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Pozol is a traditional prehispanic Mexican beverage made from fermented nixtamal dough; it is still part of everyday life in many communities due to its nutritional properties. It is the product of spontaneous fermentation and has a complex microbiota composed primarily of lactic acid bacteria (LAB). Although this is a beverage that has been used for centuries, the microbial processes that participate in this fermented beverage are not well understood. We fermented corn dough to produce pozol and sampled it at four key times to follow the community and metabolic changes (0, 9 24 and 48 h) by shotgun metagenomic sequencing to determine structural changes in the bacterial community, as well as metabolic genes used for substrate fermentation, nutritional properties and product safety. We found a core of 25 abundant genera throughout the 4 key fermentation times, with the genus Streptococcus being the most prevalent throughout fermentation. We also performed an analysis focused on metagenomic assembled genomes (MAGs) to identify species from the most abundant genera. Genes involving starch, plant cell wall (PCW), fructan and sucrose degradation were found throughout fermentation and in MAGs, indicating the metabolic potential of the pozol microbiota to degrade these carbohydrates. Complete metabolic modules responsible for amino acid and vitamin biosynthesis increased considerably during fermentation, and were also found to be abundant in MAG, highlighting the bacterial contribution to the well-known nutritional properties attributed to pozol. Further, clusters of genes containing CAZymes (CGCs) and essential amino acids and vitamins were found in the reconstructed MAGs for abundant species in pozol. The results of this study contribute to our understanding of the metabolic role of micro-organisms in the transformation of corn to produce this traditional beverage and their contribution to the nutritional impact that pozol has had for centuries in the traditional cuisine of southeast Mexico.

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Leuconostoc mesenteroides strains are common contributors in fermented foods producing a wide variety of polysaccharides from sucrose through glycosyltransferases (GTFs). These polymers have been proposed as protective barriers against acidity, dehydration, heat, and oxidative stress. Despite its presence in many traditional fermented products and their association with food functional properties, regulation of GTFs expression in Ln. mesenteroides is still poorly understood. The strain Ln. mesenteroides ATCC 8293 contains three glucansucrases genes not found in operons, and three fructansucrases genes arranged in two operons, levLX and levC-scrB, a Glycoside-hydrolase. We described the first differential gene expression analysis of this strain when cultivated in different carbon sources. We observed that while GTFs are expressed in the presence of most sugars, they are down-regulated in xylose. We ruled out the regulatory effect of CcpA over GTFs and did not find regulatory elements with a direct effect on glucansucrases in the condition assayed. Our findings suggest that only operon levLX is repressed in xylose by LexA and that both fructansucrases operons can be regulated by the VicK/VicR system and PerR. It is essential to further explore the effect of environmental conditions in Ln. mesenteroides bacteria to better understand GTFs regulation and polymer function.

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Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

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BACKGROUND: Although Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products. RESULTS: The expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h. At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level. CONCLUSION: We demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

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Prebiotics and probiotics have shown a number of beneficial impacts preventing diseases in cultured shrimps. Complex soluble carbohydrates are considered ideal for fostering microbiota biodiversity by fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPS). Here we evaluated the growth performance and microbiota composition of the white shrimp Litopenaeus vannamei after dietary intervention using agavin as a FODMAP prebiotic under farming conditions. Adult L. vannamei were raised at a shrimp farm and the effect of agavin supplemented at 2% (AG2) or 10% (AG10) levels were compared to an agavin-free basal diet (BD). After 28 days-trial, the feed conversion ratio, total feed ingested, and protein efficiency ratio was significantly improved on animals fed with AG2. At the same time, no effect on growth performance was observed in AG10. Surprisingly, after sequencing the V3-V4 regions of the 16S rRNA gene a higher microbial richness and diversity in the hepatopancreas and intestine was found only in those animals receiving the AG10 diet, while those receiving the AG2 diet had a decreased richness and diversity, both diets compared to the BD. The beta diversity analysis showed a clear significant microbiota clustering by agavin diets only in the hepatopancreas, suggesting that agavin supplementation had a more substantial deterministic effect on the microbiota of hepatopancreas than on the intestine. We analyzed the literature to search beneficial microbes for shrimp's health and found sequences for 42 species in our 16S data, being significantly increased Lactobacillus pentosus, Pseudomonas putida and Pseudomonas synxantha in the hepatopancreas of the AG10 and Rodopseudomonas palustris and Streptococcus thermophiles th1435 in the hepatopancreas of the AG2, both compared to BD. Interestingly, when we analyzed the abundance of 42 beneficial microbes as a single microbial community "meta-community," found an increase in their abundance as agavin concentration increases in the hepatopancreas. In addition, we also sequenced the DNA of agavin and found 9 of the 42 beneficial microbes. From those, Lactobacillus lactis and Lactobacillus delbrueckii were found in shrimps fed with agavin (both AG2 and AG10), and Lysinibacillus fusiformis in AG10 and they were absent the BD diet, suggesting these three species could be introduced with the agavin to the diet. Our work provides evidence that agavin supplementation is associated with an increase of beneficial microbes for the shrimp microbiota at farming conditions. Our study provides the first evidence that a shrimp prebiotic may selectively modify the microbiota in an organ-dependent effect.

