura l an d
biochemical investigation s o f matur e huma n milk . Food Microstruct. 4, 321-323 .
Cyr, T . D. , Lawrence, R . C, an d Lovering , E . G. (1989) . Determinatio n o f siz e distribution o f
fat globule s i n intravenous fa t emulsion s b y photoncorrelatio n spectroscopy./ . Assoc. Off.
Anal. Chem. 7 2 , 4 3 6 - 4 4 1 .
Donnelly, W . J., McNeill , G . P. , Buchheim , W. , an d McGann , F . C. A. (1984) . A comprehen -
sive stud y o f th e relationshi p betwee n siz e an d protei n compositio n i n natura l bovin e
casein micelles . Biochim. Biophys. Acta 789 , 136-143 .
Eigel, W . N. , Butler , J. E. , Ernstrom , C . A., Farrell , H . M. , Jr., Harwalker . V . R., Jenness, R. .
and Whitney , R . M . L. (1984) . Nomenclatur e o f protein s o f cow' s milk . /. Dairy Sci. 67 ,
1599-1631.
Farrell, H . M. , Jr., Pessen , H. , Brown , E . M., and Kamosinski , T. F . (1990). Structural insight s
into the bovin e casei n micelle : Small angl e X-ra y scatterin g studie s an d correlation s wit h
spectroscopy./ Dairy Sci. 73 , 3592-3601.
Farrell, H . M. , Jr., Brown , E . M., and Kumosinki , T . F . (1993). Three-dimensional molecula r
modeling o f bovin e caseins . Food Struct. 12 , 235-250 .
Guidry, A.J . (1985) . Mastiti s an d th e immun e syste m o f th e mammar y gland . In "Lactation "
(B. L. Larson , ed.) , pp . 229-262 . Iow a Stat e Universit y Press , Ames .
Hay ward, A . R. (1983) . T h e immunolog y o f huma n milk . In "Lactation , Physiology , Nutri -
tion, an d Breastfeeding " (M . C. Nevill e an d M . R. Neifert , eds.) , pp . 258-261 . Plenu m
Press, Ne w York .
Heald, C . W. (1985) . Mil k collection . In "Lactation " (B . L. Larson , ed.) , pp . 198-228 . Iow a
State Univ . Press , Ames .
Huston, G . E. , and Patton , S . (1990). Factors relate d t o the formatio n o f cytoplasmic crescent s
on mil k fa t g l o b u l e s . / Dairy Sci. 73 , 2061-2066 .
Institute o f Medicin e (lOM) . (1991). "Nutrition Durin g Lactation, " p . 137 . National Academ y
of Science , Washington , DC .
Jensen, R . G., Ferris , A.M. , Lammi-Keefe , C.J. , an d Henderson , R . A. (1990) . Lipid s o f
bovine an d huma n milks : A comparison . J. Dairy Sci. 73 , 223-240 .
Jensen, R . G., Lammi-Keefe , C.J. , an d Ferris , A.M . (1992) . Effec t o f mil k triacylglycero l
structure o n absorptio n an d metabolism . Inform 3 , 560 .
Jones, G . M. , Person , R . E., Clabaugh, G . A., and Heald , C . W. (1984) . Relationships betwee n
somatic cel l count s an d mil k p r o d u c t i o n . / Dairy Sci. 67 , 1823-1831 .
Kunz, C , an d Lonnerdal , B . (1989a) . Huma n mil k protein : Separatio n o f whe y protein s an d
their analysi s b y polyacrylamid e gradien t ge l electrophoresis , fas t protei n liqui d chro -
matography (FPLC ) ge l filtration, an d anion-exchang e chromatography . Am. J. Clin.
Nutr. 49 , 464-470 .
Kunz, C , an d Lonnerdal , B . (1989b) . Casei n micelle s an d casei n subunit s i n huma n milk . In
"Protein an d Non-Protei n Nitroge n i n Huma n Milk " (S . Atkinso n an d B . Lonnerdal ,
eds.), pp . 10-27 . CR C Press , Boc a Raton , FL .
62 Rober t G . Jensen' et al.

Lawrence, R . A. (1989) . "Breastfeeding, " 3r d Ed. , pp . 120-126 . C . V. Mosby , St . Louis , MO .

Lipkin, E. , Shalom , M. , Khabib , H. , Soller , M. , an d Friedman , A . (1993) . Mil k a s a sourc e o f
deoxyribonucleic aci d an d a s a substrat e fo r th e polymeras e chai n reaction . J. Dairy Set.
