Latin American applied research
versión ISSN 0327-0793
Lat. Am. appl. res. v.33 n.3 Bahía Blanca jul./sept. 2003
Influence of non-sacharomyces yeast growth on the metabolism of nitrogenous compounds in lactic acid bacteria from wine
M. E. Farías1,2, P. A. Aredes Fernandez1, O. A. Sosa1 and M. C. Manca de Nadra1,2
1 Centro de Referencia para Lactobacilos (CERELA), and Facultad de Bioquímica, Química y Farmacia,
2 Universidad Nacional de Tucumán, 4000 Tucumán, Argentina.
Abstract ¾ A comparative study of the influence of Hanseniaspora uvarum metabolism on the growth and physiology of two lactic acid bacteria involved in vinification: Lactobacillus hilgardii 5w and Oenococcus oeni X2L was carried out. At different yeast growth times in grape juice medium, fermented broth was inoculated with L. hilgardii or O. oeni and incubated for 48 h at 30oC. When O. oeni grown in the culture supernatant after 0, 3 and 8 hours of yeast growth (M1, M2 and M3 respectively), the growth rate and final biomass were not modified. With L. hilgardii a decrease of both parameters was observed. After 12, 24 and 34 h of yeast growth (M4, M5 and M6 respectively), the results for both microorganisms were similar: the growth rate increased and the final cellular mass decreased. When O. oeni was inoculated in M1, M2 and M3 media, an initial decrease of proteins directly related to amino acids production was observed. With L. hilgardii the amino acid and proteins concentrations diminished. In M4, M5 and M6 media O. oeni showed that the protein concentrations remained constant and the amino acid concentrations diminished. The results obtained with L. hilgardii were different with a higher protein consumption rate without significative amino acids modification. The amino acids increment in the first hours of O. oeni growth could regulate the protease production achieving the equilibrium between amino acids production and utilization. The high proteolytic activity in L. hilgardii could be responsible of the amino acids increase despite its consumption.
Keywords ¾ Lactic Acid Bacteria. Non-Saccharomyces Yeast. Wine.
Alcoholic and malolactic fermentations are the main processes that take place in wine winemaking. Yeasts transform sugars to ethanol, but at the same time play an important role in organic acid production and volatile end products in alcoholic beverages. The malolactic fermentation remains an imperfectly controlled process, since many nutritional and physic-chemical factors affect the growth and metabolism of lactic acid bacteria. Some of them depend on the yeast strain used, providing different amounts of amino acids, peptides and vitamins available acting as growth factors for lactic acid development, or on the presence of metabolic products which act as inhibitors. Several studies reviewed the importance of nitrogen from source, to transport, metabolic fate and influence on the fermentation carried out by the yeast Saccharomyces cerevisiae. Unfortunately, there is very little information about the nitrogen metabolism of non-Saccharomyces species of wine yeast. Recent quantitative studies on winemaking ecology showed that non-Saccharomyces species (especially Hanseniaspora uvarum (Kloeckera apiculata) and Candida stellata) survive during alcoholic fermentation at significant levels for longer periods than previously thought and grew to maximum populations of 106-107 cfu ml-1 (Fleet et al., 1984; Heard and Fleet, 1985; Pardo et al., 1989). Such growth was considered to be quantitatively significant and likely to influence the chemical composition of the wine.
We carried out a comparative study of the influence of Hanseniaspora uvarum metabolism on the growth and nitrogen physiology of two lactic acid bacteria involved in vinification. Lactobacillus hilgardii 5w, a detrimental microorganism, with negative ecological effect like hydrogen peroxide (Rodriguez and Manca de Nadra. 1995a, b) and histamine production (Farías et al., 1995; Farías et al., 1996), and Oenococcus oeni X2L, a beneficial microorganism with optimal technological properties.
II. MATERIALS AND METHODS
A. Microorganisms and Culture Conditions
Lactobacillus hilgardii 5w, Oenococcus oeni X2L and Hanseniaspora uvarum ca12 (Kloeckera apiculata) were isolated from Argentinean wines. Yeast and lactic acid bacteria were grown in basal medium containing per liter: yeast extract, 10 g; glucose, 5 g; tween 80, 1 ml and grape juice, 57 ml. When indicated, 1% KNO3 was added to the culture medium. The pH was adjusted to pH 4.8 with 0.1 M HCl before sterilization by autoclaving for 15 min at 121oC.
B. Sequential Cultures in Liquid Medium.
A stoppered 250-ml flask, containing 200 ml of basal medium, was inoculated with a yeast strain and incubated at 30oC. At different times (0, 3, 8, 12 , 24 and 34 h), 20 ml of fermented broth was collected and the number of viable cell counts determined. The broth was filtered aseptically and dispensed into 75-ml flask to which 10% of basal medium culture of the Lactobacillus hilgardii 5w or Oenococcus oeni X2L were added and incubated for 3 d at 30oC. 5ml-aliquots of culture were taken at different time intervals and the number of viable cell counts was determined. The yeast and bacteria culture supernatants were utilized for analytical measurements.
C. Analytical Measurements
Proteolytic activity and amino acids determination: At different times of growth, cells were centrifuged and the supernatant was used for proteolytic activities determinations. Using autoclaved grape juice as substrate, amino acids were determined by the modified Cd-ninhydrin method (Doi et al.,1981). After 1 h of incubation at 30oC, the reaction was stopped by the addition of 0.65 ml of 24% trichloroacetic acid (TCA). A sample of TCA supernatants (20-100 µl, depending on the concentration of amino acids) was added with 1.7 ml Cd-ninhydrin reagent (0.8 g ninhydrin were dissolved in a mixture of 80 ml 99.5% ethanol and 10 ml acetic acid, followed by the addition of 1 g CdCl2 dissolved in 1 ml of distilled water). The mixture was heated at 84oC for 5 min, then cooled and the absorbance at 507 nm determined. The absorbance for each sample was adjusted to the value produced by 1 mmol l-1 of leucine solution. The molar absorption coefficient of leucine & M(M-1 cm-1) was obtained from: & M= absorbance (1 mol l-1 sample) x (total vol/sample vol)x 103.
