C) Integridad del ADN
6.3. Bioensayos
6.3.3. Evaluación de la actividad citotóxica de los extractos con mayor actividad
AMP + PPi
ATP + Aspartate
CITRULLINE
H 20 ureas e
UREA
2 AMMONIA
+CARBON
DIOXIDE
ORNITHINE
Carbamyl phosphate 2 ADP + Pi 2 ATP C02 + NH 4+Fig. 5.9 The urea cycle (modified after Stryer, 1 981 ).
-..] 0
7 1 1 983). The evidence above strongly suggests that the urea cycle does not exist i n win e LAB. Spon holz ( 1 992) reported the production of u re a from arginine by some wine LAB (strains of Lb. brevis and Lb. buchnen) , contrary
to the findings of the current studies. However, the author did not describe the materials and methods used in the determination of urea ·or, indeed, of any of the metabo lites i nvestigated i n this study. Thus, it is not certai n whether the reported "urea" was indeed urea. From a stoichiometric poi nt of view, the production of urea fro m arginine is not likely, si nce argi n i n e is com p letely deg raded t h roug h the argin ine dei m i n ase pathway and thus, there is no arginine available to go through a urea cycle. On the othe r h and, if arginase alone of the urea cycle enzymes was present, there wou ld be a stoichiometric conversion of arginine to ornithi ne, but urea would also be present in a 1 :1 stoichi ometry u n less urease was also present. However, there is no evidence on the presence of urease in wine LAB. In the Sponholz stud
Y)
the reported "u rea" is present at very low leve ls i n relation to the amount of arginine catabolised, indicating that it may not be urea.lt should be stressed that urea, if present, would interfere with the citru lline analysis used in this study, because diacetyl monoxi me, one of the reage nts, reacts with urea to g ive a co lo ured product (Archibald, 1 944). H o wever, si nce u rea was shown to be abse nt in the fermentations, this interference can be ru led out.
The excretion of small amounts of citrulline (1 0-1 5% on a molar basis) from arginine catabolism (Figures 5.1 -5.6) was found, in addition to the production of a m m o n i a and o rn it h i n e . K u e n sch et al. ( 1 974) also noted a s l i g ht
production of citrulline associated with arginine deg radation and attributed the production of citrulline to the transformation of ornithine in the urea cycle. However, the time-course of citrulline production in the present study is not consistent with this pro posal. In fact, the time-course of citrulline formation correlated well with that of arginine degradation and o rnithine production (Figures 5.1 -5.6). This provides additional evidence against the existence of t h e u re a cycle in w i n e LAB . The productio n of citru l l i n e fro m arg i n i n e catabolism has been reported i n oth e r microorg a n isms possessin g the arginine deiminase pathway, such as Streptococcus faecalis mutants (Simon e t a / , 1 9 8 2 ) , L a ct o b a cillus s a k e I N RA 300 fro m m e at ( M a nt e l a n d
Champomier, 1 987) and Clostridium botulinum Okra B (Patterson-Curtis and
72 The physiology of citrulline excretion is not clear and only some speculations can be made at this stage. I n Lactococcus lactis subsp. lactis M L3 known to
possess the arginine deiminase pathway, arginine is transported into the cell via an antiport system in a 1 :1 stoichiometry with ornithine export (Driessen
et al. , 1 987; Poolman et al. , 1 987). This process does not require m etabolic
energy. lt is likely that this arginine-ornithine antiport process function s i n the transpo rt of arginine i nto the ce ll of wine LAB . lt i s possible that arginine uptake may also be coupled to export of citrulline via the same antiport system, but only to a small extent. The excretion of citrulline wou ld result in loss of ATP, since the excreted citrulline could not be catabolised further along the ADI pathway to produce ATP. Alternative ly, citrulline excretio n may involve a n independent transport system.
S o m e of the excreted citru lli n e can appare ntly be recatabo lised by the lactobacilli strains (Figures 5.3-5.6), but not by the Le. oenos strai n (Figures
5. 1 and 5.2). This suggests that citrulline can be transported back i nto cells by lactobacilli, but not by the leuconostoc, which raises the questio n of the physio logical differe nce between these organisms. Another questio n to be posed is why do the lactobaci lli excrete citru lline, then reassimi lat e and recatabolise it? These questions remain to be answered.
