Food-grade Lactic Acid Bacteria Research Articles

Development of a Golden Gate Assembly-Based Genetic Toolbox for Lactiplantibacillus plantarum and Its Application for Engineering Monoterpenoid Biosynthesis.

Lactiplantibacillus plantarum is a food-grade lactic acid bacterium widely used in the food and beverage industry. Recently, this probiotic organism has been applied as a biofactory for the production of pharmaceutical and food-related compounds, but existing promoters and expression vectors for the genetic engineering of L. plantarum rely on inefficient cloning strategies and are usually not well-characterized. We therefore developed a modular and standardized Golden Gate Assembly-based toolbox for the de novo assembly of shuttle vectors from Escherichia coli to L. plantarum. A collection of the most relevant genetic parts, e.g., different origins of replication and promoters, was incorporated in our toolbox and thoroughly characterized by flow cytometry and the fluorescence assay. Standardized fusion sites allow combining the genetic part freely into a plasmid in one step. This approach allows for the high-throughput assembly of numerous constructs in a standardized genetic context, thus improving the efficiency and predictability of metabolic engineering in L. plantarum. Using our toolbox, we were able to produce the aroma compounds linalool and geraniol in L. plantarum by extending its native mevalonate pathway with plant-derived monoterpenoid synthases.

Effects of lacidophilin in a mouse model of low-grade colitis and nonalcoholic fatty liver disease

Lacidophilin, which is a bacteriocin produced by food-grade lactic acid bacteria, modulates the gut microbiota structure to enhance gut health. This study investigated the effects and mechanism of lacidophilin in a mouse model of low-grade colitis and NAFLD. After 126 days of feeding and medication intervention, high-dose lacidophilin (1.2 g per kg body weight) mitigated NAFLD-associated changes in biochemical parameters and prevented hyperlipidemia and hepatic lipid accumulation. Additionally, lacidophilin exerted a protective effect by reducing damage to the intestinal barrier (ZO-1/Mucin 2), increasing the relative abundance of Lachnospiraceae and Prevotellaceae, decreasing the relative abundance of Erysipelotrichaceae in low-grade colitis and NAFLD mice. In addition, fecal metabolomics studies showed that lacidophilin could regulate both amino acid and lipid metabolism to protect against NAFLD. Flow cytometry analysis of blood samples showed that lacidophilin could regulate the balance of Th1/Th2 and Th17/Treg. Transcriptomic analysis of the liver suggests that treatment with lacidophilin helped maintain immune homeostasis and facilitate lipid metabolism. In short, lacidophilin extracts alleviated lipid accumulation and inflammation in NAFLD mice.

Flavour by design: food-grade lactic acid bacteria improve the volatile aroma spectrum of oat milk, sunflower seed milk, pea milk, and faba milk towards improved flavour and sensory perception

BackgroundThe global market of plant-based milk alternatives is continually growing. Flavour and taste have a key impact on consumers’ selection of plant-based beverages. Unfortunately, natural plant milks have only limited acceptance. Their typically bean-like and grassy notes are perceived as “off-flavours” by consumers, while preferred fruity, buttery, and cheesy notes are missing. In this regard, fermentation of plant milk by lactic acid bacteria (LAB) appears to be an appealing option to improve aroma and taste.ResultsIn this work, we systematically studied LAB fermentation of plant milk. For this purpose, we evaluated 15 food-approved LAB strains to ferment 4 different plant milks: oat milk (representing cereal-based milk), sunflower seed milk (representing seed-based milk), and pea and faba milk (representing legume-based milk). Using GC‒MS analysis, flavour changes during anaerobic fermentations were studied in detail. These revealed species-related and plant milk-related differences and highlighted several well-performing strains delivered a range of beneficial flavour changes. A developed data model estimated the impact of individual flavour compounds using sensory scores and predicted the overall flavour note of fermented and nonfermented samples. Selected sensory perception tests validated the model and allowed us to bridge compositional changes in the flavour profile with consumer response.ConclusionSpecific strain-milk combinations provided quite different flavour notes. This opens further developments towards plant-based products with improved flavour, including cheesy and buttery notes, as well as other innovative products in the future. S. thermophilus emerged as a well-performing strain that delivered preferred buttery notes in all tested plant milks. The GC‒MS-based data model was found to be helpful in predicting sensory perception, and its further refinement and application promise enhanced potential to upgrade fermentation approaches to flavour-by-design strategies.

