Studies on the Decarboxylation of Acetolactate in Milk
Products
T hesis p rese n ted b y
Dipl.-Ing. Britta Mohr
for the Degree of
Master of Science
July 1997
R e search Supervisor: D r. T .M . C o gan3
A cadem ic Supervisor: D r. R . D ev ery b
‘“N atio n al D airy P ro d u cts R e se a rc h C entre,
M oorepark,
F erm oy, Co. C ork.
bSchool o f B io lo g ical S ciences,
D u b lin C ity U n iv ersity ,
G lasnevin, D u b lin 9.
D e c la ra tio n
I hereby certify that the m aterial, w hich I n o w
su b m it fo r the assessm ent on the p rogram m e o f study
leading to the aw ard o f M aster o f Science is entirely m y ow n
w ork and has not been taken from the w ork o f others
save and to the extent that such w ork has been cited
and acknow ledged w ithin the text o f m y w ork.
Signed:
C andidate
ID N o :
D ate:
95971491
TABLE OF CONTENTS
ABBREVIATIONS
iv
ABSTRACT
1
LITERATURE REVIEW
2
Starter Cultures
2
Lactose Metabolism
3
Citrate Metabolism
4
Factors influencing Acetoin and Diacetyl Production
8
Branched Chain Amino Acids and Citrate Metabolism
10
Metabolic Engineering of Citrate Metabolism
11
Measurement of Diacetyl and Acetoin
12
Measurement of Acetolactate
13
Manufacture of Butter
14
Effect of Butter Cultures on Butter
16
Quark
17
MATERIALS AND METHODS
20
Bacteria
20
Media
20
Measurement of Diacetyl
21
Measurement of Acetoin
22
Measurement of Acetolactate
23
Measurement of Citrate
25
Measurement o f L-Lactate and Acetate
26
ALA Standard Curves
26
Model System
26
Conversion Rates
27
i
Growth Experiments
Pure Cultures
27
27
S c ree n in g
27
G row th kinetics
27
C om parison o f the tw o m ethods f o r the determ in a tio n o f A L A
28
Mixed Culture 4/25
28
L a b o ra to ry trials
28
E ffect o f solids
28
E ffect o f tem perature
29
C o m m ercia l trials
Quark
RESULTS
PARTI
29
29
31
31
ALA Standard Curves
31
Model System
33
E ffect o f oxy g en
33
E ffect o f m ilk solids
34
E ffect o f tem p eratu re
35
E ffect o f p H
37
E ffect o f m etal ions and h a e m in
39
Development of a New Method for the Determination o f ALA
40
Comparison of C u S 0 4 Method and Jordan and Cogan [1996] Method 46
Mixed Culture 4 /25
47
C om m ercial tria ls I
47
L aboratory trials
49
E ffe c t o f m ilk solids
49
E ffe c t o f tem perature
51
C om m ercial trials II
E ffe c t o f tem perature
Quark
54
54
57
PART II
59
Growth Experiments
59
Screening
59
E ffect o f o x y g en atio n on strains 999 and 1166
59
Effect o f leucine and valine on strains 999 and 1166
61
E ffect o f C u S 0 4 o n strain 999
62
Effect o f FeSO,, on strain 999
62
Effect o f haem in on strain 999
63
E ffect o f o x y g en co n centration on 1166 and 116 6 M 1
63
DISCUSSION
73
PART I
73
PART II
78
BIBLIOGRAPHY
81
ACKNOW LEDGEMENTS
92
iii
ABBREVIATIONS
ALA
a -a c e to la c tic acid
ALD
acetolactate decarboxylase
ALS
acetolactate synthase
ATP
adenosine trip h o sp h ate
BCAA
branched ch a in am ino acids
pP gal
p-D -ph o sp h o g alacto sid e galactohydrolase
C it
citrate
EM P
E m b d e n M e y e rh o f P athw ay
HPr
h eat-stable pro tein
Lb.
la cto b a cillu s
Lc.
lactococcus
LDH
lactate dehydrogenase
Ln.
leuconostoc
NAD+
nico tin am id e adenine d in u cleotide (oxidised form )
NADH
nico tin am id e adenine din u cleo tid e (reduced form )
NMR
n u c le a r m ag n etic resonance
PD H
p y ru v ate dehydrogenase
P E P -P T S
P ho sp h o en o l-p y ru v ate ph o sp h o tran sferase system
PK
pho sp h o k eto lase
PM F
p ro to n m o tiv e force
Str.
S trep to co ccu s
ABSTRACT
Studies on the Decarboxylation of Acetolactate in Milk Products
M ohr, B .1,2, R ea, M .C .1, C ogan, T .M .1 and D every, R .D .2
’D P C -M o o rep ark , Ferm oy, Co. C ork
2S chool o f B iological Sciences, D ublin C ity U n iv ersity , G lasnevin, D u b lin 9
T he effect o f d ifferent param eters on the d ecarb o x y latio n o f acetolactate (A L A ) to
diacetyl a n d a ceto in w ere studied. T he d istillatio n volum e an d the m ilk solids
co n centration h a d no significant effect on decarboxylation o f A L A , w hereas
break d o w n o f A L A increased w ith d ecreasing pH an d increasing tem perature.
O xy g en atio n in creased diacetyl pro d u ctio n fro m A L A , b u t diacetyl w as lo st from the
m odel system . O xy g en atio n did n o t have an effect on acetoin prod u ctio n from A L A .
M etal ions (C u2+, Fe2+) an d haem in caused h ig h b reakdow n o f A L A to diacetyl
during steam distillation, w ith Cu2+ b ein g the m o st effective. T he d ecarb o x y latio n o f
A L A w as a first ord er reaction.
A n ew m eth o d w as developed for th e determ in atio n o f A L A b ased on steam
distillatio n at p H 3.5 in the presence o f C u2+, w h ich caused com plete
decarb o x y latio n o f A L A to diacetyl. A L A concentrations w ere calculated from the
difference b etw een diacetyl levels in a sam ple in w h ich A L A w as com pletely
converted to diacetyl, and diacetyl levels in a sam ple w ith m inim al decarboxylation
o f A L A to diacetyl, w h ich w as achieved by distillatio n at p H 0.8. T he m ethod
co m p ared w ell to the Jo rd an and C ogan [1995] m ethod.
T rials w ere carried out at laboratory and industrial scale to im prove the
m anufacturing p ro cess fo r lactic butter. A n increase in tem p eratu re during
m anufacture increased diacetyl concentrations and converted m o st o f th e A L A to
diacetyl; it had no significant effect on aceto in concentrations.
C it+ strains o f L a cto co ccu s lactis subsp. diacetylactis, 999 and 1166, w ere grow n in
th e presence and absence o f oxygen, leucine, valine, C u S 0 4, F e S 0 4 and haem in.
E x cep t fo r oxygen, w hich increased diacetyl p ro d u ctio n and decreased grow th, there
w as no sig n ifican t effect o f these com pounds o n m etabolite production.
1
LITERATURE REVIEW
Starter Cultures
T he cultures u sed in m ilk ferm entations are called starters b ecau se th ey initiate or
start the p ro d u ctio n o f lactic acid in the m ilk. T he b a c te ria co m m o n ly found in
starter cultures are u su a lly present in raw m ilk as p a rt o f th e natural m ilk m icroflora,
and, therefore, can sour th e raw m ilk i f it is left u n d istu rb ed fo r a day or so. T his
ability w as u se d fo r m an y years in th e p ro d u ctio n o f starter cu ltu res for cheese and
other ferm en ted dairy p roducts w ithout kno w in g w h a t ex actly w as hap p en in g during
the souring o f th e m ilk or th at b acteria w ere involved. A t th e b eg in n in g o f this
century, as kno w led g e o f bacteriology grew , the b acteria in v o lv ed w ere identified
and the intentional inocu latio n o f m ilk and cream w ith th ese organism s to carry out
the ferm en tatio n b e g a n [Sandine, 1975; C o gan an d H ill, 1987].
T here are tw o types o f starter cultures, m eso p h ilic and therm ophilic. M esophilic
starter cultures h av e a optim um grow th tem p eratu re o f ~ 2 6 °C and are u sed in the
pro d u ctio n o f C heddar, G ouda, E dam , B lue, C am em b ert a n d cottage cheese,
cultured b u tte r and butterm ilk and sour cream . T h erm ophilic cultures have higher
optim um tem p eratu res (45-50°C ) th an m esophilic ones, w h ic h m ak es th em useful in
the p ro d u ctio n o f th e
so
called
“c o o k ed ” cheeses
like
S w iss
an d
Italian.
T herm ophilic cultures are also used in th e prep aratio n o f y o g h u rt [A ccolas and
A uclair, 1983; C o g an and H ill, 1987],
M esophilic cu ltu res are com posed m ainly o f L a cto co ccu s la ctis subsp. crem oris and
the closely related Lc. lactis subsp. lactis. B o th o f th ese o rg an ism s are generally
considered n o t to be able to utilise citrate (C it-), alth o u g h m an y cultures, especially
those used as b u tte r and q uark starter cultures, also contain lactococci th a t are able to
m etabolize citrate (C it+ ) and produce diacetyl. T his org an ism w as form erly called
S treptococcus diacetylactis. It w as ren am ed Lc. lactis subsp. lactis, due to the fact
that th e difference b etw een it and Lc. la ctis subsp. la ctis is a p lasm id that is
responsible fo r citrate uptake. It is n o w called C it+ Lc. la ctis subsp. lactis [K em pler
and M cK ay, 1979; S chleifer et al., 1985].
B esides C it- and C it+ lactococci, w h ich are involved in acid and flav o u r form ation
respectively, C it+ L eu co n o sto c sp. are also fo u n d in m eso p h ilic cultures, w hich
contribute to the fo rm atio n o f flavour com pounds like diacetyl from citrate [C ogan
2
and H ill, 1987]. T h e citrate utilizers are also often called gen erically arom a
producers.
T he ty p es o f aro m a b acteria present in m eso p h ilic starter cultures are u se d to further
differentiate th em . C ultures containing o nly L eu co n o sto c sp. are called L ty p e, those
containing only C it+ Lc. lactis subsp. lactis are k n o w n as D ty p e, and D L cultures
co n sist o f b o th C it+ Lc. lactis subsp. la ctis an d L eu co n o sto c sp. C u ltures w ithout
any flav o u r p ro d u cers are called 0 ty p e [C ogan and H ill, 1987].
T h erm o p h ilic cu ltu res contain Str. th erm ophilus and one or several lacto b acilli, i.e.
L a cto b a cillu s delb ru eckii subsp. bulgaricus, Lb. delbrueckii subsp. la ctis, and Lb.
helveticus [A ccolas and A uclair, 1983].
M esophilic and th erm o p h ilic starter cultures can be fu rth er divided into defin ed and
u n d efin ed or m ix e d cultures. D efin ed cu ltu res co n sist o f one to several phageu n related p u re cu ltu res w hereas m ix ed cu ltu res are n o t selected b u t h a v e evolved
from ‘g o o d ’ acid p ro d u cin g starters, w h ic h w e re first u sed in the late 19th and early
20th centuries and, since then h av e b e e n tran sferred n um erous tim es. T he n u m b er o f
strains in such cu ltu res is unknow n [Sandine, 1975; C ogan and H ill ,1987].
M ixed strain starters h av e a m ajo r advantage over defined cultures in th a t they are
quite ph ag e resistan t, due to the fact th at th ey co n tain m an y phage resista n t or phage
unrelated strains. W h en a phage attack occurs th ese strains grow , resu ltin g generally
in only a sm all d ecrease in acid pro d u cin g ability. B u t m ix ed strain starters also have
m any disad v an tag es, the m ost n o tab le b e in g th eir ability to p ro d u ce variable
am ounts o f lactic acid. T heir use h a s led to the d evelopm ent o f defin ed strain
cultures, w h ic h are easier to control [Sandine, 1975; S tadhouders an d L eenders,
1982; C o g an and H ill, 1987],
D efined stra in starter cultures can be d iv id ed into single and m ultiple stra in starters.
S ingle stra in starters consist o f one strain o f Lc. lactis subsp. crem oris or less
com m only Lc. lactis subsp. lactis. M u ltip le strain cultures co n sist o f tw o to six
(som etim es m o re) ph ag e unrelated strains o f Lc. lactis subsp. crem oris a n d Lc. lactis
subsp. la ctis [L aw rence et al., 1976].
Lactose Metabolism
L actose is a disacch arid e com posed o f galactose and glucose and is th e m ain energy
source for the lactic acid bacteria (L A B ) w h e n they are grow n in m ilk.
3
LA B h av e tw o different system s for tran sp o rtin g lactose. L eu co n o sto c sp. and
th erm o p h ilic cultures use a p ro to n m otive force (P M F ) system , w hile L actococcus
sp. u se g roup translocation, th e so-called ph o sp h o en o l-p y ru v ate phosphotransferase
sy stem (P E P -P T S ) [C ogan and H ill, 1987]. B o th system s require energy. T he exact
en erg y c o m p o u n d u sed in th e P M F -sy stem is u n know n, a lth o u g h it is likely to be
A T P as in g alacto se transport in Lc. lactis subsp. lactis [T hom pson, 1980]. Lactose
is tra n sp o rte d against a co n centration g rad ien t and o ften another m olecule is
tra n sp o rte d e ith e r into (sym port) or out o f (antiport) the cell d u ring the transport o f
lactose [C ogan an d H ill, 1987].
In th e case o f the PT S system , the energy source is PE P [M cK ay e t al., 1969]. In this
sy stem lacto se is transform ed into lactose-P as it is tran sp o rted into the cell. This is a
v ery co m p le x process involving four d ifferent p roteins, enzym e I, H P r (a heat-stable
pro tein ), fac to r III (a lactose-specific factor) and enzym e II. T he first three are
soluble
p ro tein s,
w hereas
enzym e
II
is
a
lactose-specific
m em brane-bound
c o m p o n en t [M cK ay et al., 1970].
C ultures w h ic h tran sp o rt lactose v ia the P M F sy stem h y drolyse lactose v ia Bgal to
glucose an d galactose. T he nex t steps in m etab o lism depend very m uch on the
culture itself. In Lb. helveticus glucose is m etab o lised v ia the EM P system and
galactose b y th e L eloir p athw ay w h ile all strains o f Lb. delbrueckii subsp.
bulgaricus, m o st strains o f Lb. delb ru eckii subsp. lactis and all strains o f Str.
th erm o p h ilu s excrete galactose in am ounts e q u im o lar w ith the am ounts o f lactose
used. T his is th o u g h t to be involved, at least in Str. th erm o p h ilu s, w ith lactose
transport. M e ta b o lism o f glucose is v ia th e E m b d e n -M e y erh o f p athw ay (EM P). In
L eu c o n o sto c the ferm entation o f glucose pro ceed s v ia the phosphoketolase (PK )
path w ay to e q u im o lar concentrations o f D -lactate, ethanol and C 0 2, w hile galactose
is p ro b ab ly m etab o lised v ia th e L eloir path w ay to glucose-6-P before entering the
P K p a th w a y [C ogan and H ill, 1987].
In th e lactococci, lactose-P form ed d u ring P T S tran sp o rt is hy d ro ly sed to galactose6-P and g lu co se by B -D -phosphogalactoside galactohydrolase (BPgal). G alactose-6-P
is furth er m etab o lized to trio se p h o sp h ates th ro u g h th e tagatose pathw ay w hereas
glucose is ferm en te d v ia the glycolytic p ath w ay [L aw rence et al., 1976; C ogan and
H ill, 1987],
U su ally lactate is the only p ro d u ct o f su g ar m etabolism , b ut w h e n these bacteria are
g ro w n on so m e sugars e.g. galactose o th er p ro d u cts can be form ed, e.g. form ate,
eth an o l a n d acetate. I f the m ed iu m also contains citrate, p ro d u ctio n o f acetoin and
diacetyl also occurs [C ogan and H ill, 1987]. B ased on th e discovery that Lc. lactis
4
A T C C 7962, w h ic h does n ot possess BPgal, grow s slow ly o n lactose converting only
15% o f it to L -lactate, it w as concluded that th e rapid and h o m o lactic ferm entation
o f lactose is d e p e n d en t on a functional P E P -P T S system a n d th e presence o f BPgal
[T hom as, 1976].
Citrate Metabolism
C itric acid is a tricarboxylic acid consisting o f six carbon atom s, w h ich is p resent in
m ilk in only sm all concentrations (~10m M ). M etab o lism o f citrate to flavour
co m pounds lik e acetate and diacetyl is im portant in th e p ro d u ctio n o f m any
ferm ented dairy pro d u cts such as b u tter and quark. In contrast, diacetyl is an
un d esirab le co m p o u n d in beer. T he p ro d u ctio n o f C 0 2 d u rin g m etab o lism o f citric
acid also adds tex tu re to som e products, although it is un fav o u rab le in the p roduction
o f fresh ch eeses like q uark [H ugenholtz and S tarrenburg, 1992].
T he ab ility o f cells to utilize citrate is unstable, sugg estin g th a t it is plasm id
encoded. T he existence o f such a p lasm id w as p ro v en by K em p ler and M cK ay
[1979] w h o sh o w ed th at treatm ent o f cells o f C it+ Lc. lactis subsp. lactis w ith
acridine orange resu lted in a loss o f the ability to u tilise citrate and a loss o f the
p lasm id en co d in g th e citrate transport system , citrate perm ease. T his p rotein is
active w ith in the p H range 5.0 to 6.0 in C it+ Lc. la ctis subsp. lactis and is
c onsidered to be a n integral m em brane p ro tein [D avid et al., 1990; H ugenholtz,
1993].
C itrate is n o t a n energy source for C it+ Lc. lactis subsp. la ctis [C ogan, 1982; C ogan
and H ill, 1987], alth o u g h an increase in the specific g ro w th rate o f C it+ Lc. lactis
subsp. la ctis w as found w h e n citrate w as added to a lacto se containing m edium
[H arvey
an d
C ollins,
1963a].
C itrate
also
stim ulated
the
grow th
rate
of
h e tero ferm en tativ e lactobacilli in lactose containing m e d ia [D rinan et al., 1976].
H u g e n h o ltz et al. [1993] show ed th at in lactose-lim ited co n tin u o u s cultures C it+ Lc.
la ctis subsp. la ctis w as able to g ro w on citrate as th e only energy source at low
grow th rates a t p H 5.3, generating a P M F as a resu lt o f electrogenic uptake or
c itrate/p ro d u ct exchange together w ith p roton c o n su m p tio n b y th e intracellular
oxaloacetate d ecarboxylase [Starrenburg and H ugenholtz, 1991; H ugenholtz, 1993].
A P M F as th e d riving force for citrate tran sp o rt w as also suggested by D avid et al.
[1990]. A sim ilar m ech an ism w as found for Ln. m esenteroides, w h ich generates
m etabolic en erg y from citrate m etabolism in the form o f a p ro to n electrochem ical
5
gradient across the m em brane by electrogenic exchange o f c itrate an d D -lactate
[M arty-T eysset e t al., 1996].
C itrate uptake is in h ib ited by C u2+ and F e3+, w h ich at the sam e tim e stim ulate
the p ro d u ctio n o f diacetyl, p ro b ab ly b ecau se the citrate u p tak e a c tiv ity o f th e cells is
low ered by diacetyl [K aneko et al., 1990b],
A fter tran sp o rt into the cell, citrate is cleaved to oxaloacetate an d acetate by citrate
lyase (citritase), w h ich is co n stitutively p resent in C it+ Lc. la ctis subsp. lactis, but
ind uced by citrate in L euconostoc sp. [S peckm an and C ollins, 1968; C ogan, 1981;
M ellerick and C ogan, 1981], T he fo rm er is decarboxylated to y ie ld p y ru v ate [Seitz
et al., 1963]. H ence, i f cells are grow n in the presence o f citrate in add itio n to an
energy source such as lactose or glucose, excess pyruvate is p ro d u ce d w h ic h cannot
be red u ced to lactate because o f th e n eed to recycle N A D H to co n tin u e glycolysis.
T he excess p y ru v ate n o t req u ired fo r the synthesis o f cell m ate ria l h as to be
rem oved, w h ich leads to the fo rm atio n o f the typical p ro d u cts o f c itrate m etabolism ,
acetoin a n d diacetyl. T his effect can be th o u g h t o f as a d e to x ific a tio n m echanism .
P yruvate th erefo re is a key interm ediate in citrate m etab o lism [H arvey and C ollins,
1963b; K e m p le r and M cK ay, 1981; S tarrenburg and H u g en h o ltz, 1991], It has been
found th a t C it+ Lc. lactis subsp. lactis can produce acetoin a n d d iacety l ev en in the
absence o f citrate as additional source o f pyruvate u n d er aerobic g ro w th conditions
[B ruhn and C ollins, 1970], In this case N A D H oxidase is activ e a n d is partly
resp o n sib le fo r th e reo x id atio n o f N A D H to N A D + to co n tin u e glycolysis. In
anaerobically (norm al) grow ing cells this function is carried out b y th e reduction o f
pyruvate to lactate and th e co n com itant production o f N A D + fro m N A D H by LD H .
In aerobically g ro w n cells th e p y ru v ate not required fo r th e latte r rea c tio n is u sed to
form diacetyl and acetoin.
