Biochemistry of Milk Products 1994

Milk Products
Biochemistry of Milk Products
A. T. Andrews
J. Varley
WOODHEAD PUBLISHING LIMITED

The proceeidinl2:S
RSC Industrial ,-"v."..",,_
First put)Usl!1ed ChemIstry 1994
Woodhead PUVtlishiing
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ISBN-13: 978-1-85573-775-4
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Preface
of the whole food and

gr~ltest
share of a11 human
with little or no
pretrealtmcmt, such as
are as
maJlufactwrmg processes, so only quite a smalJ
oer~ceD1:a2e would generally be tbo\lgJJt of as . In this the dairying area
exc:eptlon and most milk is still consumed as such rather than being made into
Nevertheless even only a part of the total milk production still
rep:rescmt:s an extremely It is worth that historicalJy
the oldest manufacturing industries of were to food drink products~
namely fermentation to give alcoholic beverages and the production of cheese from
milk. Both of these industries function world wide and are performed on scales
from amateur in the home, through smalJ often speciallist
indlllStries, to major multinational with turnovers of hundreds of millions
of dollars.
ago, probably in the Middle East where the

stomachs of animals were kept slaughter and used as leather-type bottles for
and transporting liquids. It is thought that the of milk in imperfectly
cleaned stomachs which still contained traces of the pepsin, aided
perhaps by lactic led to the milk and to the
realisation that the resulting curds a convenient and concentrated form of
most of the protein. Also because action of the low
by the fermentation of lactose to lactic the curds could be stored for considerable
of time. In spite of this it is still that is the most
active and fruitful area in dairy This is very apparent from the
contents of this book which of a recent on
advances in biochemistry. There are two r~h at
ImrtrOv'em4mts in starter to better quality
SU[len()f flavour and texture, developed in a shorter time to
mtnlIDlSe and to the tailoring of cheese flavour to
particular products and processes. the production of new milk coaguhmts
as alternatives to traditional rennet. Both these lean on
molecular techniques, the former to new with
eWime profiles (peptidases, and to a lesser lipases) better suited
tasks than current microorganisms, and latter to produce from
mlc:rOCtrgllllllS:ms, following manipulation and a purified prO'teiJJl8Se
speCIfiCity capable of milk without the formation of undesirable
DV-DfO(iUCts such as bitter peptlde:S.
In order to be successful in these obl,ectJves it is to understand in fine

detail at the molecular level all process and of what takes
ma1tunltiolD, iniClueJing especllllly the role of starter enz:vmc~s.
The initial papers this
extlenamg kD()wledflte in this area.
The second research covers the functional behaviour

of milk prollems. many years as in a wide
food products aJ)pli1cations because of their desirable nb"slca)
attJibtlltes and nutritional rAL""'fttl!" however have sep,ara1ion
methods improved to the extent that larile-!;calle plroauC110n of individual protlein

COIlLloonents with functional prO'f)erttes bas become
a viable rOUlle to new food ingredien1:S. such advances depend
upon a at the molecular level involved" in this
case of what molecular features make a a enable it
to form or to stabilise etc. Once these features can it then
pra1cttcaI pJropos1110n to allier via the
functional behaviour and so ultimarely to produce mUOr-lma{!e
proflleillS dc~slgnea to fulfil a task. For these reasons other invitled papers
SYIl!100'SlUlm and a number of the cover not only
prCKiUl=tjOtn and functional evaluation of natural but also the effect that
substitution of particular amino acid residues bas on functional behaviour.
This should us a of the intleractions involved, which should
in tum lead taller to with Imllro'ved pertOl'lmailce.
as the of what was the

selection of topics covered may to be that
the most active areas of are well and that the review-like
nature of most of the papers means that the coverage is much less limitled than
would be Many of the and certainly the tleClmi<llUeS
will undoubtledly be applicable not elsewhere in the milk and dairy
chemistry area but also outside it, in the and protlein
chemistry/prollein fields. These a good stalle-of-the-art
picture of current research which should be very valuable to research w01t'ker'S.
graduare students and final year undergradualles with intlerests in the
pra1cttcaI al)J)Ucattons of molecular and prorein chemistry, not
in the quality and and but also
in a much wider conllext.
We should like to thank aU those who made this possible by both their
physical and moral support, and especially all the contributors of papers
and whose excellent quality manuscripts made our task as editors
so straightforward and enil[)Va1ble.
A. T . Andrews
J.Varley
Contents
Prolteol'vsis in Lactococcus Lactis 32

A.J'.Ht'lfmlrJrIJ<7IlQjn. I.Mierau. J.Kok and
G.Venema
New Starter Cultures for Cheese Rlvenulg 47

B.A. Law
hni~meerulg Pivotal Proteins for Lactococcal PrOlteol'VSIS 56

W.M.de Vos and R.I. Siezen
ArutJIVSlS of CHY155-165RHI 72
_,._.,,:.... - R.
l~L,. BI,U1l(Jfell, J. Uusitalo and M. Penttila
Petl~ti<ulSeS from Lactococci and Secon<llary Prolteol'vS1S of Milk Proteins 83

F.Mulholland
Functional Milk Protein Products 94

D.M.Mulvihill
En~~neeri[lg Studies of f3-l.act()glclbulin 114

J.H.Morais Cabral and C.A.Batt
Functional of Chhana Products 121

A.S. Grandison and A.R.Jindal
Thermal Agjue~~atl()n of Protein Concentrates under Fluid Shear 133

Conditions
.'\tp"p"tl')n A.M.Dono.ld and L.F.Gladden
143
viii Bioc'hemistrv of Milk
The Effect of Thermisation on the Thermal Denaturation of 152

'Y-Olutamyltranspeptidase in Milk and Milk Products
S. S. POIel and B.A. Wilbey
Keeping Quality of Pasteurised and High Pasteurlsed Milk 157

B.Borde-Lekona. M.l.Lewis and W.F.Harrigan
J.:i'nnliin'" and UHT Proces~;ini 162

P.Kastanas. M.l.Lewis and A. Grandison
Ultrafiltration of Sweet Cream Buttermilk 169

H. G.Ramochandra Roo, M.J.Lewis and A. S. Grandison
Index 177
Proteolysis in Cheese during Ripening
p.
DEPARTMENT OF FOOD CHEMISTRY, NATIONAL FOOD BIOTECHNOLOGY

UNIVERSITY IRELAND
1. INTRODUCTION
The conversion of milk to cheese curd is the first

cheese varieties. all hard, and many
from a few weeks to two years or
numerous biochemical which
texture, flavour and aroma.
The of cheese nnt~ntrlO
food. the
cOI1!lpll~x of
2, CHEESE RIPENING AGENTS AND THEIR CONTRIBUTION TO

PROTEOLYSIS
in cheese varies from very limited
nrl'ltp£1llvCil,J(1. to very
mould The of nrll.tPll,lv'Cil''Cil range in size from
COlDPjarabJe in size to a range of medium
peiJf{J<I«~S to free amino acids.
P1"ll.tPll,lvtlll' agents in cheese ... .,.r."" ..~.II., on,gmate the coagulant,
the milk, starter non~starter oac:[enla hn:zvl1!les from the
first four sources are active in
mH~rO()rg'imj~)ms added to che~esc:~milk
pel)tl(jlas~~s on caseins or
pel,tHles from cheese and,
based on the known SPE~CIlICU:y pf()teina:seslp(~ptj.da:ses on the caseins in SOlut14::m.
identificiation of their tn~'T"'Hlh\,'p
The use of model ~\I~tpnl~
cheeses can be summarized as

residual cO~lgulal1lt,
resl1ltulg in the of
the C03Lgulant
sman pe1J;t1Clc~S
bactenal plrotc:nmlses and oel>t10ase:s.
Prntpn/\I.<:;s: in
contribution of to the initial Inu1 ....... U.!C"'" prcmoum;ed than in

Cheddar and Dutch varieties.
pr()(el.nases and from the starter influence nr()t~,nl\)''''l''' <:tr£\no:lv
This will focus on the SP<:~Cll1Cl1tv
peJ)ttdlasc~s in cheese on the individual and cas.eUI-mernfea pel)U<les
ISOi(atlOn and identification of from Cheddar cheese.
3. SPECIACITY OF THE PRINCIPAL PROTEINASES AND PEPTIDASES

IN CHEESE
Most of the GMPs are lost in the

DaJra-iK-c:ascem remains attached to the casein micelles and is mc:ofl)orate~d
cheese.
cleaVf~a are
5.2 in the presence

at the lower
4 Biochemistry of Milk Products
marxianus var, lac/is

l1Jvp"f1~m'J'rpfil were mtlroduce~d rf"I"'f"tltlv
since their authorities for use in
used for in many, but not countries.
lnvnlvlno a number of have shown small differences between cheese
calf rennet or recombinant Recombinant ctn/m()SlT1S
calf rennet can contain three I"'h'lltTl,nCl1In
Possible differences in SPC:~CltlCl1ty
amount of C08l2Ultant reulinc:ld

Proteolysis in Cheese during Ripening 5
Cbeeses wbicb are cooked at a

bave little coa,2ulant
on ('L,"li-CCllSellD
solution.
lDd.lgen01JS plrotf:maLses in milk bas been recogluze~d for a

pnlDClpal lD(l112.~ncmsprclteinas.e is wbicb i.s active at
bas a pH at -4.0.
of
con:Slst.lD2 of
______~t___t~______~+___ ~
LYS2S·LYSZ9 LY SIOS·His I06 LyslIl'Tyrll4 Argl!!.rAsPI84
~t~
~,
LySJorG1uI03
PRODUCTS
Proteose
Known Probable
PM fl-28 fl06-113
PPSs f29-105 fl08-lJ3
PPSs f29-107 fll4-183
PP-T f29-113 fl06-183
PPS fl-105 fl08-183
PPS fl-107 fl_113Iun.lilr ••h'\
oroteo:se Dj~D[4:>ne 8
6
of
" ..."."".+."".t'n D on the
incubated with milk
SDt:~lilClltv very similar to that of
to rates of of
to be a poor substrate for
a
are considered not to be
milk that other lysos()m,al protem.asc~s are also
present, altJl0u' gil , de!tected in milk.
The theoretical combined action of "h,,,.,...,, .......

c'eaV;U1e sites of and is shown sctlenf1atic3:lIy in
UsrCASEIN
CHYMOSIN
~
--~I~t~t--------~~~f--~t--~t~~~H~t-----,~
PLASMIN
tl-CASEIN
______ C_HY-M-O-SIN--------------~~~t--Il.T-O----iatt~~-·~~f---~
1 II 11
PLASMIN
Potential combined action of l':hvn1to""ln and plasmiin in cheese
during Ripening
their combined action could ~.I"""''''A

fact cOlmpleI10eIltalry
the C-terminal in the we
know, the action of these .... ,,'\1""''''0 on the isolated caseins has not been
studied and it is not known whether concerted manner in cheese.
those of the:rm:ophillc )ln~l71O(X}CCUS

considerable attention.
The nr.n£,ln~1 prc)telna~;e associated with the cell
Cell wall-associated
and -types .
...... ... ... ...

It s: P I It I!l Q Ll'Q&V N N A F V If' EllERY E
... ... ...

AKItI> It Q K!;Q It It. PI!: E 1\
...... ... ...... ...

ItYIIVPQLIt v A K Ii: E A R E M
......
... ...
... ......
r Y Q L I> It. Y P160 S G A \of PLOT A r E E T K
Amino acid sequence posOIucm of the

L:1t~a\l"19r. sites of cell waH-associated prC)lel,nases
SK 112 (ref. 87) and L
It Jl T K It II 11 5 5 S It It 5 I 5 0 It T YZO It Q I It If K A II l!' S It It If 4 S T F C40 K I V V II If A If E ESO
Y Y
EYSI SSSEEf,OSAEVATItEVIt TVtll)lt8YQltllOALNIIlIEFYQltFIi' QYL
y y
o PIV4NPWtlQVltRRAVPITnoPTLRREQ STIEENlltlT
y Y Y
TIL TEE It I N 11,160 4 If r It I ISO it Y 0 It r A QY
Y
IIi'YVRYLZ01
It-Casein B
ItIAItTIPIQYV I'lIi'VA
y y y Y Y Y
I If iii Q F P P 'i60 Y A It I' A A V II, S Ii' A 0 I w v VPAI A MAllRI'
PH Ii: P T VA
Y
TVQVT
Amino acid sequences of &s A and K-casein B the

po~;iti()ns of the sites of cell waU-8lSSCK:U:llted pr('telna~)eof Lactococcus lcu'ti:,
ssp. lactis NCDO
(1)
• • •• ••••
•••
121
III
III lI! 1 1 I. It I' G 1 V II $ 126 E 'l'II.IltllltIIlIlPO' Ell OO"O'l'III)II.OPII
••... • • ... • • •••...

...
•• OS...•• •1'8 ,
III'PA V Ii' P P , I
.........
II P P
. .......
I. 'I'
'I'
......
... .. ..... .
..........
...
T P I l i I. II I' I:. Ii' I. 1:.140 Q S \II H II P II Q
... • ...• .... •... •... ••... ....

1'1.. I' P 'I' MFI'
•• .......
... •... ....•... ••
s Iii Q It V I. I' 1'0ltAVP
••
•...
... PQII
.
••...
,
...
••••
••....•
MPIO'AFI.I.YQ
... 1''11.01'200
1'1
171
VII I' F
...
t'f}
I I
CleaV:loe sites of cell

H2
Proteolysis in Ripening
tvn,u'J;1,lIv contained a GIn or Ser residue and are

have
pe)Jt1<leS from casein than

cel) wall-associated has reviewed
Tlliennophl!hc Lactobacillus spp. used as starters also possess a cell
ref.
role of lactococcal be the
and intermediate-sized pel)ti(les nr,.".rU'I"<l.r{
A number of authors have imrestlgate~<1
pn)temclse:s on such
does not appear to
Cheddar as detected
.,
'" ""'" """ "
"'" "
"" " "
"
",,"
" "".,., ••""''''
1111
• • •.,.
1$)
"''''
.,.,
"193 (I (I , v t.
.
'191 (I (I P V I. G I' 'I If G I'
•
r P I I
"l02
v~.)9
."
14/
1"1
18)
1'1
1'1 v \I (I II
.."
." .. "...
".
."."
G II II L V I II " T I. v I R G ,. ,. T T P II II»
1111
1'1
111I1'1'£l .. TV
" VT TA
10
substrate are rendered inaccessible due to hY4jrCtDhobic interactions of tbe C-tenninal

of the moleCllUe.
and
Lactococcal Dei)tldtasc~s surnm.an:z.eo In

Table 1; of the relevant literature include refs
Lactococcal amtIn()pe:ptloaises
accumulate to become
either inactive in cbeese or is
the His and
Protei nases
nrr'\tp,nl\,fC!'<O in cheese varieties where such adlluncts
144} have used lactobacilli

"""r,t"",,I,,'''''''' is the same non-starter lactOlJtaCllIl,
above. For references on the enzymes of traditional adlun1cts, ie.,
"'1:1
Table 1. Peptidases of Lactococcus and Lactobacillus ~
~
e
~
Peptidase Strain Substrate MWIkDa Opt. Adivity Subunits Class Reference ~~
S·
pH "c (')
;::-
~
~
to.)
~
$::I
Endopeptidoses ;;:
S·
~
~
peptides metailo tOO -e'
~
::::
peptides 2 metaiio 101 S·
~
peptides neutral
42
Atninopeptidoses
35
,
7 metalio
metalio
3 metalio
Glu-p-NA metalio l<E
2
2 serine no
serine III
lt2
pep' 113
PCP :n
14
lactoeoccal oli~n<k)peptjdase; 2. MEP, metallool'ldopeptidase.

XAP, X·prulyi.dipeptidyamiflopcplid.'tsc.
N
Table 1. Cont'd.
Peptidase Strain Substrate MWIkDa opt. Activity Subunits Class Reference
pH "c
Amilwpeplidtues
zeillus
II Lb. delbrueckii s.'Op. 11&3 78·91 6.2-7.2 475 meta110 11.5
A!>.{PIII Lb. at:idoplii/u... R-26 38 melallo 116
mecallo 117
melallo 118
A!\{PVI 814 Lys-p-NA 95 7 50 melallo H9
AMPvn Lys-p-NA 92 37 melallo 120
AMPVIU 87 39 melallo 121
A~{PIX ACA-OC2.U 98 6 .ro I melallo 122
x.~61 165 7 .:'i>-55 2 serine 123
XAPIV X-Pro-p"NA 72 .ro serine 124
XAPV 82 serine 125
XAP 170-2(X) 6.5 45 2 serine 126
XAPvn 170-200 45 2 serine 126 ttl
bulRaricus L8l;-1.J7 ~.
XAPVIH 50 3 serine 127
~
5.J tbiol 128
50
~
E.~
~
4. general amino peplidase. XAP, X-prolyl-dipeplirl}' aminopeptidase. ~
~
~
'd"
~
l"l
;:
"'1::1
~
a~
~
t;.
s·
Table 1. Cont'd.
~
Peptidase Strain Substrate MWIkDa opt. Activity Subunits CI85S Reference
~
i-
pH "c
~.
:.:tI
"S'
Di-ITripeptidases
=."'
J:
DU,s dipeptides 25 and 34 7 129
dipeptide!> 51
DIP II ssP. H61 dipeptides 100 8 mctallo
dipeptides 49 8 50 )32
tripeptides 75 7 mctallo 10-l
TRPU Wg2 tripeptides 1m-lOS 75 55 2 mctailo
TRPm A~12 tripeptides 10.') 8.6 2 mctallo 134
X-Prodipeptidcs 43 65·7.5 mctallo 135
PRO X-Pro dipcptidcs 42 7.35-9.0 - mctaUo 136
PIpit SSP. f'ro..X-(Y) peptides 100 85, 37 2 mctallo 98
DIP I\, IJJ. delbrueckii ssP. btd$laricus B 14 dipeptides 51 50 mctallo
8. dipeptidase. TRP, trippetidasc. 10. proIidasc. 11. PIP, pttlline aminopeptidase.
w
14 Biochemistry of Mille Products
PepO
PIP
ProtHiS- Phe
PepC
HisYPhe
DIP
t
Leu-Leu
ue.gm~tlonofanYI)OUleucalol1lwpePtlde the combined action of

refs. 91, 147 and which has not

been found in lactoc'OCC:l.
4. PROTEOLYSIS IN CHEDDAR CHEESE
Isolation and identification of individual is fundamental to the cornplete

un~(lel"sta.n"mg of in cheese. combined action of the and
leads to the formation of from
pollyp·epltld4es cjompaI'abl.e in size to the intact and
to amino acids and their of
in cheese is such that fractionation is necessary to
Various fractionation schemes have been Dr()OClse4ci.
tractllOfl(lllctn :>\;Ul<:;l:UI<:;. modified from
involves of the waler··SOIIUO.re
of Kuchroo and Fox. 154
VPlnf1l'1PQ in the water-insoluble fraction can be visualized
exc:nallge ctu'OITlat()gr:aplJty on DEAE-cellulose

Individual were isolated from cblronlatoglrap,hic
fractions of a 3 month-old Cheddar cheese made with Lc.lactis lactis
nnlvvinvllninp difluoride membranes and ,rI.,.nt1.t"u,.rI
Three with slow ele~ctr·opholretl.C wlnh,t .. t"\!
fl06-209 and
also
4S were
DJrOaUCl of tbe action of
nrflrn<:>,."
evident. The
tOflmatlon of 3 further
('nlrrp'~nl'lnct to
not
plaSmltn or cell wall-associated
is not in cheese
cheese made
Unli>utII1SJne<lI), s11g~:estmg that this is
enzyme for the formation of one
in the water-insoluble fraction of Cheddar remains
These results confirm the that the of
caseins in Cheddar cheese occurs that
the microflora of the cheese contributes at this level
The water-soluble fraction is first fractionated
kDa membranes. lSI The DF retentate contains
which have been nl'llrh~,lIv pUr111f~"
The sI0'Ner-mI2rallm2 peJl~tl"C~S
16 Biochemistry of Milk
These peJlftidc~s the action of microbial eml'.Vn1eS

e.g.
cell
nrl:nn~t,.,ti at
Water: 2: 1
Homogenize ( Stom3(:her or similar apparatus )
Centrifuge ( 10,000 9 x 30 min)
WiSN WSN Fat
UF, , 0 kDa membranes

Re-extract as above
WISN
Urea-PAGE
l
Permeate
Sephad.. G-25
I, H, lilt IV, V, VI, VII, VIII, IX
~ ~ JI A'-Acids I
Sep-Pak C8 or C18
and HPlC
Fractionation scheme for cheese mtlro~en.
57-1 and
of Lactococcus
wa:lH:lSSOCllatt:~d. p:rotlein~ase
f58- 72 was also isolated from water-soluble fraction
and found to inhibit intracellular lactococcal
cbl·orrlat()~r.;lpbIY on
eN WISF
4% pH
fraction of 3 month-old Cheddar cheese
made with Lactococcus lacti.') cremoris SK 11 the of the
caseins and the N-terminal of the orincloal oetlttld4es i~lenltifi,ed. (* Undetermined
4% pH
a retentate a water-soluble extract water soluble
extracts from Cheddar 2 to and of the corTesporldit1l~ (lanes 7 to 11)
0.4
0.6
,
/
0.5 " " 0.3
" "
0.4 " 0.2
.........
0 1:
CO
N
<
0.1
-
'-"
u
a
Z
0.0
50 100 150
Tube No.
Cnrol1rlat~()gramof the OF retentate from a water-soluble extract of

