CHAPTER 7. IMPACT OF COMMON WINEMAKING TECHNIQUES ON
7.1. INTRODUCTION ,
In Chapter 6, the metabolism of arginine by lactic acid bacteria (LAB) was investigated in wine. The oenococcal strains used in Chapter 6 are commercially available, but they were pregrown in a high arginine containing medium to induce ADI pathway enzymes, and the wine used was made from a retail grape juice.
For the experiment presented here, the results obtained in the previous chapter were verified under more practical conditions. The wine used was fermented from Chardonnay grape j uice originating from a commercial vineyard and malolactic fermentation (MLF) was induced with recently developed commercial strains for direct inoculation, following the manufacturer's recommendations. Moreover, two treatments were chosen to reflect common winemaking techniques that may have an impact on arginine metabolism.
Natural or spontaneous yeast alcoholic fermentations (AF) are common In 'old-world' winemaking (Europe) and gaining popularity amongst some wine makers in the 'new-world'. e.g. U.S .A., Australia and New Zealand (Eglinton et al. 2000) . Some wine makers believe that spontaneous yeast AFs help in creating unique wines with higher complexity, depth and more diverse aromas (Goldfarb 1 994; Price 1 996; Ross 1 997; McCorkindale 1 999), although other reports indicate that wines made with spontaneous fermentations had similar or worse sensory properties than those fermented with commercial preparations of freeze dried yeasts (Gaia and Matta 1 984; Bach 1 985; Bader 1 997; Lorenzini 1 999). In any case, differences in the management of AF will lead to wines with a distinct chemical composition, regarding the content of nutrients as well as substances with a potential inhibiting effect on wine LAB growth. The difference in chemical composition of wines may not only affect growth of LAB , but could change the kinetics of arginine degradation, itself.
Storing wines on yeast lees (sedimented yeast cells) after AF is also an important technique in the making of wines, particularly in the production of white and sparkling wines (e.g. Methode Champenoise sparkling wines), and has a significant effect on the sensory character of the wines that are allowed to rest on lees (Charpentier and Feuillat 1 993; Price 1 996) . The yeast lees have been shown to stimulate wine LAB growth (Lonvaud-Funel et al.
1 988). Above all, they constitute a major pool of proteins, capable of releasing amino acids into the wine (Stuckey et al. 1 99 1 ; Ribereau-Gayon et al. 1 998a; Martinez-Rodriguez and Polo 2000). This release is caused first by yeast cell lysis and later by proteolytic activity of residual yeast enzymes (Leroy et al. 1 990; Charpentier and Feuillat 1 993; Sato et al. 1 997). Furthermore, it was shown recently that several wine LAB produce exoproteases, capable of breaking down wine polypeptides into free amino acids, and in particular, arginine (Manca de Nadra et al. 1 997 ; Manca de Nadra et al. 1 999). Therefore, the yeast lees represent a potential and significant arginine reservoir after completion of AF and may influence the kinetics of arginine degradation by wine LAB, as well.
In order to study the effect of both techniques presented on the arginine metabolism of
commercial wine LAB, the wines used for MLF were produced either by a natural yeast fermentation or by inoculation with commercial freeze dried yeast; and MLF was carried out either in wine containing yeast lees or in wines racked-off the lees.
7.2. Experimental conditions
The grape must used for this experiment was obtained from a vineyard in the Fernhill area of Hawke's B ay, New Zealand. The Chardonnay grapes were handpicked at 22 Brix and 30 mg kg-1 S 02 was added upon crushing. The must used was free run j uice obtained after 45 minutes of skin contact and had a pH of 3.22. The must was separated into two 1 0 1
batches and a temperature controlled AF was carried out at 1 3 cc. One batch was not
inoculated with yeast and allowed to ferment by the natural yeast flora, whereas the second
was inoculated with 2% (v/v) S. bayanus PC, pre-grown in the same but filtered grape j uice
with 1 g r l yeast extract added.