Optimization and cholesterol-lowering activity of exopolysaccharide from Lactiplantibacillus paraplantarum NCCP 962

Exopolysaccharides (EPSs) are biological polymers with unique structural features have gained particular interest in the fields of food, chemistry and medicine, and food industry. EPS from the food-grade lactic acid bacteria (LAB) can be used as a natural food additives to commercial ones in the processing and development of functional foods and nutraceuticals. The current study was aimed to explore the EPS-producing LAB from the dahi; to optimize the fermentation conditions through Plackett-Burman (PB) and response surface methodology (RSM); and to study its physicochemical, rheological, functional attributes, and cholesterol-lowering activity. Lactiplantibacillus paraplantarum NCCP 962 was isolated among the 08 strains screened at the initial stage. The PB design screened out four independent factors that had a significant positive effect, i.e., lactose, yeast extract, CaCl2, and tryptone, while the remaining seven had a non-significant effect. The RSM exhibited lactose, yeast extract, and CaCl2, significantly contributing to EPS yield. The maximum EPS yield (0.910g/L) was obtained at 6.57% lactose, 0.047% yeast extract, 0.59% CaCl2, and 1.37% tryptone. The R2 value above 97% explains the higher variability and depicts the model's validity. The resulted EPS was a heteropolysaccharide in nature with mannose, glucose, and galactose monosaccharides. FTIR spectrum reflected the presence of functional groups, i.e., O-H, C-H, C = O, C-O-H, and CH2. SEM revealed a porous and rough morphology of EPS, also found to be thermally stable and negligible weight loss, i.e., 14.0% at 257°C and 35.4% at 292.9°C was observed in the 1st and 2nd phases, respectively. Rheological attributes revealed that strain NCCP 962 had high viscosity by increasing the EPS concentration, low pH, and temperature with respectable water holding, oil capacities, foaming abilities, and stability. NCCP 962 EPS possessed up to 46.4% reduction in cholesterol concentration in the supernatant. Conclusively, these results suggested that strain NCCP 962 can be used in food processing applications and other medical fields. KEY POINTS: • The fermentation conditions affect EPS yield from L. paraplantarum and significantly increased yield to 0.910g/L. • The EPS was heteropolysaccharide in nature and thermally stable with amorphous morphology. • Good cholesterol-lowering potential with the best rheological, emulsifying, and foaming capacities.

Exploring the binding capacity of lactic acid bacteria derived bacteriocins against RBD of SARS-CoV-2 Omicron variant by molecular simulations

The changes in the SARS-CoV-2 genome have resulted in the emergence of new variants. Some of the variants have been classified as variants of concern (VOC). These strains have higher transmission rate and improved fitness. One of the prevalent were the Omicron variant. Unlike previous VOCs, the Omicron possesses fifteen mutations on the spike protein's receptor binding domain (RBD). The modifications of spike protein's key amino acid residues facilitate the virus' binding capability against ACE2, resulting in an increase in the infectiousness of Omicron variant. Consequently, investigating the prevention and treatment of the Omicron variant is crucial. In the present study, we aim to explore the binding capacity of twenty-two bacteriocins derived from Lactic Acid Bacteria (LAB) against the Omicron variant by using protein-peptidedocking and molecular dynamics (MD) simulations. The Omicron variant RBD was prepared by introducing fifteen mutations using PyMol. The protein-peptide complexes were obtained using HADDOCK v2.4 docking webserver. Top scoring complexes obtained from HADDOCK webserver were retrieved and submitted to the PRODIGY server for the prediction of binding energies. RBD-bacteriocin complexes were subjected to MD simulations. We discovered promising peptide-based therapeutic candidates for the inhibition of Omicron variant for example Salivaricin B, Pediocin PA 1, Plantaricin W, Lactococcin mmfii and Enterocin A. The lead bacteriocins, except Enterocin A, are biosynthesized by food-grade lactic acid bacteria. Our study puts forth a preliminary information regarding potential utilization of food-grade LAB-derived bacteriocins, particularly Salivaricin B and Pediocin PA 1, for Covid-19 treatment and prophylaxis. Communicated by Ramaswamy H. Sarma

