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January 1977T h e J o u r n a l o f P E D I A T R I C S 2 9

n f l u e n c e o f th e h e at t r e a tm en t o f h u m a n m i l k onso m e o f t s p r o t e c t i v e c o n st i t u en t s

Hum an mi lk was subjec ted to hea t t rea tments of graded sever i ty and exam ined for i t s content of

imm unog lobulins, lactoferrin, lysozym e, vitam in B,=,-and ola te-b inde r protein s, and lactoperoxidase.

Holder pasteurization (62.5 ~ C 30 minutes) red uced the IgA ti ter by 20% , and destroyed the small

content of lg M and mos t of the lactoferrin. Lys ozym e w as stable to this treatment, bu t with an increase

in temperature there w as progressive destruction, to near 100% at 100 ~ C. The sam e w as broadly true

of the capacity of milk to bind folic acid and protect i t against bacterial uptake, with vitamin B,. , the

binder was m ore labile at 75 ~ C than at 100 ~ C. The milk contained no d etectable lactoperoxidase.

J . E . F o r d P h . D . D . S c . B . A . L a w P h . D . Va l e r ie M . E . M a r s h a l l B . S c .

and B. Rei te r P h . D . , D . S c . , S h i n fi e ld , R e a d i n g , U n i t e d K i n g d o m

I T I S W I D E LY H E L D a m o n g p e d i a t r i c ia n s th a t t h ei n c i d e n c e o f i n f e c t io u s d i s e a s e i s lo w e r i n b r e a s t - f e d t h a ni n b o t t l e - fe d i n f a n ts . ' C e r t a i n l y t h e m a t e r n a l m i l k c o n f e r ss p e c if i c p r o t e c t i o n a g a i n s t m a n y p a t h o g e n i c b a c t e r i a a n dv i r u s e s b y v i r t u e o f it s c o n t e n t o f i m m u n e a n t i b o d i e s . I ta l so c o n t a i n s a v a r i e t y o f n o n s p e c i f i c f a c t o rs t h a t s t r o n g l ya n d s e l e c ti v e l y i n f l u e n c e t h e g r o w t h o f d i f f e r e n t m i c r o o r -

g a n i s m s i n v i tr o a n d m a y p l a y a n i m p o r t a n t p a r t i n t h en u t r i t i o n o f th e i n f a n t a n d i n p r o t e c t i n g i t a g a i n s t i n f e c -t io n . T h e s e n o n s p e c i f i c f a c t o r s i n c l u d e t h e i r o n - b i n d i n gp r o t e i n l a c t o f e r ri n , th e p r o t e i n s t h a t b i n d v i t a m i n B ,~ a n df o l a te , a n d l y s o z y m e a n d l a c t o p e r o x i d a s e .1

E x p r e s s e d b r e a s t m i l k is w i d e l y u s e d i n t h e n u r t u r i n g o fs m a l l - f o r - g e s t a t io n a l a g e a n d p r e m a t u r e i n f a n t s . S o m e a r eg i v e n m i l k f r o m t h e i r o w n m o t h e r s w i t h o u t a n y i n t e r m e -d i a t e h e a t t r e a t m e n t , w h i l e o t h e r s r e c e i v e m i l k f r o m d o n o r

m o t h e r s t h a t h a s b e e n h e a t t r e a t e d t o d e s t r o y c o n t a m i n a t -i n g b a c t e r i a . T h e p r e s e n t s t u d y w a s u n d e r t a k e n t o d e t e r -m i n e t o w h a t e x t e n t t h e i m m u n o g l o b u l i n s a n d o t h e rp r o t e c t i v e p r o t e i n s i n t h e m i l k a r e s t a b l e t o s u c h

hea t ing .

M A T E R I A L S A N D M E T H O D S

M i l k sa m p l e s . M i l k w a s o b t a i n e d f r o m t h e H u m a n M i l kB u r e a u a t S t . D a v i d ' s H o s p i t a l . C a r d i f f . It w a s a b u l k e d

From the Nat ional Ins t i tu te for Research in Dai ry ing .

s a m p l e r e p re s e n t in g m a i n l y m a t u r e m i l k f ro m 2 5 o r m o r ed o n o r s , a n d i t h a d b e e n s t o r e d u n f r o z e n a t a b o u t 4 ~ C f o ro n e t o t w o d a y s b y t h e t i m e i t w a s r e c e i v e d a t o u rl a b o r a t o r y. I t w a s c e n t r i f u g e d a t 2 ~ C f o r o n e h o u r a t7 5, 00 0 g , a n d t h e a q u e o u s p h a s e w a s c a r e f u l l y d e c a n t e da n d f i l te r e d t h r o u g h K l e e n e x t is s u e . T h e f a t a n d s e d i m e n tw a s d i s c a r d e d .

See related article p. 167.

Abbreviat ion usedHTST: high temperature-short t ime

Heat treatment o f m i l k . S a m p l e s o f m i l k ( 1 .5 m l ) w e r es e a l e d i n t o s m a l l g l a ss a m p u l e s a n d h e a t e d i n a b a t h o fd e t e rg e n t m a i n t a i n e d a t t h e r e q u i r e d t e m p e r a t u r e + 0 .1 oC . T h e y w e r e t h e n c o o l e d i n ic e d w a t e r a n d s t o r e d a t - 3 0 ~C un t i l t e s t ed .

