Bioquímica da Produção de Queijo

Bioquímica da Produção de Queijo

(Parte 1 de 3)

Milk Products Milk Products

Biochemistry of Milk Products

A. T. Andrews J. Varley

RSC Industrial ,-"v."",,_

The proceeidinl2:S First put)Usl!1ed ChemIstry 1994

Woodhead PUVtlishiing The authors have

This book contains information obtained from authentic and sources. material is with and sources are indicated. Reasonable efforts have been made to reliable data and but the authors and the for the of all materials. Neither authors nor the put)llsltler, this shaH be liable for any or caused or be caused

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tra<lenlaric.s and are used for and without intent to

British CaltaJclgumg in Publication Data A cata:loglle

ISBN-13: 978-1-85573-775-4 ISBN-IO: 1-85573-775-2

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

Preface of the whole food and share of a11 human with little or no pretrealtmcmt, such as are as maJlufactwrmg processes, so only quite a smalJ 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 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.

Biochemistry of Milk Products

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

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 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


Prolteol'vsis in Lactococcus Lactis

A.J'.Ht'lfmlrJrIJ<7IlQjn. I.Mierau. J.Kok and G.Venema

New Starter Cultures for Cheese Rlvenulg

B.A. Law Pivotal Proteins for Lactococcal PrOlteol'VSIS Vos and R.I. Siezen

ArutJIVSlS of CHY155-165RHI

_ ,._.,,:-R. BI,U1l(Jfell, J. Uusitalo and M. Penttila from Lactococci and Secon<llary Prolteol'vS1S of Milk Proteins


Functional Milk Protein Products

D.M.Mulvihill Studies of f3-l.act()glclbulin J.H.Morais Cabral and C.A.Batt

Functional of Chhana Products A.S. Grandison and A.R.Jindal

Thermal of Conditions Protein Concentrates under Fluid Shear

.'\tp"p"tl')n A.M.Dono.ld and L.F.Gladden viii

The Effect of Thermisation on the Thermal Denaturation of 'Y-Olutamyltranspeptidase in Milk and Milk Products

S. S. POIel and B.A. Wilbey

Keeping Quality of Pasteurised and High Pasteurlsed Milk B.Borde-Lekona. M.l.Lewis and W.F.Harrigan

J.:i'nnliin'" and UHT P.Kastanas. M.l.Lewis and A. Grandison

Ultrafiltration of Sweet Cream Buttermilk H. G.Ramochandra Roo, M.J.Lewis and A. S. Grandison


Bioc'hemistrv of Milk 152

Proteolysis in Cheese during Ripening p.


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 food. the of

2, CHEESE RIPENING AGENTS AND THEIR CONTRIBUTION TO PROTEOLYSIS nrl'ltp£1llvCil,J(1. in cheese varies from very limited to very mould The of nrll.tPll,lv'Cil''Cil range in size from

P1"l.tPl,lvtll' agents in cheese,.r."" .. on,gmate

COlDPjarabJe in size to a range of medium to free amino acids. the milk, starter oac:[enla first four sources are active in added to the coagulant, hn:zvl1!les from the based on the known identificiation of their

The use of model

Biochemistry of Milk Products on caseins or pel,tHles from cheese and, on the caseins in SOlut14::m.

cheeses can be summarized as residual resl1ltulg in the of the C03Lgulant sman 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 <:tr£\no:lv

This will focus on the in cheese on the individual and cas.eUI-mernfea pel)U<les ISOi(atlOn and identification of from Cheddar cheese.


Most of the GMPs are lost in the DaJra-iK-c:ascem remains attached to the casein micelles and is cheese. are

5.2 in the presence at the lower

4 Biochemistry of Milk Products marxianus var, lac/is were 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 amount of C08l2Ultant reulinc:ld

Proteolysis in Cheese during Ripening 5

Cbeeses w bicb are cooked at a bave little coa,2ulant on ('L,"li-CCllSellD solution.

lDd.lgen01JS plrotf:maLses in milk bas been for a pnlDClpal prclteinas.e is wbicb i.s active at bas a pH at -4.0.

of con:Slst.lD2 of

LYS2S·LYSZ9 LYSIOS·HisI06 LyslIl'Tyrll4 Argl!!.rAsPI84 ~t~ _ ~t _ t~ _ ~+ _ ~

Known PM


Proteose fl-28 f29-105 f29-107 f29-113 fl-105 fl-107


Probable fl06-113 fl08-lJ3 fll4-183 fl06-183 fl08-183 fl_113Iun.lilr •• h'\ oroteo:se 8

""."".+."".t'n of D on the

incubated with milk very similar to that of to rates of of to be a poor substrate for are considered not to be milk that other lysos()m,al 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


--~I~t~t--------~~~f--~t--~t~~~H~t-----,~ tl-CASEIN

_ C_HY-M-O-SIN--------------~~~t--Il.T-O----iatt~~-·~~f---~ 1 I 1

PLASMIN Potential combined action of l':hvn1to""ln and plasmiin in cheese during Ripening their combined action could

know, the action of these,,'\1""''''0 on the isolated caseins has not been

fact cOlmpleI10eIltalry the C-terminal in the we studied and it is not known whether concerted manner in cheese.