cellulose a linear NaCl 0 to 0.5 M. in 50 mM
3
v
VI
o 50 100 150 200

Tube No.
Gel in a 10 kDa ultrafiltration

~~~~of a water-soluble extract cheese. Freeze-dried extract
was dissolved in water and to a column x2 of G-25 which
was eluted with water rate 0.7 ml ); eluate was collected in 3.5 ml fractions
and the absorbance at nm determined Fractions were as for
further ::tn::t]v~il!'l.
and RP-HPLC
peJ1ltJ(lc~s g~eneralJlV C~l)rr~esponcled to

fI-?
an
G25-V
G25-1
GU-V,
GU-V"
G25-111
G25-1V
TIme (mll'l) Time (min)
HPLC of 10 kDa ultrafiltration pelmelate of a

and fractions thereof ob'talliled
SeDhad,ex G*25
3 a.
I I I I I I I I
0 10 20 30 40 50 80 70
Time (min)
b.
3 4
Peak No.1,
22 Biochemistry 0/ Milk Products
5. DEATH AND LYSIS OF LACTOCOCCUS IN CHEDDAR CHEESE
In Cheddar and similar cheese varieties, the starter attains maximum numbers at the end
of the The cells then die at a rate on the strain of
h,,,,,,,,,,,,II,, to of maximum numbers after 3 months}. The rate of
cells with strain. The best 'ni,r.r....""tin",
indicates that the external em:VlTle
is attached to the cell wall it has access to extracellular
Drcltelns. The endo- and to be intracellular some of them
may be located toward the of the 138 Since can
be into the bacterial celi and since most of from
<xsr or ~ -casein the cell wall-associated
lactococcal endo- or must have
to 90% of the 01l.g()(:m<10pc~ptll<1a:!)e
cyltoplasmi c. Therefore, further work on
i
its amino acid to be warranted.

If the intracellular lactococcal are to contribute to cheese np,emn2.
the cells must or become to molecules and the pel)tlcleases
must be stable the cheese environment. differences in the rate of cell
to be considerable, 156, 157 Since intracellular lactococcal pel)b(las~:!s
pnlmalnly reSl[)OnlSlble for the final stages of .....",t...",,,,,,,,,,
De):>tl(:les and free amino acids
ad'vaIlta2e()US in cheese npemlng
the ~.~ .... +.,~~.~~~
Proteolysis in Cheese during 23
2 3
2.C
CD
U
C
tD
fc 1.0
(I)
.c
C
~"'-'--214 nm
0.0 ~-----------------280 nm
o 20 40 60 80 100 120
Fraction No.
on G25 of Fraction II obtained from

permeate of a water-soluble extract of Cheddar cheese
Another feature of most of the pelptu1es isolated and characterized

both water-soluble and that their N-terminal seQiUeJilCe
commences at an established or lactcoccal wall
prc,telillase. This suggest that are not very
active in cheese.
6. SIGNIFICANCE OF NON-STARTER LACTIC ACID BACTERIA

IN CHEDDAR CHEESE
As discussed the starter reach maximum numbers in Cheddar at

and then die off. In contrast, numbers of NSLAB are very low
in cheese made in modem factories from a
these
UFP G25-11-1
UFP G25-U-2
UFP G25 -11-3
I
Time (min)
HPLC of fractions obtained

....,,·"'.. ,.·... n
located emwnrles
The cell of has not
detail. However, studies on Cheddar cheese made from raw or 'Pa~;te'lm~ed
indicate that the NSLAB in raw milk make a ............. ,''''' ....,'''' q:ual.ltative
qmmtl tatlve contribution to and
1---'"
_ _ _- 0
a.1*CASEIN
_ _ _ _ 13
_ _ _9 25-*
,. Sj'
1 _ _ _* IOZ-- •
lOS "-_ __
!
199
!:l:l
-S'
~
~'
7 ________________________,
p..CASEIN
IO'--_ _~
....--.-....,
1- _________________________ •
Pef1ttfd~~~
olriglnatmg from and p-casem isolated from Cheddar cheese etat. 142 and
Mc:S",'eel1ev et Water soluble water insoluble ---.. ------. tv
t.Il
26
to a lesser extent,
definitive studies on this
controlled microflora have eX(~IU(lea ref.
cheeses made under conditions appears warranted.
7. CONCLUSIONS
or<H!reSS has been made on the SPC~CitlCllty

and several of the small in '-u.'......u ....
The water-insoluble pelJtlcles are nrClIt1114l't":t1
ch"rm(,H~m on and the cell wall
<1trL"'\nohl h'vdrlDotlobic and

the DF
active cheese.
starter strains
lactococcal
iactococcal prC)telnaz;e
should also
8. ACKNOWLEDGEMENTS
The financial of is n1"':lltAt'IUU <>f"II"l1£\ull,.,.litt",,1i The

authors also to thank Ms Anne Cahalane for assistance in this
9. REFERENCES
L
2.
3.
4.
5.
6.
t'roteolysis in during Ripening 27
7. F.M.W. Visser, ::...==-==-::=::...z....=..:.'

8. F.M.W.
9. F.M.W.
10. F.M.W.
11. F.M.W. Visser
247.
12. B. Y. Sorokin, A. t'lckerme, and AJ. 36,
65.
13. J.e. lJe~)ma,zea.uC1, D. Le Bars and J.L. ___ ,.,,_._, 1975,
14. D. Le Bars and J.L.
15. 151.
16.
17.
18.
19.
20.
21.
701.
22. L.B. Larsen, A.
P.L.H. M(,""UJf~pnf'V
24. A. NO()men, :...:=~::.::.::.;:..;=~:.::.'
26. Intern
28.
29.
30.
1.
33.
34.
35.
36.
38.
39.
40.
1993.
45. P.L.H. S. P.E Fox and A. ::..:...==~=,1994,
46.
47. 680.
48.
49. 1986.
SO.
51.
52. Nallonal UmV1ersrtv of Ireland,
53. P.A. Lowe and EA. O. Marston, ::.::.....::::;:..::~~::::.:. 1985,
54.
55.
56.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
13579.
P. Vos, M. R. Siezen, G. Simons and
W.M.de
SO. J.
81. EA.
82. V. Monne!, D. Le Bars and J.-c.

127.
83. and M. Teuber, !iRJ!&Jllif!Qj2!Qh
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
1253.
97. 135.
98.
99.
100. l\.almm,oJ!~lwa and K.
101. KalrmnloJ!~lwa and K. Yamauchi. ~U:..!:!1lQ£!l!!m1.,
102. Ue:smcize,aud and C. Zevaco. &!lli!!:.m2h.Li!lill1J1!Q£J]Im~!QJ2~b
103.
104. MJ. Desmazeaud and C. Zevaco, M!l£!!~§sw~Y!ll

105. A. W. Bockelmann and M. ~Rh..M!£!QID.QU~~ll!Q.b
79.
106. P.S.T. Tan and W.N. n.VIUU;;:;;:'. t\ru?!:.J;a~Q!1..~~!lli!J:.:.,
107. EA. Exterkate and "" ..1 .• " , ••"."-
577.
108. 1991, 1207.
109. A. Geis and M. Teuber, tlIllllJM!f!Q!l~
75.
110.
111.
112. 1991, 49.

113.
30 of Milk Products
114. Phan Thanh and J.-C.
115. 876.
116.
I 1717.
118.
119.
120.
27.
121. G. Arora and RH. Lee, ::.:....:;:=~=.
122. E. Taskalidou. I. val'vLl\J'",
1993. 2145.
123.
124.
125.
126.
127.
128.
129.
Kalmm.og'lwa and K. am:auc.hl :....::~:::.:.....===-=== 1981
134. Jel1,l1ullgs, I. Ni Fhaolain and O'Cuinn,
135. and K. Y amauchi, ~=~~

136. M. Fhaohlin and G.
245.
137. Y. Wohlrab and W. J::S04ckellmallln. ===~=
138. P.S.T. M.P. Kostseau,
J.-c. and
140. P.S.T. T.AJ.M. van Kessel,
A.P. Bruins and W.N ......VJUU~;.,. 8~~nY!mn~l!g~Qh,
141. EA. Exterkate and A.C.
142. T .K. P.E P.
143. M.C. D.A. Krause
67.
I::SrclOme. D.A. Krause and M.W. 1991,
145. V. Monnet, G. Lambert and MJ. Desmazeaud, in 'Food

tnJr.vtTlolc)gv', P.F. Fox Elsevier Science London.
131.
146.
148. Ue!;malZealud, ;:;..:..:;;::=.~= 1978,
149. C.N. Kuchroo and P.E Fox, M!J!f!l1~~~~, 389.

Ripening
150.
151.
152.
153.
154.
155.
156.
157.
15K
159.
Manipulation of Proteolysis in Lactococcus Lactis
J. 1-1 ('l,a.."rl",,'f Ir~"'nan
Jan Kok,
DEPARTMENT OF UNIVERSITY OF '-JL"-"'''''A.'''''JLJJ. KERKLAAN

NN THE NETHERLANDS
A.A.I"">.L"LJj,'h
INTRODUCTION
Lactococci are fastidious For are on the

pre:sen~ce of small and free amino acids in the culture medium. Either because
the absence various functional genes or ref.!:ul(iltOl'Y
lactococci have amino acid . For in chf~mlca1jly
defined the various L.lactis strains the addition of 4 to 15 different amino
either in free form or as of The concentration of free
amino acids and in milk is of up to 25 % of the
and lactic acid
fenmeJltatlOns, Qjepe:nQS on their
de~~racle milk of the
oelJlUQje- carriers. These various of the

of intensive biochemical and
a set of otherwise lactococcal strains
is or deficient for one or more
These strains will be derived from Lactococcus
lactis means of a that allows the exclusion
of hetierolog,ous DNA and antibiotic resistance markers from a modified
of the of these strains may elucidate the role of prcltelnru;.es,
pel)tldlas(~s and in casein utilisation as well as their role
fermentations.
2 THE PROTEOLYTIC SYSTEM
As outlined lactococci on the efficient of casein to meet their

need for essential amino flrOWlflfl in milk. it is unclear which enzymes
and/or are indeed involved in this all and
PeJ>tlClOI~mc enzymes as well as the various and amino acid· trWIlStllOrt ..;:v..;:tp,nl~
are to be of the lactococcal
sch,em,lticluly deJ:'lct(~d in L
Manipulation LaC'tOCiOCCJIJ.S wCfis 33
CASEIN
~ .....-AA CARRIERS
- ~~ •• PROTEINASES
PEPTlDASES
PEPTlDES
AMINO ACIDS
Schematic representatl(m of Lactococcus lactis.

Extracellular casein de~;radatl(m protemlase PrtP is followed the
of amino acids and peTltt1dc~s sutlseCIUell1t intracellular cJe(lVaj~e
may result in the release
pel)tldlast~S into the culture medium.
acids.
34 Products
that some of the are not essential for org:anj,sm in

the enzymes may very well contribute to flavour <1e'vel~Dprnellt
lactococci is the extracellular de~p-aciat:ion of casein

for its on the
presence of the this 200-kDa the
lactococcal ....l'r.t...l'1,hrttl"' enzyme whose extracellular location is certain. for an 93-
Kda intracellular isolated Muset et al. 1I from L.lactis PrtP is
the lactococcal enzyme casein. All other ....l''',t'''''.hrtu'
isolated from lactococci have an almost neg~i1g1tl1e
rebitiv'elv small oeDtidc::s.
the action of PrtP are taken up the

lactococcal cell.
~et1!arate tr'an~~Dort 5l"ste:ms for amino
have been des;crilt>ed
8 amino acid residues are internalized an ATP-driven
which was shown to be essential for
triJ:)ep1tidc;~s are taken up a motive carrier
is essential for L.lactis ML3 to grow on
observation indicates when grown on at least one essential amino acid has
to be taken up this strain in the form of a di- or the fact
that as no conclusive evidence for an extracellular enzyme other than the
proteil1lase PrtP has been it is conceivable that di- and/or trit,epltidf~s gcmelrated
PrtP from casein nl'n'uiri.",,, for the amino acids reCI'Ulre:<1 PVlt1Pl1('.P! for
the of di- and/or PrtP is still l"'l'lrirUT J
An ever is isolated from Lactococcus

of course, exist between the enzymes from different
strains all lactococci seem to the same of oelptuiasles
On the basis of their these enzymes can be divided into three groups:
prclteilrtaSeS i.e. enzymes that are
and Until now, no carbO);:ypeptlldaf,e
lac1tocIOCC:f'. As described two distinct have been isolated from L.lactis:
PrtP and a 93-kDa intracellular enzyme identified in L.lactis . The other
lactococcal Two distinct of
and The main difference
is 70 kDa
of
and
are present in lac1toclOCC:1.
differ~ent lactococcal amjn~)emidru)e was Durified
L.lactis IMN-C12: active towards
tnt)eptlctc~s as well as some
h'lTcll'nJ""""rI"", The dlpeptlctruse DIP and the trilleptld.ase
enzymes of L.lactis. ~
t:l
::::
'S.
liO:
i:r
Mw (kDa) Class Substrate Leader Reference 5'
::::
Proteinases
PrtP 200 serine casein yes 3.38.43

NisP 54 serine yes 45
Neutral Droteinase 93 metallo B-casein 11
LEPI 98 metallo
70 metallo no
95 metallo LeUlLys-pNA no
50 thiol no
43 metallo no
45 metallo 28
90 serine no
26 serine no
49 metallo Leu-Leu 23
52 metallo no 24
53 23 metallo 25
43 metallo X-Pro
50 metallo Pro-X-(Y) 27 w
VI
can be considered as sut.str;ate,·s12:e~I'eC()gnlZlI:lg

diDeotides and tripepti!des. reS1Jec1tively"'~!'"''
Olll~OtJePltlCle:s. alth01Utm less as to
of this enzyme is towards N-terminal Glu- and ASlJ-rc:Sl(1ues:"'V
PCP is a enzyme that removes N-
terminal f r o m . Because of the content of
in casein (11.7 % in a-casein and 16.7 % in of deg:radlmg
are to be of utmost for the de.~ratc1ation
bacteria. In N-terminal Pro-residues can be removed
the action of the lmmo . The enzyme
does not on the presence of an N-terminal Pro-residue for its and may
therefore be as a more The from
L.cremoris . An which
cleaves off N-terminal
r~t",~nt1lv pluriiied from L.lactis
appear as the result of the action of
action of the is
pellUl1timate residue is a
pr(Jtllaase which is
in the second
The location of the enzymes has been a matter of

time. Several to be extracellular
Ho'we'v-er. a lactococcal extracellular Le. an
enzyme translocated across the has not been On the
evidence below indicates that all examined so far
are intracellular enzymes. This may also hold true for the isolated cell wall
antibodies
fractionation and 11Dlmuno~~olc1-1~lre.lltnig
location
Baankreis
of end,ope:pti(lase:s"
altllougb. in conflict with cell fractionation
may be located in the cell en\i'elooe'
which indicated that a leader is absent in
Absence of extracellular was indicated
al. : PrtP-deficient L.lactis can not utilise LelLl-CjOntainmg olll!l;Ot)ep'tldc~s
8 amino acids as their sole source in the absence of an functional ol1j~op!eptlde
In these the L.lactis cells were still di-
and extracellular activities were so low that
gerleralted to sustain of the . In an
in PrtP-deficient lactococcal
All
Manipulation of Proteolysis in La£~tOCiOCClUS 37
with the model of the lactococcal system as defl~ict(~

of casein is the prc,telnw>e
hvcirolvsis p:ro<.1[UCc~S sufficient tri- and to sustain
cell may result in the release of peJ)tlc:lles,
Pet)ti(jlas(!s into the culture medium.
3 THE GENES
The first prcltecllytic enzyme of LJactis to be characterised both blOich€~mlca1ly and

was the extracelular cell . PrtP is
pre-pro-fltrotemase which for its matunltlOn, and
delJ,enC1S on the presence of the extracellular molecular cmlJ)eJrOn
the genes the PrtM and
reside on a genes are
l~-eleJtl1eJlts, thus a . The mature active
prc,tel:nase, devoid of its sequence remains associated to the cell
means of a membrane anchor in the extreme C-terminus of the molecule.
encoded show considerable with the much
smaller serine secreted Bacillus This simliIaJrity especllulv
the the three aJtl1ino acids of subtilisin active
anaLlVSlS of the lactococcal and its as wen as the prcJrtelllasles
other lactic acid have been a second
extracellular lactococcal serine was described: NisP is
pre~-PJ~0-I:>rote1l1aSe; also the mature NisP remains associated to
the lactococcal cell The enzyme has a well defmed
function in nisin blosvnitlleslS. prcltecllytlc processing of the nisin precursor.
The gene (fn.o.....t1"ulno blOiSVJltll~~tlc operon, which is
.ru.~uv,,,~,, a of NisP to flavour de,{elllprneIlt
can not be NisP is not considered to be
i!-oleticielnt L.lactis as a host for the I.<lVU1U,Iti.

Nardi et
sequen.ces of the enzymes from both showed seven
assay similar to the one used for the
L-Jew;yl··Jj-lna~'htJlvljamide was used as a identified
from L.cremoris . An E.coli
gene from L.lactis ... ,..,.. ,. .Jv,..t.
qUfmtilties when to L. cremoris

~ ~~
,-aliBn}/'I-lS-na,ph1:hyilamtide as a substrate. The ammopet)tld.ase

E. coli the cloned gene could be identified
mClnOCIonal antibodies directed towards the pUl·iti€~d eln'7Vme·~J
a from L.lactis MG 1363 was cloned for
coInpJ.ementatlC)fl in the E. coli mutant CM89. This strain is
the when the cloned
A gene from L.lactis MG 1363 the other was

cloned antibodies directed towards the amino
from L.cremoris Tan et al..
~r,thf'!hc ol1igorlUc.leo'tld~~s based on this N-terminal amino acid sequence in a PCR
synltheslsc~d that was used as a for the
. An identical was used for the
. For the of the lactococcal pcp gene, PCR
ftP11MPlrC were Synlthe:SlSi~ on the basis of the known nucleotide sequence
from B. subtiUs and . The PCR obtained these ftM1MPlrc in

a reaction with lactococcal DNA was used as a
of L.lactis
For the of the gene the lactococcal use was made of the
fact that the N-terminal amino acid sequence of the enzyme had been determined. A 53-
ol1J~onucleotide. synttheslsc~d on the basis of this N-terminal amino acid sequence, was
used to screen a of L,lactis MG 1363 in The gene
could be gene, which is of an operon with
unknown The deduced amino acid sequence of the
lactococcal enzyme shares 47.7 % identical residues with from :::i. tvpllimurium JJ
p p
Schematic repres1entati(.n
the various genes. The PQ!;;iti4)nS are

indicated.
The gene from L.cremoris P8-2-47 was isolated from

a chromosomal antibodies raised the Dur'ltie:d
en<lo]Jep1tlwlse". The amino acid sequence of the as deduced from the
nucleotide sequence, shows a remarkable sequence with puln:'I.1"U'ntil"
t<urtherm()re, nucleotide sequence anStlVSIS
is the last in the lactococcal 0PP
an operon a omam.i!-[.rmem del>endeIlt o.llg()pelpticle tr'aru;port
. The lactococcal opp genes were Of1i~lmIUY spClnUllIle()US 0PP mutant
Manipulation Prnu1f'Jl'vS'is in Lac:toC,OCCiUS Lactis 39
of L.lactis MG1614. This strain is not

of a functional gene, and
of the . The olill:opc:pti(ie tr;aJlS]Jort
that do carry
N-terminal amino acid sequences as deduced from the genes,
N-terminal amino acid sequences of the enzymes, revealed that neither
nor . The absence
sequence in aU of these intracellular
location of these enzymes.
4 THE GENETIC TOOLS
of lactic acid bacteria can be achieved in various ways.

An obvious way concerns the introduction into or the removal from the cells of a plasmJld
pr(.teina~;e gene. This has been on numerous occasions: lactic
or acqlUlrmg
of new genes, or ch~mgmg eXlstirtg ones,
of a set of and
mtc~gr;atlclD vectors.
On the basis of the Dro'aa··nOl,t-ran!:l!e lactococcal plasmlld

has been vectors for the lsollatl()D
.Ex]ploltatton of these prc,mCtter scr!eentng vectors resulted in the characterisation at the
nucleotide level of a number of strong from the lactococcal Chl,ODlosomle
One of these promoters, was used in the construction of the eXJ)re~.sioin
. This vector was to the eXI)re~)sicm
he1:ef()101~0'llS and genes, hen ellg'·Wllue lysoZJ{m~e, B'.lic~hel'Zito'rmis
N-$lm'J'IR~IP: (X,·!2;aJactosllaru;e from guar, and the B.subtilis neutral prcltetnas.e""'v" The latter
proauc:ts are secreted L.lactis.
de1veI4[me:d on the basis of the lactococcal nl!:t<:!nt'fi1

incetpalJ.le of repJtlcaltlon
mllLltiJ)lled in strains in which is inte:gralted

in the chromosome. If the mtcegr.atlc1n vector a lactococcal chromosomal
and is introduced in a strain it will via
recombination, If the chromosomal of DNA is internal to a gene. the mt<~grliti(l.n
40 Bio.chelnistiry of Milk Products
mutants in Lactococcus lactis.

of of Emf
details see
will result in of the chromosomal . In this way mutations can be

and genes can be in the lactococcal genome.
drawback of this method is the inevitable of a selection marker.
markers are now available to overcome this . As an alternative a
for recombination has been this method a nOJrl-n~Dl1.catlve
J)lasmllc:l is introduced into the chromosome recombination InvolvInQ:
cross-over events. If the contains a chromosomal gene with a mutation the
Manipulation Pro.teolvsis in /.Ac:tOC.OCCi!(S
double cross-over the mutation in the chromosome. The for

obtamm~ a mutation in a chromosomal gene. is outlined in
3. Plasmid carries two selection markers: a gene cOIltel[,rlIltg Plrvfl,rnfnvC':tn
and the E. coli lacZ gene under control of prOtmclter
cOIltainiIllg a chromosomal insert B in 3) with a mutation, is introduced into
the strain the can either via A or B I in
The transformants will stain blue on agar A second
recombination via the A or B will excise the intt;~~ra~ted plasmllCl re'nClCerll1l~
the cells lacZ and sensitive to and result in either the WllG-I'Vne
or in gene II in Southern or n1n:::lQQ,r:lVQ
can be used to discriminate between both The absence of antibiotic resistance
markers in the modified strain offers the of modified
the above described in rounds of mllLtatlons,
strains C':!:n''1M.ltrlO mtu!t]lple for genes can be
5 MANIPULATION OF PROTEOLYSIS BY L.LACTIS
PrtP is the enzyme in the .....".t.,.",lut·.l' gleneratt~S pceptldes and amino acids
from casein. Because of its tUIllctlion, several groups have to enhance the
eXJ)re~;S10tn of the and r1.1\UV'.l.l",U enhanced could indeed be
established in of the is still a matter of
to obtain a strain with enhanced
plasmlld with a copy number than that
on~maI PJ~otemase pl.asnud"'. The of L.cremoris SKI 1 was threefold
ovc~rplroCl.ucc~Cl in L.lactis MG 1363 upon a tenfold increase of the copy number of the
This OV€:rpJ:OCluctlon of which was shown to be strain CleJ>ender:lt,
and acidification rate in v'-"UUJ'J; of the
prclteinaSie-cleticient derivative of that strain also

the pr01temase, cOIlnpared to the ':Ullrf_T'U'nl'>
strain. Ho'we'/er.
i.e. an enhanced rate in
of Leenhouts et al. indicated that enllan,Cllllg Dlrot€~Ol"SlS
of the genes in L.lactis MG 1363 does not result in a
or~:amlsml'J7. L.lactis strains with 2-3 and 8-9 of the prt genes were obtained
Cwmpbell-t,rpe mu~grjltl(l,n into the chromosome L.lactis MG1363 of a plasm'ICl t"!U·"'l·tnt'T
the prt . Vander Vossen et al. the onJ~m,:tHy oPl)ositeJy
genes in tandem in an structure, under the control of the
. The of L.lactis MG 1363 C'::::l1"1rVlrIO' this operon on a
l'!:'Il'1nr1rlt'T both genes on a
r>.H.HV~LJ;.u its beneficial effect

disputablie, increased nm,tenlv<:;l<:; may still offer a
prodU(~tioln and/or fermented
proClucmg hetieroJlog40US protteillaSles may also offer an

gene, the neutral is
in L.lactis when under the control of a lactococcal promoter in the
· The enzyme, which is in;1f t!:llll.., s~vntJlles'lsed as a pre-pro-
is to the active and secreted L.lactis.
the B.subtilis alkaline is secreted L.lactis l"!:lrr'l.TITH7
the gene cloned in similar to the one
illustrated in 3, a food L.lactis strain has now constructed that
SUIJ.tlll:Sin. To this purpose, the B. subtilis under control
was cloned in vector the lactococcal
gene a deficient transformants in
which the subtilisin gene was inserted two
suttseclUeltlt cross-overs as described assay
cllrom,oge:mc substrate
(lsl- and K-Ic:aseinc J

" ..
bre:a.kc1m/VD, the lactococcal an extlremlely

pro,teil1asles from the L.cremoris strains and SKI I, which are rettres:en1tatlves
differ in 44 out of 1902 amino acid ..,:>",.riu"",,,'J
constructed on the basis of the cloned prc.teinru,e
from L.cremoris strains and SKI . In this way, two prCttelnru,e
identified accounted for the observed differences in One of these
contained 7 amino acid differences and to the subtilisin substrate
The other contained 8 amino acid differences and is located in the C-
terminal of the This is absent in the subtilisins.
lactococcal contain an additional involved in substrate btndtn.g.
way, various L.lactis strains were some of which .....".1'1."",:>1'1 prcfteiltlas1es
new different from those of the oarental
Bnlimmberg et al. demonstrated that
of the L. cremoris SK11 nrotelflase
sp~~citlci1ty and some of these prclteulas4es
aU~JnnJte()lytic dcegn:lda1:1on as to the wild-
COIltlpOnents of the lac1toClJCc,al nl'otenlvttc in the utilisation

