Aims: This study aimed to investigate the suitability and efficacy of various encapsulation media in bioencapsulating the probiotic Lactococcus lactis subsp. lactis in Artemia franciscana nauplii. The impact of the encapsulation media on nauplii survival and probiotic recovery was also determined. Methodology and results: Various encapsulation media (sodium alginate, palm oil, starch, gum Arabic and gelatin) were prepared by dissolving the respective media in artificial sea water. Each media was prepared in four different concentrations: 0.25, 0.5, 1.0 and 2.0 g/L. To determine the suitability of encapsulation media on the survivability of A. franciscana, survival rate (SR) of Artemia nauplii was determined after 8 hours post-encapsulation. Instar II stage Artemia nauplii at 1 nauplii per mL was used for each replicate. The result revealed that A. franciscana reached 100 % SR in the encapsulation media at ≤ 0.5 g/L. All media enabled > 23 % recovery of L. lactis subsp. lactis from encapsulated A. franciscana, which is similar (p > 0.05) to the recovery of free-cells (non-encapsulated) of L. lactis subsp. lactis. Noticeably in sodium alginate (E1) treatment, the total counts of L. lactis subsp. lactis in bioencapsulated A. franciscana were the highest among others, accounting for 2.44 × 107 CFU/mL per A. franciscana tissue homogenate. Conclusion, significance and impact of study: Artemia nauplii bioencapsulated with L. lactis subsp. lactis using 0.5 g/L sodium alginate as encapsulation medium has the highest SR for nauplii and bioencapsulation efficiency, respectively. This result provides a basic guideline for Artemia bioencapsulation in fish/shrimp larval culture.
Lactococcus lactis

Aims: Lactococcus lactis is a non-colonizing, generally-regarded as safe (GRAS) lactic acid bacteria that has been frequently studied as a potential vector for bactofection. To mediate bactofection, a series of interaction between the bacteria and the host cell needs to occur. This study aims to investigate the in vitro bacterial-cell interaction between a locally-isolated L. lactis M4 strain with human colorectal cancer line, Caco-2. Methodology and results: Bacterial interaction was evaluated via adherence and internalisation assays. A 250:1 ratio of bacteria to cancer cell was selected as the optimum multiplicity of infection for all assays. After 2 h, L. lactis M4 was able to adhere to and internalise into Caco-2 cells at comparable rates to commercial strains L. lactis NZ9000 and MG1363. Conclusion, significance and impact of study Findings from this study showed that this strain has similar interaction properties with the commercial strains and would make a promising candidate for future bactofection studies and development of bacteria-mediated DNA vaccination against various diseases.
Lactococcus lactis ; Colorectal Neoplasms ; Caco-2 Cells

Streptococcus mutans is the most important causative bacteria of dental caries among the oral bacteria. Lactococcus lactis 1370 was isolated from the oral cavity of child. The effect of Lactococcus lactis 1370 on the formation of artificial plaque by Streptococcus mutans was studied. 1. The insoluble substances and bacteria were much more attached on the wall of disposable cuvette in the culture of Streptococcus mutans than in the combined culture of Streptococcus mutans and Lactococcus lactis 1370. 2. The mean weight of produced artificial plaque on the wires in the beaker was 131.7 mg in the culture of Streptococcus mutans only, whereas being reduced to 6.4 mg in the combined culture of Streptococcus mutans and Lactococcus lactis 1370 (p<0.05). The viable cell didn't show the significant difference between them after culturing. 3. When Streptococcus mutans was cultured in the media containing culture supernatant of Lactococcus lactis 1370 cultured in M17 broth containing 0.5% yeast extract and 5% sucrose, the mean weight of produced artificial plaque was 8.0 mg on the wires, whereas being 125.4 mg in the media without culture supernatant of Lactococcus lactis 1370 (p<0.05). The viable cell didn't show the significant difference between them after culturing. 4. When Streptococcus mutans was cultured in the media containing soluble polymer produced by Lactococcus lactis 1370, the mean weight of produced artificial plaque was significantly reduced compared with being cultured in the media without soluble polymer (p<0.05). The viable cell didn't show the significant difference between them after culturing. 5. The soluble polymer produced by Lactococcus lactis 1370 was glucan. 6. The glucan produced by Lactococcus lactis 1370 was water-soluble glucan containing alpha-1,6-glucose linkage as the main linkage. These results suggest that the artificial plaque formed by Streptococcus mutans is inhibited by water-soluble glucan produced by Lactococcus lactis 1370.
Bacteria ; Child ; Dental Caries ; Humans ; Lactococcus lactis* ; Lactococcus* ; Mouth ; Polymers ; Streptococcus mutans ; Sucrose ; Yeasts

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.
Escherichia coli/genetics* ; Lactococcus ; Metabolic Engineering ; Pyrazines ; Threonine

