Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K_m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.     

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K_m values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.     

Characterisation of the microflora of attiéké, a fermented cassava product, during traditional small-scale preparation

Attiéké is a fermented cassava product consumed mainly in Cote d'Ivoire. The aim of this study was to characterise the attiéké fermentation by examining products from 15 small-scale production sites at various stages of its preparation. For the preparation of attiéké, fresh cassava is grated to a pulp and inoculated with 10% of a spontaneous traditional inoculum. The inocula contained aerobic mesophiles at mean numbers of 8.2 x 10(7) cfu/g and lactic and acetic acids at mean concentrations of 0.2% and 0.1%, respectively. The mean pH was 5.0. Lactic acid bacteria were the dominant microorganisms in cassava pulp throughout fermentation with the mean numbers being 1.2 x 10(9) cfu/g after 15 h. The identification to the species level of microorganisms from one representative attiéké production of good quality showed that, at the start of fermentation, Leuconostoc mesenteroides subsp. mesenteroides was present in the highest numbers, accounting for 20% of all lactic acid bacteria. As the fermentation proceeded, this species was replaced by homofermentative lactic acid bacteria, Lactobacillus salivarius and Lactobacillus delbrueckii subsp. delbrueckii, present at 20% and 16%, respectively, and obligate heterofermentatives, Lactobacillus fermentum and Lactobacillus confusus at 12% and 10%, respectively, of total lactic acid bacteria in the flora at the end of fermentation. High numbers of acid-sensitive microorganisms, Bacillus circulans, Bacillus lentus, Enterobacter sakazakii, Enterobacter cloacae and Klebsiella pneumoniae subsp. pneumoniae, were transferred to the pulp in the inocula, but acidification to a mean pH of 4.4 with mean lactic and acetic acid concentrations of 0.59% and 0.2%, respectively, prevented their growth and reduced their numbers to less than 10(2) cfu/g at the end of fermentation. The mean numbers of Candida tropicalis, the main yeast present, remained relatively constant at about 10(5) cfu/g throughout attiéké production. The mean numbers of aerobic mesophiles decreased to below 10(2) cfu/g as a result of the steaming process. The finished attiéké had a mean pH of 4.4 and mean lactic and acetic acid concentrations of 0.6% and 0.1%, respectively.     

Effect of residual chymotryptic activity in a trypsin preparation on peptide aggregation in a β-lactoglobulin hydrolysate

Peptide composition and peptide aggregation in β-lactoglobulin (β-LG) hydrolysate were studied as related to residual chymotryptic activity in a commercial trypsin (CT) preparation. Residual chymotryptic activity produced smaller and more hydrophobic peptides in tryptic hydrolysate of β-LG, which enhanced peptide aggregation, mainly at acidic pH. The contribution of the chymotryptic peptide β-LG 15–20 to this aggregation process appeared to be very important, but other peptides (i.e., β-LG 41/43−60, 1–8 and 61−69/70+149−162) and residual α-LA may also decrease peptide solubility. When using CT mixtures in the preparation of whey protein hydrolysates, the impact of residual chymotryptic activity should not be neglected because of its influence on peptide–peptide interactions and on the resulting solubility of the hydrolyzed product.     

Suitability of TEMPO-oxidized oat β-glucan for noodle preparation

Native oat β-glucan (N-BG) and the oxidized derivative β-glucan (Oxi-BG) were incorporated into noodles and preparation characteristics (pasting, cooking, and textural properties) were investigated. N-BG caused an increase in the pasting parameters of wheat flour, and values were increased by Oxi-BG. Noodles containing either N-BG or Oxi-BG had higher cooked weights, cooked volumes, and water absorption values than controls. Raw noodles containing either N-BG or Oxi-BG showed high L values. Cooked noodles lacking BG exhibited higher L values. N-BG-containing noodles had textural properties similar to controls. Noodles prepared with Oxi-BG had higher textural parameter values than N-BG noodles, except for hardness.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

Molecular cloning and characterization of a β-glucanase from Piromyces rhizinflatus

Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. To examine the enzyme activities, CbhYW23-2 was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis was then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the high activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications.     