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Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (Topt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:ß:γ-CDs (34:62:4) from soluble starch, as well as G3-G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

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[This corrects the article DOI: 10.3389/fmicb.2021.629449.].

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The genus Weissella is composed of a group of Gram-positive facultative anaerobe bacteria with fermentative metabolism. Strains of this genus have been isolated from various ecological niches, including a wide variety of fermented cereal foods. The present study aimed to determine the relative abundance and fermentation capabilities of Weissella species isolated from pozol, a traditional maya product made of lime-cooked (nixtamalized) fermented maize. We sequenced the V3-V4 regions of 16S rDNA; Weissella was detected early in the fermentation process and reached its highest relative abundance (3.89%) after 3 h of culture. In addition, we evaluated five Weissella strains previously isolated from pozol but reported as non-amylolytic, to define alternative carbon sources such as xylan, xylooligosaccharides, and sucrose. While no growth was observed on birch xylan, growth did occur on xylooligosaccharides and sucrose. Strains WcL17 and WCP-3A were selected for genomic sequencing, as the former shows efficient growth on xylooligosaccharides and the latter displays high glycosyltransferase (GTF) activity. Genomes of both strains were assembled and recorded, with a total of 2.3 Mb in 30 contigs for WcL17 and 2.2 Mb in 45 contigs for WCP-3a. Both strains were taxonomically assigned to Weissella confusa and genomic analyses were performed to evaluate the gene products encoding active carbohydrate enzymes (CAZy). Both strains have the gene content needed to metabolize sucrose, hemicellulose, cellulose, and starch residues, all available in pozol. Our results suggest that the range of secondary enzymatic activity in Weissella confusa strains confer them with wide capabilities to participate in fermentative processes of natural products with heterogeneous carbon sources.

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Aguamiel is the sap collected from agave, while pulque is the result of the natural fermentation of aguamiel. Despite its ancestral origin and numerous publications on pulque production, little is known about the evolution and concentration of sugars and fructo-oligosaccharides in aguamiel, either during its daily accumulation or through the agave production lifetime. In this study, we examined aguamiel composition in three agave plants during their productive lifetime (4 to 9 months). After each collection, the agave pine is scraped to induce aguamiel to flow into an internally created cavity (cajete), producing a residual bagasse (metzal). We found that the concentration of agave fructans and sucrose, as well as the fructan profile, change during the aguamiel production process. During the daily collection, a small amount of agave fructans released from the pine by scraping is drawn into the cajete with the first milliliters of sap where it is then diluted with the inflow of aguamiel. The main component of aguamiel is the sucrose produced in high concentration in the leaves through photosynthesis and then hydrolyzed in the cajete as aguamiel accumulates. We also describe how the fructan profile changes during the accumulation of aguamiel in the cajete. In addition to the varying amount of sucrose that is hydrolyzed in the aguamiel accumulated, we found that fructo-oligosaccharides are either diluted, consumed, or hydrolyzed, depending on the plant and its production stage, thus yielding different fructan profiles. New fructo-oligosaccharides are, in some cases, synthesized by bacteria present in aguamiel. These profiles were also observed in aguamiel collected from ten different plants in the same production region. We also found that a considerable amount of agave fructans is lost in metzal (bagasse), the agave material that is scraped and thrown away twice a day during the production process.

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Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

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Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.

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Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.

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Leuconostoc citreum CW28 was isolated from pozol, a Mayan fermented corn beverage. This strain produces a cell-associated inulosucrase, the first described in bacteria. Its draft genome sequence, announced here, has an estimated size of 1.98 Mb and harbors 1,915 coding genes, 12 rRNAs, 68 tRNAs, 17 putative pseudogenes, and 1 putative phage.

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We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB1SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB1, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s-1. LevB1 in LevB1SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB1SacB or both enzymes (LevB1 & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB1SacB or LevB1 & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.

