The adsorption of wine components by yeasts, yeast lees, and inactivated yeast fractions is of increasing interest in winemaking, for managing fermentations, wine stabilization, and aging processes [1
]. Several studies have reported on the interaction of yeast cells with a variety of wine compounds, including anthocyanins [3
] and flavan 3-ol derivatives [2
], aromatic substances [1
], sulfur products [9
] or undesirable components, such as octanoic and decanoic acids [10
], 4-ethylphenol [11
], geosmin [14
], and some pesticides commonly used in vineyards [15
Currently, there is an increasing interest towards yeast adsorption/removal of ochratoxin A (OTA) [16
]. OTA is a mycotoxin produced as a secondary metabolite by several toxigenic molds belonging to Aspergillus
species. It possesses nephrotoxic, immunosuppressive, teratogenic, and carcinogenic (group 2B) properties [17
]. Since the vintage of 2006, with the adoption of Regulation CE 123/05, the level of OTA in commercial wines cannot exceed 2 μg/L, but many trade agreements usually require lower limits (e.g., 0.5 μg/L) [18
Several researchers studied the removal of OTA by yeasts during alcoholic fermentation [19
]. Several winemaking practices involve a prolonged contact between yeasts or yeast-derived products and wine, thus suggesting that yeast cells could play a significant role in OTA removal also at the end of the fermentation process [28
]. Núñez et al.
] and Petruzzi et al.
] studied the effect of aging on the removal of OTA in a model wine system by Saccharomyces cerevisiae
and a commercial yeast-cell wall preparation. However, in real wine OTA adsorption might be modified by other molecules that could be also adsorbed by yeast cell wall. Therefore, a topic of great concern relies upon the potential interactions between OTA, yeasts, and anthocyanins, being these last compounds largely responsible for the color of red wines [30
]. At present, only some speculations are available in the literature. For example, García-Moruno et al.
] and Caridi et al.
] suggested the existence of competition phenomena between wine polyphenols and OTA for the same binding sites on the surface of the yeast cells. Cecchini et al.
] suggested that yeast mannoproteins, known to react with polyphenols, could interact with OTA, too. Thus, the aim of this paper was to investigate the ability of inactivated S. cerevisiae
strains and a commercial yeast-cell wall preparation to reduce OTA content in Nero di Troia red wine over 84 days of aging. In addition, potential competitive or interlinking phenomena between OTA, yeast cells, and anthocyanins were evaluated through the analysis of anthocyanic compounds adsorbed on yeast cells in wines added with OTA.
Although several physical, chemical, and biological methods are available to control the levels of OTA in musts and wines [32
], parietal adsorption of toxin by yeasts, mainly S. cerevisiae
, is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated products [16
OTA and yeast cell walls could interact due to the chemical traits of both of them. OTA is a complex organic compound, consisting of chlorine-containing dehydroisocoumarin linked through the 7-carboxyl group to 1-β-phenylalanine. Phenol and carboxyl are the main functional groups involved in some different adsorption mechanisms [16
]. S. cerevisiae
cell wall is described in terms of three layers, an outer electron-dense layer, an adjacent less dense layer, and another dense layer bordering the plasma membrane. The yeast wall is composed of a three-dimensional internal skeletal layer of 1,3-β-glucan and 1,6-β-glucan (30%–40% of wall mass) stabilized by hydrogen bonds. Other important components of yeast wall are the mannoproteins (30%–40% of wall mass), which are the most highly-exposed cell-wall molecules, and can form sorption sites. These components are all interconnected by covalent bonds. Mannoproteins are bonded by a 1,6-β-glucan chain with 140 glucose residues to a 1,3-β-glucan chain of approximately 1500 sugar residues [33
The most recent winemaking technologies are focused on the use of inactivated yeast fractions (e.g., inactivated whole yeasts, yeast autolysates, yeast extracts, cell walls, or mannoproteins) to reduce the time required to obtain wines with physico-chemical and sensory characteristics similar to those aged on lees [2
]. To address this important topic, two strains of S. cerevisiae
(the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a commercial yeast cell wall preparation have been tested for their OTA-adsorption ability. Yeast cell walls revealed the highest adsorption values in comparison to thermally-inactivated cells (50 vs
. 43% toxin reduction). Probably, heat treatment caused the inactivation of the metabolism, as well as a possible damage to cell structure. This injury could in turn cause a loss of some cell wall components involved in interactions with OTA and/or to an important alteration of their three-dimensional structure, affecting the accessibility to interaction sites and OTA adsorption within the wall network thickness. Although yeast cell walls removed more OTA than whole yeasts, there is another main trait to keep in mind for the final selection and choice amongst the aging tools, i.e.