of cru;ein as a source of essential amino acids is understood. As outlined
even the role of the extracellular pf()teinal;;e PrtP is not clear: is the
of from casein? Elucidation of the role
peJrudtalK)S and in casein is facilitated the
genes and the tools that allow the construction of well defined
L.lactis mutants.
et al. described the inactivation of the gene in the chromosome of
L.lactis . This mutant strain was obtained via a double cross-
over, of the gene resistance marker. Since
in milk of the mutant relative to the strain was not it was concluded
that is not essential for in that medium. incubation of the
perttajJtep1:ide me:terlk:e'ph~llin with cell free extracts obtained from lactococci and
Manipulation
0.1 .............~......................~'-'-'~............;
~-'--'-
o 10 20 30
time Ihn
.,
Effect of a mutation on of L.lactis MGI363 on chc::mlcalJly
defmed mec:IlUltn with casein as the sole source of essential amino acids .
• , MGI363 .... ,
MGI363
flavour
manufactured with a
To assess the role of the enclop!eptlda:se in casein de~~ra(1atlon,
made the chromosomal gene of L.lactis
an resistance. Growth of this mutant in milk or a
Chc:~mlCally defined mediUltl1 with casein was not affected the close
between the opp genes and that
products be taken
-detlcl.ent derivative of L.lactis MG 1363 was COI1Lstnlctc::d J.T
mutants described the mutant grew nr\1'ft'\~llIv

in milk. none of the is essential for
lactococci to grow in milk. These raise a nUltl1ber of mtcere:stil1tg QlleS~:lOn:s:
Are these enzymes involved in the utilisation of casein or do
different function in the Iactococcal cell ? Are non·essential essential for

flavour fermentations ? Can the function of individual pepltid,lSeS
be taken over with similar and/or ? To be able to
answer these que:sttcms, a set of lactococcal strains is pre!,ently constructed in our
t"~1"1r'Vl1'UT mlultiiple mutations in chromosomal pe}:)tldase genes.
The of the tools for the food construction of lactococcal strains
with altered levels of of enzymes, now opens the to
t"fl1-°It"~llu examine the of the fermentations.
REFERENCES
1. A. '-'Aol\.J'.,Jln. ~~..MI9l1t!W1~~
2.
3.
4.
5. and T. '-'v""'.. v"'...... .t~~~nm!2b~!::"
6. K.1. Leenhouts and G. aPtlrOalcn'
3, p. 65.
,"-,. .<..I,U .."",.
7. 1. K.1. Leenttouts. and G.
8. n..UIIUll:!,», and G.
9.
10.
11.
12. 292.
13.
14.
15.
16. and A.1.
17. P.S.T.
18. T.-R.
259.
19. P.S.T.
20. F.A. de
21. L. Phan
22.
23.
24. HOSlnaD, P.S.T.

Manipulation of Proteolysis in Lac.tOCO,CCUS 45
2S. S. J. and T. :sortllaug, AmgnY!!:QlbM!gm!!JtQb

3016.
26. F.A. Exterkate and G.J.C.M. de
21. R. Baankreis and F.A. cxt(~rKalte, §:~md~!Mi&r!ltQ!Qb
28. V. 1. and J.-C.
29. B. W.
30. JenJl1i11!J~s, and G.
31.
32.
33.
34.
3S.
36.
31.
38. A.M. Le<1eI)Oc~r, and G.
39. and G. V",n.,.,-n!::l
40.
41. IJUUVU,,",. and W.M. de
42.
43.
IS.
44. A.Awade and J. Rotlert-UatldOllV
4S. J.R. Van der J. rUII"", ..
W.M.de
46. B.
41.
48.
49.
50.
51. Poolman. R.J.
52.
46
53. HaandnkmalD, P.S.T. C.J. Lee:nboiuts, J.

Submitted for put)ilcl:ttlon.
54. rynJlc.kYJlen, atnd M. ~uo]nme:n, Al?l2b~!r.Qn~~~
55.
56.
57. atnd
Enterococci', W3JShIl1lgton.
58. J.M.B.M. Vatn der
540.
59. J.M.B.M. der atnd O.
60. J. Kok atnd O.
61.
62.
63.
64.
65.
66.
67.
68. W. M. de
69.
70.
71. O.F.
72.
73. UnJ)UbJjsh(~d results.

74. atnd OJ.C.M. de
75.
76.
77.
78.
2051.
New Starter Cultures for Cheese Ripening
INSTITUTE OF FOOD RESEARCH, READING EARLEY

WHITEKNIGHTS UK
The central function of the culture lactic acid bacterta)

maturation of Cheddar cheese was established more than
1984). the secclndary Ulion-smrren microbj,al
cheese influence the final flavour to an extent Clependlent on their
and to grow in research on mechanisms of flavour
de,relorpm.ent has concentrated on and of starter bacteria
gerlenLlly and the genus The characteristics
starter cultures are the lactic acid their
resistance to and to mediate in the Cle\'eW,pmlent
of balanced in the stored cheese. These requirc~m~~nts
prcldulCeCI conflicts within the culture because can become mutually
exclusive. This paper will discuss this and which
have from the science base for
in any milk fermentation is the conversion of lactose in milk

the lactic starter culture These cultures are suppl1~~
.... rI .. .,h.. , either as undefined mixtures of many strains of the apI,roJ)riate
as pure defined strain cultures in the of

mixtures of a small number of the correct org.ani:sm.
Cheeses are made with Lactococcus lactis (subsp. cremoris or
aia,cetl~lac~tIS) where curd are below 400C
the thermophilic lactobacilli
oroduc:ine; COIlsis,tent, pure cultures of starter lactic acid

20 years with the introduction of
frozen or rr~~ze·-al'llea. cfu cultures which can
be added to the cheese vat. This tecltlnic~ue
rll1·"'....I I "
sutlerSieCI€~ the more traditional approa(:n lrIVOiIVlrU'

Fig.t. Schematic representation of the pathway for uptake and metabolism
of milk lactose to lactic acid in Lactococcus spp. used as cheese starter
cultures (from Law, 1982).
inoculation vat inoculation (re~fle'¥ed

<le\,relo'pment represc~nts a success for the tra~cJltJ,onlal
produc1tion of strains that to high cell
cel1ltnltug;al h~I""I.,.ctirla and has been
seIE:cti()fl te:chlllqtleS, and mass prOaUC1tlOn
which used non-milk media
automated fermentation control, J)aJ1icularly
nrf~vel'tjfm of over-acidification of starter cu]lur'es,
solid neutralizers or even gaseous had de'felc)Deo
teclnnclloil:Y in its own
J!w'''''JV"'-'''''' these successes there are stilI associated with the use of
lactic starter cultures which can be solved in the medium- to by
the of the 'new' a detailed of
cell metabolic The need for such
basic of starter arises from the demands
now on them in cheese factories. investment automated
chc;:esc:~m(llkirlg is now as are raw material costs, and
rn~'r01t\<: are low. To cope with the factories starter cultures
to lactic acid more than traditional cultures in less automated
units. For the time to and milk
'rennet to mill' has been reduced from > 5 h to about
factories. This allows the cheese vats to be used several times a
round the but it its own The most is
caused the existence of which attack the lactic
culture slow down acid At best this interferes with the
and and at worst leads to under-acidified
49
cheese of low (and value) which is also susrec~tib]le to invasion by

Most cheese in this latter has to or used as a
low-value in cheese.
InS1tltu1tes, or from the culture

prulge-·un:re12tted. strains for use
resilstall1t strains and the
rep.llcatlon is inhibited. The prospects
from molecular and cell
are lDCJreruilDg as lm()wled,!e nhf·nnltvn'lr. and determinants
of natural reSllstaJnce increases.
svstenls are
phtlLge·,reSJlstance pla!imic:ls have now been
J.,I~IL41U~;UC information on the
ph2lge·,res:lstance plru~mi(:ls has been derived from
n!:ltl1t"!:l,llv selected to in culture
anulClpiited. that defined traits will
prO(lU(~lDg ph2lge·'res:lstant variants which maintain other desirable
acid and flavour without
events. It is therefore to
understand the nature of the which starter cultures make to flavour
de,relclpmlent in in order to achieve this aim.
Much of the evidence derived from the industrial use of robust,

ph2lge··res.isumt. efficient staJrters now that do not
balanced flavours in hard and semi-hard cheeses that
as~JCU:lted. with 'slower' cultures which have not had to in the
of modern environments. There are many reasons for this
nhC:'P'I'\I!:ltli"n but it is assumed to be the of a combination of
(cc.mrner'cial) selection for strains grow
sele~ti(>n for strains which are not
or so that intracellular enzy~es (lD(~lu(jllDg
J>et)tloases) are not released into the cheese matrix. It is to uft(ler~.ta1l1d
altl1lOUjth not all of the mechanisms which starter cultures mediate
in flavour are of evidence which links
cheese taste and short and amino
acids from casein the extracellular staJrter
prCttellrtasles and intracellular starter Failure of the culture to
causes an accumulation of bitter release of short J>ellIUclc::s
having influences on cheese taste because only the
50 Bio,chelnistry of Milk
chymosin (coagulant) and extracellular proteinases are free to act on the casein
under such circumstances.
Evidence for the importance of families of in the

de"rel()~pment of taste intensity and savoury notes in cheese comes from a
of sources, not least from the similarity between the short hydrophilic cheese
peptides isolated by et al., (1993) and the strildngly 5alvOtlrv··tas1in2
oet~tidc~ in, for beef soup (Yamasaki & Maekawa,
more circumstantial thus, Cliffe et (1989) developed a
high-rl~so]lutic)n liquid chromatographic method to resolve low molecular weight
'Pet.tidl~ in ripening cheese and showed that accelerated by peptidase
mi:x:tur4~s was characterized by the sequential production and breakdown of
hydrophobic peptides, to in the most intensely flavoured
cheese, of which molecular weight ftl"t'kfil.",,,
up of 2-3 amino acid residues et
in concentrations that
tnaluce~rt) flavour intf'~n~11tv
2.0 100
Volume (ml)
F'aa.1. Reverse-phase chromatography of cheese-flavoured, water soluble gel

filtration G-25 fraction of high quality Cheddar cheese on a Pbarmacia Pep
RPC DR SIS column. Elution was with Solvent A (0 .. 1%, v/v. TFA in 8%0)
and Solvent B (0.1, v/v. TFA in methanol); (Cliffe et al., 1993).
The of the starter culture-derived enzymes, including those which

liberate 'Pet.tidces involved in cheese flavour is to
51
de,rel()pnlents in culture and accelerated

both at the strain and at the level of
For researchers in industrial laboratories have
method to screen commercial cultures from their vast
of their to low molecular weight N
ma1ten:aJS) in cheese and to savoury flavour notes (Law
selc::ctU12 lac-variants of these it was to show
the clean, acid flavour by the most
homofermentative cheese with a
As our increases of the enzymes and em~VlIle products that

are behind such we have the to characterize
biochc~mically and clone the more
tavouJrabJLv in the most effliClelllt, pltla2Ie-rc:~sisltant
enzymes such
such enzymes have
includulg an amlnOJ)4eJ)tlClase
et Tan
carried out on the enzymes of Lac. lactis Kaminogawa,
used cluster to demonstrate that cremoris and lactis
sub:s.pec~ies of Lac. lactis distinct groups on the basis of pel,tidase
It is therefore that there are the
characlters and of enzymes between which contribute to
their different Further studies of the enzymes of Lac.
lactis lactis are an is to be made.
ne~~lec:ted in pel)tidase
and slow
N-terminal
tnj:lep110e substrates
but desigmlted as an
residues
in aminopeptidase A
pUIjfif~ from mammalian sources Danielsen et al.
Tobe et al. Many of the serine residues in caseins are
et al., 1984) but it is that this enzyme may have

J)bc)sJ):hol'Ylated serine residues if the acidic side-chain causes
aspartate analogues. This may therefore be of oartiC\llar
bre.lkd()wn of peptides and also in releastrlg
glutamyl residue:s, Lactic cultures also
produce dipeptidyl peptidases which release from the N-terminal end
of oligopeptide substrates. Such enzymes could be involved in the release of
flavour-enhancing sequences, and attempts are already under way in a
number of European laboratories to clone them for overproduction in
commercial starters.
of starter cultures is intracellular,

cheese have included methods to
maltunlLtIOJIl.TecllmQues nmge from the use
to the of phage
bacterial host cell can involve cell wall
a enzyme known as lysin. 0ML3 is a
DrOrIatf~-ne3af~ phage that attacks Lac. lactis strains and the lysin gene from this
has been cloned in E. coli and its DNA sequence determined (Sbeannan
et The phage has now been in Lac. lactis
which were found to be unaffected growth but
re3ACllulg the phase in GMl7 In milk, the Lac lactis subsp.
cremoris strains that the gene were found to be less viable than
controls This system may be of potential in
accelel:atulg cheese ripening by early and increased cell
the peptidases to act on milk It is sug;gesited
that this autolytic could also be as a method of colltaininlg
gelleticallly manipulated lactis strains by routinely the gene
in a vector et 1989).
The cheese culture tnl1uC1tt"V and the science base are continuing to use
advances in cheese starter and molecular biology to
reconcile the for cultures and the
maintenance of taste in cheese. core research currently
involves the isolation and characterization of and
which can release amino acids and
J)e]:JtIdles from caseins. The elements coding for these em~VIl'les.
cofltr(J,mrI2 their production, are also with the aim
prot(1uc:mg new strains of starter and making available in
the enzymes gene cloning stnlLteg.ies.
overall aJ)J)roa'~1l is sunlm2lrisc~
Biotechnology of Peptides ~
:e
/;.;')
~
"'l
;:
"'l
o ~
isolate DNA controlled ..,~
ceflwall ~
prepare gene ....... c DNA -+ expression "'l
c: c:
o libraries
~
:;:
::tl
"6"
.2 .2
cac:
~.
19c:
<I.)
§ U) ~
.g 1
N-terminal
prepare enzyme
cocktails for
.
<I.) U) "-
u. (\1 u. sequence
E ----+
.2
I
OJ
purify and characterize

characterize ----+
enzymes related to flavour
Fig 3. Schematic representation of the core research activities designed to understand 1.11
v.>
and exploit enzymes of lactococci in starter cultures, and cheese ripening technology.
54 Bioc'hemistry of Milk
BibliOCfaphy
BENAJIBA, A. and MAROUX, S. (1980). Purification and characterization of

an A from intestinal brush-border memb'ranle. E:ur~'pe('ln
JoumlJ,l 107, .JU"~.Juu.
BOOTH, M., NI JEJlOONGS. P.V. and UINN, G.
Purification of a post-proline dipeptidyl amjlnOI)el)tida:se
Streptococcus cremorls AM2. JoumlJ,l of Dairy Research. 57, 89-99.
BOSMAN, B.W., P.S.T and KONINGS, W.N. Purification and
characterization of tripeptida:se from Streptococcus cremorls Applied and
Environmental Microbiology 56, 1839-1843.
CHAPOT-CHARTIER, M.-P., ROUSSAU, M., VASSAL,L. and
J.C., (1994). of two strains of Lactoccus lactis
rtpemmg. International Journal. 4, 251-269.
MARKS, J.D. and Isolation and
characl:en~;atl()n of non-volatile flavours from cheese: profile of flavour
fractions from Cheddar cheese. International Journal. 42, 1761-1765.
D., and LAW, B.A. A method for the reverse
peptides from Cheddar Cheese. Food 34, 147-160.
A.I., LAW, B.A. (1991).A time course of
proclucltlon in Cheddar cheese HPLC.
INGRAM, J. and
A.I. (1980). Proteins microvillar membrane.
Biochemical JoumIJ,1189, 591-603 .
........ ,,.,............ W.N.,
Science
F.A. and Purification and some

nrn,nerties. of a membrane-bound Amlinclpel)tidase A from cremorls.
nV/J'uc;;u and Environmental 53, 577-583.
KAMINOOAWA, T and K. (1983).
strepto~occ~i. Journal Science 67,
of multiple
mechanisms of phage defense from a nrnlrnfvlnP pllagc~-msen:sU1\re strain. Journal
Science. 72, 3429-3442.
T.R. bacteri,opllagc~-re~sis1:ant strains
...,.,........ , . ......I;" .. j,.,.U',U.TA.,&..t,..,.••
of lactic acid bacteria. Biochemical Transactions. 19, 675-681.

LAW, B.A., M. and M.E. The contribution
of starter stre~ptococci dc~velopnlent in Cheddar cheese. JoumIJ,l of
New Starter Cultures for Ripening 55
LAW, B.A., M.E. and B. (1974). The release of

intracellular from starter streptococci Cheddar cheese
nlX~ntng. Journal of Dairy Research. 41,
LAW, B.A., Cheeses. In Economic Vol. 7 Fermented
Foods. Ed.) pp147-198. Press ,LAJU'UVII.
LAW, B.A., (1984). Mechanisms of cheese ripening. In Advances in the

and Fermented Milk. and
B.A.Law, pp Applied Amsterdam.
LAW, B.A., (1990). The application of biotechnology for accelerated riJ)C~nil1l&:
of cheese. of the XXIII International Vol.2. pp
1616-1624. Mutual Ottowa.
R.J. and 0.0.
MONNET, V., PHAN THANH, L. and

ORIPON, J.-C. Purification and characterization of an amllnoJ)eplldase
from LactococcUS lactis. AM2. and Environmental
55. J~U'.J~h.J
O.W. Purification and characterization of A
from Laclococcus lactis lactis NCDO 712. General
MIC'rOIJlrotOJ~Y 137, 1207-1212.
UNDERWOOD, JURY, K. and OASSON, M. (1989).
v.n.'UUllb and DNA sequence of a LacIOCOCCUS gene.
Molecular General Genetics 214-221.
TAN, and KONINOS, W.N. (1990). Purification and characterization
of an from LacIOCOCCUS laclis cremoris and
Enll'irOlmtejrtlat Mtc'roIJlroloJI!V 56, 526-532.
AOYAOI, T. and H. (1980).
Amastatin and of
Biochemica et Acta 613, 459-
468.
.A"'-J'.L'.AJl',",,~. W.N. (1988). Purification and
Slrf~1JI(J'CO(:CUS cremoris and
with a delicious taste.

Engineering Pivotal Proteins for Lactococcal
Proteolysis
M.
DEPARTMENT OF BIOPHYSICAL """"...,.. '","LV POBOX BA

THE NETHERLANDS
1 INTRODUCTION
Recent years have seen a dramatic increase in the of tec.lm()IO~~les

that allow the of strains of Lactococcus lactis used in industrial milk
These have had a on the and
enJ~meermg of conapJ(~x metabolic conversions that are characteristic of lactococci used
as starter cullture:s. including lactose and of antimicrobial
peJ:.tid~es such as without is the of
en~:meerlIlg in the DrcltecllvtlC reactions that allow lactococci to
grow in and nroduc:e flavor precursors from
Three types of of the lactococcal system can be

distinl~ished that are all based on the of characterized genes involved in
cloned genes can be used to mutations that have been
obtained or can be modified to create
mutations that result in the elimination of functional enzymes. These no'wel'ful
aor,ro(lchles have been used to the of and amino acid traJOSDlort
systems and the multitude of present in lactococci. selected
enzymes in the system can be in hosts. This allows
not for the biochemical characteristics of those enzymes in more detail but
also for the effect on the of lactococci in milk and the of the
fermented milk the introduction of amino
selected enzymes followed their has become feasible. This
enj~meermg aotlro'!lCh allows for a detailed of structure-function rel~1t1onsl1l1PS
the construction of new enzymes with novel prc)J)ertie:s.
After a brief summary of the

this review will address the enj~mc:er]lDJ;?; enzymes in the
of lactoc1occ'}. elnve:lot:llf-iloca.ted pr()telna~;e that initiates
casein and the intracellular ammopeJ>tl<1"ase
In further for the of lactococcal nrt'.tpt'.lutll" enzymes and
their casein substrates will be discussed.
Em~ineert"!Jl Pivotal Proteins for La(:tococcal Plrntp,n/>.Jf'tfi: 57
2 ESSENTIAL FEATURES OF THE L.LACTIS PROTEOLYTIC SYSTEM
distinJ~i:shed in the cascade of reactions that allow

genlerate the amino acids to
scl1lemlatilcall!y dc~pi':ted in 1. Their intrinsic
DI·TRI AMINO ACIDS

CASEIN
PEPTIDES
PEPTIDES
~ Pep
AMINO ACIDS
repreSientlltic~n of the different

cel1l-en,veI4oDe located prolteinase~
AA: dedicated trarlSpctrt QVQtE'n'lQ for
expflanaticfn see text.
The frrst
in petlltldc~s and amino outside
the lactococcal cell pel,ticlas«:s released from
lactococcal cells also in this extracellular dei~rrulatJlon but definite
for the contribution of these has not yet been nrclvidled. Pre~serlt)v there is no
COIlvillcirl2 evidence the often-claimed presence of extracellular peI)t1d,ase:s,
as has rec.Jent!lv been . The located is essential for
in milk - strains that are deficient in this enzyme have lost the
t'l:It'1,llt'tllV to grow in milk without the addition of a second ceU-
was detected in some lactococcal strains that the
Howe'ver this serine is not to be involved
in caselllLoll'SlS because strains deficient in NisP are known to grow fast in milk. In
ad<llt1ICm, modelliing studies indicated that NisP has a very narrow substrate SpeClf1lClty
de~~rac1es its natural precursor
58 Products
pet'tidc~s
and amino acids are into the
for have been identified. The
1) that has the capiaCl1ty
of 4-8 residues is an essential of this tralflSPort machi.neJ':'j since its deletion
results in lactococci that can not grow in milk even when with DeltJtnles' Y
The di- and 1) is also for

of lactococcal strains in casein as sole nitro$l~en
~fll1!rr-p.4U. For the individual amino acids various carriers in 1) have been
identified but no mutants are available to determine their role in
J>et:ttid.es that have been into the cell are

a multitude of and intracellular
so far no have been found that are
indisperu;able for in milk as is described in a recent review on the of
at least 12 different lactococcal half of which have sequenced
It is evident that various steps in the of lactococci are essential

for in milk and hence the fast acidification of starter cultures. In adc:liUon,
the enzymes involved in the of the milk casein gelllerate
amino acids and that contribute to the flavor of the fermented
the in the last decade have been as to what
determines flavor and in what way do the lactococcal enzymes pal1icipa'te
process of flavor these can not be answered
cOInpletely but a number of have been carried out aimed at the
relative contribution of the various enzymes. These have been done with mutant
lactococcal strains that are deficient in a enzyme of the .....,...,.1'",..,,11;,1'.1"'
Because of the these have been carried out cheese and
flavor has been scored as a function of maturation time. In this way it has
been established that the located protellltaSe
COIltro,UiI12 the of cheese . In addition,
with strains that are deficient in the These
experunents have shown that inclrea~;ing in a
multiple strain starter increases the formation of bitter tipt,F'r-tc:":":> cOInp~ltible with
the cap!acilty of PepN to debitter try1pSnl-Clllge~>ted
Because of the essential role of PrtP in of lactococcal cells in milk and

the contribution of PrtP and in cheese we have mtc:!ns:iveJlv
studied these enzymes, their and their genes, and set out to these as
described below.
59
3 STRUCTURE AND MODELLING OF THE L.LACTIS SKII PROTEINASE
As as in 1976 the extracellular location of the in the

cell-wall of various L.laetis strains could be the detection of its
labelled casein as . Since many reports have aplJeared
crulIac:terizaltion of these 110-150 kDa serine pro!te1l1aSE~s"u.
An was the observation that the of

prolteiJtlSSleS from different strains differed . Different classifications of
prolteiltlaS4es have been that are based on COInp~U'llllg
the aeg;raaat1c)D casein fra~~mc~nts synthetic Sllbstratc~s""··"'''. Two main
