produccion of tequila from agave historical influences and contemporary processes

Production of tequila from agave 225

Chapter 15

Production of tequila from agave: historical influencesand contemporary processes

M. Cedeño Cruz and J. Alvarez-JacobsTequila Herradura, S.A. de C.V. Ex-Hda San Jose del Refugio, Amatitán, Jalisco,México

Introduction: Agave beverage alcoholproducts

Tequila is classically associated with Mexico,particularly with Jalisco, a state located in the westof the country. This beverage is obtained bydistillation of fermented juice from only the agaveplant (Agave tequilana Weber var. Azul ) if 100%agave tequila is required. Fermentation is carriedout by inoculated strains of Saccharomycescerevisiae or in some cases by a spontaneousprocess. Up to 49% (w/v) sugars may comefrom a source other than agave, usually sugarcane or corn syrup, if 100% agave tequila is notrequired. This tequila could not be labeled as100% agave tequila.

Different kinds of tequila are produced in 50registered companies in Jalisco. The tequilaproducts made by these companies differ mainlyin proportions of agave used, productionprocesses, microorganisms used in thefermentation, distillation equipment used and thematuration and aging times. The product knownas �silver� or white tequila must be distilled to aconcentration not exceeding 55% alcohol involume (v/v) and not less than 38% (v/v) from afermented wort containing not less than 51%sugars from the agave plant. �Gold� tequila is the

white product to which caramel color (generally)has been added. Rested tequila (Reposado) andaged tequila (Añejo) are white tequila maturedin wood containers, preferably oak casks, for atleast 2 and 12 months, respectively, accordingto present regulations. In practice, the agingperiod is normally longer and depends on thecharacteristics each company wants to give tothe final product. The products known as 100%agave, which are becoming more popular, couldbe white, rested or aged and are distilled fromfermented wort with only agave as a source ofsugars. The labels of these products must indicatethat they were obtained using only agave andunder Mexican government supervision.

Tequila is differentiated from the beverageknown as �mezcal� by the type of agave used inits elaboration. Mezcal is made from Agavepotatorum, which grows in the state of Oaxaca.Most mezcal producers use a rudimentaryfermentation and distillation process (Sánchez,1991). There is no technical reason for, or anyimprovement in the organoleptic characteristicsfrom, the worm inside the bottles of some mezcalbrands. The worm is primarily a commercial ploy.Worms are grown in agave plants and introducedmanually in the bottling line.

226 M. Cedeño Cruz and J. Alvarez-Jacobs

�Pulque� is another beverage obtained byfermentation of the juice obtained from severalspecies of agave, A. atrovirens and A. salmianaamong others, by a complex succession of yeastand bacteria that produce ethanol, a diversity ofchemical compounds, and some polymers thatgive a sticky consistency to the final product(Rzedowski, 1978; Sánchez Marroquin andHope, 1953). Pulque is sometimes mixed withfruits or vegetables, but has poor stability as it isneither distilled nor pasteurized.

Agave plants still serve as food in some statesof México; and other fermented regionalbeverages are produced (e.g., �Sotol� in the stateof Chihuahua and �Bacanora� in the state ofSonora), but only tequila and more recentlymezcal have reached international recognition.Another difference between tequila and mezcaland all other regional drinks is that both aresubject to an official standard that for tequila isNOM006-SCFI-1993 (Secofi, 1993), andproduction is supervised by the Mexicangovernment.

Origin and history of tequila

The word �tequila� is believed to originate fromthe tribe of ticuilas who long ago inhabited thehillside of a volcano bearing the same namelocated near the city of Tequila. Another possibleorigin is the Nahuatl word tequitl, which meanswork or employment, and the word tlan, whichmeans place. Therefore �tequila� would meanplace in which labor or work is done.

The most ancient information revealing theexistence of agave and its different uses is fromthe era before the Spaniards in several codicespreserved to the present time (a codex, fromthe Latin codex meaning board or writing tablet,is a manuscript volume, especially of a classicwork or scripture). The most important is theTonalmatlnahuatl codex, which notes that certaintribes had learned to cook agave plants and usedthem as food and to compensate for the lack ofwater in desert lands. Also, these tribesdiscovered that cooked agave when soaked in

water would ferment, producing a veryappreciated beverage. In fact, this primitive andrudimentary method was used for centuries toproduce beverages from agave, considered asacred plant possessing divine properties. Inother codices, such as Nutall, Laud, Borgia andFlorentine, there are many references to usesof the agave plant for soap manufacture, asource of fiber, footwear, medicine and sewingneedles as well as thread, paper and rope. Infact, Indians could distinguish the different speciesof agave by color, size, stem, leaf width and thedifferent uses given each plant (Muria, 1990).The great religious importance of agave wasapparent in those codices, as only warriors andpriests used fermented drinks in ritual cere-monies.

In prehispanic México the general name forall species of agave (or mezcal as it is also known)was Metl, which is a representation of thegoddess Mayahuel. The alcoholic drink producedwas called Iztac octli (white wine). The firstSpaniards to arrive in México referred to theplant as �maguey�, a name used for an identicalplant they had seen in the Caribbean Islands,where they first encountered new world plantsand animal life (Bottorff, 1971). It was not untilarrival of the Spaniards, who brought knowledgeof distillation techniques, that tequila took itspresent form (Goncalves, 1956).

