Β-glucanase Activity Research Articles

Minimizing molecular weight reduction of β-glucan during barley bread making

During the production of composite wheat/barley breads β-glucans are rapidly degraded by endogenous wheat and barley flour β-glucanases. Since the physiological effect of β-glucans is related to their molecular weight a protection from degradation is essential in order to retain their full effect on the mitigation of serum cholesterol and blood glucose levels. In this study we have shortened the contact time between barley flour and water while focusing on the production of good quality barley breads. Incorporation of barley flour into already fermented wheat dough or omitting fermentation and shortening proofing times by more yeast and sucrose addition enabled the production of barley bread with increased β-glucan calcofluor weight average molecular weight (Mwcalc) and good bread quality (crumb fineness, bread slice area). The use of barley components with bigger particle size like coarse barley flour or intact barley flakes further increased β-glucan molecular weight in bread. Inactivation (with scalding) or reduction (dry heat treatment) of β-glucanase activity in barley flour prior to use, on the other hand, had little effect on β-glucan molecular weight reduction during one of the optimized short bread making processes.

Effect of barley and oat β-glucan concentrates on gluten-free rice-based doughs and bread characteristics

The impact of commercial oat or barley β-glucan concentrates incorporated at different levels (1.3–3.9 % actual β-glucan concentration, flour basis) into gluten-free (GF) rice-based dough formulations differing in water content (89–141%, flour basis) on dough rheology (empirical and fundamental tests) and breadmaking performance has been investigated. The effect of the baking process on the content and molecular weight of β-glucan in the final bread has been also evaluated. The rheological properties of the dough were dramatically influenced by dough water content; i.e. optimization of dough hydration is indeed of primary importance on improving GF bread quality. The bread specific volume was negatively correlated with the dough elastic modulus (G′) and the viscosity (η0), and positively with the loss tangent. At optimum hydration, the rheological properties of barley β-glucans-enriched doughs and the quality attributes of breads derived therefrom were notably affected by the soluble fibre content; the G′ at 1 Hz increased up to ∼100% and the bread volume decreased ∼32% with respect to the values of the control dough and bread. In contrast, the impact of concentration of the oat-β-glucan preparation in the fortified doughs and bread was much less pronounced. These findings could be explained by the ability of the low molecular weight barley β-glucans to develop a gel network structure at higher concentrations, whereas the preparation of the high molecular weight oat β-glucan exhibits a more viscous-like rheological response. The added β-glucans to doughs were also quantitated in the bread crumbs, and a significant decrease in their molecular weight was noted, most likely due to the β-glucanase activity in the raw materials incorporated in the GF flour mixtures. Consequently, although the EFSA claims are achievable in gluten-free breads enriched with commercial β-glucans concentrates, control of β-glucanase activity in the raw materials may be a critical issue in exerting all the physiological benefits associated with the consumption of these bioactive polysaccharides.