of speclflcItl~es prCtteilnases that are
able to pr01temases that
The main casein sites of these two classes of have been

deternlin<~d and are covered in recent it is assumed that the
peiJJtldces glenerate:d from contain all the essential amino acids requrrt:d
in . There appears to be a of the
pr('teina~~e to cleave bonds at which residues are at the or
po~,ttl.Jns of the whilst the does not cleave such bonds but
has a for at those po~sitljJns
Studies aimed at the differences in cas:eiIltol~rtic spe~iticIty had to wait

until the structures of the two classes could be deduced from their
genes. These genes were identified and se(]IUelllCed from various strain
orotelIlase that resembles a and strain SKll that contains a
Here we will focus on the from the industrial starter

strain SKII that has the broadest cle~lVali!.e stlecitlcitv and does not generate as many
bitter from casein as the
Lactococcal strains that are able to grow in milk contain genes that are
located on . In the industrial strain L.laetis SKII the 78-kb proteirmse
has been identified and transfer studies and sut1seqlUe]tltly
mappe~'>""·'>o. Gene banks of 11 DNA in E. eoli were gelleralted
lambda vectors and screened for the of with antibodies raised
the studies in L.laetis a
10 kb was identified that contained the prt genes. The structure and function of this
was further sequence deletion and various
eXI)re~;sicln studies
60 Products
ISSl ..Nl prtM prtP ISSl ..N2
2. OrJ~aniization of the L.lactis SKII operon bar indicates a size of 1
The is characterized two divenlentlv transcribed genes, and

with oVf:rlal)plIlg plromLoters located in a with bidirectional . The
of the SKII to be the medium
Thc~refIDre, the of traJnsc:riPt:ion of both genes has been studied
and found to be controlled at the level a that is
pre:sel1Ltly the of further studies 41 •
The gene has a size of 5886 and encodes the cell en'~eh)ne.-)oc:atcoo
pf()teilnal)e PrtP. gene codes for a 33-kD maturation
r.t"'l".h,t.f' activation of the secreted
ft .. translation . This
maturation has been found in all other gene clusters identified so far and
aPI:Jeared to be a lip~Dprotein"" that has to a of
extracellular . The SKI 1 operon is flanked by two tandem
a wiclehr-di:strilout(~ elelrneIlt. and hence has a structure resembliJlg
The ava.Habllity seCluence allowed us to deduce the

nrilM!:U-V structure The has
1962 residues and starts with a residue:s. the
fuIllctic:mality of which has been shown in the secretion
Based on the N-terminal sequence of the active orc)teimu.e
SKI1 also contains a 154-residue and is hence orclduiced as a pre-
PRE PRO CATALYTIC DOMAIN SPACER MA
-187 1 SL x R 1775
•1
3. Structure of the SKll in which the different domains and the active
site residues are indicated. The 151-residue in the domain is cross-hatched.
The black and autlDPr,ocesSlrlg
sites are shown below or above the structure, (1 the
first residue of the mature and the main MA ret.re~;enlts
the membrane anchor. Further is in the text.
Engineering Pivotal Proteins for LaClfococcai Proteolysis 61
The N-terminal, 500 resitdue:s. of the mature includes the

domain that is
t"!lt!:l Ivt11" of the subtilisin now
This not includes the active site triad residues
and S433 but also the substrate and has allowed for modelllmg
cat~lIvtic domain of the SKII for kn{)wIledD'e-
The C-terminal end of the SKI1 contains a stol[)-U'aruifer

may act as a membrane anchor and secures the enzyme in the cell-elnVf~lot1e,)"
anchor is from the domain by a spacer of more than 1000
resldules, that shows no to with known function. Part of this spacer
may be involved in domain at the outside of the and
it is feasible that sequences the anchor may interact with the cell and
hence affect the fIXation of the In add,ition,
have shown that the spacer contains a that is involved in substrate and
affects 2; see 3 and
Based on the with members of the subtilase for which 3-D

structures were available subtilisins and a model of the catllvtic
domain of the SKll was . This model illustrates the
8tnlCtlllraily conserved core of subtilases that includes the active site triad residues
(D30, H94, and The also identified 10 inserts of 3-151 with
a total size of 238 additional residues not in the core structure of the subtilase
The extensions vr7 and vr9 are located in
or close to the substrate of the enzyme, as is the another extension
found to be at the surface of the domain and here denoted
see 3, 4, and The extension 238-
has a size of 151 residues and is to form a domain and
is indicated as such in 3.
loop 238-3118
4. Model of the cat:;alvtic domain of the L.lactis SKl1 proteilJlaSe details see
62
COD[lplc~x with
gel1leralted in
L. pre~;entced in 5.
Electrostatic between the chlllfge~ posItion 166 (N in SKll and
D in and 138 (K in SKI I and T in preterc~ncces of the
prclteiltlaSleS for ne,~ati.vel.v residues at the
This model can be used to the results of some of the
proteillta.se eng;ine,erntg eJtpelrnncents that are described below.
5. Model of the substrate in the catl~lvtic domain of the SKll

tra:gments. For further eXllflanaticfn see
text.
4. ENGINEERING THE SKII PROTEINASE
vJ.VlUU]!;, eJi(pr,ession and characterization of the SKIt
the a variety of and

eXI>erltme:nts have been to determine the sIDICtllre·-twoctlon
em:vme. which is the of the serine prclteiltlasles
h,..
f:". ......The ;>. used to achieve this vary from subclonin:g, o'Vel'leXI're~;si(Jfn
deletion and cassette mutaglcnesis,
enzymes. a summary will be
labora1:ory that have resulted in eng:ineeriIlg
specifl1city and the:rm()stability
Engineering Pivotal Proteins for Lac'toc.()cc.rzl Proteolysis 63
A crucial to assess the of the model of the SKII

pr()teilWte was the of the active site residues 4), This was
gen.eraltmg mutant SK11 S433A that to be devoid of
ca:l,eUllOl)'tlc ~l"tIVl1"1·". In this identified S433 as the site for
the inhibitors PMSF and DFP that bind serine residues and are known to
inhibit the lactococcal The active site mutant S433A to be useful in
elucid:atttag the and intermolecular of the SKII protemlase
.....''''''''''''...-. A similar active site mutant, has been created in the
vlf>.1ltHl1l0' e:sseJl1tutlly the same results and the nrc\nn~ct
In the course of the biochemical it appean~ct that the SKII and other
lactococcal to N- and C-terminal for the
cle~lVa,!e of the the
aut.op]l"OU~ol~(tic and may inactivate or not affect the nrrltpt'Ilvtlf'
autcJPrc)tec)lvtJic sites have been at the both ends of the SKll proteirtase
cle~lva'le in a hierarchial as deduced from the mk~nslty
and
Inactive forms of the SKI 1 obtained site-directed of

the active site or in strains without a functional gene, contained a N-
terminal extension. The N-terminal sequence of these could not be determined
Edman in contrast to the that starts with the sequence
. These results indicate that the site
position. In our current model of at this site
eluninatulg the N-terminal nn)-re:l1l(l~n
an intramolecular Recent eXlleriime~nts
teIlmlllalJ.y extended was incubated with sto:ichiolIletric
showed the removal of the
activation is an intermolecular
A well-known for the of the lactococcal is release

from the by incubation in a calcium-free It has been shown that
this is a result of intermolecular and involves C-terminal pr(]ICe~lsirlgl...;>l
In an indirect truncated obtained 3' deletions
proces~~ing site has been located between residues 1127 and 1272
pr(ICe~.sirl2 sites that lead to inactivation of the SKI 1 have pp-ro",nfh,

N -terminal site that leads to an inactive prc,teiJnasle.
at 205-219 its location
en~:meenrlg eJ(pe]~imc~nts aimed at <lelletUll2
aut:oplrot(~ol"sis site were unsuccessful since the surface
....""",,,),..0. C-terminal of an inactive de!pw(latlon ft1"rvin,,,'"
aut:OP]~ok~oh'sis site is located at bond 623-624
multiclDOV vectors the SKll and genes have been

cloned addition, constructions have been made in which the eXJ)re!;SlOIQ
of the gene was lowered the . The resultllIlg
were tested for the of active and
ove~rorlodulcti()D of could be realized. InD~ref>tinJtly
rate in milk aOl)1ean~
indicates that the casiein()lytic
for
deletlrlg the C-terminal membrane anchor gelleflitiIlg a series of 3'-

gene or a residue into the core
sequence, it was to secrete the SKll
In the case of deletions this also resulted truncated
orOtteilllasc~s with different size that retained . Since the secretion did
not affect the rate in milk it was concluded that the location per se of the
pr()teina~;e does not affect its action on . An those studies
was the to obtain amounts of the the
supemawtlt of L.lactis cells grown in
44 out of 1902 residules"''''

prolteiDiises differs cOllSidlerablyU-.l:1I
gel1lerated, inC4)rpl)ra1:in2 different segltnel1lts of the two
em:ym.es;);). A effect on the
sublstitutirtg 173 resdidues
established evidence for the presence of a spe~cifici1Y

1, that includes residues at both sides of the substrate .... fl,Ai..,....
cleft 4 and it was of interest to determine whether amino acid
substitutions would the It was predicted from the model
that the of the residues 138 and located at either side of the
binding would also affect the of the SKl1 proteilltase
eXJ1lerlltnelltally confirmed the mutant SKl1 prOtteilllaS4eS
the latter resllltulg in cba:ngulg the speicitlicity
to a
Since autoOlrott~ol',sis leads to inactivation of the protteiJtlaSle, the spe:cifici1ty and

C!h:aI"Hlii"Uare intil1llaltely coulOled. the of the engmec;~red
protteiltlasles could be altered and im[)ro'ved uellrec:s in mutant pro1teutlasces such
as the N166D and the K138DI A137G orot:eiruLse<+;
Engineering Pivotal Proteins for Lactococcal Proteolysis 65
+ + + + + +
R-P-K-H-P-I-K-H-Q-G-L-P-Q-E-V-L-N-E-N-L-L-R-F-
SK11
+t t
abed
+ +
N166D
t+ t
K748T
+ +
K138D
++ ~
~
4
+~
~
Wg2
6. of the
++ and The
The engllneering expc:rirn:ents prc'teiJnas~es also allowed to defme

2, the part of the protemtase
has no ealllVlilleJlt in subtilases and therefore can not be modelled,
It includes two residues that are in the SKI I but not in (R747L
and and contributes to the clellvaJ~e stM:cificitv towards
or small model substrates . In sUOlseqlUeJlt ellgiIleeJing eXI)Cdme~nts it
has been established that residue 748 contributes to the since
mutant K748T showed a new It is 2 folds
back to the active site and interactions with
the substrate. In since the mutant shows reduced an indirect
effect on the of the can not be excluded.
a(l()pte~ in an to the
anatlyz:ing the function of the surface SL
that contains an site A deletion mutant
which lacks the 14 residues of SL was constructed and used to introduce
various insertion cassettes for the with three mutations
mutant) or for neutral spacers of 1,4, 7, or 16 serine
appean~ that the presence of residues 205-219 is essential for orc'tecllvtic
since the mutant retained cas,emlol)rtlc
mutant was found to be defective in C-terminal autoOlrociessing
r1U,uv\.,~u an altered of site R can not be ruled out cOInoletejly
suggests that SL forms a third 3, that contnbliltes
The residues 238-388 in the SKI I constitute the insert in an

is to constitute an additional internal domain
A similar but less conserved domain is found in the S.D'VOj~enj,!s
enclop1eptiida!ie and the B.subtilis minor A mutant that lacked residues 238-288
was constructed and . This mutant strain showed a two-fold reduced
rate in milk mdlca1:mg domain is not essential for and its
removal does not inhibit .LV"'''U'\'ic" monoclonal antibodies that had been raised
the lactococcal we were able to show that the determinant
mAB-I, in the is at least located in the
"'''~._''~.~V~'~'. Since this is most to be located on the surface of the
I"'<lr·~ luf'11'" U'i.1UUIUl, it candidate for Ins(~rtU12 h~eteI'01(]lgOl1S aJlltl~~enlC
determinants. we are the L.lactis strains
expres~iinJl such mutated SKI I determinants
in new vaccination programmes.
4 ENGINEERING OF AMINOPEPTIDASE N
The N has been to the

JleIlenlti(]ln of antibodies and determination of the N-terminal sequel1lCe".;>,.;>O.J7
monomer with a size of 95 kD and shows a broad substmte spe:citlci1':y
deil:radlinJl several tri- and of the N-terminal amino acid
but has no and
are cleaved and these
nal)htJhyl.amlide derivatives have been used as chromoge:nic
purific:ation. and of
The structural gene has been identified in a lambda of L.lactis

antibodies the pUI'ifie:d . The nucleotide
sequence has been determined and its tr81lSClr1ption has been <ln~l hl~7...11""
A similar sized gene has been from L.lactis that differs in
nu(:lec,tides The
Q1
• gene shows a monocistronic org:anization
consensus prc,mc,ter sequence
pepN
7. Sequence OJt'garllza1tion of the gene.

Engineering Proteins for La('/oc.ocCc'.ll P}"ntp,(}/vl:is 67
The deduced nrilm!l1"V nOlnOJlog4JUS to Zn-
similarity to the
cornprlsU1lg residues 281-
321 shows bOl1nolcJgy Z,n--de1pel1ldeltlt neutral prc,teilflaSies
for which various 3-D structures are known. This that
residues H292 and E311 are the whilst E289 is involved in catalvsis.
The N-terminal sequence determined from the to that deduced
from the gene sequence and indicates that the a
In no transmembrane sequences were found in the deduced structure
which is with its intracellular location as revealed fractionation and
C1UUV'''E.ll caseins form a nutritiowtlly well-defined and valuable source,

their use can be extended with enzymes. in many
cases spe~ClrlC n~yc.troptloblC ~eptides are gerleraLted that cause bitter off-flavors. Because
of its broad substrate N has been for the capiacilty
to debitter a . The in such an hvcirolvsalte
before and N were isolated and sublseQluel1tly
identified acid seQ1uencinlg, amino acid corDp()Sltion analYsils.
and on line - mass In this way it could be
established that several oriJginatiIJlg from the bitter C-
terminal end of the D-c:ast:m. . In line
with this enz:ymatic is the observation the bitter score of the
bvcLrolvsate after treatment with N was less than before
treatment. this demonstrates the of it
is evident from the amounts of enzyme needed to obtain that cOlnmlercaally
viable processes a source of the enzyme that can be obtained by
In the first to gene it has been cloned in E. coli under

control of several inducible resulted in the slgmt:tcal[}t o'verprc,c.tUictU)fl
~ N~~be ~~ ~and~~~
antibodies. These antibodies did not show the to other lactococcal
prolteulS that were found with antibodies from L.lactis
and hence have been used in various localization eXl)erllIDt:m'bl;"'''
the gene has been cloned in L.lactis a copy
number vector. This in an of apI)rOJtimlately 25-fold in the
nOlnolloglJUS host. Since the geIlenltted strain contains a transferable antibiotic-resistance
gene located on the the NIZO lOO(l-fl!rd(le
marker based on cOIDplementaticln of the lacF gene was mconJOr;atec:1""""'''
reS\lltirlg pJlaslmc.t eXl)re!>sIl1Lg the gene has been obtained
contained DNA from L.lactis. A L.lactis strain harboI'ing
plasmllc.t has now been excluded from the and has pODential
to be c.tev'elonec.t for the of N.
repilacement recombination a mutant of L.lactis MG 1363 has been constructed

that contains a deletion in the chromosomal . This strain did not produc:e
aminopeJ)tidase N and was used to demonstrate that there are other broad host-
am.in(JIJ)e1pt1clasc:s in This may the observation that this
almost the same and acidification rate in milk as the
. The thus constructed strain could be used as a suitable host
in aimed at in order to further the
structure-function of aminorpel)tidlase
5. CONCLUDING REMARKS
In recent years the lactococcal cascade prc)tec.lytic processes has into

one of the best characterized systems involved metabolism. In this
paper we have reviewed the enR;ineieriIu~ enzymes in the
nr('~tI"('~Jvtll" C~lscaLae. the ceU,-en.vel~[)oe
that appear to be wlcleS})reaLCI Dr(~tec.lvtlc S'fste:ms of other lactic acid bac:teria as has
been 1"AI",,..ntll,, rf~vif~werl13 our in the structure-function
relations enzymes, these studies have resulted in or have laid the
basis for de"el(Jlpment of lactococcal starter strains with It is
eXI)ected that by these strains in industrial processes not novel or imllrO'ved
products may be de"el()t)od. but also our will be increased of the
contribution of the system to the final flavor and of fermented
DrC)dUl:;;ts. In this it is relevant to mention recent studies that
involve the substrate we have been able to
generate and novel when
incubated with a enzyme, such as the of
traos8:enlc cows mutant caseins will it illustrates an
alternative avenue that can be taken to imr,rO,Fe e]'tistin2 generate novel fermented
ACKNOWLEDGEMENTS
SUpiJ)Olted by EC contract BIOT-CT91-0263 in the framework of

-U£I01e1;['Eliotlechnoll[)gy of Lactic Acid Bacteria'.
Engineering Pivotal Proteins for Lactococcal Prot,(1olw~is 69
REFERENCES
L and B. Issue
L
2. W.M. de Vos and G. 'Genetics and of Lactic Acid
Bacteria', M.J. Gasson and W.M. de and
3. in the 21st , R.
American Chemical
4. B. Reiche and W.
5. KOC)lJe:n. S. van ~... tl~llt"(viiilr

7176.
6. ""V''''''J'''''''' and W .M. de
7. R.J. van and W.M. de
8. W.M. de Vos and E. in
9. R.I. Siezen and
10.
lL R.J. Siezen and W.M.

3555.
12. R.J. Siezen and W.M. de Vos 'Bacteriocins
of Lactic Acid Bacteria', L. de and J. Valldmmn.e,
and p.223.
13. 1.
14. W.M. de
'Genetics and Molecular and
Enterococci'. G.M. American
for Mic~rot)101iogy
15. 1. Kok and W.M. de 'Genetics and Hicltec.hn()lojn'
Bacteria'. M.J. Gasson and W.M. de
p.169.
16. W.E. Sandine and P.R. 1253.
17. J.R. Van der J. M.M. O.P .
........."'...,•., and W.M. de
18. 'Subtilisin Bds. Plenum
in press.
19. E. B. Poolman and G. VP1,\pn'l:I
20.
2L
22.
23. R. Harunkrels, ripc:nin,g' ,

AmlsteJ:-darn. The
24. T.A.J.M. van P,F.

ZUllll'endon.k, A.P. Bruins and W.N. 1"lI..UI11Ug,:s.
1430.
25.
26. T.D. Thomas and G.G. Prj1tcwlrd.
245.
27. S. Sla!l1gen, and G.J.C.M. de
28.
29.
30. 179.
31.
32.
33.
34. F.A. Exterkate and J. Staian(IUaC~rs. Neth. Milk
35.
36.
37. W.M. de
38. W.M. de
169.
39. W.M. de P. G. Simons and S.
3398.
40. P. HllJline:nD~~rg, P. Vos and W.M. de
41. HllJLmenDc:~rg. P. Lav'ernlan. R. van KralnerlDUJrg and W.M.
42. G. Simons and
43.
44. W.M. de Vos and

1890.
45.
46. F.A. Extf!rkalte. A.C. W.M. de Vos

and R.J. Siezen and stabilization of enz'vme:s' van den
A. Harder and R.M. Hul1lela:u
p. 231.
47. Bn;line:nbc:~rg.
P, 1. van Alen-l:SoefJn~er
F.A.Exterkate and W,M. de
48. Bn;line:nbc:~rg. W.M.de Vos and R.J. in
49. J. Kokand G.
50. O.E. Mills and T.D. Thomas

209.
51. H. Laan and W.N. "''''VluUfi,<>'
52. P. M. " .........."'.
and W.M. de
53. P. BruineIlber'g,
54. J. HUJ~enJloltz.
55. results.
55. M. .1'U".IU'''.
56. F.A. Exterkoate. W.M. de

Vos and R.J.
57. H. J.
L. J!iQ£ll£O.L..
58. P.S.T. Tan and W.N. ","VI.uUfi,>:I,
526.
59. R. Baankreis and W.M. de
2555.
60. P.S.T. W.M.de
61. 107.
62. 1989. 0 355 036
63. AleO-Hoenrtlltler and W.M. de
64. L"''''''''.''''h T. "'''':,U,"""U.
Protein Engineering and Preliminary X-Ray Analysis of
CHY155·165RHI Loop Exchange Mutant
J. ,P. , J.
T. L. Blundell l ,
DEPARTMENT OF
THE BIOTECHNICAL LABORATORY, SF-02 I, FINLAND
1 INTRODUCTION
the determination of the three-dimensional
structure the target in
this process. a
common fold can learnt from
the
of one
of the
have been
of
of bovine
I1W1ChelmS1Jry of Milk Products
2 PROTEIN ENGINEERING
CBDCOSIN II D R. It G Q B SilL
RBIZOPOSPBPSIN I G It A It It G G G G JI L
JDIDO'l'BIAPBPSllt II G Y B - A P G '1' L
MOCORPBPSllt D G G G Q L
are to
structural differences
Protein Engineering PrPl'imi,Ulrv X -Ray 75
was
by
The Eeo RI-Mse
mutation was cloned into the vector in
correct orientation. The DNA was then cleaved with Eeo
and combined with the Eeo RI
4) to the mutant
• The correct insert was
the M13 chain
CHYMOSIN
155
MET ASP ARG ASN GLY GLN --- --- GLO SER MET LEU
5' ATG GAC AGG AAT GGC CAG GAG AGC ATG CTC 3'
5' ATC GGC AAG GeT .lAG MC GGA 00'1' GGe GGA GAG CTC 3'
ILE GLY LYS ALA LYS ASN GLY GLY GLY GLY GLU LEU
155
RHIZOPUSPEPSIN
Hind III IP t
(feoR 1/ Pst/~-_-L-Jr~.. romopAMHl 04
(11.9 Kb)
feaR I
vector for
reesei
3' GACAAGAGCCAAATGTA
GCCGTTCCGATTCTTGCCTCCACCGCCTCT
CGAGTGCACCCCC 5'
61-mer used to introduce the
CHYMOSIN
WILD TYPE
Rut
the
the
of the
at the 101
as the sole
in the
Protein Engineering and Preliminary X-Ray o/CHY155-165RHl Loop Exchange Mutant 77
The milk
while the amount
.... ,u"" • ..:;gi;)..:;. The
acetamide. Transformants with

T. reesei gave
no growth).
MYCEUAL _ _ _ _ _
'" PROTOPlASTS
7
Life of Trichoderma reesel
1. 40% Ammonium Sulphate Precipitation
2. Ion Exchange Chromatography using Q Sepharose Fast Flow

4 1200 .....c
3
1000 ..:;
.1
D
800
.
....,
l\1l'i
C
CO
N
C
2 600 ..:e
C
0
Butter A: 10 rrH Tm.
BufferS: tOmMTm.
• ZMNaCI
400
200
..
.5
IlII
/I.
Co.)
A280
0
0 20 40 60 80 100
0 i! Milk Ootting Acthlity
:E
fraction No.(14ml)
3. Gel Filtration using S 1 00

.....
1.2
1.0
3000
..
.!
Jl
;S
....,
0.8 ~ 2000 I
! 0.6
l
l
Buffer. 100 mM ItIOAc, 150 mM Naa. pH 6.0
flow rate: 1.5 mllmin.
0.4
0.2
1000
.u•
:! III
0.0 l J \.J ~ o ;I
Al80
Milk Ootting Activity
o 50 100 150 f
fraction No.(10ml)
4. Affinity Chromatography using Affi-Prep<ll with V-dl-P-F-F-V-dl

2 3000 .....
...e§ Starting buffer. 30mM Nll-tolTfllilJte pH 4.0
e .15%0i0_
2000 :e
C i A : SampIll(SmI) _sloaded at 0.5 mllmin.
~ 'Z
B : 30 mM Na-fOOT\8te pH4.0 ... t3fi Oio_
C C c: 6.S,lmMEOTA
A B
c 1000 .r... Collected in Z vol. of 50 rrH ~phate
s
Co.)
pH 1 mM EOTA, 0.75 lot NaO.
+L~~~--~~~~a-~~"*-+O ;I A280
10 20 30 40 f Milk Ootting Acthlity
fraction No.(lml)
The four
ammonium
Protein Engineering and Preliminary X-Ray of CHYJ55-J65RHI Loop Excjfuln!~e Mutant
kDa 1 2 3 4 5 6 7 8 9 10
66.0
45.0
36.0
29.0
24.0
20.1
11 % SDS~PAGE the mutant stained with Coomassie

blue. Lanes 1 and 9: low molecular markers~ lanes 2 and 10: calf rh"YfTll"l<:tn
B standard; lane 3: lane 4: spent medium; lane 5: ammonium
sUilphate fractionation and lane 6: active material from Q Fast Flow
active material from the S 100 column; lane
Bio<chelnistry of Milk Products
after the
to obtain the
model
software
on
is
Protein f<."ntunp'prIl10 and Preliminary X-Ray o!CHYI55·165RHl Loop t:xc.fulnl~e Mutant 81
similar to that of