There are only two main regions for tequilaproduction in México. The oldest, the Tequila-Amatitán region that comprises the Amatitánvillage, developed at the end of the seventeenthcentury. The second region, the Jalisco High-lands, appeared in the last decade of the 19thcentury (Luna, 1991). The first tequila productionprocess with a commercial purpose wasestablished in the city of Tequila around the endof the 18th century. The main consumers werein the mining zones located in the state of Jalisco.The Spaniards tried to suppress consump-tionof tequila in order to reduce competition forbrandy and other wines imported from Spainwith a decree signed by Carlos III forbidding itssale and production under the pretext that itsconsumption was the cause of several illnesses.


The results were negative. The governor of theregion later issued a decree imposing a tax ontequila in order to enrich the royal coffers, thuspermitting its sale in all of New Spain. By the endof the 19th century, expansion of the tequilaindustry, helped by the railway, was evident.However it was not until the first casks wereexported to the US that tequila was knownbeyond México�s borders.

The Agave plant

According to Granados (1985), the genus Agave,which means �noble� in Greek, was defined byLinneaus in 1753 when he described the plantA. americana as the first agave species knownto science. Agave plants, which are oftenconfused with cacti, belong to the familyAgavaceae and are succulent plants with spirally-arranged leaves forming a rosette. Some havedefinite trunks, but more often they are nearlystemless. The leaves are bluish green in color,over 1 m long in mature plants, and end in asharpened brown thorn. As Backman (1944)pointed out, the widespread distribution of some300 species, combined with the fact that theplants require approximately 8-12 years to matureand hybridize very easily, make the taxonomicand phylogenetic study of the genus Agaveextremely complicated and very difficult. Thefamily Agavaceae includes 20 genera and nearly300 species. Of these, around 200 are found inMéxico.

Several agave species are important from aneconomic point of view. Fiber derived from A.foucroydes, grown in the state of Yucatán, isknown worldwide for its use in producing ropesand carpet. More recently, the pulp remainingafter removal of the fiber has found use in animalfeed. A. salmiana and A. atrovirens are valuedfor pulque production and A. potatorum formezcal production. Finally, A. tequilana Webervar. Azul, named around 1900 by the Germanbotanist Weber (Diguet, 1902), is used toproduce tequila.

Cultivation and harvest

The blue agave, as it is known, is the only speciesout of hundreds of Agavaceae with theappropriate characteristics for tequila production.These include a high inulin concentration, lowfiber content and the chemical compoundspresent in the plant that contribute to the finaltaste and flavor of tequila to give the beverageits particular character. Some have attempted toproduce tequila in other states with other agavespecies, but without success. The blue agave iscultivated in the state of Jalisco in the two regionswith the right climate and soil composition forits growth, namely the Highlands and the Tequila-Amatitán region. The temperature conditions forgood agave yields are a minimum of 30C, anoptimum of 260C and maximum of 47°C. Soilshould be fertile but not very deep, 30 to 40 cm.Good drainage is required to avoid effects offlooding, which are very harmful fordevelopment of the agave. The plants must beplanted at 800 to 1700 m above sea level wherethe annual rainfall is about 800 to 900 mm. Thecorrect planting time is immediately before therainy season, from June to September, so thatthe plants do not suffer from water stress duringthe first year of growth.

Propagation is accomplished by thevegetative route in agave. Sexual reproductionvia seeds is not usual. Asexual bulbils develop inthe inflorescence at the base of the flowers,producing small plants that after some timedetach themselves from the floral peduncle andfall to the soil where they root. Another modeof asexual propagation is by suckers, which area characteristic type of lateral bud or branchdeveloping at the base of the main stem. Plantsdeveloping near each mother plant areseparated at the age of 3-4 years. These babyplants are called �first-class seed� because theyare better and healthier (Sánchez, 1991). Inpractice, people use the word �seed� to refer tosuch young plants, but from a botanical point ofview these are rhizome shoots or suckers(Valenzuela, 1992). Plant cell culture is used


experimentally by some agave producers, but isnot being used commercially due to unavailabilityof trained technicians and laboratory facilities tosmall agave producers. Some developments arebeing carried out to improve agave plants, or toobtain plants resistant to pests; and these newplants will be ready in the next two years (CIATE,1993).

Land for agave cultivation must be clearedand deep-ploughed, sometimes twice. Agave isplanted approximately 2-4 m apart in straight linescalled ruts. Sowing is done by hand in holes 15cm in depth. Plant density is around 2000-4000plants per hectare, depending on the plantationsystem used; and yields can be between 30,000and 200,000 kg/ha, assuming that the weight ofa harvested plant varies from 15 to 50 kg. Thisvariation is caused by differences in soilconditions, quality of plants sown, rainfall, pestsand fertilization. Sometimes agave is sownintercalated with nitrogen-fixing crops such aspeanuts, beans, chickpeas or soybeans. After theagave has been in the soil for a year, visualinspection is carried out to replace sick or deadplants with new ones. This operation is called re-seeding. The percentage of dead plants dependson soil and plant characteristics, but is generallyfrom 8 to 15% (Pérez, 1990).

Agaves regularly host borer insects that livein the stems, leaves and fruits during the larvalstage. These include butterflies of the familyMegathymidae and moths of the family Prod-oxidae. Also, the fungi Diplodia theo-bromae andColletotrichum agavae may cause seriousdamage to agave leaves (Halffter, 1975; Agri-cultural Research Service, 1972).

