RESUMEN
Probiotic production in commercial culture media is expensive, so, it is necessary to design culture media based on "low-cost" components like agro-industrial by-products. Therefore, this study aimed to design an agro-industrial by-product-based culture media using whey, sugarcane molasses, and palm kernel cake as components to produce Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10 isolated from Nile tilapia (Oreochromis niloticus) associated gut microbiota. Higher bacterial concentrations were achieved at high whey concentrations and low concentrations of sugarcane molasses and palm kernel cake (PKC) using agitation. The optimal conditions were whey, 3.84% w/v; sugarcane molasses, 7.39% w/v; PKC, 0.77% w/v; and agitation speed, 75 RPM. Bacterial growth under optimal conditions was compared to that in commercial Brain-Heart Infusion (BHI) broth. L. lactis A12 showed similar growth in the optimal media and BHI. The estimated cost of the culture media based on component prices was USD $ 3.01/L, which is 86.93% lower than BHI broth (USD $ 23.04/L). It was possible to design a "low-cost agro-industrial by-product-based culture media to produce L. lactis A12 and the two Priestia species under monoculture conditions.
Asunto(s)
Medios de Cultivo , Probióticos , Probióticos/metabolismo , Animales , Medios de Cultivo/química , Lactococcus lactis/metabolismo , Lactococcus lactis/crecimiento & desarrollo , Suero Lácteo/microbiología , Suero Lácteo/metabolismo , Cíclidos/microbiología , Cíclidos/metabolismo , Cíclidos/crecimiento & desarrollo , Microbioma Gastrointestinal , Melaza , Alimentación Animal , SaccharumRESUMEN
Target delivery of therapeutic agents with anti-inflammatory properties using probiotics as delivery and recombinant protein expression vehicles is a promising approach for the prevention and treatment of many diseases, such as cancer and intestinal immune disorders. Lactococcus lactis, a Lactic Acid Bacteria (LAB) widely used in the dairy industry, is one of the most important microorganisms with GRAS status for human consumption, for which biotechnological tools have already been developed to express and deliver recombinant biomolecules with anti-inflammatory properties. Cytokines, for example, are immune system communication molecules present at virtually all levels of the immune response. They are essential in cellular and humoral processes, such as hampering inflammation or adjuvating in the adaptive immune response, making them good candidates for therapeutic approaches. This review discusses the advances in the development of new therapies and prophylactic approaches using LAB to deliver/express cytokines for the treatment of inflammatory and autoimmune diseases in the future.
Asunto(s)
Enfermedades Autoinmunes , Lactococcus lactis , Humanos , Lactococcus lactis/metabolismo , Interleucinas/metabolismo , Citocinas/metabolismo , Enfermedades Autoinmunes/tratamiento farmacológico , AntiinflamatoriosRESUMEN
Staphylococcus aureus (SA) can thrive in a wide variety of hosts and environments, causing clinical infections and foodborne intoxications. In Brazil, SA is commonly isolated from traditional soft cheeses, especially those prepared from unpasteurized milk. In this research, the isolate S. aureus SABRC1 was evaluated for virulence traits under different conditions, including co-inoculation with Lactococcus lactis MC5 (isolated from "Fresh Minas Cheese"), which produces antibacterial peptides. Results from experiments with Caco-2 culture indicated S. aureus SABRC1 was able to adhere (42.83 ± 1.79%) and to invade (48.57 ± 0.41%) the intestinal cells. On the other hand, L. lactis MC5 presented anti-staphylococcal activity as indicated by agar assays, and it was also able to antagonize intestinal cell invasion by S. aureus. Moreover, Reverse Transcriptase-PCR experiments showed virulence genes of S. aureus SABRC1 (hla, icaA and sea) were differentially expressed under diverse culture conditions, which included Brain Heart Infusion modified or not by the addition of glucose, sodium chloride, milk or cheese. This suggests the virulence of S. aureus SABRC1 is influenced by compounds commonly found in daily diets, and not only by its genetic repertoire, adding a novel level of complexity for controlling infection by this pathogen.