The time-cou rse study fou nd that g lucose and arginine were metabolised concurrently in heterofermentative wine LAB (Figures 5. 1 -5.7). Concom itant degradation of glucose and arginine has also been reported i n Str. faecalis
ATCC 1 1 700 (Si mon et al. , 1 982). Concu rrent uti lisation of gl ucose and
arg i n i n e indicates that g lucose does not repress the synthesis of arg i nine deiminase pathway enzymes. Lack of glucose repression has been shown w i t h Lb. buch n e ri N C D O 1 1 0 ( M a n c a d e N ad ra et al. , 1 98 6 a ) a n d Mycoplasma ferm e n ta n s P G 1 8 ( O i s o n e t al. , 1 9 9 3 ) . C o nv e rs e l y , a
s e q u e nt i a l m e tabo l i s m of g l u c o s e a n d a rg i n i n e i s fo u n d i n s o m e homofe rmentative LAB such as Lactococcus lactis H 1 (Crow and Thomas,
1 982) and Lb. plantarum fro m fis h (Jo nsso n , e t al. , 1 983). Seq u e ntial
uti lisation of g lucose and arg i n i n e suggests that g lucose does rep ress the synthesis of the arginine deiminase pathway e nzymes. This repression h as been demonstrated in Lb. leichmannii ATCC 4797 (Manca de Nadra et al. ,
7 3 Although glucose and arginine were found t o b e metabolised concurrently i n this i nvestigation, when a m i xture o f g lucose, fructose a n d arg i n i n e was supplied, the arginine was not catabolised until the fructose level was low (<1 g/L) in the heterofermentative wine LAB (Figures 5.6 and 5.7). This does not appear to h ave been reported in othe r microorganisms. This fi nding suggests that fructose represses the synthesis of the arg i n i n e deimin ase pathway enzymes. The mechanism of the repression of arginine catabolism by fructose is not known .
lt is known that the ATP pool size is an important signal in the regulatio n of the arginine deimi nase pathway i n Pseudomonas a eruginosa PA0 1 , Str. faecalis ATCC 1 1 700 and other bacte ria (Mercen ier et al. , 1 980; Simon e t a/. , 1 982; Cunin e t al. , 1 986). Repression of the ADI pathway occurs u nder
conditions which promote the energy status of the cells; for instance, the presence of a fermentable substrate or other energy sources or both, as well as aerobic and anae robic respirati on (Cu ni n e t al. , 1 9 86). Conve rsely ,
energy depletion resu lts in i nduction of the pathway enzymes. Hence, t h e sequential utilisation of fructose and arginine in heterofermentative win e LAB could conceivably be due to the generation of additional ATP from fructose
metabolism relative to that available from glucose fermentation.
Acco rding to Kandler ( 1 983) , fructose can act as a hydrogen acceptor for reoxidation of NADH and NADPH by the reduction of fructose to m an n itol during h eterofe rmentation, and mannitol fo rmation fro m fructose h as been demonstrated in h eteroferm entative wine LAB (Pi lone e t al. , 1 99 1 ). The
acetyl phosphate formed from the phosphoketolase reaction could therefore be converted to ATP and acetate, rathe r than being reduced to eth a n o l . Consequently, a n additional ATP i s generated when fructose is reduced. l t i s perh aps t h i s additio nal ATP p e r mole of substrate oxidised that may h ave repressed arginine catabolism observed in this study (Figures 5.6 and 5.7). Further work would be required to substantiate this hypothesis. Alternatively, t h e d iffe re nce betwee n t h e fructose and g l ucose effects on arg i n i n e metabolism may be a consequence of differences in the mechanism o r rate of transport of the two sugars.
This study indicates that arginine catabolism does not seem to affect malic acid degradati on or vice versa. Comp lete uti lisatio n of malic acid was achieved with Le. oenos OENO, but not with the lactobacilli. The inco m plete
7 4 utilisation of malic acid by the lactobaci lli may not necessari ly be due to arg i n i n e catabol i s m , s i nce i ncomplete degradatio n of malic acid by the lactobaci lli also occu rred i n AJ MRS contai ning 0.5 g/L arg i n i n e (Section 3.2.2) and in synthetic media containing only 0. 1 7 g/L arginine (unpublished data). In a wine containi ng 4 g/L malic acid and 1 .3 g/L arginine, Sponholz ( 1 992) found that malic acid was completely degraded by Lb. brevis 822, but
i ncompletely by a strain of Lb. buchneri, while arginine was almost u sed up
by both strains. This reflects strain variation in the ability to degrade malic acid, rather than the influence of arginine catabolism.