Ehrler (1967) discovered that unlike stomatain most plants, agave stomata close during theday and open by night. This prevents loss ofwater through transpiration during hot daylighthours, although it results in a hotter leaf surfacethan most plants could tolerate. The thick cuticleoverlying the epidermis, which is quite evidentin tequila agave, apparently prevents damage tothe leaf from high temperature. This waxy cuticleproduces turbidity in tequila because it dissolvesin the distillation step and produces a haze in

the final product when it is diluted or cooled.One way to avoid this is to treat tequila withactivated charcoal and to filter it through purecellulose filter pads. This, unfortunately, resultsin the loss of some aromas.

Agave fertilization is based on soilcomposition, plant age, and the type of chemicalcompound used. The normal procedure is to useurea as a nitrogen source in amounts of 30-70 gper plant added directly into the soil. In someareas, phosphorus and potassium fertilization isalso required. As some agave regions are alsoinvolved in cattle, swine, and chicken breeding,manure is sometimes employed for fertilization(GEA, 1992).

The average maturity time for agave is 10years. Every year following planting, fields arecultivated to loosen the soil and weed and pestcontrol is carried out. Each plant maturesindividually. Harvest begins at eight years. Theleaves are cut from the base and left in the fieldto recycle nutrients. The harvested plants freeof leaves look like large pineapples and weighfrom 20 to 90 kg. They are transported to thedistillery. Only the better plants, meaning thoseof good size and high inulin content (measuredas reducing sugars) are harvested; but on the12th year, all plants remaining (the weakestplants) are cut. These are called �drag� and aregenerally discarded. Agave composition variesseasonally, but an average would be (wet basis)27% (w/w) reducing sugars, a juice content of0.572 ml/g and a pH of 5.2.

Tequila production

Tequilas differ greatly depending on agave qualityand origin (Highlands or Tequila-Amatitánregions). The production process also stronglyinfluences quality of the final product. Somedistilleries still employ rudimentary productionmethods, just as they did several decades ago(Pla and Tapia, 1990). Most companies, however,employ technological advances that improveprocess efficiency and consistency; and somehave implemented a ISO-9000 standard. What-


ever processes are used, tequila manufacturecomprises four main steps: cooking, milling,fermentation and distillation.

Processing harvesting agave

Agave is transported from the fields to thefactories as soon as possible to avoid weightlosses, because today most distilleries pay byweight and not by inulin content. The heads areunloaded from the truck in the receiving area ofthe factory and must be protected from the sunand rain in order to avoid withering and fungalgrowth. Although the agave has already beeninspected during growth and at harvest, it isexamined again to reject visually unacceptableplants or those damaged by pests. At this point,a representative sample of agave is taken forlaboratory analysis. Modified AOAC (1990)procedures are used to determine reducingsugar content (after acid hydrolysis of inulin)along with pH, moisture, dry weight, juice andash content.

Agave heads usually weigh between 20 and60 kg, although some can reach 100 kg. Theyare cut to sizes that facilitate uniform cookingand handling. Different agave cutting systemsexist, but the use of axes and a specialized toolcalled �coa� are the most popular. The heads arecut in halves or quarters, depending on theweight, and the pieces are arranged manually inan oven or autoclave. Band saws can also beused to cut the agave heads. Band saws are fasterand less labor-intensive than the manualprocedure, but the belts break frequently becauseof the resinous consistency of agave. Somefactories tear uncooked agave first with a knifeand place the resulting pieces mixed with waterinto autoclaves to be cooked.

Other raw materials for tequila production

When producing 100% agave tequila, the onlysource of carbohydrate is the inulin hydrolyzedfrom agave in the cooking step. For other kinds

of tequila the law permits the use of other sugarsin amounts of up to 49% by weight in wortformulation. There are no legal specificationsregarding the type of adjunct sugar sources tobe used in tequila manufacture; and theoreticallyany kind of fermentable sugar can be used forthe formulation of wort. In practice and from aneconomical point of view, only cane sugar,Piloncillo, cane molasses and acid- or enzyme-hydrolyzed corn syrup are employed. Canesugar is received in 50 kg bags and stored in adry, cool place for subsequent utilization.Piloncillo consists of brown cones of crystallizedcomplete cane juice, sometimes individuallywrapped in corn or cane leaves and packed insacks. Cane molasses is also used but it is difficultto handle and there is risk of spoilage over along storage period. Acid- or enzyme-hydrolyzed corn syrup may be used to formulatethe wort. All sugars used in wort formulation areroutinely analyzed by measuring solids contentand reducing and fermentable sugar content.

The cooking step: hydrolysis of inulin

Cooking the agave serves three purposes. First,the low pH (4.5) together with the hightemperature hydrolyze inulin and other comp-onents of the plant. The correct composition ofthese compounds is still unknown. In addition,cooked agave has a soft consistency thatfacilitates the milling operation.