Pette [1949] p ro p o sed a hyp o th etical substance w hich c o u ld act as an interm ediate
for b o th a ceto in an d diacetyl p ro d u ctio n from pyruvate. T his h y p o th etical substance
w as later show n to b e acetolactic acid (A LA ), a very u n sta b le co m p o u n d [De M an
and P ette, 1956]. A L A is form ed from tw o pyruvate m olecu les. Juni [1952]
suggested a condensation
o f pyru v ate w ith “ active” acetald eh y d e
(hydroxy-
ethylthiam ine pyrophosphate). T his m ech an ism w as later co n firm e d by 13C nuclear
m agnetic reso n an ce (N M R ) [V erhue and Tjan, 1991]. T h e enzym e th a t catalyses this
reaction, acetolactate synthase (A L S), is expressed co n stitu tiv ely [C ogan, 1981;
Snoep et al., 1992], requires th iam in e pyrophosphate (T P P ) and M g 2+ o r M n 2+ and is
inhibited by citrate [K obayashi and K alnitsky, 1954;
H arv ey a n d C ollins, 1961;
B rauen and K eenan, 1972; Snoep et al., 1992], although C ogan [1981] found th a t in
som e strains o f C it+ Lc. lactis subsp. lactis A LS can b e p artly in d u ce d by citrate.
6
A L S h as a p H optim um o f about 6.0 [Juni, 1952]. In C it+ Lc. la ctis subsp. lactis
A L S has a h ig h K M for p y ruvate, 50m M as co m p ared to lO m M in L euconostoc sp.
[Snoep et al., 1992; M aru g g et al., 1994]; its activity is h ig h er in L a cto co cci sp. than
in L eu co n o sto c sp. A L S is allosteric in C it+ Lc. lactis subsp. lactis, w hereas it can
be allosteric or obey M ich aelian k inetics in L e u co n o sto c sp. [M onnet et al., 1994a].
D ue to its h ig h K,n fo r pyruvate in C it+ Lc. lactis subsp. lactis, A L S is only active
w h en th e in tern al p o o l o f py ru v ate is high, as it is d uring co -m etab o lism o f citrate
an d a ferm en tab le carbohydrate. T herefore toxic excess p y ru v ate can be rem oved
w ith o u t co m p etitio n w ith other enzym es such as lactate dehy d ro g en ase (LD H ) or
py ru v ate dehydrogenase (P D H ). P D H has a K,n o f Im M fo r p y ru v ate and therefore
has a m u ch h ig h er affinity fo r pyru v ate th an A L S , w h ereas A L S has a higher
activity. T he tw o enzym es are also n o t ex p ressed sim u ltan eo u sly . P D H is only
p ro d u ced aero b ically [Snoep et al., 1992; S m ith et al., 1993; M o n n e t et al., 1994a].
Lc. lactis subsp. lactis p roduces tw o different A L S w h ich p erfo rm different
functions. T h e gene encoding one o f th e A L S is p art o f an o p e ro n involved in the
biosynthesis o f b ran ch ed ch ain am ino acids (B C A A ), th e second gene for A LS
produces a sim ilar enzym e. T he bio sy n th etic A L S h as a p H o p tim u m o f 8.0 and
contains F A D w hile the catabolic enzym e has an o p tim u m p H o f 6.0 and does not
contain FA D . F o rm er is reg u lated by tran scriptional atten u atio n , and the latter
expressed in a co n stitutive fashion, suggesting th a t Lc. la ctis subsp. lactis can
pro d u ce d ifferen t ty p es o f A L S u n d e r varying g ro w th co n d itio n s [M arugg et al.,
1994]. T his also explains w hy S noep a t al. [1992] did n o t fin d any feed back control
o f th e pH 6.0 enzym e from B C A A .
Juni [1952] found th a t in E n tero ro b a cter aerogenes, a ceto in is produced from
pyruvate v ia A L A by acetolactate decarboxylase (A L D ). T h e enzym e had a pH
o ptim um o f ab o u t 6.0 and 75% o f its activity w as c o n serv ed w ith in th e p H range o f
5.4 to 6.9. P halip et al. [1994] found th at A L D o f Lc. la ctis subsp. lactis consisted o f
six identical subunits o f 26 50 0 D a and w as activated by B C A A , M n 2+ and Z n2+ b u t
n o t M g 2+. T he enzym e show ed allosteric properties in th e absence and M ichaelian
kinetics in th e p resen ce o f leucine. In C it+ Lc. lactis subsp. lactis A L D has a h ig h
K m fo r A L A (60m M ) co m p ared to L eu co n o sto c sp. (0 .3 m M ) [M onnet et al., 1994a].
A L A and aceto in are o p tically active com pounds. In C it+ Lc. lactis subsp. lactis the
a ceto in p ro d u ced is d ex trorotatory w hereas
in E n tero b a cter aerogenes it is
levorotatory [S peckm an and C ollins, 1968; C ollins a n d S peckm an, 1974]. It has also
b een fo u n d th a t only one o f the optical isom ers o f com m ercial A L A is attacked by
the A L D o f C it+ L c. lactis subsp. lactis. T his indicates th a t A L A is enzym atically
b ound d u ring decarb o x y latio n to acetoin, otherw ise th e resu lt o f decarboxylation
w o u ld be a racem ic m ix tu re [C ollins and Speckm an, 1974], A n o th er m echanism for
7
the p ro d u ctio n o f aceto in w as p roposed by K a m iy a et al. [1993]. T hey found that
u n d er an aero b ic co n d itio n s a m ajor p o rtio n o f th e A L A is co n v erted to acetoin
w ith o u t in v o lv em en t o f an enzym e. T hey su ggested th at A L A is decarboxylated to
an u n k n o w n interm ed iate X , w hich th en is co n v erted to a c eto in under anaerobic
c o n ditions or to diacety l i f enough ox y g en is present. D e c a rb o x y la tio n is supposed
to be the rate d eterm in in g step because sim ilar reactio n rate constants fo r the
con v ersio n o f A L A to a ceto in and to diacetyl w ere found.
A ceto in is e ith e r ex creted as an end-product o r it is fu rth er reduced to 2,3b u tanediol, a rea c tio n cataly sed by butanediol d eh y drogenase [H ugenholtz, 1993].
B u tanediol dehy d ro g en ase is constitutively p resen t in several strains o f C it+ Lc.
lactis subsp. la ctis an d is partly repressed by citrate in som e strain s [Cogan, 1981].
T here are d iffe re n t opin io n s o n how diacetyl is produced. U n til th e 1960s, it w as
b eliev ed th at diacety l w as form ed v ia ox id atio n o f aceto in [Jo n sso n and P ettersson,
1977]. T his th eo ry w as th en replaced by tw o m ain theories. S om e w orkers believe
th at d iacety l w as p ro d u ce d directly in the cell v ia c o n d en satio n o f acetyl coenzym e
A w ith “a c tiv e ” acetaldehyde involving th e enzym e diacetyl synthase [C huang and
C ollins, 1968; S peckm an and C ollins, 1968; 1973; Jo n sso n an d P ettersson, 1977;
K aneko et al., 1990a]. D espite exhaustive studies, no c o n v in cin g evidence for the
existence o f th is en zy m e has ever b een found in L A B . T he o th er th eo ry claim s that
diacetyl w as fo rm e d o u tsid e th e cell by oxidative d e c arb o x y latio n o f excreted A L A
[De M an an d P ette, 1956; Seitz et al., 1963; V erhue an d T jan, 1991]. It cannot be
excluded th a t b o th m ech an ism s occur sim ultaneously. R e c e n t resu lts w ith Ln. lactis
in d icate a th ird m ech an ism , suggesting th at the estab lish ed path w ay for acetoin
synthesis from p y ru v ate a n d A L A catalysed by A L S and A L D is also responsible for
th e en zym atic fo rm a tio n o f diacetyl [Jordan et al., 1996], T he level o f diacetyl
p ro d u ced is u su a lly m u ch sm aller th an th at o f aceto in [W alsh and C ogan, 1973].
O ne p h e n o m e n o n th a t can occur, w h ich is desirable in b e e r ferm entations but
un d esirab le in dairy ferm entations, is the irreversible red u c tio n o f diacetyl to acetoin
b y acetoin d ehydrogenase. T his enzym e has a pH op tim u m o f 5.5 and is stim ulated
b y C u2+ and h a e m in [S eitz et al., 1963; Jo n sso n a n d P ettersson, 1977; K aneko et al.,
1990a]. It is c o n stitu tiv e ly p resen t in several strains o f C it+ Lc. lactis subsp. lactis
and p artly rep re sse d b y
grow th on citrate in
8
som e
strains
[C ogan,
1981].
Mg1" or Acetate
M n^
C itrate
CO,
COz
f
f
_► [Acetaldehyde-TPP]
Oxaloacetate — ^ ► P y ru v a te
C itrate
lyase
O xaloacetate
decarboxylase
. Acetyl•' CoA
A cetolactate
synthase
D iacetyl
synthase
Acetolactic Acid
fcX oA S H
O,
A cetolactate
CO,
decarboxylase
► TPP
A cetoin d eh y drogenase
B u tanediol reductase
-------------<
^ Acetoin ^ ----- -p.__________
2,3-Butanediol
NADCP4)
NAD(P)H
NAI)(P")
J
Diacetyl
NAD(P)H
F ig .l: C itrate M etab o lism by C it+Z. ¡actis subsp. lactis
Factors influencing Diacetyl and Acetoin Production
O ne facto r affecting th e am o u n t o f diacetyl and acetoin p ro d u ce d fro m A L A by C it+
Lc. lactis subsp. lactis is pH . In a ferm en tatio n co n tro lled at p H 4.5, the m axim al
specific rate o f citrate utilisatio n , the b io co n v ersio n y ield and th e ratio o f diacetyl to
acetoin w ere increased co m p ared to a ferm entation co n tro lled at p H 6.5. In contrast,
th e specific grow th rate and specific rate o f lactose ferm en tatio n w ere low er at the
low er p H value. T he p H acts in directly by increasing th e p ro p o rtio n o f n o n ­
d issociated lactic acid, w h ich is considered in h ib ito ry to g row th and lactose
ferm en tatio n [C achon a n d D ivies, 1994],
T em perature also has an effect. T he rate o f grow th o f C it+ Lc. la ctis subsp. lactis
and lactic acid p ro d u ctio n w ere h alv ed at 18°C com pared to 30°C , and m ore diacetyl
w as pro d u ced at 18°C th an at 30°C w hereas acetoin p ro d u ctio n w as unchanged at
b o th tem peratures. O ne reaso n fo r th is could be the effect tem p eratu re has on the
p rincipal enzym es in v o lv e d in p y ru v ate m etabolism . W h ile L D H and A L S activities
w ere relativ ely unaffected b y a change in tem perature, N A D H oxidase activity w as
9
h ig h er a t 18°C th a n at 30°C w hereas aceto in dehy d ro g en ase activity w as decreased
at 18°C co m p ared to 30°C [B assit et al., 1995].
E xperim ents in v estig atin g th e effect o f p H and tem p eratu re o n synthetic A L A
show ed sim ilar results; the specific rate o f A L A d ecarb o x y latio n in creased w ith
decreasing p H an d increasing tem perature, w ith diacety l and aceto in p ro d u ctio n
co rresponding to A L A degradation. A L A d ecarb o x y latio n w as a first order reactio n
[M onnet et al., 1994b].
T he am o u n t o f diacetyl produced from A L A during ferm entation o r in a m odel
system w ith sy nthetic A L A is also d e p e n d en t o n th e redox po ten tial o f the
surrounding m edium . A h ig h redox poten tial, as found at the b eg in n in g o f a lactic
ferm entation, resu lts in production o f diacety l and acetoin. T he lo w red o x po ten tial
at the e n d o f a ferm entation only allow s the p ro d u ctio n o f acetoin. H ence, the
am ount o f diacetyl can be increased by k e e p in g th e red o x potential at a h ig h level,
e.g. by b u b b lin g o x y g en th rough the culture o r by continuous stirring [Jônsson and
P ettersson, 1977; B assit et al., 1993;, M o n n et e t al., 1994b].
A n o th er rela te d factor is th e initial oxy g en co n cen tratio n in th e m edium . A t 0% 0 2
satu ratio n at 30°C , little diacetyl w as p ro d u ced b y C it+ Lc. lactis subsp. lactis
(O.Olm M diacety l com pared to 2.4m M acetoin). A ceto in p ro d u ctio n increased to
5.4m M w h e n 0 2 w as increased to 100% satu ratio n w h ile diacetyl p ro d u ctio n w as
increased by factors o f tw o , six and e ig h teen at initial 0 2 concentrations o f 21, 50
and 100% , respectively. T he increase in th e ratio o f diacetyl to acetoin w ith
increasing 0 2 co ncentrations w as linear. T h e reaso n for these fin d in g s is th at the
specific activities o f A L S and N A D H o x id ase are lo w er at low er 0 2 concentrations.
N A D H o xidase rep laces L D H , acetoin d eh y d ro g en ase and butanediol dehydrogenase
in the re o x id a tio n o f N A D H allow ing a c cu m u la tio n o f aceto in a n d diacetyl [B assit et
al., 1993].
W hen th e co m b in ed effect o f 0 2 and tem p e ra tu re w as studied, it w as ev id en t that
the effect o f 0 2 w as m ore im portant th an th e effect o f tem perature in p ro d u ctio n o f
acetoin a n d d iacetyl, w hereas th e o p p o site w as true for acidification. M axim al
diacetyl con cen tratio n s and a m axim al ratio o f diacetyl to acetoin w ere reached at
18°C a n d 100% 0 2 saturation, m axim al a c eto in concentrations at 26°C and 100% 0 2
satu ratio n and a m ax im al acidification rate a t 30°C and 0% 0 2 satu ratio n [B assit et
al., 1994],
T he ad d itio n o f C u2+, F e2+ or haem in also in creases the am ount o f diacetyl produced
by C it+ Lc. la ctis subsp. lactis. A ll th re e are co n sid ered to stim ulate th e activity o f
diacetyl synthase. It w as observed th a t C it+ Lc. lactis subsp. lactis pro d u ced diacetyl
10
a n d a ceto in fro m glucose in the absence o f citrate w h e n grow n aerobically in the
p resen ce o f C u2+ and haem in [K aneko et al., 1990a; 1990b].
Branched Chain Amino Acids and Citrate Metabolism
D airy strains o f Lc. lactis subsp. Jactis are u n ab le to synthesise th e three branched
chain am ino acids (B C A A ) v alin e (val), leucine (leu) an d isoleucine (ile) in contrast
to th eir n o n -d airy counterparts w hich can grow in th e absence o f all three B C A A
[G odon et al., 1993; C hopin, 1993]. T he reaso n s fo r this system atic B C A A
aux o tro p h y in d airy strains o f Lc. lactis subsp. lactis are unknow n. C hopin [1993]
explains th e aux o tro p h y for B C A A in dairy strains as a consequence o f their
ad aptation to g ro w th in m ilk and dairy products. M ilk contains a significant am ount
o f p ro te in a n d sm all am ounts o f free am ino acids. Since B C A A are the m ost
freq u en t am ino acids o f Lc. lactis subsp. lactis pro tein s w hile n o t being particularly
ab u ndant in m ilk , G odon et al. [1992; 1993] su g g est th e existence o f a selective
p ressu re fo r au x o tro p h y , m eaning th at m ay b e an interm ediate o f th e B C A A pathw ay
is toxic fo r th e cell or perturbs th e reg u latio n o f oth er pathw ays such as th e anabolic
p athw ay for p an to th en ate and the catabolic p athw ay for aceto in an d 2,3-butanediol,
w h ich are b o th linked to the B C A A p ath w ay , the fo rm er via a -k e to iso v a le ric acid,
w h ich is a p rec u rso r o f valine, the latter v ia A L A , the c o m m o n interm ediate o f
leucine and v a lin e biosynthesis [U m barger and D avis, 1962],
The genes fo r th e biosynthesis o f B C A A in L. la ctis subsp. lactis are organised in a
large cluster, w h ic h is divided into tw o units. B o th u n its are n ecessary for leucine
bio sy n th esis, w hereas only the second is n eeded for the synthesis o f isoleucine and
valin e [R en au lt e t al., 1995; G odon et al., 1992]. C ho p in [1993] suggests th at the
tw o u n its fo rm a single operon. T his o rganisation in a single operon, in contrast to
other b a c te ria w h ere those genes are m o re scattered, allow s a co-o rd in ated regulation
o f the e x p re ssio n o f B C A A genes. It has b een show n by several w orkers that the
ex p ressio n o f B C A A genes is c o n tro lled by tran scrip tio n al atten u atio n [R enault et
al., 1995; G o d o n et al., 1992; C hopin et al, 1993].
It is in terestin g to note that the gene en coding A L D is situated in th e sam e operon as
the B C A A genes. A L D transform s A L A , the first interm ediate fo r the biosynthesis
o f leucine a n d valine, to aceto in and is po sitiv ely controlled by th e availability o f
leucine and p o ssib ly valine in th e cell. A L D is a key enzym e in a new class o f
reg u lato ry m ech an ism s, a m etab o lic shunt, w h ich controls the flu x o f A L A tow ards
b io sy n th esis o r catabolism [C hopin, 1993; R enault et al., 1995].
11
T his special reg u lato ry m echanism m akes it possible to iso late A L D negative
m utants, w h ic h are o f considerable interest for th e p ro d u ctio n o f lactic b u tter and
quark since th ese m utants are unable to form acetoin from A L A , w h ich can th en be
decarb o x y lated to diacetyl under the proper conditions. T he iso latio n w orks on the
basis th a t w ild type Lc. la ctis subsp. lactis cannot grow in th e p resen ce o f leucine
and sim ultaneous absence o f valine, because h ig h leucine co ncentrations in the cell
activate A L D , w h ich converts available A L A to acetoin rath er th a n to leucine and
valine. Since th e cells n eed valine, they die in its absence. A L D negative m utants
cannot p ro d u ce acetoin from A L A enzym atically, im p ly in g th a t A L A is still
available fo r v alin e biosynthesis. T herefore th e m u tan ts survive in a m edium th at
contains leucine but n o t valine [G oupil et al., 1995; C hopin, 1993].
A c o m p ariso n o f th e genom es o f Lc. lactis subsp. la ctis deriv ed from m ilk and
p lan ts show ed th at th e o peron resp o n sib le for B C A A b io sy n th esis is p resent in the
auxotrophic d airy strains b u t th at som e genes are inactive. T he rem aining active
genes m ig h t h a v e a role o th er th an B C A A biosynthesis; this ro le cannot be essential
th o u g h b ecau se deletion o f th e operon in prototrophic strains d id n o t affect their
v iab ility [G odon et al. 1993]. O ne o f th o se rem aining active genes m ig h t be the gene
encoding A L D , w h ich is activated by B C A A in dairy strains o f Lc. lactis subsp.
la ctis despite th eir in ability to synthesise those am ino acids [M onnet et al., 1994a].
Metabolic Engineering of Citrate Metabolism
M utations o c c u r frequently in bacteria and som etim es resu lt in desirable properties
in the m utant. M cK ay and B aldw in [1974] for exam ple iso lated a strain o f Lc. lactis
subsp. lactis w h ich fo rm e d abnorm ally large colonies o n agar. W h en exam ined m ore
closely, it w as d isco v ered th at th e m u tan t grew as fast in m ilk and bro th as the parent
strain but w as slo w er in acid production. It also consum ed six tim es as m uch oxygen
as the p a re n t strain and p ro d u ced large am ounts o f a ceto in and som e diacetyl. The
reaso n for th ese ab n orm alities w as an enzym atic defect; th e m u ta n t p ossessed only
lo w am o u n ts o f L D H and w as therefore unable to red u ce p y ru v ate to lactic acid,
w h ich resu lted in an excess o f pyruvate in the cell. T his m im ics th e situation in C it+
Lc. lactis subsp. lactis, w h ic h produce excess pyruvate fro m citrate on top o f the
pyruvate fro m sugar m etabolism . K u ila and R an g an ath an [1978] tried to induce
m u tatio n s in C it+ L. lactis subsp. lactis using U V radiation. T his resulted in tw o
ty p es o f m u tan ts; ty p e I w as a high diacetyl producer, w hereas type II produced
greater am ounts o f acid. M u tan ts o f ty p e I w ere L D H n egative o r at least im paired
and th erefo re disp o sed o f the excess pyruvate by p ro d u cin g m ore acetoin and
12
d iacetyl. S om e o f the type I m u tan ts also h ad an im paired a ceto in dehydrogenase.
T ype II m u tan ts show ed a slight increase in L D H activity. T he prod u ctio n o f
diacetyl and aceto in can also be m an ip u lated by m etabolic en gineering. G asso n et al.
[1996] suggested three sites, L D H , A L D and A L S genes, fo r m anipulation. B y
elim in atin g L D H in a C it- Lc. lactis subsp. lactis, th ey pro d u ced a strain that
generated sim ilar am ounts o f acetoin during sugar ferm en tatio n as C it+ Lc. lactis
subsp. lactis grow ing o n both sugar an d citrate. In activ atio n o f the gene encoding
A L D in creased prod u ctio n o f diacetyl b y preventing d ecarb o x y latio n o f A L A to
acetoin a n d therefore increasing the o p p ortunity for its o x id ativ e d ecarb o x y latio n to
diacetyl. F in ally th ey to o k advantage o f the fact that Lc. lactis subsp. lactis can
p ro d u ce d ifferent ty p es o f A L S. T hey substituted the A L S no rm ally active in the
diacetyl p ro d u ctio n pathw ay, w h ich has a lo w affinity fo r p y ru v ate and therefore
only w o rk s in situations o f excess p yruvate, w ith a n A L S th at co nverts py ru v ate to
A L A during B C A A biosynthesis, w hose affinity fo r py ru v ate is hig h er. T his enzym e
is n o t tran scrib ed w h en B C A A are p rese n t in the m edium , b u t ch anging the p rom oter
for the genes resu lted in constitutive p roduction o f th is type o f A L S , therefore
allow ing A L S activity in the p resen ce o f B C A A and in creasin g the p ro d u ctio n o f
diacetyl and aceto in in several C it' Lc. la ctis subsp. lactis strains.
