Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

Effects of malting on β‐glucanase and phytase activity in barley grain

The effects of malting on β‐glucan and phytate were investigated in one naked and one covered barley by a full factorial experiment with three factors (steeping temperature, moisture content and germination temperature) each with two levels. Analysis of total content of β‐glucan in the malted samples showed small changes after steeping at the high temperature (48 °C), while steeping at the lower temperature (15 °C) gave a significantly lower content. This trend was even stronger for β‐glucan unextractable at 38 °C. Analysis of the activity of β‐glucanase for the samples steeped at 15 °C showed a strong increase over the time of germination, while those steeped at 48 °C had a much slower development. The other two factors influenced the outcome to a small extent, mainly because the steeping temperature was the most important factor overall where any changes in β‐glucan and β‐glucanase were observed. When β‐glucan was extracted at 100 °C, a larger yield was obtained, and this was influenced by the steeping temperature in a much stronger way than for β‐glucan extracted at 38 °C. Determination of average molecular weight for β‐glucan extracted at 100 °C gave a lower value for samples steeped at 15 than at 48 °C. The design did not have any large effects on phytate degradation and phytase activity. However, it indicated that selective control of the enzymes might be possible, since phytase activity was barely affected by the parameters studied, while β‐glucanase was heavily affected. © 2002 Society of Chemical Industry     

Phytate content is reduced and β‐glucanase activity suppressed in malted barley steeped with lactic acid at high temperature

The effect of different steeping conditions on phytate, β‐glucan and vitamin E in barley during malting was studied by a full factorial experiment with three variables (steeping temperature, barley variety and steeping additions). Addition of lactic acid to the steeping water induced a reduction of phytate during steeping and germination, especially in combination with the high steeping temperature (48 °C). Furthermore, lactic acid and high temperature steeping inhibited β‐glucanase development, resulting in a well‐preserved β‐glucan content after germination. When steeping was conducted without addition of lactic acid, the low steeping temperature (15 °C) promoted development of both phytase and β‐glucanase activity during germination. A slightly higher level of tocopherols and tocotrienols was observed in samples steeped at 15 °C than in samples steeped at 48 °C. However, addition of lactic acid reduced the amount for both temperatures. When lactic acid bacteria were added to the steeping water none of the parameters studied differed from samples steeped with water only. The results show the possibility of combining phytate degradation with a preserved β‐glucan content during malting and can thus be of interest for development of cereal products with improved nutritional value. Copyright © 2004 Society of Chemical Industry     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

Characterization of the Supermolecular Structure of Polydatin/6‐O‐α‐Maltosyl‐β‐cyclodextrin Inclusion Complex

Polydatin is the main bioactive ingredient in many medicinal plants, such as Hu‐zhang (Polygonum cuspidatum), with many bioactivities. However, its poor aqueous solubility restricts its application in functional food. In this work, 6‐O‐α‐Maltosyl‐β‐cyclodextrin (Malt‐β‐CD), a new kind of β‐CD derivative was used to enhance the aqueous solubility and stability of polydatin by forming the inclusion complex. The phase solubility study showed that polydatin and Malt‐β‐CD could form the complex with the stoichiometric ratio of 1:1. The supermolecular structure of the polydatin/Malt‐β‐CD complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffractometry (XRD), thermogravimetric/differential scanning calorimetry (TG/DSC), and proton nuclear magnetic resonance (¹H‐NMR) spectroscopy. The changes of the characteristic spectral and thermal properties of polydatin suggested that polydatin could entrap inside the cavity of Malt‐β‐CD. Furthermore, to reasonably understand the complexation mode, the supermolecular structure of polydatin/Malt‐β‐CD inclusion complex was postulated by a molecular docking method based on Autodock 4.2.3. It was clearly observed that the ring B of polydatin oriented toward the narrow rim of Malt‐β‐CD with ring A and glucosyl group practically exposed to the wide rim by hydrogen bonding, which was in a good agreement with the spectral data.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Effect of High Hydrostatic Pressure‐Temperature Combinations on the Activity of β‐Glucanase from Barley Malt

β‐Glucanase from barley malt is known to be thermolabile but important in the mashing process. Therefore, the potential of increasing the thermostability of β‐glucanase in ACES buffer (0.1 M, pH 5.6) by high hydrostatic pressure has been investtigated. Inactivation of the enzyme as well as changes of the conversion rate in response to combined pressure‐temperature treatments in the range of 0.1–900 MPa and 30–75°C were assessed by analyzing the kinetic rate constants. A significant stabilization of β‐glucanase against temperature‐induced inactivation was detected at 400 MPa. With increasing pressure up to 600 MPa the catalytic activity of β‐glucanase was progressively decelerated. However, for the overall depolymerization reaction of β‐glucans in ACES buffer (0.1 M, pH 5.6) a maximum was identified at 215 MPa and 55°C yielding approximately 2/3 higher degradation of β‐glucan after 20 min as compared to the maximum at ambient pressure (45°C).     