Asunto(s)
Queso , Lactococcus lactis , Infecciones Estafilocócicas , Humanos , Animales , Staphylococcus aureus , Virulencia , Queso/microbiología , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Células CACO-2 , Técnicas de Cultivo de Célula , Expresión Génica , Leche/microbiologíaRESUMEN
Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.
Asunto(s)
Bacteriocinas , Lactococcus lactis , Frutas/química , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , ARN Ribosómico 16S/genética , Secuencia de Bases , Bacteriocinas/metabolismo , Antibacterianos/metabolismoRESUMEN
Lactic acid bacteria are distributed in nature, isolating themselves from diverse ecosystems and presenting a wide metabolic biodiversity. In Corrientes (Argentina), artisanal cheeses and their specific environment are an important source of autochthonous lactic acid bacteria. The objective of this work was to establish associations between the phenotypic characteristics of strains of Lactococcus lactis subsp. lactis native from Corrientes with climatological data of the Province and the characteristics of the soil and the landscapes. Physiological and biochemical characterization data of Lactococcus lactis subsp. lactis isolated from the dairy environment and from different localities of Corrientes will be used. The strains were space-located through Google Earth, flood and drought events were evaluated using Standardized Precipitation Evapotranspiration Index, and soil composition data (A and Bt horizons) in the study areas were obtained from the experimental station National Institute of Agricultural Technology - Corrientes. A statistical analysis was applied to these results (Infostat Software, Di Rienzo et al. 2008). The resulting consists in three conglomerates, differentiating strains from soils coming from "flooded landscapes" and those from "sandy hills landscape". The analysis by main components highlighted the preference of strains from flooded landscapes by a saline-alkaline environment, affecting during periods of drought, and strains from sandy hills landscape by a low medium in salts and acid soil, directly during period of high humidity resulting from previous floods.
Asunto(s)
Lactococcus lactis , Biodiversidad , Ecosistema , Microbiología de Alimentos , Lactococcus lactis/metabolismo , SueloRESUMEN
Lactococcus spp. are industrially crucial lactic acid bacteria (LAB) used to manufacture lactic acid, pickled vegetables, buttermilk, cheese, and many kinds of delicious dairy foods and drinks. In addition to these, they are also being used as probiotics in specific formulations. However, their uses as probiotics are comparatively less than the other LAB genera. The present communication hypothesizes to validate the probiotic potentiality of two new Lactococcus lactis subsp. lactis strains for their future uses. These native food fermenting strains were characterized for in vitro acid tolerance, tolerance to simulated gastric and pancreatic juices, autoaggregation and co-aggregation, hydrophobicity, haemolytic activity, bile salt deconjugation, cholesterol removal, antimicrobial spectrum, and antibiotic sensitivity. The in vivo live bacterial feeding of these strains for 30 days was done in Swiss albino mice either singly or in combination with prebiotic inulin and evaluated for hypocholesterolemic activity, immune enhancement, and gut colonization efficiency and compared with the commercial probiotic consortia. The study revealed that the strains could survive in human gut bile concentration, gastric pH conditions at pH 2.0, 3.0, and 8.0 for 6 h, had a broad antibacterial spectrum, and cholesterol binding efficacy. The strains could survive with higher colony-forming units (CFU/mL) when amended with sodium caseinate. The strains had autoaggregation ranges from 15 to 25% over 24 h and had a significant co-aggregation with both lactic acid and Gram-positive and Gram-negative bacterial strains related to human illness. The strains also showed solvent and media-specific hydrophobicity against n-hexane and xylene. The live bacterial feeding either singly or in combination with prebiotic inulin resulted in a significant reduction of LDL (low-density lipoprotein), VLDL (very low-density lipoprotein) cholesterol and triglyceride (TG), and a significant increase in HDL (high-density lipoprotein) cholesterol level, and improved gut colonization and gut immunomodulation. The results prove that these non-haemolytic, non-toxic strains had significant health benefits than the commercial probiotics consortium with the recommended prebiotics mix. Thus, these new Lactococcus lactis subsp. lactis strains could be trialled as a new probiotic combination for human and animal feeds.
