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INTERNATIONAL JO URNAL OF
F o o d M icro b io lo g y
ELSEVIER
In te rn a tio n a l Jo u rn a l o f F o o d M icro b io lo g y 68 (2001) 1 1 5 -1 2 3
w w w .e ls e v ie r.c o m /lo c a te /ijf o o d m ic ro
Influence o f acetate and C 0 2 on the TM A O -reduction reaction by
Shewanella baltica
J. Debevere, F. D evlieghere*, P. van Sprundel, B. D e M eulenaer
U n iversity o f G hent, D e p a rtm e n t o f F o o d T e c h n o lo g y a n d N utrition, C o u p u re Links, 653, 9 0 0 0 G hent, B elgium
R eceiv ed 10 O c to b e r 2 0 0 0 ; re c e iv e d in re v ised form 15 Jan u a ry 2001 ; a c c e p te d 2 9 J a n u a ry 2001
A b stra c t
In this work, the TM AO-reduction by Shewanella baltica, one o f the representative spoilage organisms in modified
atmosphere packaged marine fish fillets, and the effect o f acetate and C 0 2 on this reduction were studied in vitro. The
growth o f S. baltica and the corresponding evolution o f some compounds (acetate, lactate, pyruvate, glucose and
trim ethylam ine (TMA)) were followed during storage at 4°C in two types o f broths. The first medium was a defined medium
(pH = 6.8) to which lactate or pyruvate was added as hydrogen donor. Pyruvate show ed to be m ore efficient as H-donor for
S. baltica than lactate, as growth was much faster when equim olar amounts o f pyruvate instead o f lactate w ere present.
Although the growth o f S. baltica, when pyruvate is used as H-donor and no acetate is added, was not much inhibited by the
C 0 2-atmosphere, C 0 2 had a pronounced effect on the studied reactions as it partly inhibited the reduction o f pyruvate to
acetate. The effect of acetate on this reaction was, on the other hand, not significant.
To simulate the reactions occurring in situ, a buffered fish extract (pH = 6.8) was used. In spite o f the neutral pH, the
growth o f S. baltica in this m edium was highly inhibited by relatively small concentrations o f acetate ( < 0.3%). W hen 0.1%
o f acetate was added to the fish extract, less acetate was formed and lactate was m ore slowly consumed in comparison to the
experiments without the addition o f acetate. The consumption o f lactate and the production o f acetate were almost
completely inhibited when the fish extract contained 0.25% o f acetate. Apparently, the addition o f acetate inhibited the use
o f lactate as H-donor. After extended storage times (17 days at 4°C) TMA production started. M ost probably, alternative
H-donors were used by S. baltica, from w hich the pathway seems to be less energy efficient. This can be deduced from the
exceptional growth inhibition o f S. baltica by small amounts o f acetate. However, when practical storage times for fish (e.g.
6 days at 4°C after packaging) are considered, growth and TMAO-reduction by S. baltica was completely inhibited during
this period by 0.25% o f acetate. © 2001 Elsevier Science B.V. All rights reserved.
K e y w o rd s: F ish ; S h ew a n ella b a ltic a ; T M A O ; T M A ; A cetate; M o d ifie d atm o sp h ere p a ck a g in g
1. Introduction
—
S p o ila g e o f fish is m a in ly d u e to m ic ro b ia l p ro C o rre sp o n d in g author. T el.: + 3 2 -9 2 6 2 -6 1 7 7 ; fax: + 3 2 -9 2 2 5 -
55]0
F
E -m a il a d d re ss: F ran k .D ev lieg h e re @ ru g .ac .b e
(F. D cv lie g h ere).
T
r
,
.
,
,
.
/vr - . « A \ •
..
m ain c o m p o n e n t re sp o n sib le fo r a n u n p le a s a n t ‘ fish y ’
o d o u r (D a in ty , 1996). W h e n o x y g e n le v e ls are de-
0 1 6 8 - 1 6 0 5 / 0 1 / $ - see fro n t m a tte r © 2001 E lse v ie r S cien ce B .V . A ll rights reserved.
P II: SO 1 6 8 -1 6 0 5 ( 0 1 ) 0 0 4 8 4 - 6
.
cesses. In m a rin e fish , trim e th y la m in e (T M A ) is the
116
J. D e b e v e re e t al. / In te r n a tio n a l J o u rn a l o f F o o d M ic ro b io lo g y 6 8 ( 2 0 0 1 ) 1 1 5 -1 2 3
p le te d , T M A O serv es as a te rm in a l e le c tro n a c c e p to r
fo r a n a e ro b ic re s p iratio n a n d is re d u c e d to T M A
(E a s te r e t al., 1983). T M A O -re sp ira tio n is o f im p o r­
v o la tile b ases a n d T M A w as, in d e e d , in h ib ite d b y a
tre a tm e n t w ith an a c e t ic a c id /N a - a c e ta te b u ffe r (1 0 %
( v / v ) - s p r a y p H 5 .6 o f fre sh c o d fille ts ( G adus
ta n c e to b a c te ria l g ro w th d u rin g s p o ila g e o f m a rin e
fish , b u t few d e ta ils are k n o w n a b o u t su b s tra te p re f­
e re n c e s , c a ta b o lic p ro c e sse s a n d e n e rg y c o n se rv a tio n
d u rin g T M A O -d e p e n d e n t a n a e ro b ic g ro w th o f sp e ­
c ific sp o ila g e o rg an ism s. C a rb o n s o u rc e s u tiliz e d by
m o rh u a ) sto re d a t 7 ° C , u n d e r m o d ifie d atm o sp h ere .
