Agave Juice Research Articles

Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study.

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like—rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47–50% w/w) and non-cellulosic polysaccharides (16–22% w/w), and whole leaves were low in lignin (9–13% w/w). Of the dry mass of whole Agave leaves, 85–95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41–48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.

Fermentative capacity of Saccharomyces and non-Saccharomyces in agave juice and semi-synthetic medium

Various consortia of yeasts and bacteria involved in the natural fermentation process of tequila have been identified, particularly non-Saccharomyces yeasts. This study evaluates the fermentative capacity of two non-Saccharomyces yeasts (isolated from traditional mezcal fermentation): Kluyveromyces marxianus (DU3) and Pichia kluyveri (GRO3), and assesses their production of volatile compounds. The values found are compared with those of the same attributes of a Saccharomyces cerevisiae (AR5) isolated from tequila fermentation. The fermentations were performed in two different media, agave juice (JA) and a semi-synthetic medium (M11). The study also compared free and immobilized yeast fermentations in the JA medium in order to evaluate the potential benefits of immobilization on the yeast behaviour. This study demonstrated the potential of non-Saccharomyces yeasts, which fermented the agave juice in the same manner as S. cerevisiae but with higher ester production. Furthermore, K. marxianus produced more higher alcohols than S. cerevisiae. This could lead to tequila with different aroma profiles. Results were different in the synthetic medium, thus showing sensitivity to the composition of the medium. No significant differences between yeast fermentations with free and immobilized cells were detected, except for ethanol yield.

Sugars and Fructans Separation by Nanofiltration from Model Sugar Solution and Comparative Study with Natural Agave Juice

The fructan separation from a model sugar solution and natural agave juice was studied using a stirred-cell nanofiltration unit operated in concentration mode. Hydrophilic cellulose membrane with MWCO of 1000 Da was used. The experimental conditions were varied to predict the influence of pressure (0.14–0.350 MPa) and feed concentration (0.15–0.25 g/mL) on the initial permeate flux and solute retained fraction (SRF) values of the process. Response surface plots (p < 0.05) showed that the permeate flux and SRF increased significantly with the pressure and decreased with feed concentration. The permeate flux varied from 0.5 to 4.1 L · h−1 · m−2. The fructan retained fraction in model sugar solution varied from 0.85 to 0.97 whereas fructose, glucose and sucrose presented similar SRF values ranging from 0.38 to 0.65. Promising results were obtained when natural agave juice was used.

Bioconversion ofAgave tequilanafructans by exo-inulinases from indigenousAspergillus nigerCH-A-2010 enhances ethanol production from rawAgave tequilanajuice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme(®) (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.

Volatile compound production in Agave duranguensis juice fermentations using four native yeasts and NH4Cl supplementation

Nitrogen has a significant effect on the fermen- tation rate and the chemical composition of alcoholic bev- erages. Nitrogen deficiency during fermentation of Agave juice for mescal production can lead to slow fermentations and end-products with low aromatic compound variety. In this study, the effects of NH4Cl supplementation on volatile compound formation in Agave duranguensis juice fer- mented at 28 C with the native yeast strains Saccharomyces cerevisiae ITD00185, Hanseniaspora uvarum ITD00108, Torulaspora delbrueckii ITD00110 and Kluyveromyces marxianus ITD00211 were analysed. Nitrogen content in the Agave juice unsupplemented with NH4Cl was low. In the control treatments, the four yeasts consumed nitrogen at approximately the same rate, almost completely finishing by 24 h. Nitrogen supplementation increased biomass produc- tion with S. cerevisie, H. uvarum and T. delbrueckii but not with K. marxianus. K. marxianus consumed the total assimilable nitrogen more slowly than the other strains in the supplemented fermentations. In addition, the volatile com- pound profile differed between the studied yeasts. Volatile compound production by S. cerevisiae, H. uvarum and K. marxianus was higher in the supplemented fermentations, compared to the unsupplemented ones. In T. delbrueckii, the initial volatile compound concentrations remained unchan- ged, or decreased for some compounds, with nitrogen supplementation. The initial acetic acid and vanillin con- centrations decreased with all strains tested and nitrogen supplementation. Furthermore, the concentration of higher alcohols increased with S. cerevisiae and H. uvarum in the NH4Cl-supplemented fermentations, but they decreased with T. delbrueckii and K. marxianus. In conclusion, the addition of an inorganic nitrogen source promotes microor- ganism metabolism, increases biomass formation and ben- efits the fermentation process.

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.