In the pre-Hispanic era, agave cooking wascarried out in holes filled with stones heatedusing wood for fuel. The stones retained the heatfor the time needed to cook the agave.Nowadays some distillers have replaced thestuffed stone holes with brick ovens and heatingis conducted by steam injection after the cut,raw agave has been introduced into the oven.Oven cooking is slow, and steam injection lastsaround 36-48 hrs to obtain temperatures of 1000C. After that period, the steam is shut off andthe agave is left in the oven for a further twodays to complete the cooking process. Duringthis step, a sweet liquid called �cooking honey�


is collected and used later as a source of freesugars, mainly fructose. Also during this stepsome of the sugars are caramelized; and someof the compounds that contribute significantlyto the aroma and flavor in wort formulation aredue to its high content of fermentable sugars(>10% w/v). Finally, the oven door is opened toallow the cooked agave to cool. The agave isthen ready for milling.

In most distilleries brick ovens have beenreplaced by steel autoclaves. Autoclaves havesuperior efficiency and allow good pressure andtemperature control, enabling a homogeneousand economic cooking. In a typical autoclavecooking operation, steam is injected for 1 hr sothat the condensed steam washes the agave. Thiscondensed liquid is called �bitter honey� and isdiscarded because it contains waxes from theagave cuticle and has a low sugar content (<1 %w/w). Steam is injected for an additional 6 hrs toobtain a pressure of 1.2 kg/cm2 and a temp-erature of 121 °C. At the end of that time theagave remains in the autoclave for another 6 hrswithout additional steam, cooking slowly in theremaining heat. This step produces a syrup witha high sugar concentration (>10% by weight)that is later used to formulate the initial wort. Tocalculate the yield and efficiency of this step, theamount of cooking honey and its reducing sugarcontent as well as the cooked agave aremeasured.

The main difference between autoclaved andoven-baked agave is that careful control ofcooking time, temperature and steam pressuremust be maintained in autoclaves to preventovercooking or burning the agave. Overcookinggives a smoky taste to the tequila, increases theconcentration of furfural in the final product andreduces ethanol yield due to the caramelizationof some of the fermentable agave sugars. This iswhy some factories with both cooking systemsreserve the ovens for their better-qualityproducts. Although it is easier to obtain well-cooked agave in an oven than in an autoclave,there is no difference in terms of flavor andfermentability between agave cooked inautoclaves or in ovens if both are correctlycontrolled.

Extraction of agave juice: milling

Milling has gone through three historical stages.In ancient days cooked agave was crushed withwood or steel mallets to extract the juice. Later,a rudimentary mill consisting of a large circularstone 1.3 m in diameter and 50 cm thick wasused. Driven by animals, the stone turned in acircular pit containing cooked agave andextracted the juice. The resulting juice wascollected by hand in wood basins and carried tofermentation tanks. By the 1950s modernsystems were implemented in which cookedagave was passed through a cutter to beshredded (except in factories that did thisoperation before cooking); and with a comb-ination of milling and water extraction, sugarswere extracted. The mills used for agave aresimilar to those used in the sugarcane industrybut are smaller in size (normally 50 cm wide).This system is still employed in most distilleries.

Juice obtained in milling is mixed with thesyrup obtained in the cooking step and with asolution of sugars, normally from sugarcane (ifthe tequila to be produced is not 100% agave),and finally pumped into a fermenter. Althoughthe amount of sugar employed as an adjunct isregulated by law and must be less than 49% byweight at the beginning of the fermentation, eachfactory has its own formulation.

The milling step generates a by-product calledbagasse, which represents about 40% of the totalweight of the milled agave on a wet weight basis.Bagasse composition (dry weight basis) is 43%cellulose, 19% hemicellulose, 15% lignin, 3%total nitrogen, 1% pectin, 10% residual sugarsand 9% other compounds. The bagasse, mixedwith clay, is used to make bricks; but it is also thesubject of research to find alternative uses.Examples are use of bagasse as an animal feedor as a substrate on which to grow edible fungi.Attempts are also underway to recover itscomponents (cellulose, hemicellulose andpectin) using high-efficiency thermochemicalreactors (Alonso et al., 1993), to obtain furfurals,make particle board, or enzymes (cellulase andpectinase). Many of these projects are at the


laboratory stage, and there is not enoughinformation to evaluate feasibility.

In milling, as in all steps of the tequila process,a sugar balance is computed to determine theyield. If the yield decreases, the extractionpressure in the mill and the water/agave ratioare increased to improve efficiency.

Fermentation

Wort formulation

To produce 100% agave tequila, only agave maybe used and the initial sugar concentration rangesfrom 4 to 10% w/v, depending on the amountof water added in milling. When other sugarsare employed, they are previously dissolved andmixed with agave juice to obtain an initial sugarconcentration of 8-16%, depending on sugartolerance of the yeast strain. Wort formulationin most of the distilleries is based solely onprevious experience. A few distilleries base wortformulation on composition of raw materials andnutritional requirements for yeast growth andfermentation. In these distilleries responsesurface methodology is the preferred methodto optimize nutrient concentrations, using ferm-entation efficiencies and the taste of the resultingtequila as responses (Montgomery, 1984). Tocomplement nutritional deficiencies of agavejuice and sugars employed in the growth andfermentation steps, urea, ammonium sulfate,ammonium phosphate or magnesium sulfatecould be added. Because the pH of the agavejuice is around 4.5, there is no need foradjustment and the same wort composition isused for both inoculum growth and fermentation.