Extracellular secretion in Bacillus subtilis of a cytoplasmic thermostable β-galactosidase from Geobacillus stearothermophilus

β-Galactosidase catalyzes the hydrolysis of β-galactosides into monosaccharides and is widely used in dairy processing. This study reports the extracellular secretion of a cytoplasmic thermostable β-galactosidase from Geobacillus stearothermophilus IAM11001 in Bacillus subtilis. This enzyme has potential applications in the dairy industry. It was not secreted in B. subtilis by mediation of 3 general secretory signal peptides, but was secreted extracellularly when it was fused to a twin-arginine signal peptide of B. subtilis phosphodiesterase. Defined and rich culture media were used for recombinant enzyme production, and the extracellular target enzymatic activity reached about 44% of the total enzymatic activity synthesized at 18 h of cultivation in Luria-Bertani medium. As a control of secretion, when the signal peptide coding sequence was absent from the N terminus of the target gene bgaB, the extracellular target enzymatic activity obtained under the same condition of cultivation accounted for less than 7% of the total enzymatic activity synthesized. Results also showed that coexpression of the B. subtilis proteins TatAd and TatCd was indispensable for the secretion of the target enzyme.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Dihydroactinidiolide from thermal degradation of β-carotene

The formation of dihydroactinidiolide by thermal degradation of β-carotene was studied. A comparison of yields of dihydroactinidiolide in commercial β-carotene and β-carotene derived from crude palm oil was investigated. Thermal degradation of commercial β-carotene promoted the formation of dihydroactinidiolide with the highest yield, 61.21%. Thermal degradation of recovered β-carotene yielded 29.23% of dihydroactinidiolide. The lower recovery of β-carotene was due to the mixture of compounds in the extract. Further investigation indicated some other useful aroma compounds formed from this thermal degradation were β-ionone, 3-oxo-β-ionone, and β-cyclocitral.The outcome provided wide opportunities in utilizing crude palm oil as natural source of β-carotene to produce aroma compound.     

Optimisation of conditions for the preparation of β-carotene nanoemulsions using response surface methodology

Response surface methodology (RSM) was used to optimise the conditions for preparing β-carotene nanoemulsions. The effects of β-carotene (0.2–1.8%, w/w) and emulsifier (6.9–13.1%, w/w) concentrations, the homogenization pressure (79.1–140.9 MPa) and temperature (34.5–65.5 °C) on the particle size and stability of the nanoemulsions were studied. Results showed that the experimental data could be adequately fitted into a second-order polynomial model with multiple regression coefficients (R²)$(R^2)$ of 0.921 and 0.981 for the particle size and stability, respectively. Homogenization pressure and β-carotene concentration and the quadrics of β-carotene concentration (P<0.05)$(P<0.05)$ had a significant effect on the particle size of the nanoemulsions. Homogenization temperature and pressure, β-carotene concentration, the quadrics of emulsifier concentration and the interactions between β-carotene and emulsifier concentrations and between homogenization temperature and emulsifier concentration (P<0.05)$(P<0.05)$ had a significant effect on the stability of the emulsions. The optimum conditions for preparing β-carotene nanoemulsions were predicted to be: homogenization pressure, 129 MPa; homogenization temperature, 47 °C; β-carotene concentration, 0.82%; emulsifier concentration, 8.2%.     

An attempt to use a barley fibre preparation containing β-glucan in the production of functional canned meat

Nutritional value and physical and sensory quality characteristics of medium-grounded model canned meat products containing barley fibre preparation Vitacel BG300 (0.0%, 3.0% and 6.0%) were assessed. The results showed that regardless of the type of thermal treatment (pasteurisation and sterilisation), the addition of barley fibre did not significantly (P > 0.05) alter the content of the main nutrients (protein, fat and chlorides) in the block of canned meat product. Increasing the dose of barley fibre from 3.0% to 6.0% significantly increased (P < 0.05) the thermal loss in both pasteurised and sterilised canned meat products. The colour of the blocks of both pasteurised and sterilised canned meat products with barley fibre was significantly (P < 0.05) darker, and the instrumental hardness differed from the corresponding control products. Despite the beneficial nutritional effect of the Vitacel BG300 barley fibre preparation, its use significantly deteriorated the sensory quality of canned meat products.     