Oral delivery of the intracellular domain of the insulinoma-associated protein 2 (IA-2ic) by bacterium-like particles (BLPs) prevents type 1 diabetes mellitus in NOD mice

Antigen-specific immune tolerance, which possesses great potential in preventing or curing type 1 diabetes mellitus (T1DM), can be induced by oral vaccination with T1DM-related autoantigens. However, direct administration of autoantigens via oral route exhibits a low tolerance-inducing effect as a result of the digestion of protein antigens in the gastrointestinal tract (GIT) and therefore, a large dosage of autoantigens may be needed. In this study, bacterium-like particles (BLPs) made from food-grade lactic acid bacteria were used to deliver the intracellular domain of the insulinoma-associated protein 2 (IA-2ic). For this purpose, BLPs-IA-2ic vaccine in which IA-2ic bound to the surface of BLPs was constructed. BLPs enhanced the stability of the delivered IA-2ic based on the stability analysis in vitro. Oral administration of BLPs-IA-2ic significantly reduced T1DM incidence in NOD mice. The mice fed BLPs-IA-2ic exhibited a significant reduction in insulitis and preserved the ability to secrete insulin. Immunologic analysis showed that oral vaccination with BLPs-IA-2ic induced antigen-specific T cell tolerance. The results revealed that the successful induction of immune tolerance was dependent on the immune deviation (in favor of T helper 2 responses) and CD4+CD25+FoxP3+ regulatory T cells. Hence, oral vaccination with BLPs-IA-2ic shows potential for application in preventing T1DM.

Four in one-Combination therapy using live Lactococcus lactis expressing three therapeutic proteins for the treatment of chronic non-healing wounds.

Diabetes mellitus is one of the major concerns for health care systems, affecting 382 million people worldwide. Among the different complications of diabetes, lower limbs chronic ulceration is a common, severe and costly cause of morbidity. Diabetic foot ulcers are a leading cause of hospitalization in diabetic patients and its rate exceed the ones of congestive heart failure, depression or renal disease. Diabetic non-healing ulcers account for more than 60% of all non-traumatic lower limb amputations and the five-year mortality after amputation is higher than 50%, being equal to several types of advanced cancer. The primary management goals for an existing diabetic foot ulcer are to achieve primary healing as expeditiously as possible and to achieve a reduction of the amputation rate in the patients. Unfortunately, approximately a quarter of patients do not partially or fully respond to the standard of care. Advanced therapies for chronic wounds are existing, however, recent guidelines including the latest reviews and meta-analyses of the scientific and clinical evidence available from current treatment strategies and new therapeutic agents revealed that there is a lack of clinical data and persistent gap of evidence for many of the advanced therapeutic approaches. In addition, no pharmacological wound healing product has gained authority approval for more than 10 years in both US and EU, constituting a highly unmet medical need. In this publication we present data from a live biopharmaceutical product AUP1602-C designed as a single pharmaceutical entity based on the non-pathogenic, food-grade lactic acid bacterium Lactococcus lactis subsp. cremoris that has been genetically engineered to produce human fibroblast growth factor 2,interleukin4 and colony stimulating factor 1. Designed to address different aspects of wound healing (i.e. fibroblast proliferation, angiogenesis and immune cell activation) and currently in phase I clinical study, we show how the combination of the individual components on the wound micro-environment initiates and improves the wound healing in chronic wounds.