T h e t i m e s a n d t e m p e r a t u r e s o f h e a t i n g w e r e a s f o l l o w s :5 6 ~ C 3 0 m i n u t e s ; 6 2 .5 ~ C 3 0 m i n u t e s ( h o l d e r p a s t e u r i z a -t ion) ; a nd 65, 70, 75, 80, 85, 90, 95, an d 100 ~ C a l l for 15m i n u t e s . F o r s o m e o f th e t e s ts m i l k w a s h e a t e d b y a s m a l l -s c a l e l a b o r a t o r y p r o c e s s w h i c h c l o s e ly s i m u l a t e d h i g ht e m p e r a t u r e - s h o r t t i m e p a s t e u r i z a t i o n . : '

Estimation of imm unoglobulins.T h e r a d i a l i m m u n o d i f -f u s i o n t e c h n i q u e o f M a n c i n i a n d a s s o c ia t e s w a s u s e d

Vol. 90, No. 1, pp. 2 9-35

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3 0 For d et al. The Journal of PediatricsJanuary 1977

Ta b l e I . I m m u n o g l o b u l i n a c t iv i ty ( m g / m l ) i n u n h e a t e da n d h e a t - t r e at e d h u m a n m i l k

Heattreatment IgA IgM lgG

Non e 0 .50 0 .10 Non e de tec ted56 ~ 30 min 0 .48 0 .10 Non e de tec ted62.5 ~ 30 min 0 .39 0 Non e de tec ted70 ~ 15 min 0 .24 0 Non e de tec ted80 ~ 15 min 0 .10 0 Non e de tec ted

Ta b l e I I . To t a l l a c to f e r r i n a n d u n s a t u r a t e d i r o n - b i n d i n gc a p a c i ty in u n h e a t e d a n d h e a t - t r e a t e d h u m a n m i l k

Heattreatment

UnsaturatedLactoferrin* iron-binding capacityt

(mg/ ml) ( gFe ml)

No ne 3.4 3.7 3.4 3.1 3.156 ~ 30 rnin - 3.0 - -Ho lde r pas teu- 1.2 1.5 1.3 0.7 0.8

r iza t ion(62.5 ~ 30m i n )

65 ~ 15 rn in 1.0 1~0 0.6 0.4 0.470 ~ 15 rain 0.2 0.1 0.1 0 075 ~ 15 rain 0.2 0.2 0 0 0

*Duplicate determinations were made on each test sample.tEach value represents a different preparation of milk.

w i t h B o e h r i n g e r l o w -l e v e l I C L i m m u n o g l o b u l i n p l a t e s

( B o e h ri n g er , M a n n h e i m G m b H ) . T h e a n t i s e r a w er e r a is e d

a g a i n s t s e r u m i m m u n o g l o b u l i n s . I g A o c c u r s a s s e c r e t o r y

I g A i n m i l k , b u t t h e cr o s s r e a c t i o n w i t h s e r u m I g A

a n t i s e r u m w a s s u f f i c i e n t f o r t h e p u r p o s e o f th i s s tu d y. T h e

v a l u es a r e e x p r e s s e d a s m i l l i g r a m s o f i m m u n o g l o b u l i n

a c t i v it y p e r m i l l i l i te r o f m i l k .

M easurem en t o f i ron -b ind ing capac i ty.To t a l l a c t o f e r r i nw a s m e a s u r e d b y t h e r a d ia l i m m u n o d i f f u s i o n m e t h o d o f

F a h e y a n d M c K e l v e y, 7 u s i n g r a b b i t a n t i s e r u m a g a i n s t a

p u r i fi e d p r e p a r a t i o n o f la c t o f e r r i n w h i c h c o n t a i n e d o n l y

t r a c e a m o u n t s o f l y s o z y m e . 4U n s a t u r a t e d i r o n - b i n d i n g c a p ac i ty w a s d e t e r m i n e d b y

t i t r a t i n g t h e m i l k a g a i n s t a s t a n d a r d s o l u t i o n o f f e r r ic

n i t r i l o t r i a c e t a t e . ~

M easurem en t o f v it amin -b ind ing capac i ty.T h e c a p a c i t yo f t h e m i lk to b i n d a d d e d v i t a m i n w a s m e a s u r e d b y

a d d i n g e x c e s s o f [ G -3 H ] c y a n o c o b a l a m i n o r [ G -:~ H] fo l i c

a c i d to a s a m p l e o f t h e te s t p r e p a r a t i o n , a n d t h e n

s e p a r a t i n g f r ee a n d p r o t e i n - b o u n d v i t a m i n b y g e l f il tr a -

t i o n ? ~

Growth t e s t s w i th scherichia coli 0 1 0 1 : K ? : H ? N C T C9 7 0 3 ) . T h e m i l k s a m p l e s w e r e f i r s t d i a l y z e d a g a i n s t b u f f e r

o f p H 7 .0 c o n t a i n i n g 0 . 0 5 M p h o s p h a t e a n d 0 . 0 1 M

Ta b l e I l L I n f l u e n c e o f d if f e r e n t h e a t t r e a t m e n t s o f h u m a nm i l k o n i ts c a p a c it y t o b i n d a d d e d [~ H] c y a n o c o b a l a m i n

a n d [ :~ H ] f o l i c a c i d

Heat treatment

Unsaturated bindingcapacity (ng/ml)