Performance of selected emulsifiers and their combinations in the preparation of β-carotene nanodispersions

In this work, the characteristics of β-carotene nanodispersions prepared with sodium caseinate (SC), Tween 20, decaglycerol monolaurate (ML750) and sucrose fatty acid ester (L1695) using solvent displacement technique were compared. The mean particle size of the nanodispersions ranged from 30 nm to 206 nm, depending on the emulsifier used. SC-stabilized nanodispersions had the largest particles size, while those prepared with Tween 20 had the smallest. The mean particle size and size distribution of the nanodispersions generally remained unchanged after 8 weeks of storage time at 4 °C, but the β-carotene content in all nanodispersions decreased over time. The β-carotene in SC-stabilized nanodispersions was the most stable against oxidation among all samples, most likely due to the small specific surface area of the particles, physical barrier of SC protecting the encapsulated β-carotene against free radicals and the antioxidative activity of caseins. The combined effect of SC and ML750 or L1695 on the mean particle size and β-carotene retention was also investigated. Nanodispersions prepared with SC-ML750 or SC-L1695 showed larger mean particle sizes compared to those prepared with ML750 or L1695 alone. However, the stability of β-carotene in these systems was improved owing to the antioxidative activity of SC. This work demonstrated the importance of emulsifier in determining the characteristics and stability of β-carotene nanodispersions.     

Isolation and Identification of a Novel β-Carotene Degrading Microorganism from Sea Buckthorn Juice

This study was designed to isolate and identify a novel β-carotene cleaving microorganism from sea buckthorn juice and to detect the carotenoid-derived aroma compounds catalyzed by the microbial enzyme. The strain was characterized based on morphological and biochemical properties, as well as part of its 16S rDNA sequence analysis. It was then identified using Basic Local Alignment Search Tool (BLAST) and classified as coagulase-negative Staphylococcus pasteuri (GeneBank Accession No. KP171185). β-Ionone and β-cyclocitral were formed as the main flavor products of β-carotene in submerged culture. When cell-free culture supernatant was applied for the conversion, nearly complete degradation of β-carotene (2 mg) was observed after 12 h. β-Ionone and β-cyclocitral were the main aromas, whereas β-ionone-5,6-epoxide, 2-hydroxy-2,6,6-trimethyl cyclohexanone and dihydroactinidiolide were formed in minor quantities. Moreover, the carotenoid-cleavage enzyme activities of S. pasteuri were partially characterized and partially purified enzyme possesses high activity in adverse industrial condition.     

Molecular structure of large-scale extracted β-glucan from barley and oat: Identification of a significantly changed block structure in a high β-glucan barley mutant

Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200–300 kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC–PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the ¹H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics.     

Development of a monoclonal antibody-based competitive ELISA for detection of β-conglycinin,an allergen from soybean

Soybean β-conglycinin is one of the major food allergies for children and young animals. In order to detect immunoreactive β-conglycinin from soybean and soybean products, monoclonal antibodies against β-conglycinin were prepared using a conjugated chicken ovalbumin with a synthetic peptide that corresponded to one epitope sequence of β-conglycinin as the immunogen. The generated monoclonal antibodies, named as 6G₄, 3B₇, and 5F₁₀, were identified as being of IgG₁ isotype and exhibited high specificity to β-conglycinin. A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody 6G₄ was established to determine β-conglycinin and showed an IC₅₀ value of 4.7 ng/mL with a detection limit of 2.0 ng/mL. The recovery tests of β-conglycinin indicated that the monoclonal antibody 6G₄-based competitive ELISA gave reliable reproducibility. Therefore, the produced monoclonal antibody 6G₄ and the developed competitive ELISA could provide a valuable tool for sensitive determination of β-conglycinin and for future studies on food allergies related to soybean β-conglycinin.     

Characterisation of inclusion complex of trans-ferulic acid and hydroxypropyl-β-cyclodextrin

The inclusion complex of trans-ferulic acid (FA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) was prepared by the freeze-drying method and its characterisation was investigated by different analytical techniques including UV–visible spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffractometry, and scanning electron microscopy. All these approaches indicated that FA was able to form an inclusion complex with HP-β-CD, and the FA/HP-β-CD inclusion compounds exhibited different spectroscopic features and properties from FA. The stoichiometry of the complex was 1:1. The calculated apparent stability constant of the FA/HP-β-CD complex was 166.3 M⁻¹, and the water solubility of FA was significantly improved by phase solubility studies. Moreover, the irradiation-induced decomposition of FA in aqueous solution was markedly reduced by complexation with HP-β-CD. The results showed that HP-β-CD was a proper excipient for increasing solubility and stability of FA.