Characterization of a lactic acid bacterium-derived β-glucosidase for the production of rubusoside from stevioside

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5–6.5 and 30–40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher β-glucosidase activity toward the β-linked glucosidic bond of stevioside than toward other β-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The β-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this β-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Synthesis and characteristics of a rebaudioside-A like compound as a potential non-caloric natural sweetener by Leuconostoc kimchii dextransucrase

Stevioside (ST) is currently considered as a highly-demanded natural and zero-caloric replacer of sucrose with several health-promoting properties. Nonetheless, its bitter aftertaste limits its use in the food industry. Herein, glucosyl steviosides were synthesized using primarily a food-grade lactic acid bacteria, Leuconostoc kimchii dextransucrase and conversion yield (%) was 40.3%. A glucose moiety was transferred stereo-selectively to ST by α-1,6-linkage and this is the first report about obtaining rebaudioside A (Reb-A) like glucosyl stevioside-2 (STG-2). Glucosyl steviosides revealed greatly improved stability up to 120 °C and remained stable over 32.1% and 58.12% in the pH (1.4) compared with 30.55% of ST. Moreover, the glucosylated steviosides improved the stability, reaching 95% after 30 days and Reb-A like compound (STG-2) especially exhibited higher stability in commercial beverages. Furthermore, the glucosyl steviosides showed over 1.92- and 2.24-fold decreases than that of enzymatically modified ST in the glucose generation rate test.

High ZnPP-forming food-grade lactic acid bacteria as a potential substitute for nitrite/nitrate to improve the color of meat products

Zinc protoporphyrin IX (ZnPP)-forming food-grade lactic acid bacteria (LAB) were screened from various sources for their ability to improve the color of meat products. The effects of salt and nitrite on the ZnPP-forming ability of these bacteria were also investigated. Finally, these bacteria were applied in salt-added minced meat to assess their ability to improve the color. Twenty-five LAB were screened for their ZnPP-forming ability in pork. Most of the strains exhibited maximum growth anaerobically in 3% salt at 30 °C and grew well at pH 5.5 and 6.5. Moreover, 3% salt slightly retarded ZnPP formation; however, nitrite completely inhibited ZnPP formation in all the ZnPP-forming LAB. Thirteen LAB (avoiding duplication and non-food-grade) could form ZnPP in salt-added minced meat, resulting in improvement of the bright red color, high ZnPP autofluorescence, and increased fluorescence intensity. Finally, considering the safety, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, and Leuconostoc lactis were suggested as promising candidates to improve the color of meat products.

Anti-listerial activity of thermophilin 110 and pediocin in fermented milk and whey

Listeria monocytogenes is a pathogenic bacterium responsible for foodborne illness worldwide. Antimicrobial peptides, or bacteriocins, produced by food-grade lactic acid bacteria can serve as preservatives to prevent Listeria's growth in various foods, including dairy products. This study investigated the anti-listerial activities of bacteriocin-producing lactic acid bacteria, Streptococcus thermophilus B59671, and Lactobacillus plantarum 076. In vitro studies showed that the concentration of pediocin produced by L. plantarum 076 (2560 AU/mL) inhibited the growth of a six-strain cocktail of L. monocytogenes. However, the concentration of thermophilin 110 produced by S. thermophilus B59671 (320 AU/mL) only delayed the growth by ~2 h. Higher concentrations of thermophilin 110 (≥640 AU/mL) suppressed Listeria growth for up to 22 h. Pasteurized skim milk fermented with a co-culture of S. thermophilus B59671 and L. plantarum 076 reduced the number of L. monocytogenes cells by > 4 Log CFU/mL due mainly to the activity of pediocin. The anti-listerial activity was not observed in whey samples collected from pasteurized skim milk fermented with this co-culture but was detected when raw milk was the substrate. Two additional whey preparations, the by-products from commercial bovine and goat raw-milk cheeses, also inhibited Listeria growth and reduced the number of cells following storage at 4 °C for one week. This study showed that a concentrated preparation of thermophilin 110 has potential as an anti-listerial compound. It demonstrated the prospect of using a co-culture of S. thermophilus B59671 and L. plantarum 076 to prevent Listeria contamination in dairy foods. Additionally, results showed that metabolites with antimicrobial activities may be generated during the fermentation of raw milk due to indigenous microflora.

Lactobacillus fermentum CQPC08 protects rats from lead-induced oxidative damage by regulating the Keap1/Nrf2/ARE pathway.