After completion of AF, the wines were cold-settled for 48 h. The composition of both wines after AF and cold stabilization is shown in Table 7 . 1 . For use in experiments, the only modification was the addition of arginine to increase the concentration in both batches to 750 mg r I , followed by repeated cold-settling of the wines. Then, 5 I of each batch were racked-off and transferred into another container. The remaining 5 1 in the original carboys were used as yeast lees treatment. From each of the four batches, 4x
1 1
aliquots were taken and transferred into 1 I glass bottles for induction of MLF with 2 commercial freeze-dried strains (strains EQ54 and VFO, in duplicate; yeast lees batches were stirred before the division).The inoculations were carried out according to the manufacturer's recommendation with a modification. The inoculation size was doubled to compensate for the high ethanol content of the wine and potential loss of viability of the starter culture. The growth of bacteria
during MLF was not monitored regularly, but verified at certain times during cultivation by viable cell counts (upon inoculation and t = 8, 20 and 35 days). From an initial inoculum of 9.2 x 1 05 and 2.3 x 1 06 cfu mr l , up to 3 .2 - 7.2 x 1 07 cfu rnI- 1 were formed. Samples were
taken periodically, centrifuged ( 1 0,000 g for 5 min) and the supematant was frozen (- 1 8°C) until analyzed. The analysis of arginine, citrulline and malic acid are described in 2.4.4. Specifically, arginine and citrulline were determined by HPLC (2.4.4.5).
Table
7.17.3.
a
b
Characterization of Chardonnay wines after natural AF or AF by
s. bayanus PC at l 3 0e. Type of AF Compound PC Natural Ethanol 14. 1 % (v/v) 13.9 % (v/v) pHa 3 .4 1 3 .42 Malate 3 .03 g r l 2.7 g r l Glucose 80 mg rl 1 1 0 mg r l Fructose 0.882 g r l 2.06 g rl Arginine 1 0 mg rl 20 mg r l Urea < 0. 1 mM < 0. 1 mM Ammonia < 0. 1 mM < 0. 1 mM free S02 n.d.b n.d.b after degassing none detected
Results
Figure 7. 1 and Figure 7.2 show MLF by o. oeni strains EQ54 and VFO under several conditions as described in section 7.2. Arginine degradation by both strains was not noticeable or greatly delayed in comparison with the degradation of malic acid in all treatments. During the entire duration of the experiment, arginine degradation did not occur in only two treatments without yeast lees: after natural AF and MLF by strain EQ54 (Figure 7 . 1 , A), and after AF by S. cerevisiae PC and MLF by strain VFO (Figure 7.2, C). In treatments with yeast lees, the arginine concentrations increased after inoculation with both o. oeni strains EQ54 and VFO compared to respective treatments without yeast lees. Equally, the citrulline concentrations found at the end of incubation periods were higher in yeast lees treatments than in the respective treatments without yeast lees.
I 0 I _r-. e.o
E.
Q,I c: 'c 'en '"' < 900 750 600 450 300 1 50 900 750 600 450 300 1 50D -
- - - -- --- ---
.- (:tY::=-�c-@"-'�-0-- 30Time [d)
Figure 7.1
MLF by O. oeni EQ54 in Chardonnay wine. A, wine from natural AF; B ,wine from natural A F with remaining yeast lees; C, wine from A F b y S. bayanus PC ; D, wine from AF by PC with remaining yeast lees. Arginine (D), citrulline (0), and malic acid (6). Error bars represent standard error.
I o I 900 750 600 450 -:: 300 e.o 1 50 E '--' � 900 'c 750 'en '"' < 600 450 300 30 25 I 20 i 6. 1 5 0 I c I 10 _r-. '- ...-I >< e.o _ E "- '--' e.o 5 30
,_5 �
25 'u "3 < .!: ,� U '; 20 1 0 � 1 5 1 50 5Figure 7.2
MLF by O. oeni VFO in Chardonnay wine. A, wine from natural AF; B , wine from natural AF with remaining yeast lees; C, wine from AF by S. bayanus PC; D, wine from AF by PC with remaining yeast lees. Arginine (D), citrulline (0), and malic acid (6). Error bars represent standard error.Arginine to citrulline conversion ratios were calculated by using the difference between arginine and citrulline concentrations at the beginning and end of experiments (Table 7 .2). In treatments where arginine degradation occurred, the remaining amount of arginine was rather homogenous. Therefore, higher final citrulline concentrations led to higher arginine to citrulline conversion ratios.
