Saparbekova А.А., (b.s.c., docent), Astafyeva Y.A. (master),

Esenbayeva A. (student)

South Kazakhstan state university, Shymkent

Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations

Several studies have indicated that the contribution of non-Saccharomyces yeasts leads to a more complex aroma and an improved wine quality. However, natural multistarter cultures remain uncontrolled processes and multistarter cultures need to be used under better-defined conditions. For these reasons, studies have evaluated the possibility of using controlled multistarter cultures to improve the quality of wines. In this context, our knowledge of the metabolic interactions between S. cerevisiae and non-Saccharomyces wine yeasts under winemaking conditions needs to be improved [1].

The natural fermentation of grape must is usually started by low-alcohol-tolerant apiculate yeasts (Kloeckera/Hanseniaspora) that predominate the first stages of fermentation. After 3–4 days, they are replaced by elliptical yeasts (Saccharomyces cerevisiae) that continue and finish the fermentation process. In addition, during the various stages of fermentation, it is possible to isolate other cultures belonging to other yeast genera, such as Candida, Torulaspora, Kluyveromyces and Metschnikowia. Recently, there has been a re-evaluation of the role of non-Saccharomyces yeasts in winemaking. Their presence and permanence throughout inoculated and non-inoculated fermentations are well documented, as well as their contributions to the analytical composition and the sensorial characteristics of wine [2].

In the present study, we report on the fermentation behaviour and the metabolic interactions of mixed and sequential cultures of S. cerevisiae and three potential non-Saccharomyces wine yeast starter species, Hanseniaspora uvarum, Torulaspora delbrueckii and Kluyveromyces thermotolerans. Multistarter fermentations of Hanseniaspora uvarum, Torulaspora delbrueckii and Kluyveromyces thermotolerans together with Saccharomyces cerevisiae were studied. In grape musts with a high sugar content, mixed trials showed a fermentation behaviour and analytical profiles of wines comparable to or better than those exhibited by a pure culture of S. cerevisiae. Sequential trials of T. delbrueckii and K. thermotolerans revealed a sluggish fermentation, while those of H. uvarum exhibited an unacceptable increase in ethyl acetate content (175 ml l⁻¹) [3].

The results of the principal enological characters of microfermentations of our H. uvarum/S. cerevisiae trials (Table 1) show that despite its consistent production of volatile compounds in pure culture, H. uvarum did not cause an increase in volatile acidity in mixed cultures.

Table 1 - Principal enological characters of multistarter fermentations at 20 °C

Culture trial	Ethanol (vol.%)	Volatile acidity (g l^−^1)	Glucose residue (g l^−^1)	Fructose residue (g l^−^1)	Assimilable N compounds used (mg l^−^1)
Pure S. cerevisiae	15.9±0.1	0.71±0.01	0.0	0.0	236.7±6.4
Pure H. uvarum	4.5±0.1	1.24±0.12	53.8±2.0	51.8±2.3	235.0±1.0
Mixed S. cerevisiae/ H. uvarum	15.8±0.1	0.71±0.05	0.0	0.0	193.9±4.2
Sequential S. cerevisiae/ H. uvarum	16.0±0.1	0.66±0.01	0.0	0.0	194.9±5.3
Pure K. thermotolerans	13.6±0.8	0.45±0.05	0.0	30.2±0.5	236.0±4.9
Mixed S. cerevisiae/ K. thermotolerans	15.9±0.1	0.60±0.14	0.0	0.0	262.2±0.9
Sequential S. cerevisiae/ K. thermotolerans	15.6±0.2	0.51±0.04	0.0	2.9±2.0	259.9±1.0
Pure T. delbreueckii	10.7±0.8	0.22±0.04	31.3±8.4	70.5±14.5	148.8±6.0
Mixed S. cerevisiae/ T. delbreueckii	15.7±0.1	0.40±0.05	0.0	1.9±0.5	145.4±0.3
Sequential S. cerevisiae/ T. delbreueckii	15.6±0.1	0.32±0.04	0.0	5.5±3.7	208.8±0.6

This behaviour was also confirmed in sequential cultures, where a prolonged cell survival of H. uvarum was seen. Both mixed and sequential cultures did not show sugar residues, highlighting the correct evolution of the fermentations. Furthermore, in all multistarter trials, less nitrogen was used than in pure cultures, indicating the absence of competition for assimilable nitrogenous compounds between the apiculate and the S. cerevisiae yeasts. In both the K. thermotolerans/S. cerevisiae and the T. delbrueckii/S. cerevisiae multistarter trials, acetic acid production was lower than that seen for pure S. cerevisiae cultures, confirming the peculiar characteristic under these conditions, of the low acetic acid production of these yeasts. In contrast to the mixed fermentations that did not exhibit sugar residues, sequential trials exhibited fructose residues, confirmed by a feeble reduction of final ethanol concentration compared with the pure cultures of S. cerevisiae showing a sluggish fermentation. Moreover, in both mixed and sequential cultures of T. delbrueckii/S. cerevisiae, lower assimilable nitrogenous compound consumption was seen than that exhibited by pure S. cerevisiae cultures, showing a similar behaviour to the H. uvarum strain. In contrast, all of the K. thermotolerans/S. cerevisiae trials exhibited an increase in nitrogen consumption, suggesting possible competition at the critical nitrogen concentrations during mixed and sequential fermentations [4].