In this experiment, Lactobacillus fermentum CQPC08 (LF-CQPC08) isolated from traditionally fermented pickles was used to study its mitigation effect on lead acetate-induced oxidative stress and lead ion adsorption capacity in rats. In vitro experiments showed that the survival rate in artificial gastric juice and the growth efficiency in artificial bile salt of LF-CQPC08 was 93.6% ± 2.2% and 77.2% ± 0.8%, and the surface hydrophobicity rate was 45.5% ± 0.3%. The scavenging rates of hydroxyl radical, superoxide anion, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were 47.8% ± 0.9%, 63.9% ± 1.2%, and 83.6% ± 1.5%, respectively, and the reduction power was 107.3 ± 2.8 μmol L-1. LF-CQPC08 could not only adsorb 76.9% ± 1.0% lead ions in aqueous solution but also reduce the lead content in serum, liver, kidneys, and brain tissue of Sprague-Dawley (SD) rats, as well as maintain the cell structure and tissue state of the liver and kidneys. In addition, by examining the indicators of inflammation and oxidation in the serum, liver, and kidneys of SD rats, we found that LF-CQPC08 can reduce the proinflammatory factors interleukin (IL)-1 beta (1β), IL-6, tumor necrosis factor alpha, and interferon gamma in the body, increase the level of anti-inflammatory factor IL-10, enhance the activity of antioxidant enzymes such as superoxide dismutase and catalase and glutathione levels in serum and organ tissues, and reduce the production of reactive oxygen species and accumulation of lipid peroxide malondialdehyde. LF-CQPC08 can also activate the Keap1/Nrf2/ARE signaling pathway to promote high-level expression of the downstream antioxidants heme oxygenase 1 (HO-1), NAD(P)H : quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). As food-grade lactic acid bacteria, LF-CQPC08 has great potential and research value in removing heavy metals from food and alleviating the toxicity of heavy metals in the future.

Selection of Riboflavin Overproducing Strains of Lactic Acid Bacteria and Riboflavin Direct Quantification by Fluorescence.

Riboflavin (vitamin B2) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic acid bacteria (LAB) can synthesize riboflavin and this capability is strain-dependent. In the last years, a growing interest has focused on the selection of riboflavin-overproducing food-grade LAB for the vitamin biofortification of fermented foods, as well as for the formulation of innovative functional products.In this chapter we report fast and inexpensive techniques in order to (1) screen LAB isolates able to produce riboflavin from different matrices, (2) select spontaneous roseoflavin-resistant riboflavin overproducing strains, and (3) quantify vitamin B2 in culture media by fluorescence detection.These protocols could be useful to select new overproducing strains and/or species from different ecological niches, as well as to optimize the conditions for vitamin bioproduction.

Purification and characterization of novel exopolysaccharides produced from Lactobacillus paraplantarum KM1 isolated from human milk and its cytotoxicity

BackgroundMicrobial origin polysaccharides have gained popularity due to lesser toxicity, better degradability and selectivity as compared to their synthetic counterparts and can be used as emulsifier, stabilizer, thickener, texturizer, flocculating and gelling agent. Here main emphasis on exopolysaccharide production from potential lactic acid bacteria that has GRAS status. ResultsThis work was aimed at isolating, purifying and characterizing an extracellular polysaccharide (EPS) produced by a foodgrade lactic acid bacteria Lactobacillus paraplantarum KM1. L. paraplantarum KM1 was isolated from human milk and identified by conventional and molecular techniques. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KX671558. L. paraplantarum KM1 was found to produce EPSs in lactose containing MRS medium, and the maximum yield (47.4 mg/ml) was achieved after 32-h incubation. As evident from TLC and HPLC analyses, the polysaccharide was found to be a heteropolymer-containing glucose, galactose and mannose as main sugars. Different oligosaccharides namely hexoses were obtained after partial hydrolysis of the polymer using MALDI-ToF-MS. The total molecular weight of all polysaccharides present was 348.7 kDa with 100 °C thermal stability as well as water soluble in nature. Cell cytotoxicity revealed that the purified EPS was safe for consumption; thus, it can be used in various food industries as emulsifying and texture agent. ConclusionsThe present study highlighted that exopolysaccharides could be harnessed to improve food products in terms of texture, emulsifying agents, pharmaceutical industry (antioxidants, antitumour, anti-inflammatory and antiviral agents) and as safety purposes.

