Adaptive Evolution of Lactococcus Lactis to Thermal and Oxidative Stress Increases Biomass and Nisin Production.

High values of agitation and temperature lead to stressful conditions in the fermentations of Lactococcus lactis due to its aero-tolerant and mesophilic nature. Here, the adaptive laboratory evolution (ALE) technique was applied to increase biomass and nisin production yields by enhancing L. lactis subsp. lactis robustness at higher growth temperature and aeration rates. In two separate ALE experiments, after 162 serial transfers, optimum agitation and growth temperature of L. lactis were shifted from 40 rpm and 30 °C to 200 rpm and 37 °C, respectively. Oxidative and acid resistance were enhanced in the evolved strain. Whole-genome sequencing revealed the emergence of five single-nucleotide polymorphisms in the genome of the evolved strain in jag, DnaB, ArgR, cation transporter genes, and one putative protein. The evolved strain of L. lactis in this study has more industrial desirable features and improved nisin production capability and can act more efficiently in nisin production in stressful conditions.

Generation of Lactose- and Protease-Positive Probiotic Lacticaseibacillus rhamnosus GG by Conjugation with Lactococcus lactis NCDO 712.

Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L. rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L. lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L. rhamnosus LAB49 effectively degraded ß-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GMOs), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture.IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.

Importance of the leader peptide sequence on the lanthipeptide secretion level.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides. Their precursor peptide comprises of an N-terminal leader peptide and a C-terminal core peptide. Here, the leader peptide is crucial for enzyme recognition especially for the modification enzymes and acts furthermore as a secretion signal for the lanthipeptide exporter. The core peptide is the target site for the posttranslational modifications and contains dehydrated amino acids and lanthionine rings. Nisin produced by the Gram-positive bacterium Lactococcus lactis is one of the best-studied lanthipeptides and used as a model system to study their modification and secretion processes. Nisin is secreted as a precursor peptide. Here, we present an in vivo secretion analysis of NisT in the absence of the modification machinery allowing the secretion of leader peptide mutants and their impact solely on the secretion activity of NisT. Additionally, we created leader peptide hybrids to provide new insights, how the secretion is effected by unnatural leader peptides. The focus on the secretion activity of the transporter alone enabled us to determine the recognition site of NisT within the leader peptide of nisin.

Broadening the antimicrobial spectrum of nisin-producing Lactococcus lactis subsp. Lactis to Gram-negative bacteria by means of active packaging.

Cast films obtained from polyvinyl alcohol (PVOH) blended with casein hydrolysates (HCas) in a weight ratio of 1:1 were employed to carry nisin-producing L. lactis and phytic acid in order to broaden the antimicrobial spectrum of L. lactis to Gram-positive and Gram-negative spoilage and pathogen bacteria. For this purpose, the effect of the antimicrobial activity of various film formulations and combinations of films on the growth of E. coli at 37 °C for 24 h was studied. The film system that showed antimicrobial activity against Gram-negative bacteria consisted of phytic acid and L. lactis incorporated in separate films. When the active agents were in the same film the viability of L. lactis decreased considerably and it did not exert antimicrobial activity against the bacterium. Therefore, the combination of L. lactis and phytic acid in separate films was chosen as the reliable system, and the effect of its activity on the growth of Gram-negative bacteria (E. coli, Salmonella enterica, and Pseudomonas fluorescens) and Gram-positive bacteria (Listeria monocytogenes) in liquid culture medium was tested at refrigeration temperature (4 °C), and with simulated breaks in the cold chain (14 °C and 24 °C). The survival of L. lactis in coexistence with these bacteria was also studied. The film system exerted an antimicrobial effect against the Gram-negative bacteria tested, and the activity depended on the bacteria and the temperature assayed. With regard to the antimicrobial activity against L. monocytogenes, phytic acid improved the antimicrobial capacity of L.lactis. The survival of L. lactis was maintained at 7-8 log (CFU/mL) culture in liquid medium throughout the storage period. The films developed were intended to be used as coatings in the design of a double-sided active bag for a non-fermented dairy product. The bags were filled with homemade preservative-free pastry cream, and the microbiological shelf life and evolution of pH of the packaged ready-to-eat food stored at 4 °C was studied for 20 days. The results showed a reduction in the growth of spoilage bacteria and therefore an increase in the shelf life of the packaged product. The films developed could be applied in the design of packages for perishable dairy foods in order to increase their microbiological shelf life.

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

BacSJ-Another Bacteriocin with Distinct Spectrum of Activity that Targets Man-PTS.

Lactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L.lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L.lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ.

Construction of Genetically Modified Lactococcus lactis Producing Anti-human-CTLA-4 Single-Chain Fragment Variable.