Cyanocobalamin [ Fo licacid

No ne 43.4 21.9HT ST pas teur iza t ion 26 .2 -62.5 ~ 30 rai n 22.6 19.765 ~ 15 m in 18.4 16.470 ~ 15 m in 14.4 14.275 ~ 15 m in 14.0 11.580 ~ 15 m in 15.0 10.285 ~ 15 m in 18.4 4.290 ~ 15 m in 21.6 2.495 ~ 15 m in 22.8 1.5100 ~ 15 m in 18.6 1.5

Ta b l e I V. I n f l u e n c e o f d i f f e re n t h e a t t r e a t m e n t s o f h u m a nm i l k o n i t s l y s o z y m e a c t i v i t y

Heat treatment

Lysozyme activity(~g egg w hite lysozymeequivalent/ml milk)*

Non e 98 99HTS T pas teur iza t ion 103 10762.5 ~ 30 mi n 103 105

70 ~ 15 m in 62 6880 ~ 15 m in 35 3790 ~ 15 min 7 7100 ~ 15 m in 2 3

*Each value represents a different preparation of milk.

N a H C O 3 , a n d t h e n t e s t e d a s d e s c r i b e d b y R e i t e r a n d

col leagues . l~ '

Tes t s on bac t e r i a l up take o f v i t amins .T h e m i l k p r ep a -

r a t i o n s w e r e t e s t e d f o r t h e i r c a p a c i t y t o i n h i b i t u p t a k e o f

[ G -3 H ] c y a n o c o b a l a m i n i nE, coli ( N C T C 9 7 03 ) , a n d o f[G-:~H] fo l ic a c id inBifidobacter ium bif idus( N C D O 1 4 5 2 ) ,

a s d e s c r i b e d b y F o r d )Dete rmina t ion o f l y sozyme ac t iv i ty.Ly s o z y m e a c t i v i t y

w a s a s s e s s e d f r o m t h e r a t e o f l y si s o f a s u s p e n s i o n o f d r i e d

c e l l s o f Micrococcus lysodiecticus, a s d e s c r i b e d b yL i t w a c k . 11 C r y s t a l l i n e e g g w h i t e l y s o z y m e ( W o r t h i n g t o n

B i o c h e m i c a l C o r p . ) w a s u s e d a s t h e s t a n d a r d .Dete rm ina t ion o f i ae tope rox i f l a se .L a c t o p e r o x i d a s e w a s

m e a s u r e d b y f o l l ow i n g t h e o x id a t i o n o f o - d i a n i s i d i n e b y

H~O., at 37 ~ C. - '

R E S U L T S

Immunog lobu l in s . Ta b l e I s h o w s t h e e f f e c t s o f h e a t

t r e a t m e n t . T h e c o n t e n t o f I g A - - t h e p r e d o m i n a n t c l a ss in

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Hours of incubation

Fig. 1. Inf luence of hea t t rea tm ent o f human mi lk , and the effec t of adding i ron , on growth ofEscher i ch ia co l i. - o - =Raw m i lk ; - - -o . . . r aw mi lk + Fe ; - o - = 6 2 .5 ~ C 3 0 m i nu t e s ; - -A - - = H TST; - O - = 7 0~ 15 m i nu t e s; - l - =80 ~ C 15 minu tes; ---A . . . 80 ~ C 15 minu tes + Fe.

h u m a n m i l k - w a s n o t s i g n if i c an t l y r e d u c e d b y h e a t i n g a t

5 6 ~ C f o r 3 0 m i n u t e s . P a s t e u r i z a t i o n b y t h e h o l d e r p r o c e s s

( 6 2. 5 ~ C 3 0 m i n u t e s ) r e d u c e d t h e c o n t e n t b y 2 0 , b u t a t

h i g h e r t e m p e r a t u r e s t h e p e r c e n t a g e o f d e s t ru c t i o n

i n c r e a s e d s h a r p l y. T h e c o n t e n t o f I g M w a s v e r y l o w ; i t

s u r v i v e d h e a t i n g a t 5 6 ~ C b u t n o t a t 6 2 .5 ~ C . I g G w a s n o t

d e t e c t e d i n t h e m i l k .

Iron binding capacity. Ta b l e I I s h o w s t h e c o n t e n t o fl a c t o f e r r i n a n d t h e s a t u r a t e d i r o n - b i n d i n g c a p a c i t y i n

s o m e o f th e m i l k p r e p a r a ti o n s . H o l d e r p a s t e u r i z a t i o n

i n v o l v e d c o n s id e r a b l e l o s s b o t h o f l a c t o fe r r i n a n d o f

u n s a t u r a t e d i r o n - b i n d i n g c a p a c i t y, a n d h e a t i n g a t 7 0 ~ C

c a u s e d v i r t u a l l y c o m p l e t e d e s t r u c t i o n .