Measurement of Diacetyl and Acetoin
T he first m ethods u sed to m easure diacetyl and acetoin w e re gravim etric ones, w hich
inv o lv ed th e fo rm ation o f a nickel d im ethylglyoxim e co m plex b etw een diacetyl and
h y d ro x y lam in e. A ceto in had to be oxidised to diacetyl p rio r to th e reaction
[M ichaelian and H am m er, 1935]. N e w er m ethods w ere subsequently developed
in clu d in g colorim etry [Prill and H am m er, 1938; W esterfeld, 1945], polarography
[Ferren e t al., 1967], gas liquid chrom atography [T hornhill an d C ogan, 1984], and
head sp ace gas chrom atography [M onnet et al., 1994b].
T he m eth o d u su ally u sed to m easure acetoin, the W esterfeld pro ced u re [1945], is n o t
specific b ecau se aceto in is oxidised to diacetyl during the assay, and therefore
separation o f acetoin from diacetyl is required w hen b o th com pounds are present.
T w o o f th e separation m ethods used are salting-out ch ro m atography [S peckm an and
C ollins, 1968b] and steam distillatio n [W alsh and C ogan, 1974]. In the laboratory in
w h ich th is study w as carried out, steam d istillation is th e routine m eth o d used. The
first 10m l fractio n collected contains all the diacetyl and m o st o f the acetoin,
w hereas the second 10m l fraction contains - 2 5 % o f the aceto in [W alsh and C ogan,
1974]. T h e m eth o d o f W alsh and C ogan, w hich is a m o d ificatio n o f the colorim etric,
13
P rill and H am m er m ethod, and w h ich is specific fo r diacety l, is u se d to quantify
diacetyl in the first fraction, w hile acetoin c a n b e m e a su re d b y th e W esterfeld
m eth o d [1945] in the second fraction.
A p ro b lem occurs i f th e m ixture also contains A L A , since th is co m p o u n d is easily
converted
to
diacety l
and
aceto in
by
h e a t (e.g.
steam
d istillatio n
or
gas
c h rom atography), w h ic h can lead to false results. T he b rea k d o w n d u rin g distillation
can be red u c e d to 2% b y ad justing the pH to 9.0 w ith N a O H p rio r to distillation
[V eringa et al., 1984]. A red u c tio n in the break d o w n o f A L A to diacetyl to 0.2%
during d istillatio n at p H 1.0 w as reported by C ronin and R isp in [1996].
Measurement of Acetolactate
A L A is an in term ed iate in the bacterial p roduction o f a c eto in a n d diacetyl. It is an
unstable co m p o u n d an d easily decarboxylated, either o x id ativ e ly to diacetyl or nonoxidatively to a ceto in [D e M a n and P ette, 1956].
O n th e one hand, th is is a disadvantage in q u an tificatio n but, o n th e other hand, it
opens up th e p o ssib ility o f u sin g decarboxylation as a m ea n s to m easu re A L A , i.e.
the co m pound is m easu red as th e difference in th e levels o f a c eto in before and after
decarboxylation. T here are different w ays to do this; one m eth o d u ses h eat to break
dow n A L A [U m barger and B row ne, 1958; Jordan and C o g an , 1988], other m ethods
use acids su ch as HC1 or a c o m b in atio n o f heat and acid [V erin g a e t al., 1984; Jordan
and C ogan, 1995]. A fter th o se m ethods, the W esterfeld p ro ce d u re [1945] can be
used to qu an tify th e am o u n t o f th e acetoin pro d u ced by b rea k d o w n o f A L A . In those
m ethods th e aceto in m easu red before decarboxylation is n o t th e true am ount o f
acetoin b u t the sum o f a ceto in and diacetyl. H o w ev er, th is is n o t norm ally a
problem , as th e levels o f a ceto in pro d u ced by cultures are m u ch g reater than th o se o f
diacetyl.
A n o th er m eth o d rep o rted by G ollop
e t al.
[1987], u ses oxidative
decarb o x y latio n o f A L A to diacetyl to m easure A L A . T his m eth o d requires
quantitative o x id atio n o f A L A to diacetyl w h ich w as o b tain e d b y heatin g in the
presence o f F e 2+ and F e 3+. T he resulting diacetyl w as th en sep arated by an air
distillatio n and assay ed b y th e m eth o d o f P rill and H a m m e r [1938]. A L A w as th en
q uantified from th e difference b etw een a sam ple d istilled in th e presence and
absence o f F e2+ and F e 3+.
14
Manufacture of Butter
Tw o types o f b u tte r are p roduced, sw eet cream b u tter an d sour c ream butter, w hich
is also called lactic, cu ltu red or ripened cream b u tter. T h e tw o ty p es differ
considerably in taste. T h e b lan d flavour o f sw eet c ream b u tte r originates in the
flavour o f th e m ilk c o n stitu en ts, particularly th e m ilk fat and th e changes caused by
th e p a ste u risa tio n o f th e cream , w hereas the flavour o f lac tic b u tte r is dom inated by
the p roducts fo rm ed b y th e starter organism s d u ring ferm en ta tio n o f th e cream , e.g.
lactate, acetate a n d diacetyl. T he starter is u su ally a m ix e d -strain culture o f the L or
D L type.
L actic b u tter can b e m an u factu red b y tw o processes as sh o w n in F igure 2. D uring
th e trad itio n al p ro cess, th e m ilk is separated and th e resu ltin g c ream pasteurised. The
cream , co n taining 3 5-40% fat, is th en in o cu lated w ith th e starter culture and
incubated at 2 1 °C u n til th e p H reaches 4.5 to 4.8, w h e n th e ferm en tatio n is com plete.
T he rip en ed cream is co o led to 5°C and churned. T his resu lts in b u tter and sour
butterm ilk. T he u se s fo r so u r b u tterm ilk are lim ited; in co n trast, sw eet butterm ilk is
m u ch m ore usefu l as an in g red ien t in dairy products. It is o b tain e d during churning
o f sw eet cream in th e p ro d u c tio n o f sw eet cream butter. So an altern ativ e process for
the p ro d u ctio n o f lactic b u tter, the so-called N IZ O p ro cess, w as dev elo p ed [V eringa
e ta l., 1976].
T he alternative m e th o d fo r th e m anufacture o f lactic b u tte r d iv id es the process into
three in d ep en d en t steps, n am ely the pro d u ctio n o f a lactic acid culture concentrate,
the p ro d u ctio n o f a ro m a com pounds and the p ro d u ctio n o f sw eet cream butter
[V eringa et al., 1976]. L actic acid culture concentrate is p ro d u ced from w hey by
ferm en tatio n w ith Lb. h elveticu s w h ich produces large am o u n ts o f lactic acid. The
w h ey culture is th e n u ltrafiltrated and concentrated b y ev a p o ra tio n [V eringa et al.,
1976; V an den B erg, 1991]. T he aro m a com pounds are p ro d u ce d b y ferm entation o f
m ilk w ith a p a rticu la r culture. A fter th e end o f the ferm en ta tio n th e m ilk is cooled
d ow n to 5°C , lactic acid culture concentrate is added an d th e m ix tu re is aerated for
15m in to 2h [V an d e n B erg, 1991], L actic acid culture co ncentrate and aeration
enhance th e d e c arb o x y la tio n o f A L A to diacetyl. In th e altern ativ e process, sw eet
cream is churned to th e granule stage w ith the release o f sw eet butterm ilk. A fter
separating the b u tte r g ran u les from th e butterm ilk, th e sta rte r m ix tu re to g eth er w ith
the lactic acid cu ltu re concentrate are w orked into the b u tte r granules to obtain a
p ro d u ct w h ich c an n o t b e distin g u ish ed from lactic b u tte r m ad e b y the traditional
process [V an d en B erg, 1991].
15
TRADITIONAL
A LTER N A TIVE
M ETHOD
I
METHOD
PASTEURISATION
PAST EURJSATION *
PASTEURISATION
I
Inoculation
Inoculation
I
S — ^
production |
o l b e lle a d d
and aroma
CONCENTRATE
compounds
and aroma
(LACTIC ACID)
compounds
tf=¥
tr—
H
RIPENED CREAM
(lat.lactic acid,arom a
com pounds,bacteria)
I churning
SWEET
BUTTERMILK
n Isweeti butter
•ih
I
/
RIPENED CREAM
BUTTER
CULTURE
production
o( lactic a d d
\
SOUR BUTTERMILK
i—
*
i -
¡.w orking
I
SOUR AROMATIC
BUTTER
Fig. 2: M eth o d s fo r th e p ro d u ctio n o f lactic b u tte r [V eringa et al., 1976]
16
T he n e w pro cess h as several advantages over the o ld er p ro cess. T he m ajo r advantage
is the p ro d u ctio n o f sw eet rather th an sour b u tte rm ilk as a by-product. A dditionally,
there are few er ox id ativ e defects on cold storage o f th e b u tte r due to a low er copper
co n ten t in the “ sw eet cream ” lactic butter co m p ared w ith traditionally produced
lactic butter, a n d it is less likely to develop ran c id fla v o u r because o f the low er
co n ten t o f free fatty acids in the sw eet cream granules. A p art from that, the
rheological p ro p erties o f the butter can be better co n tro lled b ecau se the choice o f the
m o st suitable tem p e ra tu re treatm ents in the sw eat cream b u tte r is w ider. In addition,
the o p tim u m tem p eratu re for arom a pro d u ctio n can be ch o sen and starter cultures
w ith a tem p eratu re o p tim u m unfavourable fo r the p ro p ertie s o f cream can be used
[V eringa et al., 1976]. A pro blem th at can occur is th a t resid u al A L A , w h ich has not
b e e n b ro k en d o w n d u rin g aeration o f the starter/lactic a c id m ixture, is converted to
aceto in rath er th a n to diacetyl during storage.
T he m o st co m m o n starter type for the p ro d u ctio n o f b u tte r is 4/25, w h ich is a D
culture co n tain in g C it+ Lc. lactis subsp. la ctis as flav o u r producer. T his strain lacks
A L D and, as a resu lt, overproduces A L A , w h ic h is th en su b sequently b ro k en dow n
to diacetyl d u ring aeratio n at low pH . T he starter also p ro d u ce s acetaldehyde, w hich
gives a n u n w elco m e flav o u r to the butter. C on seq u en tly th e L eu co n o sto c containing
culture ( F rl9 ) is also ad d ed w h ich is able to red u ce acetaldehyde to ethanol w hich
has n o effect on flavour. T he m ost im portant aro m a c o m p o u n d s in th is type o f butter
are diacetyl, a n d p ro b ab ly also acetate and lactate [B abel, 1944; V an den B erg,
1991].
Effect of Butter Cultures on Butter
T he m ain p u rp o se o f the starter cultures in b u tte r is to produce lactic acid and
diacetyl. L actic acid low ers the pH giving the b u tter a distin ct acid taste w hile
diacetyl is th e a ro m a com pound com m only associated w ith lactic butter. D iacetyl is
p ro d u ced by the so -called aro m a bacteria, u su ally C it+ Lc. la ctis subsp. lactis. M any
strains o f these b a c te ria g row poorly in m ilk p ro d u cin g little acid and arom a. The
addition o f acid th o u g h , especially citric acid, results in a n increase in the p roduction
o f diacetyl and acetoin, w h ich indicates a k in d o f co -o p eratio n or “ sy m b io sis” o f the
tw o ty p es o f b a c te ria p rese n t in b utter starter cultures, i.e. th e acid producers and the
aro m a p ro d u cers [K luyver, 1933], A ddition o f sy nthetic diacetyl to b utter results in
an u n satisfacto ry h a rsh and unnatural flavour acco rd in g to B abel and H am m er
[1944],
17
Quark
Q uark, also spelled quarg, to d istin g u ish it from th e subatom ic p articles, is a fresh,
unripened soft cheese. It is related to such cheeses as cream cheese and B a k e r’s
cheese and is o ften co nfused w ith cottage cheese [K osikow sky, 1977; K roger, 1980;
Sohal et al., 1988; Jelen and R enz-S chauen, 1989]. T he greatest p ro d u ctio n o f quark,
w h ich is c a lled tv orog in E astern E urope, is in G erm any, a lth o u g h pro d u ctio n o f
q uark is sp reading to o ther countries n o w due to its surprising v ersatility in cooking
[M ann, 1987].
Q uark consists essentially o f coagulated, flocculated casein w ith a high w ater
content. It is p ro d u ced from m ilk by acid and/or rennet co a g u la tio n fo llo w ed by a
separation o f th e w hey. Its co m p o sitio n varies, dependent on th e c o m p o sitio n o f the
m ilk. I f it is m ad e from skim m ilk it is alm ost fat free b u t it can c o n ta in up to 12%
fat. T h e p ro tein co n ten t varies fro m 14 to 18% and the m a in fla v o u r com ponent is
diacetyl [K osikow sky, 1977; K roger, 1980].
T he starter cu ltu res u sed in its p ro d u ctio n are m ixed D L cultures, co n taining m ainly
Lc. la ctis subsp. lactis and Lc. la ctis subsp. crem oris as acid p ro d u cers and C it+ Lc.
lactis subsp. la ctis and L e u co n o sto c as aro m a producers [Law , 1981].
The p ro d u ctio n o f q uark can be divided into three steps, i.e. in o cu latio n , incubation
and separation. P asteu rised sk im -m ilk is m ixed w ith 1 to 2% starter culture and
in cubated at 20 to 22°C fo r lo w -tem perature incubation (preferred) or at 25 to 30°C
for h ig h -tem p eratu re incubation. S ixty or n in ety m inutes a fter in o cu latio n w ith the
starter culture, ren n e t is added to enhance p rotein stabilisation. A t th is tim e th e pH is
ab o u t 6.3. A t th e end o f the in cu b atio n period, usually 16 to 18h later, th e coagulum
w ill h av e reach ed a p H o f 4.6 to 4.7. T raditionally, w hey sep aratio n w as achieved by
cu ttin g th e curd into sm all cubes (10-15cm ) and filling it into b ag s p laced on drip
tables. In the early 1960’s a co n tin u o u s separator w as in tro d u ced to rem ove the
w hey fro m th e b ro k en coagulum . T his m o d ificatio n o f a classical dairy centrifuge
opened th e door fo r fu rth er dev elo p m en ts and im provem ents [K roger, 1980; Jelen
and R en z-S ch au en , 1989].
Several
refin em en ts
w ere
m ad e
to
im prove
y ield,
sh e lf
life
and
quality
characteristics. O ne o f the n ew pro ced u res is the C entri-w hey m eth o d , w here the
w h ey o b tain ed in the q uark m an u factu re is heated to 95°C , cooled and the denatured
w h ey p ro te in is rem o v ed w ith a self-cleaning separator and added to th e m ilk for the
nex t days processing. O ther m eth o d s are the L actal and the U ltrafiltratio n m ethods
[Jelen and R enz-S chauen, 1989; K roger, 1980].
18
A t p resen t a n e w strategy for the prod u ctio n o f qu ark is being investigated. T he new
process is b ased o n th e sam e idea as th e N IZ O m ethod fo r b utter m aking, i.e.
separation o f acid p ro d u ctio n from flav o u r production. T his w ould allo w better
control over the quality o f the end p ro d u ct and prod u ctio n o f consum er-tailored
flav o u red varieties.
A m ajo r p ro b le m o f q uark is its short s h e lf life o f 14 days; this is m ainly due to the
grow th o f co n tam in an ts and the developm ent o f bitterness during storage. The
co n tam in atio n p ro b lem has been reduced recen tly by the use o f the therm isation
process d u ring w h ic h th e m ilk undergoes a h ig h -tem perature treatm ent. B itterness is
caused by th e accu m u latio n o f b itter peptides fro m hydrolysis o f th e C -term inal end
o f B-casein, w h ich are form ed during ripening. B ittern ess can b e reasonably reduced
by decreasing th e am ount o f rennet added. In contrast to cheese, th e starter cultures
in q u ark h av e alm o st no influence o n bitterness a n d there is no co rrelation betw een
m icrobial contam inants and bitterness [Sohal et al., 1988].
A n o th er p ro b le m th a t can occur during th e m anufacture o f q uark is floating o f the
curd, due to C 0 2 p roduction from citrate m etabolism . T his can be av o id ed by
m ak in g th e q uark w ith Lc. lactis subsp. crem oris or Lc. lactis subsp. lactis as lactic
acid pro d u cers, th e n “d ressin g ” it w ith cream cultured w ith C it+ Lc. lactis subsp.
lactis as a separate source o f diacetyl.
F lav o u r d efects m ay occur w h e n diacetyl is red u ced by b acteria containing diacetyl
reductase (aceto in dehydrogenase), w h ich converts diacetyl into th e flavourless
com pound acetoin. T his can happen d u ring m anufacture as w ell as during storage o f
the fin ish ed product, b u t h igh diacetyl red u ctase starter cultures sh o u ld be avoided
[L a w ,1981],
19
AIMS AND OBJECTIVES
T he aim o f th is study w as to investigate the relationship b e tw e en the breakdow n o f
A L A and th e p roduction o f acetoin and diacetyl, as w ell as to dev elo p im proved
m ethods for the determ ination o f A L A and diacetyl in a m ix tu re o f the three
com pounds.
A nother ob jectiv e w as to screen strains o f C it+ L a cto co ccu s la clis subsp. lactis for
diacetyl pro d u ctio n and to investigate the effect o f 0 2 and various ad ditives on the
m etab o lism o f selected strains.
20
MATERIALS AND METHODS
Bacteria
T he bacteria u sed fo r screening w ere C it+ Lc. la ctis subsp. lactis strains from the
D PC collection o f strains. T he strains studied m ore clo sely w ere C it+ Lc. lactis
subsp. lactis strains 999 and 1166, 1166M 1, w h ic h is an aceto lactate decarboxylase
negative (A L D ') m u ta n t o f 1166, m ixed culture 4/25 and strain 4 /2 5 A , w hich is an
ALD" strain o f C it+ Lc. lactis subsp. lactis isolated fro m m ix e d cu ltu re 4/25, and
Lb. casei strain 4191 an d its A LD " m u tan t F 207M 3. S trains 999, 1166 and 4/25A
w ere obtained fro m the D P C collection o f strains; strain 1 166M 1, 4191 and F207M 3
w ere obtained fro m th e C entre de R echerche International A n d ré G aillard , Y oplait,
Ivry-sur-Seine, France. M ix ed culture 4/25 is the starter culture u se d com m ercially
for the p ro d u ctio n o f lactic butter.
Media
R eco n stitu ted S k im M ilk (R SM )
R S M w as p rep ared fro m skim m ilk po w d er at various co n cen tratio n s and either
heat-treated at 9 0 °C fo r 3 0 m in or sterilised at 121 °C fo r 5m in.
L actic a c id co n cen tra te
The lactic acid co n cen trate w as o b tained from N IZ O , N eth erlan d s. It h as a p H o f 3.1
and a lactic acid c o n ten t o f 150g/l. F o r certain experim ents, lactic acid concentrate
w as added to R S M a t a ratio o f 3 :2 to im itate conditions d u ring th e m anufacture o f
lactic butter.
21
L -M l 7 B roth:
T ryptone
5g
Soytone
5g
M eat d ig est
5g
Y east ex tract
2.5g
A scorbic acid
0.5g
M ag n esiu m sulphate
0.25g
D isodium -B -glycerophosphate
19g
dissolved in 950m l d istilled w ater. A fter au toclaving at 121°C for 15m in and cooling
to 50°C , 50m l o f lOg/lOOm l lactose solution is added.
L itm u s m ilk.
S kim m ilk p o w d e r
1OOg
L itm us so lu tio n
10m l
D istilled w ater
1000m l
d issolved in 1000m l o f d istilled w ater. T he litm u s m ilk w as sterilised in 100m l
D uran bo ttles a t 121 °C fo r 5m in.
Measurement of Diacetyl
D iacetyl w as m ea su red b y th e m ethod described b y W a lsh and C o g an [1974], w hich
is a m o d ificatio n o f th e P rill and H am m er [1938] m ethod.
R eagents:
1. H y d ro x y lam in e: 17.5g hy d ro x y lam in e hy d ro ch lo rid e (N H 2O H .H C l)
m ade up to 5 00m l w ith d istilled w ater.
2.
A ceto n e-p h o sp h ate: 38g K 2H P 0 4.3H 20 and 4 0m l acetone m ad e up to
20 0 m l w ith d istilled w ater.
22
3.
A lk alin e tartrate: 80g N aK tartrate.4H 20 and 24m l 3 5 g /l 00m l
N H 3 m ade up to 200m l w ith distilled w ater.
4.
F erro u s sulphate: 5g F e S 0 4.7H 20 m ade up to 100m l w ith lm l/lO O m l
H 2S 0 4.
P rocedure:
A v o lu m e o f sam ple w as steam distilled using a B iichi steam distillatio n apparatus.
T he first and second 10m l o f distillate w ere collected sep arately in graduated test
tubes. T he first 10m l w ere used for the d eterm in atio n o f diacety l and the second
10m l fo r th e m easu rem en t o f acetoin. A ll the diacetyl an d m o st o f th e aceto in are
p rese n t in th e first 10m l; th e second 10m l contain about 2 5% o f th e acetoin [W alsh
and C ogan, 1974],
To 5m l o f the first 10m l o f distillate, 1m l o f hyd ro x y lam in e w as added. T he sam ple
w as m ix e d and h eated in a w aterbath to 75°C fo r 20m in. It w as th en cooled in air.