Lactic acid bacteria are human commensal organisms that have immunomodulatory and metabolism-promoting effects. In addition, due to the increasing demand for biopharmaceuticals, genetically modified lactic acid bacteria (gmLAB) that produce recombinant proteins are expected to be used as microbial therapeutics and next-generation probiotics. In this study, we constructed a gmLAB strain that produces anti-human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) single-chain fragment variable (CTLA4scFv) for possible use in a cancer treatment strategy using gmLAB. CTLA-4, an immune checkpoint molecule, suppresses the anti-cancer immune response; thus, inhibition of CTLA-4 signaling is important in cancer therapy. In this study, we designed a CTLA4scFv composed of a heavy and light chain of the variable region from anti-human CTLA-4 antibody connected by a flexible peptide linker. CTLA4scFv was expressed using nisin controlled gene expression (NICE) system, a lactococcal inducible gene expression system, and the DNA sequence encoding CTLA4scFv was inserted downstream of the PnisA promoter of the gene expression vector pNZ8148#2. Furthermore, expression of recombinant CTLA4scFv was confirmed by Western blotting, and the immunoreactivity of recombinant CTLA4scFv against human CTLA-4 protein was examined using ELISA. We speculate that gmLAB producing bioactive CTLA4scFv will become an attractive approach for cancer treatment.

Suppression of lactate production by aerobic fed-batch cultures of Lactococcus lactis.

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.

Genomics and transcriptomics analysis reveals the mechanism of isobutanol tolerance of a laboratory evolved Lactococcus lactis strain.

Isobutanol, in spite of its significant superiority over ethanol as a biofuel, remains commercially non-viable due to the non-availability of a suitable chassis which can handle the solvent toxicity associated with its production. To meet this challenge, we chose Lactococcus lactis which is known for its ability to handle environmental stress and carried out Adaptive laboratory evolution (ALE) in a continuous stirred tank reactor (CSTR) to evolve an isobutanol tolerant strain. The strain was grown for more than 60 days (> 250 generations) while gradually increasing the selection pressure, i.e. isobutanol concentration, in the feed. This led to the evolution of a strain that had an exceptionally high tolerance of up to 40 g/l of isobutanol even though a scanning electron microscope (SEM) study as well as analysis of membrane potential revealed only minor changes in cellular morphology. Whole genome sequencing which was done to confirm the strain integrity also showed comparatively few mutations in the evolved strain. However, the criticality of these mutations was reflected in major changes that occurred in the transcriptome, where gene expression levels from a wide range of categories that involved membrane transport, amino acid metabolism, sugar uptake and cell wall synthesis were significantly altered. Analysing the synergistic effect of these changes that lead to the complex phenotype of isobutanol tolerance can help in the construction of better host platforms for isobutanol production.

Hydroxypropyl ß-cyclodextrin improving multiple stresses tolerance of Lactococcus lactis subsp. lactis.

L. lactis is known as industrial starter in the fermentation of dairy and meat products, and it plays an important role in human health as an edible probiotic. During industrial production, L. lactis often experiences different stresses that delay the growth and decrease the survival in some serious conditions. In this study, the protective effects of hydroxypropyl ß-cyclodextrin (HP ß-CD) on L. lactis under multiple stresses were investigated. The microbial cells were treated with different stresses including heat, NaCl, cold, and H2 O2 stresses, and the results were showed by measuring the OD600 or spot plating method. The growth and tolerance were improved when HP ß-CD was added during different stress conditions, better than that of trehalose. Besides, the scanning electron microscopic and fluorescence spectrum studies showed that HP ß-CD could combine with L. lactis to protect the cell structure, suggesting that HP ß-CD may act as a protective agent of L. lactis. Therefore, HP ß-CD could be considered as a potential protective agent to be applied in food industry, and its protective mechanism on L. lactis still needs further investigation.

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

ß-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

Generation of an engineered food-grade Lactococcus lactis strain for production of an antimicrobial peptide: in vitro and in silico evaluation.

BACKGROUND: Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. RESULTS: Peptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureus E. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 µ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. CONCLUSIONS: Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.

Harnessing Adaptive Evolution to Achieve Superior Mannitol Production by Lactococcus lactis Using Its Native Metabolism.

Mannitol can be obtained as a by-product of certain heterolactic lactic acid bacteria, when grown on substrates containing fructose. Lactococcus lactis, a homolactic lactic acid bacterium, normally does not form mannitol but can be persuaded into doing so by expressing certain foreign enzyme activities. In this study, we find that L. lactis has an inherent capacity to form mannitol from glucose. By adaptively evolving L. lactis or derivatives blocked in NAD+ regenerating pathways, we manage to accelerate growth on mannitol. When cells of the adapted strains are resuspended in buffer containing glucose, 4-58% of the glucose metabolized is converted into mannitol, in contrast to nonadapted strains. The highest conversion was obtained for a strain lacking all major NAD+ regenerating pathways. Mannitol had an inhibitory effect on the conversion, which we speculated was due to the mannitol uptake system. After its inactivation, 60% of the glucose was converted into mannitol by cells suspended in glucose buffer. Using a two-stage setup, where biomass first was accumulated by aerated culturing, followed by a nonaerated phase (static conditions), it was possible to obtain 6.1 g/L mannitol, where 60% of the glucose had been converted into mannitol, which is the highest yield reported for L. lactis.