I n h i b i t i o n o fE . c o l l F i g . 1 s h o w s t h e i n f l u e n c e o f h e a t

t r e a t m e n t o f m i l k , a n d o f s u p p l e m e n t a t i o n o f th e m i l k

w i t h ir o n , o n i ts c a p a c i t y t o s u p p o r t g r o w t h o fE . c o i l

G r o w t h i n ra w m i l k w a s v e r y p o o r , t h e v i a b l e c o u n t

i n c r e a s i n g b y o n l y o n e l o g c y c le i n 6 h o u r s . A d d i t i o n o f

i r o n ( 5 ~ g / m l ) t o r a w m i l k c a u s e d a m a r k e d i n c r e a s e i n

g r o w t h , a s d i d h e a t i n g t h e m i l k a t 8 0 ~ C f o r 1 5 m i n u t e s .

A d d i t i o n o f i ro n t o th i s h e a t e d m i l k g a v e n o f u r t h e r

g r o w t h i n c r e m e n t . I n m i l k p a s t e u ri z e d b y t h e h o l d e r o r

H T S T p r o c e s s t h e re w a s m o r e g r o w t h a t t h re e h o u r s t h a n

in t he r aw m i lk , bu t by s i x hou r s t h e r e w as l i t t le d i f f e r -

e n c e .Vitamin binding cap aci ty.Ta b l e I l i s h o w s t h e e ff e c t s o f

h e a t t r e a t m e n t o f m i l k o n i t s c a p a c i t y t o b i n d a d d e d

c y a n o c o b a l a m i n a n d f o l i c a c i d .

B , = , - b i n d i n g c a p a c i t y d e c l i n e d p r o g r e s s i v e l y w i t h a n

i n c r e a s e i n p r o c e s s i n g t e m p e r a t u r e , t o a m i n i m u m a t 7 5 ~

C . F u r t h e r i n c r e a s e i n t e m p e r a t u r e r e v e r s e d t h i s t re n d , s o

t h a t t r e a t m e n t a t 9 5 ~ C w a s l e s s d e s t r u c t i v e t h a n a t 6 5 ~

C .T h e s i t u a t i o n w i t h f o l a t e b i n d e r w a s d i f f e r e n t . H o l d e r

p a s t e u r i z a t i o n d e s t r o y e d 1 0 o f t h e u n s a t u r a t e d b i n d e r ,

a n d w i t h i n c r e a s e i n p r o c e s s i n g t e m p e r a t u r e t h e r e w a s a

p ro g r e s s i v e i n c r ea s e i n t he lo s s , t o a bo u t 90 a f t e r 95 ~ C

1 5 minu t e s ,Inhibition of vitamin uptake in bacteria.F i g . 2 s h o w s t h e

i n f l u e n c e o f h e a t t r e a t m e n t o f m i l k o n i ts c a p a c i t y t o

i n h i bi t th e u p t a k e o f c y a n o c o b a l a m i n b yE . c o i l I n t h e

a b s e n c e o f m i l k , u p t a k e o f c y a n o c o b a l a m i n w a s n e a r l y

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3 2 For d et aL The Journal of PediatricsJanuary 1977

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[ H] C y a n o c o b a t a m i n a d d e d p e r 5 m lE. colic u l t u r e , n g

Fig. 2 , Inf luence of heat t rea tm ent of hum an milk on i ts capaci ty to inhib i t uptake o f cyanocobalam in inEseherichia coilTo 5 ml por t ions of bacter ia l cul ture were ad ded graded amou nts of [:~H] cyanoco balam in wi th: (1) no fur ther addi t ion;(2) 0.2 ml m ilk unhea ted, o r (3) heated 62.5 ~ C 30 m inutes , or (4) 70 ~ C 15 minute s, or (5) 80 ~ C 15 min utes, or (6) 90 ~ C15 minu tes, or (7) 100~ C 15 minu tes. After incu bat ion for 30 minu tes at 37 ~ C the bacterial cells were harves ted bycentr i fugation and analyzed for [:~H] cyanocob alamin.

p r o p o r t i o n a l to t h e a m o u n t o f t he v i t a m i n a d d e d t o t h eb a c t e r i a l c u l t u r e , w h e r e a s i n t h e p r e s e n c e o f 0 . 2 m l o fu n h e a t e d m i l k t h e r e w a s n o s i g n i f i c a n t u p t a k e u n t i l t h ea m o u n t o f a d d e d v i t a m i n e x c e ed e d 5 n g . T h e r a w m i l kh a d t h e p r o p e r t y o f s e q u e s t e r i n g 2 5 to 5 0 n g o f c y a n o c o -b a l a m i n p e r m i l l i li t e r a n d p r o t e c ti n g i t a g a i n s t u p t a k e b yE. eoli. H e a t t r e a t m e n t r e d u c e d t h i s c a p a c i t y o f t h e m i l k t op r o te c t c y a n o c o b a l a m i n a n d , a s w i t h t h e u n s a t u r a t e db i n d i n g c a p a c i t y ( Ta b l e I I I ) , t h e e f f ec t w a s g r e a t e s t a t t h e

i n t e r m e d i a t e t e m p e r a t u r e s .