T h e tre a tm e n t r e s u lte d in a p H -d ro p fro m 6.63 to
6 .2 9 an d a to ta l v o la tile a c id ity in th e tre a te d co d
fille ts o f 2 6 0 m g a c e tic a c i d / 100 g). H o w e v e r, th e
s h e lf life e x te n d in g e ffe c t o n fish o f th e a p p lie d
a cetic a c id /a c e ta te b u f f e r s o lu tio n co u ld , n e x t to its
S h e w a n e lla sp ec ies g ro w n o n L B -b ro th o r a m in im a l
sa lts m e d iu m a t 2 0 -3 0 ° C in c lu d e g lu c o se , la cta te ,
p y ru v a te , p ro p io n a te , eth an o l, a c e ta te , fo rm a te a n d a
n u m b e r o f c a rb o x y lic a n d a m in o a c id s (S c o tt an d
N e a lso n , 1994). L a c ta te has b een s u g g e s te d to b e the
n a tu ra l H -d o n o r fo r T M A O -re d u c tio n d u rin g fish
sp o ila g e b e c a u s e its c o n c e n tra tio n is h ig h (a b o u t 25
m M ) in fish m u sc le , a n d it d isa p p e a rs a s T M A O is
re d u c e d (S trO m an d L arsen , 1979, S trO m e t ah,
1979). T h e re d u c tio n o f T M A O in th e p re s e n c e o f
la c ta te b y S h e w a n e lla sp ec ie s w a s su m m a riz e d as
fo llo w s (R u ite r, 1971):
C H 3C H O H C O O H + ( C H 3) 3N 0
- > C H 3C O C O O H + ( C H 3) 3N + H 20
(1 )
p ro d u c t in h ib ito ry e f f e c t o n th e T M A O -re d u c tio n
re a c tio n , p o ssib ly a ls o be e x p la in e d b y th e k no w n
a n ti-m icro b ial e ffe c t o f acetate b y a c id ific a tio n o f th e
c y to p la sm a , c a u sin g p ro te in d é n a tu ra tio n an d en erg y
lo ss d u e to th e a c tiv a tio n o f A T P -d e p e n d e n t p ro to n
p u m p s lo ca te d in th e c e ll m e m b ra n e (M a rsh a ll e t ah,
2 0 0 0 ). T h e re fo re , th e a im o f th is w o rk w as to stu d y
in v itro th e b io c h e m ic a l re a c tio n s in v o lv in g the
T M A O -re d u c tio n b y S h e w a n e lla b a ltic a a n d th e in ­
flu e n c e o f a c e ta te a n d C 0 2 o n th is reactio n . In a
d e fin e d m e d iu m , th e e ffe c t o f C 0 2 a n d a c e tate on
re a c tio n s (2 ) a n d (3 ) w ere in v e s tig a te d sep a rately .
T h e se c o n d p a rt o f th e e x p e rim e n t w a s p e rfo rm e d in
a fish e x tra c t to b e tte r sim u la te th e n a tu ra l e n v iro n ­
m e n t o f th e m ic ro o rg a n ism .
T h e p y ru v a te is o x id iz e d a c c o rd in g to:
2. M aterials and m ethod s
C H 3C O C O O H + ( C H 3) 3N O
- > C H 3C 0 0 H + ( C H 3) 3N + C 0 2
(2 )
2.1. S tra in
T h e o v e ra ll re a c tio n can be w ritte n as fo llo w s:
T h e a p p lie d stra in w a s iso la te d fro m re frig e ra te d
( r = 4 ° C ) m o d ifie d a tm o sp h e re p a c k a g e d (60%
C H 3C H O H C O O H + 2 ( C H 3) 3N O
(3 )
C O 2- 3 0 % O 2- 1 0 % M 2) co d fille ts a t th e e n d o f the
s h e lf life an d w as p re v io u s ly c a lle d S h e w a n e lla -lik e
(B o sk o u a n d D e b e v e re , 1997). L ater, th e stra in w as
S in c e a c e ta te h as b een p ro p o s e d as a n e n d p ro d u c t
d u rin g th e T M A O -d e p e n d e n t re s p ira tio n o f sp e c ific
sp o ila g e b a c te ria su c h as S h e w a n e lla sp ecies (R u ite r,
id e n tifie d b y L M G as S. b a ltic a u sin g th e fo llo w in g
re fe re n c e strain s: S. b a ltic a N C T C 1 0 7 3 5 1 (Z ie m k e
et al., 1998) an d S. b a ltic a L M G 2 2 5 0 T. T h e stra in
1971; R in g O e t a h , 1984), see V e n k a te sw a ra n e t al.
(1 9 9 9 ) fo r th e ta x o n o m y o f th e g e n u s S h e w a n e lla ,
th e a d d itio n o f a c e ta te in th e fish tissu e c a n be
w as c h o se n as it w a s re p re se n ta tiv e fo r th e sp o ilag e
flo ra o f m o d ifie d a tm o sp h e re p a c k a g e d fre sh m a rin e
- * C H 3C 0 0 H + 2 ( C H 3) 3N + H 20 + C 0 2
e x p e c te d to in h ib it th e T M A O -re d u c tio n by b a c te ria l
trim e th y la m in e o x id e re d u c ta se s. N e x t to an in h ib i­
fish s to re d at 4°C (B o sk o u a n d D e b e v e re , 1997). T he
stra in w a s s to re d on M a rin e A g a r (3 7 .4 g /1 M arin e
B ro th 2 2 1 6 , D IF C O 0 7 9 1 -1 7 a n d 15 g / 1 A g a r no. 1,
tio n o f H 2S -p ro d u c in g b a cte ria, B o sk o u a n d D e b e v e re ( 2 0 0 0 ) n o tic e d th at th e p ro d u c tio n o f total
O X O ID L I I ) slan ts a t 6°C w h ic h w ere re n e w e d
e v e ry m onth.
