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Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6 T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to β-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1 → 3)-, (1 → 4)- or (1 → 6)-linked α-GOS but exhibited a preference for formation of (1 → 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1 → 3)-, (1 → 4)- or (1 → 6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.

The influence of environmental pH on biological activity of Bifidobacterium longum CRL 849 grown in MRS-raffinose was evaluated. At pH 6.0, 5.5 and 5.0, raffinose was completely consumed by this microorganism, showing different consumption rates at each pH value (between 3.03 and 0.76 mmol l(-1) h(-1)). At pH 4.5, the growth was lowest. The removal of raffinose was due to the alpha-galactosidase (alpha-gal) activity of this bifidobacteria, which was highest at pH 6.0-5.5 (1,280-1,223 mU ml(-1)). The production of beta-glucosidase (beta-glu) showed a similar pattern to alpha-gal activity with major values. The yield of organic acids produced during raffinose consumption was also highest at pH 6.0-5.5. The results of this study will allow the selection of the optimum growth conditions of B. longum CRL 849, with elevated levels of alpha-gal to be used in the reduction of nondigestible alpha-oligosaccharide in soy products and beta-glu activities involved in isoflavone conversion to bioactive forms when used as starter culture.

In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control. Growth was similar under all conditions assayed except at pH 4.5. These microorganisms were able to eliminate raffinose, a nondigestible alpha-oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l(-1) h(-1) (pH 6.0-5.0). The removal of raffinose was due to the high alpha-galactosidase (alpha-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml). The yield of organic acids produced during raffinose consumption was also highest at this pH. The results of this study will allow selection of the optimum growth conditions of L. fermentum with elevated levels of alpha-gal to be used in the reduction of NDO in soy products when used as starter cultures.

Stirred, pH-controlled anaerobic batch cultures were used to evaluate the in vitro utilisation by canine gut microflora of novel alpha-galactooligosaccharides synthesised with an enzyme extract from a canine Lactobacillus reuteri strain. Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates for the prebiotic properties of the synthesised oligosaccharide (galactosyl melibiose mixture-GMM). Addition of Lactobacillus acidophilus was used as control for the evaluation of the synbiotic properties of the oligosaccharide with L. reuteri. Populations of predominant gut bacterial groups were monitored over 48 h of batch culture by fluorescent in situ hybridisation, and short-chain fatty acid (SCFA) production was measured. GMM showed a higher increase in bifidobacteria and lactobacilli population number and size as well as a higher decrease in clostridia population number and size compared to the commercial prebiotics (FOS, melibiose, raffinose). This prebiotic effect was further increased by the addition of L. reuteri followed by a change in the SCFA production pattern compared to GMM alone or GMM with L. acidophilus. The observed change in SCFA production was in accordance with the fermentation properties of L. reuteri, suggesting that the novel synbiotic had a significant effect on the canine gut microflora fermentation.

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degrees C ( K(M) 0.55 mM, V(max) 0.80 micromol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l-1 D-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l-1 D-glucose or raffinose was used as co-substrate together with 50 g l-1 D-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation.

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked beta-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of beta-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. beta-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also.

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of alpha-galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean alpha-protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the alpha-galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.