Fructan Hydrolysis Research Articles

Saccharification of fructans and simultaneous alcoholic fermentation for sustainable production of agave distillates

ABSTRACT In a world where climate change and sustainability are at the forefront, innovative solutions are more pressing than ever. In the production process of distillates, the critical stage is the cooking of agave for thermal hydrolysis of fructans. This stage determines the efficiency of the process, incurs the highest production cost, and has a detrimental impact on the environment. In this study, we investigate the saccharification of agave fructans in fresh juice using fructanases produced in situ by Kluyveromyces marxianus as an alternative to traditional agave cooking. furthermore, we explore both sequential and simultaneous alcoholic fermentation processes using a native mixed culture to improve the overall yield and obtain a distillate with enjoyable sensory characteristics. The results show that saccharification and simultaneous fermentation allow for obtaining a fermented product containing an average of 7% alcohol. Gas chromatography and mass spectrometry analysis shows that the distillate is essentially an ethanol – water mixture with a broad profile and high concentration of volatile compounds. The composition of the distilled product provides a well-balanced sensory experience that is highly valued by consumers. Hence, for the first time, a novel, efficient, low-cost, and sustainable process for producing agave distillates has been successfully designed.

Brassinosteroids alleviates wheat floret degeneration under low nitrogen stress by promoting the distribution of sucrose from stems to spikes

The trade-off between yield and environmental effects caused by nitrogen fertilizer application is an important issue in wheat production. A reduction in fertile florets is one of the main reasons for decreased yield under low nitrogen application rate. Brassinosteroids (BRs) have been found to play a role in nitrogen-induced rice spikelet degeneration. However, whether BRs play a role in wheat floret development and the mechanisms involved are not clear. Therefore, different nitrogen gradient experiment and exogenous spraying experiment were conducted to investigate the role and mechanism of BRs in wheat floret development under low nitrogen stress. The results showed that as the nitrogen application decreased, the endogenous BRs content of spikes decreased, photosynthesis weakened, and total carbon, soluble sugar and starch of spikes decreased, leading to a reduction in the number of fertile florets. Under low nitrogen stress, exogenous spraying of 24-epibrassinolide promoted photosynthesis, stimulated stem fructan hydrolysis and the utilization and storage of sucrose in spikes, directing more carbohydrates to the spikes and increasing the number of fertile florets. In conclusion, BRs mediate the effects of nitrogen fertilizer on wheat floret development, and under low nitrogen stress, foliar spraying of 24-epibrassinolide promotes the flow of carbohydrates from the stem to the spikes, alleviating wheat floret degeneration.

Agave cupreata fructans, encapsulation as pro and prebiotics

The agave or maguey belongs to the agaváceas family, with long and fibrous leaves, among this species is the Agave cupreata, endemic to the state of Guerrero, the agave has various properties, among them is the high content of fructooligosaccharides (FOS) derived from the hydrolysis of fructans. It has been shown that it possesses prebiotic properties, due to its β bonds it cannot be hydrolyzed by intestinal tract secretions. Therefore, they beneficially affect the host and stimulate the growth of lactic acid bacteria (LAB), which are colonizing the intestinal microbiota and preventing the proliferation of pathogenic bacteria. Therefore, the objective is to design a prebiotic and probiotic with agave fructans. With Bifidobacterium lactis Bb12 and L. acidophilus La5, encapsulate them and evaluate their antimicrobial effect against Escherichia coli and Salmonella typhi. Materials and methods: an extraction of agave juice was carried out in a 1:1 ratio between grams of sample and water, in crude and cooked agave, later it was inoculated and fermented with LAB for 4 days, it was decided to encapsulate to evaluate the antimicrobial effect. against Escherichia coli and Salmonella typhi. Results: the antibiogram indicated that E. coli is resistant to ampicillin, however the cooked and crude agave juice inhibited to E. coli, the same way cooked and crude agave juice, inhibited the growth of S. typhi, but it did not show resistance to antibiotic. Conclusion: the agave juice inoculated with (BAL) has an inhibitory action against Escherichia coli and Salmonella typhi.

Microbial inulinase promotes fructan hydrolysis under simulated gastric conditions.

Fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) have emerged as key contributors to digestive discomfort and intolerance to certain vegetables, fruits, and plant-based foods. Although strategies exist to minimize FODMAP consumption and exposure, exogenous enzyme supplementation targeting the fructan-type FODMAPs has been underexploited. The objective of this study was to test the hydrolytic efficacy of a food-grade, non-genetically engineered microbial inulinase preparation toward inulin-type fructans in the INFOGEST in vitro static simulation of gastrointestinal (GI) digestion. Purified inulin was shown to undergo acid-mediated hydrolysis at high gastric acidity as well as predominantly inulinase-mediated hydrolysis at lower gastric acidity. Inulinase dose-response simulations of inulin, garlic, and high-fructan meal digestion in the gastric phase suggest that as little as 50 inulinase units (INU) and up to 800 INU per serving promote fructan hydrolysis better than the control simulations without inulinase. Liquid chromatography-mass spectrometry (LC-MS) profiling of fructo-oligosaccharides (FOS) in the gastric digestas following inulinase treatment confirms the fructolytic activity of inulinase under simulated digestive conditions. Altogether, these in vitro digestion data support the use of microbial inulinase as an exogenous enzyme supplement for reducing dietary fructan-type FODMAP exposure.

High fructan barley lines produced by selective breeding may alter β-glucan and amylopectin molecular structure

Six cross-bred barley lines developed by a breeding strategy with the target to enhance the fructan synthesis activity and reduce the fructan hydrolysis activity were analyzed together with their parental lines, and a reference line (Gustav) to determine whether the breeding strategy also affected the content and molecular structure of amylopectin and β-glucan. The highest fructan and β-glucan content achieved in the novel barley lines was 8.6 % and 12 %, respectively (12.3-fold and 3.2-fold higher than in Gustav). The lines with low fructan synthesis activity had higher starch content, smaller building blocks in amylopectin, and smaller structural units of β-glucans than the lines with high-fructan synthesis activity. Correlation analysis confirmed that low starch content was associated with high amylose, fructan, and β-glucan content, and larger building blocks in amylopectin.

Achieving of high-diet-fiber barley via managing fructan hydrolysis

High fructan content in the grain of cereals is an important trait in agriculture such as environmental resilience and dietary fiber food production. To understand the mechanism in determining final grain fructan content and achieve high fructan cereal, a cross breeding strategy based on fructan synthesis and hydrolysis activities was set up and have achieved barley lines with 11.8% storage fructan in the harvested grain. Our study discovered that high activity of fructan hydrolysis at later grain developmental stage leads to the low fructan content in mature seeds, simultaneously increasing fructan synthesis at early stage and decreasing fructan hydrolysis at later stage through crossing breeding is an efficient way to elevate grain diet-fiber content. A good correlation between fructan and beta glucans was also discovered with obvious interest. Field trials showed that the achieved high fructan barley produced over seven folds higher fructan content than control barley and pull carbon-flux to fructan through decreasing fructan hydrolysis without disruption starch synthesis will probably not bring yield deficiency.

Physiochemical changes and nutritional content of black garlic during fermentation

This study was conducted to understand the reactions involved in black garlic fermentation, and thereby to determine its maturity stage. Black garlic was produced from fresh garlic after fermentation at 70–75 °C and 80–90% relative humidity up to 12 days in this study. The enzymatic hydrolysis of fructans and oligosaccharides, and endophytic bacterial action were likely to be the dominant processes at the initial stage, and followed by non-enzymatic browning Maillard reaction. Fructose was initially detected at the highest concentration and started to decrease after 10 days together with the exponential increase of 5-hydroxymethylfurfural (5-HMF) content. The predominant acids were citric and succinic acids, whereas potassium was the most abundant minerals. The loss of pungent smell could be attributed to the degradation of sulfur containing compounds. The pH of fresh garlic was 6.61, and gradually decreased to <4.0 after 10 days. Fresh garlic lost about 22% of mass after aged in a high humidity environment. There was significant difference in the bioactivities of fresh and black garlic, but no difference among black garlic fermented for >8 days. Garlic fermentation involved multiple reactions including enzymatic hydrolysis, endophytic bacterial action, and non-enzymatic browning reaction to achieve maturity after 8 days.

Identification of genes related to hydrolysis and assimilation of Agave fructans in Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 by denovo transcriptome analysis.