differences are
with the same sequence in the
the is identical in
mutant, this indicates a
side-chain environment on the
at residues 161 and
the mutant exhibits
in the of
molecule stabilises the
bonds with the
atoms of the residues
the native rnlzopuspepSln
a water molecule in the
The main-chain atoms

mutated ball and
the water molecule (W) which stabilises the
conformation of the mutant; the
structure
been
has
3 CONCLUSION
in the
the environment
Such studies will
~.\J~~'.U.
the evolution of on
structure and function.
References
1.
2. M.S. Johnson and T.L.
3. G. A.
N.. Andreeva,
4. R.R. Bott, E.A • s.

• H. Cohen and D.R.
877.
5.
6. J. Ott and F.
8764.
7. P.G.
8. and A.R. Coulson,

5463.
9. A.T. Jones, 1 268.
Peptidases from Lactococci and Secondary Proteolysis
of Milk Proteins
BIOTECHNOLOGY AND ENZYMOLOGY INSTITUTE OF FOOD

EARLEY WHITEKNIGHTS
ROAD, READING
1. INTRODUCTION
Lactic acid such as an essential role in the manufacture

of cultured such as cheese. for the
acidification the the in the such products, and
are also considered to be involved in the formation of the characteristic
flavour notes in cheeses The economic of cultured
prol(luc:ts. and manufacturers demands for starter cultures that will produce
COJ'lISlst:ent "'.."'.n ..."t in the modem has led
tn-(leoltn 1IlIve5.tlg:atlcm of these and
The lactococci used for cheese manufacture are nu1:riti.omlllv fastidious orJ!:anJ.sms.
requin:ng, amonJ!:st other sources of amino acids in order to
aCflle\re the rate of for the acidification reaUlr(~
in cheese manufacture. can be met by two routes:
1. by or small t>et.tidles in the

extracellular
2. if there are insufficient small nutrieJlts.

the media to amino acids and peJJ.tidc~s a traJlsPC)rtable
To achieve lactococci have de\i'elooed "'..""I'<)"'.lu1".", .... ,..."'"" .. _ COl1lSlS1ttn2 of a

mixture of enzymes, both nrl'ltpl'llvt,(, pet)tldOlvtlC. and several amino and
nrCltVfl1lmp' these essential nutrients to
Milk as a medium has a limited of free amino Kolstad and

Law reviewed data that milk cannot ......".. ''''16 all the free amino acids such as
Hisitidllne, .....,..,"'. . Ul ..... Glutamate and for and lactococci
prOltell}S to
3.0-
of which the caseins about 80 %. The four different types

(j and IC, and are organised into micelles to form
gerlentlly been that the caseins are the
lacC:OCCICCl for growth in milk.
As well as their in I:.l"-""I.IU. prolteolytic SYStelllS of starter cultures

have also been in the matunitlCln plrooessc~s where it is
thought that the lactococcal pr01teolytlc an essential
role in the formation of amino pc):ltidles irnootrtalrlt in cheese
..... '.......". either or as the precursors of flavour notes.
2. PRIMARY HYDROLYSIS OF MILK PROTEINS
The first in of milk oro,teulS lactococci is the action of the cell

wall associated protein,ase is an extracellular event and the
proteiniase has been well cllaraCl:en~ied. genetically and in the
decade and the of several reVle~/S essential role this
was demonstrated' shown by proteiniase
the to
nmlithre strains when grown on milk. This in
the media was with the essential free
proteInlase gene into the mutant
It has been that since an essential role in the rapid

growth of then the from the
action of the would source the amino nutrients
the lactococci after the use of the available free amino acids and
small Table 1 shows the forming casein-derived pc):ltidc;,s
protemlase in in vitro studies. These do the
reaum~ by it is difficult to determine
are made available. It was demonstrated that optimal growth
cremoris HP the presence of both (j-and IC-
casein in the media and it was further that IC-casein may
be a source of the essential amino acid histidine It is that
one of the formed IC ·casein is hlSllOUle
Peptidases from Lac'toCI'Jcci and Secondary Prntonl',,<,i~ of Milk Proteins 85
the lactococcal proteit1lase on
I1e-Val
IC-casein
IC-casein Thr-Val-Gln-Val-Thr-Ser-Thr-Ala-Val
References 1\
3. SECONDARY HYDROLYSIS
nrtlm!:»!", h'.lIirt"\h,,!lC! shows the that will go on to

n1Jtnents, an examination of
nitI'og~m
TeQuirc:d to determine how this is likely to be
It was in 1991 that the cell wall associated proteinlase

known at that time to be associated with lacltOC()cCl pO~~sessed
reauin:d to all the pOtentIal l1-calsem (8).
total hvdlrol'vsis lac1tOC()CCi in
and in seclL)ndary hvdlro)'llsis of
A
cornplementary range of ph'1'SlOJloglcal, aPt)rO,lChlCS are
em:nlo'ved to examine the roles J)eV'U<UlseS both on
flpc~nu1lg processes in cheese.
86
4. LACTOCOCCAL PEPTIDASES
a substantial advancement in our kn()wledll~e

occurred. Table 2 shows the current list
characterised from lactococci. A corresporiouig CC)mlpleJmelrlt
been found in lactobacilli. A common proble:m
is the have been carried out
associated lactococcal
DIJ>eJ)ltloa,ses and the TfllDepltloases.
these J)e)JttlO:::lSes
on the nutntlOmUlY lmT\nr1'~nt
The first lactococcal was the gene

The X-Pro encodes for was
considered to be one with the casein
h".irl'\h,~i~ due to the rich nature 17% of the amino
have demonstrated that Proline as a free acid does not
enter the cell and is more to be the dll'tnJ)eptioe
Once these cross the membrane an mtlracc:Uutlar
pro1lloclse. which can cleave most of the X-Pro dlJJ'epltlo~~s
lactococci An extracenular enzyme with the
cOlltalmnlg Oltpet)tHl,es was therefore which concert with the
prc.l1oase would and other amino acids to cell. The initial
PUlrltllcatllon of this enzyme gave a cell wall location of the enzyme a location
~1I11innl·tpt1 to some extent an immuno-histochemical All other
PUJ1tl(;aUOns of the enzyme, indicate a intracellular a
of the gene. No sequence was
membrane anchor cross the membrane.
information exists on role of this enzyme in the of lactococci. In one
a deletion mutant in Lactococcus lactis lactis NCD0763
able to grow in milk at 60% of rate of the wild whilst a seoond
stated a deletion mutant did not effect the of lactococci
It should be that none of the formed shown in

Table 1 have an X-Pro sequence at the N-terminal and therefore these peJ)'tlOC~S
would not be available as substrates for Other ex()pe]pti(lasc~s
n"""""n.II\1 an en(lOpept1lda~)e are reo'um:~ before these J)et~tl(ltes
can be SUD!stn:ltes
of Milk Proteins
isolated from Lactococci
Enzyme- Action Reference
X-OOO Leu 1l~14
X-OOO Phe 15-16
GAP* X-OOO Asp, Ser 17-19
PCP X-OOO 19
DIP* x-o 20-22
TRP* X-OO 23-25
ox-OO Pro 26-31
PRO o·x Pro 32-33
PIP X-OO Pro 34
... WX-YZ ... 35
LEPI ... WX-YZ ... 36
LEPII ... WX-YZ ... 37
-Abbreviation for ref

* Gene cloned and
enzyme
is not yet clear.
in milk (41).
ne(!~atl'/e mutant has
hex.ape:pti(Je SUDstrates but still
ttlflouj;!~h a
88 Biolcnelnistry of Milk Products
tetr:apelptloe. the mutant did show a reduced

nep:Ul\/e
involved in oliJ~opeptide pr(JICe~!sil1ie;
essential nutrients can
cornpensate for the absence of this peI)tIdase
Whilst this essential for growth on milk its

involvement in may It's ability to
hydrophobic amino acids such as to a lesser extent ph~~n}rJal,arune,
have made it a candidate as a enzyme. A
that the addition of was able to reduce bitterness in a
casein (41). has been over extlres:sed
to date have been in cheese
ammopet.tio;ase is also in some str:ains of lactococci.

protelIllase with a broader mnge than
on aI1:lLlvsis: of
gene and the Imlmuln0I111st~JChemlStI·y show it to be an intrncellular enzyme
Growth studies strains are but have
with a for N-terminal and

aS1)~artate C()nta.inirlg pc~ptujes,
has also been described. This metaIlo enzyme was
rpnnrlf'ci to be membrane-bound It to have a
wallllextolcelllul:ar location from immunohistochemical The
enzyme, has also been from an intr:aceUular extrnct and was
shown to have the same N-terminal amino acid sequence as the extracellular form.
The gene this sequence has been cloned and and
shows no or membrane sequences to an extrnceUular
location Glutamate is an essential amino acid in but the role of
this enzyme in its has to be determined. Growth in milk
Pp.r:~A-lnegat1\re mutants have not been rPnnrtf"':t1
This enzyme may also have a Sle;lnifllCaJlt de\'elo'pm,ent of flavours in

ripc~nirle; cheese. One of the DroOUC:ts a recogllw:d
role in cheese is not
understood. Fractionation studies have demonstrated that is the most
nre:valent amino acid in the water soluble fraction in matured Cheddar cheese.
fraction is considered to contain the that make the e;reatest
contribution to the of the flavour Cheese trials
...... n''3t'nl... starter strains may wen evidence on the role this em~vltle
Peptidases from Lac'toc/'}cci and ,'iprnnn'arv PrntpnlvsiJ; of Milk Proteins 89
Several peP1uO,lses Ctl[)tlD,lC Oipeptl,Oes or tnpepOldes have

been report~~ • ,L.U- L.. . . . . .
genes have ..."""',.,.... 'Ilu

eXlJeCtea mtlracc~JlUllar
location of enzymes.
in milk (41). No
ext:lenmelnts, have been repom~
An obvious candidate for further extracellular hvcirolvS1S of the casein derived

of which
transport
emlopeptlOases have now ,.pnnri~~rl (19,
has been cloned and seQluellc~d. The
location
SU2,ge5,tea a extracellular location
'eD(J-nle2::tt1\i'e mutants did not show any differences
The function of an intracellular enclopeptlldal>e
J)e1JttlOC~S for is debatable. Lactococci
5. CURRENT POSITION ON PEPTIDASES.
At our current level of l<nC.wlt~2Ie. a number of observations H"'A ..........'&
of the lactococcal to utilise casein-derived J)eJ]IUOI;}S

have to be
source of amino acids
the nutrients requln~

on the
suggests that should not be able to trar.:SJ)Olrt
the peJltt10c~s p]rOOuc~~ from the of the extracellular cell wall associated
prc)teina~.e on the caseins. Lactococci have two distinct
a motive force Oepenclent 01l1lnpeptlOe trallsplortc~r
From the characterisation the maximum size
90 Bio,chelllistry of Milk Products
In vitro studies on the

prCttei.nas:e on caseins have shown
The for both the
In
that both are essential

of the needs to occur extraoelh;llarly
This raises the second on the involvement of lactococcal pel)t1CIaSC~S

What is the true location of these pe(:,tid:ase:s?
evidenoe for the extracellular of any of the is ool1lt1i(~tinjg.
immunohistochemical evidence for and indicates that these
emr.vnle~ are oeU wall/membrane associated and some have been
PUlltU~ from cell wall! membrane These same enzymes,
have also been from intracellular extracts and all the
evidenoe are intracellular. The of some
mechanism of the should not ruled out.
there two other candidates as a 36 IDa amino'peJ:)tidase
isolated from the cell wall of cremoris ACI in 1985 and the
......"',...ntll'" plunlled 23 kDa oell wall from Lactococcus lactis
no further information on the first of these
enzymes is one of the first lactococcal to be
described.
pe~'tld.ase has been shown to be an essential enzyme when

milk. Some studies double petJUdase-negative
pel·tOl·mf~ but still no difference in
With the wide
An exa,mple
the formation
Lactococci possess
nWllrOlv~trlO pC~'Ptides to prOi(1u(:e gJutaJmate.
case, may be necessary, QUaldrulPle pet,uoase-negatlve mutant

before any restriction of
6. FUTURE AREAS OF RESEARCH
As noted one of the next areas of research will un(10ubtecUv

de,'elc,pmient of the aimed at renl0Vmg
of the lactococci to of one the
eSSlentlal amino acids. A word be noted. It is
Peptidases from LaCtOCl'JCCI ."PI'/}YUlfl'MI Pf'Oteoivsis of Milk 91
generally assumed that these petlltl(1(lSeS
also have a sigllific:ant
restricted could be due to this

an hydrolyse c(lSein peptides.
autotr()phic oJrgantslills, which do not nrt'ltPl'llvt11" system to
amino also contain Slglllt1C:a:nt peJ)tidase activities (G. W. Niven,
personal Other metabolic functions for the in
lactococci should not be discounted.
Another issue that has to be addressed in is variance. The

lactococcal cell wall associated has several natural
variants. These variants have some different on casein which
are considered in cheese manufacture Whether or not
variation occurs in has not been examined. An of this
could be the the lactococcal to cleave N-1:errninal gllutama1te
diJJepl:ide~s. The isolated from Laclococcus lactis cremoris
does not cleave Glu-Ala whilst a similar from Laclococcus lactis
lactis NCDO 712 is able to cleave this and other glutarIlate:-C(llntainillg
To address this more substantial SpeClf14Clty
Staillda.rdlsed COfl(11t10ns, and in some cases . with more
of individual between different
(inj:lustri~llly 1m1'\nri~nt''l\ strains are studies at the level
ratlionale for these differences. Linked to this are
peI)tl(1.ase activities individual strains. Little work
cornp~l.fil1tg the amount of enzyme found in different strains.
A third area for future expres~.ion of the peptidases.

the lactococcaiproteil1lase
selected pep'tide·colltanung
7. CONCLUSIONS
In a substantial made in 1Ol0wled2e of

lactococcl, both blo~:helillically
role of the individual peI)tid(lSes,
seciondlary hurll ..nl'l1<"'" of casein petlltlCU~S
This work is in by the EC BRIDGE on the HIOttechnc.loJ!~y

of Lactic Acid Bacteria. Contract No. BIOT-CT91-0263.
92 Bioc'hemistry of Milk Products
REFERENCES
1.
2. 1. Kok, _~-==~~~,
3. Pritchard and T. Coo'l~tr,
4. P.S.T. B. Poolman and W.N. AUtUU!,;), _~~~:.&.,
5. R.C. T. D. and B.E. Ter:zagfll, &&~~A.UI::Iri:':"
141.
6. 1. 1.M.B.M. van der Vossen and G. V~n~ft'\'2
94.
7. F.A. Exterkate and G.l.C.M. De Veer, 1987,
471.
8. E.l. B. Pool man and W.N. 1991,
2447.
9. V . .ly,l.v•.un....., D. Le Bars and J .-C. W~.Ml~:ll2!~~,
,,",,>',. .J U I < .
127.
10. 1.R.
526
AIm1.
L.P. Thanh and J.-C.
A. and J.-C.
17. F.A.
577.
18. G.W.
19. R.
20. I.K. 1981,
159.
21. A. Van
43.
22. S. Movahedi and F.
23. B.W. P.S.T.
1839.
M. I. Ni Fhaolain and G.
25. T. 1993,
26. B. A. Geis and M.
27. C. 357.
Peptidases from Lactococei and Secondary Proteolysis of Milk Proteins 93
Fha.olain, P. V. and G.
Pritc::hard. L.~U~!2Illi;~ 1991,

Bocllcelmiann, M.
38.
Crus and J.-C. "-I£ .....n.ru.
~ ... u ....
LK. Q. Y. Susuki and K. YarnaUchl,
3035.
Jemrung;s, 1. Ni Fbaolain and G.
Extc~rka1te, ::C~AmU~~lL. 1991, 317.
Kallmnc.ga"/a and K.
Karmn()gavva and K. Yarrlaucltll. ~UL.J.....m~~,
K01JSSC~U. C.~Y. Boquien,

"'''V1UUF;,.3. AW2L.lm.Yml!D.t.J~!l&I.2l..., 285.
HaandrlClo1nan, K.l.
2049.
44. J.W. Aston and L.K ..........."'Ul"'.

229.
45. A.I.
46. I. j.Y.f.llV.lQU,
Poolman and G. vpnf>m:::t

50. A. W. Bockelmann and M.
79.
51. F.A. Exterkate and G.J.C.M. De 108.
52. F.A. J;vt/;orv!lItf'>
Functional Milk Protein Products
FOOD CHEMISTRY DEPARTMENT, UNIVERSITY \,..-VLLJr:,UlC. CORK, IRELAND
1 INTRODUCTION
However, the
of processes used
nrlodllct:S the most
The of bovine milk has been well

documentedl.; falls into two main based
on 4.6 at > 8°C. Under these conditions
80\ of the and is referred to as
casein while remains the 15\
whey pr'ot,e1.n with the remainder
K-casein from
of K-casein
and some cheese
the caseins and
differ very
~. caseins are insoluble at their isoelectric
4.6) while in the ionic environment of
are soluble at their isoelectric
ii. Addition of crude

rennets, to milk and
results in its remain
soluble.
iii. Caseins are heat stable while the
are heat labile. On milk at ) 72°C
become denatured and interact with caseins to
iv. In milk the caseins occur as macromolecular

with molecular of and mean
of 100 nM, known as micelles, that also
salts,
citrate, rc+crron
calcium
are in solution
These differences between casein and are
in industrial methods used for the recovery of
functional milk products.
an overview for the
of functional,
PRODUCTION OF CASEINS AND CASEINATES
Caseins skim milk as this ensures

that the fat to minimize flavour defects
The first
the process is casein to
it insoluble. This may be achieved
a. skim milk with a mixed or defined-
and at 22 to 14-16 h;
the added starter ferments some of the lactose to
lactic acid and a casein network or is formed
as the pH of the milk under
conditions to the isoelectric pH the casein.
b. dilute (1-2
pressure into milk
direction
c. skim milk at < 10 0 e with a cation in
the form in a reaction column; this
cations in the milk by H+ to a pH of 2.
acidified milk is then mixed with untreated
Bio,cht?lnistry of Milk
the final desired pH of 4.6.

d. any of a number of which can
milk at its natural
; the first stage
K-casein to
, while the second
rennet-altered casein
of about 30°C.
The casein destabilized milk is then
the 50-60°C direct steam
1 min ensure
of the curd
first three
of an acid curd and
referred to as lactic
caseins,
results in of a as
rennet casein this curd retains
colloidal calcium pn.ospn.ate
salts,
Dried casein is hot as it emerges from the

drier and the moisture of individual varies.
it or cool and blend the
u-u-,-a-...; mills to
by the end-
The caseins can be from milk at its normal

addition of ethanol about the concentration of
to induce decreases to about
reduced The use of ethanol to
for industrial has been
described 2 • An alternative the direct addition of
ethanol to milk to induce casein is the use of
ethanol to dissolve the lactose skim milk
an insoluble residue which as a total
When milk or milk concentrate

is frozen and stored at are cryo-
destabilized and is thawed. The
of this of casein has also
Functional Milk
,5 When casein is
40°C it retains micellar
to
similar
With
characteristics it casein
micelles and whey is referred to as
microfiltration as nominal cut-offs in the
0.1 to 10 of membranes of this type
Acid caseins are insoluble in water but wet

acid casein casein will dissolve in alkali
under suitable conditions water-soluble caseinates.
Sodium caseinates, acid casein with
NaOH followed the water-soluble casein most
used in HowAvAr, other bases and
conditions may be used to a wide range of
caseinates 9 •
for many
scale based
acid pH
It is also
of
of the
There are a number of incentives for cost

methods for the fractionation of caseins on
scale, e.g.
1. surface
emulsifier
2. Human milk contains 8- and K-caseins but no a-caseins;

98 Bioichelntstry of Milk Products
hence, B-casein should be an attractive for

bovine milk-based infant formulae.
3. K-Casein is for the
micelles and available in sufficient
be a useful additive for certain milk
4. All the and indeed all milk
which have
A number of methods
B-casein-rich and
industrial scale have been
the ionic and/or
characteristics of the caseins. B-Casein is the most
of the caseins and strong temperature
at 4°C it in monomeric form but
as the This
calcium
B-casein remains soluble
A method for the isolation of
milk or calcium caseinate at SoC was
Famelart
B-casein from
the
the
is the serum or
the curd formed acid
the
cheese
the manufacture
factors
and the
on the
Functional Milk 99
as
the
This,
in
to convert
use in both human and
animal
contain
(
; the chief is lactose
70% of the solids 14 •
concentration of minerals than rennet
of the colloidal calcium component of the casein
micelles Cheese derived from whole
milk has of milk fat than that
derived from residual milk fat is also a
function of and other pre-treatments
to remove this component from the to
recovery_
whey
concentrated at
and maintained at low
microbial and
and other
alter the
A brief review of the the various methods known to be in

commercial or advanced scale use for the recovery of
follows:
is
the
the
methods non-
be cooled
nuclei then
15-18c:>C to
more
first
moisture. The
more lactose in
a drier 16 •
used
sweet
for use
has been shown to be cost-effective for up

from cheese
for removal minerals
and ion
is concentrated to
temperatures less than 70°C (to
The concentrate is cooled in a
lactose The lactose is
mother
a
to
can be delactosed before

demineralization. In is concentrated
to 50-60\ to induce
40-60\ of the removed
Residual lactose in the
to 43-S0°C. The mother is
and then demineralized
eXChange processes at up to 33\
minerals.
on a
a commercial
diafiltration
of
in a solution
membrane that allows of
molecules. The retained (retentate) flows over the
while under the influence of pressure water flows
membrane, together with low molecular
solutes (the is retained
membrane and concentrated relative to other
solutes in the retentate. Fat and suspended solids
are also retained.
several
include:
with
of low molecular
(dilution of retentate with
sanitation and related
volumes of permeate.
J1i()·chl!.~mlj,try of Milk Products
colloidal calcium to remove insoluble cheese

curd or casein fines, fat and calcium
These pre-treatments increase
nTAU'A~,r of the membranes
concentrates.
Because of
of UF (
concentration
fouls) it
The
above 50°C to
are continuous
which enables
to be
be achieved
0.65:1.0; above this value
such that the flux rate becomes
ratios be achieved
retentate at the stages
dilutes the retentate, decreases the
permeates, washes out lactose and minerals.
called diafiltration and is to achieve
to total solids up to :1.
In the final retentate is
and some further concentration is
Low temperature (
in the concentrate is the normal
results in the of
molecules and therefore

on the At pH values lower
4.6 , have a
as cations can be adsorbed
At pH values above their isoelectric
have a net ~~~~~~ and behave as
adsorbed on Media with
and surface have been
for the from dilute
solutions upon the pH Two ion
exchange processes for
the manufacture of WPI.
The "Vistec U in a
stirred tank T02~rn.T~o.27 a series of
that are (1) is
to reactor and
to ex(::nang~;u: t (2 )
lactose and other unadsorbed (3 )
the resin is > 5.5
with alkali to the 4)
the solution of
the tank
and spray
Functional Milk Protein Products
UF treatment of the eluate fraction is

essential for concentration of the
use either cationic
ion exchangers and
column reactors.
to the S column
the acidic
solutes have
alkali to elute
eluate
dried
anionic ion
the
with Released
are as WPI, as for the
S process.
processes recover 85% of the
conditions and the recovered concentrates
and low lactose and
However several
of the
and are
from their
104
individual
ion ex.ch.an.ge
have been available for
about functional
or other of the
there is an isolation on an
industrial
in bovine
than a-lao However,
human does not is the most
of the bovine milk human infant
a-Ia would
but renatures
transformed into
the form it