Effect of pH, aeration and feeding non-sterilized agave juice in a continuous agave juice fermentation

Continuous cultures have been used since the 1950s in beer and wine fermentations due to their higher productivities compared to traditional batch systems; nevertheless, the tequila industry has not taken advantage of the possible improvements that continuous fermentations could offer. In this work, the effect of pH, aeration and feeding of non-sterilized medium, on the fermentative capability of two Saccharomyces cerevisiae strains (S1 and S2) cultured in continuous fermentation, using agave juice as the fermentation medium, were studied. In continuous cultures, the control of the medium pH (set point at 4) did not have a significant effect on fermentation efficiency compared to fermentations in which the pH was not controlled (pH 2.5 +/- 0.3). Conversely, aeration of the cultures of both strains improved biomass production and consumption of reducing sugars and ammonium. The aeration also significantly augmented ethanol production only for S1 cultures (P < 0.05). Furthermore, the feeding of medium, either sterilized or not, did not show significant differences on the production of ethanol for S1 cultures. Higher concentrations of acetoin, succinic acid and diacetyl were found in the cultures fed with non-sterilized medium. Compared to S2, S1 has a better fermentative performance in continuous non-sterilized medium fermentations. Not controlling the pH during the cultures could prevent the possibility of microbial contamination as a result of the extreme medium acidity (pH 2.5 +/- 0.3). This work showed the possibility of scaling up agave juice continuous fermentation feeding non-sterilized medium with no control of pH.

Effect of Agave tequilana age, cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.

Quantitative Characterization of Nonstructural Carbohydrates of Mezcal Agave (Agave salmiana Otto ex Salm-Dick)

Fructans are the reserve carbohydrates in Agave spp. plants. In mezcal factories, fructans undergoes thermal hydrolysis to release fructose and glucose, which are the basis to produce this spirit. Carbohydrate content determines the yield of the final product, which depends on plant organ, ripeness stage, and thermal hydrolysis. Thus, a qualitative and quantitative characterization of nonstructural carbohydrates was conducted in raw and hydrolyzed juices extracted from Agave salmiana stems and leaves under three ripeness stages. By high-performance liquid chromatography (HPLC), fructose, glucose, sucrose, xylose, and maltose were identified in agave juice. Only the plant fraction with hydrolysis interaction was found to be significant in the glucose concentration plant. Interactions of the fraction with hydrolysis and ripeness with hydrolysis were statistically significant in fructose concentration. Fructose concentration rose considerably with hydrolysis, but only in juice extracted from ripe agave stems (early mature and castrated). This increase was statistically significant only with acid hydrolysis.

Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice

The production of aroma compounds during tequila fermentation using four native yeast strains isolated from agave juice was quantified at controlled (35 degrees C) and uncontrolled temperatures (room temperature) by gas chromatography (FID). Three of the four strains were identified as Saccharomyces cerevisiae (MTLI 1, MALI 1 and MGLI 1) and one as Kloeckera apiculata (MALI 2). Among the aroma compounds produced, acetaldehyde has the highest accumulation at the controlled temperature and before 50% of sugar was consumed. The S. cerevisiae strains produced ethyl acetate in almost the same quantity at a concentration of 5 mg/L and the K. apiculata produced six-times more (30 mg/L) than the S. cerevisiae strains, independent of the fermentation temperature. The rate and amount of 1-propanol, amyl alcohols and isobutanol production were affected by the type of yeast used. The K. apiculate strain produced 50% less of the higher alcohols than the Saccharomyces strains. The results obtained showed that indigenous isolated yeasts play an important role in the tequila flavor and suggest that mixtures of these yeasts may be used to produce tequila with a unique and desirable aroma.

Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice

Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juice supplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classified as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classified as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxic compounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenyl ethyl acetate and ethyl acetate. The methanol concentration was significantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic profile than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.

Kinetics of infective juvenile production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture

The effects of culture medium formulations on the kinetics of infective juvenile (IJ) production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture, were studied at the cylindrical-bottle scale using six culture media containing agave juice from Agave spp. among other ingredients. The IJ production kinetics was well modelled through a re-parameterised 3-parameter Gompertz model with kinetic parameters: IJ-lag phase lambda (IJ) (day), maximum IJ-stage production rate m (max )(day(-1)), and IJ-multiplication factor (C (IJ)/C (IJ,0))(max)(-). The variation of lambda (IJ) was not very important within fermentations (10.3-16.2 days); nonetheless, important effects were observed on m (max) (32.8-241.2 days(-1)) and (C (IJ)/C (IJ,0))(max) (66(-) to 611.4(-)). Particularly, maximum values of m (max) and (C (IJ)/C (IJ,0))(max) were obtained in medium A4 (0.276 l l(-1) agave juice, 17 g l(-1) yeast extract, 12 g l(-1) dried egg yolk, 0.025 l l(-1) corn oil). Also, the maximum IJ concentration (249,444 per ml) was achieved in A4-fermentations.

Monoxenic production of the entomopathogenic nematode Steinernema carpocapsae using culture media containing agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp). Effects of the contents of nitrogen, carbohydrates and fat on infective juvenile production

The production of infective juvenile stages (IJ) of the entomopathogenic nematode Steinernema carpocapsae in the presence of its symbiotic bacterium Xenorhabdus nematophilus was carried out in orbitally agitated bottles. Four complex culture media (M1-M4) were used, containing from 8% to 28% (by vol.) agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp) as the main carbohydrate source. After 20 days of fermentation, a maximum viable IJ concentration of 249,000 IJ/ml and an initial nematode population multiplication factor of x620 were achieved when medium M4 was used (aguamiel concentration in this medium was 28% by vol.). M4 medium contained (w/v): 0.3% total nitrogen, 3.2% total carbohydrates and 3.0% total fat. According to the results obtained, total carbohydrates concentration appeared to be of great importance in obtaining high IJ concentrations.