F i g . 3 s h o w s t h e r e s u l t s o f a s i m i l a r e x p e r i m e n t o n

f o l a t e u p t a k e b yB. b i f idus . F o l i c a c i d a d d e d a l o n e w a st a k e n u p q u i c k l y a n d a l m o s t c o m p l e t e l y b y t h e b a c t e r i a ,a s w a s f o l ic ac i d a d d e d w i t h 0 . 2 m l o f m i l k t h a t h a d b e e n

h e a t e d a t 1 0 0 ~ f o r 1 5 m i n u t e s . I n t h e p r e s e n c e o f 0 . 2 m lo f u n h e a t e d m i l k t h e re w a s n o u p t a k e o f fo l i c a c i d u n t i lt h e a m o u n t a d d e d e x c e e d e d 2 ri g. Wi t h i n c r e a s i n g s e v er i t yo f h e a t t r e a t m e n t o f t h e m i l k i t s c a p a c i t y t o p r o t e c t f o l ica c i d a g a i n s t b a c t e r i a l u p t a k e d e c l i n e d p r o g r e s s i v e l y.

Ly s o z y m e a c t iv i t y. Ta b l e I V s h o w s t h e l y s o z y m e a c t i v i t yi n s o m e o f th e m i l k p r e p a r a t i o n s . H o l d e r p a s t e u r i z a t i o nc a u s e d n o l o s s o f a c t i v it y b u t t r e a t m e n t a t 7 0 ~ C a n dh i g h e r t e m p e r a t u r e s c a u s e d p r o g r e s s i v e l y i n c r e a s i n gd e s t r u c t i o n o f t h e e n z y m e . O n l y a b o u t 3 % o f t h e a c t i v i t ysu rv ived hea t ing a t 100 ~ C .

L a e t o p e r o x i d a s e . I n t h e m a t u r e m i l k u s e d in t h e p r e s e n t

s t u d y t h e c o n t e n t o f l a c t o p e r o x i d a s e w a s l e s s t h a n 0 . 0 5

u n i t s p e r m i l l i l i t e r , t h e l o w e r l i m i t o f s e n s i t i v i t y o f t h ea s sa y m e t h o d e m p l o y e d .

D I S C U S S I O N

l m m u n o g i o b u l i n s . S z 611 6s y a n d a s s o c i a te s ' :' f o u n d t h a tt r e a t m e n t o f h u m a n m i l k a t 6 5 ~ C f o r 3 0 m i n u t e s c a u s e dn o r e d u c t i o n i n a n t i b o d y t i t e r a g a i n s tE. coi l t ype 055 ,w h e r e a s w e f o u n d a 2 0 % f a l l i n I g A a n d d e s t r u c t i o n o fI g M . T h e y f u r t h e r o b s e r v e d a f a l l i n a n t i b o d y t i t e r o n

s t o ra g e o f t h e m i l k a t 2 5 ~ C , w h i c h t h e y a t t r i b u t e d t ob a c t e r i a l e n z y m e a c t i v i t y. We f o u n d n o s u c h f a l l i n m i l ki n o c u l a t e d w i t h a s t r o n g l y p r o t e o l y t i c s t r a i n o fPs e u d o -

m o n a s f l u o r e s c e n sa n d s t o r e d a t 4 ~ C f o r 4 7 h o u r s , d u r i n g

w h i c h t i m e t h e v i a b l e c o u n t i n c r e a s e d f r o m l 0 :~ t o l 0 ~ p e rmil l i l i ter.

T h e i r o n a n d vi t a m i n b i n d er s . H u m a n m i l k , u n l i k e c o wm i l k , is ri c h i n t h e i r o n - b i n d i n g p r o t e i n l a c t o f e r r i n , w h i c hi n it s u n s a t u r a t e d f o r m i n h i b i t s t h e g r o w t h o f s e v e r a lp a t h o g e n s , a p p a r e n t l y b y d e p r i v i n g t h e o r g a n i s m s o fi ron . ' . .. .. B reas t - f ed in fan t s a r e ma rke d ly r e s i s t an t t oi n f e c t i o u s g a s tr o e n t e r i t is c a u s e b yE. coli a n d B u l l e n a n dassoc ia t e s ~ sugges t t h a t t h i s r e s i s t ance i s l a rge ly co n t r i b -u t e d b y l a c t o f e r r i n a c t i n g i n c o m b i n a t i o n w i t h s p e c i f i ca n t i b o d y. T h e f o l a t e a n d v i t a m i n B , ., i n h u m a n m i l k , l i k et h e ir o n , a r e s t ro n g l y b o u n d t o m i n o r w h e y p r o t e i n s a n d ,

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Volume90 H e a t t r e a t m e n t o f h u m a n m i l k3 3N u m b e r 1

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1 2 5[3 H]Folie acid p er 5 ml B.bif idusculture, ng

Fig. 3. Inf luence of heat t rea tm ent of hu ma n m ilk on i t s capacity to inhibi t uptake o f fol ic ac id inBifidobacterium bifidus.To 5 ml po rtions of bacte rial culture were adde d grad ed a mo unts of [:~H] olic acid with: (1) no further a ddition ; (2) 0.2ml milk u nhe ated, or (3) hea ted 62.5 ~ C 30 min utes, or (4) 70 ~ C 15 minu tes, or (5) 80 ~ C 15 min utes, or (6) 90 ~ C 15min utes, or (7) 100 ~ C 15 min utes. After in cub ation for 30 m inute s at 37 ~ C the b acterial cells were harvested bycentrif ugatio n and analyz ed for [3H] folic acid.