МЕТОДЫ ПОДГОТОВКИ РАСТИТЕЛЬНОГО СЫРЬЯ К БИОКОНВЕРСИИ В КОРМОВЫЕ ПРОДУКТЫ И БИОЭТАНОЛ

An analysis of literature data the following preprocessing methods starch, pentosane- and cellulosic feedstock for enzymatic hydrolysis polysaccharides: hydrothermal, organosolvent obtaining technical cellulose, organosolvent with sulfuric acid, acid-catalyzed steam explosion, alkali, bisulfite (SPORL) and acid-catalyzed using a disk mill is presented.Recommendations about improvement of technological schemes of obtaining fodder protein products of bran together with other grain waste are provided. Technological scheme should include additional process fine grinding of bran and grain waste and hydro-thermodynamic treatment (HTDO) at the next technological mode: a.d.s ≥ 15%; t = 86–100 °C; processing time ≤ 1 h; using a thermostable α-amylase (Zymajunt-340С* or Amylase HT-4000 or Amileks 4T). The effectiveness of a subsequent enzymatic hydrolysis of partially decomposed starch and non-starch polysaccharides of starchy raw material is ensured by the enzymatic Начало формыКонец формыcomplex including Glucoamylase, GC 220, containing cellulase, cellobiase, xylanase and β-glucanase activities, and Novozymes 188, containing cellobiase.The most effective methods for preprocessing of pentosan - and cellulosic feedstock to enzymatic hydrolysis of cellulose in a 2% suspension of lignocellulosic residue are allow obtained an overall yield of sugars and ethanol at least 90% of the theoretical. The following preprocessing methods are considered to be as the most promising: organosolvent with H2SO4 and steam explosion with SO2 and the subsequent washing of the extruded biomass with water and oxidation-alkaline solution and mechanochemical: bisulfite (SPORL) and acid catalysis units with installation of hot grinding (IHG). It is established, that in the first two methods allowed the lignin content in the lignocellulose is to 16-18%, and the third up to 30%. Acid-catalyzed hydrolysis method with elements of SPORL technology with the use of installation hot grinding (IHG) is recommended for laboratory and experimental-industrial tests in the hydrolysis industry.Cellulose from lignocellulosic residue is efficiently hydrolysed by Сelluclast (20 FPU/g glucan), and Novozyme 188 (β-glucosidase : cellulase 1,75–2,0 : 1) or Spezyme CP, GC-220 (15 FPU/g glucan), Multifect Xylanase (β-glucosidase:cellulase 2,0 : 1).

Effect of Steam Explosion on In Vitro Gas Production Kinetics and Rumen Fermentation Profiles of Three Common Straws

To investigate the effect of steam explosion on in vitro gas production (GP) and rumen fermentation profiles of common straws, in vitro cultivation was conducted for 96 h with the rumen fluid collected from steers. Different types of straw had various chemical compositions, which were affected by steam explosion (P<0.01). Steam explosion increased (P<0.01) the rate and volume of GP, lag time disappeared and asymptotic GP decreased, which were also affected (P<0.01) by the type of straw. The type of straw influenced (P<0.05) the final pH, while steam explosion exerted an effect (P<0.01) on the ammonia-nitrogen concentration. The proportions of individual volatile fatty acid (VFA), except acetate (A), differed (P<0.05) among the feeds. Steam explosion increased total VFA production and the proportion of propionate (P), while decreased the proportions of A, isobutyrate and valerate as well as the ratio A/P (P<0.01). The type of straw had an effect (P<0.05) on the activities of avicelase and carboxymethyl cellulase (CMCase), while steam explosion increased (P<0.01) the activities of avicelase, CMCase, β-glucanase and xylanase. The available energy concentrations and digestibilities differed (P<0.01) in the feeds and were increased (P<0.05) with steam explosion processing. The interaction straw type×treatments was significant (P<0.05) for most monitored parameters. These results suggest that steam explosion could improve rumen fermentability and energy utilisation of straw, being an effective pre-treatment method in feed industry.

누룩곰팡이 분리균의 다양성 및 당화능 분석과 독소생산능 조사

Nuruk samples collected from various regions in Korea were investigated in terms of fungal contents and diversity. In measurement of colony forming unit (CFU) in Nuruk suspensions on DRBC agar, Nuruk samples MS4, MS8, and MS10 were among the highest fungal density, with 1,278.9±21.6 (×10 4 ), 1,868.0±27.7 (×10 4 ), and 775.1±19.2 (×10 4 ) were among the samples showing the highest fungal density. CFU per 20 mg Nuruk, respectively. The majority of fungal components were yeasts, including Pichia anomala, P. kudriavzevii, Kluyveromyces marxianus, and Saccharomycopsis fibuligera, whereas Aspergillus oryzae and Rhizopus oryzae, the representative Nuruk fungi, were predominant only in the low fungal density Nuruks (MS2, MS5, and MS11). Saccharification capability of the fungal isolates was assessed by measurement of amylase activity in the culture broth. The highest amylase activity was found in A. niger and A. luchuensis, followed by S. fibuligera. A. oryzae and R. oryzae showed fair amylase activity but significantly lower than those of the three fungal species. R. oryzae was suggested to play an additional role in degradation of β-glucan in crop component of Nuruk since R. oryzae was the only fungus that showed β-glucanase activity among the fungal isolates. To confirm the safety of Nuruk, aflatoxigenicity of the isolated Aspergillus was estimated using the DNA markers norB-cypA, aflR, and omtA. All of the isolates turned out to be non-aflatoxigenic as evidenced by the deletion of gene markers, norB-cypA and aflR, and the absence of aflatoxin in the culture supernatants shown by TLC analysis.