at their
to fractionate
acidified to
< 0.023% ash;
The may also be fractionated

FeCl 3 at hexame~talptlos>ptla1:e~NaCl 37 or
I
to recover WPI used to

All the are
but on continued
has a
a pressure of 2000 in
was denatured and
while there was no This
as a method of
to simulate human
that are of
Many of these may

as isolation proced,ur'es
of commercial viz.
(LPO) is a broad
concentrations in bovine
in human milk.
LPO has attracted considerable interest since it has been
shown to be involved in the antibacterial of various
secretions. In milk the antibacterial system consists of LPO,
H2 0 2 and -SCN. The active is n~VBnB~~ (OSCN-)
or some oxidation contains
and some
but no H2 0 2 ;
antibacterial system,
e the action glucose
necessary to
-SCN.
Commercial in LPO involves: (1) activation of
cold sterilization of milk or in the
(2) addition of
to
in
650 M44.
of
the best
I of are:
serotransferrin, ovotransferrin and lactotransferrin.
Human and 2-4
25% of the total
in the colostrum milk contain 1
and 0.02-0.35 Because the concentration
of Lf in human than that in bovine
there is considerable interest in bovine
infant formulae with bovine Lf. Bovine
lactotransferrin has also been considered for use in food as an
in feed as the
and mediator or iron
Lfs have been isolated from the milks of several species
Bi(J'CIu;'mi~,try of Milk
and some of the have industrial scale

As stated conditions
cation Lf and LPO and
separate elution.
for the isolation of
), one of the defence

are in mammary secretions,
of mammalian Bovine
contains - 10% but level decreases to
about a week
In situations where it is not to feed colostrum
neonatal ruminants and an source of Ig is
and therefore interest in the of
for this Calf milk
enriched with Ig are
able to absorb from the

defensive in
infection. There is
of breast for
to
be fed on
and energy
milk and consequently formulae
"milk concentrate" oreoared
acid from colostrum and
for use in such
The final contained 75%
and not IgA, which is
are
in
interest in
e.g. it
of aromatic
nutrition of
CO-PRECIPITATE PRODUCTION
The methods described above are used to and
and fractions
can be
, to temperatures
their
of the milk to
a combination of added acidification 62 -
in this are referred to as
Yields of 92-98% of total
to < 80% for acid or rennet
produced these methods have poor
for the manufacture
have been
milk to
milk to
isoelectric
the isoelectric or
are similar manner to
these also be converted to
the addition of base.
PRODUCTION OF MILK PROTEIN CONCENTRATES
Skim milk may also
diafiltration to
contain a range
the casein is in a
while the
form 70 ,
since
CHEMICALLY, PHYSICALLY AND ENZYMATICALLY MODIFIED MILK
PROTEINS
least one commercial

modification to
In this
.
temperatures of ~
concentrate solution,
solids of which about 45 to
which is to a pH in
108 Biochemistry oj Milk Products
in a dried state
than about 2\ of the
diameter. The
is marketed under
and due to its
it is fat substitute
• A number based fat
have been described74 . ? 5 .
in the
evaluation
GENETICALLY ENGINEERED MILK PROTEINS

of casein and
in the literature in the
method
the range
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1978,
29. J.
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30. B.P. Robinson, J.L. Short and K.R. Marshall, N.Z. J.
Sci. Technol., 1976, 114.
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32. R.J. Pearse International Brussels,
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Proc. Preserv., 1982, 5S.
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1237.
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743.
38. K.K. Fox, V.H. uv~~~u,~~,~, L.P. Posati and M.J.
J. Sci. , 1363.
Functional Milk Protein
39. J.N. G. Klarenbeek and M. Adamse, Neth. Milk

41.
40. R. Ha'vaE;n1, Y. Kawamura and S. Ku.nug~, J. Food Sci., 1987,
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1980, 200.
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Elsevier
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2486.
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Functional Milk 113
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Protein Engineering Studies of 8-LactogJobulin
A. Batt2
ITHE EDINBURGH CENTRE FOR MOLECULAR RECOGNITION AND

DEPARTMENT OF THE UNIVERSITY OF
EDINBURGH
DEPARTMENT OF FOOD CORNELL

NY USA
I INTRODUCTION
The of this article is to review some of the site-

work which has been ~O.~~Av_a.~
BLG,
behaviour of this
2 BACKGROUND
For many years the abundant milk

(BLG) has been the ect of detailed
where it has served convenient test wide
of all of this work no
function has ascribed to it several
have been 2 when the structure of one
and shown to be
(RBP},3,4 this
function of BLG was as a
structures of
which bear close 3-dimensional
similarities to BLG and RBP.IO
fold appears to occur much more
tncJu~,nt. For Pervais &
sequences of BLG with those of a-I
HC) and
Protein Engineering Studies 115
I and the continues to grow. ( 1) .
N·Tenninus
A B c
Schematic of the sheet

structure of the fold. The arrows represent the
the hatched box is the a-helix and the
ions of two of the characteristic sequence motifs are
shown at the start of strand A and the end of strand E
Now, the contains about 20 small

(160-180 residues) most of which are found in
secretions like and mucous. 13-15 The
of the are able to bind small,
molecules in their internal
form of or transduction function is
has so far been found which has
that
16
.................... , in this
indications of
may be more
15
BLG binds retinol than does RBP

17,18 and model and retinol show
not that the can
accommodate retinol, but also that there may be more space
at the inner end to allow somewhat molecules to be
bound and solvent. There are
several molecules to
BLG 17,19 studies
on to indicate an alternative
retinol and
observations from work in
form soaked in
indication is
clear that
acid-stable Aschaffen-
this fact to prepare
to 2 at which
are denatured. FUrther, BLG survives
the the stomach to appear intact in
intestine. to pass the
stomach unscathed and is thus a candidate for invest-
as a carrier of small, toxic subst-
ances at least as far as the intestine.
A of other have been identified

to the behaviour in there
group at
associated
with the between 119
and 121. Titration has revealed a
group with a of histidine and,
furthermore, a series of distinct conformational changes
exist between 3 and 10, as monitored rota-
and
That some of
on
erature~ but more
known to the food
incor-
irreversible
needs to be addressed
since this heat treatment but also
the and Calorimetric
studies on the thermal denaturation of BLG
range of and, more
the denaturation process at
6 5 and 3 mg mIl, is most
result of such calorimetric measurements on the denatur-
to whey
based upon temperatures up to 90°C have been
abandoned since denaturation occurs at around 70·C.
Protein f<:)""inl"l"yi119 Studies of f3-Lactoglobulin 117
One obvious direction in

not in the
components themselves.
considerable
with the of trans-

animals.
Recent advances in molecular

have allowed mutations to be introduced into a
's amino acid sequence in such a way as to mimic,
and to some extent, up natural selection. More
mutations which would not
selective in nature can be introduced in a
directed and deliberate manner. Several of mutation
can be considered: those which are introduced to
UU~L'.LU~.35 those which are introduced to
and those which are
to a mechanism or a
In almost every case, unlike the
ations where the odd millenium matters
site-directed
3-dimensional structure
for
are
3 THB STRUCTURB OF LACTOGLOBULIN
We have determined the structure of two Y

(orthorhombic spacegroup
and a re-_n..t_e__rp~_retation
blocks, spacegroup
)22 of BUG at pH 7.8 to 2.
and are at
resolution. We have found that resolution
the data obtainable in the but
that a resolution espec-
of the Y form, can be measured. However, there is a
considerable amount of thermal diffuse which
indicates that the molecules in the
greater motion than be It is
not yet clear whether this also reflects intramolecular
associated with conformational
Data have been collected on the Y form to 1. resolution
at the SERe and
these data alone or in

conjunction with lower resolution data has not gone
and we have recollected both the low-to-medium
resolution shell (2. resolution on lattice Y collected
on an area detector), have all of the
atom data and recalculated a map at a nominal 2. res-
olution. This is in the
of the recent results on the Z form.
The structure of the lattice Z form of the BLG was

several years that of the Y
form. 3 Whilst both forms indicated a conserved core
barrel or
in refined coord-
inates of the Y form, resolution
indicated that some details were incorrect. Whilst
and much of the Y-form we
have carried out a medium resolution structure redet-
ermination of the Z form which has the
lem in both forms. The basic scheme of the
structure for the 2 and remains
but the chain
has been altered between residues 30
and 60 a and defined loop has become
smaller and the extra 5 residues removed at this are
drawn back the strands to increase the size of the
around residue 30. This has a dramatic effect upon
the distribution of the residues, and makes the
of the molecule derived the
~.39 from one which shows a severe
between the residues and their
to Fort-
of affects the results or
of the mutations which are discussed
below.
Because of the number of distinct

of BLG which exist, the various conformational
observed as the is raised between 3 and 8
observable in molecular detail. There are
available for the structure at pH 6.5 of the X form, and the
structure at this tackled in Leeds
A.C.T.North and the at
lower and this in
turn of the x-ray diffraction data
obtainable. For 3,
A cartoon of the sheet

in the current, medium resolution structure of
viewed 'into' the The exact of
the various stretches of structure may still
alter as the structure is refined.
space group P63, a=b=68.5, C=143. ) have

found to diffract modest resolution between
and, unlike the pH forms, the resolution
does not with radiation. The
structure of the 3 form of BLG, for which we have
collected will allow us to compare it
pH, protease labile forms and
into the reasons for the remarkable acid
4 CLONING AND EXPRESSION
Amongst the several

of BLG, two
from Cornell used ~~-= BLG with an
additional N-terminal methionine
vector .40-42 Some 15t of the cell was BLG
but most of this was as inclusion bodies which
solubilisation in with
renaturation. However, the BLG so obtained is
from the obtained from milk.

Totsuka ~.43 used a different the
for
of
secreted in its
been
we have also used
time under control of the
p~91, to native
ovine culture super-
natant. MUtants of this in M13
and subcloned into the 44,45
5 BINDING STUDIBS
BOO mayor may not be

1 but it be to bind to
the same way as it . in the
central The direct 22
however, indicates a ~.u~,.~,~
the helix and the outer surface of the
licated in the interaction with retinol, however, is a
residue: in the modelled BOO-retinol com-
3 Lys141 in the X-ray model. MUtants K70M
and K141M were the former exhibited a
marked decrease in as observed fluor-
escence. Further, K70M lacked the characteristic blue
shifted spectrum for the Schiff base
which was observed for K141M and the
wild-type BOOs. Final mass
metry which revealed the presence of the retinol coval-
bound to a which contained the
residue. Clear
Because of the acid and
~uv~~u more convenient alternatives are

available. a to carry small,
insoluble or labile molecules is an exercise of
considerable interest if not
The first of such a is to model the small
molecule into the which residues need
to be altered creation of a different
chains may of incor-

or these have
be overcome in the case
6 THE SULPHYDRYL GROUP
The events to the ma.cromolecular association

observed with BLG appear to be mediated
disulfide interactions. A number of indirect
of which
therefore stab-
ilize We have
obtained evidence both from natural BLG and
modified forms of the to support these observ-
ations. Porcine BLG which does not contain a free thiol
does form 46 BLG
does not of
bovine BLG, is
not a chain-reaction
without a free thiol The mutants
Cl19S and C121S were whilst the
former was it was not secreted from the yeast
whilst no such was encountered
with the latter. This is in agreement with the X-ray work
where no evidence of either the alternative or a mixture
of is obtained. 3 ,22,45
in Cornell, a recombinant form of BLG was
eliminate the free thiol group. Whilst the
intent was to create a variant without the
this variant could not be
A C121A variant constructed
mediated was but
ation and renaturation failed to
were
conditions where all other BLG
acid-stable
ence between the yeast and ~~== exoreSS10n
AS an we chose to form the free thiol

into a third bond the substitution of
another amino acid with TwO mutations were
in a rational manner on the basis of
observations and then
simulation to test the
their thermal Both
substitutions to to
the formation of a new bond. However,
the simulated molecular behaviour of the two
proposed mutant forms of BLG differed both with to
fluctuation in the of the backbone atoms and
also the molecular radius of both
the L104C mutation to be the closer to the wild-
structure.
Bach mutant in sufficient quant-

to test its the recombinant
system at Cornell
bodies. The a denaturation
followed a renaturation presence of both
reduced and oxidized The refolded BLG
on the basis of its acid
filtration. yields
but this was sufficient of the
A number of indirect measures of
structure circular dichroism and in-
trinsic fluorescence revealed that the recombinant BLG was
from the isolated from bovine
small of the recombinant
which are not yet suitable but
which appear similar in form to the
milk.
A formation of the
correct of the
model and of
I formal of their existence was necessary.

Formation of disulfide in the correct arrange-
ment, as was confirmed At
the resolution which could be acheived
a third disulfide was identified which consisted
to the introduced either at
or at 132 (A132C). In contrast to wild-
BLG, which at temperatures >65°C , SDS-PAGB
showed that neither of the mutant
The conformational of the L104C and A132C mutant
thermal denaturation had also·been
increased some 8-10 0 C) with the
Furthermore, the A132C BLG exhibited
denaturation
to that of both wild-
L104C BLG.47
Protein Engineering
to reduce thermal aggreg-

ation the way toward
Gelation is the controlled of monomers res-
in the formation of a network that entraps
water. BLG can form thermoset it does so
I
at concentrations >10% and at temperatures above

upon the conditions of ionic
concentration and of cation. This limits the
usefulness (and hence the value) of BLG as a food
ient where the is to increase the
solution. The characteristics of bovine
have been enhanced the selective introduction of
substitutions to increase the free thiol content
of the 48 A recombinant version of bovine BLG A
modified , and the
variants. Titration showed that, as
the number of free thiols increased
that additional disulfide
not formed.
90°C was
F82C and a
wild- BLG at a much lower concentration
wild- BLG. R40C could not be to a sufficient
concentration (>5%) without the formation of insoluble
free thiol content also
enhanced the molecular
as observed of milk. An
result, however, was that the introduction of
an extra free thiol also increased the of these
variants were more

These latter observations may in
the of BLG process.
cause of these differences in
in the subtle modifications to the
overall structural of the mutant when
to the wild-type (native) version.
mutant BLG
was selected
a but also
formation of a is critical for this
and failure of to form is considered a defect. A
was formulated milk fortified with
skim milk and mutant BLG was added to a

final concentration of 0.075%_ These solutions were
heated either at 85°C or 70°C, the former the normal
The heated solutions were then
formulations at 70°C when to

which wild- BLG was added. 50 This reduced
resulted from the formation of a much
which was of water. Extensive
studies have been carried out to the functional
of concentrates used for yogurt
manufacture; however there is still a need to
formulate novel The introduction of
additional free thiols to BLG appears to its
for manufacture and
8 CONCLUSIONS
The annual literature on BLG is extensive and covers too

~V~.~D, few of which have so far involved
of the for this review. of
these studies involve the behaviour of BLG alone, in
mixtures or in the more mixtures
but under conditions similar to those found
The studies above show that site-
directed modification of BLG can have both and
useful consequences on the
additional efforts to
between the structural elements of BLG and its functional
should prove fruitful.
9 ACKNOWLEDGEMENTS
The John Cho, Sam-pin

Linda Paterson,
North and Carl Holt for
with and discussions of the work
whiCh was the Science
Council, the European Commission
the National Promotion and Research Board, the New
York State Science and the US
Research Office and the National Science Foundation. This
work has been facilitated a NATO Travel Award.
Protein Engineering Studies of fJ-Lactoglobulin 125
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Protein Engineeking, 1993, in press.
48 S.P. Lee, Y. Cbo and C.A. Batt, J AQr Food Chem , 1993, 41, 1343
49. S.P. Lee and C.A. Batt, Food Texture, 1993, ~, 73
50. S.P. Lee, D.S. Kim, S. Watkins and C.A. Batt, Biosci Biotecbnol
Biochem , 1993, in press
Functional Properties of Cbbana Whey Products
R.
DEPARTMENT OF FOOD SCIENCE AND THE UNIVERSITY OF
READING
1 INTRODUCTION
Chhana is a traditional Indian product used in the cOllttec'tJ.OI1Lery mdustJrv

pro!dw;cd from cow's milk a combination
acidification to pH 5.4
product .. chhana .. contains about 60 g
which leads to ponution problems. It is calculated that approx:ima:tely
powder could be prodw;ed annually from chhana in
popularity of chhana..based is to other countries.
In a and Grandilwn.
chhana with protein content
(reverse OSDI08J.s, ultrafiltration and diajlltr,moll),
or freeze has been demonstrated. The chemical COllllpo:sltl{:.n
fractions was shown to be somewhat different to commercial cheese
(Jindal and In the of cbbana
more denatured, and the relative of caseins was much
The aim of this was to the of chhana
in relation to commercial cheese and hence to assess their as
functional in the food tnrtnQl1rv
2 MATERIALS
Chhana derived from Channel Island was obtained from HOlmbclY

Halwa Limited (Southall, Commercial cheese
35 and 55% were obtained from
3 METHODS
Cbbana concentrates were prepared by reverse osmosis (RO), ultrafiltration

or UF fonowed tubular polysulphone membranes
Powder Solids K
Protein Protein* Fat Ash Lactose
UFSD 944 42.0 268 172 45.0 449
DFSD 944 64.1 409 393 33.6 105
UFSD 948 55.0 351 48 57.7 454
UFSD 980 34.6 221 459 29.5 270
UFFD 965 34.1 217 453 29.0 266
DFSD 945 63.4 404 140 45.3 320
DFFD 937 62.8 401 131 44.9 NO
DFSD 953 90.7 579 113 38.2 155
DFFD 961 91.4 584 114 38.5 NO
ROSDW 956 3.3 21 60 65.2 141
ROSDS 946 3.1 20 13 65.6 828
ROFDS 922 3.0 19 13 64.0 807
.An values . . . as g kg'"l; NO .. Not r . -, * .. Protein Nx 6.38
UltJ'atiltratiion; DF .. Dia:tiltration; RO .. Reverse Osmosis
Dried FOS - Freeze Dried
meJmbl'ane:a, or ES 625 UF meJmbranea, Paterson International,

WllitchlUI"C.h.. ---~-~-r Powders were prepared by spray or freeze and
the gross composition is shown in the table 1. Fat removal by
centnn:aga1tlon was carried out on the whcys prior to meJmbrane in most
cases. Due to the variability of the of fat separation, and the high of
concentration of the the fat levels were impossible to control accurately.
emulsifying activity and eJmulsion

properties <patel and
tOall!UDg
solution (Brookfield and heat gelation <patel and Stripp, 1988)
of chhana products were measured over the pH range and results were
compared to data for commercial cheese powders of similar protein contents.
4 RESULTS
Protein solubilities of chhana varied from 57.. 1000/0 depending on

pH. Results for some chhana and commercial cheese whey are shown in
1. Protein solubility was lower in the isoelectric for all
POvideJ:8. but was quite outside this range. For the low protein 001W<.1ers
the solubilities were over the whole pH range. In
."._____ --. the results for chhana and cheese products were very similar.
Functional Properties of Chhana 129
EA (a measure of the to fonn and ES

emUlslloDl;) were measured a standard oil-in·water emulsion test a final
concentration of 0.1 % Results for chhana and cheese
were very similar indeed over the pH range. An example 2.
• 35% Cheese
100
a= 35% Chhana
• 55% Cheese
90
0- 58% Chhalla
+ 12.5% Cheese
80
• 2% ROSDS
~ 70
2 4 5 6 7 8 9 10
solubilities of chhana and cheese over
------ 35% Cbhana

---a-- 35 % Cheese
2. 4 6 8 10
pH
v. "' .....0 , . T of cheese and chhana

130 Products
The toalllU1lg C~ln:lj::'1tv and half-life foams were measured

spargD'lg of solutions COllltaililing 0.1 % protein Both were lower for
powders than for cheese wbey products 3 and 4), but
this was probably related to their fat contents. to
produ(~e a'~CCI'tab'le foams the chhana
of solutions of chhana
VtCl't"nC!1tv powders were similar to cheese pr()(1lllCIS at
but below 4.0 chhana pro(1uctsgave rise to solutions.
concentrate solutions COI1Wmtllg

7.5% for 30 min. chhana products did not
form at all at the and formed only very weak at the lower
and were very much inferior to cheese in this Gel of
chhana and cheese are in Table 2.
800 ---..-35<7'0 chhaDa
-
--a-- 35<7'0 cheese
600 58<7'0 chua
:;:
'\j S5<7'O cheese
~
c:.
~
:.I 400
~
;;; 200
3 '} I I)
pH
~=-...... Foam cat:-aclnes of solutions of cheese and chhana whey
concenlrates.
35<7'0 c::hbaDa
35<7'0 cbeese
S8C11 cbbaa
SSCII c::beese
-
]
'} 10
:;;....:;,;~=--.:. :S~lb1l1tv of foams DrOld.uc~ed cheese and chhana

concenlrates.
Functional Properties of Chhana Whey 131
Gel strc:mgltIl of chhana and cheese concentrates solution.