a s w i t h l a c t o f e r r i n , t h e r e i s a l a rg e e x c e s s o f u n s a t u r a t e db i n d e r p r o te i n s . T h e f r ee v i t a m i n s a r e t a k e n u p a v i d l y b ys e v e r al s p ec i e s o f b a c t e r i a a n d i t h a s b e e n s u g g e s t e d t h a t ,

i n p r o t e c t i n g t h e v i t a m i n s a g a i n s t b a c t e r i a l u p t a k e , t h eb i n d e r s m a y s t r o n g l y i n f l u e n c e t h e e c o l o g y o f t h e i n t e s -t i n al m i c r o fl o r a a n d t h e v i t a m i n n u t r i t i o n o f t h e s u c k l i n gi n f a n t. ~ A n i m a l e x p e r i m e n t s i n d i c a t e t h a t t h e p r o t e i n -b o u n d v i t a m i n s m a y b e e f f ic i en t ly a b s o r b e d f r o m t h e

i n t e s t i n e b o t h b e f o r e a n d f o r s o m e t i m e a f t e r t h e c e s s a t i o no f t r a n s p o r t o f i n t a c t p r o t e i n a c r o s s t h e n e o n a t a l g u t

e p i t h e li u m ? ' . . . . .

I t is i n t e r e s t i n g t h a t t h e B l ~ - b i n d e r w a s m o s t l a b i l e a ta r o u n d 7 5 ~ C ( F i g . 2 ; Ta b l e I I I ) . T h e s a m e w a s t r u e f o r

sow m i lk , wh ic h a l so i s r i ch in B~2-b inde r. I t s eemsp o s s i b l e t h a t s o m e o f t h e d e s t r u c t i o n a t t h e s e i n t e r m e d i a t et e m p e r a t u r e s w a s c a u s e d b y a m i l k e n z y m e w h i c h w a si t s e lf d e n a t u r e d a t th e h i g h e r t e m p e r a t u r e s . I f so , t h e np r o l o n g e d s t o r a g e o f m i l k i n a d o m e s t i c r e f r i g e r a t o r m i g h tc a u s e a p p r e c i a b l e l o ss . A s s u m i n g a d o u b l i n g o f t h e r a t e o ft h e e n z y m i c a c t i v it y p e r 1 0 ~ C r i se i n t e m p e r a t u r e , s t o r a g ea t 1 0 ~ C f o r 1 6 h o u r s w o u l d b e a s d a m a g i n g a s h e a t i n g a t70 ~ C fo r 15 min u te s .

Ly s oz y m e . H u m a n m i l k i s r ic h i n l y s o z y m e , c o n t a i n i n g

a b o u t 3 9 m g / d l , a s a g a i n s t o n l y 1 3 # g / d l i n c o w m i l k . ' I ta c ts d i r e c tl y a g a i n s t m a n y b a c t e r i a a n d i t al s o a c t s

i n d i r e c t l y b y p o t e n t i a t i n g t h e b a c t e r i c i d a l a c t i v i t y o fi m m u n e a n t ib o d ie s .' -' T h e p u r i f i e d e n z y m e i s h i g h l y s t a b l ea t a c i d p H : a t p H 4 . 5, h e a t t r e a t m e n t f o r 3 m i n u t e s a t 1 00 ~C c a u s e s n o l o ss o f ac t iv i ty. - T h i s f i n d i n g h a s b e e n w i d e l yq u o t e d , a n d s o i t n e e d s t o b e e m p h a s i z e d t h a t , i n m i l k a t

i t s n a t u r a l p H , t h e e n z y m e i s h e a t l a b i l e .L a e t o p e r o x i d a s e . C o w m i l k i s r i c h i n l a c t o p e r o x i d a s e

w h i c h , in t h e p r e s e n c e o f t h i o c y a n a t e a n d p e r o x i d e ,inh ibi t s a var ie ty o f ba cter ia an d vi ruse s . I '- '- '~ -':~G o t h e f o r s

a n d M a r k l u n d ~ f o u n d t h a t t he c o n c e n t r a t i o n i n h u m a nm i l k w a s c o m p a r a t i v e l y s m a l l ; i t d e c l i n e d s t e e p l y w i t ht i m e a f t e r p a r t u r i t i o n a n d a t s ix d a y s a v e r a g e d o n l y 0 . 5u n i t / m l , a s a g a i n st 40 u n i t s / m l i n co w m i l k . We f o u n d n op e r o x i d a s e i n o u r m i l k s a m p l e s , p o s s i b l y b e c a u s e o f t h el o n g - t i m e i n t e r v a l b e t w e e n c o l l e c t i o n a n d t e st . O u rs a m p l e s h a d b e e n c o l l e ct e d o v e r o n e t o t w o d a y s a t 4 ~ Ca n d b u l k e d , w h e r e a s G o t h e f o r s a n d M a r k l u n d -'~ r e p o r t e do n i n d i v i d u a l s a m p l e s t h a t h a d b e e n t e st e d w i t h 4 h o u r s o f

co l l ec t ion .G e n e r a l . M o s t p e d i a t r i c i a n s w o u l d t a k e t h e v i e w t h a t

h e a t d e n a t u r a t i o n o f t h e p r o t e c t i v e p r o t e i n s i n m i l k i s