Temporal restriction of enzyme supplementation in barley-based diets has no effect in broiler performance

It is now well established that exogenous microbial β-glucanases effectively contribute to improve the nutritive value of barley based diets for broilers while they are predominantly effective in the first stages of the production cycle. Here, the performance of broilers fed on barley-based diets supplemented with a β-glucanase enzyme mixture, in part or during the entire length of the production cycle, was evaluated. Six hundred 1-day-old Ross 308 chicks were randomly divided into 4 groups. Animals were fed on a barley-based diet supplemented with a commercial enzyme cocktail for the entire duration of the trial (35 days) or exclusively during the first 11 or 23 days of the experiment. Animals of the control group were fed on the basal diet not containing the microbial biocatalyst mixture. Body weight and feed intake were recorded weekly and weight gain and feed conversion ratios were determined. At the end of the trial, 2 animals were slaughtered to collect gastrointestinal contents to evaluate viscosity and enzymatic activity. Weight/length of gastrointestinal compartments were also recorded. Data revealed that all β-glucanase supplemented groups outperformed non-supplemented birds at day 35. Significant differences in body weight were detected at day 11, suggesting that response to enzyme supplementation occurs, primarily, during the initial stages of broiler growth. In addition, final body weight of animals fed on diets supplemented during the first 11 or 23 days of the experiment was identical to animals supplemented during the entire trial (P>0.05). Together the data suggest that in barley-based diets exogenous enzymes act primarily in the earliest periods of broilers growing period and that enzyme supplementation may be restricted to the first 11 days of the production cycle without negatively affecting animal performance. Zymogram analysis allowed detecting β-glucanase activity in the crop of non-supplemented birds at day 35. This activity does not arise from endogenous enzymes present in the feed suggesting colonization of the crop with a β-glucanase secreting microbial population. Although this activity was not sufficient to reduce digesta viscosity in the upper portions of the GI tract when compared with birds exposed during the entire period of the experiment to the exogenous enzymes, it might have contributed to attenuate the antinutritive effects of β-glucans at later stages of animal growth.

Viscosity based quantification of endogenous β-glucanase activity in flour

High molecular weight (MW) is a key parameter for cereal β-glucans physiological benefits like decreased serum cholesterol and attenuated post prandial blood glucose. However, the activity of endogenous flour enzymes during bread production results in a decrease of β-glucan MW. The depolymerization of a standard β-glucan solution by different flour extracts (wheat, barley and rye) was followed by measuring the viscosity decrease with a Rheometer. The slope of the inverse viscosity (1/η) against degradation time was used to quantify β-glucanase activity by comparison with slopes obtained with known concentrations of the β-glucanase Lichenase. Results correlated well with depolymerization rates estimated by HPSEC. The viscosity based method is rapid (20min per sample), accurate (≤6% variation), and a powerful screening tool for identifying flour fractions with low β-glucanase activity, treatments that can inactivate β-glucanases in flour, or the development of β-glucanase inhibitors for the use in e.g. bread making.

Immobilization of Piromyces rhizinflata β-Glucanase on Poly(Dimethylsiloxane) and Si Wafer and Prediction of Optimum Reaction for Enzyme Activity

EglA, a β-1,4-glucanase isolated from the ruminal fungus Piromyces rhizinflata, shows promise in a wide range of industrial applications because of its broad substrate specificity. In this study, EglA was immobilized on different supporting materials including poly(dimethylsiloxane) (PDMS), Si wafer, textured Si wafer, and indium tin oxide-coated (ITO-coated) glass. The binding abilities of PDMS and Si wafer toward EglA were significantly higher than those of the other supporting materials. The optimized temperature and pH conditions for EglA immobilized on PDMS and on Si wafer were further determined by a response surface methodology (RSM) combined with a central composite design (CCD). The results indicated that the optimum pH and temperature values as well as the specific β-glucanase activity of EglA on PDMS were higher than those of free-form EglA. In addition, EglA immobilized on PDMS could be reused up to six times with detectable enzyme activity, while the enzyme activity of Eg1A on Si wafer was undetectable after three cycles of enzyme reaction. The results demonstrate that PDMS is an attractive supporting material for EglA immobilization and could be developed into an enzyme chip or enzyme tube for potential industrial applications.