J. D e b e v e re e t al. / In te r n a tio n a l J o u rn a l o f F o o d M ic r o b io lo g y 68 ( 2 0 0 1 ) 1 1 5 -1 2 3
2.2. E x p e r im e n ta l se t-u p
T h e g ro w th o f S. b a ltic a a n d th e c o rre sp o n d in g
e v o lu tio n o f ac e ta te , la cta te , p y ru v a te , g lu c o se an d
trim e th y la m in e (T M A ) w a s fo llo w e d d u rin g sto rag e
a t 4°C in tw o ty p e s o f in o c u la te d bro th s.
T h e first m e d iu m w a s a d efin e d m e d iu m b a s e d on
th a t d e s c rib e d b y E a ste r e t al. (1 9 8 3 ) a n d G rah a m
an d W a rd (1 9 8 8 ). It c o n ta in e d 5 g / 1 b a c te rio lo ­
g ical p e p to n e (O X O ID , L 3 4 ), 2.5 g / 1 D - ( + ) g lu c o s e (S IG M A ), 7 .2 g / 1 T M A O 2 H 20 (S IG M A ),
2 0 g / 1 N a C l (V E L ), 1 g / 1 K 2H P 0 4 (S IG M A ), 1
g / 1 M g S 0 4 • 7 H 20 (S IG M A ) an d 1 g / 1 N H 4C1
(S IG M A ). T o th is m e d iu m , 60 m M o f lactate
(S IG M A ) o r 65 m M p y ru v a te (S IG M A ) w a s a d d e d
a s h y d ro g e n d o n o r. F o r e ac h h y d ro g e n d o n o r, e x p e ri­
m e n ts w e re p e rfo rm e d w ith th re e d iffe re n t c o n c e n tra ­
tio n s ( v / v ) o f a c e ta te (0 % , 0 .5 % a n d 1.0% ) a n d in
tw o g a s a tm o sp h e re s (1 0 0 % o f N 2 o r 5 0 % o f N 2 a n d
5 0 % o f C 0 2). 5 0 % o f C 0 2, co m b in e d w ith a
h e a d sp a c e o f 1 / 1 , re s u lte d in 253 m g /1 a n d 263
m g / 1 d is so lv e d C 0 2 in th e d e fin e d m e d iu m w h e n
la c ta te o r p y ru v a te w e re , re s p e c tiv e ly , u se d as H d o n o r. A fte r th e a d d itio n o f th e ap p ro p riate am o u n t
o f a c e ta te , th e p H w a s a d ju ste d to 6.8 b y m e a n s o f
117
v o lu m e ratio o f 1) a n d a u to c lav e d a t 121°C fo r 15
m in . T h e p H w as a d ju s te d to 6.8 a fte r a u to c lav in g
w ith filte r ste rilise d 2 N N a O H o r 2 N HC1.
2.3. In o c u la tio n p r o c e d u r e a n d sa m p lin g
T h e S. b a ltic a s tr a in w as su b c u ltu re d in M arin e
b ro th 2 2 1 6 (D ifc o ) f o r 48 h a t 30°C . A se c o n d
s u b c u ltu re in M a rin e b ro th (D ifc o ) (0.1 m l in 10 m l)
w a s in c u b a te d fo r 1 6 h at 30°C . T h e in o cu lu m w as
th e n s to re d fo r 8 h a t 4°C to allo w th e te s t stra in to
a d a p t to th e c h illin g te m p e ra tu re . A fte r th e a d a p ta ­
tio n p e rio d , th e s te rilis e d g lass ja r s , fille d w ith p re v i­
o u sly c o o le d (4°C ) liq u id m e d iu m , w ere in o c u la te d
w ith S. b a ltic a to a lev el o f IO 6 c f u / m l . A fte r
in o c u la tio n , th e ja rs w e r e im m e d ia te ly g as p a c k a g e d
(1 0 0 % o f N 2 o r 5 0 % o f N 2 + 5 0 % o f C 0 2) as
d e s c rib e d b y D e v lie g h e re e t al. (1 9 9 8 ) a n d sto re d at
4°C . T o fo llo w th e e v o lu tio n o f th e c h e m ic a l an d
m ic ro b ia l p a ra m e te rs in the b ro th d u rin g tim e , 4 -m l
sa m p les w e re ta k e n a t re g u la r tim e in te rv als by
m e a n s o f ste rile d is p o s a b le 10 -m l sy rin g es.
2.4. M ic r o b ia l d e te rm in a tio n
The
e v o lu tio n
of
th e
num ber
of
S.
b a ltica
10 N N a O H so lu tio n (V E L ).
T o sim u la te th e re a c tio n s o c c u rrin g in situ , a fish
e x tra c t w a s u se d as th e se c o n d liq u id m ed iu m . It w as
( c f u / m l ) w a s fo llo w e d d u rin g tim e in th e b ro th , by
d ilu tin g th e sa m p le s, i f n e c essary , w ith P ep to n e
p re p a re d as p re v io u s ly d e sc rib e d b y B o sk o u an d
D e b e v e re (1 9 9 7 ). T h e T M A O -c o n te n t w a s, a fte r d e ­
te rm in a tio n o f th e in itial c o n te n t, a d ju ste d to a lev el
N a C l) a n d p la tin g th e m in d u p lica te o n M a rin e A g a r
w ith a S p ira l P la te r (M o d e l D , S p iral S y ste m s, C T,
o f 80 m g T M A O - N /1 0 0 m l b y th e a d d itio n o f
T M A O • 2 H 20
(S IG M A ). A d d itio n a lly , 7 g / 1
K H 2P 0 4 (S IG M A ) a n d 7 g / 1 K 2H P 0 4 (S IG M A )
w e re a d d e d to b u ffe r th e m e d iu m (D alg aard , 1995).