for 30 min)
3.5 91 ' 27
4.0 201 20
4.5 207 27 NM
5.0 199 33 NM NM
5.5 142 22 NM NM
6.0 181 NM NM NM
7.0 172 NM NM NM
8.0 315 NM NM NM
All
5 CONCLUSIONS
• Protein of chhana varied from 57-100% over the

range 2.5-9.0 and behaviour was very similar to cheese
.Chhana DOllVde:rs had cornparable e!nul:s:dJmg _n__-t.""Q to cheese
• Foalm1t'10' J)lropertil~ of chhana OO~yders were inferior to commercial cheese

pt'DQUclts, but this was probab,Iy due to fat levels.
• of solutions of chhana
\ / U:!I'''Q9Ch! oo\'vae:rs was similar to eatlllVaJlenl cheese
proauclts above but was very 2.5..4.0.
• Chhana pt'odUc~ts formed very weak on to at acid and did

not form at an in the range 6.0-9.0.
These studies indicate that there is considerable l)OlienbaI for the use of chhana
produc~tsas functional food mgt'eCSlc:mts.
6 REFERENCES
M.J. James and P.O. Briliish Food ManUl'aCtlllm1lg Research MlOci.ation,

Research report No. 1988
A.R. Jindal and A.S. Gramctison, "-'--"'~~~~""" 511.
A.R. Jindal and A.S. Urandl1lon, ~~~~~ 79.

P.D. Patel and In 'Developments in Food Proteins' Vol. 5, ed. B.F.J.

......_ ............ Elsevier Applied London, 1981,
P.D. Patel and A.M. British Food Manufacturing Research As8lOCllattOn.,

Research report No. 1988.
S.l S.C. R.J. Hart and C.L. Walters, British Food Manufacturing
Research Association, Research report No. 1986.
Thermal Aggregation of Whey Protein Concentrates
under Fluid Shear Conditions
M. .lJ'V,l,lU,lU , and
UNIVERSITY OF v.n.~ •• V'."".-"'Jl';', MADINGLEY

UK
DEPARTMENT OF CHEMICAL ENGINEERING, UNIVERSITY OF

",-,.n.".......'.,.".."..."I.#,. PEMBROKE CAMBRIDGE CB2 UK
1 INTRODUCTION
functional
of
not well
for texture
and
both to be
such as
include
Mild heat treatment,

sufficient to
the
the
is
or at
....." ............ v' ..... which
.lCUJ-J'I.J-.LI,l(;I.J-. As a resul t
.,., .................. "'" aggregates are
134
when electrostatic
the isoelectric pH
in the formation of
in the of collisions
orientation and
both mechanisms
but the
final
assumed to on a balance shear-induced
aggregate formation and shear-controlled aggregate
processes
The overall thermal shear
conditions can therefore
(i)
step aggregate
processes.
Textural characteristics of
shear conditions are to be
the size distribution. From a
control of the size
described here
and process
of a commercial
shear.
2 MATERIALS AND METHODS

A commercial, ultra-filtrate
concentrate (UF-WPC), Carbelac (
Products Ireland) was used. The
of the is shown in table 1. Solutions
were made up in distilled water to a concentration
7 %w/w in solution (natural pH 6.3).
Thermal Aggregation of Whey
WPC
Component I%w/w
35%
50%
Fat 7
Minerals 4
Moisture 4
Shear treatment of WPC was carried out in a

stainless steel couette .nn.~.r1'ia. The consisted
of two concentric I in which
(o.d. 106 rom) was inside the outer
(i.d. 110 rom) an annular 2 rom. Shear
rates could be between 0 and variation
of shear rate across the was calculated less than
4%. For the shear rates showed
that on the whole the flow was in
the form of vortices oresent. Simultaneous
thermal treatment the couette in a
constant temperatures
could be and 90·C.
measure of
of the coefficient of
I mean size).
electron
a method described
were onto a which was then sputter
coated with in a Polaron Sputter Coater.
Aggregates were then viewed at 30 keY in an
Environmental Electron (ESEM)
in the conventional SEM mode.
3 RESULTS AND DISCUSSION
viewed in the
been formed
at 80·C
have
consistent
136
at this
show
of smaller
(a)
(b)
aggregate
untreated 7%w/w
which have at
are shown in
of the
(CUS) versus the
d.
WPC
Protein (:or.rcentrates 137
detected Cd;:; The existence

is not a commercial
used here. Most UF-WPC
30 and 50% and as a result the
of the WPC is limited to between 77
material of both lactose
examination
small amount of
based on
of
were observed
resolution
found in
assumed to
artefacts
100
90
80
70
---
dP
60
--x--
50
tJl
0
()
40 --0-
30 --Il--
-0--
---
20
--e-
10
0
1 10 100 1000
d
Particle size distribution for

at 80·C
(see
a PSD was
I
.53.
was slow. After
to have increased
for times
and 15 there was a dramatic increase in
I
aggregate size; after 15 minutes of

138 Biolchelnistry of Milk
d :: 27.9 t S 30 minutes aggregate size

continued and after 30 minutes of
had increased to 43.0 this
however there was
aggregate
The time course described above is

consistent with the process described
the . It is assume that
(t S 10 ), when aggregate
denaturation is the rate
times (t > 10 )
interactions
are rate
The effect of shown in

3, for 7%w/w at 80·C.
of minutes
were formed at 1480
rates. This is consistent with the
trequenC'1E!S, and thus faster
rates. However, for
under shear rate
size
was
of the PSD, but as the time of
exposure to shear increased the size distribution became
narrower, as evidenced the decrease in . However,
the size of formed this shear
with those a similar
at 80·C and minutes see
3), suggests that under low
Protein Cnr.rrp"trntps under Fluid Shear COIuJitj'ons 139
-
I
45
40
35
30
25
fro 20
15
10
5 10 15 20 25 30
t
Effect of shear rate on aggregate size and

for WPC at
shows
obtained in the of
difference in scale).
( x 0. 1 0 290 0 S40 <> 800 ,.1 t:,. 1480
conditions some kind of which make

them more resistant , i.e. the cross-links
the due to either
The size described

above is between shear
controlled (t S 5 minutes) I
when the processes are

so increases with shear.
However, as the aggregate
mechanisms become increase with fluid
shear. As a result, formed under shear
conditions tend to sizes than those formed in a low
shear environment.
0.95
0.9
40 0.85
0.8
35
0.75
0.7
30
0.65
25 0.6
15 20 25 30 35 40 45 50 55 60
t
Effect of shear rate on aggregate
is
Effect of process temperature on

for WPC at
30 minutes
ARJ?reJJ.'atilm of Whey Protein Concentrates under Fluid Shear Conditions 141
i.e.
is time course.
temperature
size increases. This is consistent with
and the concomitant increase
which leads to faster
I
increased aggregate
4 CONCLUSIONS
concentrates in laminar
time course of
with a two step process; (i) an
denaturation was assumed to be rate
(ii) a faster
interactions
were assumed to be rate
v.~n¥A. nprT'p~Rpn with shear.
formed under conditions of lowest
in the absence of
size with shear was
oreaK-UD mechanisms.
temper.ature. This was
of
kinetics and increased
this work demonstrates that
aaareaat1o,n processes can controlled careful
of the usual process1ng such as
and fluid shear, and hence desired
can be formed.
ACKNOWLEDGEMENTS
also like
and ICI for the ESEM,
Dr. P. Meredith and Mr.
electron m1.cr'oarra:ontS
REFERENCES
1. T. A. Evans and C.
Harris, Elsevier
11, 435
2. C. V. Morr I
Proteins' ed. P. .
Ltd. t 1982, Vol
3. H. G. Kessler and H. J.
1991, 165
142 Bio<chelnistiry of Milk
4. Donovan,
5. 1981, .3..5., 47
6. 1990, .l.a
.w......~.......to..lIoI..I--.....-.::d.-Io<.oU.l~, I ( 11) I
7. 1992, (3),380
8. K. Creamer, J.....
9.
10.
11. R.
12 E. Barbu
77
13. P. Kratochvil, P. Munk and P Bartl,
Commun., 1961, 945
14. R. H. Schmidt, 'Protein
J. P. A C S . Ser. 147,
p 131
15. A. J. 1992
16. C. D. Nelson ~~~~~~~~I 1985,
ll, 1434
17. T. G. M. van S. G. Mason,
1977,
18. Reich
19. S.
20. D. J.
1271
21. G I. 1923, 223A ,
289
22. R. R. Fisher and C. E. Glatz, ~~~U-~~~I 1988,
777
23. G. D. Danilatos,
24. C. V. Morr,
25. C. V. Morr I
I 1973,
Debittering of a-Casein Hydrolysates by a Fungal
Peptidase
SCHOOL OF CONSUMER STUDIES, TOURISM AND HOSPITALITY

UNIVERSITY OF
UK
1 SUMMARY
2 INTRODUCTION
with casein
defect in taste renders the
foods thus its
produced by the
(1- and
144 Biojc:helnist,ry of Milk
addition of
published and
extracts.
increased
immediate use in
strict
are
on a-casein. A
for
al (1993),
3 MATERIALS AND METHODS
Whole bovine casein was obtained at

4.6 from skimmed milk. It was then the
urea method of et al(1952)8
that was washed and
acid before use. A the a-casein
(
140
and an :S(w/w)
40 0 C for 4 hours and the
was measured as the 12% soluble
the 2 4,6-trinitrobenzene-sulfonic acid
was terminated in each case
bath for 10 minutes and this was
at 4000 rpm for 15 minutes to remove
formed.
in the incubator for

there, which allowed any
recorded.
of 100
of the
an for 17
Debiltering a Fungal Peptidase 145
produce the debittered hydrolysate (DBB). Each DBB and CBB

were boiled after 17 hours to stop enzyme activity and then
frozen at -200C until
.( One uni t of Dr~c)'t:E~asre was defined as tbat amount
of III>n'7vmlll> of tyrosine
per minute under assay conditions [pH 7 37oC,
2% casein solution (wlv) ). 1 XsIg = .6 mulg.)
Preparation of the Peptide Fraction (PF).
The 70% ethanol soluble fractions (PF) of both
controls and debittered were prepared using a
variation of the method Cliffe et al (1993).7
Fermentation ethanol Witham, Essex, UK.)
was added to ml of a final volume of
100 ml followed by at room temperature
and then 15 minutes. After
rotary evap'or'at1oln at 400C, 1 mm Bg and
~.~~.~'W water a 3 fold
achieved.
Size-Exclusion Chrgmatqgraphy psing Sephadex G-25 (fine).
The Sephadex G-25 was obtained from Sigma Chemical Co.
Ltd., Poole, Dorset, UK. (2.5 ml of the PF) were
applied under eluent to the (1.6 x 40 cm) and eluted
with distilled water at a flow rate of 16 ml/hour.
Fractions were collected at 20 minute intervals and the
of eluent absorbance at 280 nm, was recorded on a
chart recorder. The fractions were pooled as shown in figures
1 and 2, to at 40 0 C and re-dissolved
in 1 ml of water. for BPLC analysis were
filtered 0.2 ~ filter discs.
TAsting gf FrActigns.
The concentrated fractions were tasted only by

two due to the size and after each sample
the 's mouth was distilled water. Tasting
results are in terms of bitterness, with bland describing
no detectable taste. with flavour after
were described as savoury if no bitterness could
be tasted.
Reyersed Phase High Performance Ligyid Chrgmatpgrapby (RP-
HPLC) pf Fractipns.
The BPLC used was a Perkin Elmer series 410 BPLC pump
fitted with an 5 ~ reversed phase column (4.6 x
250 mm) and a UV at a wavelength of 220 nm. Each
sample (10 ~l) was and eluted with 0.06%
trifluoroacetic acid /BPLC grade water as a mobile phase,
at a flow rate of 1.0 ml/min. The concentration of the mobile
modifier (0.056% methanol) was increased
from 0-71% over 55 and then to 91% over a
further minutes.
146 Htolclu!lntstry of Milk Products

The 1) show that after 17 hours the
(N) concentration was 5 mM for
produced by papain (CRHp) and
hydrolysate produced by the
This is a small increase in soluble
-50 mM increase for the debittered
DBRb. Thus the control
to be
when
cnroma.~orqr'am~
control
) after 4
of the
la.
This loaded
onto the column TCA
soluble N concentration an equally
increased EtOR soluble N
concentration). have five
fractions molecular
and the in Table 2 the
bitter fraction same fraction number
3.
fraction
results, soluble
the a-casein by
action over time and the final
a-CASEIN a-CASEIN
HYDROLYSIS HYDROLYSATE HYDROLYSATE BY
TIME BY PAPAIN BACILLUS PROTEASE
(BOURS)
ivalent Units
0 0 0
1 10.4 13.2
I 2 12.4 20.9
3 14.0 24.0
4 15.8 27.9
..... :.r.J"U'I!;I action terminated after 4 hours.
DEBITTERING CONTROL DEBITTERED CONTROL DEBITTERED
TIME (CBRp) (DRRp) (CBRb) (DRHb)
17 hours 20.6 67.9 29.9 83.0
Debittering of a~Casein Hy(,(ro/~",sales a Fungal Peptidase 147
(I)
.
•
III
I
N
(0)
8 16 112 128 144
[igum 1 filtration of the

Papain control (a) and debittered (b) ,
a-casein fractions.
(I)
I
B
o 1.
figure 2 Sephadex G-25 filtration chromatograms of the
Bacillus protease bitter control (a) and debittered
( b), a-casein fractions.
148 Biochemistry of Milk
Table 2 collected after Sephadex G-25

gel filtration of the control and debittered
hydrolysate ) .
PAPAIN, BACILLUS PROTEASE,
FRACTION a-CASEIN HYDROLYSATE a-CASEIN HYDROLYSATE
NUHBER PEPTIDE FRACTION PEPTIDE FRACTION
PF PFb
CCBTROL CCB:rBCL
'C2fg) 'llB2fg) 'C2fb)
some
1 bland not bland flavour not
bitter
2 bitter not bitter not
very
3 bitter
slightly
4 bitter
5 bland
6 bland bland
7 bland bland
Figure 38 is the RP-BPLC peptide map associated with the most
bitter fraction 3(8) of the CPFb filtration
chromatogram and it also shows only late running
hydrophobic to be The higher absorbance
values of peaks are with there being a higher
concentration of peptide material in the CPFb.
The Sephadex G-25 filtration (
1b), for the fraction of the produced by
papain action then debittered by Aspergillus peptidase
(DBPFp), shows clearly a shift in the peptide canpc.s
towards the later material. The fraction 5 has
been confirmed by further to be tyrosine and number
7 to be tryptophan. These acids are not eluted in order
of molecular weight because the use of water as an eluent may
effect ionic interactions. The presence of increased amounts
of amino acids well with the (50 mM)
increase in soluble observed in the TNBS assay results
(Table 1). it was found that the most strongly
flavoured was fraction 3 2)
which to
most bitter The flavour with
fraction could not really be described as a pleasant 'savoury'
but was however not bitter to taste. RP-BPLC analysis of
the DBPFp fraction 3(C) (figure 3C) gives a peptide map
showing running peaks. These peaks represent
hydrophilic peptides and their presence, combined with the
Fungal Peptidase 149
100
C!III!
91
•
CD
Q
,,'" .. ' "

,,,'
71
50 !:t
.-
:I:
........
.....
.....
c
en
0
C!III!
Retentloo fI .. halautes)
C
100
C!III!
91
•
CD
Q
..•
c
71
50 !:t
.
:I:
........
.....
....
N C
•.
en
0
CIt
C!III!
......
..I
c
0
Reteatloo fI.. (mlnules)

Reversed Phase BPLC
pa11:tEtrn of
J.J.C:a.¥\~.I."'UO,
DrcKlu4:::ecl by
150 Bio(:hemistry of Milk Products
• 100
Ratentlo. TI.. (minutes)
D
100
iiIII!
91
••
c:::t
71
50 e:.
.-
:a
....
...
........
c:::t
en
CD
iiIII!
30
Ratlntlo. TI .. (minutes)
a 151
reduction in late , accounts for the loss of

bitter flavour in
5 CONCLUSIONS
The food
been shown
of has
is
6 REFERENCES
1. Y. and J. S01ms, 1976,
4, 71.
2. T. Ma1!:ot.a R. and T. Bata, Agric. Biol. Chem.,
1970 (8) , •
3. K.M • e.L. Lim and W. Manson, 1974,
.il, 283.
4. Bill Van Leeuwen,
5. F. Tsukasaki and K.
5), 1225.
6. E.
7. and F. Int. DaiU J"
8. J.B. Custer and T.L •
9.
.
Dairy Res., 1988, ~, 585.
10. FOX, Milchwissenschaft, 1982, ll,
11. and B.A. 1990, 73.
The EtTect of Thermisation on the Thermal
Denaturation of ),-Glutamyltranspeptidase in Milk
and Milk Products
Patel and R. Witbey

READING
1 INTRODUCTION
ThlernlOUlDue. D~)vcltlrcltrc[>hjc crgani!!tms such as Pseudomonas
extremlelv heat stable. levels Ds\'chlrotr'ODltls in
gen:eraltlon 'Of 'Off-flavours milk and proltucl:S, and
in the nan,(1lulg
Grcwth 'Of Ds'\'chlrctroDl1S
at lcw temlperatulre
Th4~rmclaibtle DS\fChlfCtl'CDltls may also
theirmi sati 'On) 'Of thermisaticn included in recent
"thermised milk" means raw milk which has been heated fcr at least
temlperuture between 57°C and 68°C and after such treatment shcws a
as described in Part IV 'Of Schedule 5 and
such milk nct be treated as reacticn if
that taken fcr that test a 'Of 10]1. g 'Of P -
Dltl'CDJlenOJlrnl 'Of milk....
EC 2.3.2.1. has been identified as a suitable

a.8S(:8SI11g heat treatments above the minimum conditicns 3-5 fcr
denaturaticn characteristics similar 'Of lact'OperoiXldlase
is associated with membtane material and increases the
deactl\l'atl()n is a functicn 'Of the heat treatment and the water
·!·h,"........ " ....,'" is kn'Own t'O reduce the 3.1.3.1) in

milk. This was carried 'Out tc I... ..,"'~l"" ..."..,
has 'On the deactivaticn 'Of GGTP in milk
2 MATERIALS & METHODS
Raw commercial bulked milk was 'Obtained fr'Om Cliffcrds Dairies Ltd (HI'3.c~Jlelti.
48% fat cream was fr'Om the milk at SO°C a Lister selJ,al1llt'Or
Juni'Or heat rated IIh fcr milk. The warm
The Effect ofThermisation the Thermal Denaturation of "Y·Glutamyltranspeptidase 153
cream was standardised to 18% and used ImIDe.:!lately or cooled and sutJlSeClluelltly used
in the ice cream formulation as listed in table L
Table 1: Formulations of the milk orcKiucts. eXJ)fcssed as g per
Milk Cream Ice Cream Mix

Raw milk HXlO
Skimmed milk 620
48% cream 380 285
Skim milk nnlJlJtipr I 73
Sucrose 130
4.6
Water 507.4
*t Emulsifier-stabiliser
Medium heat skim milk !AJ,TU,",'I. SllppJJea
Unn<l~'tea I-J'rnt1f ...'tc!
Edmunds.
Heat treatment of the pr(xlucts

excnan,ger rated at
the heat were characterised
In the first series of tre,umem:s,

treatments at 55° • (jJO and 65°C f()f a miJ:lim,um
in a cold store at 2°e On the folliowin2
thermised from each pretrccltm.ent
a range from to sooe
hold not less 15 In the second series
at 65°e were carried out in a similar manner,
treatments were carried out in tnj:.llailte.
The GGTP at 37°e

-nitroanilide from 8 buffer over a 5
absorbance at 410 Mean activities were then converted into
of that untreated and the
treatments.

The effect of the beat treatments on the activities of the cream and ice cream mix are
shown in 1 - 3. heat treatments from both pretreaLtmc~nts and the first
series of heat treatments were included in the treliltn1lent" curve on each
154 Biochemistry Products
Activity
100 o Control
" gs>
• «!
80 A(B
2~control
60 (J 2$
I'l 2..t)SO+6SO
40
20
0
50 60 70 80 90
Temperature
Figure 1 %of
Activity
100 o Cr control
" Cr 55"
• Cr 60"
80
A Cr 65"
o Cr 2-control
60 (J Cr 2-65"
I'l Cr 2-65"+65"
40
20
0
50 60 70 80 90
Temperature
Figure 2 ~l"tii1.liru\ with

The Thermal Ve",atu,ratiIJf'l 155
Activity
100 o Ie control
• Ie 55"
A Ie 60"
80
A Ie 65"
Ie 2-controJ
60 [J Ie 2-65"
D Ie 2-65"+65"
40
20
o~~~~~~~~~~~~~~~~~~~~~~~~
50 60 70 80 90
Temperature
Figure 3 Reduction in GGTP in ice cream as of on;gmal with
mClreasmg heat treatment tenlperatlJre.
The pre'trea,tmfmts
the corresportduig
trials there was a low level of
treatment at 76°C and no
The effect of heat treatments on GGTP

with up to 11 % reduction in
prcKiucinlg a reduction in
in the rate of derlatlJlratlon
increase in the
that found for milk.
78°C for ISs minimum.
The transition in the ice creanl mixes was also

this was not as clear-cut as for the milk and Cfeanl.
cn~mg:e01vef tenlperatlufe was in line with for heat treatment of a range of
produc:ts 6 where aw was found to be the most iml)Ortant factor COlltrclIlir12 the rate of
deactivation of OOTP on heat treatment.
pre:trea.trrlen'ts were to make small reductions in the of

treatment, but these had no effect on the UJ tlmate
was lost Thus for a test based on the absence of
appear to have no effect. Thermisation
apJ)ro"imatelly 10% estimations. Below
for the ice cream then the
thermisation is not in the
the are to sweetened
Carter et reporu~ overall deactivation rates of not more than 1.1 %

with ice cream mixes at 65.6°C. Thus at at or the
transition the should be a more heat labile indicator
such as alkalule pll1ospb~ltase.
Agnclllltlire. Fisheries & Food for their
REFERENCES
1. The Milk and Dairies (St('JfI(iara~tsaltton ImrJl{)rtJztininl Rjegulatw,ns 1992 • SI
1992 No.
2. Milk HMSO.
3.
4.
5.
6. Thermal inactivation of garnma-glutun~fl tr:anspeJ)tidase

EnJ~ro'co£:cus.lW;;(;Uljr" in milk-based systems. .lV£&, 'Il~.
7.
8.
9.
10.
Keeping Quality of Pasteurised and High Pasteurised
Milk
J.

READING
Introduction
There is much interest in eX1~en(1111g
wi thout a cooked flavour. M":::III",t-,...,re
are: raw milk heat treatment conditions, extent of poI5t-prc)cesa:lng
contamination and Of these,
contamination is considered . However it were
beneficial to do so, this could be reduced. Therefore, it was
fel t opportune to quali ty under condi tiona where
I
prC)CedUll:'es were used to contamination to very low

There is evidence about the effects of conditions on
the temperature from 12 to 80 and 95 or
time from 15 to 30 or 45 s was to reduce
(1). Also t condi tiona of for 1 s was
activate spore germination and decrease quality (2),
for 5 s was reported to inhibit spore added
double cream (3). In terms of it would be to use
,u_I;lI~,I.'lW conditions which did not a cooked flavour in milk.
StCltr~re temperature will also have a marked influence on keeping quality.
imttro'tres as the temperature is reClUCieCl.
The aim this work was to treat milk at
for 2 s, in the virtual absence of PPC and to de1:el::m.ille
at two different storage temperatures { 2 and
Blparimeatal Coaditiou8
Raw milk from same batch was at two sets of conditions:
for 15 s and for 2s. This latter process was selected because it was
on the threshold of cooked flavour.
An APV Junior UBT was used to process the milk: it has been modified
to allow by use of the hot water set, or
temperatures by using steam.
Po~st-'pa:stE!ur'isiilti.on contamination was reduced circulating hot water at
about for at least 10 min, the section beforehand.
All temperature instruments are calibrated and certified at
intervals.
pasteurised milk was collected in 8terilin bottles, in a .Qltl.,u.:i ....
flow cabinet. from each heat treatment were stored at and

Standard plate counts (8pe) and Aerobic spores counts (ABC) were determined
158 lJwlcht!.·ml~,try of Milk Products
at weekly intervals, for three weeks,

Five separate samples were analyzed for
Once analyzed, samples were discarded.
WASTE CHILLED WATER
HEATER
A-Feed C-Pre Heater E-Cooler G-Second Cooler

B-Flow D-Homoqenizer F-Holdinq Tube H-Final Product
PIG I PASTBORIZATIO. PLAHT SCKBKB
Results
SPC and ASe counts for raw milk are shown in Table 1
Table I SPC and ABC COUDts in raw milt, log (ctu/ml)
SPC counts and ASC counts for the heat treated milks are shown in Tables
2 and 3 for a period of 22 days. Two for each combination of heat
treatment and storage for 36 days before being
analyzed. The results are 5.
Table 2 SPC counts during storage, log (cfu/ml)
Keeping Quality Pasteurised Milk 159
fable 3 ABC couats during storage, log (cfu/ml)
pH values for heat treated milks are shown in Table 4.
fable 4 pH of raw m1lk and changes during storage
RAW 6.7 6.1 6,6 6,1 6.61
PASTEUR 11512 I 115110 I 72/2 I 72110

• day, 6,1 6.7 6,6 6,7
6,8 6,8 6,8 6,8
Clay 3 6.1 6.7 6.5 6.7
6,1 6,6 6,6 6.6
day 7 6,8 6,7 6,6 6,5
6.8 6,1 6,7 6,7
dayS 6,8 6.8 6,7 6,7
6,8 6,8 6,8 6.7
day " 6,7 6,5 6,1 6.6
6,6 6.5 6,8 6,7
day 14 6,8 6,7 6,1 6,1
6.8 6.1 6,1 6,6
day 16 6.1 6,7 6,6 6.4 ""
6,1 6.7 6.7 6.6