Yeasts

Some companies do not inoculate a specificstrain of S. cerevisiae and instead allow naturalfermentation to proceed. Others inoculate thewort with fresh packages of baker�s yeast or acommercial dried yeast to obtain initial

populations of 20-50 x 106 cells/ml. The driedyeasts were originally prepared for wine, beer,whisky or bread production; and sometimes thequality of the tequila obtained using these yeastsis not satisfactory, with large variations in flavorand aroma. To achieve high yields and maintaina constant quality in their tequila, somecompanies have been using yeast strains isolatedfrom a natural fermentation of cooked agavejuice. Nutrients are added; along with specialconditions such as a high sugar concentrationor temperature. These isolated and selectedyeast strains have been deposited in nationalmicrobial culture collections, the most importantbeing the Biotechnology and BioengineeringDepartment Culture Collection of CINVESTAV-IPN, located In México City.

Inoculum growth

When an inoculum is used, it is grown in thelaboratory from a pure culture of a strain of S.cerevisiae maintained on agar slants in lyophilizedform or frozen in liquid nitrogen. All laboratorypropagation is carried out under asepticconditions using a culture medium with the sameingredients used in the normal process butenriched to promote cellular growth. Theinoculum is scaled-up with continuous aerationto produce enough volume to inoculatefermentation tanks at 10% of the final volume.Populations of 200-300 x 106 cells/ml are norm-ally achieved. Strict cleanliness is maintained inthis step as bacterial contamination is highlyundesirable. When contamination is detected,antibiotics or ammonium bifluoride are used asantimicrobial agents. Once an inoculum is grown,it is maintained by mixing 10% of the volume ofan active culture with fresh agave juice andnutrients. Although inoculation with commercialyeast greatly improves yield and turnover time,some companies prefer a more complex (interms of the microbial diversity) fermentation.While yields might be lower and turnover timehigher, the range of microorganism producesmore compounds contributing to a more


flavored tequila. It is also important to recognizethat a change in taste and flavor could negativelyaffect the market for a particular brand of tequila.

Fermentation of agave wort

Once a wort is formulated with the requirednutrients and temperature is around 30 oC, it maybe inoculated with 5 to 10% (volume) of apreviously grown S. cerevisiae culture with apopulation of 100-200 million cells/ml.Otherwise, microorganisms present in the wortcarry out the fermentation. If an inoculum is notadded, the fermentation could last as long asseven days. With an inoculum the fermentationtime ranges from 20 hrs in the faster process tothree days in the slower one.

Production of ethyl alcohol by yeast isassociated with formation of many fermentationcompounds that contribute to the final flavor ofthe tequila. These are organoleptic compoundsor their precursors produced either insubsequent maturation of the wort before it goesto the distillation step, in the distillation process,or in the barrels if tequila is aged. The factorsinfluencing formation of the organolepticcompounds in alcoholic beverages have beenreviewed by many authors (Engan, 1981; Berry,1984; McDonald et al., 1984; Ramsay and Berry,1984; Geiger and Piendl, 1976). Experience inthe tequila industry is that the amount oforganoleptic compounds produced is lower infast fermentations than in slow fermentations. Asa consequence, the flavor and general quality oftequila obtained from worts fermented slowly isbest. The rate of fermentation depends mainlyon the yeast strain used, medium compositionand operating conditions. The wort sugar contentdecreases from an initial value of 4-11% to 0.4%(w/v) reducing sugars if an efficient yeast strainis employed. Otherwise, the residual sugarcontent could be higher, increasing the prod-uction costs.

Fermentation vessels vary considerably involume, depending on the distillery. Theircapacity ranges from 12,000 liters for small tanks

to 150,000 liters for the largest ones; and theyare constructed of stainless steel in order to resistthe acidity of the wort. Ethanol production canbe detected almost from the onset; and a pHdrop from 4.5 to 3.9 is characteristic of thefermentation. The alcohol content at the end offermentation lies between 4 and 9% v/v,depending on the initial sugar concentration.Alcohol losses may be significant because manyfermentation tanks are open, allowingevaporation of alcohol with carbon dioxide.Some of the largest distilleries have a coolingsystem that keeps fermentation temperaturewithin a tolerable range for yeast, but smallproducers do not have these systems. Thefermentation temperature can exceed 40 oC,causing the fermentation to stop with anaccompanying loss of ethanol and flavors thatconsequently decreases yields and affects thequality of the tequila. Fermentations carried outwith pure agave juice tend to foam, sometimesrequiring the addition of silicones. In worts withadded sugars, foaming is usually not a problem.

Non-aseptic conditions are employed infermentation, and in consequence bacterialactivity may increase. The size of the bacterialflora depends on a number of factors includingthe extent to which bacteria grow during yeastpropagation (if used), the abundance of bacteriaon the raw materials and hygiene standards inthe distillery. There is no doubt that the activityof these bacteria contributes to the organolepticcharacteristics of the final product. Occasionally,the size of the bacterial population in fermentingwort may become too large (>20 x 106 cells/ml), in which case the bacteria use the sugars,decreasing ethanol yields and sometimesexcreting undesirable compounds. The samecompounds used in the propagation step maybe used here to decrease common bacterialcontaminants found in tequila worts. Lactobacillus,Streptococcus, Leuconostoc, and Pediococcusare the most common contaminants, but Aceto-bacter may be found in fermented worts that areleft inactive for a long time prior to distillation.

