The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable ␣-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r , 53,514). The observed homology between the deduced amino acid sequences of the enzyme and ␣-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 ␣-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C for p-nitrophenyl-␣-Dgalactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal ␣-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea ␣-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.␣-Galactosidases (␣-Gals) are known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). ␣-Gals catalyze the hydrolysis of 1,6-linked ␣-galactose residues from oligosaccharides and polymeric galactomannans (19,27,28). In the sugar beet industry, ␣-Gals have been used to increase the sucrose yield by eliminating raffinose, which prevents the crystallization of beet sugar (31). Raffinose and stachyose in beans are known to cause flatulence. ␣-Gal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (6).We have studied the substrate specificity of ␣-Gals from eukaryotes by using galactomanno-oligosaccharides, such as 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose (Gal 3 Man 3 ) and 63 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose (Gal 3 Man 4 ). The structures of these galactomanno-oligosaccharides are shown in Fig. 1. Mortierella vinacea ␣-Gal I (11) and yeast ␣-Gals (32) are specific for Gal 3 Man 3, having an ␣-galactosyl residue (designated the terminal ␣-galactosyl residue) attached to the O-to-6 position of the nonreducing end mannose of ␤-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 ␣-Gal (12) and Penicillium purpurogenum ␣-Gal (27) show a preference for Gal 3 Man 4 , having an ␣-galactosyl residue (designated as the side chain ␣-galactosyl residue) attached to the O-to-6 position of the third mannose from the reducing end of ␤-1,4-mannotetraose. The M. vinacea ␣-Gal II (28) acts on both substrates to almost equal extents. These facts indicate that eukaryotic ␣-Gals were classified into three groups based on the substrate specificity of these galactomanno-oligosaccharides.Genes encoding ␣-Gals have been cloned ...

For the fermentation of vinegar using onion, acetic acid bacteria and yeast strains with high fermentation ability were screened. Among them, Saccharomyces cerevisiae 1026 was selected as a starter for ethanol production and Acetobacter orientalis MAK88 was selected as a vinegar producer. When the two-stage fermentation of onion vinegar was performed at 28 °C, the titratable acidity reached 4.80% at 24 h of fermentation. When semi-continuous fermentation proceeded to charge-discharge consisting of three cycles, the acetic acid content reached 4.35% at 48 h of fermentation. At this stage, the fermentation efficiency, acetic acid productivity, and specific product formation rate were 76.71%, 17.73 g/(L·d), and 20.58 g/(g·h), respectively. The process in this study significantly reduced the fermentation time and simplified the vinegar production process. The content of total flavonoids and total polyphenols in onion vinegar were 104.36 and 455.41 μg/mL, respectively. The antioxidant activities of onion vinegar in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS+) radical scavenging activity, and reducing power were 75.33%, 98.88%, and 1.28, respectively. The nitrite scavenging abilities of onion vinegar were 95.38 at pH 1.2. The onion vinegar produced in this study showed higher organoleptic acceptability than commercial onion vinegar.

Production of oligosaccharides from Leuconostoc lactis CCK940 was optimized using a response surface methodology with a central composite design. Culture temperature and the concentrations of sucrose and maltose were used as the main factors. The predicted optimum conditions for the production of oligosaccharides were a culture temperature of 30 °C, a sucrose concentration of 9.6% (w/v), and a maltose concentration of 7.4% (w/v). Using these optimal conditions, Leuconostoc lactis CCK940 was cultured using a fermenter to produce oligosaccharides, and the resulting oligosaccharides with a degree of polymerization greater than 4 were purified by Bio-gel P2 gel permeation column chromatography and then lyophilized. When macrophages were treated with the purified oligosaccharides at concentrations of 0.1–10 mg/mL, no cytotoxicity towards the macrophages was observed. However, nitric oxide production levels were similar to those following treatment with 1 μg/mL lipopolysaccharide. The mRNA expression levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, and inducible nitric oxide synthase were all also increased in a dose-dependent manner following treatment with the oligosaccharides. These data suggest that oligosaccharides produced by Leuconostoc lactis CCK940 could be used as an immune enhancer of macrophages.

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