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3 4 Fo rd e t a l . The Journa l of Pedia tr icsJanuary 1977

u n d e s i r a b l e , b u t t h a t i n t h e p r a c t i c a l s i t u a t i o n i t i su n a v o i d a b l e . T h e y m a y w e l l b e c o r r ec t i n t h i s v i e w, a n dc e r t a i n l y i t w o u l d b e i r r e s p o n s i b l e t o a d v o c a t e t h e f e e d i n g

o f r a w m i l k w i t h o u t s t r i n g e n t c o n t r o l o f i t s b a c t e r io l o g i cq u a li t y. B u t w e m a y r e a s o n a b l y w o n d e r w h e t h e r a n y h e a tt r e a t m e n t t h a t i s a d e q u a t e t o e n s u r e s t e r i l it y i n m i l k t a k e na n d s t o r e d u n d e r s a n i t a ry c o n d i t i o n s m i g h t n o t a c t u a l l yi n c r e a s e t h e r i s k o f e n t e r i c i n f e c t i o n in t h e i n f a n t . E v e n i nb r e a s t - f e d i n f a n t s t h e m i l k c o n t a i n s b a c t e r i a ,T '-'~p r e s u m -

a b l y d e r i v i n g f r o m t h e m o t h e r ' s n i p p l e s a n d f i n g e r s , a n d i tm i g h t w e l l b e t h a t w i t h c a r e f u l h y g i e n e i n t h e c o l l e c t i o na n d s t o r a ge o f d o n o r m i l k t h e b a c t e r i a l c o u n t c o u l d b em a i n t a i n e d w i t h a c c e p t a b l e li m i t s . T h e i d e a l s h o u l d b e t oc o ll ec t c l e a n m i l k , w i t h m i n i m a l c o n t a m i n a t i o n b y s k i no r g a n i s m s a n d e n t e r o b a c te r i a , a n d t o s to r e it i m m e d i a t e -

l y i n a f r e e z e r u n t i l i t i s g e n t l y t h a w e d f o r f e e d i n g t o t h ei n f a n t. B u t i n m a n y m a t e r n i t y u n i ts a n d b r e a s t m i l kco l l ec t ion cen te r s t he p rac t i ce mu s t f a l l sho r t o f t h i s i dea l .M i l k f r o m d o n o r s l i v i n g a t h o m e m a y h a v e b e e n c o l l e ct e do v e r a p e r io d o f 2 4 h o u r s 9 r lo n g e r , w i t h o n l y p e r f u n c t o r ya t t e n t i o n t o h y g i e n e , a n d s t o r e d i n t h e d o m e s t i c r e f r i g e r -a t o r b e f o r e b e i n g b r o u g h t t o t h e c o l le c t i o n c e n t e r. U n d e rt h e s e c i r c u m s t a n c e s e x t e n s i v e g r o w t h o f p s y c h r o t r o p h i cb a c t e r i a is t o b e e x p e c te d a n d h e a t t r e a t m e n t o f t h e m i l k i sc l e a r l y n e c e s s a r y, h o w e v e r r e g r e t t a b l e i t m a y b e . E x p e r i -e n c e i n H u n g a r y -'~ e d to t h e r e c o m m e n d a t i o n t h a t d o n o rm i l k s h o u l d b e h e a t e d a t 6 5 ~ C f o r 3 0 m i n u t e s i f i t sb a c t e r i a l c o u n t e x c e e d e d a p r e d e t e r m i n e d l e v e l . T h i s

w o u l d i n v o l v e a l a rg e a m o u n t o f r o u t i n e b a c t e r i o l o gi ct e s ti n g a n d i t w o u l d f u r t h e r p r o l o n g t h e i n t e r v a l b e t w e e n

c o l l ec t i on a n d f e e d i n g o f t h e m i l k . B u t s o m e s u c h a t t e m p tt o p r o v i d e a G r a d e A q u a l i t y o f m i l k fo r f e e d i n g r a w t ow e a k a n d p r e m a t u r e b a b i e s m i g h t b e p r a c t ic a b l e , a n d i t i s

c l e a r l y i m p o r t a n t t o e s t a b l i s h w h e t h e r i t w o u l d b e b e n e f i -c i a l. M e a n w h i l e , t o t h e e x t e n t t h a t h e a t i n g o f t h e m i l k i sp r a c t i c e d , i t s h o u l d b e n o . m o r e s e v e r e o r p r o l o n g e d t h a ni s r e q u i r e d f o r t h e d e s t r u c t i o n o f l i k e l y p a t h o g e n s . F o rb a t c h p r o c e s si n g o f s m a l l q u a n t i t i e s o f m i l k t h e h o l d e rp r o c e s s ( 6 2 .5 ~ 3 0 m i n u t e s ) w o u l d s e e m t o b e t h e m e t h o do f c h o i c e .

The authors are indebted to Drs . D. E. H. Tee andS. Wil l iamson, King 's College Hospi ta l , London, for the im mu -nologic assays and helpful discussion; to Sister V. Lew arne, of theHu ma n Milk B ureau, Univers i ty Hospi ta l of Wales, for k indlyarranging the supp ly of breas t mi lk , and to Mrs . Valer ie Bines fortechnical help.