Fructans are the main sugar in agave pine used by yeasts during mezcal fermentation processes, from which Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 were isolated. De novo transcriptome analysis was carried out to identify genes involved in the hydrolysis and assimilation of Agave fructans (AF). We identified a transcript annotated as SUC2, which is related to β-fructofuranosidase activity, and several differential expressed genes involved in the transcriptional regulation of SUC2 such as: MIG1, MTH1, SNF1, SNF5, REG1, SSN6, SIP1, SIP2, SIP5, GPR1, RAS2, and PKA. Some of these genes were specifically expressed in some of the yeasts according to their fructans assimilation metabolism. Different hexose transporters that could be related to the assimilation of fructose and glucose were found in both the transcriptomes. Our findings provide a better understanding of AF assimilation in these yeasts and provide resources for further metabolic engineering and biotechnology applications.

Ohmic heating pretreatment accelerates black garlic processing

Black garlic (BG) is obtained by the Maillard reaction under controlled conditions of temperature and humidity. This study describes the traditional processing (TT-BG) and the use of ohmic heating (OH) as pre-treatment (OH-BG) to improve time processing. Physicochemical composition and sensory properties were of particular interest in this study. The OH as a pretreatment causes the hydrolysis of fructans to fructose, which are degraded during storage under traditional treatment conditions (70 °C/94% RH) through the Maillard reaction causing the faster formation of compounds responsible for the color of black garlic compared to black garlic without pre-treatment. This reaction cuts down the time to attain the characteristic color of black garlic from 30 to 12 days. The 5-hydroxymethyl-2-furaldehyde (HMF) content was 3.6 times higher in OH-BG than in TT-BG. The polyphenol content and antioxidant capacity did not show differences between TT-BG and OH-BG, but their levels were superior to commercial sample. Both products (OH-BG and TT-BG) were sweet (36.8 and 31.6 °Bx, respectively) and had an excellent organoleptic acceptance (75 and 70%). These results highlight the potential of ohmic heating pretreatment in reducing the time to obtain good quality BG.

Small Differences in SUC Gene Sequences Impact Saccharomyces cerevisiae Invertase Activity and Specificity toward Fructans with Different Chain Lengths.

Saccharomyces cerevisiae (S. cerevisiae)invertase is encoded by a family of closely related SUC genes. To identify and understand the molecular basis for differences in substrate specificity, we examined 29 SUC alleles from industrialS. cerevisiaestrains and cloned alleles with small sequence differences into an invertase-negative strain. Our study showed that an F102Y substitution in Suc-enzymes lowers yeast invertase activity toward fructo-oligosaccharides (FOS) by 36% and the specificity factor by 43%. By contrast, an A409P substitution in Suc-enzymes resulted in an increased capacity of the yeast to hydrolyze FOS and Fibruline by 17 and 41%, respectively, likely because of a change in the loop conformation resulting in a wider active site. Bread dough fermentation experiments revealed that sucrose and fructan hydrolysis during fermentation is influenced by this natural variation in SUC sequences. Our research thus opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that may ultimately allow controlling fructan hydrolysis.

Characterization of the Extracellular Fructanase FruA in Lactobacillus crispatus and Its Contribution to Fructan Hydrolysis in Breadmaking.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.

Variability in yeast invertase activity determines the extent of fructan hydrolysis during wheat dough fermentation and final FODMAP levels in bread

Consumption of fructan-containing cereal products is considered beneficial for most people, but not for those suffering from irritable bowel syndrome (IBS), as they should avoid the consumption of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (acronym: FODMAP). Controlling fructan levels in cereal products is not trivial. However, controlling yeast invertase-mediated hydrolysis of fructan during dough fermentation might offer a handle to modulate fructan concentrations. In this work, the variability in invertase activity and substrate specificity in an extensive set of industrial Saccharomyces cerevisiae strains is investigated. Analysis showed a high variability in the capacity of these strains to hydrolyse sucrose and fructo-oligosaccharides. Industrial yeast strains with a high activity towards fructo-oligosaccharides efficiently reduced wheat grain fructans during dough fermentation to a final fructan level of 0.3% dm, whereas strains with a low invertase activity yielded fructan levels around 0.6% dm. The non-bakery strains produced lower levels of CO2 in fermenting dough resulting in lower loaf volumes. However, CO2 production and loaf volume could be increased by the addition of 3% glucose. In conclusion, this study shows that variation in yeast invertase activity and specificity can be used to modulate the fructan content in bread, allowing the production of low FODMAP breads, or alternatively, breads with a higher soluble dietary fibre content.