Viscosity reduction of cassava for very high gravity ethanol fermentation using cell wall degrading enzymes from Aspergillus aculeatus

Cassava is an important feedstock for bioethanol production; however, its use in very high gravity (VHG) fermentation is limited by the high viscosity of mash due to the presence of plant cell wall derived polysaccharides. In this study, viscosity reduction of cassava root mash, chips, and pulp was achieved using a mixture of cell wall degrading enzymes prepared from solid state fermentation of Aspergillus aculeatus BCC17849. Proteomic analysis showed the mixture contained endo- and exo-acting cellulases, hemicellulases, and pectinases from various glycosyl hydrolase families. Enzymatic pretreatment of cassava substrates with the enzyme mixture containing endoglucanase, FPase, xylanase, polygalacturonase, β-glucanase, and mannanase activities at 45°C, pH 5.0 for 2h reduced viscosity to the operating level of <500mPas, equivalent to the final viscosity of 3.0–51.3% of the initial levels. Simultaneous saccharification and fermentation of the pretreated root mash (32% initial solid) by Saccharomyces cerevisiae with a conventional high-temperature liquefaction process and an uncooked process using a raw starch degrading amylase at 32°C for 96h led to a final ethanol concentration of 19.65 and 17.54% (v/v), respectively. The results demonstrated potential of the enzyme for improving process efficiency and economics in VHG bioethanol production.

Effects of malting on molecular weight distribution and content of water-extractable β-glucans in barley

In the beer industry β-glucans are extensively studied non-starch polysaccharides due to their ability to increase the viscosity of solutions and to form gels. The current study was designed to determine the total and water-soluble β-glucan contents of barley during malting. Total and water-soluble β-glucans were analyzed from two different malts that originated from the same barley but varied in germination time from 36h (malt A) to 72h (malt B). Water-soluble β-glucans were also characterized using high-performance size-exclusion chromatography with triple-detector analysis (HPSEC-TDA) to evaluate the variation in molecular weight distributions, intrinsic viscosity, radius of gyration, Mark–Houwink parameters and polydispersity and thus the overall structural changes during malting. Total β-glucan content decreased from barley to malt due to the action of β-glucanase and was greatest in malt B (where 92% of β-glucans were degraded) which highlights the influence of germination time. β-Glucan solubility increased during malting, again particularly in malt B, where most of β-glucans became soluble. The β-glucanase activity also affected the molecular weight of the polymers which ranged from 298·103g/mol in barley to 293·103 and 218·103g/mol in malts A and B respectively. The molar mass of the most abundant fraction decreased from barley (256·103g/mol) to malt A (112·103g/mol) and malt B (89·103g/mol), again highlighting the effect of the longer germination time. Proceeding from barley to malt, the cumulative molar mass distribution function confirmed that the weight fraction of polymers below 200·103g/mol increased, while the high molecular weight fraction (between 200·103g/mol and 400·103g/mol) decreased. Moreover, the presence of a higher molecular weight fraction (14–16%) beyond 400·103g/mol which does not change during malting was observed. The Mark–Houwink constants α and log k confirmed the random coil conformation of soluble β-glucans and showed an increase in the compactness of the macromolecules from barley to malts.

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.

Simultaneous enhanced catalytic activity and thermostability of a 1,3-1,4-β-glucanase from Bacillus amyloliqueformis by chemical modification of lysine residues

The thermostablility and enzymatic activity of 1,3-1,4-β-glucanase (BglA) from Bacillus amyloliquefaciens was improved by modifying five (out of 12) ε-amino groups in lysine residues with nitrous acid. The optimal modification condition for BglA was determined as 30mM nitrous acid at, 40°C for 30min. The optimally-modified BglA had higher specific activity and T 50 value, which were 3,370U/mg and 70°C, respectively. Its half-life values at 50 and 60°C were extended and reached 58.5 and 49.5min, respectively. Circular dichroism analysis showed that the secondary structures in modified BglA were almost the same with that of wild-type BglA. Thus, modification of lysine residues can simultaneously improve the activity and thermostability of β-glucanase which are ideal targets for further protein engineering.