Increasing fermentation efficiency at high sugar concentrations by supplementing an additional source of nitrogen during the exponential phase of the tequila fermentation process.

In the tequila industry, fermentation is traditionally achieved at sugar concentrations ranging from 50 to 100 g x L(-1). In this work, the behaviour of the Saccharomyces cerevisiae yeast (isolated from the juices of the Agave tequilana Weber blue variety) during the agave juice fermentation is compared at different sugar concentrations to determine if it is feasible for the industry to run fermentation at higher sugar concentrations. Fermentation efficiency is shown to be higher (above 90%) at a high concentration of initial sugar (170 g x L(-1)) when an additional source of nitrogen (a mixture of amino acids and ammonium sulphate, different than a grape must nitrogen composition) is added during the exponential growth phase.

Generation of Maillard Compounds from Inulin during the Thermal Processing of Agave tequilana Weber Var. azul

During the cooking process of Agave tequilana Weber var. azul to produce tequila, besides the hydrolysis of inulin to generate fermentable sugars, many volatiles, mainly Maillard compounds, are produced, most of which may have a significant impact on the overall flavor of tequila. Exudates (agave juice) from a tequila company were collected periodically, and color, Brix, fructose concentration, and reducing sugars were determined as inulin breakdown took place. Maillard compounds were obtained by extraction with CH(2)Cl(2), and the extracts were analyzed by GC-MS. Increments in color, Brix, and reducing sugars were observed as a function of time, but a decrease in fructose concentration was found. Many Maillard compounds were identified in the exudates, including furans, pyrans, aldehydes, and nitrogen and sulfur compounds. The most abundant Maillard compounds were methyl-2-furoate, 2,3-dihydroxy-3,5-dihydro-6-methyl-4(H)-pyran-4-one, and 5-(hydroxymethyl)furfural. In addition, a series of short- and long-chain fatty acids was also found. A large number of the volatiles in A. tequilana Weber var. azul were also detected in tequila extracts, and most of these have been reported as a powerful odorants, responsible for the unique tequila flavor.

Provenance and use wears of Pre-Hispanic obsidian scrapers from Metztitlan, Hidalgo, Mexico

The research was performed with archaeological obsidian scrapers, collected at the Metztitlan area, Hidalgo, Mexico. The provenance of the raw material was determined by NAA. SEM technique was applied to identify use wears on pre-Hispanic tools and experimental replicas. The Metztitlan scrapers made of the obsidian of Zacualtipan, Hidalgo, Mexico, were probably used for Agave juice extraction in the Late Postclassical period.

Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ' pulque ', a traditional Aztec alcoholic beverage

Dextransucrase was produced from a Leuconostoc mesenteroides isolated from pulque, a traditional Aztec alcoholic beverage produced from agave juice containing sucrose as the main carbon source. Almost all the dextransucrase activity (87%) was associated with the cells, and was unusually high (1.04 U mg−1 of cells). The culture medium composition was optimized through a Box-Behnken method resulting in a process yielding 2.2 U ml−1 of insoluble glucosyltransferase activity. The enzyme had a molecular weight of 166 kDa. Optimal temperature was 35°C with a half-life of 137 min at the same temperature. As with dextransucrase from the industrial strain L. mesenteroides NRRL B-512F, the enzyme showed Michaelis–Menten kinetic behavior with excess substrate inhibition (Km and Ki values of 0.026 M and 1.23 M respectively); produced soluble linear dextran with glucose molecules linked mainly in α(1–6) with branching in α(1–3) in a proportion of 4:1 as shown by NMR studies; and produced a high yield of isomalto-oligosaccharides in the presence of maltose.

Comparative study of extracellular and intracellular β‐glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β‐glucosidases of this yeast were purified by ion‐exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β‐glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β‐glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β‐glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)‐β, (1 → 6)‐β and (1 → 4)‐α linkage configuration. The intracellular enzyme possesses (1 → 6)‐α‐arabinofuranosidase activity and extracellular enzyme (1 → 6)‐α‐rhamno‐pyranosidase activity. The two β‐glucosidases are competitively inhibited by glucose and by D‐gluconic‐acid‐lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β‐glucosidases for the liberation of the bound aroma is discussed.

Notes: Taxonomic Position of Additional Zymomonas mobilis Strains

The strains CP3 and CP4, isolated from fermenting sugar-cane juice in Brazil, and the strain Ag11, isolated from fermenting agave juice in Mexico, belong in Zymomonas mobilis subsp. mobilis. The existence of a separate Zymomonas mobilis subsp. recifensis for the former two is not justified. The strains NCIB 8777 and NCIB 10565 from sick cider and apple pulp, respectively, in England, belong in Zymomonas mobilis subsp. pomaceae. Protein electropherograms allow a quick and accurate identification in Zymomonas.

Protein made on agave juice base

Protein made on agave juice base