W ith in lO m in, 0.5m l o f acetone phosphate w ere added. A fter m ixing, 1.5m l o f
alkaline tartrate w ere added, the sam ple w as m ix ed ag a in and im m ed iately 0.2m l o f
F e S 0 4 w ere added. A fter 15-20m in the A 530 w as read against a reag en t blank.
In d ep en d en t aqueous solutions o f diacetyl (lO m M ) w ere sto red at 4°C . A standard
curve w as o b tained by distillin g and analysing different co n cen tratio n s o f diacetyl in
the sam e w ay as th e sam ples. T he standard curve w as linear up to an A 530 o f a t least
2. O nce th is h a d b een estab lished, it w as only n ecessary to d istill one standard in
duplicate in su bsequent tests.
Measurement of Acetoin
A ceto in w as determ in ed b y the W esterfeld [1945] m ethod.
R eagents:
1. 0.5 g / 100m l creatine
2.
2.5M N a O H
3.
5 g /l0 0 m l 1-naphthol in 2 .5 M N aO H
23
Procedure:
To 5m l o f the seco n d 10m l o f steam distillate, o r an a liq u o t m ad e up to 5m l w ith
distilled w ater, 1ml creatin e solution w as added, fo llo w ed by 1ml o f 1-naphthol, and
the sam ple w as m ixed. A fter exactly 60m in in a w a te rb a th at 21 °C the A 525 w as read
against a reag en t blank.
Tw o in d ep en d en t aq u eo u s stock solutions o f a ceto in (lO m M ) w ere stored at 4°C. A
standard curve w as obtain ed by distilling a n d assaying d iffe re n t concentrations o f
acetoin. T he stan d ard cu rv e w as linear up to an A 525 o f a t least 1.25. O nce this had
been established, only one standard in duplicate w as su b sequently u sed at each
analysis.
Measurement o f Acetolactate (ALA)
Since A L A is easily decarboxylated to aceto in in acid ic solutions, it can be
determ ined fro m th e d ifferen ce in the con cen tratio n o f a ceto in before and after
decarboxylation w ith HC1 [Jordan and C ogan, 1995].
R eagents:
1.
0 .5 M H C 1
2.
5 0 m M p h o sp h a te bu ffer pH 6.5 o r 125m M p h o sp h a te b u ffer pH 7.5
3.
0.5 g /10 0m l creatine
4.
2 .5 M N a O H
5.
5g /100m l 1-N aphthol in 2.5M N aO H
Procedure:
To p o rtio n o f th e sam ple, usually 50 o r 1OOf-il o f liq u id sam ple or 50 or lOOmg o f
solid sam ple, 0 .4m l o f 0.5M HC1 w ere added to in d u ce decarb o x y latio n o f A L A to
acetoin. A second p o rtio n o f sam ple w as treated w ith 0.4 m l o f distilled w ater instead
o f HC1. S u fficien t 5 0m M ph osphate buffer w as th en ad d ed to each test tu b e to m ake
up the volum e to 5m l. T his m inim ises a u to d ecarb o x y latio n o f A L A to acetoin in the
24
w ater-treated sam ple. T he sam ples w ere h eld at 4°C for 16 to 30h an d acetoin w as
th en determ in ed according to the W esterfeld [1945] m eth o d as d escribed above.
A m o d ific a tio n h a d to be m ade for sam ples containing th e lactic acid concentrate,
w h ic h d ecreased th e pH o f the sam ples to 3.2. D istille d w a te r w as u sed instead o f
ph o sp h ate bu ffer for sam ples treated w ith HC1, an d the c o n cen tratio n o f phosphate
bu ffer w as in creased to 125m M for sam ples treated w ith w ater, in ord er to m ain tain
the correct pH fo r decarboxylation o f A L A on th e o n e h an d and to m inim ise
au to d ecarb o x y latio n o n the other.
U sin g th is m ethod, A L A and acetoin co n cen tratio n s can be determ ined at th e sam e
tim e. T h e am o u n t o f A L A p resent in a sam ple is o b tain e d by subtracting the
con cen tratio n o f aceto in in th e w ater-treated sam ple from the co n centration o f
acetoin in th e H C l-treated sam ple, tak in g into account th at only 84% o f A L A is
con v erted to aceto in by this procedure [Jordan an d C ogan, 1995]. Since during the
W esterfeld m eth o d aceto in is converted to diacetyl, any diacetyl p rese n t in a sam ple
h as to be determ in ed separately by the W alsh and C o g an [1974] m ethod and
subtracted fro m th e w ater-treated sam ple to ob tain th e tru e v alu e fo r acetoin.
A n alternative m eth o d for the determ in atio n o f A L A w as developed, based o n the
fact th a t A L A can be d ecarboxylated oxid ativ ely to diacetyl u n d er the influence o f
m etal ions, lo w p H and heat.
R eagents:
1. O .lm l/lO O m l H 2S 0 4
2. C itric acid (0.2M )/N a2H P 0 4 (0.4M ) bu ffer at p H 3.3
3. lO m M C u S 0 4 in solu tio n (2)
To 3m l o f sam ple 5.5m l o f bu ffer and 1.5m l o f C u S 0 4 solution w ere added; for
sam ples w ith lo w diacetyl concentrations the vo lu m e w as increased 3-fold (final
C u2+ c o n cen tratio n 1.5m M ). The p H o f this m ixture w as 3.5. T h e m ixture w as steam
distilled, w h ic h resu lted in com plete ox id atio n o f A L A to diacetyl (see results). T he
first 10m l o f distillate w ere collected and assayed for diacetyl b y the W alsh and
25
C ogan [1974] m ethod. T he am ount o f diacetyl p resen t in th e sam ple before
o x id atio n w as determ in ed by steam d istillation in w ater in ste a d o f
the C u S 0 4
solu tio n at p H 0.8. A p H 0.8 w as achieved b y ad d in g 6M H 2S 0 4. A L A w as then
calculated as th e difference in the di acetyl levels m easu red before and after
oxidation.
A n aceto in standard w as tested w ith every set o f sam ples assayed for A L A by the
Jo rd an and C o g an [1995] m ethod, w hereas a diacety l standard w as used w ith
sam ples assay ed fo r A L A u sin g the C u S 0 4 m ethod.
Measurement of Citrate
C itrate w as determ in ed b y the m ethod o f M arier and B o u let [1958].
R eagents:
1.
5.5 6 g /1 0 0 m l trichloracetic acid (TCA )
2.
P y rid in e
3. A cetic anhydride
Procedure:
A 0.5m l sam ple w as ad d ed to 4.5m l o f the TC A . T h e m ixture w as sh ak en vigorously
and left standing fo r at least 30m in to desorb and solubilize any citrate attached to
the casein. T he extract w as th en centrifuged fo r 5 m in a t 14000rpm in an E p p e n d o rf
m icrofuge. T o 1m l o f supernatant, 1.3m l o f p yridine an d 5.7m l o f acetic anhydride
w ere added. T h e te st tu b es w ere p laced im m ediately in a w ater b ath at 30°C for
30m in to d issip ate th e h e a t developed in the m ixture a n d allo w unifo rm colour to
develop. T h e A 428 w as read against a reagent blank w ith in another 30m in. A standard
curve m u st be carried o u t at each analysis since the relationship betw een A 42g and
c o n centration is variab le a n d curvilinear.
26
Measurement of L-Lactate and Acetate
A 2m l sam ple w as ad d e d to 2m l o f lOg/lOOml T C A , m ix ed a n d left standing for at
least 30m in. T he e x tract w as centrifuged for 5m in at 14000rpm in a n E p p e n d o rf
m icrofuge. L actate and acetate w ere m easured in the su pernatant u sin g B oehringer
enzym atic test kits. S am ples treated th at w ay decreased the p H o f th e bu ffer solution
used in the te st kits b y 0.1 o f a pH unit.
ALA Standard Curves
An
ALA
sto ck
so lu tio n
w as
obtained
by
hydro ly sin g
2-acetoxy-2-m ethyl-
acetoacetate (A L A do u b le ester) w ith tw o equivalents o f fre sh ly p rep ared 0.1M
N aO H by m ix in g gen tly at room tem perature for 30m in. R S M (lO g/lOO g) or a
m ixture o f R S M and lactic acid concentrate w ere added to the h y d ro ly sed ester to
give concentrations fro m 0-1 OmM A L A . T his stock so lu tio n w as th en u sed for
standard curves.
T he p H w as adjusted w ith N aO H (6M ) or lactic acid (lO g/lO O m l) to give p H values
from 3.3 to 8.0. F iv e o r 20m l o f sam ple w ere distilled and th e first 10m l o f steam
distillate assayed for diacetyl.
Model System
The effects o f o xygen, m ilk solids, tem perature, pH , m etal ions (F e 2+ and C u2+) and
h aem in on th e b rea k d o w n o f A L A to diacetyl and acetoin w ere studied in a m odel
system . T he m odel sy stem consisted o f a B rau n ferm enter co n tain in g R S M o r the
3:2 m ixture o f R S M a n d lactic acid concentrate and 1.2m M A L A . T he contents o f
the ferm enter w ere stirred at 500rpm and, w here th e effect o f 0 2 w as studied, the
m edium w as sp arged w ith 0 2 (lO psi). A L A , acetoin and diacetyl w ere m onitored
27
over tim e. S am ples for diacetyl w ere adjusted to p H 6.5 w ith N a O H before
distillation.
Conversion Rates
C onversion rates o f A L A to a c eto in and diacetyl w ere d e te rm in e d by p lo ttin g the
concentrations o f eith er c o m p o u n d against A L A concentrations. T h e con v ersio n rate
w as obtained by m u ltip ly in g the slope o f the linear reg ressio n line b y 100.
Growth Experiments
Pure Cultures
S creen in g
O ne h u n d red and th irty fo u r strains from th e laboratory c o lle ctio n w ere screened for
citrate utilisatio n and diacetyl production. T he strains, stored at -80°C , w ere grow n
in L -M 17 b ro th o v er n ig h t at 30°C . A few drops o f th e fu lly g ro w n culture w ere th en
u sed to inoculate litm u s m ilk, w h ic h w as incubated at 30°C u n til clotted. R S M
(10g/100m l) w as in o cu lated (lm l/1 0 0 m l) w ith the fresh ly clo tted culture. P ortion
(40m l) o f the in o cu lated m ilk w as th en transferred to a 500m l sterile D u ran bottle
and oxygenated w ith 0 2 fo r lm in . T he bottles w ere th en tig h tly capped and
incubated at 30°C fo r 16h. T he rem aining 60m l w as u sed as a n o n -oxygenated
control and in cubated in the sam e w ay. A fter 16h in cu b atio n the cu ltu res w ere iced
dow n and assayed fo r diacetyl, citrate and pH . F o r th e estim atio n o f diacetyl 10m l,
or 10g in case o f a clo tted sam ple, w as distilled. The p H w as n o t ad ju sted p rio r to
distillation.
G row th kinetics
A fter the screening strains 999 and 1166 w ere selected fo r m ore detailed studies.
Strains 999 and 1166 w ere ex am ined for their grow th ch aracteristics and m etab o lite
p ro d u ctio n under o x y g en ated an d non-oxygenated conditions. T he cultures, stored
at -80°C , w ere g ro w n o v ern ig h t in L -M 17 b ro th at 30°C . T h is b ro th culture w as used
28
to inoculate R S M (100g/l), w h ich w as then in cu b ated at 30°C u n til clotted. H eat
treated R S M (10 0 g /l) w as inoculated at lm l/lO O m l w ith the c lo tted m ilk culture,
m ixed w ell a n d d istrib u ted in 100m l volum es in 500m l D u ra n bottles. H a lf o f the
bottles w ere sp arg ed w ith 0 2 for 3m in and th e n tig h tly closed. T h e o ther h a lf w ere
used as n o n -o x y g en a ted controls.
T he effects o f leu cin e and valine (lO m M ) on strains 999 an d 1166, an d th e effects o f
m etal ions (F e+I~, C u ^ , 100p,M) and h aem in (10|J,M) on strain 999 w ere studied
u nder b o th o x y g en ated an d non -o x y g en ated conditions.
Strains 1166 an d 1166M 1 w ere also studied u n d e r 0% , 2 1% and 100% 0 2. T he
cultures w ere p rep a re d as described above a n d sparged w ith 0 2, a ir or N 2 fo r 6m in.
0 2 con cen tratio n s w ere m easu red u sin g a K n ick 0 2 m eter.
The bottles w ere in cu b ated at 30°C in a w ater b a th u n til the p H reach ed 4.7. A t
regular in terv als b o ttles w ere tak e n out, iced d o w n and assayed fo r A L A , acetoin,
diacetyl, citrate, lactate, acetate an d pH .
C om parison o f the tw o m ethods f o r the d eterm ination o fA L A
To com pare the C u S 0 4 m eth o d for th e d eterm in atio n o f A L A w ith th e Jo rd an and
C o gan [1995] m eth o d , strains 4/25A , 1166, 1166M 1 a n d 4191M 3 w ere grow n as
described above. S am ples w ere tak en at reg u lar intervals and an aly sed fo r A L A w ith
the C u S 0 4 m eth o d and the Jo rd an and C o g an [1995] m ethod.
Mixed Culture 4/25
L a b o ra to ry trials
E ffect o f solids
M ixed culture 4/25 w as stored in the com m ercial containers at -20°C . H eat-treated
R S M (16, 19 and 23g solids/1 OOg) w as in o cu lated w ith th e culture follow ing the
instructions o n the container. T he culture w as sp lit into 50m l v o lu m es in 250m l
bottles and in cu b ated at 21°C . T he p H w a s m on ito red co n tin u o u sly u sin g the
29
C IN A C hardw are and softw are (IN R A , G rignon, France) a n d sam ples w ere tak e n at
regular intervals fo r citrate, A L A , diacetyl and acetoin. A L A an d diacetyl w ere
determ in ed w ith th e C u S 0 4 m ethod.
E ffect o f tem p eratu re
T he culture w as g row n at 23°C for 18h o r until th e pH d ro p p ed b e lo w 5.0. T he pH
w as m o n ito red co n tin u o u sly using th e C IN A C hardw are and softw are (IN R A ,
G rignon, France). A t the en d o f grow th, a sam ple w as tak e n fo r citrate, A L A , acetoin
and diacetyl. T he culture w as th en div id ed into three separate ferm enters. L actic acid
culture co ncentrate w as ad d ed (ratio culture to lactic acid culture concentrate 2 to 3)
and th e
cu ltu res w ere stirred at 11, 23 and 30°C for 2h. S am ples w ere tak e n at
reg u lar intervals an d a n aly sed for A L A , acetoin and diacetyl. A L A w as determ ined
by th e Jo rd an an d C ogan [1996] m ethod.
C o m m ercial trials
C ulture
4/25
w as
g ro w n
for
18h at 23 °C
in
lOOOlitres R S M
w ith
solid
co ncentrations ranging fro m 17 to 23g/100m l in three co m m ercial plants. Sam ples
w ere tak e n p rio r to the ad d itio n o f lactic acid concentrate a n d subsequently during
aeratio n o f th e m ixture a t various tem peratures, and an aly sed fo r A L A , aceto in and
diacetyl. T he sam ples ta k e n p rio r to the ad dition o f lactic acid concentrate w ere also
analysed fo r citrate. A e ra tio n tim es varied from 35m in in p la n t C over 5 3 m in in
p lan t A to 2 .5 h in p lan t B.
Quark
Q uark sam ples w ith vary in g A L A concentrations w ere o b tain ed fro m the C entre de
R echerche In tern atio n al A ndré G aillard, Y oplait, Ivry-sur-S eine, France. In order to
m o n ito r A L A b rea k d o w n during storage at 4°C , sam ples w ere analysed in triplicate
for A L A and diacetyl u sin g the n ew m ethod during storage at 4°C .
30
RESULTS
PA R T I
ALA Standard Curves
T he effects o f vo lu m e (5 and 20m l), p H (pH 3.3-8.0) and A L A co n centration (0lO m M ) on th e co nversion o f A L A to diacetyl during d istillatio n in R S M (lO g
solids/lO O g) or a m ixture o f R S M (lO g solids/lO O g) and lactic acid concentrate w ere
investigated. T he results are show n in T ables 1 and 2. In R S M , the distillatio n
vo lu m e h a d n o effect on th e co nversion o f A L A to diacety l i f th e p H w as >5.0.
B elo w p H 5.0, co nversion increased w ith d ecreasing p H an d w as greater w h en 5m l
in stead o f 20m l w ere distilled. T he A L A co n centration seem ed to h av e an effect on
the b reak d o w n d u ring distillation; break d o w n w as h ig h er in standard curves from 0
to 2 .5 m M th an in standard curves from 0 to lO m M (T able 1). W h en th e individual
standard curves w ere ex am in ed m ore closely, it appeared th a t the b reak d o w n w as
n o t lin ear over the con cen tratio n range fro m 0 to lO m M , w ith h ig h er breakdow n
rates o ccurring at low er A L A concentrations. A n exam ple o f a standard curve at pH
6.0 is sh o w n in F igure 3.
ALA, mM
Fig. 3: S tandard curve for A L A in R S M at p H 6.0. 2 0 m l w as distilled
31
In a m ix tu re o f R S M and lactic acid co n centrate, the d istillatio n volum e did not
appear to h av e as g reat an effect on A L A b reak d o w n (T able 2). A s in the case o f
R S M alone, b rea k d o w n rates w ere greater at low er p H values. In th ese experim ents,
th e reg re ssio n lin es w ere linear w ith r2 values > 0.90.
T he resu lts fro m th e A L A standard curves w ere d ifficu lt to reproduce from one day
to another, alth o u g h the sam e procedure w as fo llo w ed each tim e. It w as evident,
how ever, th at th e p H at w h ich a sam ple is distilled has a m ajo r effect on A L A
b reak d o w n d u rin g distillation, w ith h ig h er b reak d o w n rates occurring at lo w pH , and
th at th e b rea k d o w n o f A L A is n o t lin ear o v er th e con cen tratio n range from 0 to
lO m M .
T able 1: B re a k d o w n o f A L A to diacetyl d u ring distillatio n in R S M
V o lu m e
d istilled (m l)
ALA
(m M )
pH
n
A verage
B reak d o w n (% )
sd
5
0-1.2
3.3
1
38.6
5
0-1.2
4.5
4
16.0
5
0-1.2
5.5
3
7.91
3.38
5
0-1.2
6.8
3
4.92
0.82
5
0-10
4.5
2
4.32
0.66
20
0-2.5
3.3
1
11.3
20
0-2.5
4.3
1
16.0
20
0-2.5
4.5
16
9.95
5.77
20
0-2.5
4.7
3
9.50
2.10
20
0-2.5
4.9
2
8.28
3.82
20
0-2.5
5.0
5
7.35
2.88
20
0-2.5
6.0
7
6.24
1.11
20
0-2.5
7.0
6
4.27
1.67
20
0-2.5
8.0
4
5.31
1.33
20
0-10
4.5
9
5.90
2.69
20
0-10
4.7
2
7.89
0.76
20
0-10
4.9
2
4.30
0.51
20
0-10
5.0
5
4.10
1.12
20
0-10
6.0
5
2.51
1.28
20
0-10
7.0
4
3.30
1.20
20
0-10
8.0
2
4.36
0.18
4.43
T able 2: B re a k d o w n o f A L A to diacetyl during distillation in R S M and lactic acid
co ncentrate
A verage
B reakdow n (% )
sd
4
37.6
4.29
1
29.4
pH
n
5
(m M )
0-2
3.3
5
0-2
4.5
5
0-2
6.5
3
5
0-2
9.0
1
15.2
20
0-2
3.3
1
46.3
20
0-2
4.0
3
29.1
20
0-2
4.5
3
9.85
3.35
20
0-2
6.0
2
4.67
0.00
20
0-2
6.5
4
7.21
1.84
20
0-2
7.0
2
6.87
2.17
V o lum e
distilled (m l)
ALA
8.33
0.63
8.88
Model System
E ffe c t o f oxygen
T he m ed iu m u se d for th e o x y g enation experim ents w as a m ix tu re o f R S M (16g
solids/1 OOg or 19g solids/lO O g) and lactic acid concentrate. T he p H o f the m ixture
w as 3.3. T h e m ix tu re w as h e ld at 21°C , stirred and sp arged w ith 0 2 and the
co nversion o f A L A to aceto in and diacetyl w as co m p ared to a non-oxygenated
control.
The first a e ra tio n trials resu lte d in conversion rates o f A L A to diacetyl o f 44.1%
(16g
solids/lO O g
RSM )
an d
35.4%
(19g
solids/lOO g
R S M ),
w hich
w ere
co nsiderably lo w er th a n in the non-oxygenated controls w h ic h sh o w ed conversion
rates o f 52.6 a n d 55.7% , respectively (T able 3). T he u n e x p e cte d lo w er conversion
rates in th e o x y g en ated sy stem w ere due to the fact th a t som e d iac e ty l w as carried
out fro m th e ferm en ter by th e air leaving the system . In o rd er to qu an tify the am ount
o f d iacetyl th at w as lo st th is w ay, th e air leaving the sy stem w as led through either
one o r tw o b o ttles each filled w ith 1 litre o f distilled w ater. A d d in g th e am ount o f
diacetyl m ea su red in the first bottle o f w ater to the d iacety l in th e system increased
con v ersio n rates fro m 44.1 to 67.7% in the m ilk w ith th e lo w er so lid s concentration
(1 6g solids/lO O g) and fro m 35.4 to 72.3% in R SM w ith 19g solids/lO O g. A dding a
33
second bo ttle containing 1 litre o f w ater to trap diacetyl leaving th e first bottle d id
n o t increase th e recovery. C onversion o f A L A to acetoin in the low er con cen tratio n
o f R S M tested (16g solids/1 OOg) w as low er in the oxygenated system th an in the
control, 12.9% co m p ared to 30.1% . A t th e h igher level o f m ilk solids (19g
solids/1 OOg) there w as no difference in the average breakdow n o f A L A to aceto in
w h e n the system w as
oxygenated. A ddition o f th e con v ersio n rates o f A L A to
diacetyl and aceto in show th a t > 80% o f the A L A added w as recovered as diacetyl
a n d acetoin.