T o e stim a te th e e ffe c t o f a c e ta te on th e g ro w th o f S.
b a ltic a a n d its p ro d u c tio n /c o n s u m p tio n o f the
a b o v e m e n tio n e d c h e m ic a l c o m p o u n d s, ex p e rim e n ts
w e re p e rfo rm e d w ith 0 % , 0 .1 % , 0 .2 5 % an d 0 .5 % o f
a d d e d a c e ta te (S IG M A ). A fte r th e ad d itio n o f th e
a p p ro p ria te am o u n t o f a ce ta te , th e p H o f th e m e d iu m
w a s also ad ju ste d to 6.8 b y m e a n s o f 10 N N a O H
s o lu tio n (V E L ).
E x p e rim e n ts w e re p e rfo rm e d in 6 0 0 -m l ja r s p r o ­
v id e d w ith a T eflo n ® v a lv e a n d a cen tral o p e n in g
w h ic h w a s c lo s e d w ith a silic o n e sep tu m (D e v lie g h e re et al. 1998). T h e g la ss ja r s w ere filled w ith
300 m l o f liq u id m e d iu m (re su ltin g in a g a s / p r o d u c t
P h y sio lo g ic a l S alt so lu tio n (0 .1 % p e p to n e , 0 .8 5 %
U S A ). T h e p la te s w e re ae ro b ic a lly in c u b a te d fo r 2
d ay s a t 30°C .
2.5. C h e m ic a l d e te rm in a tio n s
T rim e th y la m in e -N (T M A -N ) c o n te n t w a s d e te r­
m in e d in d u p licate w ith th e sp e c tro p h o to m e tric
m e th o d (D y e r, 1945) a s m o d ifie d b y B o sk o u and
D e b e v e re (2 0 0 0 ).
T h e p H w as m e a s u re d in d u p lica te w ith an In gold
sh arp p o in t e le c tro d e a n d a k n ic k p H -m e te r.
C o n c e n tra tio n s o f g lu c o s e , aceta te , p y ru v a te and
la c ta te in d iffe re n t m e d ia w e re d e te rm in e d b y m ean s
o f Io n M o d e ra te d P a rtio n H ig h P e rfo rm a n c e L iq u id
C h ro m a to g ra p h y (F e m a n d e z -G a rc ia an d M c G reg o r,
1994). S a m p le s w ere p re v io u s ly h e a te d fo r 15 m in at
80°C a n d c e n trifu g e d (E p p e n d o rf C e n trifu g e 5415 C )
fo r 10 m in a t 10,000 rp m . T h e su p e rn a ta n t w as
J. D e b e v e re e t al. / In te rn a tio n a l J o u rn a l o f F o o d M ic ro b io lo g y 6 8 ( 2 0 0 1 ) 1 1 5 -1 2 3
118
filte re d th ro u g h a 0.22 jxm M ille x (M illip o re ) filter
b efo re in jectio n . T h e H P L C ap p a ra tu s co n siste d o f a
H P L C p u m p (G IL S O N 3 0 7 ), a p re c o lu m n (B io rad ),
a se p a ra tio n co lu m n (A m in e x P IP X -87H , B io -rad )
w h ich w a s th e rm o sta tic a lly re g u la te d a t 35°C , a R e ­
fractiv e In d e x d e te c to r (G IL S O N 132) a n d an in te ­
g ra to r (S h im a d z u C -R 6 A c h ro m ato p a c). T h e m o b ile
p h ase w a s filte re d (2 2 |x m ) a n d d e a e ra te d 5 m M
H 2S 0 4 a t a flo w ra te o f 0.5 m l / m i n . R eten tio n
tim es fo r aceta te, g lu c o se , p y ru v a te and la c ta te w ere,
14.7, 8.7, 9.6 an d 12.2 m in , re sp e c tiv e ly . S tan d ard
c u rv es w e re d e riv e d fo r e v e ry c o m p o u n d th a t w as
u sed fo r th e c a lc u la tio n o f th e e ffe c tiv e c o n c e n tra ­
tions. A ll an aly ses w e re p e rfo rm e d in d u p licate.
T o e stim a te th e p re c is io n o n th e ch e m ic a l d e te r­
m in a tio n s, a s a m p le w a s in d e p e n d e n tly a n a ly s e d in
d u p lic a te six tim es. T h e sta n d a rd d e v ia tio n o f the
c h e m ic a l d e te rm in a tio n s a m o u n te d to 1.8 % fo r ac­
etate , 3 .0 % fo r p y ru v a te , 6 .7 % fo r la c ta te, 5 .9 % for
g lu c o se a n d 9 .6 % f o r T M A .
3. R esults and d iscu ssion
3.1. E x p e r im e n ts in th e d e fin e d m ed iu m
F ig . 1 illu s tra te s th e e ffe c t o f C 0 2 an d a c e ta te on
th e g ro w th o f S. b a ltic a a t 4 °C in d e fin e d m edia
10
E
3
75
g5
0
5
15
10
20
25
Time (days)
10
5,5
0
5
10
15
20
25
30
35
40
Time (days)
F ig. I. In flu en c e o f a ce ta te c o n c e n tra tio n an d atm o sp h ere ( ♦
100% o f N 2 a n d 0 % ( v / v ) acetate, ■ 100% o f N 2 a n d 0.5% ( v / v ) acetate,
▲ 100% o f N 2 an d 1% ( v / v ) a c e ta te , X 5 0 % N 2 + 5 0 % o f C 0 2 and 0% ( v / v ) acetate, * 50 % N 2 + 5 0 % o f C 0 2 an d 0 .5 % ( v / v ) acetate,
#
50% N 2 + 5 0 % o f C 0 2 and 1% ( v / v ) a c e ta te ) o n th e gro w th o f S. ba ltic a a t 4°C in a d e fin e d m e d iu m (p H = 6 .8 ) w ith lactate (A ) or
p y ru v a te (B ) as H -d o n o r.