Abscisic acid associated with key enzymes and genes involving in dynamic flux of water soluble carbohydrates in wheat peduncle under terminal drought stress

Remobilization of stem water soluble carbohydrates (WSC) can supply crucial carbon resources for grain filling under drought stress, while the regulatory metabolism associated with abscisic acid (ABA) is still limited. Two cultivars, LJ196 (drought-tolerant) and XD18 (drought-prone), were pot-grown under well-watered (WW) and drought-stressed (DS) conditions. Concentrations of WSC components and ABA, and fructan metabolizing enzymes and genes were investigated in peduncle after anthesis. When compared with those under the WW, LJ196 remained higher grain yield and grain-filling rate than XD18 under the DS. During the early period of grain filling (0–14 DAA), DS increased concentrations of total WSC and its components, but thereafter substantially reduced them. The gene expression levels and enzymatic activities of fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH) showed similar trends, whereas those of fructan: fructan 1-fructosyltransferase (1-FFT), and sucrose: fructan 6-fructosyltransferase (6-SFT) were depressed and declined over the period of examination. LJ196 still showed higher levels of ABA and fructan metabolizing. The ABA concentration under the DS was positively and significantly correlated with total WSC and fructan concentration, and expression levels of these enzymes and genes as well, with more prominently with those of 6-FEH. Presumably, ABA could enhance fructan hydrolysis by strongly up-regulating the gene expression and enzymatic activity of 6-FEH to accelerate WSC remobilization. However, stem WSC induced by DS could be not fully remobilized to grains, due to its weaker correlation with grain-filling rate and finally indicating lower grain yield. The findings would provide useful information for wheat production under water-deficit environments.

Novel process to obtain agave sap (aguamiel) by directed enzymatic hydrolysis of agave juice fructans

Kluyveromyces marxianus and Saccharomyces paradoxus, which are the native microorganisms found in aguamiel, were used successfully for exo- and endo-fructanase synthesis to hydrolyze agave fructans important in agave sap production. We studied culture media for enzyme synthesis, as well as the physicochemical properties and enzyme activity, and we optimized the bioprocess parameters and the enzyme extract mixture to maximize agave fructan hydrolysis. The results showed that the best culture medium for fructanase synthesis contained 14.0, 20.0, and 1.0 (g-L−1) of sucrose, peptone, and K2HPO4, respectively. The best enzyme-to-substrate ratio for agave juice containing 90 g-L−1 of fructans was 10:1. Under constant operating conditions of 45.25 °C, 65.47 rpm, and pH 4.28, a product with 93.62 g-L−1of total reducing sugars (TRS) and fructooligosaccharides (FOS) with a polymerization degree (PD) from 3 to 10 hexose units was obtained using a mixture of 98.4% K. marxianus and 1.6% S. paradoxus enzyme extracts. The resulting agave sap preserves the natural flavor and tastes closest to natural agave sap (aguamiel); hence, the results show, for the first time, a resolution of the technical challenge for stable agave sap production and confirm the importance of using native strains to evolve from spontaneous to directed fermentation.

Determination of Fructan (Inulin, FOS, Levan, and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients): Single-Laboratory Validation, First Action 2018.07.

Traditional enzyme-based methods for measurement of fructan were designed to measure just inulin and branched-type (agave) fructans. The enzymes employed, namely exo-inulinase and endo-inulinase, give incompletely hydrolysis of levan. Levan hydrolysis requires a third enzyme, endo-levanase. This paper describes a method and commercial test kit (Megazyme Fructan Assay Kit) for the determination of all types of fructan (inulin, levan, and branched) in a variety of animal feeds and pet foods. The method has been validated in a single laboratory for analysis of pure inulin, agave fructan, levan, and a range of fructan containing samples. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo-inulinase, endo-inulinase, and endo-levanase, followed by measurement of these sugars using the PAHBAH reducing sugar method which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are specifically hydrolyzed and removed by borohydride reduction. The single-laboratory validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit®), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX®) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included working range, target selectivity, recovery, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. The method is robust, quick, and simple.

Inulin enrichment of gluten free breads: Interaction between inulin and yeast.

