Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration J. Arrizon1,2 and A. Gschaedler1 1 Centro de Investigacio´ n y Asistencia en Tecnologı´a y Disen˜ o del Estado de Jalisco A.C., Guadalajara, Jalisco, Me´ xico 2 Departamento de Ingenierı´a Quı´mica, Centro Universitario de Ciencias Exactas e Ingenierı´as, Universidad de Guadalajara, Guadalajara, Jalisco, Me´ xico Keywords Abstract fermentation rate, nitrogen source, tequila, volatile compounds. Aims: To study the effect of the addition of different nitrogen sources at high sugar concentration in the tequila fermentation process. Correspondence Methods and Results: Fermentations were performed at high sugar concentra- ) J. Arrizon, Centro de Investigacio´ ny tion (170 g l 1) using Agave tequilana Weber blue variety with and without Asistencia en Tecnologı´a y Disen˜ o del Estado added nitrogen from different sources (ammonium sulfate; glutamic acid; a de Jalisco A.C., Normalistas 800, Colonia mixture of ammonium sulfate and amino acids) during the exponential phase Colinas de la Normal, Guadalajara, Jalisco C.P. 44270, Mexico. of growth. All the additions increased the fermentation rate and alcohol effi- E-mail: [email protected] ciency. The level of synthesis of volatile compounds depended on the source added. The concentration of amyl alcohols and isobutanol were decreased while 2006/0105: received 25 January 2006, revised propanol and acetaldehyde concentration increased. 7 July 2006 and accepted 11 July 2006 Conclusions: The most efficient nitrogen sources for fermentation rate were ammonium sulfate and the mixture of ammonium sulfate and amino acids. doi:10.1111/j.1365-2672.2006.03142.x The level of volatile compounds produced depended upon types of nitrogen. The synthesis of some volatile compounds increased while others decreased with nitrogen addition. Significance and Impact of the Study: The addition of nitrogen could be a strategy for improving the fermentation rate and efficiency in the tequila fer- mentation process at high sugar Agave tequilana concentration. Furthermore, the sensory quality of the final product may change because the synthesis of the volatile compounds is modified. (Ceden˜o 1995). The agaves are cooked in autoclaves or Introduction brick ovens. Most of the assimilable nitrogen is degraded ‘Tequila’ is a Mexican alcoholic beverage obtained by dis- by Maillard reactions because of heating (Mancilla-Mar- tilling and rectifying fermented agave juice and is pro- galli and Lopez 2002), making the juice poor in available duced in a territory protected by a guarantee of origin. nitrogen content. Normally, for tequila fermentation, the There are two main types of tequila: tequila 100% agave juice is diluted with water to reach 12–14°Brix ) obtained exclusively from sugars of the Agave tequilana degrees (80–100 g l 1 of agave sugar) after milling, this Weber blue variety and tequila produced using 51% of condition corresponds to industrial agave sugar fermenta- sugars from A. tequilana Weber blue variety and 49% tion (IASF). In some cases, an inorganic nitrogen source sugars from other sources like sugar cane, ‘sugar-loaf’, is added at the beginning of fermentation. It has been molasses, or hydrolysed corn syrup. The process is divi- found that when tequila is produced at high A. tequilana ded into four main phases: cooking, milling, fermenting sugar fermentation (HASF), the addition of a mixture of and distilling. The agave is harvested 8 years after plant- ammonium sulfate and amino acids at the exponential ing and contains an average of 27% reducing sugars phase of growth, caused an increase in the fermentation ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1123–1131 1123 Nitrogen addition in tequila fermentation J. Arrizon and A. Gschaedler rate and alcohol efficiency (Arrizon and Gschaedler line, tryptophan and valine). All of them equivalent to 2002). A similar behaviour has been observed in wine fer- 88Æ36 mg of total assimilable nitrogen. The composition mentations at high sugar concentration with additions of of the mixture of amino acids used was described in a ammonium (Mendes-Ferreira et al. 2004) or ammonium previous work (Arrizon and Gschaedler 2002). and amino acids (Beltran et al. 2005). These additions have an impact on volatile compound production. When Inoculation conditions additions were performed in the early steps of fermenta- tion, a decrease in higher alcohols and an increase in Cells were grown for 12 h at 30°C with shaking ) acetaldehyde and ethyl acetate were observed (Beltran (250 rev min 1) in 500-ml Erlenmeyer flasks with 200 ml ) et al. 2005). In case of tequila, no studies have been per- of must, reaching cell populations of 200 · 106 cells ml 1. formed about the impact of the addition of nitrogen at The must consisted of A. tequilana Weber blue variety juice ) high sugar concentration on volatile compound