Bioprotective potential of lactic acid bacteria

Abstract Acidification in lactic-fermented foods is realized by lactic acid bacteria as an added starter culture or by autochthonous strains. These microbial strains possess different prominent features that define the technological, organoleptic, nutritional, and microbial safety aspects of the product. The bioprotective effect of the bacterial strains may be related to antagonistic properties against food spoilage and/or pathogenic strains. The aim of the present study is to determine the antimicrobial properties of three different food-grade lactic acid bacteria in order to use them as bioprotective cultures. Our findings show that the Lactobacillus pentosus, Enterococcus faecalis, and Pediococcus parvulus exerted a bacteriostatic effect on Escherichia coli and Bacillus cereus, whereas the Saccharomyces cerevisiae growth was not inhibited, which made them susceptible agent for co-culture systems.

An oral vaccine against CVA16 (Coxsackievirus A16) was developed by constructing a recombinant<i> Lactococcus lactis</i>

Purpose: To develop an oral vaccine against CVA16 (Coxsackievirus A16) by constructing a recombinant Lactococcus lactis that expresses VP1 from CVA16.Method: An oral CVA16 vaccine was prepared by expressing CVA16 VP1 protein with Lactococcus lactis. CVA16 VP1 gene was incorporated into a Lactobacillus expression vector, namely, pNZ8148, and then expressed in NZ9000, a food-grade lactic acid bacterium which serves as a carrier for oral vaccines.Results: There was statistically significant difference in CVA16-specific IgG antibody level between NZ9000-pNZ8148-CVA16-VP1 group (0.49 ± 0.05) and control group (0.05 ± 0.00) when the antiserum was diluted 1:10 (t = 19.84; p < 0.05). Furthermore, the level of CVA16-specific IgA antibody in NZ9000- pNZ8148-CVA16-VP1 group (0.17 ± 0.02) was significantly higher than in control group (0.05 ± 0.00) following antiserum dilution of 1:10 (t =12.08; p < 0.05).Conclusion: A CVA16 oral vaccine made from Lactobacillus elicits protective antibodies against CVA16. Thus, it is a potential as oral vaccine against CVA16 but further studies in vivo are required to ascertain its safety and effectiveness.
 Keywords: Coxsackievirus A16, Hand, foot and mouth disease, Lactococcus lactis, Oral vaccine, Enterovirus 71

A Lactococcus lactis-vectored oral vaccine induces protective immunity of mice against enterotoxigenic Escherichia coli lethal challenge

Enterotoxigenic Escherichia coli (ETEC) is a global primary pathogenic bacterium causing diarrhoea in human and a wide variety of neonatal animals. Lactococcus lactis as non-pathogenic and food-grade lactic acid bacteria has already been explored as a vector for mucosal vaccine. Here, the current study was undertaken to evaluate the live recombinant L. lactis (rL. lactis) vaccine expressing the trivalent enterotoxin protein STa-LTB-STb and the F5 fimbrial antigen (SLS-F5) with OmpH of Yersinia enterocolitica in protection against ETEC. Western blot confirmed the expression of fusion protein SLS-F5-OmpH in nisin-controlled expression (NICE) system. Mice orally immunized with rL. lactis-SLS-F5-OmpH were observed to produce high levels of mucosal SIgA and serum IgG antibodies, while also inducing increases in the production of CD4+ and CD8+ T cells, lymphocyte proliferation, and secretion of cytokines. Moreover, orally immunized mice produced complete protection after ETEC challenge. The above results suggested that rL. lactis-SLS-F5-OmpH has the potential as a candidate for oral vaccine against ETEC.

Targeting Melanoma Hypoxia with the Food-Grade Lactic Acid Bacterium Lactococcus Lactis.