In contrast to other distilled beverages, theorganoleptic characteristics of tequila come from


the raw material (cooked agave) as well as fromthe fermentation process. In most of theprocesses used in the tequila industry,fermentation is spontaneous with the partici-pation of microorganisms from the environment,mostly yeasts and a few bacteria. This peculiaritybrings about a wide variety of compositions andorganoleptic properties. However, the specialcharacteristics of the wort make it a selectivemedium for the growth of certain kind of yeastssuch as Saccharomyces and to a lesser extent,acetic and lactic bacteria. In experiments isolatingmicrobial flora from musts of various origins,different microorganisms were isolated. In mostcases, this difference in flora is responsible forthe wide variety of organoleptic characteristicsof tequila brands (Pinal, 1999). Where a singlepurified strain is used, the final flavor and aromaare more neutral since the bouquet created bythe contribution of several stains is richer thanthat obtained from only one type of yeast.Moreover, when yeast produced for bakeryapplications is used, the final product is also moreneutral. It is also recognized that when non-100%agave tequila is made, a poorer bouquet isobtained because since a more defined mediumyields a more defined product.

Organoleptic compounds generated duringfermentation

Fusel oil

As in many other alcoholic fermentation proc-esses, higher alcohols are the most abundantcompounds produced along with ethanol. Wehave found (in decreasing order of abundance)isoamyl alcohol, isobutanol, active isoamylalcohol and phenylethanol. It has beenestablished that production of isoamyl andisobutyl alcohols begins after the sugar level islowered substantially and continues for severalhours after the alcoholic fermentation ends. Incontrast, ethanol production begins in the firsthours of the fermentation and ends withlogarithmic yeast growth (Pinal et al., 1997).

The most important factor influencing theamount of isoamyl alcohol and isobutanol is theyeast strain. It was found that a native strainisolated from tequila must produces a higheramount of such compounds when comparedwith a strain usually employed in bakeries. Theseresults agree with those reported for Scotchwhisky (Ramsay and Berry, 1984) and beer(García et al., 1994).

The carbon:nitrogen ratio also has a significantinfluence on higher alcohol production. Intequila musts, which contain mainly fructose(.95%) as a carbon source and an inorganicnitrogen source (ammonium sulfate), it wasfound for both native and bakery yeast strainsthat low carbon:nitrogen ratios result in lowamounts of isoamyl alcohol: 19 mg/L in bakerystrains and 30 mg/L for native strains vs 27 and64 mg/L, respectively, for high carbon:nitrogenratios. A similar relationship exists for isobutylalcohol production (Figures 1 and 2).

Temperature is a third factor affecting isobutyland isoamyl alcohol production with highertemperatures (e.g. 38 vs 32°C) yielding higherconcentrations of those alcohols. On statisticalanalysis it was found that in addition to the directeffects of yeast strain, carbon:nitrogen ratio andtemperature, the interaction of these threefactors also had an impact on higher alcoholconcentration in tequila (Pinal et al., 1997). Theseresults are consistent with the fact that with highcarbon:nitrogen ratios there is a tendency to useamino acids as a nitrogen source, which impliesthe production of fusel oil as a by-product (bythe Erlich pathway). On the other hand, variablessuch as the type of nitrogen source (urea orammonium sulfate) or the amount of inoculumused for fermentation had little or no effect onthe production of higher alcohols. Paretodiagrams involving all the variables tested aredepicted in Figures 3 and 4.

Methanol

Another characteristic compound present intequila is methanol. It is the general idea that


Figure 2. Higher alcohol production in tequila wort by a native yeast strain.

Figure 1. Higher alcohol production in tequila wort by a bakers yeast strain.


Figure 4. Pareto diagrams for isobutyl alcohol production in tequila.

Figure 3. Pareto diagrams for isoamyl alcohol production in tequila.

methanol is generated through hydrolysis ofmethylated pectins present in the agave plant.Nevertheless, it is also believed that some yeaststrains, natural or inoculated, have pectin methylesterases. Some authors describe variousamounts of methanol in musts with the samecomposition but fermented with different strains(Téllez, 1999).

Aldehydes

Along with the production of ethyl acetate, theoxidation of ethanol also generates acetal-dehyde, an intermediate in the production ofacetic acid. It is well known in commercial prac-tice that an oxidation process instead offermentation begins after the sugar concen-


tration declines, thus provoking the increase inthe acetaldehyde level. However, there is noformal report of such phenomena in tequila, asit is described in beer (Hammond, 1991).

Organic acids

Small organic acids (up to six carbons) and largermolecules (fatty acids) are produced duringfermentation. The smaller molecules can beproducts of intermediate metabolism of thenormal microbial flora; and their productiondepends on the presence of oxygen. The largerfatty acids are synthesized for membranestructures during cell growth and can also appearat the end of the fermentation when lysis takesplace. The presence of octanoic and decanoicacids in the final product has been describedparticularly for tequila.

Esters

Esters are very important compounds in theirparticular contribution to flavor and aroma, sincethey have the lowest organoleptic thresholdvalues (Ramsay and Berry, 1984). In particular,ethyl acetate is the most abundant and importantcompound of this family. Ethyl acetate has beenreported to be the second most abundantcompound in tequila after isoamyl alcohol. Thequantity of this compound present in the finalproduct can vary widely, since it is synthesizedfrom acetic acid (in form of acetyl-CoA) andethanol. Acetic acid can also be produced bythe oxidation of ethanol when the fermentationhas ceased and an oxidative process starts onthe surface of the fermentation tank bySaccharomyces and many other yeasts such asBrettanomyces. Therefore, long fermentationperiods (a current practice in the tequila industry)yield high ethanol oxidation. In addition, in openfermentation tanks with worts at low pHcontaining alcohol, ethanol is also transformedto acetic acid (itself a precursor of ethyl acetate)by bacteria of the genera Acetobacter. Besides

ethyl acetate, the presence of several other estershas been described including ethyl and isoamylesters. Some of the most important esters foundin silver tequila are listed in Table 1.

