Reduction of D-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene.

The D-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of d-lactate from pyruvate to l-lactate, we expressed the l-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The ldhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of ldhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to ldhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both l-LDH activity and l-lactate productivity during fermentation, decreasing the d-/l-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased l-lactate concentration and decreased d-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high l-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of d-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods.

Development of Bile Salt-Resistant Leuconostoc citreum by Expression of Bile Salt Hydrolase Gene.

Probiotic bacteria must have not only tolerance against bile salt but also no genes for antibiotic resistance. Leuconostoc citreum is a dominant lactic acid bacterium in various fermented foods, but it is not regarded as a probiotic because it lacks bile salt resistance. Therefore, we aimed to construct a bile salt-resistant L. citreum strain by transforming it with a bile salt hydrolase gene (bsh). We obtained the 1,001 bp bsh gene from the chromosomal DNA of Lactobacillus plantarum and subcloned it into the pCB4170 vector under a constitutive P710 promoter. The resulting vector, pCB4170BSH was transformed into L. citreum CB2567 by electroporation, and bile saltresistant transformants were selected. Upon incubation with glycodeoxycholic acid sodium salt (GDCA), the L. citreum transformants grew and formed colonies, successfully transcribed the bsh gene, and expressed the BSH enzyme. The recombinant strain grew in up to 0.3% (w/v) GDCA, conditions unsuitable for the host strain. In in vitro digestion conditions of 10 mM bile salt, the transformant was over 67.6% viable, whereas only 0.8% of the host strain survived.

In vitro digestion and fermentation properties of linear sugar-beet arabinan and its oligosaccharides.

This study was conducted to investigate the prebiotic effects of linear arabino-oligosaccharides (LAOS) and debranched (linear) sugar beet arabinan (LAR) for the development of new prebiotics. LAOS were prepared from LAR by enzymatic hydrolysis with endo-arabinanase from Bacillus licheniformis, followed by removal of the arabinose fraction by incubation with resting cells of Leuconostoc mesenteroides. The resulting LAOS contained DP2 (28.7%), DP3 (49.9%), DP4 (20.1%), and DP5 (1.16%). A standardized digestibility test showed that LAOS and LAR were not digestible. Individual cultures of 24 strains of gastrointestinal bacteria showed that LAOS and LAR stimulated growth of Lactobacillus brevis, Bifidobacterium longum, and Bacteroides fragilis. In vitro batch fermentation using human fecal samples showed that LAOS had higher bifidogenic properties than LAR; LAOS increased the population of bifidobacteria which produced short-chain fatty acids (SCFAs). LAOS was fermented slowly compared to fructo-oligosaccharides and this may permit SCFA production in the distal colon. This study demonstrates that LAOS prepared from LAR are promising dietary substrates for improvement of human intestinal health.

Isolation of dextran-hydrolyzing intestinal bacteria and characterization of their dextranolytic activities.

The aim of this study was to isolate dextran-hydrolyzing bacteria from the human intestines and to identify their dextranolytic enzymes. For this, dextranase-producing microorganisms were screened from fecal samples by using blue dextran-containing media. Colonies producing a decolorized zone were isolated and they were grouped using RAPD-PCR. 16S rRNA gene sequencing analysis revealed the isolates were Bacteroides (B.) thetaiotaomicron, B. ovatus, B. vulgatus, B. dorei, B. xylanisolvens, B. uniformis, and Veillonella (V.) rogosae. Thin layer chromatography analysis showed that the dextranases exhibit mainly endo-type activity and produce various oligosaccharides including isomaltose and isomaltotriose. Zymogram analysis demonstrated that enzymes localized mainly in the cell membrane fraction and the molecular weight was 50-70 kDa. When cultured in a dextran-containing medium, all strains isolated in this study produced short-chain fatty acids, with butyric acid as the major compound. This is the first study to report that human intestinal B. xylanisolvens, B. dorei, and V. rogosae metabolize dextran utilizing dextranolytic enzymes.

An improved process of isomaltooligosaccharide production in kimchi involving the addition of a Leuconostoc starter and sugars.

Isomaltooligosaccharides (IMOs) are α-(1→6)-linked oligodextrans that show a prebiotic effect on Bifidobacterium spp. This study sought to improve IMO synthesis during lactate fermentation in kimchi by inoculating the kimchi fermentation mix with a starter and sugars; the psychrotrophic Leuconostoc citreum KACC 91035 strain with high dextransucrase activity was used as a starter and sucrose (58 mM) and maltose (56 mM) were added as the donor and acceptor for the glucose-transferring reaction of the dextransucrase, respectively. With the addition of both the starter and the sugars and incubation at 10°C, IMOs were produced in kimchi after 3d. Without the starter, the IMO production rate and maximal concentration in kimchi were 15.05 mM/d and 75.27 mM, respectively, whereas with the starter, the rate and concentration increased to 22.04 mM/d and 110.19 mM, respectively. In addition, the sucrose-maltose mix gave an appropriate level of sweetness by releasing fructose and prevented unfavorable polymer synthesis by IMO production. This result suggests that lactic acid bacteria expressing a highly active glycosyltransferase can be used for the synthesis of beneficial oligosaccharides in various fermented foods.

Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi.

The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312bp, a low G+C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the ß-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB.

Isolation and characterization of biogenic amine-producing bacteria in fermented soybean pastes.

Biogenic amines (BAs) are produced primarily by microorganisms found in fermented foods and are often implicated in food poisoning. BA-producing bacteria found in fermented soybean pastes were isolated and characterized using a decarboxylating medium and multiplex PCR analysis. Two BA-producing bacteria were isolated from traditional soybean pastes: one was a histamine-producing Clostridium strain, and the other was a tyramine-producing Pseudomonas strain. The Clostridium strain was determined to be a potent histamine producer among the cultures tested. Synthesis of tyramine by Pseudomonas sp. T1 was observed for the first time in this study.

Optimized substrate concentrations for production of long-chain isomaltooligosaccharides using dextransucrase of Leuconostoc mesenteroides B-512F.

Isomaltooligosaccharide (IMO) is a promising dietary component with prebiotic effect, and the long-chain IMOs are preferred to short chain ones owing to the longer persistence in the colon. To establish the optimal process for synthesis of long-chain IMOs, we systematically examined the reaction condition of dextransucrase of Leuconostoc mesenteroides B-512F by changing the ratio of sucrose to maltose (varying as 1:4, 1:2, 1:1, and 2:1) and amount of each sugar (from 2% to 20%). As a result, a ratio of 2:1 (sucrose to maltose, 10:5% or 20:10%, w/v) was determined as an optimal condition for long-chain IMO synthesis (DP3-DP9) with relatively higher yields (70-90%, respectively).