produc- (60 g l 1) that was filtered and sterilized (121°C, 15 min). tion. In this study, a comparison of the fermentation The reactor was inoculated with 200 ml, which provided ) behaviour at HASF conditions and volatile compound an initial population in the reactor of 20 · 106 cells ml 1. production, was evaluated with the addition of nitrogen in the exponential phase of growth, using nitrogen Fermentation conditions sources of different nature: inorganic nitrogen (ammo- nium sulfate), organic nitrogen (glutamic acid) and a Batch cultures were carried out under semi-anaerobic mixture of inorganic and organic nitrogen (ammonium conditions in 3-l fermentors containing 2 l of must. sulfate and amino acids). Finally, the concentrations of Fermentation was run with constant low stirring ) ethanol, methanol, higher alcohols, ethyl acetate and acet- (200 rev min 1)at35°C. The medium was saturated with aldehyde, produced from fermentations with additional sterilized air before inoculation. The nitrogen addition nitrogen sources were compared with the concentrations from each of the different sources was carried out after ) allowed by the Mexican Tequila standard (NOM-006- 6 h of culture at 170 g l 1 of agave sugar. Each fermenta- SCFI-2005, 2006). tion was run in triplicate to statistically analyse the vola- tile compounds and fermentation kinetics. Materials and methods Monitoring of fermentation Yeast strain Samples were taken every 2 h during the first 16 h of fer- The strain of Saccharomyces cerevisiae (MG) used in this mentation and every 4 h until a total of 72 h of culture study was isolated from the tequila industry and con- to determine yeast biomass and the concentrations of served in our laboratory collection (Centro de Investiga- sugar, ethanol and volatile compounds. cio´n y Asistencia en Tecnologı´a y Disen˜o del Estado de Jalisco A.C.). Analysis Biomass Culture media Yeast biomass was determined by measuring dry weight. The must used is an A. tequilana Weber blue variety The cellular dry weight was obtained by harvesting the juice. It was filtered, sterilized (121°C, 15 min), and dilu- cells from 12 ml of the culture medium by centrifugation ted with distilled water to reach sugar concentrations of (3000 g,20°C, 20 min), rinsed with the same amount of ) 170 and 80 g l 1 (high and normal industrial sugar con- distilled water, and desiccated at 108°C until constant ) centration respectively). In all cases, 1 g l 1 of ammonium weight was obtained. sulfate was added at the beginning of fermentation. Nitrogen addition in the exponential phase of growth Reducing sugars (6 h of fermentation) was performed at high sugar A. The reducing sugar concentration in the medium was ) tequilana concentration (170 g l 1) and was compared determined using 3,5-dinitrosalicylic acid reagent (Miller with fermentations without nitrogen addition (170 and 1959). ) 80 g l 1). The composition of the nitrogen sources added ) ) was: 416Æ55 mg l 1 of ammonium sulfate, 928Æ60 mg l 1 Ethanol ) of glutamic acid and 437Æ12 mg l 1 of a mixture of Samples were first distilled, and the resultant ethanol con- ammonium sulfate and amino acids (alanine, arginine, as- centration was measured using a dichromate reagent (Bo- partate, glutamate, glutamine, leucine, phenylalanine, pro- ehringer and Jacob 1964). ª 2006 The Authors 1124 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1123–1131 J. Arrizon and A. Gschaedler Nitrogen addition in tequila fermentation Volatile compounds Statistical analysis The fermentation samples were distilled and the analysis of volatile compounds was carried out in a Hewlett-Pack- Five treatments were applied. Two treatments which con- ard 5890 gas chromatograph (Palo Alto, CA, USA) with a sist of IASF and HASF without nitrogen addition and flame ionization detector equipped with an HP-Innowax three treatments at HASF conditions with different nitro- PEG column (60 m · 320 lm2). The column was condi- gen sources added (ammonium sulfate; glutamic acid; a tioned at 50°C which was maintained for 6 min, then mixture of ammonium sulfate and amino acids). The var- increased at the rate of 10°C per min to 160°C which was iables statistically evaluated were: fermentation kinetics ) followed by a more rapid increase of 20°C min 1 until it and the level of volatile compounds produced. anova reached 220°C. Injector and detector temperature was tests were carried out with the Statgraphics (Manugistics maintained at 250°C. Sample size was 1 ll and the carrier Inc., Rockville, MD, USA) software. gas was nitrogen. Results Parameter calculation Alcohol efficiency was calculated from the ratio of the Effect of IASF and HASF conditions without nitrogen average ethanol produced at the end of fermentation and addition on production of volatile compounds theoretical ethanol production from the biochemical con- version of the sugar consumed.