Table 7.2
Arginine to citrulline conversion ratios (%, w/w) from MLFs with O. oeni EQ54 and VFO in Chardonnay wine (Standard error in parentheses).Treatment O. oeni EQ54
Natural � n.a. c
Natural AF, leesb 3.7 (±0. 1 5)%
AF by S. bayanus PC 1 . 1 (±0.23)%
AF by S. bayanus PC , lees 2 .7 (±0. 1 8)%
AF
� MLF carried out in wine containing yeast lees C
not applicable, no arginine degradation
7.4. Discussion
O. oeni VFO 2.7 (±O.22)% 4.6 (±O.29)% n.a. 3.4 (±0. 1 8)%In this Chapter, the potential of common wine making techniques to increase carcinogenic ethyl carbamate precursor citrulline formation during malolactic fermentation (MLF) was investigated. Particularly, the realization of MLF on the yeast lees was considered in view of the possible role of yeast lees in supplying free arginine to wine lactic acid bacteria (LAB) . In fact, Rollan et al. ( 1 993) demonstrated extracellular proteolytic activities in four strains of 0. oeni isolated from wine. The exoproteases degraded polypeptides in white wine (Manca de Nadra et al. 1 997) and red wine (Manca de Nadra et al. 1 999), releasing predominately arginine into the medium along with other amino acids. According to these
studies, exoproteases could be a common feature of O. oeni and thus, growth and proteolytic
activity of O. oeni would have the potential to increase the concentration of free arginine in wines. Sponholz et al. ( 1 99 1 ) reported that white wines stored on yeast lees did not have increased ethyl carbamate concentrations compared with wines racked-off the lees. In the same publication, however, Sponholz et al. ( 1 99 1 ) reported that wines after MLF always had higher ethyl c arbamate concentrations than those without. Therefore, it seems likely that the white wines with yeast lees analyzed by Sponholz et al. did not undergo MLF.
In this experiment, when comparing fermentations where arginine degradation occurred, wines containing yeast lees always had higher citrulline concentrations than wines racked off the less. S pecifically, the arginine to citrulline ratios were 145% higher when comparing wines fermented with S. bayanus PC and MLF by strain EQ54; and 70% higher when comparing naturally fermented wines and MLF by strain VFO. This demonstrates the potential of the yeast lees to increase ethyl carbamate precursor citrulline formation during MLF by increasing the availability of free arginine for degradation by wine LAB.
As well, wines produced by natural AF had higher arginine to citrulline ratios than wines fermented with the commercial yeast strain. This resulted in a 37% higher ratio when
comparing MLF of strain EQ54 in wines with yeast lees ; and a 35% higher ratio when comparing MLF of strain VFO in wines with yeast lees. The reason for the increased citrulline formation is not clear, since all wines were adjusted to the same arginine concentration prior to inoculation with malolactic bacteria. Moreover, the increase in arginine concentration during the first days of MLF (presumably due to proteolytic activity) was higher in the wines fermented by the commercial yeast preparation. The difference observed might come from the direct effect of an unknown substrate formed during natural fermentation on the regulation of arginine metabolism, leading to a greater accumulation of citrulline. It is possible, too, that a change in arginine metabolism was caused by growth differences which in turn depend on the medium composition. However, the data collected from viable cell counts carried out at different stages of incubations did not support this suggestion and therefore, this aspect remains to be studied in future work.
Finally, strain specific differences in the ability to form citrulline by the wine LAB used were found. Compared with strain EQ54, the arginine to citrulline ratios of strain VFO were 24% and 26% higher in wines with yeast lees fermented either by S. bayanus PC or by natural fermentation.
From the results of this experiment, it can be concluded that the presence of yeast lees increases the citrulline formation potential of MLF and this effect is suggested for wine produced from natural AF, as well. Strain specific differences in the ability to excrete citrulline seem to exist and therefore, studies of the citrulline formation potential may be included in the selection of starter bacteria. The results obtained confirm conclusions made in Chapter 6: arginine degradation by oenococci is avoidable by inactivation of the bacteria after malic acid depletion. It is not possible to assess when degradation of arginine exactly took place because between the sample taken at t = 35 days and t = 1 20 days no other samples were taken and analyzed. However, it is clear that apart from the treatment with strain EQ54 in wine fermented by S. bayanus PC (treatment without yeast lees), where arginine degradation commenced 5 to 1 0 days after depletion of malic acid, the delay between malic acid depletion and the beginning of arginine degradation was approximately 20 days.
It is not known why arginine was not degraded in two of the treatments. The metabolism of the bacteria may have been inhibited by the prevailing conditions after MLF. It is also possible that arginine degradation was only delayed in these treatments and would have been observed later on. In any case, the results suggest that it is unsafe to allow bacterial metabolism to occur freely in wines after complete malolactic conversion. Compared with the experiments described here, where wines were sterile filtered before inoculation with commercial oenococci, the danger of arginine degradation is greater in practical winemaking all the more, considering that naturally occurring wine LAB may be active, too.