day 18 6,1 6,6 6.7 6,4
6.1 6,7 6,7 6,5
day 21 6.7 6,6 6,7 6,1
6.7 6.1 6.7 6.2
day 23 6,7 6.7 6.1 6,1
6.1 6.7 6,7 6,3
I day25 6.1 6.1 6.7 6,4
6,8 6.1 6.1 6.0
day 36 6.7 6,6 6,6 6.2
6,6 6.6 6.6 5,9
"the mIlk was aefectecl sour since tnlS day
DisCU8SiOD aDd Conclusions

The of the raw milk was excellent. The mean SPC
ASC was 1730
The heat treatments used had little effect on ASC and achieved about 1
decimal reduction for SPC.
The results for ASC and SPC during storage are i n . 2 and 3.
8 15 22
STORAGE DAYS
rig 2 ABC COUDt for milk daring storage
8 15 22
STORAGE DAYS
rig 3 SPe COUDt for milk during storage
Keeping Quality Pasteurised Milk 161
Milk pasteurised at for 15 s and became unacceptable

after about 15 days. However milk stored at was still acceptable after
samples analyzed after 36 days also acceptable total
counts, with values well below . This is well below
is considered to be on the threshold of acceptability.
Milk treated at for 2 s and stored both at lOGC and was still
acceptable after 22 There was little difference in the quality of the
milks (Table 5).
Table 5 SPe and ABC COUDta after 36 days atorage
SPC ABC
Z·C 10·C z·c 10·C
7Z·C 1. 78 XXX 3.40 XXX
3.18 XXX 2.82 XXX
115·C n.q. 1. 30 2.91 3.06
1.00 4.20 2.82 2.30
after 36 days storage were sti 11

advantage in using the harsher
Statistical of the results showed that variations in ABC counts

were most af1:ec1ted by heat treatment conditions and storage time and less
so by temperature. On the other hand SPC counts were influenced by
all three
Only bacteria were found in samples analyzed by gram staining
after storage.
There is scope for the keeping quality by reducing post ...
PAlstE!Ur'isistjlon contamination. attention should be paid to the role
by raw milk quality and storage temperature.
Rafereacea
Kessler, H.G. and Horak, F.P., (1984), l1Ll£nmJi.!iUS~U,
A.H., Griffiths, M.W.
H!1~Wl!m.l!.ShlbUt 38, 641-644.
, Hunois .. Y., Philipps, J.D. and Muir, D.D., (1986),
ru&h![!!Jum!~lU., 41, 403-405
Fouling and UHT Processing

READING
ABSTRACT
has been designed and ~~·nal~~"~+'A~ a

and a cooler. It has a variable rate it is
t'\~''''~t1~+-a to between 100 and few seconds. Flow
tellll'P~erlilt\Jlre's are monitored through a system. A
has been which allows real calculations
heat transfer • The can be used to
monitor the extent of fouling and cleaning the of cleaning
for UHT processes.
INTRODUCTION
is the adhesion of material on surfaces of heat

milk there are two distinct
which is formed between has
content 30-40%,
are
are calcium
of the
passage,
DESIGN OBJBCTIVBS
and 163
3000
.... ~
~
~
.t"L..I"L
-0--
...n.
1.J
...!J..
~ ..... -- J'"L"" ..., '"' ...
2000
1000
o
10 20 30 50 90
Figure 1. The Overall Heat Transfer Coefficient (OHTC) the sterilizer when soft water is heated
to a temperature 140°C proving its long- and short-term stability.
The effects of often

become obvious when some time
two or three of the
progress of the a few hours more. This
means that a will be and
therefore the will be . The
cost of milk for a with even a small
commercial heat £100 whereas with the
miniature UHT In order to minimise the
amount of that a maximum
flowrate necessary.
The effects of on and on flowrate have
to be measured in order to be able to measure the
cnanqes of overall heat transfer coefficient which has been
chosen as the characteristic parameter of the process.
DBSCRIPTION OF THB APPARATUS
The
exchaDqers
cooler
The is hot water, heated
is controlled with an accuracy
The water comes an external source and has no
tendencies. The dimensions of the
1. The can raise the
from ambient (lS±2 up to 8SOC when
The he.ter (sterilizer) is heated pressure
(O-S 1. At the steam exit of the heat
a thermostatic It is to
and use the heat
~
21
1) Product
21 Water Tank
31 Three·way valve
41
5'
61 Pre-heater
Valve
7) Hot Water In
6 8t Hot Waler Out
9) POSition of Thermocouple
10) Main Heater
Steam Outlet
Steam Inlet
13) Position of Thernloc:ouple
141 Position of Thern~oc:ouple
un Position or Thefl'1rloc;oIJlpie
161 Cooler
17, Cooling Water Inlet
18) Cooling Water Outiet
1 19) Valve
Valvel
, 19
201
211 Position0'
Flow Meter
Pressllre
24... ~.r." w": Transdure IPll
.. 221
231
Position of Pressure
Transdme (P2)
posirion of PressufC
lransdUle (P3)
tl:!
24) Posltion 0# Press! ~.
Transdure Cf4)
~
5'
.a.
1 ~
lOj:
"'tI
a
~
Figure 2. Block Diagram of the miniature UHT plant. The flow-path of the product is the dotted tine. a
Fouling and UHT Processing 165
Table 1. Dimensions of the Heat

Preheater Heater Cooler
0.445 0.985 0.48
(10') m) 72.3 72.5 72.3
2.667 2.667 2.667
(10.3 m) 0.254 0.254 0.254
2 2
at alower
controlled a pressure
pressure there is a
condensed to drain from the
rotate the heater and have it in a
horizontal . The dimensions of heater are
1. The Pd~~dQe tube is extended before the heat excnan,ge:r
for more than
in the heat excn.anigetr
heat
flow is counter-current
tube in order to reduce the
from the milk
raise the temperat,ur'e
maximum
which the instruments The heat
to allow tubes removed and
of the heater is before each
and the external surface of the tubes is tested for
or corrosion. The outer surface of the shell is
to minimise heat losses.
The cooler uses mains water as shell-side medium with a
of 1 0 ° C with no
The dimensions of cooler are
tubes when more is
moves under the action of a multi-lobe
pump which with a needle
pressure valve maintains inside
to avoid and . Before
there
maintains the same diameter

2.667' and number of bends is to a
material of construction is 316 stainless steel.
166 Bio,che1nist,ry of Milk
INSTRUMENTATION
The telmpE:!rel.tl.llre
at the
heater
exit of
the
via a
pressure
in
accurate The
calibrated with water and milk at ambient
additional calibration was done with water at
in order to check if the difference in te~mt)el~a1~u:re
the calibration line and it was found that it
3000
2000 ~--------~~----------------------------------~
1000
o
o 50 100 150 200
TIME (min)
Figure 3. The Overall Heat Transfer Coefficient (OHTC) of the steriliser when reconstituted
skimmed milk (pH:6.761 is heated to UHT temperature (140°C). The OHTC
calculated Equation
allows real
Coefficient (U)
water and
(1)
U=
where
G
CLEANING
A combination of the standard

_~,,~~_u and the Almas-Lund nrot:.OCOl
achieve maximum removal
• At the end of each run the st:.anaara
and before the start
when the
flow is
the
to
there are
It has been observed
at a rate at the
its value at the .,,_~~._
REFERENCES
1. Tissier J.P, Lalande M. device for

_~n~iu; milk on surface,t
Progress, 2, 4, p.218-229
2. Sandu C, Almas K. (1984 "A
heat in
and
.. ed.
3.. Almas __ ~wu.u~ and Characterisation
of stainless 23,
p.29-39.
Ultrafiltration of Sweet Cream Buttermilk
J.

READING
1. INTRODUCTION
Even buttermilk is utilised in the as a source of SNF for

repJlace:meJllt of skim its full in view
of lecithin content Membrane over the last
two decades. the factor in the of ultrafiltration
( in the is the fall in flux with time due to concentration
pol;artsatl()n ( a n d of the . Membrane UF of
has been limited information is available
proces~;ing of hllt1t""'1"nr'll11r
There is a distinct difference in the flux between buttermilk and sweet

The concentration and state of some constituents such as
DroiteulS and calcium in these seems to be for these
differences. The aim of this is to compare the characteristics and CP
UF of skimmed milk and sweet and to assess the ootlentlal
of UF for utilisation of buttermilk.
- To determine the difference in flux between buttermilk and sweet
To determine the reje:cu()O characteristics of the cOl1rloonen,ts in buttermilk.
To determine the effect of various col1rtoonel1lts ( concentration and on flux

and of membranes.
170 Hto,che:lnist,ry of Milk Products
2. MATERIALS AND METHODS
A Paterson International tubular ultrafiltration

membranes (po,lyetbelrsuIph()ne, area 0.8 and molectUar
was used. All the eX]JertJrnel1lts mode
retl1rning both retentate and perlrneate
Ultrafiltration was carried out at inlet pressure of 0.5 MPa and outlet
pressure of 0.2 MPa. The retentate flow rate was maintained at 1360
Rej~lQ!l : The relc:~ti()n of any feed cornpemelnt in a membrane process is defined

as
Cf= Concentration of a corDpemelllt in the feed

Concentration of a in the nPT'mp",tp
Water flux after

FI=
Initial flux of pure water Tnt',,",,,u,,t'\ the membrane
This deslgmltes gradiellt between the Up~itream ( retentate and

downstream (penm:~te the membrane. In it is calculated from the
average pressure at the inlet and outlet the module and in
units of MPa.
: This deslgn:a.tes uressure difference between inlet and

result from an increased flow rate of
=
If ltl"ati,rtra,tinn of Sweet Cream Buttermilk 171
3. RESULTS
of buttermilk COI1DPared to other oroClucts during UP under

in 1.
-Sw.etwhay f::rWbol.milk
milk -Acid wll.y
+Sw.et cr..m butt.milk
5 10 15 20 25 30 35 40 45 50 55 60
Time ( min,)
1. Flux of some UP under total

mode
of UP under
- Sweet whey + Buttermilk

tOO
-....
1:.
80
<':'
E 60
-
X
:J
u: 40
20
5 10 15 30 45 60 20 180 240 300
and buttermilk ofUF

under total
FI in buttermilk and sweet whey UF under total mode is ClelPlCteC1 in

3.
•5
.i
3. I=<nlllltrUy UF under total

mode
Cl1clD21Og the transmembrane pressure on flux of buttermilk and

sweet in 4 and 5 res1JeC1tivelv
Pressure drop=O.3 MPa
10
5 10 15 20 25 30
Time
4. Effect transmembrane DreSSUlre on flux of buttermilk

-0.25 Mpa +0.35 Mpa *0.45 Mpa 1-------------.
40 Pressure drop= 0.3 MPa
20
10 15 20 25 so
Time (min.)
5. Effect of transmembrane pressure on flux pattern of sweet whey
Flux rates of buttermilk and skimmed milk during batcb concentration are in
6.
50r----------------------------------------,
CF = Concentration Factor
40
30
~ 20
u::
10
15 30 45 60 75 90 105 120 135 150

Time (min.)
6. Batcb concentration of buttermilk and skimmed milk

ReJlectlon of major components in buttermilk UF in total and batch

concentration are in table 1 and table 2.
0 30 60 i 120 150
Fat 0.75-0.77 0.80-0.83 0.83-0.85 0.85-0.88 0.86-0.88 0.86-0.88
Protein 0.95-0.97 0.96-0.98 O. 0.94-0.96 0.95-0.97
Lactose 0.29-0.32 0.1-0.14 10-0.12 08-0.10 0.09-0.11 0.08-0.11
Table 1. R~iiection of major components in buttermilk UF in total

mode
Tillie (lIIln.)
0 60 120 150
Fat 75 0.81-0.8) 0.83-0.14 0.85-0.88 0.86-0.90 0.92-0.96
Protein 0.95-0.98 0.94-0.97 0.93-0.95 94-0.97 95-0.98 0.97-0.98
Lactose 0.48-0.50 0.2)-0.:11 0.15-0.22 0.09-0.14 0.06-0.10 0.01-0.02
Table 2. Rejection of major COI1Il00nents in buttermilk batch COl1lcel1ltralion
4., DISCUSSION AND CONCLUSIONS
The initial flux was lower for buttermilk than for sweet but flux stabilised
Buttermilk gave rise to lower flux rates to skimmed milk
cOIlDooIsition. The initial flux rates of sweet and acid were
than skimmed milk and whole milk. the in flux

with time was also in products. an extended run of five hours
cornpc,sitj,on. the flux much faster in buttermilk than sweet
rel;Jltiol[}shlLP between FI and flux rates for buttermilk.
the reduction in flux rates was associated with
decreased flux reduction is by in sweet
FI for buttermilk was 0.4 after 5 minutes to 0.32 after 60 minutes.
this time flux did not that CP was the process.
In sweet FI was 0.5 after 5 minutes with a decline over
the next 55 The flux also fell this that flux was
controlled An increase in transmembrane pressure in buttermilk form
0.25 MPa to MPa had no effect on flux rates. in sweet
traJtlSnlenlbniUle pressure from 0.25 MPa to 0.35 MPa increased
that with buttermilk the formation of a CP
cOlrpl€~ with the first few minutes of was
reslponslblle for lower initial flux. batch the rate of
concentration of buttermilk was lower than for skimmed milk. The CF achieved in
buttermilk concentration was smaller because of lower flux
rates. of lactose was ) in buttermilk at the
beg;inn,lng of the process, but was to 0.1 after 1 h OPf~ratLon.
5. REFERENCES
1. J.L. Maubois. Llt....l~~=--'-.1--~~ 55.
2. R.S. Patel and H.
3. P.S. n.M. Barban,o. and M.A. Rudan, L&....oI~~~". ...... v ..... , 604.
Subject Index
Acid milk, 2 Casein (continued)

Adjunct cheese starters, 1,10 industrial production,
peptidases, 10 micelles, 3,95,97
proteiDases, 10 milling, 96
Affinity chromatography rennet, 96
recombinant chymosin, 78 solubility, 95
Alkaline phosphatase, 152,156 thermal stability. 95,98
Amino acids Caseins (bovine)
in 1,33,49 amino acid sequences, 7-8
in milk, 32 as1- , 2,3,4,5,6,15,26,42,84,94,143
6-Aminohexanoic acid, 2 Ctt2- , 3,4,5,6,84,94
Aspartic proteinases, 73 13- , 2-6,15,16,26,42,67,84,94,
loop structures, 73-75,80-82 97-98,143
Aspergillus oryzo.e peptidase, 143,144, &- ,6
150 K- , 3,4,6,42,84,,94,96,98
'1. ,
Bacillus subtilis proteinase, 143,144, para-K- , 3,4,96
147,15Q J3-Casomorphins, 98,108
Bacteriophage, 47-49 cathepsin
Bitter peptides, 6.49.50,58,67,143-151 Cheese
Bovine serum albumin, 94,104 adjunct starters, 1,10
Brevibacterium linens, 1 amino acids 1,33,49
bitter peptides, 6,49,50,58,143-151
Carboxypeptidase, 15,34 coagulant, 1,1,4,50,72-82,96,98
Casein curd, 1,96,98
bioactive peptides, 98,108 flavour, 41,43,47,49-51,58,83,
bitter peptides, 143-151
debittering, 143,144,146 non-starter flora, 1,2,10,14,23,24,26
fractiooation, 145,146,148 peptides, 1,15-22,25,144
cryodestabilisation, 96 fractionation, . . .., ......... 1 ""'lI""T
drying, 96 ripening
emulsifying properties, 97 biochemistry of,
ethanol precipitation, 96 flavour development, 83,88
foaming properties, 97 e:lvc:olvs,Js in, 1,95
fractionation, 97 llPOlVsis. I
genetic 95 proteolysi.s 1-10,15-22,26,50
human milk, 97 starter bacteria, 1,32-44,47-53,95
hydrolysates, 143-146 varieties
hydrophilic peptides, 148 blue mould, 1
Cheese (COlltinlled)
varieties (colltinl1ed) Dtpc~cllase, 13-14,34,35,81,89
EIe<~oc:HaI'fSis. 91,100
Dutch,
Gouda, 2,10 Fermented dairy products, 41
Mesbanger, 2 Flavour peptides, 50,51,56,58,83,88,
Mozzarella, 143,144,148
surface 3,14 Fungal 143
whey, 94,96,98-106 Gel filtration

Chbana whey, 121-132 bitter 143,145,141
concentrates, 121,128 cheese peptides, 16,19,21,144
ohv~)icaJ protterbc~s, 128-131 recombinant 18
powders, Genes in lactic bacteria
comparison with cheese whey, Campbell-type 39,41
129-131 chromosomal integratioo, 39
com.post1tJon, 128 expr'essic,n, 39,41,59,62-66
emllllsif:Ving pro~lertic~, 129 food-grade selection markers, 40,42
foaming orooe111es. replacc::ment recombinatioo in, 40
gelation properties, 130 soec:1fv1l1l2 peptidases, 31-39,66-68
128,129
130 Uluconlc acid-a-lactooe, 1,10
-Y-Glutamyltranspeptidase, 152-156
Glycomacropeptide, 94,96,105,106
action on ~'1Ul), HPLC

alternative coalgul2lOts, bitter peptides, 143,145
amino acid seq'JeDc:e. casein hydrolysates, 143,149
combined plal,milll,6 cheese peptide:s. .M\.),-J:..., •
.L..JJ ...iJU
DNA seqllenc:ing,
eX1)res~;ion in Trichoderma 15-19 Immunoglobulins, 94,104-106
loop structures, role in infant 106
protein engmecmng, role in 106
recombinant, Infant feed formulas, 105,106
anaJlYSIS of, 19 action 00 4
crystallisatioo 19 loo-e~cnallgec~omarography
purification, 18 cheese peptides, 18
specificity , recombinant chymosin, 78
stability, 74 whey proteins, 101,104
structure, 12-15,80-82
crystallography, 80 Lactalbumin, 103
coatgul:ants (see also chymosin, rennet) Lactalbunlin, 94,104
(1-
Lactic acid, 41,95

resll<tuaLl. in 4 Lactobacillus, 1,9,12-14,47,86
Colloidal calcium phosphate, 95 Lactococcus 7,8
Colostrum, 105,106 amiJllOJ)C'I)tldlase, 34,35,66-68,83-91
Cop'rec~pita1:e, 101 cell
Subject Index 179
Lactococcus laeds (continued)

end<)JJepticW;e, 10-1 U__ "","UI">. 114,115,120
enzyme enf!:meemle: seccmdarv struCt1lre, 118, 119
growth reqlLlire~ments, J"" ....""-........ c;,.... ~.v site-directed mU1:ageneslls, 114-126
expression. 119-120
122
catalytic domain, 61-63

function, 60-62
homology to su1>1111s1n,
macUvaltion. 63
inhibitors, 63
loop struCt1lres, 61,63,65,66
membrane anchor. 61
Pi-type,
7,8,9,16,42,59,60,62,64
r ... 7,8
··LYUC.
punUca1t1on, 63
release from cell wall, 63,64
site directed 62-66
spec::lDClity 61,62,64,65,84,85
64,65 Microfilttation,
Milk
proteinase ~f"ihl'inO'
62-66 cooked flavour, 157
heat 157
keeI)1De: quality, 157-161
~-Lactoglobulin, 94,104,114-126 mlclrol>llolo!l:tCal quality. 157-161
acid 123 pas1:euri.saU4J:n plant, 158
agf/:ref!;aU()n mechanism, 121 pH 159
cbymosin 123 post-pt'QCf$S1tlg contamination, 157
conformational structut'e. 157-161
crystal struct11re, temperature, 157-161
denaturation, 1
functional 124 conlpo~.iUon. 162
gelation,
incorporation into cheese 116
in vivo 114 miniture proces!iine:
UpocaJin 115 plant clesLll1D:g,
loop structures, 118 plant per1fornl8Dc:e,
molecular 115-124 Milk
nrinrlGru structure, 116 compoS11Don,94
Milk protein (continued) Psychrotrophic (cout.)

concentrate, 96,107 off-flavour production, 152
functional 94, 107,108 stability of UHT milk, 152
engillleeJin2. 108
variants, 94 Rennet, 3,4,95,98,106
hetE::rogjeneity 94 Reverse 99,127
hydrolysates, 108,143
modification, 107 Sodium caseinate, 97
Milk retinol-binding protein, 114 Spblero:sil process, 103
Mucor pusillus, 73 Starter
Nisin, 37,56,57
NisP, bacteriophage, 47-49,52
cultures, 47-53,58,83
Papain, 143,144,147,149 lactic acid production, 47,48,56
PCP, 35,36,87 proteinases, 7-9,15,49
Penicillium camemberti, 1 inactivation of, 63
Penicillium roqu,eforti, 1 purification, 63
PepA, 11,14,35,36,87,88 site-directed mutagenesis, 7,32-44,
PepC, 11,14,34,35,37,87,88
PepN, 10,11,14,22,34,35,38,56,58, specificity of, 7-9,15,61-64,84,85
66-68,87 peptidases, 2,9-14,42,49
PepO, 14,22,34,35,38,43,87 functions of, 42,67
PepP, site-directed 32-44,
Pepsm,4 52,53,56-68
PepT, Strt,vtococ_r:us. 7,10,47
Peptide transport, 10,34,56,58,89
PepX (PepXP), Thermisation, 152-156
43,86,87 cream, 153-155
Phenylketonuria, 106 ice-cream mix,
PIP, 13,14,35,36,87 milk, 152-155
Plasmin, M.J.,,LV.,"'''' sweetened desert 156
action on 5-6 Thrombin, 6
combined action with chymosin, 6 Tripeptidase (PepT), 10,13,14,34-36,
inhibitors, 2 38,43,87,89
specificity, 5,6,15
Prolidase, 13,14,35,36,86,89 Ultracentrifugation, 97,101
Proteinase also PrtP, plasmin) Ultrafiltration
acid,im milk, 2 buttermilk,
aspartic, 73 case:ina1:e, 98
genetic engineering, 74 concentration polarisation, 169,113
indiginous milk, 5,6,95 fouling index, 170,172,175
loop structures, 73-75,80-82 membrane flux, 169-115
microbial, 4 membrane 101,133,169
. starter 7-9,15,16,49,73-82 permeate, 170
Proteose-peptones, 5,16,94 retentate, 170
PrtM, 33,34,39,59,60 skiDlmilk, 96,107 ,169,171,174,175
PrtP,7,8,15,33-35,39,41,42,56-68,91 whey, 101,102,104,127,169,171-175
Psychrotrophic microorganisms, 152 whole milk, 171,175
control of growth, 152
Subject Index 181
Vistec process, 102
Wbey
acid,98
temperature 140
composition, 104
demineralisatioo, 100,104 bioactive neotides.
dried, 98-101
powder, 99-101
pr04~SSln2. 99
reverse 99
sweet, 98
Sync~esjIS. 124
uses, 99 functional _n_hAC!
Wbey protein, 94-97 101,102
aggJ"e2a1t1on. 133 pressure, effects 104
products, 98
mechanism, 134 properties
microstructure, 135 solubility ,
particle E:LU""''''I. thermal stahilitv
11111 111111111111111111111
9 781855 737754

Biochemistry of Milk Products 1994

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Biochemistry of Milk Products 1994

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Milk Products

Biochemistry of Milk Products

WOODHEAD PUBLISHING LIMITED

First put)Usl!1ed ChemIstry 1994

This book contains information obtained from authentic and

Trademark notice: Product or {'.nt'nnr·~t.. names may be trademarks or ree..lste:red

British CaltaJclgumg in Publication Data

Printed in the United .t","U10V'UIH Llg;htnmg Source UK Ltd

of the whole food and

ago, probably in the Middle East where the

In order to be successful in these obl,ectJves it is to understand in fine

The second research covers the functional behaviour

methods improved to the extent that larile-!;calle plroauC110n of individual protlein

as the of what was the

Prolteol'vsis in Lactococcus Lactis 32

New Starter Cultures for Cheese Rlvenulg 47

hni~meerulg Pivotal Proteins for Lactococcal PrOlteol'VSIS 56

Petl~ti<ulSeS from Lactococci and Secon<llary Prolteol'vS1S of Milk Proteins 83

Functional Milk Protein Products 94

En~~neeri[lg Studies of f3-l.act()glclbulin 114

Functional of Chhana Products 121

Thermal Agjue~~atl()n of Protein Concentrates under Fluid Shear 133

The Effect of Thermisation on the Thermal Denaturation of 152

Keeping Quality of Pasteurised and High Pasteurlsed Milk 157

J.:i'nnliin'" and UHT Proces~;ini 162

Ultrafiltration of Sweet Cream Buttermilk 169

DEPARTMENT OF FOOD CHEMISTRY, NATIONAL FOOD BIOTECHNOLOGY

The conversion of milk to cheese curd is the first

2, CHEESE RIPENING AGENTS AND THEIR CONTRIBUTION TO

cheeses can be summarized as

contribution of to the initial Inu1 ....... U.!C"'" prcmoum;ed than in

3. SPECIACITY OF THE PRINCIPAL PROTEINASES AND PEPTIDASES

Most of the GMPs are lost in the

5.2 in the presence

marxianus var, lac/is

amount of C08l2Ultant reulinc:ld

Cbeeses wbicb are cooked at a

lDd.lgen01JS plrotf:maLses in milk bas been recogluze~d for a

The theoretical combined action of "h,,,.,...,, .......

their combined action could ~.I"""''''A

those of the:rm:ophillc )ln~l71O(X}CCUS

...... ... ... ...

... ... ...

...... ... ...... ...

Amino acid sequence posOIucm of the

It Jl T K It II 11 5 5 S It It 5 I 5 0 It T YZO It Q I It If K A II l!' S It It If 4 S T F C40 K I V V II If A If E ESO

Amino acid sequences of &s A and K-casein B the

••... • • ... • • •••...

... • ...• .... •... •... ••... ....

CleaV:loe sites of cell

tvn,u'J;1,lIv contained a GIn or Ser residue and are

pe)Jt1<leS from casein than

substrate are rendered inaccessible due to hY4jrCtDhobic interactions of tbe C-tenninal

Lactococcal Dei)tldtasc~s surnm.an:z.eo In

144} have used lactobacilli

lactoeoccal oli~n<k)peptjdase; 2. MEP, metallool'ldopeptidase.

Peptidase Strain Substrate MWIkDa opt. Activity Subunits Class Reference

DIP I\, IJJ. delbrueckii ssP. btd$laricus B 14 dipeptides 51 50 mctallo

8. dipeptidase. TRP, trippetidasc. 10. proIidasc. 11. PIP, pttlline aminopeptidase.