T able 3: E ffect o f 0 2 o n con v ersio n (% ) o f A L A 3 to diacetyl and a ceto in in a m odel
system
C onversion (% ) to
D iacetyl
average
A ceto in
sd
n
average
sd
n
R S M 16g so lid s/1 OOg
n on -o x y g en ated R S M
52.6
8.25
5
30.1
1.98
7
oxygenated R S M
44.1
8.72
5
12.9
8.56
5
oxygenated R S M + 1L o f w ater
67.7
6.47
2
n o n -oxygenated R S M
55.7
9.24
5
24.0
13.1
5
oxygenated R S M
35.4
2.48
2
24.5
3.62
2
oxygenated R S M + 1L o f w ater
72.3
0.00
1
oxygenated R S M + 2 x 1L o f w ater
73.8
4.02
2
R S M 19 g so lid s/1 OOg
* measured by the Jordan and Cogan [1995] method
E ffe c t o f m ilk so lid s
T he conversion o f A L A to acetoin and diacetyl w as studied in R S M (13, 16, 19 and
23g solids/1 OOg) a n d lactic acid concentrate, m ixed in a ratio 3:2, at 21°C . The
results are show n in T able 4. T he solids level o f the m ilk d id n o t affect the
co nversion o f A L A to aceto in and diacetyl to any great extent. B reak d o w n to
diacetyl w as greater th an b reakdow n to acetoin on all occasions.
34
Table 4: Effect of concentration of milk solids on the conversion of ALAa to acetoin
and diacetyl a t 21°C
C onversion (% ) to
M ilk
(g/lOOg)
13
A cetoin
D iacetyl
(% )
^ALA
(h-*)
21.8
69.0
56.6
0.2652
average
sd
16
8.22
average
sd
19
23
A L A u sed
30.1
1.98
average
24.0
sd
13.1
average
25.5
8.84
sd
8.65
56.3
9.11
55.7
9.24
54.5
3.84
3.11
63.0
8.38
64.9
7.14
53.9
2.43
n
6
0.0340
0.3422
7
0.0354
0.3352
5
0.0773
0.2597
4
0.0190
a measured by the Jordan and Cogan [1995] method
D uring these ex perim ents it becam e apparent, th at th e b reak d o w n o f A L A over tim e
w as n o t linear, b u t fo llo w e d an exponential function, im p ly in g th at b rea k d o w n w as a
first order reaction:
CALA
= a x e k>“
w here cALA is the A L A co n centration (m M ), a the coefficient, k the specific
b reakdow n rate (h '1) and t th e tim e (h).
T he specific b reak d o w n rate, k, is also show n in T able 4; it increased fro m 0.2 6 5 2 h ''
in R S M o f 13g solids/lO O g to 0.3352h_1 in R S M o f 19g solids/lO O g, b u t dropped
ag ain to 0.259711'1 in R S M o f 23g solids/lOOg.
E ffe c t o f tem perature
T he effect o f tem p eratu re on the b reakdow n o f A L A w as studied o v er a tem perature
range fro m 7 to 37°C in R S M o f 16g solids/1 OOg and lactic acid concentrate, m ixed
in a ratio o f 3 :2. C o n v ersio n rates to aceto in and diacetyl, and specific b reakdow n
rates are show n in T able 5.
35
Table 5: Effect of temperature on breakdown of ALAa
C onversion (% ) to
T em perature
A cetoin
D iacetyl
^ALA
(h-‘)
average
0.71
27.7
0.0896
sd
0.26
(°C)
7
13
average
sd
18
average
sd
21
average
sd
23
average
sd
26
30
37
38.0
17.5
9.68
8.63
30.5
9.59
38.5
12.9
average
45.9
0.3291
48.0
4.48
6.80
2
0.0378
34.7
3.36
50.1
0.1834
0.0354
48.9
2
0.0164
10.5
4.87
3.93
0.1376
0.3422
51.1
2
0.0103
47.9
0.04
2.09
sd
sd
1.79
2.45
27.6
average
34.9
26.2
average
sd
2.01
n
7
2
0.0312
0.6026
3
0.0263
1.0869
3
0.3592
2.5574
3
0.5097
a measured by the Jordan and Cogan [1995] method
The specific rate o f A L A b reakdow n (kALA) increased w ith increasing tem perature
w ith the ex cep tio n o f a sm all decrease at 23°C . T he sam e w as m ore or less tru e for
the conversion o f A L A to acetoin w hereas co nversion to d iacety l decreased slightly
at tem peratures > 26°C .
T he relationship b e tw e en kAI A and tem perature is no n -lin ear, but can be expressed as
an exponential fu n ctio n (Fig. 4). A n A rrhenius p lo t o f th e relatio n sh ip betw een k ^
and tem perature w as linear. T his p lo t is used in th erm o d y n am ic s to determ ine the
activation energy (E A) o f a chem ical reaction:
E A = -m x R
w here EA is th e ac tiv atio n energy, m is the slope and R is the general gas constant
[8.314J/(m olxK )].
T he activ atio n en erg y fo r th e b reakdow n o f A L A w as 8 2.7kJ/m ol or 19.8kcal/m ol.
36
T e m p e r a t u r e , “C
1 /a b so lu te T e m p e r a t u r e , K
Fig. 4: (A ) E ffect o f tem p eratu re o n the specific b reak d o w n rate, k, o f A L A , w ith
erro r bars. (B ) A n A rrh en iu s p lo t o f the sam e data.
E ffect o f p H
P relim in ary trials o n th e b reak d o w n o f A L A as a fu n ctio n o f p H w ere carried o u t in
250m l D u ran bo ttles filled w ith a m ix tu re o f R S M (16g solids/1 OOg) and w ater (ratio
3:2) containing 1.2m M A L A over a p H range from 4.0 to 6.5. T he pH w as adjusted
w ith lactic acid (3.33M ) in T rial 1 and HC1 (1M ) in T rial 2, to determ ine i f the ty p e
o f acid in flu en ced th e rate o f breakdow n. T he b o ttles w ere stirred on a m u lti-stirrer
u n it in a w ater b a th at 21°C . A L A b reak d o w n did n o t appear to fo llo w a first order
reactio n ex cept at p H 4.0. C o rrelatio n coefficients (r2) fo r the h ig h er p H v alues w ere
low , b ecau se o f lo w b rea k d o w n rates (T able 6). Specific b reakdow n rates w ere
determ in ed and p lo tted against p H and resu lted in an exponential curve (Fig. 3). T he
type o f acid u sed d id n o t sig n ifican tly influence A L A breakdow n.
A th ird trial w as carried out in a ferm enter u sin g th e sam e reactio n m ixture, w h ich
w as stirred at 500rpm a t 21°C ; th e p H ranged from 3.0 to 6.0 and w as ad ju sted w ith
HC1 (1M ). A t p H 3.0, 3.5 an d 4.0, the correlation coefficients o f an exponential
curve fit fo r A L A b rea k d o w n over tim e w ere >0.97, indicating ex cellent agreem ent.
37
A t p H values above 4.0, little breakdow n occurred and co rrelatio n coefficients w ere
b e lo w 0,6 (T able 6), T he relationship b etw een kALA and p H w as exponential, b u t kALA
values w ere slig h tly low er th an in T rials 1 and 2 (Fig. 5).
T able 6: Specific b reakdow n rates (kALA) and correlation co efficients (r2) for A L A a
b reak d o w n a t varying pH values
T rial 1
pH
^ ala (h"1)
T rial 3
Trial 2
r2
kALACh'1)
r
2
W
h ' 1)
r2
3.0
0.2646
0.9796
3.5
0.1367
0.9787
4.0
0.0912
0.9449
0.0975
0.8615
0.0587
0.9722
4.5
0.0504
0.8056
0.0564
0.8401
0.0219
0.5739
5.0
0.0442
0.8424
0.0438
0.7966
0.0137
0.3726
5.5
0.0265
0.6855
0.0398
0.8957
6.0
0.0195
0.6998
0.0120
0.5469
0.0377
0.5263
6.5
0.0207
0.6824
0.0075
0.4000
11m easured by the Jordan and C ogan [1995] m ethod
3
4
5
6
7
PH
Fig. 5: E ffect o f p H o n th e specific b reak d o w n rate o f A L A (k); th e pH w as adjusted
w ith lactic acid in T rial 1 ( O ) , and in T rial 2 ( ♦ ) and T rial 3 ( ■ ) w ith HC1.
38
E ffe c t o f m e ta l ions a n d haem in
T he effects o f C u S 0 4 (0.1, 0.2 and 2m M ), F eC l3 (0.2m M ) and haem in (0.01 and
0.1 m M ) on th e b reak d o w n o f A L A to diacetyl w ere studied in R S M (1 3g
solids/1 OOg) at p H 6.5 a t 21 °C. Figure 6 show s a ty p ical result.
1.4
1.2
1.0
0.8
%
e 0.«
0.4
0.2
0.0
0
20
40
60
80
100
120
Time, min
Fig. 6: A L A b rea k d o w n ( ■ , □ ) and diacetyl p ro d u ctio n ( A , A ) in R S M (13g
solids/lO O g) a t p H 6.5 a t 21°C in th e p resen ce (full sym bols) and absence
(open sy m b o ls) o f 0.2m M C u S 0 4.
O ver a p erio d o f 2 h little or no breakdow n o f A L A occurred in the presence and
absence o f C u2+. H o w ev er, ~ 4 tim es m ore diacetyl w as detected in th e presence o f
C u2+ th an in th e control. B reakdow n o f A L A to diacety l w as in stantaneous and
diacetyl levels d id n o t increase above the in itial value. T his indicates th a t A L A was
converted to diacety l during m easurem ent rath e r th an in th e ferm enter, m o st likely
during distillatio n , d u e to the com bined effect o f h eat and th e o x idising agent.
S im ilar resu lts w e re o b tain ed for F eC l3 a n d haem in. B reak d o w n o f A L A to diacetyl
fo r all additives is sh o w n in T able 7.
39
Table 7: Breakdown® of ALAb to diacetyl in the presence of various additives
A d d itiv e
C o n centration
(m M )
n
B reakdow n
H aem in
0.01
0.10
0.10
0.20
2.00
0.20
/
5
4
1
3
1
2
4
(% )
9.3
26.7
21.1
22.7
67.3
11.3
5.6
C uS04
F e C l3
C ontrol
sd
1.80
4.18
3.92
0.94
0.59
a Breakdown was calculated from the values at time 0
b ALA was measured by the Jordan and Cogan [1995] method
B reak d o w n w as g reatest in th e presence o f 2m M C u S 0 4 (67.3% ), follow ed by
O .lm M h aem in (26.7% ). T here w as only a sm all d ifference b e tw e en th e breakdow n
rates in the p resen ce o f 0.1 and 0.2m M C u S 0 4 (21.1% and 22.7% ); breakdow n w as
sim ilar w h e n O.O lm M haem in and 0.2m M F eC l3 w here u sed (9.25% and 11.3% ). In
the control, 5.6% o f A L A w as co n v erted to diacetyl.
Development of a New Method for the Determination of ALA
A n alternative m eth o d fo r th e determ ination o f A L A , p rev io u sly described by
G ollop et al. [1987], in volves the oxidative d ecarboxylation o f A L A to diacetyl by
h eatin g th e sam ple fo r lO m in at 80°C in the presence o f 0 .1 5m M each o f FeC l3 and
F e S 0 4 follow ed b y “ air” distillatio n at 60°C . W hen th is m eth o d w as investigated,
u sin g steam distillatio n at 100°C, only 15.7% o f the A L A w as reco v ered as diacetyl.
T he p H o f th e m ixture o f R S M and F eC l3/F e S 0 4 w as 5.5 instead o f 4.0 as
reco m m en d ed by G ollop et al. [1987], T he effect o f differen t concentrations o f acid
and F e w as investigated. T he results (T able 8) show ed considerable v ariation w ith
th e h ig h est b reak d o w n (48.7% ) at pH 0.8 in the presence o f 3.5m M each o f F e2+ and
F e 3+. In addition, som e o f th e reg ressio n coefficients o f diacetyl on A L A w ere low,
in d icatin g p o o r reproducibility.
40
Table 8: Breakdown of ALA to diacetyl during steam distillation at various pH in
the p resen ce o f iron
A cid
F inal F e
con cen tratio n
(m M )
Final acid
concentration
(m M )
PH
n
S lo p e3
r>
5
0.157
0.837
4
1.5
1.5
5.5
h 2s o
0.15 F e 5"
0.15 F e3+
0.15 F e‘+
h 2s o
4
7.14
3.5
5
0.360
0.728
h 2s o
4
7.14
3.5
5
0.316
0.967
Ô.Ï5 F e i+
h 2s o
4
7.14
3.5
5
0.437
0.922
3.5 Fei+
3.5 F e3+
HC1
350
0.8
5
0.487
0.999
HC1
0.15 F ei+
0.15 F e3+
* R egression o f diacetyl on A L A
T he results in th e m odel system (T able 7) show th at C u S 0 4 has a greater effect on
the b reak d o w n o f A L A to diacetyl th an F eC l3. T o find the o ptim um con cen tratio n o f
C u S 0 4, A L A (2m M and Im M ) in R S M (lO g/lOO g) w as d istilled in th e p resen ce o f 0
to 7m M C u S 0 4 (in 18.8m M H 2S 0 4) at p H 3.5. T he results sh o w th at at C u S 0 4
concentrations > lm M , th e conversion o f A L A to diacetyl w as essentially 100% (Fig.
7). A c o n cen tratio n o f 1.5m M C u S 0 4 w as ch o sen as the optim um .
100
80
O
©
©
O
—
©
V
cs
o
I
O
>/■)
o’
O
p
— ’<
O
>n
—
<
O
p
O
cn
v-|
O
©
cn
C uS04, mM
Fig. 7: R eco v ery o f A L A as diacetyl at a range o f C u S 0 4 concentrations; closed
bars: Im M A L A , open bars: 2m M A L A
41
S tandard curves containing 0.25-4m M A L A w ere distilled in th e p resen ce and
absence o f 1.5m M C u S 0 4 (in 18.8m M H 2S 0 4) at pH 6.5, 3.5 a n d 0.8. T he pH
attained by th e m ix tu re o f R S M (norm al pH 6.5) and C u S 0 4 in H 2S 0 4 w as 3.5. The
other pH v alu es w ere obtained by adjusting w ith 2.5M N a O H and 6M H 2S 0 4 to
obtain p H v alu es o f 6.5 and 0.8, respectively. T ran sfo rm atio n o f A L A to diacetyl
w as g reatest at p H 3.5 in the presence o f C u S 0 4 (Fig. 8). In a d d itio n it w as linear
and reproducible; the error bars show the standard dev iatio n o f 10 trials. A t A L A
concentrations > 4 m M b reak d o w n rates decreased due to in co m p le te reco v ery o f the
large am ounts o f diacetyl in th e first 10m l o f steam distillate. T he lo w e st breakdow n
occurred a t p H 0.8 in th e absence o f C u S 0 4. R egression analysis fo r all conditions is
show n in T able 9. It w as co ncluded th at A L A sh o u ld be d e te rm in e d at p H 3.5 in the
presence o f 1.5m M C u S 0 4, in w h ich com plete oxidative d e c arb o x y la tio n o f A L A to
diacetyl occurs, a n d diacetyl at p H 0.8 in th e absence o f C u S 0 4, w h ere <2%
oxidative d ecarb o x y latio n o f A L A to diacetyl occurs.
ALA, mM
Fig. 8: A L A standard curves at p H 6.5 ( • , O ), 3 .5 ( 1 , □ ) and 0.8 ( ♦ , < > ) , in the
p resen ce (closed sym bols) an d absence (open sym bols) o f 1.5m M C u S 0 4
42
Table 9: Effect of pH and Cu2S 0 4 on the conversion of ALA to diacetyl
+C uS04
pH
0.8
3.5
6.5
n
slope
r2
n
-C u S 0 4
slope
9
9
9
0.260
1.025
0.690
0.996
0.994
0.970
6
5
7
0.0103
0.1970
0.0749
2
r
0.782
0.805
0.973
C hanging th e p H fro m 3.5 to 3.0 in the presence o f 1.5m M C u S 0 4 d ecreased the
conversion o f A L A to diacetyl by - 1 0 % . A t p H 4.0, b reak d o w n o f A L A to diacetyl
rem ained at 98% , b u t it d ropped by 20% , w h e n the p H w as in creased to 5.0 (Fig. 9).
0
1
2
3
4
5
6
7
pH
Fig. 9: B reakdow n o f A L A to diacety l in the presence o f 1.5m M C u S 0 4 at different
pH values. E rro r bars sh o w th e standard deviation o f three trials.
A s th e p H o f th e m ix tu re o f sam ple and C u S 0 4 appeared to be fairly critical,
replacem ent
o f th e
H 2S 0 4 w ith
a b u ffer
w as
investigated.
A
citric
acid
(0.2M )/N a2H P 04 (0.4M ) bu ffer a t p H 3.3 resu lted in a p H o f th e m ix tu re o f R S M
(norm al p H 6.5) a n d C u S 0 4 o f 3.5. T his bu ffer also resu lted in 100% co nversion o f
A L A to diacetyl (resu lts no t sho w n ) and w o rk ed w ell over the ran g e o f p H values
expected in a ty p ical g ro w th curve fo r C it+ Lc. lactis subsp. lactis (~ 6.6 to -4 .7 ). A
m ixture o f 7m l b u ffe r and 3m l R S M at p H 6.6 resu lted in a p H o f 3.54, w hereas
m ixing 7m l o f b u ffer an d 3m l o f R S M at pH 4.7 resulted in a p H o f 3.47.
W hen A L A stan d ard curves w ere rep eated u sin g the citric acid /N a2 H P 0 4 b u ffer and
substituting a m ix tu re o f F e 2+ and F e 3+ (both 1.5m M ) fo r C u2+, rec o v e ry o f A L A as
diacetyl w as 96.3% , in d icatin g th a t th e type o f transition elem en t is p ro b ab ly n o t too
critical under the co n d itio n s ch o sen (data n o t show n).
43
E xperim ents w ere conducted to determ ine i f th e A L A , w h ich w as not converted to
diacetyl, w as c o n v erted to acetoin. S tandard curves o f A L A w ere distilled in the
presence an d absence o f 1.5m M C u S 0 4 at p H 0.8, 3.5 and 6.5, and diacetyl
m easu red by th e W a lsh and C ogan [1974] procedure in the first 10m l o f distillate
and aceto in b y th e W esterfeld [1945] p ro ced u re in the second 10m l o f distillate. In
the presence o f C u2+, A L A w as p referentially d ecarboxylated to diacetyl at p H 3.5
and 6.5 and to acetoin at p H 0.8, w hereas in the absence o f C u2+, A L A w as
tran sfo rm ed to a ceto in (Fig. 10). A s expected, tran sfo rm atio n to diacetyl w as 102%
at p H 3.5 in th e p resen ce o f C u2+ (T able 10). T he sum o f diacetyl and aceto in found
after d istillatio n at b o th pH 3.5 and 6.5 in the presence o f C u2+ w as greater th an the
am ount o f A L A in itially p resent in th e distillatio n flask (124% recovery). T his
p ro b lem d id n o t occur at p H 0.8 in b o th the absence and presence o f C u2+, w here
recovery o f A L A w as 95% and 96% , respectively, and at pH 3.5 a n d 6.5 in the
absence o f C u2+, w here recovery o f A L A w as 99.1% a n d 84.4% , respectively. The
o v erestim atio n o f the sum o f acetoin and diacetyl in th e presence o f C u2+ w as not
due to in terferen ce o f Cu2+ w ith the assays fo r acetoin and diacetyl, since the
separate m easu rem en ts o f diacetyl an d aceto in them selves w ere unaffected by the
presence o f 1.5m M C u S 0 4. A lso, no a ceto in w as transform ed to diacetyl
during
d istillatio n and m easu rem en t (data n o t show n).
5
B
5
s
«(J
C
S
5
A L A , mM
A L A , mM
Fig. 10: T ran sfo rm atio n o f A L A to diacety l (A ) and acetoin (B) at p H 0.8 ( • , O ),
3.5 ( ■ , □ ) and 6.5 ( A , A ) in th e p resen ce (closed sym bols) and absence
(o p en sym bols) o f 1.5m M C u S 0 4 (diacetyl w as m easured in th e first 10ml
o f d istillate; acetoin w as m easu red in the second 10ml o f distillate).
44
Table 10: Conversion of ALA to diacetyl and acetoin at pH 0.8, 3.5 and 6.5 in the
p resen ce and absence o f 1.5m M C u S 0 4.