those of the:rm:ophillc considerable attention. The

It P I It Ll'Q&V


ItYIIVPQLIt v r P160

Amino acid sequence

A r sites of cell waH-associated prC)lel,nases SK 112 (ref. 87) and L


associated with the cell Cell wall-associated and -types .

posOIucm of the

8 Biochemistry of Milk Products T It S It It T It Q I If A I S It It If 4 T F C40 I V V If A If E



It-Casein B

ItIAItTIPIQYV y y y Y I If Q F P P 'i60 Y A It I' A A V S A 0 I w v P T



Amino acid sequences of &s A and K-casein B the of the sites of cell waU-8lSSCK:U:llted of Lactococcus lcu'ti:, ssp. lactis NCDO


• •• ...
•• •
1'8 I'PA OS
• • .... •
• •

• • • • •• 'l'I.IltllltIlIlPO' Ell O"O'l'I)I.OPI

• ••
•• • ... , . ....
I' MFI' s I' 1'0ltAVP PQII MPIO'AFI.I.YQ 1''1.01'200

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 pn)temclse:s on such does not appear to Cheddar as detected

1'1 1'1

'191 I' I' r .,., .

"" . "... ."

. ". " v v II»

" 111I1'1'£l TV VT TA substrate are rendered inaccessible due to hY4jrCtDhobic interactions of tbe C-tenninal of the moleCllUe.


Lactococcal 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.,

Table 1. Peptidase


Atninopeptidoses pep' PCP

Peptidases of Lactococcus and Lactobacillus Strain Substrate peptides peptides peptides

Glu-p-NA lactoeoccal 2. MEP, metallool'ldopeptidase. XAP, X·prulyi.dipeptidyamiflopcplid.'tsc.

MWIkDa Opt. Adivity pH



Subunits 2 e ~

Class Reference metailo tOO -e' metaiio 101 S· neutral ~ metalio metalio metalio metalio l<E serine no serine I lt2 113

Table 1. Cont'd. Peptidase Strain Substrate MWIkDa opt. Activity Subunits Class Reference pH "c

Amilwpeplidtues zeillus

I Lb. delbrueckii 1&3 78·91 6.2-7.2 475 meta10 1.5

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 ACA-OC2.U 98 6 .ro I melallo 122 165 7 .:'i>-5 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 XAPVIH bulRaricus L8l;-1.J7 50 3 serine 127

5.J 50 tbiol 128 ~

~ 4. general amino peplidase. XAP, X-prolyl-dipeplirl}' aminopeptidase. ~

'" d ~ ;:

Table 1. Cont'd. Peptidase Strain Substrate MWIkDa opt. Activity pH "c


DU,s dipeptides 25 and 34 7 dipeptide!> 51

DIP I ssP. H61 dipeptides 100 8 dipeptides 49 8 50 tripeptides 75 7

TRPU Wg2 tripeptides 1m-lOS 75 5 TRPm tripeptides 10.') 8.6

X-Prodipeptidcs 43 65·7.5

PRO X-Pro dipcptidcs 42 7.35-9.0 - PIpit f'ro..X-(Y) peptides 100 85, 37

DIP I\, IJJ. delbrueckii ssP. btd$laricus B 14 dipeptides 51 50 8. dipeptidase. TRP, trippetidasc. 10. proIidasc. 1. PIP, pttlline aminopeptidase.

Subunits CI85S mctallo mctallo 2 mctailo 2 mctallo mctallo mctaUo 2 mctallo mctallo


)32 10-l a ~ t;. s·

14 PepO

DIP t Leu-Leu

Biochemistry of Mille Products

ProtHiS-Phe PepC

HisYPhe the combined action of

Proteolysis in Cheese during Ripening 15 refs. 91, 147 and which has not been found in lactoc'OCC:l.


Isolation and identification of individual is fundamental to the cornplete 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 modified from involves of the waler·· 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 wlnh,tt"\!

fl06-209 and also 4S were nrflrn<:>,." DJrOaUCl of tbe action of evident. The tOflmatlon of 3 further to not plaSmltn or cell wall-associated is not in cheese cheese made Unli>utII1SJne<lI), 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

The sI0'Ner-mI2rallm2

These WiSN

Biochemistry of Milk the action of microbial eml'.Vn1eS e.g.

cell at

Water: 2: 1 Homogenize ( Stom3(:her or similar apparatus )

Centrifuge ( 10,0 9 x 30 min)

WSN Fat Re-extract as above UF, , 0 kDa membranes

Permeate l SephadG-25


I, H, lilt IV, V, VI, VII, VIII, IX

~ ~ J I A'-Acids I

Sep-Pak C8 or C 18 and HPlC

Fractionation scheme for cheese

57-1 and of Lactococcus f58-72 was also isolated from water-soluble fraction and found to inhibit intracellular lactococcal on

Proteolysis in Cheese during Ripening eN WISF made with Lactococcus lacti.')

4% pH fraction of 3 month-old Cheddar cheese cremoris SK 1 the of the caseins and the N-terminal of the orincloal oetlttld4es (* Undetermined

18 Biochemistry of Milk Products

4% pH a retentate a water-soluble extract extracts from Cheddar 2 to and of the water soluble (lanes 7 to 1)

0.6 , /

(Parte 1 de 3)