J. D e b ev e re e t a l . / In te r n a tio n a l J o u rn a l o f F o o d M ic ro b io lo g y 6 8 (2 0 0 1 ) I ¡ 5 - 1 2 3
(p H = 6 .8) c o n ta in in g lac ta te (F ig . 1 (A )) o r p y ru v a te
(F ig . 1(B )) as H -d o n o r. P y ru v a te a p p e a re d to be
m o re e ffic ie n t as H -d o n o r fo r S. b a ltic a th a n lactate,
as g ro w th w as m u ch fa ste r w h e n e q u im o la r a m o u n ts
o f p y ru v a te in stea d o f la c ta te w e re p re s e n t as H d o n o r. P o ssib ly , th is d iffe re n c e in g ro w th ra te c o u ld
a lso b e e x p lain ed b y th e a n ti-m ic ro b ia l e ffe c t o f
la c ta te , w h ic h is, h o w e v e r, v e ry lim ite d a t th e e x p e ri­
m e n ta l p H o f 6 .8. W h e n la c ta te w a s p re s e n t as
H -d o n o r, a g as c o n c e n tra tio n o f 5 0 % o f C 0 2 o r th e
a d d itio n o f a c e ta te (0 .5 % o r 1.0 % ) c o m p le te ly in h ib ­
ited th e g ro w th o f S. b a ltic a fo r 25 d ay s. T h is w as
n o t th e c ase in the m e d iu m c o n ta in in g p y m v a te .
H o w e v e r, w h en 1% o f a cetate w a s p re s e n t, n o g ro w th
w a s d e te c te d w ith in 33 days. T h e g ro w th -in h ib itin g
e ffe c t o f a c e ta te a n d C 0 2 o n S h e w a n e lla sp ec ie s w as
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
alread y p re v io u sly d e s c r ib e d b y B o sk o u a n d D e b e ­
v ere (2 0 0 0 ).
T h e e v o lu tio n o f th e c o n c e n tra tio n o f T M A -N ,
acetate, la c ta te , g lu c o s e an d p y ru v a te a t ex p e rim en tal
co n d itio n s w h e re g r o w th o f S. b a ltic a o c c u rre d is
sh o w n in F ig . 2. W h e n n o a c e ta te o r C 0 2 w a s added,
an d w h e n la cta te w a s a d d e d as a H -d o n o r (Fig.
2 (A )), 1 m o le o f la c ta te w as co n su m e d , re s u ltin g in 2
m o le s o f T M A a n d 0 .6 5 m o le s o f ace tate . R in g 0 et
al. (1 9 8 4 ) a lso d e s c r ib e d o n ly a p a rtia l a c c u m u latio n
(40 m o l% ) o f a c e ta te w h e n la c tate w a s u se d b y S.
p u tr e fa c ie n s as su b s tra te . W h e n p y ru v a te w a s ad ded
as H -d o n o r (F ig . 2 (B )), th e re a c tio n w as m o re sy m ­
m etric (1 m o le o f p y ru v a te and T M A O reacted,
re su ltin g in 1 m o le o f T M A a n d ac e ta te ). A t the
sam e tim e , sm a ll a m o u n ts o f la c tate w e re accu m u -
0,00
16,00
119
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
T im e ( d a y s )
T im e ( d a y s )
D
140
0,00
2,00
4,00
6,00
8,00
T im e ( d a y s )
10,00
12,00
14,00
16,00
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
T im e (d ay s)
F ig. 2. E v o lu tio n o f the co n ce n tratio n s (m M ) o f T M A -N ( ♦ ) , a ce ta te ( ■ ) , lactate ( a ) , g lu c o se ( • ) a n d p y m v a te ( * ) in a d e fin e d m edium
a t 4 °C in o c u la ted w ith S. b a ltic a in 100% o f N 2 and 0 % ( v / v ) a ce ta te a n d lactate as H -d o n o r (A ), 10 0 % o f N 2 and 0 % ( v / v ) ace ta te and
p y m v a te as H -d o n o r (B ), 5 0 % N 2 + 5 0 % C 0 2 an d 0 % ( v / v ) a ce ta te and p y m v a te as H -d o n o r (C ), 100% o f N 2 and 0.5% ( v / v ) ace ta te and
p y m v a te as H -d o n o r (D ).
J. D e b e v e re e t al. / In te r n a tio n a l J o u r n a l o f F o o d M ic ro b io lo g y 6 8 (2 0 0 1 ) 1 1 5 -1 2 3
120
9.5
9
8.5
X
Q.
8
7.5
7
6.5
6
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
T im e (days)
Fig, 3. In flu e n c e o f a ce ta te c o n ce n tratio n an d atm o sp h ere (❖
100% o f N 2 , 0 % ( v / v ) ace ta te and la c ta te as H -d o n o r, H 100% o f N 2 , 0%
( v / v ) a ce ta te , a n d p y ru v a te as H -d o n o r, a 100% o f N 2 , 0 ,5 % ( v / v ) ace ta te and p y ru v a te as H -d o n o r, X 5 0 % N 2 + 50 % o f C 0 2 , 0%
( v / v ) a ce ta te an d p y ru v a te as H -d o n o r) o n th e pH e v o lu tio n a t 4°C in a d e fin e d m e d iu m (p H = 6 .8 ) in o c u la te d w ith S. baltica.
la te d (9 m M ) p ro b a b ly b e c a u se o f a fe rm e n ta tiv e
p ro d u c e d (68 m M ) th a n w h e n n o C 0 2 w as p re s e n t in
th e h ead sp a ce . B a c te rio lo g ic a l p e p to n e , a so u rce fo r
m e ta b o lism fro m g lu c o se (5 m M o f c o n su m p tio n ).