-C u S 0 4
+C uS04
pH
D iacety l
slope
r2
A cetoin
slope
r2
D iacetyl
r2
slope
0.8
3.5
6.5
0.260
1.015
0.869
0.702
0.220
0.366
0.017
0.197
0.043
0.996
0.993
0.985
0.999
0.939
0.744
A cetoin
slope
r2
0.879
0.805
0.672
0.931
0.794
0.801
0.998
0.935
0.996
T o fin d out, w h a t caused the overestim ation, diacetyl and aceto in (0.5-4m M ) w ere
d istilled at p H 3.5 and 6.5, and tw o 10m l fractions o f each w ere collected. A ll
fractions w ere assayed for diacetyl and acetoin, respectively, using absolute
(undistilled) standards for the calculations. T he results (Tables 11) w ere analysed by
linear regression.
T able 11 : R eco v ery o f acetoin and diacetyl standards after d istillatio n at pH 3.5
and 6.5
p H 6.5
p H 3.5
F raction
1
2
A ceto in
slope
r2
0.4569
0.2440
0.989
0.999
D iacetyl
slope
r2
0.841
0.041
0.997
0.966
A cetoin
slope
r2
0.3624
0.2279
0.999
0.999
D iacetyl
slope
r2
0.850
0.038
0.999
0.970
R ecovery o f diacetyl standards w as 84% in the first fraction and 4% in the second
fraction, w h ile 46% and 2 4% o f the aceto in standards w as reco v ered in the first and
second fraction, respectively. W hen A L A is distilled in th e presence o f C u2+, it
p referentially breaks dow n to diacetyl. T he m ajority o f th is diacety l com es over into
the first 10m l o f distillate, b u t - 4 % com es over into the second 10m l o f distillate
(data n o t show n). T he diacetyl in th e second 10m l fraction o f distillate, w h ich is
usually u sed fo r the determ ination o f acetoin, reacts in th e sam e w a y as acetoin w ith
the W esterfeld [1945] reagents. T herefore, diacetyl that has alread y b een accounted
for in th e first 10m l fraction by using a distilled standard, is calcu lated a second tim e
as acetoin, and since only 24% o f th e a ceto in standard com es o v er in the second
10m l fraction, th e concentration o f diacetyl in that fraction is actu ally m ultiplied ~4fold. T his explains the ov erestim atio n o f diacetyl and aceto in fro m A L A in the
45
presence o f C u 2+ at p H 3.5 and 6.5, w here A L A is p referen tially transform ed to
diacetyl and th erefo re large am ounts o f diacetyl are present.
Comparison of C u S 04 Method and Jordan and Cogan [1995]
Method
T he m eth o d u su a lly used for the d eterm in atio n o f A L A is th e Jo rd a n and C ogan
[1995] m eth o d , w h ic h is carried out on u n d istilled sam ples. To determ ine h o w the
C u S 0 4 m eth o d co m p ared w ith this m ethod, the p ro d u ctio n o f A L A by tw o A D C
negative strains o f L. lactis subsp. lactis, 4/25A and 1166M 1, and one A D C ' strain
o f Lb. casei, 4 1 9 1 M 3 , and strain 1166, w h ich is the A D C p o sitiv e parent o f strain
1166M 1, w as m o n ito re d u sin g b o th m ethods. F ig u re 11 show s th e correlation o f the
tw o m ethods. T h e r2 v alu e w as 0.958, indicating excellen t ag reem ent b etw een both
m ethods. T he C u S 0 4 m eth o d o verestim ated A L A by 5.7% co m p ared to the Jordan
and C o gan [1995] m ethod. H ow ever, th e latter m eth o d is n o t accurate i f large
am ounts o f a c eto in a n d sm all am ounts o f A L A are p rese n t an d since m easu rem en t o f
A L A b y th e C u S 0 4 m eth o d results in 100% con v ersio n o f A L A to diacetyl it is
concluded th e C u S 0 4 m eth o d is m ore reliable.
J o r d a n an d C o g an M eth o d
Fig. 11: R e g re ssio n analysis o f the C u S 0 4 m eth o d on th e Jo rd an and C o g a n [1995]
m eth o d . T he dotted line is the reg ressio n line and th e co m plete line is the
e x p e cte d line i f b o th m ethods gave identical results.
46
Mixed Culture 4/25
Commercial trials I
T rials w ere carried o u t in three com m ercial p lan ts p roducing b u tter according to the
N IZ O process. S am ples w ere tak e n fro m th e culture tan k before ad d itio n o f th e
lactic acid concentrate and assayed fo r citrate, A L A , acetoin and diacetyl. T he
results are show n in T able 12. In p lan ts A and C, h ig h solids levels w ere u sed to
grow th e culture and th e culture d id n o t use th e citrate com pletely, w h ereas in both
trials in p lan t B effectively all th e citrate w as u tilised during th e ~ 1 8 h incu b atio n
period. L ittle or no A L A w as detected in p lan ts B and C, both o f w h ich h a d h ig h
concentrations o f a ceto in (5.5-10.2m M ). T h e failure to detect A L A w as p o ssib ly due
to the Jordan and C o gan [1995] m eth o d u se d in w h ich sm all am ounts o f A L A cannot
be
detected
in
th e
presence
o f h ig h
concentrations
of
acetoin.
D iacetyl
concentrations varied, b u t w ere gen erally lo w (0.07-0.22m M ).
T able 12: C oncentrations o f the im p o rtan t p aram eters o f culture 4/25 in 3 plants
after overn ig h t grow th and b efo re ad d itio n o f lactic acid concentrate
P lant A
P lant C
P la n t B
T rial
T rial
67
94
99
72
ALA, m M
NDb
0.32
0.61
0
A cetoin, m M
ND
5.50
6.22
10.2
D iacetyl, m M
ND
0.07
0.15
S olids2, g/lOOg
21
17
17
C itrate used, %
0.22
23
a from plant records
b not determ ined
A fter the lactic acid concentrate w as added, th e m ixture w as aerated for d ifferent
lengths o f tim e in the different p lan ts, an d sam ples w ere tak en fo r A L A , aceto in a n d
diacetyl during the aeration period. T he tem p eratu re at w hich the m ix tu re o f culture
and lactic acid concentrate w as aerated w as generally lo w (<15°C ). It c a n be seen in
F igures 12a, b and c, th at th e co n cen tratio n s o f th e th ree com pounds did n o t change
from th eir initial level during the a eratio n period, except for one erratic p o in t in T rial
1 in p lan t B, w h ich w as p ro b ab ly th e resu lt o f an error in m easurem ent. A L A
47
co ncentrations w ere Im M in p lan t B, 2 m M in plant A and 4 .7 m M in P lant C.
A ceto in con cen tratio n s w ere ~ 0 .5 m M in p la n t A and ~ 2 .5 m M in p lan ts B an d C.
D iacetyl con cen tratio n s w ere generally low , b u t nev er increased b e y o n d 0.5m M .
3 r
0
0
20
40
60
Time, min
Fig. 12a: B re a k d o w n o f A L A ( ■ ) and p ro d u ctio n o f acetoin ( ♦ ) an d diacetyl ( A )
by m ix e d culture 4/25 in p lan t A
Ë
0
étm
0
-Ébàr-^
50
A
Ù.
100
À A
150
200
Time, min
Fig. 12b: B reak d o w n o f A L A ( ■ ) and p ro d u ctio n o f acetoin ( ♦ ) and diacetyl ( A )
b y m ix e d culture 4/25 in p lan t B (1: open sym bols, 2: clo sed sym bols).
48
01
0
A
A
10
*
20
A___
30
40
Time, min
Fig. 12c: B reak d o w n o f A L A ( ■ ) and p ro d u ctio n o f acetoin ( ♦ ) and diacetyl ( A )
by m ix e d culture 4/25 in p lan t C.
Laboratory trials
E ffe c t o f m ilk so lid s
M ix e d culture 4/25 w as grow n in R S M (16, 19 and 23g solids/1 OOg) at 21°C . pH ,
A L A , acetoin, diacety l and citrate w ere m on ito red over tim e. Increasing solids
concentrations slow ed dow n the decrease in pH , p robably due to the greater
b uffering capacity o f th e h ig h er m ilk solids concentrations. A L A and acetoin
pro d u ctio n w ere slightly slow er in the m ilk w ith th e h ighest solids concentration,
w hereas diacetyl p ro d u ctio n w as virtu ally unaffected. C itrate w as u tilised at sim ilar
rates in th e th ree m ilks, b u t th e initial level o f citrate increased w ith increasing m ilk
solids (Fig. 13a, 13b, 13c).
49
10
7.0
S
s
œ
a.
<
hJ
<
0
5
10
15
20
25
30
35
T im e, b
Fig. 13 a: E ffect o f milk solids on pH (no sym bol) and A L A p ro d u ctio n ( ■ ) o f
m ixed culture 4 /25, 16 (black), 19 (re d ) and 23 (blue) g so lid s/100g.
Fig 13b: E ffect o f milk solids on acetoin ( ■ ) and diacetyl ( A ) p ro d u ctio n o f m ixed
cu ltu re 4 /25, 16 (black), 19 (red) and 23 (blue) g solids/100g.
10
20
30
40
T im e, h
Fig. 13c: E ffect o f milk solids on citrate utilisation; 16 (black), 19 (red ) and 23
(blue) g solids/lOOg
50
E ffe c t o f tem perature
M ixed culture 4/25 w as incubated at 2 3 °C in R S M (1 6g solids/1 OOg) until it
decreased the p H o f the m ilk to <5.0. T able 13 show s citrate u tilisatio n and A L A ,
acetoin and diacety l prod u ctio n o f the m ix ed cu ltu re 4/25 after ~ 1 8 h o f grow th in
three trials. M o st o f th e citrate initially p resen t in the m ilk w as used. T he culture
produced sim ilar am ounts o f acetoin and diacetyl in th ree trials and m ore acetoin
th an diacetyl w as produced. T he am ount o f A L A p ro d u ced varied b etw een the three
trials.
Table 13: C itrate u tilisatio n and A L A a, aceto in a n d diacetyl p ro d u ctio n o f m ixed
culture 4/25
T rial 1
T rial 2
T rial 3
84
85
95
A L A a(m M )
1.57
0.59
3.65
A ceto in (m M )
4.30
3.75
3.96
D iacetyl (m M )
0.11
0.16
0.21
C itrate u sed (% )
3 m easured by the ordan and C ogan m ethod
A fter ~ 1 8 h , the cultures w ere divided into th ree ferm enters, lactic acid concentrate
w as ad d ed in th e ratio 3:2 and A L A breakdow n a n d aceto in and diacetyl p ro d u ctio n
w ere m o n ito red fo r 2 h at 11, 23 and 30°C . A L A a n d a ceto in w ere determ ined by the
Jordan and C ogan [1996] m ethod. Figure 14 sh o w s the resu lts o f the three trials. T he
am ount o f A L A b reak d o w n varied in th e three tria ls b u t th e trends w ere the sam e.
T he b reak d o w n w as g reatest at 30°C, follow ed by 23°C , and no b reak d o w n occurred
at 11°C. T he aceto in levels d id n o t change m u c h d uring the 2h p e rio d at 11 or 2 3 °C
but at 30°C a sm all increase from the initial v a lu e occurred. A t 11°C, the diacetyl
level did n o t increase significantly from the in itial c o n cen tratio n (0.2-0.4m M ), at
23°C it reach ed 1.4 to 1.8m M , w hile at 30°C it reach ed ~2m M . C o n v ersio n o f A L A
to diacetyl varied from 61 to 70% at 23°C and from 58 to 66% at 30°C. W ith the
exception o f trial 3, co nversion o f A L A to diacety l w as low er at 30°C than at 23 °C,
because a t th e h ig h er tem perature som e A L A w as converted to acetoin (Fig. 15).
s
E
«
5
*
0
0
20
40
60
80
100
120
T im e , m in
T im e, m in
Fig. 14a: B re a k d o w n o f A L A (closed sym bols) and p ro d u ctio n o f diacetyl (open
sym bols) a n d aceto in (closed sym bols) at 11 ( ■ ) , 23 ( ♦ ) a n d 30°C ( A ) in
T rial 1.
0
20
40
T im e , m in
60
80
100
120
T im e , m in
Fig. 14b: B re a k d o w n o f A L A (closed sym bols) and p ro d u c tio n o f diacetyl (open
sym bols) and aceto in (closed sym bols) at 11 ( ■ ), 23 ( ♦ ) and 30°C ( A ) in
T rial 2.
«
5
i<
0
20
40
60
80
100
120
T im e , m in
T im e , m in
Fig. 14c: B re a k d o w n o f A L A (closed sym bols) and p ro d u c tio n o f diacety l (open
sym bols) and a ceto in (closed sym bols) at 11 ( ■ ), 23 ( ♦ ) a n d 30°C ( A ) in
T rial 3.
52
2.0
■ y = -0.6362X + 1.9364 r2 = 0.9091
A
1.6
- v
A y = -0.5771x + 1.7125 r2 = 0.9596
\
§
a
It
cs
5
1.2
0.8
A''Us
0.4
IP
0.0
1
0
2
ALA, mM
F ig. 15a: B re a k d o w n rate s o f A L A to diacetyl at 23 °C ( ■ ) and 30 °C ( A ) in T rial 1.
2.0
A
1.6
£
E
1.2
£
5
0.8
■
y = -0 .7 0 3 6 x + 3.6048 r = 0 .9 5 1 6
A
y = -0 .6 6 4 2 x + 3.182 r2 = 0.9709
^ \ >
*
'A'
0.4
AH
0.0
2
4
ALA, mM
Fig. 15b: B reak d o w n rates o f A L A to diacetyl a t 23°C ( ■ ) a n d 30°C ( A ) in T rial 2.
2 .4
■ y = -0.61 l x + 2.5443 r2 = 0.9239
5
2.0
£
1.6
I*
o
1.2
Q
0.8
A y = -0.6 6 0 8 x 4 - 2 .5 8 8 6 r2 = 0.954
'V
CD
A ''!
K
0 .4
A
0.0
0
1
2
3
4
ALA, mM
Fig. 15c: B re a k d o w n rates o f A L A to diacetyl at 23°C ( ■ ) and 30°C ( A ) in T rial 3.
53
Commercial trials II
E ffe c t o f tem perature
T rials w ere carried out in a com m ercial p lan t p ro d u cin g b u tte r according to the
N IZ O process. T he culture w as grow n in R S M containing 17g/100g solids. Sam ples
w ere tak e n fro m th e culture tan k before addition o f the lactic acid concentrate and
assay ed for citrate, A L A , acetoin and diacetyl. A L A w as m easu red by the C u S 0 4
m eth o d , diacetyl b y distillatio n at p H 0.8 and aceto in by the W esterfeld [1945]
procedure. T he resu lts are show n in Table 14.
T able 14: C oncentrations o f th e im portant param eters o f culture 4/25 after overnight
g ro w th an d before ad dition o f lactic acid concentrate.
C itrate u sed (% )
T rial 1
T rial 2
99.9
94.1
A L A a (m M )
1.98
1.43
A c e to in (m M )
3.24
2.20
D iacetyl (m M )
0.003
0.040
a measured by C uS04 method
A fter th e lactic acid concentrate w as added, th e tem perature o f th e m ixture o f culture
and lactic acid concentrate w as increased to ~ 35°C by p u m p in g the m ixture through
a h e a t exchanger. S ignificant increases in A L A break d o w n and diacetyl p roduction
b eg an as so o n as th e tem p eratu re reached 20°C (F igure 16), b u t diacetyl production
ceased after 6 0m in in T rial 1 and continued to increase slow ly in T rial 2 up to
120m in. A L A w as converted to diacetyl at a rate o f 60 and 64% in T rials 1 and 2
resp ectiv ely (Fig. 17). D ecarboxylation w as alm o st com plete in 120m in and there
w as n o increase in th e levels o f acetoin. T he culture w as dilu ted w ith lactic acid
concentrate in th e ratio 3:2. T his resu lted in an apparent increase in th e initial level
o f A L A for w h ic h there is no obvious explanation. T here w as an im m ediate increase
in the initial levels o f diacetyl. T his w as p robably due to the lo w p H (3.5) w hich is
attain ed o n ad d itio n o f the lactic acid concentrate (T able 14 and F ig. 16).
0
20
40
60
80
100
120
Time, min
Fig. 16a: B reak d o w n o f A L A ( ■ ) an d pro d u ctio n o f aceto in ( ♦ ) a n d diacetyl ( A )
after add itio n o f th e lactic acid concentrate in T rial 1. T h e tem p eratu re ( □ )
mM
o f the m ix tu re is also show n.
20
40
60
80
100
120
Time, min
Fig. 16b: B reak d o w n o f A L A ( ■ ) and p ro d u ctio n o f a c eto in ( ♦ ) an d diacetyl ( A )
after ad d itio n o f th e lactic acid concentrate in T rial 2. T h e tem p eratu re ( □ )
o f the m ix tu re is also show n.
55
0.9
0.8
0.7
S
-
0.6
5;►> 0.5
U
|
0.4
0.3
0.2
-
0.1
-
0.0
■
0.0
'
__!_______ I-----------1
0.5
1.0
1.5
2.0
ALA, mM
Fig. 17a: B reakdow n rate o f A L A to diacetyl in T rial 1.
0
0.5
1
1.5
ALA, mM
Fig. 17b: B reakdow n rate o f A L A to diacetyl in T rial 2.
Quark
Six q uark sam ples w ith varying A L A concentrations w ere analysed fo r A L A and
diacetyl w ith th e C u S 0 4 m ethod over a p erio d o f ~3 w eeks storage at 4°C. T he
results are show n in F igure 18. F o r practical reaso n s it w as n o t p o ssib le to m easure
the levels a t day 0. A L A decreased during storage in all sam ples, w hereas diacetyl
increased, w ith th e ex ception o f sam ple Z , th e control. M o st A L A w as contained in
sam ple M , fo llo w ed by A , H , D and Y in d ecreasin g order. The standard deviation
b etw een trip lic ate sam ples w as <6% . The rates o f A L A b reakdow n in sam ples Y, D,
H , A and M w ere 6.9, 11.3, 14.6, 16.9 and 16.8|xm ol/L /day respectively. R egression
o f th e diacetyl values o n the A L A values in each sam ple gave r2 values o f > 0.94 and
co nversion rates o f 50, 44, 40, 37 and 32% fo r sam ples Y , D, M , A and H
respectively.
57
1.0
H
1.0
0.8
0.6
0.4
0.2
0.0
10
20
30
10
20
Time, days
Time, days
30
1.0
0.8
0.6
§
a
0.4
0.2
0.0
10
20
30
10
1.0
20
30
Time, days
Time, days
M
1.0
D
0.8
sa
0.4
0.2
0.0 L
10
20
Time, days
30
0
10
20
Time, days
30
Fig. 18: E ffect o f storage at 4°C on A L A and diacetyl in quark. C lo sed sym bols
rep re sen t diacetyl, open sym bols A L A .
58
PART II
Growth Experiments
Screen in g
O ne h u n d red and th irty fo u r strains o f C it+ L c. la ctis subsp. Iact is w ere screen ed for
citrate u tilisatio n an d diacetyl p roduction u n d e r o x y g en ated and n o n -oxygenated
conditions. T ab le 16 sum m arises the results o f th o se strains w h ich p ro d u ced
> 0 .2 m M d iacety l u n d e r oxygenated conditions. T h is c u t-o ff p o in t w as chosen,
because it w as th e am o u n t o f diacetyl p ro d u ced b y strain 4/25A , the A D C " strain
u sed com m ercially fo r th e prod u ctio n o f diacetyl in th e m an u factu re o f lactic butter.
O xygenated cu ltu res p ro d u ced 2.3 to 20 tim es m o re diacety l th a n n o n -oxygenated
cultures. A ll strains g rew slightly slow er u n d e r o x y g en ated th a n u n d e r nono x ygenated co n d itio n s. T w o strains, 999 and 1166, w ere selected fo r m ore detailed
studies, since th ey p ro d u ce d the greatest am ounts o f diacetyl.
E ffect o f o x yg en a tio n on stra in s 999 a n d 1166
O xygenation o f strains 999 and 1166 fo r 3 m in b efore g ro w th in R S M slow ed dow n
the su b seq u en t rate o f p H decrease, lactate p ro d u ctio n and citrate u tilisatio n
com pared w ith n o n -o x y g en ated control cultures (Fig. 19). In strain 999, th e rate o f
acetate
p ro d u ctio n
d ecreased
slightly
u n d er
oxygenated
conditions.
A cetate
prod u ctio n w as n o t determ ined fo r strain 1166. B o th strains pro d u ced - 1 0 tim es
m ore diacetyl in o x y g en ated than in n o n -o x y g en ated cultures, w h ereas acetoin
p roduction w as u n affected b y oxygen. B o th strains p ro d u ced in co n sisten t am ounts
o f A L A in o x y g en ated and non -o x y g en ated cultures. T his is due to p ro b lem s w ith
the d e term in atio n o f A L A b y the Jo rd an and C ogan [1996] m ethod. W h en A L A
co ncentrations are close to o r less th a n 10% o f a ceto in concentrations, negative
values fo r A L A can b e obtained w h ich explains th e erratic pattern s o f A L A
production.