The amino acid concentration was determined as follow:
Protein determination: The proteins of the samples were determined by the reaction with Coomassie Brilliant Blue G-250 (Bradford, 1976). Calibration was carried out using bovine serum albumin (BSA) (Sigma Chemical Company).
III. RESULTS AND DISCUSSION
Figure 1 shows Hanseniaspora uvarum ca12, growing in basal medium. The end of exponential growth phase was achieved in 12 h incubation at 30oC. After 34 h growth, a decrease in the cell number was observed (from 3.1 x 107 cfu ml-1 in 24 h to 5.0 x 106 cfu ml-1 in 34 h incubation).
Fig. 1. Growth of Hanseniaspora uvarum in basal medium. Narrows indicate time to which the supernatant was taken for bacteria sequential inoculation. (0 h: M1; 3 h: M2; 8 h: M3; 12 h: M4; 24 h: M5 and 34 h: M6).
Table 1 shows the growth kinetic parameters obtained when Lactobacillus hilgardii 5w and Oenococcus oeni growth in the filtered (0.2 m m porous size membrane) basal culture medium, after Hanseniaspora uvarum growth at 30oC. When Oenococcus oeni was grown in the culture supernatant after 0, 3 and 8 hours of yeast growth (M1, M2 and M3), the growth rate and final biomass were similar (approximately 0.035 h-1 and 3 x 108 cfu ml-1, respectively). The growth rate of Lactobacillus hilgardii in the basal medium without yeast growth was 0.11 h-1, while the growth rate after 8 h of yeast growth was 0.055 h-1. As the time of yeast growth was increased from 0 to 8 h, there was a corresponding decrease in the bacterium growth from 2.5 x 107 to 1.25 x 107 cfu ml-1. After 12, 24 and 34 h of yeast growth (M4, M5 and M6), an increase of the growth rate with diminution of the final cellular mass of both microorganisms was observed.
When Oenococcus oeni was inoculated in M1, M2 and M3 media, an initial decrease of proteins directly related to amino acids production was observed (Fig. 2A). 6 et al. (1996); Manca de Nadra et al. (1999) and Farías and Manca de Nadra (2000) characterized and purified extracellular proteases produced by Oenococcus oeni isolated from wine. Protein consumption during the first 12 h growth could be related to time of protease production by Oenococcus oeni. Its activity is observed up to 24 h growth as showed by amino acids increase. Approximately 14 mmol l-1 amino acids were produced by O. oeni after 48 h growth in the different media. The protein consumption rates were 0.07, 0.05 and 0.09 m g l-1 h-1 in M1, M2 and M3 media respectively.
Table 1. Kinetic of Lactobacillus hilgardii growth in media obtained after Hanseniaspora uvarum growth.
a Culture media after 0 h (M1), 3 h (M2), 8 h (M3), 12 h (M4), 24 h (M5) and 34 h (M6) Hanseniaspora uvarum growth at 30oC.
Values are means of three independent experiments ± Standard Deviation.
Fig. 2. Oenococcus oeni (A) and Lactobacillus hilgardii (B) growth in the media obtained after 0, 3 and 8 hours (M1, M2 and M3 respectively) of yeast growth. (£ ) Protein and (¢ ) amino acid concentrations during 0 h (a, e), 12 h (b, f), 24 h (c, g) and 48 h (d, h) bacteria growth.
With Lactobacillus hilgardii the amino acid and protein concentrations diminished (Fig. 2B). Approximately 5 mmol l-1 amino acids were consumed after 48 h growth, independently of the media. Protein consumption rates were 0.009, 0.005 and 0.01 m g l-1 h-1 in M1, M2 and M3 respectively. L. hilgardii showed proteolytic activity from 24 h.
After 12, 24 and 34 h yeast growth (M4, M5 and M6 media), Oenococcus oeni showed that the protein concentrations remained constant and the amino acid concentrations diminished (Fig. 3A). The amino acids increment in the first hours of Oenococcus oeni growth could regulate the protease production achieving the equilibrium between amino acids production and utilization.
The results obtained with Lactobacillus hilgardii were different showing a higher protein consumption rate (0.03, 0.04 and 0.03 m g l-1 h-1 in M4, M5 and M6 media respectively) without significative modification of amino acids concentration (1 mmol l-1) (Fig. 3B). In the same conditions, an increase in ammonia concentration from amino acids was observed (unpublished data). From these results it is possible to infer that the amount of liberated amino acids by protease action is higher than its utilization.
Fig. 3. Oenococcus oeni (A) and Lactobacillus hilgardii (B) growth in the media obtained after 12, 24 and 34 hours (M4, M5 and M6 respectively) of yeast growth. (£ ) Protein and (¢ ) amino acid concentrations during 0 h (a, e), 12 h (b, f), 24 h (c, g) and 48 h (d, h) bacteria growth.
We can conclude that the modifications produced by Hanseniaspora uvarum growth in the media affect both growth and nitrogen metabolism of Oenococcus oeni and Lactobacillus hilgardii. The different response of the lactic acid bacteria could be related to the different nutritional requirements and enzymatic regulatory mechanism of each microorganism.
This work was supported by grants from Fundación Antorchas, Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Argentina.
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