Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF.

Lactobacillus spp., as probiotics, have shown efficacy in the inhibition of pathogenic bacteria in the intestinal tract. In this study, we investigated the antibacterial activity of Lactobacillus casei NA-2, which was isolated from northeast sauerkraut in China. The results of co-culture suggested that L. casei NA-2 could inhibit the growth of Bacillus cereus, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli O157:H7. Moreover, L. casei NA-2 could adhere to the four pathogenic bacteria potentially associated with its exopolysaccharide (EPS). EPS from L. casei NA-2 was then isolated and its activity determined. The results showed that EPS inhibited the biofilms of B. cereus, S. aureus, S. typhimurium and E. coli O157:H7, with the highest inhibition ratios of 95.5% ± 0.1%, 30.2% ± 3.3%, 14.3% ± 0.6%, and 16.9% ± 5.4%, respectively. Moreover, EPS was able to disperse B. cereus, S. aureus, S. typhimurium and E. coli O157:H7 by 94.1% ± 1.2%, 31.8% ± 8.6%, 40.8% ± 3.3% and 49.6% ± 3.8%, respectively. Results showed that EPS from L. casei NA-2 have potential antibacterial properties by inhibiting biofilm formation and dispersing pathogenic bacteria. In conclusion, the antibiofilm property of the EPS on the surface of L. casei NA-2 is one of the possible reasons for antibacterial activity of L. casei NA-2.

Background: Exopolysaccharides (EPSs) secreted from lactic acid bacteria are carbohydrate polymers with reported biological activities. In this study, we extracted and characterized the composition as well as antioxidant and biofilm-inhibitory properties of EPS from Lactobacillus coryniformis NA-3 isolated from northeast Chinese sauerkraut (Suan Cai). Methods: Lactobacillus coryniformis NA-3 was identified with 16S rDNA amplification and Neighbor Joining (NJ) phylogenetic analysis. EPS derived from Lactobacillus coryniformis NA-3 (EPS-NA3) was analyzed, including compositions by high-performance liquid chromatography (HPLC), functional groups by Fourier-transform infrared spectroscopy (FT-IR) and glycosidic bond configuration by Hydrogen-1 Nuclear Magnetic Resonance (1H NMR). Antioxidant activity of EPS was evaluated with hydroxyl and superoxide radical-scavenging. Anti-biofilm activities of EPS-NA3 were checked through inhibition and dispersion. Results: The monosaccharide composition of EPS included α-rhamnose, α-mannose, α-galactose, and α-glucose in a ratio of 2.6:1.0:5.0:3.3. The free radical-scavenging abilities of EPS-NA3 were 37.77% ± 1.56% and 78.87% ± 3.07% on hydroxyl and superoxide reactive oxygen species respectively. Moreover, EPS-NA3 attenuated the formation of Bacillus cereus and Salmonella typhimurium biofilms by inhibition ratios of approximately 80% and 40% respectively. Additionally, treatment with EPS-NA3 dispersed established biofilms of B. cereus and S. typhimurium by approximately 90% and 20% respectively. Conclusion: These results suggest that EPS-NA3 may be developed as antioxidant and anti-biofilm agents for industrial and clinical applications due to its capacity of scavenging free radicals, inhibition of bacterial biofilm formation, and dispersion of established biofilms.

Exopolysaccharides (EPS) from Lactobacillus spp. have been found to have biological activities. Our previous work demonstrated the antibiofilm activity of EPS from Lactobacillus casei NA-2 (L.casei...

A pullulanase gene (Pul3YH5) of 2568 bp, which encodes a protein containing 855 amino acid residues, was cloned from the alkaliphilic bacterium Exiguobacterium acetylicum YH5. The pullulanase (Pul3YH5) contains the YNWGYDP motif of type I pullulanase as well as four conserved glycoside hydrolase sequences of the GH13 (α‐amylase) family. When the pullulanase gene was cloned and expressed in Escherichia coli BL21 (DE3) plysS, the recombinant pullulanase had a molecular mass of ˜100·0 kDa. It was optimally active at 50°C and pH 6·0, alkali‐tolerant and displayed excellent stability (>93·0%) over a broad pH range (4·0‒10·0) when incubated for 30 min without substrate. The enzyme activity of Pul3YH5 was significantly enhanced in the presence of Co²⁺, Fe²⁺ and Mn²⁺ and was inhibited by Cu²⁺, SDS, β‐mercaptoethanol and EDTA. The enzyme displayed the highest specificity for pullulan (K_m = 0·12 ± 0·02 mg ml⁻¹), followed by soluble starch (K_m = 0·69 ± 0·04 mg ml⁻¹). Substrate hydrolysis demonstrated that pullulanase from E. acetylicum liberated maltotriose from pullulan, although hydrolytic activity was also detected with soluble starch, amylopectin, β‐limited dextrin and glycogen. These enzymatic properties indicate that Pul3YH5 is alkali‐tolerant pullulanase and that Pul3YH5 could be useful in the detergent industry. Significance and Impact of the Study Pullulanases have great potential in various industries, ranging from food (high fructose and glucose syrups) to washing detergent industries. In this study, the gene encoding the novel pullulanase from E. acetylicum YH5 was cloned and sequenced, then expressed in E. coli. The properties of the recombinant enzyme in E. coli were also determined. The pullulanase from E. acetylicum YH5 is alkaline tolerant and has a high optimum temperature, making it a candidate for applications in the detergent industry.