Melanoma is the most aggressive form of skin cancer. Hypoxia is a feature of the tumor microenvironment that reduces efficacy of immuno- and chemotherapies, resulting in poor clinical outcomes. Lactococcus lactis is a facultative anaerobic gram-positive lactic acid bacterium (LAB) that is Generally Recognized as Safe (GRAS). Recently, the use of LAB as a delivery vehicle has emerged as an alternative strategy to deliver therapeutic molecules; therefore, we investigated whether L. lactis can target and localize within melanoma hypoxic niches. To simulate hypoxic conditions in vitro, melanoma cells A2058, A375 and MeWo were cultured in a chamber with a gas mixture of 5% CO2, 94% N2 and 1% O2. Among the cell lines tested, MeWo cells displayed greater survival rates when compared to A2058 and A375 cells. Co-cultures of L. lactis expressing GFP or mCherry and MeWo cells revealed that L. lactis efficiently express the transgenes under hypoxic conditions. Moreover, multispectral optoacoustic tomography (MSOT), and near infrared (NIR) imaging of tumor-bearing BALB/c mice revealed that the intravenous injection of either L. lactis expressing β-galactosidase (β-gal) or infrared fluorescent protein (IRFP713) results in the establishment of the recombinant bacteria within tumor hypoxic niches. Overall, our data suggest that L. lactis represents an alternative strategy to target and deliver therapeutic molecules into the tumor hypoxic microenvironment.

Surface display of uropathogenic Escherichia coli FimH in Lactococcus lactis: In vitro characterization of recombinant bacteria and its protectivity in animal model

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are very common, leading to high patient morbidity and substantial medical costs. The development of non-antibiotic strategies such as food-grade lactic acid bacterium can be recognized as an attractive and safe alternative way against UTI. Here, we report the construction of Lactococcus lactis (L. lactis) strain genetically modified to produce FimH virulence factor of UPEC on the cell surface. We showed the FimH inserted into the pT1NX vector is actively synthesized on L. lactis. The L. lactis-pT1NX-FimH exhibited an auto-aggregation phenotype in liquid cultures and formed robust biofilm on abiotic surface compared to vector-only bacteria. Then, we developed protective biofilms with L. lactis strains and examined their inhibitory effect for exclusion of uropathogenic biofilm formation. In the natural protective biofilm assays, L. lactis-pT1NX-FimH resulted in significant reduction in the pathogen load when compared to the L. lactis-pT1NX. Evaluation of the colonization ability in the bladder showed that L. lactis expressing FimH survived better in the mice bladder than L. lactis harboring vector. Protection assay against UPEC infection was investigated using a UTI mouse model. L. lactis-pT1NX-FimH displayed high effectiveness in the protection of the bladder as compared to the control group after UPEC challenge. The results suggest that genetically engineered L. lactis-pT1NX-FimH can be used as a safe alternative way for control of biofilm formation in UPEC. Furthermore, the possibility of using L. lactis-pT1NX-FimH as a new promising strategy against UTIs caused by UPEC strains is proposed.

Cheese whey to biohydrogen and useful organic acids: A non-pathogenic microbial treatment by L. acidophilus

The burgeoning organic waste and continuously increasing energy demands have resulted in significant environmental pollution concerns. To address this issue, the potential of different bacteria to produce biogas/biohydrogen from organic waste can be utilized as a source of renewable energy, however these pathogenic bacteria are not safe to use without strict contact isolation. In this study the role of safe food grade lactic acid bacteria (Lactobacillus spp.) was investigated for production of biogas from cheese waste with starting hexose concentration 32 g/L. The bacterium Lactobacillus acidophilus was identified as one of the major biogas producers at optimum pH of 6.5. Further the optimum inoculum conditions were found to be 12.5% at inoculum age of 18 h. During the investigation the maximum biogas production was observed to be 1665 mL after 72 hours of incubation at pH 6.5. The biogas production was accompanied with production of other valuable metabolites in the form of organic acids including pyruvate, propionate, acetate, lactate, formate and butyrate. Thus this research is paving way for nonpathogenic production of biohydrogen from food waste.