R E F E R E N E S

1. Hanson LA, and W inberg J : Breas t mi lk and defenceagains t infect ion in the new born, Arch Dis C hi ld 47:845,1972.

2. Reiter B, and O ram JD : Bacterial inhil~itors in m ilk andother biological fluids, Nature (Lond ) 216:328, 1967.

3 . Ford JE: Some observations on the poss ible nutr i t ionals ignif icance of v i tam in B~-an d fo la te-binding prote ins inmilk, Br J N utr 31:243, 1974.

4. Reiter B: Bacterial inhibitors in milk and other biologicalf lu ids wi th specia l reference to the complement /ant ibodytransferr in / lac toferr in and lactoperoxidase/ th iocyanate hy-drogen peroxide systems,in Skinne r TA and Hugo W B,editors: I nhi biti on and ina ctivatio n of vegetative m icrobes.Proceeding s of Society of App lied Bacteriology Sym p SeriesNo. 5, New York, 1976, Aca dem ic Press, Inc., pp 31-60.

5 . Frank l in JG: A s imple laboratory-scale HT ST milk pas teur-izer, J Dairy Res 32:281, 1965.

6 . Manc in i G , Ca rbona ra O , and Heremans , JF : Imm uno-chemical quant i ta t ion of ant igens by s ingle radia l immu-nodiffusion, I mm unoc hem istry 2:235, 1965,

7 . Fahey JL, and McKelvey EM: Quant i ta t ive determinat ionof serum imm unoglob ul ins in ant ibod y-agar p lates, Jlm m un ol 94:84, 1965.

8. Bates GW , Billups C, and Sa ltma n P: The k inetics andmechanism of i ron (111) exchange between chela tes andtransferrin, J Biol Chem 242:2810, 1967.

9 . Ford JE, Scott KJ, Sansom BF, and Taylor PJ: Absorpt ionof (~Co) cyano cobalamin by suckl ing piglets, Br J N utr34:469, 1975.

10. Reiter B, Brock JH, and Steel ED: The bac teriostatic effectof lac toferr in on a serum suscept ible and serum res is tants t ra in ofE. coli Imm uno logy 28:83, 1975.

11. Litwack G: Pho tome tric dete rm inatio n of lysozyme activity,Proc Soc Exp Biol Med 89:401, 1955.

12. Ora m JD, and Reiter B: The inh ibitio n of Streptococci bylactoperoxidase, thiocyan ate and hydro gen peroxide,Biochem J 100:373, 1966.

13. Sz6116syE, Marja i E, and Lantos J : Bacter ia l c ontam inat ion

and heat t rea tm ent of mother ' s mi lk , Acta M icrobiol AcadSci Lung 21:319, 1974.

14. Oram JD, an d Rei ter B: Inhibi t ion of bacter ia by lac toferr inand other iron-c helating agents, Biochem Biophys Acta170:351, 1968.

15. K irkpatrick CH , Green 1, Rich RR, and Schade AL :Inhibi t io n of growth o f Candida a lbicans by i ron-unsatu-rated lactoferrin, J Infect Dis 124:539, 1971.

16. BuU en J , Rogers H J , and Leigh L: I ro n-bin ding prote ins inmilk and resistance toEscherichia coliinfect ion in infants ,Br Med J 1:69, 1972.

17. Ford JE, Knag gs GS, Scott KJ, and Salter DN: Fola tenutr ition in the kid, Br J N utr 27:571, 1972.

18. Izak G, Galewski K, Rachm ilewitz M, and G rossowicz N:The absorpt ion of milk-b ound pteroylglutamic acid f romsma ll intestine segments, Proc Soc Ex p Biol Med 140:248,1972.

19. Cha ndan RC, Shah ani KM, and Hol ly RG: Lysozymecontent of hum an m ilk , Nature (Load) 204:76, 1964.

20. Hill IR, and Po rter P: Studies of bactericidal activity forEscherichia coliof porcine serum and colos t ra l immun oglo-bul ins and the role of lysozyme w ith secre tory lgA, Im mu -nology 26:1239, 1974.

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22. Belding ME, Kle bano ff S , and Ray C G: Peroxidase-med iated virucid al systems, Science 167:195, 1970.

23. Bj6rck L, Ros6n CG, Marshall V, and R eiter B: An tibac -terial activity of the lactoperoxidase system in milk against

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Pseudomonads and o the r g r am nega t i ve ba c t e ri a A p p lM icrobiol 30:199 1975.

24. Gothefors L an d Ma rklund S: Lactoperoxidase ac t iv i ty inhum an mi lk and in sa l iva of the newborn infant Infec t

lmm uno l 11:1210 1975.25. Wy at t RG and Ma ta LJ : B acter ia in colos trum and mi lk of

26.

27.

Gua tamalan Indian women J Trop Pedia t r 15:159 1969.Kl ine K: Die Bakter ienf lora ungekochter FrauenmilchM~inch Me d Woch ensc hr 104:1764 1962.Moln~ir M Fran k M and Osv~ith G: V6rell~tto ~llomgtson

Sverv ezett N6si TejgyiJt6s n6dszere l~s Jelent6s6ge OrvH6 til 110:355 1969.