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Blastose, a natural disaccharide found in honey, is usually found as a byproduct of fructo-oligosaccharide synthesis from sucrose with fructosyltransferases. In this study, we describe a novel two-step biosynthetic route to obtain blastose, designed from a detailed observation of B. subtilis levansucrase (SacB) acceptor structural requirements for fructosylation. The strategy consisted first in the synthesis of the trisaccharide O-ß-d-Fruf-(2↔6)-O-α-d-Glcp-(1↔1)-α-d-Glcp, through a regioselective ß-d-transfructosylation of trehalose (Tre) which acts as acceptor in a reaction catalyzed by SacB using sucrose or levan as fructosyl donor. In this reaction, levansucrase (LS) transfers regioselectively a fructosyl residue to either C6-OH group of the glucose residues in Tre. The resulting trisaccharide obtained in 23% molar yield based on trehalose, was purified and fully characterized by extensive NMR studies. In the second step, the trisaccharide is specifically hydrolyzed by trehalase, to obtain blastose in 43.2% molar yield based on the trisaccharide. This is the first report describing the formation of blastose through a sequential transfuctosylation-hydrolysis reaction.

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Pulque is a traditional Mexican alcoholic beverage produced from the fermentation of the fresh sap known as aguamiel (mead) extracted from several species of Agave (maguey) plants that grow in the Central Mexico plateau. Currently, pulque is produced, sold and consumed in popular districts of Mexico City and rural areas. The fermented product is a milky white, viscous, and slightly acidic liquid beverage with an alcohol content between 4 and 7° GL and history of consumption that dates back to pre-Hispanic times. In this contribution, we review the traditional pulque production process, including the microbiota involved in the biochemical changes that take place during aguamiel fermentation. We discuss the historical relevance and the benefits of pulque consumption, its chemical and nutritional properties, including the health benefits associated with diverse lactic acid bacteria with probiotic potential isolated from the beverage. Finally, we describe the actual status of pulque production as well as the social, scientific and technological challenges faced to preserve and improve the production of this ancestral beverage and Mexican cultural heritage.

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Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

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Cyclodextrin glycosyltransferases (CGTase) are reported to selectively catalyze α(1 → 4)-glycosyl transfer reactions besides showing low hydrolytic activity. Here, the effect of the anomeric configuration of the glycosyl acceptor on the regioselectivity of CGTase catalyzed glycosylations was investigated. For this purpose, the α and ß anomers of p-nitrophenyl-D-glucopyranoside were used as glycosyl acceptors, Bacillus macerans and Thermoanaerobacter sp. CGTases were used as biocatalysts and ß-cyclodextrin as the glycosyl donor. As expected, p-nitrophenyl-α-D-glucopyranosyl-(1 → 4)-O-α-D-glucopyranoside was produced when p-nitrophenyl-α-D-glucopyranoside was used as acceptor with B. macerans CGTase. Surprisingly, when p-nitrophenyl-ß-D-glucopyranoside was used as glycosyl acceptor, besides the expected α(1 → 4)-glycosylation products both α(1 → 3)- and α(1 → 6)-transfer products were also obtained. This unexpected change in B. macerans CGTase regioselectivity leading to α(1 → 4)-, α(1 → 3)- and α(1 → 6)-glycosylation products was also observed for Thermoanaerobacter sp. CGTase with the ß anomer. It is shown, applying time course analyses, that all isomers can be synthesized efficiently by adequate selection of enzyme and reaction conditions. In fact, when using Thermoanaerobacter sp. CGTase the yield of p-nitrophenyl-ß-D-isomaltoside (the α(1 → 6)-transfer product) was the highest at long reaction time (19% yield). The previously unknown capacity of α(1 → 6)-glycosidic linkages formation by CGTases demonstrates an unexpected broader regioselectivity of CGTases in glycosyl-transfer reactions as well as an acceptor dependent transfer selectivity.

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A controlled acid thermal hydrolysis process of fructans from agave (Agave tequilana Weber var. azul) was designed to produce a mixture of functional prebiotic fructooligosaccharides and sweetening power. Despite its highly branched structure, first-order kinetic behaviour with respect to substrate concentration was found with an activation energy of 95kJ/mol, similar to the value found for other linear fructans such as chicory inulin. Fructose equivalent (FE) an analogous parameter to dextrose equivalent (DE) used in the starch industry was introduced to characterise fructan hydrolysis; maximum oligosaccharide production was observed when fructose equivalent (FE) reached 27-48 with a structural profile analysed by high-performance anion-exchange chromatography HPAEC-PAD. After hydrolysis, glucose and fructose may be eliminated through a biological purification step involving the addition of Pichia pastoris cells, which selectively consume these sugars but are unable to metabolise fructooligosaccharides.

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In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.

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