Table 1. Most abundant esters found in silvertequila.1

Ester %

Ethyl acetate 17.77Ethyl decanoate 2.78Ethyl lactate 2.74Ethyl octanoate 1.92Ethyl dodecanoate 0.95Ethyl butanoate 0.63Isoamyl acetate 0.58Ethyl propanoate 0.57Ethyl hexanoate 0.48Ethyl hexadecanoate 0.48

1Estarrón et al., 1999.

Distilling

Distillation involves the separation and concen-tration of the alcohol from the fermented wort.In addition to ethanol and other desirablesecondary products, wort contains solid agaveparticles consisting mainly of cellulose and pectin,and yeast cells in addition to proteins, mineralsalts and some organic acids. Although a greatnumber of types and degrees of distillation arepossible, the most common systems used in thetequila industry are pot stills and rectificationcolumns. The pot still is considered the earliestform of distilling equipment. It is of the simplestdesign, consisting of a kettle to hold thefermented wort, a steam coil, and a condenseror a plate heat exchanger. Pot stills are oftenmade of copper, which �fixes�, according toThorne et al. (1971), malodorous volatile sulfurcompounds produced during fermentation.Batch distillation using pot stills is carried out intwo steps. First the fermented wort is distilled toincrease the alcohol concentration to 20-30%by volume, separating out the first fraction calledheads, and the last fraction, called tails.


Composition of these fractions varies dependingon many factors including the yeast strainemployed, wort nutrient composition,fermentation time and distillation technique; butin general, heads are rich in low boiling pointcompounds such as acetaldehyde, ethyl acetate,methanol, 1-propanol, 2-propanol, 1-butanol, and2-methyl propanol, which give a very pleasantflavor and taste to tequila. Heads are normallymixed with the wort being distilled. The tailscontain high boiling point components such asisoamyl alcohol, amyl alcohol, 2-furaldehyde,acetic acid and ethylactate, giving a strong tasteand flavor to the tequila; and when theconcentration is above 0.5 mg/ml, the finalproduct becomes unpleasant. This fraction is notused.

In the second step, the liquid obtained fromthe first stage is re-distilled in a similar pot still inorder to obtain a final product that is 110° proofif it is sold in bulk (reducing transport costs) or80° proof if it is to be bottled. Some companiesobtain high proof tequila and dilute it withdemineralized water or water purified by reverseosmosis.

In continuous distillation systems, thefermented wort enters the feed plate of thecolumn and flows downward, crossing a seriesof trays. Steam is injected from the bottom in acoil and strips the wort of its volatile components.Vapors condense higher in the column,depending on component volatility, allowingliquids to be drawn off or recycled at the variousplates as appropriate. Sometimes tequilaobtained in this way is mixed with tequila frompot stills to balance the amount of organolepticcompounds because in general, tequila obtainedthrough continuous columns has less aroma andtaste than tequila obtained from pot stills.

The presence of methanol in tequila is still asubject of discussion because whether methanolis produced only by a chemical reaction or incombination with a microbial hydrolysis has notbeen satisfactorily demonstrated. The chemicalreaction is demethylation of agave pectin by thehigh pH during cooking and the first distillationsteps. The microbial reaction could be thehydrolysis of agave pectin by the enzyme pectin

methyl esterase produced by somemicroorganisms during the fermentation step, butthis has not yet been demonstrated. Preliminaryresults favor the first theory, but some researchis still needed on this matter because it isimportant to maintain the methanol concen-tration within the limits established by the officialstandard, which is 300 mg methanol per 100 mlof anhydrous ethyl alcohol.

Effluent disposal

The discharge from pot stills or distillationcolumns is known as stillage, slops or vinasse;and in a typical tequila distillery 7 to 10 liters ofeffluent are produced per liter of tequila at 100°proof. Tequila stillage has a biological oxygendemand (BOD) of 25 to 60 g/L. In addition tothe dissolved salts (mainly potassium, calcium,and sulfate ions) and the low pH (<3.9) of thestillage, there are significant disposal or treatmentproblems. A general solution to the disposalproblem does not exist because every factoryhas its own production process and is locatedeither in a city or near agave fields. As a result ofthe difficulties of treating vinasses and due to theirhigh concentrations of dissolved matter, a hostof utilization schemes have been proposed.Some of the methods indicated below are underinvestigation and others are in use.

Recycling reduces the volume of waste to betreated. Stillage can be recirculated, mixing 5 to10% of the total volume of the waste obtainedwith clean water to substitute for the dilutionwater used to prepare the initial wort. This canbe carried out for a number of cycles, usually nomore than five, because the concentration ofdissolved salts increases and could affect thefermentation process. Also, great care must betaken with the final taste and flavor of the tequilabecause some components present in stillagecould affect the organoleptic characteristics ofthe final product. Currently, only one tequilacompany uses this system.