59
Table 15: C itrate utilisatio n a n d diacetyl p ro d u ctio n b y C it+ lactococci under
o x y g en ated an d n o n -oxygenated conditions after 16h at 30°C in 10% R SM
N o n -o x y g e n a te d
O xygenated
DPC
pH
S train N o
% C itrate
D iacetyl
u tilised
mM
pH
% C itrate
D iacetyl
Increase
utilised
mM
F old
4/25 A
5.63
73
0.199
5.65
99
0.082
2.40
1166
4.76
100
0.422
4.51
100
0.033
12.79
999
4.7 4
100
0.416
4.57
100
0.026
16.00
990
5.28
100
0.408
4.98
100
0.047
8.68
1006
4.70
100
0.297
4.53
100
0.044
6.75
2392
5.48
100
0.295
5.03
100
0.033
8.94
1165
4.65
100
0.289
4.51
100
0.039
7.41
1002
4.64
100
0.287
4.52
100
0.046
6.24
1003
4.64
100
0.286
4.51
100
0.042
6.81
2345
4.99
100
0.284
4.89
100
0.039
7.28
2342
4.91
100
0.282
4.83
100
0.031
9.10
1008
4.74
100
0.281
4.59
100
0.014
20.07
1007
4.66
100
0.28
4.51
100
0.049
5.71
938
4.95
100
0.276
4.72
100
0.036
7.67
2311
5.43
100
0.276
5.10
100
0.022
12.55
2337
4.87
100
0.269
4.71
100
0.033
8.15
1005
4.65
100
0.268
4.51
100
0.044
6.09
2346
5.04
100
0.267
4.90
100
0.031
8.61
2382
5.15
100
0.267
4.93
100
0.028
9.54
2351
5.05
100
0.265
4.93
100
0.037
7.16
2315
5.73
100
0.264
5.38
100
0.026
10.15
2393
4.88
100
0.262
4.67
100
0.042
6.24
2288
4.92
100
0.259
4.70
100
0.023
11.26
2349
5.04
100
0.258
4.98
100
0.031
8.32
2332
4.72
100
0.257
4.58
100
0.031
8.29
918
4.41
100
0.255
4.34
100
0.031
8.23
2327
5.89
99
0.254
5.54
100
0.022
11.55
2280
4.96
100
0.251
4.71
100
0.025
10.04
2380
5.23
100
0.251
4.74
100
0.027
9.30
2287
5.00
100
0.249
4.73
100
0.018
13.83.
2391
5.27
100
0.246
4.78
100
0.094
2.62
2339
4.89
100
0.245
4.71
100
0.03
8.17
2322
5.71
100
0.244
5.47
100
0.057
4.28
2328
4.89
100
0.242
4.73
100
0.027
8.96
1004
4.64
100
0.241
4.51
100
0.046
5.24
937
5.25
100
0.239
4.97
100
0.041
5.83
2310
5.83
96
0.237
5.50
100
0.027
8.78
60
Table 15 co n tinued
Oxygenated
DPC
pH
Strain No
N on-oxygenated
% Citrate
Diacetyl
utilised
mM
pH
% Citrate
Diacetyl
Increase
utilised
mM
Fold
2316
5.77
100
0.233
5.53
100
0.03
7.77
1160
4.65
100
0.231
4.51
100
0.025
9.24
8.37
2286
4.94
100
0.226
7.74
100
0.027
2313
5.83
99
0.226
5.48
100
0.032
7.06
2272
5.57
100
0.224
5.32
100
0.048
4.67
2279
4.91
100
0.224
4.67
100
0.024
9.33
2395
4.83
100
0.224
4.63
100
0.099
2.26
2338
4.94
100
0.222
4.73
100
0.035
6.34
2312
6.05
86
0.221
6.01
84
0.036
6.14
2307
5.60
10
0.214
5.28
100
0.026
8.23
2308
5.62
100
0.211
5.4
100
0.051
4.14
2333
4.97
100
0.211
4.76
100
0.027
7.81
2383
4.82
100
0.211
4.67
100
0.08
2.64
2329
4.92
100
0.209
4.79
100
0
925
5.90
83
0.207
5.72
100
0.045
4.60
2334
4.75
100
0.2
4.68
100
0.068
2.94
2340
5.01
100
0.2
4.77
100
0.029
6.90
E ffect o f leucine a n d valine on stra in s 999 a n d 1166
T he addition o f lO m M leucine to an o x y g en ated cu ltu re o f strain 999 in R S M
resulted in a d ecrease in lactate p ro d u ctio n and citrate u tilisa tio n (Fig. 20). T he rate
o f p H decrease w as also retarded. A cetoin, diacetyl an d acetate p ro d u ctio n w ere only
affected to a sm all ex ten t and no A L A w as detected.
U nder n o n -o x y g en ated conditions, the effect o f leucine w as sim ilar, except th at A L A
w as detected. In the absence o f leucine, A L A c o n cen tratio n s w ere erratic, due to the
lim itations o f the Jo rd an and C o g an [1996] m eth o d d e scrib ed earlier.
L eucine (lO m M ) seem ed to h av e an in h ibitory effect o n the rate o f p H decrease,
lactate p ro d u ctio n and citrate utilisation b y strain 1166 u n d e r oxygenated conditions,
but w hen the ex p erim en t w as rep eated and o n ly p H
m easured, this effect
disappeared, in d icatin g that there w as a p ro b le m w ith th e grow th o f th e culture
sh ow n in F igure 21. S lightly m ore acetoin w as p ro d u ced in the absence o f leucine;
61
diacetyl p ro d u ctio n w as u n affected and A L A w as only d etected in the first tw o hours
o f incubation.
U n d er n o n -o x y g en ated conditions, citrate u tilisa tio n b y strain 1166 w as slightly
slow er in the presence o f leucine th an in its absence, but the p H decrease and lactate,
aceto in and d iacety l p ro d u ctio n w ere virtu ally unaffected. S m all am ounts o f A L A
w ere detected b o th in th e presence and ab sen ce o f leucine (Fig. 21).
S train 999 grew , p ro d u ced lactate and u tilised citrate m ore slow ly in the p resen ce o f
lO m M valine th an in its absence, under o x y g en ated and n o n -o x y g en ated conditions
(Fig. 22). T he rates o f acetoin and diacety l p ro d u ctio n w ere slig h tly less in the
presence o f valin e th an in its absence u n d e r b o th oxygenated and non -o x y g en ated
conditions, b u t final concentrations w ere sim ilar. T he rate o f acetate p ro d u ctio n w as
alm ost unaffected u n d er o x ygenated co n d itio n s in the p resen ce o f valine, b u t
decreased u n d e r n o n -o x y g en ated conditions.
A d d itio n o f valine (lO m M ) to a culture o f strain 1166 d id n o t affect grow th,
utilisatio n o f citrate an d p ro d u ctio n o f lactate, acetoin, diacetyl an d acetate u n d er
oxygenated and n o n -o x y g en ated conditions. N o A L A w as detected (Fig. 23).
E ffe c t o f C 11S O 4 on stra in 999
T here w as no effect o f C u S 0 4 (O .lm M ) on th e p ro d u ctio n o f lactate and aceto in and
u tilisatio n o f citrate b y strain 999 u n d er b o th o x ygenated an d n o n -o x y g en ated
conditions (Fig. 24). D iacetyl con cen tratio n s w ere m arginally h ig h er in the presence
o f C u S 0 4; A L A co ncentrations w ere v e ry lo w and therefore the am ounts detected
usin g the Jo rd an and C o gan [1996] m eth o d w ere erratic.
E ffe c t o f FeSC >4 on stra in 999
S im ilarly F e S 0 4 (O .lm M ) h a d little i f any effect on grow th o r any o f th e m etab o lites
m easu red u n d er b o th o x y g en ated and n o n -o x y g en ated conditions (Fig. 25).
62
E ffe c t o fh a e m in on stra in 999
A d d itio n o f h a e m in (10p,M ) to an o xygenated culture o f strain 999 had little effect
o n grow th, citrate utilisatio n , and acetate and lactate production. D iacetyl production
w as faster at th e b eg in n in g o f g row th in the presence o f haem in, b u t th e final
d iacety l levels w ere sim ilar in b o th the p resen ce and absence o f haem in. A cetoin
p ro d u ctio n in creased in th e presence o f haem in. A L A w as only detected in the
absence o f h a e m in d u ring the first 3h o f incubation.
U n d er n o n -o x y g en ated conditions sim ilar resu lts w ere obtained, b u t diacetyl
p ro d u ctio n w as h ig h er in th e presence th an in th e absence o f haem in. A L A w as
detected only in the ab sen ce o fh a e m in , b u t the co n cen tratio n s w ere erratic (Fig. 26).
E ffe c t o f oxygen co n cen tra tio n on 1166 a n d 1166M 1
T he effect o f 0 2 concentration, 0% (N 2), 21% (air) and 100% ( 0 2), on strains 1166
an d 1 166M 1, an A L D " m u tan t o f 1166, w as studied (Fig. 27). In b o th strains, citrate
u tilisatio n and th e decrease in p H w as m ore rap id , an d hence g row th w as also faster,
w h e n the cultures w ere g ro w n under N 2, follow ed, in turn, by air and 0 2. In both
strains, th ere w as little effect o f the three g ases o n aceto in production, except that
low er am ounts w ere p ro d u ced by the m u ta n t co m p ared to the parent. A L A w as only
pro d u ced by the m u ta n t an d w as u n affected by exposure to the gas except at the end
o f grow th. D iacetyl p ro d u ctio n increased w ith increasing 0 2 concentrations, but the
increase w as sm aller in the m utant th an in th e p aren t culture. In th e parent culture 0 2
(100% ) caused a ~ 1 2 fo ld increase in diacetyl com pared to cultures grow n u n d er N 2
o r air. A ir had little effect on diacetyl synthesis, w h e n com pared to cultures grow n
u n d er N 2. T he am ounts o f diacetyl p ro d u ced w ere h ig h er in the m u tan t th a n in the
p a re n t strain at all 0 2 concentrations.
63
1166
10
ai-
L a c ta te, m M
100
0.1
T im e , b
Fig. 19a: E ffect o f 0 2 o n pH ( ■ , □ ) and lactate ( A , A ) and acetate ( • , ° )
pro d u ctio n by strains 999 and 1166 in R S M (lO g/L ); closed sym bols
rep resen t oxygenated cultures, open sym bols n o n -o x y g en ated cultures.
4
6
10
12
T im e , h
T im e , h
Fig. 19b: E ffect o f 0 2 o n acetoin ( ■ , □ ), A L A ( A , A ) and diacetyl ( • , ° )
p ro d u ctio n b y strains 999 and 1166 in R S M (lO g/L ); closed sym bols
rep resen t oxygenated cultures, open sym bols n o n -o x y g en ated cultures.
S
s
Ë
u
T im e , h
T im e , h
Fig. 19c: E ffect o f 0 2 on citrate utilisatio n by strains 999 and 1166 in R S M (lO g/L );
closed sym bols represent oxygenated cultures, o p en sym bols nonoxy g en ated cultures.
64
100
100
102
10 s
e
E
1
4.5
0.1
0.1
4
6
10
4
12
6
10
12
T im e , h
T im e , h
Fig. 20a: E ffect o f leucine o n p H ( ■ , □ ) and lactate ( A , A ) p ro d u ctio n by strain
999 in R S M (lO g/L ) u n d er oxygenated (A ) and n o n -o x y g en a ted (B)
conditions; clo sed sym bols represent the p resen ce o f leucine, open sym bols
its absence.
10
12
T im e , h
Fig. 20b: E ffect o f leu cin e o n a ceto in (■ , □ ), A L A ( A , A ) and diacetyl ( • , ° )
p ro d u ctio n by strain 999 in R S M (lO g/L ) u n d e r oxy g en ated (A ) a n d n o n ­
o x y g en ated (B ) con d itio n s; closed sym bols rep resen t the presence o f
leucine, o p en sy m b o ls its absence.
sE
2
E
T im e , h
T im e , h
Fig. 20c: E ffect o f leu cin e o n citrate utilisatio n ( ■ , □ ) an d acetate p ro d u ctio n
( A , A ) by stra in 999 in R S M (lO g/L ) under o x y g en ated (A ) and n o n ­
o x ygenated (B ) co n d itions; closed sym bols rep resen t the p resen ce o f
leucine, o p en sy m b o ls its absence.
65
4.j
0.1
4.5
4
6
8
10
12
2
14
4
6
8
10
12
14
T im e , h
T im e , h
Fig. 21 a: E ffe c t o f leucine on pH ( ■ , □ ) and lactate ( A , A ) p ro d u ctio n by strain
1166 in R S M (lO g/L ) u n d er oxygenated (A ) and non -o x y g en ated (B)
c o n d itions; clo sed sym bols rep resen t th e presence o f leucine, op en sym bols
its absence.
4
6
8
10
12
14
T im e , h
T im e , h
Fig. 21b: E ffect o f leucine on acetoin ( ■ , □ ) , A L A ( A , A ) and diacetyl ( • , O )
p ro d u ctio n b y strain 1166 in R S M (lO g/L ) under oxygenated (A) and n o n ­
o x y g en ated (B ) conditions; closed sym bols represent the presence o f
leucine, o p e n sym bols its absence.
10
S
S
=
s
T im e , h
T im e , h
Fig. 21c: E ffect o f leucine on citrate utilisatio n by strain 1166 in R S M (lO g/L ) under
o xy g en ated (A ) and n o n -oxygenated (B) conditions; closed sym bols
rep resen t th e p resen ce o f leucine, open sym bols its absence.
66
100
10 5
s
-J 0.1
4
6
8
10
12
0.1
14
2
4
6
8
10
12
14
T im e , b
T im e , h
Fig. 22a: E ffect o f v alin e on p H (■ , □ ) and lactate ( A , A ) p ro d u ctio n by strain
999 in R S M (lO g/L ) under o x ygenated (A ) and n o n -o x y g en ated (B)
conditions; closed sym bols represent th e presence o f valine, open sym bols
its absence.
0
2
4
6
8
10
12
14
0
2
4
T im e , h
6
8
10
12
14
T im e - h
Fig. 22b: E ffect o f v alin e o n acetoin ( ■ , □ ) , A L A ( A , A ) and diacetyl ( • , ° )
pro d u ctio n by strain 999 in R S M (lO g/L ) under o x ygenated (A) and n o n ­
ox y g en ated (B ) conditions; clo sed sym bols rep resen t th e presence o f
valine, o p e n sym bols its absence.
o.i
0
0
2
4
6
8
10
0
12
2
4
6
8
10
12
14
T im e , h
T im e , h
Fig. 22c: E ffect o f v alin e on citrate u tilisatio n ( ■ , □ ) and acetate p ro d u ctio n ( A , A )
by strain 999 in R S M (lO g/L ) u n d e r oxygenated (A) and n o n -oxygenated
(B ) conditions; closed sym bols represent the p resen ce o f v aline, open
sym bols its absence.
67
100
10 S
S
0.1
4
6
8
10
12
T im e , h
T im e , h
Fig. 23a: E ffect o f v alin e on p H ( ■ , □ ) and lactate ( A , A ) p ro d u ctio n by strain
1166 in R S M (lO g/L ) u n d e r oxygenated (A ) and n o n -o x y g en ated (B)
co n ditions; closed sym bols rep resen t th e p resen ce o f v aline, o p en sym bols
its absence.
S
E
&
■
V
w
«
5
0.0001
10
12
T im e , h
Fig. 23b: E ffe c t o f valine o n aceto in ( ■ , □ ) a n d diacetyl ( • , O ) p ro d u ctio n by strain
1166 in R S M (lO g/L ) u n d er oxygenated (A ) and n o n -o x y g en ated (B)
conditions; closed sym bols rep resen t the p resen ce o f v aline, open
sym bols its absence.
100
sE
10
too
12
2
E
aT
«u1
n
S
E
0.1
T im e , h
T im e , h
Fig. 23c: E ffect o f valine on citrate u tilisatio n (■ , □ ) a n d acetate p ro d u ctio n ( A , A )
b y strain 1166 in R S M (lO g/L ) under oxygenated (A ) a n d non -o x y g en ated
(B ) conditions; closed sym bols represent th e p resen ce o f v aline, open
sym bols its absence.
68
100
10 S
E
A
J
-
4
T im e , h
6
I
10
T im e , h
Fig. 24a: E ffect o f C u S 0 4 on p H ( ■ , □ ) an d lactate prod u ctio n ( A , A ) by strain
999 in R S M (lO g/L ) u n d er o x y g en ated (A ) and n o n -o x y g en ated (B)
co n d itions; closed sym bols rep re sen t th e presence o f C u S 0 4, open
sy m b o ls its absence.
T im e , h
T im e , h
Fig. 24b: E ffe c t o f C u S 0 4 on acetoin ( ■ , □ ), A L A ( A , A ) and d iacety l ( • , O )
p ro d u ctio n b y strain 999 in R S M (lO g/L ) under oxygenated (A ) and n o n ­
o x y g en ated (B) conditions; c lo se d sym bols represent the presence o f
C u S 0 4, open sym bols its absence.
s
S
E
E
«
T im e , h
T im e , h
Fig. 24c: E ffect o f C u S 0 4 on citrate u tilisa tio n ( ■ , □ ) by strain 999 in R S M (lO g/L )
u n d er o x ygenated (A) and n o n -o x y g en a ted (B) conditions; clo sed sym bols
rep resen t th e presence o f C u S 0 4, o p en sym bols its absence.
69
0
2
4
6
8
0
10
2
4
6
8
10
T im e , h
T im e , h
Fig. 25a: E ffe c t o f F e S 0 4 on p H ( ■ , □ ) and lactate ( A , A ) p ro d u ctio n b y strain
999 in R S M (lO g/L ) under oxy g en ated (A ) and n o n -o x y g en ated (B)
conditions; closed sym bols rep resen t th e presence o f F e S 0 4, open
sym bols its absence.
T im e , h
n m e ,n
F ig. 25b: E ffect o f F e S 0 4 on acetoin (■ , □ ), A L A ( A , A ) and diacetyl ( • , ° )
p ro d u ctio n by strain 999 in R S M (lO g/L ) under o xygenated (A ) and non-,
o x y g en ated (B) conditions; closed sy m b o ls rep resen t th e p resence o f
F e S 0 4, op en sym bols its absence.
T im e , h
T im e , h
Fig. 25c: E ffect o f F e S 0 4 on citrate u tilisa tio n (■ , □ ) and acetate p roduction ( A , A )
b y strain 999 in R S M (lO g/L ) u n d er oxygenated (A ) and non-oxygenated
(B ) conditions; closed sym bols represent the presence o f F e S 0 4, open
sym bols its absence.
70
4
6
T im e , h
T im e , h
Fig. 26a: E ffect o f h a e m in on p H (■ , □ ) and lactate ( A , A ) prod u ctio n b y strain
999 in R S M (lO g/L ) under oxygenated (A ) and non -o x y g en ated (B)
co n d itio n s; clo sed sym bols represent th e presence o f haem in, op en sym bols
its absence.
■i o.oi
2
4
6
8
10
2
T im e , h
4
6
8
10
T im e , h
Fig. 26b: E ffect o f h a e m in on acetoin ( ■ , □ ) , A L A ( A , A ) and diacetyl ( • , ° )
p ro d u ctio n by strain 999 in R S M (lO g/L ) u n d er oxygenated (A ) and n o n ­
o xy g en ated (B ) conditions; closed sym bols represent the p resen ce o f
haem in , o p en sym bols its absence.
100
2
4
6
2
8
4
6
8
T im e , h
T im e , h
Fig. 26c: E ffect o f h a e m in on citrate u tilisa tio n (■ , □ ) and acetate p ro d u ctio n
( A 5A ) b y strain 999 in R S M (lO g/L ) u n d er oxygenated (A ) and n o n ­
o x y g en ated (B ) conditions; closed sym bols rep resen t the p resen ce o f
haem in, o p e n sym bols its absence.
71
2
4
6
10
4
12
6
10
12
T im e , h
T im e , h
Fig. 27a: E ffect o f 0 2 (■ ), air ( A ) and N 2 ( ♦ ) o n p H o f strain 1166 (A ) and
1 166M1 (B) in R S M (lO g/L).
10
5
E
T im e , h
T im e , h
Fig. 27b: E ffect o f 0 2 ( ■ , □ ), air ( A , A ) and N 2 ( ♦ , O ) o n aceto in (closed sym bols)
and A L A (open sym bols) p ro d u ctio n o f strain 1166 (A ) an d 1166M 1 (B ) in
R S M (lO g/L ).
sE
T im e , h
T im e , h
Fig. 27c: E ffect o f 0 2 ( ■ , □ ), air ( A , A ) and N 2 ( ♦ , O ) o n citrate u tilisatio n (closed
sym bols) and diacetyl p ro d u ctio n (open sym bols) o f strain 1166 (A ) and
1166M 1 (B) in R S M (lO g/L).
72
DISCUSSION
Part I
D iacetyl is a n im p o rta n t flavour co m pound in m an y ferm en ted dairy products,
especially lactic b u tter, quark, cultured b u tte rm ilk an d co ttag e cheese. It is produced
from
ALA
by
oxidative
decarboxylation;
n o n -o x id a tiv e
or
enzym atic
d ecarb o x y latio n o f A L A results in the p ro d u ctio n o f aceto in , w h ic h is n o t im portant
in d eterm in in g flavour. D uring the m anufacture o f lactic b u tte r b y th e N IZ O process,
A L A is p ro d u ce d b y th e strain o f C it+ Lc. la ctis subsp. lactis, p resen t in m ixed
culture 4/25. B ecau se A L A is easily d e carb o x y lated in acid solutions, especially in
the p resen ce o f h eat, it can interfere w ith the d e term in atio n o f aceto in and diacetyl.
In itial ex p erim en ts in th is study w ere therefore aim ed a t d eterm in in g the effect o f pH
on th e d e c arb o x y la tio n o f A L A to diacetyl d uring distillatio n . A lth o u g h the
rep ro d u cib ility o f th e A L A standard curves w as v ery poor, th e results (T ables 1 and
2) clearly sh o w th a t distillatio n at p H 6.0 results in m in im al b reak d o w n o f A L A to
diacetyl. T he rates increase considerably at p H v a lu e s <6.0 an d slightly a t p H values
> 6.5, su g g estin g th a t in m easuring diacetyl, th e p H o f sam ples should be adjusted to
pH 6.0 to 6.5 b efo re distillation, to red u ce th e d ec arb o x y la tio n o f A L A to diacetyl.