Lactococcus lactis (L. lactis) is an important gram-positive bacterium in dairy products. It is a rare cause of opportunistic infections with only four cases of Lactococcus peritoneal dialysis (PD) peritonitis reported in the literature. In Korea, L. lactis infection was first reported in a liver abscess patient in 2010; however, PD peritonitis with Lactococcus has not been reported in Korea. Recently, we experienced a case of Lactococcus-associated polymicrobial PD peritonitis. The patient was initially managed with broad-coverage antibiotics; however, owing to a poor response, the PD catheter was removed and the patient was switched to hemodialysis. We discuss this case and review the literature.
Anti-Bacterial Agents ; Catheters ; Dairy Products ; Humans ; Korea ; Lactococcus ; Lactococcus lactis ; Liver Abscess ; Opportunistic Infections ; Peritoneal Dialysis ; Peritonitis ; Renal Dialysis

Lactococcus lactis is a gram-positive cocci used extensively in the dairy industry, but considered an unusual pathogen in humans. Among its five subspecies, L. lactis subsp. lactis in particular has rarely been reported as a pathogen. We report a case of septic shock caused by L. lactis subsp. lactis in an adult patient. A 64-yr-old male patient was admitted to outpatient clinics, with chief complaints of fever and chills for one week after convalescent hospital admission. He had severe ileus requiring surgery. He had a peripherally inserted central catheter from convalescent hospital, which was immediately removed. From two sets of blood and catheter tip cultures, we identified L. lactis subsp. lactis using the Vitek 2 system (bioMerieux Inc., USA), and confirmed this result by 16S rRNA sequencing. The patient was empirically treated with ciprofloxacin, and he recovered and was discharged.
Adult* ; Ambulatory Care Facilities ; Catheter-Related Infections* ; Catheters ; Chills ; Ciprofloxacin ; Fever ; Gram-Positive Cocci ; Hospitals, Convalescent ; Humans ; Ileus ; Lactococcus lactis* ; Lactococcus* ; Male ; Shock, Septic*

Lactic acid bacteria (LAB) are important industrial microorganism used in the production and preservation of food-stuffs. Recently, considerable advances have been made in the genetics and molecular biology of LAB. These have resulted in the construction of food-grade gene expression systems for these bacteria. This paper aims to review the essential features for food-grade systems, food-grade selection markers, food-grade controlled gene expression and food-grade inducible signaling molecule, and recent developments on food-grade cloning and expression systems for LAB. These gene expression systems have great potential for studies on gene expression and regulation in LAB and a variety of bioprocessing application in industrial fermentations.
Food Industry ; methods ; standards ; Food Microbiology ; Gene Expression Regulation, Bacterial ; Lactococcus lactis ; genetics ; growth & development

OBJECTIVETo clone and secretion express cholera toxin B subunit (CTB) in food-grading Lactococcus lactis expression systems.

METHODSctB fragment that encoding CTB was amplified by polymerase chain reaction (PCR) using the genomic DNA of Vibrio cholera strain 569B as template and was inserted into two secretion expression vector pSQZ and pSQ to construct food-grading expression system L.lactis MBP71/pSQZ-ctB and L.lactis MBP71/pSQ-ctB. The expressed CTB was detected by Western-blot assay.

RESULTSThe ctB fragment was successfully amplified from Vibrio cholera strain 569B and inserted into two secretion expression vectors pSQZ and pSQ to construct food-grading expression system L. lactis MBP71/pSQZ-ctB and L. lactis MBP71/pSQ-ctB. Western-blot assay demonstrated that CTB was secretion and expressed from L.lactis MBP71 harboring vectors pSQZ-ctB and pSQ-ctB, and the quantity of CTB secreted by L. lactis MBP71/pSQ-ctB was about 2 microg/ml, higher than that of L. lactis MBP71/pSQZ-ctB.

CONCLUSIONCTB was successfully secreted and expressed by food-grading L. lactis expression systems.

By activity-guided fractionation, gliotoxin was isolated as an antibacterial metabolite of the fungus Penicillium decumbens which was derived from the jellyfish Nemopilema nomurai. Gliotoxin was further evaluated for antibacterial activity against several piscine and human MDR (multidrug resistance) pathogens. Gliotoxin showed significant antibacterial activity against Gram-positive piscine pathogens such as Streptococcus iniae FP5228, Streptococcus iniae FP3187, Streptococcus parauberis FP3287, Streptococcus parauberis SPOF3K, S. parauberis KSP28, and Lactococcus garvieae FP5245. Gliotoxin showed strong activity especially against S. parauberis SPOF3K and S. iniae FP5228, which are resistant to oxytetracycline. It is noteworthy that gliotoxin effectively suppressed streptococci which are the major pathogens for piscine infection and mortality in aquaculture industry. Gliotoxin also showed strong antibacterial activity against multidrug-resistant human pathogens (MDR) including Enterococcus faecium 5270 and MRSA (methicillin-resistant Staphylococcus aureus) 3089.
Aquaculture ; Enterococcus faecium ; Fungi ; Gliotoxin ; Humans ; Lactococcus ; Methicillin-Resistant Staphylococcus aureus ; Mortality ; Oxytetracycline ; Penicillium ; Staphylococcus ; Streptococcus

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co²⁺, Mn²⁺, and Zn²⁺ but was strongly inhibited by Ni²⁺and Cu²⁺. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.
Glutamyl Aminopeptidase ; Lactococcus lactis/genetics* ; Biological Transport ; Culture Media ; Glutamic Acid