Impact of flour particle size and autoclaving on β-glucan physicochemical properties and starch digestibility of barley rusks as assessed by in vitro assays

Abstract Barley rusks are traditional bakery items that existed as food staple of the Cretan—Mediterranean diet and a natural source of cereal β-glucans that have well-recognized hypocholesterolemic and hypoglycemic properties, largely attributed to their rheological behavior. The aim of this work was to evaluate the influence of flour particle size and hydrothermal treatment on the potential nutritional functionality of barley rusks. Rusks were made using either a coarse ( d 50 350 μm) or a fine ( d 50 200 μm) barley flour stream without (control) or with prior autoclaving at two moisture levels (11–12 and 13–14%). HPLC-SEC-RI profiles of β-glucans isolated from barley flours and rusk revealed a shift of their molecular weight (MW), from above 1×10 6 to 0.17–0.28×10 6 , upon rusk making due to endogenous β-glucanase activity. However, the MW of β-glucans in the products was preserved upon complete thermal inactivation of β-glucanase, merely by flour autoclaving at 13–14% moisture. The ‘physiological impact’ of barley rusks was assessed by measuring the amount of solubilized β-glucans (0.5–0.7 g/100 g rusk) and the viscosity (2–7 mPa s) of the rusk extracts, using an in vitro digestion protocol. Both autoclaving and increasing flour particle size significantly ( p in vitro digestion assay, lower enzymic starch degradation was noted for the coarse flour rusks compared to the products made from the fine flour (18–37% vs. 32–53% after 5 h of digestion). Flour autoclaving also led to significant ( p p =0.003) between the degree of starch enzymic degradation and the viscosity of the rusk extracts was found. Overall, flour particle size and autoclaving of flour could be key processing factors for making barley-based baked items with improved nutritional–physiological function.

Intestine-specific expression of the β-glucanase in mice

Guan, L.-Z., Xi, Q.-Y., Sun, Y.-P., Wang, J.-L., Zhou, J.-Y., Shu, G., Jiang, Q.-Y. and Zhang, Y.-L. 2014. Intestine-specific expression of the β-glucanase in mice. Can. J. Anim. Sci. 94: 287–293. The β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was cloned into a specific expression plasmid (MUC2-GLU-LV). Transgenic mice were prepared by microinjection. Polymerase chain reaction and Southern blot analysis of genomic DNA extracted from the tail tissue of transgenic mice showed that the mice carried the β-glucanase gene. Northern blot analysis indicated that β-glucanase was specifically expressed in the intestine of the transgenic mice. The β-glucanase activity in the intestinal contents was found to be 1.23±0.32 U mL−1. The crude protein, crude fat digestibility of transgenic mice were increased by 9.32 and 5.09% (P&lt;0.05), respectively, compared with that of the non-transgenic mice, while moisture in feces was reduced by 12.16% (P&lt;0.05). These results suggest that the expression of β-glucanase in the intestine of animals offers a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.

Intestine-specific expression of the β-glucanase in mice

Guan, L.-Z., Xi, Q.-Y., Sun, Y.-P., Wang, J.-L., Zhou, J.-Y., Shu, G., Jiang, Q.-Y. and Zhang, Y.-L. 2014. Intestine-specific expression of the β-glucanase in mice. Can. J. Anim. Sci. 94: 287-293. The β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was cloned into a specific expression plasmid (MUC2-GLU-LV). Transgenic mice were prepared by microinjection. Polymerase chain reaction and Southern blot analysis of genomic DNA extracted from the tail tissue of transgenic mice showed that the mice carried the β-glucanase gene. Northern blot analysis indicated that β-glucanase was specifically expressed in the intestine of the transgenic mice. The β-glucanase activity in the intestinal contents was found to be 1.23±0.32 U mL-1. The crude protein, crude fat digestibility of transgenic mice were increased by 9.32 and 5.09% (P<0.05), respectively, compared with that of the non-transgenic mice, while moisture in feces was reduced by 12.16% (P<0.05). These results suggest that the expression of β-glucanase i...