T his p H is lo w e r th a n th e p H o f 9.0 reco m m en d ed b y V e rin g a et al. [1984], T hese
results also co n tra st w ith those o b tained by Jo rd an [1987], w ho stated th a t during
d istillatio n at p H v alu es as low as p H 4.5, < 5% b rea k d o w n o f A L A to diacetyl
occurred. T he rea so n s fo r these contradictory resu lts are n o t clear, but, in th e case o f
V erin g a e t al. [1984], could be due to th e type o f d istillatio n apparatus used. In the
p resent study, A L A standard curves w ere carried o ut in the sam e w ay and using the
sam e d istillatio n ap paratus as in the study o f Jo rd a n [1987]. T here is no apparent
ex p lan atio n fo r the differences in b reak d o w n fo u n d in Jo rd a n ’s [1987] and this
study. T here w as no significant d ifference in th e b rea k d o w n o f A L A to diacetyl
b etw een sam p les m ad e up in m ilk and sam ples m ad e up in a m ixture o f m ilk and
lactic a c id co n centrate, w h en both w ere adjusted to th e sam e p H (T ables 1 and 2),
73
indicating th a t th e p H is one o f the m ain factors d eterm in in g th e b reak d o w n o f ALA.
T he resu lts also sh o w th a t the con v ersio n o f A L A to diacety l d u rin g distillatio n is
n o t linear o v e r th e concentration range u sed (0 to lO m M A L A ), w ith greater
break d o w n o c c u rrin g at low er A L A concentrations. B reak d o w n w as reasonably
linear fro m 0 to 2 m M A L A (Fig. 3). T he no n -lin earity o f A L A b rea k d o w n does not
pose a p ro b le m as far as cultures o f C it+ Lc. lactis subsp. la ctis are concerned,
because th ese cu ltu res produce A L A in co ncentrations th a t lie a t the low er and,
therefore, rea so n a b ly linear part o f the standard curve. L ater in th is study, a paper by
C ronin a n d R isp in [1996] w as discovered, w h ich reported rates o f break d o w n o f
A L A to d iacety l as lo w as 0.2% after distillatio n at p H 1.0. C ro n in and R isp in ’s
[1996] results w e re confirm ed in this study w ith b reak d o w n rates o f ~ 1% after
distillatio n at p H 0.8 (T able 9). T herefore, distillatio n at p H 0.8 is m o re suitable than
distillatio n at p H 6.0 to 6.5 for th e d eterm in atio n o f diacetyl in sam p les containing
ALA.
O nce th e c o n d itio n s fo r m inim al b reak d o w n o f A L A to diacetyl w ere determ ined, a
m odel sy stem w a s set up to m o n ito r th e co nversion o f synthetic A L A to diacetyl and
aceto in o v er tim e in m ilk and a m ixture o f m ilk and lactic acid concen trate under
various co n d itio n s. T his m odel sy stem m im ick ed the situ atio n w h ic h occurs in the
m an u factu re o f lac tic butter, w here the m ixture o f culture a n d lactic acid concentrate
is aerated to en h an ce th e b reakdow n o f A L A to diacetyl. T he p aram eters studied in
the m o d el sy stem w ere the levels o f 0 2 and m ilk solids, the tem p eratu re, th e pH and
the ad d itio n o f m etal ions (Fe2+ and C u2+) and haem in. 0 2 u n ex p ected ly decreased
the am ounts o f diacetyl produced fro m A L A , com pared to a control w h ich w as ju st
stirred. T his w as due to th e oxy g en atio n m ethod used. T rapping th e exiting 0 2 in
w ater sh o w ed th a t about 30 to 50% o f th e total am ount o f diacety l pro d u ced w as
recovered. L ittle a ceto in w as lo st th ro u g h aeration, because 80 to 98% o f the A L A
added to th e sy ste m w as recovered as diacetyl plu s acetoin. A s a resu lt o f these
ex perim ents, it w a s d ecided to incorporate 0 2 into th e m ed iu m by stirring alone.
T his is su fficie n t fo r the sm all v o lu m es u sed at laboratory scale b u t at industrial
scale, w h ere b ig g er volum es are involved, aeration by stirring w o u ld pro b ab ly result
74
in in co rp o ratio n o f insufficient 0 2 into th e m ilk, w h ich w o u ld th en lead to
in su fficien t co nversion o f A L A to diacetyl.
In th e N IZ O p ro cess for th e m anufacture o f lactic butter, it is reco m m en d ed th at
m ix ed culture 4/25 is grow n in m ilk w ith a solids co n centration o f 16g solids/1 OOg.
T h e rea so n fo r this is, th at higher solids in h ib it th e grow th o f th e starter culture and
th erefo re th e p ro d u ctio n o f A L A [V eringa et al., 1976; V a n den B erg, 1991],
E x p erim en ts
in
th e
m odel
system
w ere
set up,
to
determ ine
i f different
co ncentrations o f solids affected A L A breakdow n. N o sig n ifican t effect on the
b rea k d o w n o f A L A to acetoin and d iacety l w as found.
A L A b reak d o w n w as a first order rea c tio n an d its specific b reak d o w n rate increased
as the absolute tem perature increased (Fig. 4A ). A n A rrhenius p lo t o f th is data (Fig.
4B ) w as linear and the activation energy w as 19.8kcal/m ol, w h ic h agrees w ith the
25k cal/m o l found by M onnet et al. [1994c]. T he specific rate o f A L A breakdow n
d ecreased w ith increasing p H (Fig. 3), w h ich also agrees w ith the results o f M o n n et
e t al. [1994c]. T he type o f acidulant (i.e. lactic acid or HC1) d id n o t affect th e rate o f
A L A breakdow n.
In m any cu ltu red dairy foods, the level o f diacety l req u ired to give organoleptically
acceptable pro d u cts is low (l-5 m g /k g ). T herefore, it is im p o rtan t to be able to detect
th e level o f A L A in a p ro d u ct post-m an u factu re, in order to determ ine the potential
o f the p ro d u ct to develop diacetyl fro m A L A during storage, p articu larly w here
A L D " m utants, w h ich produce up to 3m M A L A , are used. M o n n et et al. [1997] have
show n, th a t th e rate o f degradation o f A L A decreases w ith decreasing tem perature.
H ow ever, ou r results (Fig. 18) show th at ev e n w h e n q u ark is stored at 4 °C , A L A is
spontaneously decarboxylated to diacetyl d u ring storage at a rate o f 30-50% , In
quark and o th er dairy products, p ro d u ced w ith starter cultures containing C it+ Lc.
lactis
subsp.
la ctis, A L A
concentrations
can be
lo w
com pared to
acetoin
co n centrations, w h ich m akes the determ in atio n o f A L A by th e Jo rd an and C ogan
[1995] m eth o d nearly im possible, b ecau se th e difference b e tw e en sam ples treated
75
w ith HC1 (A L A decarboxylated to acetoin) a n d sam ples treated w ith w ater (no
decarb o x y latio n o f A L A ) is sm aller th an th e v a ria tio n b e tw e en d u p licate sam ples.
To o v ercom e th is problem , a n ew m eth o d for the m ea su rem e n t o f A L A w as
developed, in w h ic h A L A w as oxid ativ ely d e carb o x y lated to diacetyl, rath er th an to
acetoin. G o llo p e t al. [1987] used a m ixture o f 0.1 5 m M each o f Fe2+ and F e3+ in
co m b in atio n w ith low pH and h e a t to achieve d ecarb o x y latio n o f A L A to diacetyl.
T he m ec h a n ism o f m etal ion catalysed fo rm a tio n o f diacety l from A L A is n o t clear.
G ollop e t al. [1987] tho u g h t th at a co m p lex b etw een th e enediol, form ed after
decarb o x y latio n o f the A L A and a m etal io n - 0 2 co m p lex w as involved. Initial
e x perim ents w ith the m eth o d o f G ollop et al. [1987] did n o t resu lt in a satisfactory
c o n v ersio n o f A L A to diacetyl. This co u ld have been due to th e use o f steam
d istillatio n rath e r th an ‘a ir’ distillatio n as reco m m en d ed b y G ollop et al. [1987]. T he
results w ith th e m o d el system (T able 7) show ed th at C u2+ w as a b e tte r ‘ox id iser’ o f
A L A th a n F e 2+. D istillin g a sam ple containing A L A in th e p resen ce o f 1.5m M C u2+
at pH 3.5 resu lte d in 100% co nversion o f A L A to d iacety l (T able 9), and these
results p ro v id e d th e basis for the n ew m eth o d fo r m easu rin g A L A . A t pH values
<3.0 and > 4.0, significant reductions in th e am ounts o f diacetyl p ro d u ce d from A L A
w ere found. T h u s th e p H at w h ich the sam ples are d istilled is quite im p o rtan t and the
use o f a citric acid/phosphate b u ffer gave th e sam e re su lt as ad justing th e p H to 3.5
w ith H 2S 0 4. U se o f the buffer is m ore desirable in p ractice b ecau se it w ill ensure
b e tte r co n tro l o f th e pH . S ubstituting 1.5m M e a ch o f F e 27 F e 3+ fo r the copper,
resu lte d in - 1 0 0 % breakdow n o f A L A to diacetyl also. T h is resu lt show s th at the
ty p e o f m etal io n is n o t im portant as long as the o ther co n d itio n s (heat, low pH ,
m etal io n co n cen tratio n ) are optim al fo r A L A breakdow n. B y d istillin g a sam ple at
pH 0.8 in the absence o f C u2+, th e tru e level o f diacety l in th e sam ple can be
determ ined. T he difference in th e sam ple in w h ich all th e A L A is converted to
diacety l a n d th e true diacetyl level, is the A L A concentration. T h e C u S 0 4 m ethod
o v erestim ated A L A by 5.7% co m p ared to the Jo rd an an d C ogan [1995] m ethod.
H ow ever, due to th e lim itations o f the latter m ethod, this d ifference is considered n o t
to be significant. T he reason th at the C u S 0 4 m eth o d fo r m easu rin g A L A is superior
to th o se m eth o d s b ased on co nversion o f A L A to acetoin, is due to the sm all
76
am ounts o f diacety l (< 0.06m M ) relative to the large am ounts o f aceto in (~4m M )
p ro d u ced by C it+ Lc. la ctis subsp. lactis.
T rials w ere carried out in th ree com m ercial plants p ro d u cin g b u tte r according to the
N IZ O process. E ith er lo w levels or no A L A w ere detected before addition o f the
lactic acid concentrate, p ro b ab ly due to the h ig h levels o f aceto in present in the
sam ples. T hese analyses w ere carried o u t by the Jo rd an and C o g an [1995] m ethod.
W h en the lactic acid concentrate w as added, A L A w as detected, b u t no A L A
b reak d o w n occurred and, therefore, no diacetyl w as p ro d u ced d u rin g aeration (Fig.
12). T h is co u ld b e due to th e lo w tem perature o f th e m ixture o f starter and lactic acid
co ncentrate in all three plants. C itrate w as n o t used co m pletely b e fo re addition o f the
lactic acid concentrate w h ere m ilk w ith solids concentrations > 1 7 g /1 0 0 g w as used
(T able 12). It is reco m m en d ed by V a n den B erg [1991] to use m ilk w ith 16g
solids/1 OOg to g ro w th e 4/25 starter culture, since h ig h er m ilk solids in hibit its
grow th. T herefore, lab o rato ry scale trials w ere carried out, in w h ic h th e grow th o f
the culture in R S M co n tain in g 16, 19 and 23 g solids/1 OOg at 21 °C w as com pared
(Fig. 13a, b, c). A s expected, the rate o f p H decrease w as slow er in the m ilk w ith
h ig h er solids levels, p ro b ab ly due to higher buffering capacities. S urprisingly, A L A
and aceto in prod u ctio n w ere n o t affected by the m ilk solids level to any great extent.
O ne w o u ld have ex p ected th a t increased levels o f b o th co m p o u n d s w ould be
p ro d u ced in the m ilk w ith the h ig h er solids levels because o f th e increased levels o f
citrate. T he rea so n w hy th is does n o t h appen is unclear. D iacetyl p roduction w as
slightly greater at the h ig h est level o f solids. T his m ay b e due to h ig h er levels o f ions
w h ic h co u ld decarboxylate A L A .
B reak d o w n o f A L A increases w ith increasing tem p eratu re (Fig. 4). L aboratory trials
w ere carried out, to d eterm in e i f a n increase in tem p eratu re o f th e m ix tu re o f culture
4/25 and lactic acid co ncentrate w o u ld increase A L A b reakdow n and, therefore,
d iacety l p ro duction. A s expected, th ere w as no A L A b reak d o w n a t 11°C (Fig. 14),
w h ic h is close to the tem p eratu re a t w h ich com m ercial p lan ts aerate the m ixture o f
starter culture and lactic acid concentrate. A L A b reak d o w n occurred at 23°C b u t w as
greater at 30°C . S im ilar resu lts w ere obtained fo r diacetyl. R eg ressio n analysis o f
77
diacetyl o n A L A show ed good correlation coefficients (Fig. 15). A ceto in levels w ere
u n affected by the increase in tem perature.
D u e to th ese resu lts, further industrial trials w ere carrie d out, in w h ich the
tem p eratu re at w h ich the m ixture o f culture and lactic a c id concentrate w as aerated
w as ra ise d to 30 to 37°C . A s expected, th is increase in tem p e ra tu re accelerated A L A
b rea k d o w n and con seq u en t diacetyl p roduction; little a c eto in w as pro d u ced from
A L A at the h ig h er tem peratures (Fig. 16) T he c o n v ersio n rate s o f A L A to diacetyl
(~ 60% ) co rresponded w ell to the results o b tained in th e lab o rato ry trials a t 30°C
(Fig. 15 + 17).
Part II
B a ssit e t al. [1993] show ed th at grow ing C it+ Lc. la ctis subsp. lactis under
o x y g en ated co n ditions increased the level o f d iacety l p roduced. T his w as confirm ed
fo r all 134 strains o f C it+ Lc. lactis subsp. la ctis in th e p rese n t study (T able 15).
T w o strains, 999 and 1166, w ere selected fo r m ore d etailed study, because, under
o x y g en ated conditions, th ey pro d u ced tw ice th e am o u n t o f diacety l as strain 4/25 A.
T he latte r strain w as chosen as the reference strain, b ecau se it is a natural ALD"
m utant, an d p ro d u ces h ig h am ounts o f A L A , w h ich c an n o t be enzym atically
c o n v erted to aceto in b u t w h ich can be oxidatively decarb o x y lated to diacetyl under
th e rig h t conditions.
In C it+ Lc. la ctis subsp. lactis, A L D is po sitiv ely co n tro lled b y th e three branchedch ain am ino acids, leucine, valine and isoleucine, im plying th at in the presence o f
any o f th ese am ino acids, m ore a ceto in w ould b e p ro d u ced th a n in th eir absence
[M onnet e t al., 1994a]. H ow ever, w ith strains 999 and 1166 th ere w as no significant
d ifference in the am o u n t o f acetoin o r diacetyl p ro d u ced in th e presence o f leucine or
v alin e u n d e r either o x y g en ated or non -o x y g en ated co n ditions (Fig. 20, 21, 22, 23).
T he levels o f leucine and valine u se d w ere ~ 2 5 0 0 tim es h ig h er th an th e levels
p rese n t in th e m ilk. In th is p art o f th e study the determ in atio n o f A L A w as a problem
78
occasionally, because the m eth o d u sed to d etect A L A [Jordan a n d C ogan, 1995]
o ften resu lted in n egative values. In th is m ethod, A L A is d etected as the difference
in th e total level o f aceto in determ in ed after decarb o x y latio n w ith HC1 and the
a m o u n t o f ‘free’ acetoin. N e g a tiv e v alues can be o b tain ed i f lo w levels o f A L A and
h ig h levels o f aceto in are present.
K aneko et al. [1987, 1990] rep o rted , th at grow ing C it+ Lc. la ctis subsp. la ctis in
R S M in the presence o f m etal io n s (C u2+, Fe2+, F e3+ and M o 6+) o r h a e m in increased
the p ro d u ctio n o f diacetyl d uring grow th, w ith C u2+ bein g the m o st effective. T he
resu lts in th e p resen t study sh o w th a t synthetic A L A is co n v erted to diacetyl during
distillation, w h en m etal ions (C u2+, F e2+) or h a e m in are p rese n t in the sam ple, w ith
h a e m in and C u2+ b ein g m ore effective th an F e2+. T his suggests th a t th e results o f
K aneko e t al. [1987, 1990] co u ld b e due to an artefact o f the m eth o d o f analysis used
b y them , i.e. h ead space gas chro m ato g rap h y after h eatin g to 80°C fo r 30m in. T he
diacety l th a t w as
m ea su red
c o u ld
have
b een p ro d u ced
fro m
ALA
d u ring
m easurem ent, rather th a n sh o w a tru e effect o f the m etal ions and h a e m in on p ro d u ct
form ation. U nfortunately, A L A w as n o t m easu red in th e studies o f K aneko e t al.
[1987, 1990]. In th e p rese n t study, C u2+, F e2+ an d h a e m in did n o t significantly
increase diacetyl p ro d u ctio n fu rth e r in the oxygenated cultures; w hereas in th e n o n ­
o x y g en ated cultures, diacetyl p ro d u ctio n w as increased to som e ex ten t b u t o n ly in
the p resen ce o f h aem in (Fig. 24, 2 5 , 26). T his increase w as genuine and n o t caused
b y d istillatio n in th e p resen ce o f h aem in , because no A L A , w h ich co u ld b e converted
to d iacetyl, w as d etected in th e cultures g row n in th e p resen ce o f haem in.
T he effect o f different 0 2 levels (0% , 2 1% and 100% ) o n A L A , aceto in an d diacetyl
p ro d u ctio n by C it+ Lc. la ctis subsp. lactis strains 1166 and 1166M 1, its A L D "
m u tant, w as studied (Fig. 27). A L D ' m utants sh o u ld pro d u ce m ore A L A .and
diacetyl at the expense o f acetoin, since the enzym atic d ecarb o x y latio n o f A L A to
aceto in is prevented. T he m u ta n t d ecreased th e p H at a slow er rate th an the parent,
p o ssib ly b ecau se its acid p ro d u cin g ability w as p artially dam ag ed by th e procedure
used to create th e m u tan t [M onnet e t al. 1997]. N o A L A w as p ro d u ced b y the paren t
strain, w hereas the m u tant, as ex p ected, p ro d u ced h ig h am ounts o f A L A . L ess
79
aceto in w as p ro d u ce d by th e m u tan t th a n by th e parent. T h e aceto in pro d u ced by the
m u tan t cannot be due to A L D activity a n d m u st therefore be due to chem ical
decarboxylation. Increasing 0 2 concentrations in h ib ited the g row th o f both the
p aren t and the m u tan t, as indicated by the lo w er rates o f p H decrease, and, therefore,
also slow ed d o w n citrate utilisation. A L A p ro d u ctio n w a s u n affected b y increasing
0 2 levels. T he co n v e rsio n o f A L A to diacetyl w as p ro m o te d by increasing 0 2
concentrations in b o th th e parent and the m utant, b u t th e increase w as b ig g er in the
paren t th an in th e m utant. A ceto in p ro d u ctio n w as un affected b y different levels o f
In conclusion, it h a s b een show n that b reakdow n o f A L A to aceto in and particularly
to diacetyl can b e influ en ced by pH , tem perature, oxid isin g agents and heat and th at
it is a first order reaction. T hese results w ere used to develop a n e w m ethod for the
detection o f A L A , in w h ich th e com bined effects o f low pH , h ig h tem perature and
C u2+ w ere u sed to o b tain m ax im u m b reak d o w n o f A L A to diacetyl. It w as also found
that, under th e co n d itio n s u sed com m ercially for the p ro d u ctio n o f lactic butter, no
breakdow n o f A L A occurred and no diacetyl w as p roduced. Increasing the
tem perature d u rin g the a eratio n o f the m ixture o f starter culture and lactic acid
concentrate to ~ 3 5 °C w as show n to increase diacetyl p ro d u ctio n and reduce A L A
concentrations in the fin ish ed p ro d u ct to zero. E x p erim en ts w ith quark show ed
considerable b reak d o w n o f A L A to diacetyl during storage at 4°C and, therefore,
p o ssib le u n d esirab le changes in flavour d u ring storage. G row th o f C it+ Lc. lactis
subsp. lactis strain s w as n o t significantly influ en ced b y the addition o f leucine,
valine, C u2+, F e 2+ o r haem in, w hereas 0 2 increased diacety l production. Studies w ith
an A L D ' m u ta n t o f 1166 show ed the expected increase in A L A and diacetyl
production.
80
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A cknow ledgem ents
I w ish to th an k m y su p erv iso r in M oorepark, D r. T im C ogan, and esp ecially M ary
R ea and F in b a r D rin a n fo r th eir help a n d guidance d u rin g m y project.
I w o u ld also lik e to th a n k T eagasc for th e studentship an d th e u se o f th e ir facilities.
T h e support o f D e rm o t Q uill a n d Jerry R yan, N e n a g h C o-O p, L ar C um m ings,
W aterford C o-O p a n d D enis K ennealy, T ipperary C o-O p is also v ery m u ch
appreciated.
I w ish to th an k D r. C h risto p h e M onnet, at IN R A , G rignon, F ran ce fo r v aluable
discussion.
M y th anks is also d u e to all th e m em b ers o f th e B acterio lo g y D epartm ent,
M oorepark.
92