T h is a c c u m u la tio n also illu stra te s th a t p y ru v a te w ill
b e p re fe rre d o v e r la c ta te as H -d o n o r b y S. b a ltic a .
e a sily c o n v e rtib le a m in o acid s, can in d e e d also fu n c ­
tio n as H -d o n o r fo r th e T M A O -re d u c tio n re a c tio n , as
d e sc rib e d b y R in g O e t al. (1 9 8 4 ) fo r S. p u tr e fa c ie n s
a n d b y E a ste r e t al. (1 9 8 2 ) fo r S h e w a n e lla spp.
A lth o u g h th e g ro w th o f S. b a ltic a , w ith p y ru v a te
a s H -d o n o r a n d n o a c e tate a d d e d , w as n o t m u c h
in h ib ite d b y th e C 0 2 a tm o sp h ere , C 0 2 h a d a p ro ­
A c e ta te g re atly d e la y e d re a c tio n (2 ) as th e s ta rt o f
th e T M A p ro d u c tio n w a s p o stp o n e d fro m d ay 3 to
d ay 25 (F ig . 2 (D )). H o w e v e r, a t th e e n d o f the
n o u n c e d e ffe c t on th e stu d ie d re a c tio n s (F ig . 2 (C )).
C 0 2 p a rtly in h ib ite d re a c tio n (2 ), as at the e n d o f th e
e x p e rim e n t, o n ly 4 0 m M o f a c e ta te w as p ro d u c e d
re a c tio n , sim ila r a m o u n ts o f T M A a n d a c e ta te w ere
p ro d u c e d as w h e n n o a c e ta te w a s a d d e d (F ig . 2(B )).
(in ste a d o f 58 m M , w h e n n o C 0 2 w a s ad d ed ) a n d 41
m M o f p y ru v a te w as co n su m ed . H o w e v er, u n d e r th is
c o n d itio n , H -d o n o rs o th e r th a n p y ru v a te w e re a p p a r­
F o r all te s te d c o n d itio n s, th e p H sta rte d to in c re a se at
th e m o m e n t T M A p ro d u c tio n w as n o tic e d w h ile at
th e e n d , th e p H d e c re a se d (F ig . 3). T h e p H d rop at
e n tly u se d , b e c a u se sim ila r a m o u n ts o f T M A w e re
9,5
u
ÙB
O
0,00
5,00
1 0 ,0 0
15,00
2 0 ,0 0
25,00
30,00
T im e (d a y s)
F ig . 4. In flu e n c e o f a ce ta te c o n ce n tratio n an d atm o sp h ere ( ♦
100% o f N 2 and 0% ( v / v ) a ce ta te , ■ 100% o f N 2 a n d 0 .1 % ( v / v ) acetate,
a 1 00% o f N 2 a n d 0 .2 5 % ( v / v ) a ce ta te , X a ir and 0 % ( v / v ) acetate, $
a t 4 °C in a b u ffe re d fish e x tra c t (p H = 6.8).
100% o f N 2 a n d 0 .5 % ( v / v ) a cetate) o n th e g ro w th o f S. baltica
J. D e b e v e re et al. / In te rn a tio n a l J o u rn a l o f F o o d M ic r o b io lo g y 68 (2 0 0 1 ) 1 1 5 -1 2 3
th e e n d o f th e re a c tio n c a n b e e x p la in e d b y th e
sig n ific a n t la c ta te p ro d u c tio n b y S. b a ltic a a t th e end
o f the e x p erim en ts.
3.2. E x p e rim e n ts in f i s h e x tra c t
P re v io u s in v e s tig a tio n s (K im e t al., 1995; B o sk o u
a n d D e b e v e re , 2 0 0 0 ) h a v e d e m o n stra te d th a t d ip p in g
in a n a c e tate so lu tio n re s u lte d in a n e x te n sio n o f th e
s h e lf life o f re frig e ra te d fish fillets. T o in v estig ate
th e e ffe c t o f a n a c e ta te tre a tm e n t on th e T M A O -re ­
121
0 .5 % o f a c e ta te , n o g ro w th w a s o b se rv e d a t 4°C
w ith in 2 9 d ay s. A t a n e u tra l p H , w h ic h is th e case in
th is stu d y (p H = 6 . 8 ) , th e a n ti-m ic ro b ia l e ffe c t o f
a c e ta te (p K = 4 .7 5 ) i s n o rm a lly v ery lo w d u e to the
lo w fra c tio n o f u n d is s o c ia te d m o le c u le s. A t p H = 6.8
th e ra tio o f u n d is s o c ia te d to to ta l a c id is 0 .0 0 8 4 . It is
th e re fo re re m a rk a b le th a t th e g ro w th -in h ib itin g effect
is th a t p ro n o u n c e d a t su c h lo w c o n c e n tra tio n s o f
acetate.
W h e n n o a c e ta te
w as a d d e d , u sin g la c tate as
d u ctio n re a c tio n b y S. b a ltic a in fish , ex p e rim e n ts
w e re p e rfo rm e d in a fish e x tra ct. T h e g ro w th o f S.
b a ltica in 10 0 % N 2 w a s h ig h ly in h ib ite d b y re la ­
H -d o n o r, T M A O w a s re d u c e d , re su ltin g in 65 m o l%
o f a c e ta te ( 15 m M o f la c ta te re su lte d in 10 m M o f
a c e ta te a n d 30 m M o f T M A ) (F ig . 5 (A ) a n d (B )).