Evaluations of some of the volatile compounds confirmed that apiculate yeasts consistently formed higher amounts of ethyl acetate in pure cultures (Table 2). In mixed and sequential cultures, apiculate yeasts influenced the final amounts of ethyl acetate in wines.

However, in mixed cultures, the levels of ethyl acetate concentrations achieved could contribute to the fruity notes and add to the general complexity, while the sequential cultures surpassing the threshold taste level (150 mg l⁻¹) produced a sour-vinegar off odour.

The amounts of ethyl acetate produced by apiculate yeasts appear to be linked to their level of contribution to the fermentation (more in sequential than in mixed cultures). These results are of interest considering the possible use of apiculate yeasts in winemaking. A different behaviour was shown by H. uvarum regarding acetoin production in multistarter trials.

Table 2 - Secondary products of multistarter fermentations at 20 °C

Culture trial	Acetaldehyde (mg l^−^1)	Ethyl acetate (mg l^−^1)	Acetoin (mg l^−^1)
Pure S. cerevisiae	99.7±15.3a	59.6±5.3a	11.9±3.7a
Pure H. uvarum	72.5±2.1b	1606.0±22.6b	87.7±3.4b
Mixed S. cerevisiae/ H. uvarum	69.0±2.0b	111.5±18.0c	8.5±6.8a
Sequential S. cerevisiae/ H. uvarum	59.6±2.2b	180.9±18.1d	6.9±1.3a
Pure K. thermotolerans	18.7±1.4c	77.3±0.3a	3.4±1.4a
Mixed S. cerevisiae/ K. thermotolerans	63.7±6.6b	69.4±1.3a	7.5±2.4a
Sequential S. cerevisiae/ K. thermotolerans	36.2±10.1c	75.0±7.6a	2.2±0.6a
Pure T. delbreueckii	122.3±2.1d	61.7±13.9a	7.3±0.8a
Mixed S. cerevisiae/ T. delbreueckii	66.9±8.6b	59.7±5.6a	5.4±0.8a
Sequential S. cerevisiae/ T. delbreueckii	32.5±7.2c	60.5±5.8a	2.7±1.0a

Pure cultures of H. uvarum were high producers of acetoin (Table 2), confirming the results of previous studies. However, in contrast to this ethyl acetate production, in multistarter fermentations the presence of apiculate yeasts did not cause an increase in acetoin, which was probably metabolised by the actively fermenting S. cerevisiae yeast strain.

The amounts of acetaldehyde, an important by-product of fermentation, did not appear to be negatively influenced by mixed or sequential cultures of H. uvarum, since lower amounts were exhibited in the presence of apiculate yeasts.

As expected, K. thermotolerans and T. delbrueckii multistarter trials did not cause substantial modifications in the secondary products evaluated, as compared with the pure S. cerevisiae cultures. Moreover, the constant reduction of acetaldehyde content in multistarter fermentations of these two species compared with pure culture of S. cerevisiae is also worth highlighting.

In conclusion, multistarter fermentations of H. uvarum, T. delbrueckii and K. thermotolerans are influenced by the level and persistence of these yeasts during fermentation. Prolonged persistence and high levels of these yeast species during multistarter fermentations could result in stress conditions, stuck or sluggish fermentations.

List of used literature

1 Jackson, R., 1994. Wine Science. Principles and Applications. Academic Press, San Diego, USA, pp. 178–219.

2 Zironi, R., Romano, P., Suzzi, G., Battistuta, F., Comi, G., 1993. Volatile metabolites produced in wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera apiculata and Saccharomyces cerevisiae. Biotechnol. Lett. 15, 235–238.

3 Bisson, F.L., 1999. Stuck and sluggish fermentations. Am. J. Enol. Vitic. 50, 107–119.

4 Mora, J., Barbas, J.I., Mulet, A., 1990. Growth of yeast species during the fermentation of musts inoculated with Kluyveromyces thermotolerans and Saccharomyces cerevisiae. Am. J. Enol. Vitic. 41, 156–159.