Production and molecular structure of heteropolysaccharides from two lactic acid bacteria.

In the dairy industry, exopolysaccharides (EPS) produced in situ from lactic acid bacteria are of great interest because of their contribution to product texture. Some EPS cause ropiness which might be linked to specific physical and chemical EPS properties. EPS show a broad variety of chemical structures and, because analysis is rather complex, it is still a major challenge to establish structure-function relationships. The aim of this study was to produce EPS with different degree of ropiness, perform in-depth structural elucidations and relate this information to their behaviour in aqueous solutions. After cultivation of Streptococcus thermophilus DGCC7919 and Lactococcus lactis LL-2A and subsequent EPS isolation, both EPS showed similar macromolecular properties, but pronounced differences in monosaccharide composition and glycosidic linkages. Our data suggests that mainly the side chains in the EPS from LL-2A might be responsible for a higher ropiness than that observed for EPS from DGCC7919.

Higher nisin yield is reached with glutathione and pyruvate compared with heme in Lactococcus lactis N8.

There are different studies that aim to enhance the production of nisin by Lactococcus lactis since its chemical synthesis is not possible. In this study, glutathione (GSH) and pyruvate, which are known to reduce the oxidative stress of cells, have been shown to trigger the production of nisin at both transcriptional and translational levels in L. lactis cells grown under aerobic condition. Presence of GSH and pyruvate caused more nisin yield than the heme-supplemented medium. Moreover, the expression of genes that encode stress-related enzymes were apparently upregulated in the presence of GSH and pyruvate. It can be concluded that GSH and pyruvate contribute to the defense system of L. lactis cells and so that higher biomass was obtained which in turn enhance nisin production. Antioxidant effect of GSH and pyruvate was known; however, their stimulating effect on nisin production was shown for the first time in this study.

Suppression of lactate production by using sucrose as a carbon source in lactic acid bacteria.

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9 g-lactate·g-cell-1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.

Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source.

We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NAD+ that is consumed during glycolysis. Glucose suppresses NAD+ regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NAD+ regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture.

Fluorescent detection of nisin by genetically modified Lactococcus lactis strains in milk and a colonic model: Application of whole-cell nisin biosensors.

Detection of bioactive peptides in complex ecosystems like intestinal environment is a difficult task. In this study, we developed two new bioreporters for nisin based on Lactococcus lactis NZ9000 transformed with the vector pNZ:Nis-aFP or pNZ:Nis-mCherry, that encoded for the anaerobic fluorescent protein evoglow-Pp1 (aFP) or the fluorescent protein mCherry, respectively. The biosensors were used to study nisin A production by L. lactis INIA 650 in milk and in a colonic model. The use of L. lactis NZ9000 pNZ:Nis-aFP as a biosensor allowed the detection of nisin produced by L. lactis INIA 650 in milk, but not in the in vitro colonic model. In milk, this reporter was induced by direct addition of 10 ng/ml nisin while, in the colonic model, nisin concentrations of 50 ng/ml were necessary. However, the reporter system based on pNZ:Nis-mCherry showed a higher sensibility, detecting nisin concentrations of 1 ng/ml produced by L. lactis INIA 650 in colonic media using agar diffusion or cross streak bioassays.

Metabolic engineering of Lactococcus lactis for high level accumulation of glutathione and S-adenosyl-L-methionine.

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0 mg/L) and GSH (17.3 mg/L) was achieved after 17 h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.

A food-grade engineered Lactococcus lactis strain delivering Helicobacter pylori Lpp20 alleviates bacterial infection in H. pylori-challenged mice.

OBJECTIVE: To construct a food-grade bacterium producing and delivering H. pylori Lpp20 antigen and evaluate its immune efficacy against H. pylori challenges with aim to develop anti-H. pylori oral vaccines and functional foods. RESULTS: Lpp20 was expressed as a 22 kDa protein in Lactococcus lactis, constituting 11.2% of the cell lysate proteins, and recognized by mouse antisera. Mice orally gavaged with the engineered bacterium had elevated serum IgG levels and lowered urease activity of stomach following H. pylori challenges. CONCLUSIONS: This study firstly reports a food-grade L. lactis strain delivering Lpp20 to mucosal immunization sites, demonstrating a novel efficient production and safe utilization mode of Lpp20, offering a promising vaccine candidate and health food sources.