Inulin can improve the nutritional quality of gluten free (GF) bread and have a prebiotic activity. However, breadmaking might frustrate the enrichments efforts due to inulin loss. In this study we aimed at studying the inulin enrichment of GF bread. Two different yeasts [having normal (Y1) or reduced (Y2) invertase activity] were used to leaven the breads enriched with five marketed inulins, which differed for the degree of polymerization (DP) and the manufacturer. Inulin replaced 10% of the rice flour and had low, intermediate or high DP, which ranged from 2 to 20; ≈20; ≥20, respectively. Fructan hydrolysis occurred during leavening of Y1-GF breads, reaching losses up to 40% after baking, depending on the diverse DP of the inulin-forming fructans. Inulin loss was less relevant in Y2-GF breads (up to 5% after baking) than Y1-GF breads. Crumb texture was not negatively influenced by inulin presence, even if this was high (e.g., Y2-GF breads). Information collected within this study may provide further insight to better optimize a GF bread formulation in view of inulin enrichment.

Purification, characterization, and functional analysis of a novel 6G&1-FEH mainly hydrolyzing neokestose from asparagus.

Asparagus (Asparagus officinalis L.) accumulates inulin- and inulin neoseries-type fructans. Fructose released by the hydrolysis of fructans is an energy source for emerging asparagus spears. Plant fructans are hydrolyzed by fructan exohydrolases (FEHs), whose presence in asparagus has not yet been fully characterized. Here, we describe for the first time the purification and characterization of an FEH from asparagus, and the functional analysis of its gene. The purified enzyme was predicted to exist as a dimer (approximately 130 kDa) consisting of two polypeptides with a molecular mass of approximately 68 kDa. N-terminal sequences of the purified enzyme were matched with the amino acid sequences of aoeh4a and aoeh4b cDNAs isolated from asparagus (cv. Gijnlim and Taihouwase). Native enzymes obtained from asparagus roots and recombinant enzymes produced by Pichia pastoris showed fructan 1-exohydrolase (1-FEH) activity via the hydrolysis of inulin-type fructan. Unlike other 1-FEHs, these enzymes showed minimal hydrolysis of 1-kestose but efficiently hydrolyzed neokestose. Therefore, the enzyme was termed 6G&1-FEH. Gene expression studies in asparagus roots showed that aoeh4 increased during root storage at 2 °C and spear harvesting. These findings suggest that 6G&1-FEH may be involved in fructan hydrolysis in asparagus roots to provide an energy source for emerging asparagus spears.

Transcript Profiling of Genes Encoding Fructan and Sucrose Metabolism in Wheat Under Terminal Drought Stress

Maintaining photosynthetic performance and remobilization of assimilates stored in vegetative tissues are strategies of superior wheat genotypes under drought stress conditions. To better understand the response of vegetative tissues to drought stress at the grain filling period, transcript profiling of genes encoding fructan and sucrose metabolism were studied in the stem (penultimate internode) and root of two drought-tolerant genotypes. Based on a preliminary screening, the cultivars T-65-7-1 (the mutant line) and Tabasi (wild type) were selected for further study with respect to the parameters associated with photosynthesis and stem remobilization under rain-fed conditions. The expression of photosynthetic genes, chlorophyll content and relative water content were sharply reduced in the T-65-7-1 compared to Tabasi, as a result of drought-induced leaf senescence. Under drought stress, fructan remobilization in the stem and root of T-65-7-1 was significantly higher than Tabasi, which was due to the over-expressed genes involved in the synthesis and hydrolysis of fructan, as well as the synthesis, hydrolysis and transport of sucrose. The stem and root tissues depicted similar assimilate remobilization behaviours under drought stress. The grain yield reduction was less in T-65-7-1 than Tabasi under drought stress during the grain filling period, therefore, the remobilization of assimilates to the grains was a more effective strategy than maintenance of photosynthesis under drought stress conditions during the grain filling period. This research provides valuable molecular indicators for selecting drought-tolerant wheat genotypes with high fructan content and increased remobilization in wheat breeding programs.

Effect of Drought Stress on the Expression of Genes Involved in Synthesis and Hydrolysis of Fructan During Remobilization of Assimilates in Wheat Root

Effect of Drought Stress on the Expression of Genes Involved in Synthesis and Hydrolysis of Fructan During Remobilization of Assimilates in Wheat Root

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10g L-1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6g L-1, showing a high theoretical yield of 92.3%.