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

BackgroundIncorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.Methodology/Principal FindingsHere we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.Conclusion/SignificanceOur study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

Malting of barley with combinations of Lactobacillus plantarum, Aspergillus niger, Trichoderma reesei, Rhizopus oligosporus and Geotrichum candidum to enhance malt quality

Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, β-glucan- and arabinoxylan-rich cell walls have to be degraded. β-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced β-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt β-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on β-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality.

TpBGL2codes for aTetrapisispora phaffiikiller toxin active against wine spoilage yeasts

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a β-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding β-glucanase of T.phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

Effects of a β-glucanase enzymatic preparation on yeast lysis during aging of traditional sparkling wines

Scientific researches on characterization of the commercial enological preparation of Lallzyme MMX® containing β-glucanase and its influence on autolysis of different yeast strains typically employed in the production of sparkling wine are lacking. The aim of the present work was to plug a gap in this field, studying the β-glucanase activity of Lallzyme MMX® and its interactions with BCS103® and EC1118® yeast strains. The results showed that β-glucanase was slightly inhibited by ethanol, but its residual activity at wine pH was sufficient for the purposes. Kinetic parameters showed a better enzyme–substrate complex formation for the EC1118® strain. The influence on yeast lysis during 12months of bottle-aging was monitored, demonstrating that enzyme addition did not substantially influence either the content and progression of total proteins, or foam characteristics. However, scanning and transmission electron microscopy images and free amino acid analysis indicated β-glucanase improved cell wall degradation of both selected yeasts, evidencing a lower autolytic capacity of the BCS103® strain. Our study demonstrated that addition of β-glucanase catalyzed cell disorganization and promoted release of yeast components into sparkling wine, with strain-dependent effects. Therefore, employment of β-glucanase rich Lallzyme MMX® might effectively accelerate some aging characteristics of traditional sparkling wines.

Effect of the use of dilute alkaline prior to Bacillus subtilis-based biocontrol steeping and germination conditions on red sorghum malt β-glucanase activities and residual β-glucans

Malting is the ideal stage to deal with β-glucans. Their hydrolysis is very important as the diffusion of both hormones and hydrolytic enzymes in the endosperm of germinated grain depend on it. A high malt β-glucanase activity is not a guarantee of an extensive hydrolysis of β-glucans. When Bacillus subtilis is used to control mould growth, red sorghum malt β-glucanase activity (measured using carboxymethylcellulose as the substrate) was improved without significantly affecting the hydrolysis of malt β-glucans. Thus, in order to reduce the residual β-glucans content, soaking in 0.2% NaOH was combined with a biocontrol. Soaking in 0.2% NaOH is recognized as capable of improving grain hydration by opening-up the endosperm cell walls. The combined use of 0.2% NaOH with B. subtilis-based biocontrol treatments during red sorghum malting, leads to malt with increased β-glucanase activity and a significant reduction of residual β-glucans when compared with the 16 h biocontrol steeping without prior steeping in 0.2% NaOH. β-glucanase activity increases with increased germination temperature and time while, conversely, the residual β-glucans content of the malts decreases. Indeed, while the level of β-glucanase was not vastly different between the malts obtained after steeping in distilled water and those obtained after 8 h steeping in 0.2% NaOH followed by 8 h resteeping in distilled water (NaOH + H2O treatment), their residual β-glucans levels differ significantly. B. subtilis-based treatment leads to malt with improved β-(1-3)- and β-(1-4)-glucanase activities without significantly improved malt β-(1-3),(1-4)-glucanase activity. While malts obtained after 84 h germination weren't significantly different in terms of malt β-(1-3),(1-4)-glucanase activities for all steeping treatments, the use of 0.2% NaOH steeping prior to resteeping led to malts with improved β-glucans content. Combining the steeping in dilute alkaline and biocontrol enables taking advantage of the dilute alkaline effect on residual β-glucans content, due probably to the opening-up of the cell walls and the improvement of water uptake, and that of the biocontrol (improvement of β-glucanase synthesis).