Effects of pH and dissolved oxygen on the synthesis of γ‐glutamyltranspeptidase from Bacillus subtilis SK 11.004

BACKGROUND: γ‐Glutamyltranspeptidase (GGT; EC 2.3.2.2) is a widely distributed enzyme that is of interest in the food industry. In this study the effects of pH and dissolved oxygen (DO) on GGT synthesis from Bacillus subtilis SK 11.004 were investigated. RESULTS: GGT production increased to 0.5 U mL^?1 when the pH value was controlled at 6.5. The control of a single DO level revealed that the highest specific growth rate (3.42 h^?1) and GGT production rate (0.40 U g^?1 mL^?1) were obtained at DO levels of 40 and 10% respectively. To satisfy the different oxygen demands at different stages of cell growth and GGT synthesis, a stage DO level control strategy was designed as follows: 40% from 0 to 4 h, 30% from 4 to 6 h and 10% from 6 to 18 h. Furthermore, the maximum biomass (2.27 g L^?1) and GGT production (3.05 U mL^?1) could be obtained using a fermentation strategy combining a constant pH value with stage DO level control. CONCLUSION: The proposed fermentation strategy resulted in a 13.7‐fold increase in GGT production. This finding should be of great importance for the industrial production of GGT. Copyright © 2011 Society of Chemical Industry     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Species identification of Wickerhamomyces anomalus and related taxa using β‐tubulin (β‐tub) DNA barcode marker

Wickerhamomyces anomalus is used in food and feed processing, although the species has been reported as an opportunistic human pathogen, predominantly in neonates. Neither phenotypic nor the most frequently applied genotypic marker (D1/D2 LSU ribosomal DNA) provide sufficient resolution for accurate identification of this yeast. In this study, the β‐tubulin gene was used for species identification by direct DNA sequencing and as marker in a species‐specific PCR assay. The results showed that all examined W. anomalus strains were clearly distinguished from the closely related species by comparative sequence analysis of the β‐tubulin gene. In addition, the species‐specific primers were also developed based on the β‐tubulin gene, which was employed for polymerase chain reaction with the template DNA of Wickerhamomyces strains. A single 218 bp species‐specific band was found only in W. anomalus. Our data indicate that the phylogenetic relationships between these strains are easily resolved by sequencing of the β‐tubulin gene and combined with species‐specific PCR assay. Copyright © 2012 John Wiley & Sons, Ltd.     

Variety and germination time effect on total β‐glucan,water‐insoluble β‐glucan,water‐soluble β‐glucan components and β‐glucanase levels in improved sorghum varieties SK5912, KSV8 and ICSV400 before and after malting and their relationships to wort viscosity

The effects of variety and germination time on β‐glucan components – total β‐glucan (TBG), water insoluble β‐glucan (WIBG) and water soluble β‐glucan (WSBG) and β‐glucanase (BG) levels – before and after malting in improved sorghum varieties SK5912, KSV8 and ICSV400 and their relationships to wort specific viscosity (SV) were studied. This study was part of efforts to aid local malting and brewing industries in the application of sorghum varieties that are abundantly available to reduce costs. At the fifth day of germination, variety ICSV400 had the lowest TBG, WIBG and WSBG levels in its raw and malt samples. Variety SK5912 had the highest TBG, WIBG and WSBG levels in its raw samples, while variety KSV8 had the highest levels of TBG, WIBG and WSBG in its malt samples. Similarly, variety ICSV400 malts developed the highest BG levels, while the KSV8 malts gave the lowest level. The effect of variety, germination time and variety × germination time interaction was significant (p < 0.05) on the TBG, WIBG and BG levels and was not significant on the WSBG levels. Weak and significant correlation of TBG levels with SV (0.25, p < 0.05 for SK5912; 0.24, p < 0.05 for KSV8; and 0.31, p < 0.05 for ICSV400) was observed in all the samples, suggesting that the low β‐glucan levels may not be primarily and solely responsible for any viscosity impediments associated with sorghum worts during run‐off. With improvement in the effective utilization of sorghum, ICSV400 appeared the most suitable variety for malting and brewing in Nigeria.Copyright © 2016 The Institute of Brewing & Distilling     

Optimisation of cross‐linking β‐cyclodextrin and its recycling efficiency for cholesterol removal in milk and cream

This study was carried out to investigate the optimum conditions of cross‐linking β‐cyclodextrin (β‐CD) and recycling for cholesterol removal in milk and cream. The cross‐linked β‐CD was prepared with a 15% adipic acid solution, and the water solubility of the β‐CD was measured for the optimum conditions based on mixing temperature, mixing time, cross‐linking temperature, cross‐linking reaction time and cooling time. In the results of this study, optimum conditions were 80 °C mixing temperature, 2 h mixing time, 60 °C cross‐linking temperature, 24 h cross‐linking reaction time and 48 h cooling time. After determining the optimum conditions, the recyclable yields of the cross‐linked β‐CD ranged from 90.01% to 55.17% in six recyclings and the percentage of cholesterol removal by 15% cross‐linked β‐CD was over 90% until eighth recycling. On the basis of the results, this study suggests that 15% adipic acid‐added cross‐linked β‐CD maximised recyclable yield and that cholesterol removal was improved during recycling.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-β1,3-GLUCANASE FROM GREEN MALT

An endo-β1,3-glucanase from a green malt extract was purified by DEAE- and CM-cellulose ion exchange chromatography followed by molecular sieve chromatography on BioGel P-100. A final enzyme preparation had two protein components on disc electrophoresis, one of which was in-active. The enzyme had a pH optimum of 5·0 for activity on laminarin and 5·8 on carboxymethyl pachyman. The activity was stable up to 60°C and was stimulated by NaCl. The isoelectric point of the enzyme was 9·8.