Hydrolysis Of Raffinose Family Oligosaccharides Research Articles

Immobilization of fungal α-galactosidase on magnetic nanoparticles and hydrolysis of raffinose family oligosaccharides (RFO) in soymilk

Immobilization of enzymes is a very attractive technique to develop efficient and cost-effective bioprocesses. In present investigation, immobilization of partially purified α-galactosidase from Penicillium aculeatum APS1 (APS1αgal) was carried out on chitosan coated magnetic nanoparticles, Fe3O4. Immobilization conditions such as enzyme concentration, amount of chitosan coated magnetic nanoparticles (CMNP), pH and time for immobilization were optimised and maximum immobilization yield of 45 ± 2% and activity yield of 47 ± 3% were achieved. Immobilized particles were characterised by thermal analysis and Fourier transform infrared spectroscopy (FT-IR). The surface morphology of particles was studied by scanning electron microscopy (SEM). Moreover, biochemical properties of free and immobilized enzymes were compared. Thermostability and storage stability of APS1αgal were found to be improved after immobilization. Reusability of immobilized APS1αgal was assessed and it was found to retain 65% activity after 12 consecutive cycles. Further experiments were conducted to assess the ability of immobilized APS1αgal for removal of raffinose family oligosaccharides (RFO) in soymilk. During batch hydrolysis, about 80% removal of RFO was achieved. Hydrolysis of RFO was confirmed by TLC analysis. Immobilized APS1αgal was able to carry out hydrolysis of RFO up to 45% even after 7 cycles. Overall, results of this study show the potential of immobilized APS1αgal for efficient removal of non-digestible RFO in soymilk.

CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits.

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Characterization of a protease-resistant α-galactosidase from Aspergillus oryzae YZ1 and its application in hydrolysis of raffinose family oligosaccharides from soymilk

The α-galactosidase gene (galC) was cloned from Aspergillus oryzae YZ1 and expressed in Pichia pastoris. The galC (2319 bp) containing two introns encoded a protein of 726 amino acids. The activity of the α-galactosidase (GalC) increased 1-fold after coding sequence optimization. Purified GalC exhibited a single protein band (100 kDa) in SDS-PAGE. The optimum pH and temperature of GalC were pH 4.66 and 50 °C, respectively. Like many GH36 family α-galactosidases, GalC displayed its activities towards raffinose and stachyose. The Km values for pNPG, raffinose and stachyose were 2.16, 4.63 and 8.54 mM, respectively. The GalC retained about 90% activity within the pH range 3.0–8.0. The activity of GalC was inhibited by Cu2+, while Ca2+ increased the enzyme activity. Different concentrations of glucose, mannose, galactose, xylose and sucrose slightly affected the activity of GalC. The GalC displayed strong resistance to trypsin, α-chymotrypsin, and proteinase K. Under simulated gastric conditions, GalC maintained most of its native activity after pepsin treatment for 3 h. The GalC could also effectively degrade raffinose and stachyose in soymilk. The GalC with high hydrolysis efficiency towards raffinose family oligosaccharides (RFOs) and strong resistance to proteases is considered to have great potential in food and feed industries.

Levansucrase and sucrose phoshorylase contribute to raffinose, stachyose, and verbascose metabolism by lactobacilli

Raffinose family oligosaccharides (RFOs) in food are considered anti-nutritional factors. This study elucidated the role of α-galactosidase (α-Gal), levansucrase, and sucrose phosphorylase for conversion of RFOs by lactobacilli. Quantification of gene expression by reverse-transcriptase quantitative PCR revealed that expression of levansucrase and sucrose phosphorylase by Lactobacillus reuteri is increased more than 100 fold when sucrose or raffinose are available. Fava bean (Vicia faba) or field pea (Pisum sativum) flours were fermented with α-Gal positive L. reuteri or α-Gal negative Lactobacillus sanfranciscensis. Isogenic strains lacking levansucrase activity, a L. reuteri ftfA mutant and a L. sanfranciscensis levS mutant, were used for comparison. During growth in pulse flours, L. sanfranciscensis accumulated melibiose and α-galactooligosaccharides (α-GOSs); the levansucrase-negative strain did not grow. L. reuteri metabolized raffinose, stachyose, and verbascose by levansucrase activity and accumulated α-GOSs as metabolic intermediates. Oligosaccharide metabolism in the levansucrase-negative mutant was slower, and accumulation of α-GOSs was not observed. The use of sorghum sourdough fermented with L. reuteri LTH5448 and bean flour in gluten-free baking converted RFOs to α-GOSs by levansucrase and invertase activities. In conclusion, the elucidation of the role levansucrase in RFO metabolism by lactobacilli allowed the conversion or hydrolysis of RFOs in food fermentations.

Continuous hydrolysis of raffinose family oligosaccharides in soymilk by fluidized bed reactor

Soybean is an important pulse crop for human and animal nutrition. However, raffinose family oligosaccharides (RFOs) found in soybean seeds specifically hinder its consumption as it is not digested by normal human carbohydrases and is fermented by intestinal microflora, leading to flatulence. Immobilized spores of Aspergillus oryzae were grown for 5 days in calcium alginate gel beads, then harvested and employed in RFOs hydrolysis in batch mode, repeated batch and continuous mode in a fluidized bed reactor. Effective hydrolysis of 244.4 mg/60 ml was obtained after 6 h incubation. And in continuous mode 465.6 mg/100 ml was obtained at a flow rate of 25 ml/h. The use of immobilized A. oryzae cells for hydrolysis of RFOs is a promising solution to overcome flatulence and to increase the consumption of soy products.

Enzymatic removal of flatulence-inducing sugars in chickpea milk using free and polyvinyl alcohol immobilized α-galactosidase from Aspergillus oryzae

The treatment of chickpea milk was carried out in batch, repeated batch and continuous reaction by soluble and polyvinyl alcohol (PVA) immobilized Aspergillus oryzae alpha-galactosidase for the removal of raffinose family oligosaccharides (RFOs). In the batch mode of treatment 96 and 92% of RFOs hydrolysis was observed by soluble and immobilized enzyme, respectively. In repeated batch experiments, immobilized enzyme showed 70% RFOs hydrolysis up to sixth cycle. Polyvinyl alcohol immobilized alpha-galactosidase in fluidized bed reactor showed highest reduction of 94% at a flow rate of 30 ml/h. The results obtained from the present study are very interesting for industrial use of PVA-immobilized enzyme.

Immobilized alpha-Galactosidase in the Biochemistry Laboratory

This laboratory experiment was designed to demonstrate the application of immobilized galactosidase in food industry to hydrolyze raffinose family oligosaccharides in soymilk. This laboratory experiment was conducted for postgraduate students of biochemistry and developed for graduate and undergraduate students of biochemistry, biotechnology, chemical engineering, and food technology courses. The students were able to compare the hydrolysis of raffinose family oligosaccharides by free and immobilized-galactosidase. This experiment illustrates the basic concept of immobilization and its application.