76, 2025-2032 .
Mandyla, H. , an d Xanthou , M . (1986) . Functio n o f leukocyte s i n huma n milk . In "Huma n
Lactation. 2 . Maternal an d Environmenta l Factors " (M . Hamosh an d A . Goldman, eds.) ,
pp. 533-540 . Plenu m Press , Ne w York .
Mulder, H. , and Walstra , P . (1984). "The Mil k Fa t Globule, " Common. Agric . Bur., Farnha m
Royal, Bucks , England .
Neubauer, S . H. , Ferris , A.M. , Lammi-Keefe , C.J. , Green , K . W., Hinckley , L . S., an d
Jensen, R . G. (1995) . Compositio n an d bacteria l an d somati c cel l content s i n th e mil k
of wome n wit h an d withou t insulin-dependen t diabete s mellitus . Manuscrip t i n prepa -
ration.
Neville, M . C, Keller , R . P., and Casey , C . (1994) . Calciu m partionin g i n huma n an d bovin e
milk.y. Dairy Sci. 77 , 1964-1975 .
Ogra, P . L., and Ogra , S . S. (1988). Cellular aspect s o f immunologi c reactivit y i n huma n milk .
In "Biolog y o f Huma n Milk " (L . A. Hanson , ed.) , pp. 171-184 . Rave n Press , Ne w York .
Patton, S. , an d Huston , G . E . (1986) . A metho d fo r th e isolatio n o f mil k fa t globules . Lipids
21, 170-174 .
Prentice, A. , Prentice , A. , an d Lamb , W . (1985) . Mastiti s i n rura l Gambia n mother s an d th e
protection o f th e breas t b y mil k antimicrobia l factors . Trans. R. Soc. Trop. Hygiene 79 ,
90-95.
RoUema, H . S. (1992) . Casei n associatio n an d micell e formation . In "Advance d Dair y
Chemistry-1: Proteins " (P . F. Fox , ed.) , pp . 111-140 . Elsevier , Ne w York .
RUegg, M. , an d Blanc , B . (1981) . T h e fa t globul e siz e distributio n i n huma n milk . Biochim.
Biophys. Acta 666 , 7-14 .
Riicgg* R » an d Blanc , B . (1982) . Structur e an d propertie s o f th e particulat e constituent s o f
human milk . A review . Food Microstruct. 3 , 2 5 - 4 7 .
Senyk, G . F. , Barbano , D . M., an d Shipe , W . F . (1985) . Proteolysi s i n mil k associate d wit h
increasing somati c cel l counts . / Dairy Sci. 68 , 2189-2194 .
Simonin, S. , Riiegg , M. , an d Sideropoulis , D . (1984) . Compariso n o f th e fa t globul e siz e
distribution o f breas t mil k fro m mother s deliverin g ter m an d preterm . Am. J. Clin. Nutr.
40, 820-826 .
Stewart, P . S., Puppiome , D . L., an d Patton , S . (1972) . Th e presenc e o f microvill i an d othe r
membrane fragment s i n th e non-fa t phas e o f bovin e milk . Z . Zellforsch. 123 , 161-167 .
Timmen, H. , and Patton , S . (1988). Milk fa t globules : Fatt y aci d composition , siz e and i n viv o
regulation o f fa t liquidity . Lipids 23 , 685-689 .
Verdi, R . J., an d Barbano , D . M. (1991) . Propertie s o f protease s fro m mil k somati c cell s an d
blood leukocytes./ . Dairy Sci. 74 , 2077-2081 .
Verdi, R . J., Dellavalle , M . E., Barbano, D . M., and Senyk , G . F . (1984). Relationshi p betwee n
milk somati c cel l count, tru e protein , casein , nonprotei n nitrogen , an d tyrosin e value./ .
Dairy Sci. 6 7 (Suppl . 1) , 7 0 - 7 1 .
Verdi, R . J., Barbano , D . M., Dellavalle , M . E., an d Senyk , G . F . (1987) . Variabilit y i n tru e
protein, casein , nonprotei n nitrogen , an d proteolysi s i n hig h an d lo w somati c cel l milks .
) . Dairy Sci. 70 , 230-242 .
Walstra, P. , an d Jenness , R . (1984) . "Dair y Chemistr y an d Physics, " Chap . 14 . Wiley , Ne w
York.