T h e se re s u lts are in a g re e m e n t w ith th o se o b ta in e d in
tiv e ly sm all a m o u n ts o f a c e ta te (F ig . 4). T h e tim e to
re a liz e a tw o lo g in c re a se a t 4°C w a s p ro lo n g e d from
3 to 8 d ay s, a n d 16 d a y s b y th e a d d itio n o f 0 .1 % and
0 .2 5 % , re s p e c tiv e ly , o f aceta te. A t co n c e n tra tio n s o f
th e d e fin e d m e d iu m . N o a d d itio n a l a c e tate w as p ro ­
d u c e d a t th e m o m e n t th a t all lactate w as co n su m ed .
H o w e v e r, T M A p ro d u c tio n co n tin u e d , su g g estin g
th a t o th e r H -d o n o rs, s u c h as sim p le a m in o a c id s like
60
80
70
50
60
40
50
30
40
30
20
20
10
10
0
0 ,00
0
2,00
4,00
10,00
6,1
12,00
14,00
0,00
2,00
4,00
6,00
T im e (days)
10,00
12,00
14,00
T im e (days)
80
70
60
40
S
B
50
40
30
20
10
0
0 ,0 0
5,00
10,00
T im e (days)
15,00
20,00
0,00
5,00
10,00
15,00
20,00
25,00
30,00
T im e (days)
Fig. 5. E v o lu tio n o f th e c o n c e n tra tio n s (m M ) o f T M A -N ( 0 ), a ce ta te ( ■ ) , la c tate ( a ) in a buffered fis h e x tra c t a t 4 °C inoculated w ith S.
b a ltic a in 100% o f N 2 a n d 0 % ( v / v ) ace ta te (A ), air and 0 % ( v / v ) a ce ta te (B ), 100% o f N 2 and 0.1% ( v / v ) ace ta te (C ) and 100% o f N 2
and 0.2 5 % ( v / v ) a ce ta te (D ).
122
J. D e b e v e re e t al. / In te r n a tio n a l J o u rn a l o f F o o d M ic ro b io lo g y 68 (2 0 0 1 ) 1 1 5 -1 2 3
se rin e a n d cy stein e, w h ic h are c o m m o n e x tra c tiv es
o f fish , w e re at th a t m o m e n t u se d as H -d o n o rs fo r
fu rth e r T M A O -re d u c tio n . A m in o a c id s su c h as serin e
a n d cy ste in e are c o m p le te ly o x id iz e d to C 0 2 w ith o u t
th e fo rm a tio n o f a c e ta te (R in g O e t al., 1984). T h is
e x p la in s th e stop in a c e tate fo rm a tio n a t th e m o m e n t
th a t all la c ta te is c o n su m e d , w h ic h w a s also re p o rte d
fo r S. p u tr e fa c ie n s N C M B 1735 in a d e fin e d m e d iu m
b y R in g O e t al. (1 9 8 4 ). H o w e v e r, at th e m o m e n t th a t
all la c ta te w as co n su m e d , le v e ls o f 3 0 m M T M A -N
(c o rre sp o n d in g w ith 4 2 m g T H A - N /1 0 0 m l) w ere
a lre a d y p ro d u c e d , w h ic h is w e ll o v e r th e m a x im u m
lim it o f 15 m g T M A - N / 100 g in m a rin e fish sto re d
o n ic e (C o n n e ll, 1975). T h e re fo re , th e T M A O -re d u c ­
tio n w ith H -d o n o rs o th e r th a n la c ta te is p ro b a b ly
o n ly o f lim ite d im p o rtan c e in p ra c tic e . H o w e v er, to
stu d y th e m e c h a n ism o f T M A O -re d u c tio n b y S.
tra tio n s ( < 0 .3 % ) c a u s e a d e la y o f th e T M A O -re d u c ­
tio n re a c tio n b y S. b a lt ic a in w h ic h lac ta te is u sed as
a H -d o n o r. W h e n p r a c tic a l sto ra g e tim e s fo r fish
(e.g. 6 d ay s a t 4°C a f t e r p a c k a g in g ) are c o n sid ered ,
g ro w th a n d T M A O -r e d u c tio n b y S. b a ltic a w a s c o m ­
p le te ly in h ib ite d d u r in g this p e rio d w h e n 0 .2 5 % o f
a cetate w as p re s e n t i n th e g ro w th m e d iu m . M o st
p ro b ab ly , in th e p r e s e n c e o f a c e ta te H -d o n o rs oth er
th a n la c ta te h a v e to b e d ra w n o n b y S’, b a ltica ,
re s u ltin g in le ss e n e rg y e ffic ie n t p ro c e s se s a n d c o n ­
seq u e n tly , in th e in h ib itio n o f g ro w th . A lth o u g h C 0 2
p a rtly in h ib ite d r e a c tio n ( 2), its c o n trib u tio n to the
in h ib itio n o f th e r e a c tio n m e c h a n ism w a s o f m in o r
im p o rtan c e . O n th e c o n tra ry , as c o u ld b e d eriv ed
fro m F ig . 1, th e e ff e c t o f C 0 2 o n th e g ro w th o f S.
b a ltic a w a s o f m a jo r im p o rta n c e w h e n la cta te, the
n atu ral H -d o n o r fo r T M A O -re d u c tio n d u rin g fish
sp o ila g e , w as a v a ila b le .
b a ltic a , ex te n d e d e x p e rim e n ts w ith o th e r H -d o n o rs
(o r re a c tio n -in te rm e d ia te s) su c h as p y ru v a te , a re n e c ­
essary .
