Aguamiel Fermentacion

1. Introduction

Mead is a traditional honey-derived beverage containing 8 to 18% (v/v) of ethanol, which results from alcoholic fermentation by yeasts of diluted honey. This beverage has progressively gained economic importance (Sroka and Tuszynski, 2007), due to the therapeutic/ nutriceutical properties attributed to honey and by an increasing demand for gourmet products.Mead fermentation is a time-consuming process, oftentaking severalmonths to complete,depending on the type of honey, yeast strain and honey-must composition (Navrátil et al., 2001). Inmost of themead-producing countries, alcoholic fermentation is a result of the growth of indigenousmicroorganisms naturally present in honeys and always surviving on the substrates and equipment used (Ashenafi, 2006; Bahiru et al., 2001). In these cases, alcoholic fermentation is even more unpredictable and very often at the end of fermentation, mead is completely spoiled by contaminating yeasts and bacteria which makes it undrinkable. More recently, selected strains isolated from honey/honey-wine (Pereira et al., 2009; Teramoto et al., 2005) or commercial yeasts starter cultures (Sroka and Tuszynski, 2007; Ukpabi, 2006; Wintersteen et al., 2005) have been used to reduce the risks of contamination. However, despite the use of starter cultures for honey-must inoculation several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast (Pereira et al., 2009). These problems could be due to inappropriate yeast strains that are not suitable for the specific composition/conditions in honey-musts, or the result of several stress conditions: high osmotic stress, lack of essential nutrients such as a deficiency in available nitrogen (Maugenet, 1964; McConnell and Schramm, 1995), low mineral concentration, low pH (Sroka and Tuszynski, 2007) and lowbuffer capacity (Maugenet, 1964). Some of these problems in mead alcoholic fermentation are similar to those still found inwinemaking such as sluggish or stuck fermentations (Ingledew and Kunkee, 1985; Kunkee, 1991; Salmon,1989) aswell as the generation of undesirable by-products such as S-off-flavors (Spiropoulos et al., 2000). Previous research suggested that the major cause of these problems inwines is the limited nitrogen content of some natural grape-juice (Giudici and Kunkee, 1994; Hallinan et al., 1999; Spiropoulos et al., 2000; Spiropoulos and Bisson, 2000; Vos and Gray, 1979). There is a lack of scientific information about honey-must fermentations but it is accepted by mead makers that mead quality improvement includes the development of the proper additive formulation and optimization of fermentation conditions. The numerous research studies conducted in other fermented beverages may be in some extent helpful to control honey-must fermentation. Nevertheless, research is still needed on the physiology and metabolism of Saccharomyces cerevisiae under the particular harsh honey-musts environment. Thus, the aimof this studywas to optimizemead alcoholic fermentation, by modulating honey-must composition, in order to reduce fermentation length and to obtain a consistently higher quality and microbiologically stable product. The first task was to improve the preparation of honey-must before the various supplements have been tested. The amounts of nitrogen tested were based on previously published research (Mendes-Ferreira et al., 2004), which are in agreement with the previous European legislation for grape-must supplementation (300 mg/L supplied as diammonium phosphate or ammonium sulphate) and with current legal limit of 1 g/L. Acidity has extensive and important functions in alcoholic beverages. Organic acids, such as tartaric andmalic acid have important functions for organoleptic characteristics, and stability of these types of beverages (Boulton et al., 1996). Moreover, these acids in equilibrium with their salts act as buffers, maintaining the pH values within an appropriate range. In syntheticmedia the addition of tartaric acid helps to prevent sluggish or stuck fermentation due to its buffering capacity and because it keeps pH within the optimal for yeast development (Torija et al., 2003).Therefore, addition of organic acids was considered in amounts approved by European legislation for wine production, not only for the important organoleptic functions of acidity but also to act as buffer compounds in honey-must fermentation. In parallel, the effects of honey-must composition, and specifically the available nitrogen, on the fermentation performance and formation of aroma compounds were evaluated.

2. Materials and methods

2.1. Yeast strain and culture maintenance conditions

S. cerevisiae UCD522 from the Enology Culture Collection, Department of Viticulture and Enology, University of California, Davis, USA was used in this study. The yeast culturewas routinelymaintained at 4 °C on slants of Yeast Peptone Dextrose agar (YPD), containing per litre: glucose 20 g, peptone 10 g, yeast extract 5 g and agar 20 g. Before use, the culture was transferred to a new slant of YPD for 24 h, at 25 °C.

2.2. Honey

In this study, we used honey derived from plants of the Ericaceae (heather) family (Erica spp.) purchased froma local beekeeper from the north-eastern region of Portugal. The botanical origin of the honey was confirmed by relative frequency excluding pollen from nectar-less plants. Counts were done based on a total of at least 500 pollen grains and pollen-typing according to the methodology previously described

(Valdés et al., 1987). Before honey-must preparation, and to confirm characteristics and satisfactory quality of honeys in accordance to the legal limits established in the Council Directive (2001/110/EC, 2002), the following parameters were analyzed: moisture (%), pH, acidity, hydroxymethylfurfural (HMF), electrical conductivity, ash (%) and sugars according to validated standard methods (AOAC, 1990).

2.3. Preparation of honey-must for fermentation

To obtain an alcoholic beverage with approximately 11% of ethanol, honeywas diluted with natural spring-water obtained fromthemarket (37 g:100 mL) and mixed to homogeneity. Any insoluble solids were removed the mixture by centrifugation (10,000×g for 10 min Sorval centrifuge) to obtain a clarified honey-must. Then the following parameters were determined: °Brix, pH, total acidity and assimilable nitrogen concentration. Based on the results obtained by these determinations, adjustments in nitrogen content and titrable acidity were done in order to optimize yeast performance during honey-must fermentation for mead production. Consequently, the following fermentations were

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