Act ivities fo r arg i n i n e dei m i n ase pathway e n zym es were n ot fo u nd i n h o mofe rme ntative wi n e LAB (Chapte r 4). The results prese nted i n the p re s e nt chapte r co nfi rm that arg i n i ne catabo lism does not occu r in the homofermenters cu ltu red in the synthetic medium containing low levels of g lucose (1 g/L) and high levels of arginine (5 g/L) (Figure 5.8) . This suggests t h at homoferme ntative wi n e LAB are i n d e ed incapable of catabolising arg i n i n e i n contrast to othe r homofermenters (Crow and Thomas, 1 982; J o n s s o n e t al. , 1 9 8 3 ; M a n c a de N ad ra e t al. , 1 9 8 6 b ; M a n t e l a n d
Champomier, 1 987). The reason for this is u nclear.
C o m p a ri s o n s o f c e l l m as s ( O D ) rev e a l t h e p o o re r g rowth o f t h e h o mofe rm ente rs (Fig ure 5.8) compared to that of the hete roferme nters, particularly in the case of the lactobacilli (Figures 5.2-5.4), when cultured in t h e synth etic m edi u m co ntai n i n g 1 g/L g l ucose and 5 g /L arg i n i n e . Furthermore, growth and arginine catabolism continued after the exhaustion of g lucose with Le. oenos OENO (Figures 5. 1 and 5.2). With the lactobacilli,
g rowth also conti nued while citrulline was reutilised after the exhau stion of g lucose and arginine (Figures 5.3 and 5.4) . These observations sugge st that arginine and citrulline catabo lism may co ntribute energy for g rowth of the heterofermenters. A more thorough study, therefore, was carried out on the g rowth responses and e n e rg etics of arginine and citrulline catabolism in wine LAB and the results are presented in Chapter 6.
The fi ndi ngs of t h i s research h ave important o e n o log ical i mp licatio ns . Ammo nia formed from arginine catabolism leads to a n increase in t h e pH value and the exte nt of such a pH rise depends on the amount of arginine p re s e nt i n the m ed i u m . The effect of ammonia formation from arg i n i n e metabolism on pH increase may b e more significant than t h e contribution of
7 5 malic acid degradation at high arginine levels. lt is possible to find wines with arg i n i ne over 2 g/L (Spo n h olz, 1 99 1 ) and g rape juice contain i ng 1 0 g/L arginine (Eggenberger, 1 988) . The pH increases can be considerable and cause wine spoilage if the arginine level is high.
The fi n d i n g of citru l l i n e excreti o n in t h is study i s also o e n o lo g i cally significant, since citrulline is a precursor of the carcinogenic ethyl carbamate (Ough et al. , 1 988). An experime nt on the formation of ethyl carbamate and
citru lline excretion from arginine catabolism was subsequently conducted in synthetic wine and wine inoculated with heterofermentative wine LAB. The results of this investigation are presented in Chapter 7.
Carbamyl phosphate, another intermediate of arg i n i n e catabolism and a precursor of ethyl carbamate (Ough et al. , 1 988), was not determined i n the
time-co u rse stu dy. This is because carbamyl phosphate is c h e m i cally u n st a b l e and n o re l i a b l e ready-t o - u s e m et h o d is ava i l a b l e for t h e dete rmi nation of this compound. Carbamyl phosph ate i s decomposed to phosph ate , ammonia and carbon dioxide at pH levels fro m 2-4; and to phosp hate and cyanate at pH leve ls from 6-8, the cyanate bei ng further decomposed to ammonia and carbon dioxide (Jones, 1 962 ; Alien and Jones, 1 96 4 ) . Carba myl p h osp h ate i s co m m o n ly determ i n ed by d i ffe re ntial phosphate analysis using the Fiske-SubbaRow method (Leloir and Cardini, 1 957). Howeve r, this method i s not applicab le fo r the determinatio n of carbamyl phosphate excreted (if any) in the synthetic medium, since a high conce ntratio n of potassi u m phosphate (4 g/L) i s p rese nt in the medium (Table 5. 1 ). lt remains to be investigated whether carbamyl phosphate can be excreted.