W h e n 0 .1 % o f a c e ta te w a s a d d e d to th e fish
e x tra c t (F ig. 5 (C )), less a c e ta te w a s fo rm e d a n d
la c ta te w a s m o re slo w ly c o n su m e d in c o m p a riso n to
R eferences
th e e x p e rim e n ts w ith o u t th e a d d itio n o f a c e ta te . T h e
c o n su m p tio n o f lactate a n d th e p ro d u c tio n o f a cetate
B oskou, G ., D e b ev e re , J., 2 0 0 0 . S helf-life e x te n sio n o f cod fillets
w e re a lm o st c o m p le te ly in h ib ite d w h e n th e fish e x ­
tra c t c o n ta in e d 0 .2 5 % o f a c e ta te (F ig . 5 (D )). A p p a r­
e n tly , th e a d d itio n o f a c e ta te in h ib ite d th e u se o f
la c ta te as H -d o n o r, H o w e v e r, th e a d d itio n o f a c e ta te
d id n o t in flu e n c e th e fin a l c o n c e n tra tio n s o f T M A
p ro d u c e d b y S. b a ltic a . P ro b a b ly , a lte rn a tiv e H d o n o rs w ere u se d b y S. b a ltic a , b u t th is p a th w a y
se e m s to be less e n e rg y e ffic ie n t, w h ic h c a n be
d e d u c e d fro m th e e x c e p tio n a l g ro w th in h ib itio n o f
sm all a m o u n ts o f acetate.
B oskou, G ., D e b ev e re , J.,
1997. R ed u c tio n o f trim eth y lam in e
oxide b y Sh e w a n e lla s p p . u n d e r m o d ifie d atm o sp h eres in
vitro. F o o d M icro b io l. 14, 5 4 3 -5 5 3 .
w ith an a ce ta te b u ffe r s p ra y p rio r to p a ck a g in g u n d e r m o d ified
atm o sp h eres. F o o d A d d it. C ontam . 17, 1 7 -2 5 .
C onnell, J.J., 1975. C o n tro l o f Fish Q u a lity . F ish in g N e w s B ooks,
F a m h a m , S u rra y , U K .
D ainty, R .H ., 1996. C h e m ic a l/b io c h e m ic a l d e te ctio n o f spoilage.
Int. J. F o o d M icro b io l. 3 3 , 1 9 -3 3 .
D a lg aa rd , P., 1995. Q u a lita tiv e and q u a n tita tiv e ch arac te risa tio n o f
sp o ila g e b a c te ria from p a c k e d fish. Int. J. F o o d M icrobiol. 26,
3 1 9 -3 3 3 .
D ebevere, J.M ., B oskou, G ., 1996. E ffe ct o f m o d ified atm osphere
on the T V B /T M A -p r o d u c in g m ic ro flo ra o f c o d fillets. Int. J.
F o o d M icro b io l. 31, 2 2 1 - 2 2 9 .
D e v lie g h ere, F., D e b ev e re , J ., V an Im p e , J., 1998. C oncentration
o f c arb o n d io x id e in th e w a te r-p h a se as a p a ra m e ter to m odel
4. C onclusions
the e ffe c t o f a m o d ified a tm o s p h e re o n m ic ro -o rg a n ism s. Int.
J. F o o d M icro b io l. 43, 1 0 5 -1 1 3 .
D yer, W .J., 1945. A m in e s in fis h m uscle: I. C o lo rim e tric d e te rm i­
D e b e v e re a n d B o sk o u (1 9 9 6 ) d e m o n s tra te d th a t
th e s h e lf life o f m o d ifie d a tm o sp h e re p a c k a g e d fish
fille ts c o u ld b e p ro lo n g e d b y s p ra y in g fish fillets
w ith a 10% a cetic a c id / a c e ta t e s o lu tio n (p H = 5.6).
S u c h a tre a tm e n t re s u lte d in an in c re a se d c o n c e n tra ­
tio n o f ac e tic a c id / a c e ta t e a t th e su rfa c e o f th e fish
fillets a n d lo w e re d th e su rfa c e p H o f th e fish fillets.
T h e a c tu a l p a p e r d e m o n stra te d , o n th e o th er h a n d , in
s itu th a t ev en at n e u tra l p H ( 6 .8) lo w a c e ta te c o n c e n ­
nation o f trim eth y lam in e a s th e p ic ra te salt. J. Fish. R es. B oard
C an. 6 (5), 3 5 1 -3 5 8 .
E aster, M .C ., G ib so n , D .M ., W ard , F .B ., 1982. A cond u ctan ce
m e th o d fo r th e assay a n d s tu d y o f b a cterial trim ethylam ine
o x id e red u ctio n . J. A ppl. B a c te rio l. 52, 3 5 7 -3 6 5 .
E aster, M .C ., G ib so n , D .M ., W ard , F .B ., 1983. T h e in d u c tio n and
location o f trim eth y lam in e-N -o x id e re d u c ta se in A ltero m o n a s
sp. N C M B 400. J. G en. M icro b io l. 129, 3 6 8 9 -3 6 9 6 .
F e rn a n d e z-G arcia , E ., M cG re g o r, J .U ., 1994. D e term in a tio n o f
o rg a n ic acid s d u rin g the ferm e n ta tio n an d c o ld sto ra g e o f
y o g h u rt. J. D a iry Sei. 77 (1 0 ), 2 9 3 4 -2 9 3 9 .
J. D e b ev e re e t al. / In te r n a tio n a l J o u rn a l o f F o o d M ic ro b io lo g y 68 (2 0 0 1 ) 1 1 5 -1 2 3
G ra h a m , J.C ., W ard , F .B ., 1988. P u rificatio n an d p ro p e rties o f
trim eth y lam in e-N -o x id e re d u c ta se fro m S h e w a n e lla sp. N C M B
4 0 0 . J. G en. M icro b io l. 133, 3 7 9 -3 8 6 .
K im , C .R ., H e a m sb c rg e r, J.O ., V ick ery , A .P ., W h ite , C .H ., M ar­
s h all, D .L ., 1995. E x ten d in g s h e lf life o f re frig erated c atfish
fillets u sin g s o d iu m ace ta te and m o n o p o ta ssiu m p h o sp h ate . J.
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