, yeasts or yeast cell walls elimination. Removing the yeast cell wall fraction from wine is more difficult than eliminating whole yeasts, which only require filtration through a filter of 0.45-μm pore size, common in the cellars [7
Concerning the removing percentages of OTA found in this research, these values were in line with previous reports. For example, Piotrowska et al.
] added a thermally inactivated biomass of S. cerevisiae
yeast to wines from white grape and blackcurrant juices and decreased after 24 h the content of OTA by ca
. 60%. Similarly, heat-treated yeast cells have been used by Var et al.
] to remove OTA (a maximum of 30.45% within 4 h) from white wine, but in both studies the time of contact between the yeast and the OTA was shorter than in our research.
An important issue to be addressed is if OTA adsorption could compete with the removal of some wine components, namely anthocyanins.
Although numerous studies have clearly proven the interaction of yeast cells with a variety of compounds, the deep understanding of phenomenon is a challenge since it is driven by a complex interaction solute/solvent, solute/surface, surface/solvent physic-chemical interactions, and solvent cohesion. The affinity for the surface (initial adsorption) as well as the maximum adsorbed amount relies upon the number of binding sites, the accessibility to them, a possible conformational rearrangements of the solute when dealing with polymers and lateral interactions between adsorbed species [2
The results of this research suggested that OTA and anthocyanins adsorption were not competitive phenomena, as they probably acted in different ways and impacted on different targets on yeast cell wall. At a molecular level, cell wall mannoproteins play a major role in the adsorption of toxin [16
], although other parietal components could be involved. For example, some authors reported that a mixture of chitin and β-glucan as well as their hydrolysates removed OTA by 64% to 74% [37
]. Similarly, anthocyanins adsorption by yeasts is attributed to cell walls. Morata et al.
] studied the adsorption of anthocyanins by the cell wall of different strains of Saccharomyces
spp. throughout fermentation, and found that some strains showed removal rate two-fold higher than the others. Unfortunately, information concerning anthocyanin interactions with mannoproteins throughout wine aging remains scarce and is missing for β-glucans and chitins.
A different explanation for the not competitive nature of adsorption phenomenon between OTA and anthocyanins could rely upon the possibility of partial intracellular penetration of these compounds into the whole yeasts [7
] and a consequent interaction with the plasma membrane lipids [38
]. This hypothesis has been also suggested by Pradelles et al.
] to explain the adsorption of geosmin by autolysed cells. The passage of anthocyanins through the cell wall to the periplasmic space and their interaction with the plasma membrane could also explain the lower adsorption by whole cells than the commercial yeast cell wall preparation.
We could also suggest that the adsorption of anthocyanins on yeast cells in a complex polyphenolic environment does not rely upon a simple adsorption mechanism [6
]. Mechanisms of adsorption and complexation (with other biopolymers such as proteins and polysaccharides) could interact, with significant effects by hydrophobic attraction and hydrogen links.
The last issue relies upon the effect of yeast cell wall on the qualitative traits of wine. It is well known that the addition of adsorbing tools such as yeast cells to wine causes important reductions in anthocyanin content, leading to color losses [13
]. Thus, the selection of suitable yeasts could also address this trait and avoid the use of strains with high adsorption levels of anthocyanins.
Further investigations are required to elucidate some critical issues, like the effect of the concentration of cell wall material and yeasts; hereby, we focused on a standardized protocol and used the weight as a way to compare the treatments. Another issue that should be addressed is if the preliminary treatment of yeasts or cell walls could affect the adsorption properties towards OTA and anthocyanins.