Types Of Galacto-oligosaccharides Research Articles

Screening novel β-galactosidases from a sequence-based metagenome and characterization of an alkaline β-galactosidase for the enzymatic synthesis of galactooligosaccharides

βgalactosidases have wide industrial applications in lactose hydrolysis and transglycosylation reactions. Therefore, there is a need to mine novel and high-quality β-galactosidases with good tolerance and novel features from harsh environments and genomic databases. In this study, an Escherichia coli β-galactosidase-deficient host, ΔlacZ(DE3)pRARE, was constructed by the CRISPR-Cas9 system for screening active β-galactosidases. Of thirty selected β-galactosidases, twelve novel enzymes showed β-galactosidase activity, four of which were purified for further study. BGal_375 exhibited maximal activity at pH 8 and 50 °C. The concentrations of two types of galactooligosaccharides, tri- and tetra-saccharides, produced by BGal_375, reached 64.53 g/l and 8.32 g/l, respectively. BGal_375 displayed a Km value of 1.65 mM and kcat value of 53 s−1 for p-nitrophenyl-β-d-galactopyranoside (pNPG). BGal_137, BGal_144-3, and BGal_145-2 showed promising hydrolytic activity for pNPG. BGal_137 is a homodimer while BGal_144-3, BGal_145-2, and BGal_375 were all monomeric. This study provided an efficient solution for the identification of new β-galactosidases from metagenomic data, and an alkaline β-galactosidase efficient for the synthesis of galactooligosaccharides was obtained, which is important for potential industrial applications.

Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.

Assessment of in Vitro Digestibility of Dietary Carbohydrates Using Rat Small Intestinal Extract.

There are few studies on the assessment of digestibility of nondigestible carbohydrates, despite their increasingly important role in human health. In vitro digestibility of a range of dietary carbohydrates classified as digestible (maltose, sucrose, and lactose), well-recognized (lactulose, fructooligosaccharides (FOS), and two types of galactooligosaccharides (GOS) differing in the predominant glycosidic linkage), and potential (lactosucrose and GOS from lactulose, OsLu) prebiotics using a rat small intestinal extract (RSIE) under physiological conditions of temperature and pH is described. Recognized and potential prebiotics were highly resistant to RSIE digestion although partial hydrolysis at different extents was observed. FOS and lactulose were the most resistant to digestion, followed closely by OsLu and more distantly by both types of GOS and lactosucrose. In GOS, β(1 → 6) linkages were more resistant to digestion than β(1 → 4) bonds. The reported in vitro digestion model is a useful, simple, and cost-effective tool to evaluate the digestibility of dietary oligosaccharides.

Galacto-oligosaccharides and lactulose as protectants against desiccation of Lactobacillus delbrueckii subsp. bulcaricus.

Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was dehydrated on desiccators containing silica gel in the presence of 20% w/w of two types of galacto-oligosaccharides (GOS Biotempo and GOS Cup Oligo H-70®) and lactulose, until no changes in water desorption were detected. After rehydration, bacterial growth was monitored at 37°C by determining: (a) the absorbance at 600 nm and (b) the near infrared spectra (NIR). Principal component analysis (PCA) was then performed on the NIR spectra of samples dehydrated in all conditions. A multiparametric flow cytometry assay was carried out using carboxyfluorescein diacetate and propidium iodide probes to determine the relative composition of damaged, viable, and dead bacteria throughout the growth kinetics. The absorbance at 600 nm and the position of the second derivative band at ∼1370 nm were plotted against the time of incubation. The efficiency of the protectants was GOS Biotempo > GOS Cup Oligo H-70® > lactulose. The better protectant capacity of GOS Biotempo was explained on the basis of the lower contribution of damaged cells immediately after rehydration (t = 0). PCA showed three groups along PC1, corresponding to the lag, exponential and stationary phases of growth, which explained 99% of the total variance. Along PC2, two groups were observed, corresponding to damaged or viable cells. The results obtained support the use of NIR to monitor the recovery of desiccated microorganisms in real time and without the need of chemical reagents. The use of GOS and lactulose as protectants in dehydration/rehydration processes was also supported.

Stability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus in the presence of galacto-oligosaccharides and lactulose as determined by near infrared spectroscopy

The ability of two types of galacto-oligosaccharides (GOS) and lactulose to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze-drying was evaluated on the basis of their capacity to form glassy structures, using near infrared (NIR) spectroscopy.After freeze-drying in the presence of protective agents, microorganisms were stored at 4°C at different relative humidities (RH) (from 11 to 80%) up to 21days. The loss of viability was determined by plate count and referred to viability before freeze-drying. For each storage condition, glass transition temperatures (Tg) were determined by differential scanning calorimetry (DSC). In parallel, near infrared spectra registered in the 900–1700nm region provided information about viability and RHs that could be correlated with the results obtained by plate counts and DSC.The comparison of NIR spectra of freeze-dried samples stored at the same RH but differing in the time of storage allowed the identification of spectral features related with viability.The spectral differences observed on samples stored in the same conditions (same time of storage and same protective agent) but differing in RHs were ascribed to the formation of amorphous and rubbery states.Based on the spectral information, a multivariate based classification model (PLS-DA) was defined to determine whether samples are in a rubbery or an amorphous state. As the physical state of starters is related with their shelf-life, this model provided support for the use of NIR spectroscopy as a quick method to assess the proper storage of starters.

Enzymatic Production of Galactooligosaccharides by β-Galactosidase from Bifidobacterium longum BCRC 15708

The production of galactooligosaccharides (GOSs) by transgalactosylation using beta-galactosidase from Bifidobacterium longum BCRC 15708 was studied. Other than lactose, galactose, and glucose, two types of GOSs, tri- and tetrasaccharides, were formed after beta-galactosidase action on 40% lactose. Trisaccharides were the major type of GOS formed. Generally, an increase of the initial lactose concentration in the reaction mixture resulted in a higher GOS production. A maximum yield of 32.5% (w/w) GOSs could be achieved from 40% lactose solution at 45 degrees C, pH 6.8, when the lactose conversion was 59.4%. The corresponding productivity of GOSs was 13.0 g/(L.h). Transgalactosylation activity of beta-galactosidase from a test organism showed a relatively lower sensitivity toward glucose and galactose than that from other organisms. The addition of 5% or 10% glucose or galactose to the reaction mixture did not significantly (p>0.05) reduce the transgalactosylation reaction of beta-galactosidase.