Direct land application as irrigation water andfertilizer in agave fields is under careful evaluation


to determine the optimum loading rates and theeffects on the agave over the long time it takesto reach maturity. Evaporation or combustion ofstilllage could provide fertilizer or potash, but thehigh cost of such a process is a serious limitation(Sheenan and Greenfield, 1980). The productionof biomass and biochemicals including fodderyeast is a possibility, but the remaining liquor stillhas a high BOD (Quinn and Marchant, 1980).Stillage may be used as a food supplement forcattle, but it has an undesirable laxative effect onanimals. Biological, aerobic, or anaerobictreatment offers a real means of disposal, butthe cost is likely to be as high as the fermentationcosts themselves (Speece, 1983; Maiorella,1983). Ultimately, tequila vinasses should beviewed as a raw material rather than a waste,and a strategy should be devised that maximizeseconomic and social benefits and reducesrecovery costs.

Maturation

Distillation is the final stage of tequila productionif silver or white tequila is the desired product.For rested or aged tequila, maturation is carriedout in 200 liter white oak casks or in larger woodtanks. The time legally required is two monthsfor rested tequila and 12 months for aged tequila.Tequila is generally matured for longer periods,depending on the characteristics each companydesires for its particular brand.

As tequila ages in barrels, it is subject tochanges that will determine its final quality.Thickness and quality of the stave, depth of thechar, temperature and humidity in the barrellingarea, entry proof (40-110° proof), length ofstorage and number of cycles for the barrel (inMéxico barrels may be re-used several times) allaffect the final taste and aroma of the tequila.Fusel oil content decreases during maturationowing to the adsorbant nature of the char,smoothing the final product. Complex woodconstituents are extracted by the tequila,

providing color and the particular taste. Reactionsamong certain tequila compounds yield newcomponents; and oxidation reactions changesome of the original components in tequila andthose extracted from the wood. As a result of allof these changes, the concentration of acids,esters and aldehydes is increased, while theconcentration of fusel oils decreases as tequilareposes in barrels.

After aging and dilution with demineralizedwater (if necessary) the color of the tequila maybe adjusted to the desired value by the additionof caramel. Alternatively, some companiesblend different batches of tequila to obtain astandardized final product.

Government inspectors supervise the entireaging process. Prior to bottling, tequila is filteredthrough cellulose filter pads or polypropylenecartridges. Sometimes afterwards a pretreatmentwith charcoal is used to eliminate turbidity.

Future developments

Research and future developments in theproduction of tequila and agave cultivation cantake many directions, but the implementation ofany change must allow quality of the final productto be maintained and provide substantialimprovement in the process. There are severalkey areas where important developments couldtake place. Development of new varieties ofagave endowed with resistance to pests orextremely dry environments, higher inulincontent, low wax content in the leaf cuticle andsuperior growth rates would be of benefit.Mechanization would improve aspects ofcultivation and harvest of agave. In addtition,optimization of the cooking and fermentationsteps would improve yields and reduce theamount of waste, while yeast strain selectioncould improve ability to ferment musts with highconcentrations of sugars. Finally, low costalternatives for waste (bagasse and stillage)treatment are needed.


Summary

Tequila is a beverage obtained from thedistillation of fermented juice from the agaveplant (Agave tequilana. Weber var. Azul). Theuse of agave to produce drinks in México datesfrom long ago when certain tribes used it forreligious ceremonies. It was not until the arrivalof the Spaniards, who brought distillationtechnique knowledge, that tequila acquired itspresent form. The agave plant, which is generallyconfused with cacti, is propagated throughtraditional methods or by means of plant cellculture. It is grown for 8 to 10 years before itcan be harvested and sent to the distillery.

The tequila production process comprisesfour stages. In the first step, agave is cooked tohydrolyze the polymers present in the plant,mainly inulin, into fermentable sugars. In somefactories this step is accomplished using stoneovens and in others it is carried out in autoclaves.The second stage is sugar extraction fromcooked agave through milling; and the agavejuice obtained through this step can be mixedwith sugars from other sources, normally sugarcane, if 100% agave tequila is not desired. Thethird and most important stage is fermentationin which sugars are transformed into ethanol andother compounds such as esters and organicacids. These, along with other substances derivedfrom the cooked agave, give the characteristicflavor and taste to tequila. It is of great importanceto have a good yeast strain and nutritionallybalanced wort for tequila production, as lossescould be as high as 35% of the total productionif inefficient yeast is used or nutrients do notappear in the right proportions. In the last stage,fermented wort is distilled, normally using potstills or in some cases rectification columns, toobtain the final product. At the end of thedistillation process white tequila is obtained.Maturation in white oak barrels is required forrested or aged tequila. The minimum maturationtimes are 2 and 12 months, respectively, forrested and aged tequila as required bygovernment regulations. At every step of theproduction process, most companies employ

several quality control analyses in order toensure the quality of the product and theefficiency of the process. Some major producersof tequila are certified through an ISO-9000standard. Tequila production is governed by theofficial norm NOM-006-SCFI-1993, which mustbe followed by all tequila producers to guaranteea good quality final product.

Acknowledgement

The authors would like to express their gratitudeto Tequila Herradura, S.A. de C.V., for its supportin writing this chapter.

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produccion of tequila from agave historical influences and contemporary processes

Environment