Asunto(s)
Lactobacillales , Lactococcus lactis , Probióticos , Simbióticos , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias , Inulina , Ácido Láctico/metabolismo , Lactococcus lactis/metabolismo , Lipoproteínas LDL , RatonesRESUMEN
In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.
Asunto(s)
Infecciones por Birnaviridae/prevención & control , Proteínas de Peces/metabolismo , Inmunidad Innata , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , Interferón Tipo I/metabolismo , Lactococcus lactis/metabolismo , Probióticos , Salmo salar/microbiología , Animales , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/microbiología , Infecciones por Birnaviridae/virología , Línea Celular , Proteínas de Peces/genética , Explotaciones Pesqueras , Interacciones Huésped-Patógeno , Virus de la Necrosis Pancreática Infecciosa/crecimiento & desarrollo , Virus de la Necrosis Pancreática Infecciosa/inmunología , Interferón Tipo I/genética , Lactococcus lactis/genética , Lactococcus lactis/inmunología , Proteínas de Resistencia a Mixovirus/metabolismo , Salmo salar/genética , Salmo salar/inmunología , Salmo salar/virología , Carga Viral , eIF-2 Quinasa/metabolismoRESUMEN
Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.
Asunto(s)
Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/metabolismo , Chaperonina 60/administración & dosificación , Chaperonina 60/metabolismo , Tolerancia Inmunológica/efectos de los fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Mycobacterium leprae/enzimología , Administración Oral , Animales , Proteínas Bacterianas/genética , Chaperonina 60/genética , Citocinas/metabolismo , Femenino , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Lactococcus lactis/genética , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Organismos Modificados Genéticamente/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.
Asunto(s)
Proteínas de Peces/metabolismo , Infecciones por Flavobacteriaceae/prevención & control , Flavobacterium/patogenicidad , Interferón gamma/metabolismo , Lactococcus lactis/metabolismo , Oncorhynchus mykiss/microbiología , Probióticos/administración & dosificación , Administración Oral , Animales , Línea Celular , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/metabolismo , Infecciones por Flavobacteriaceae/microbiología , Flavobacterium/inmunología , Interacciones Huésped-Patógeno , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/inmunología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/metabolismo , FilogeniaRESUMEN
BACKGROUND: It is well known that alcohol can trigger inflammatory effects in the gastrointestinal tract (GIT), interfering with mucosal homeostasis. OBJECTIVES: This study evaluated the effectiveness of Lactococcus lactis treatment in controlling the increase in molecular biomarkers related to allergic inflammation and the effect on the diversity and abundance of the Enterobacteriaceae family in the GIT after high-dose acute administration of ethanol. METHODS: Mice received ethanol or saline solution by gavage for four consecutive days, and 24 h after the last administration, the animals were given L. lactis or M17 broth orally ad libitum for two consecutive days. The animals were subsequently sacrificed and dissected. RESULTS: L. lactis treatment was able to restore basal levels of secretory immunoglobulin A in the gastric mucosa, serum total immunoglobulin E, interleukin (IL)-4 production in gastric and intestinal tissues, and IL-10 levels in gastric tissue. L. lactis treatment encouraged the diversification of the Enterobacteriaceae population, particularly the commensal species, in the GIT. CONCLUSION: This research opens a field of studies regarding the modulatory effect of L. lactis on immunological and microbial changes induced after alcohol intake.
Asunto(s)
Enterobacteriaceae/metabolismo , Etanol/metabolismo , Inmunoglobulina E/metabolismo , Interleucina-4/metabolismo , Lactococcus lactis/metabolismo , Administración Oral , Consumo de Bebidas Alcohólicas , Animales , Citocinas/metabolismo , Etanol/administración & dosificación , Femenino , Tracto Gastrointestinal , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina E/sangre , Inflamación/metabolismo , Interleucina-4/sangre , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The present study aimed to evaluate the effects of resveratrol, a nutraceutical polyphenol, and Lactococcus lactis (bacteria probiotic), on metabolic parameters and hepatic proinflammatory markers expression. C57BL/6 mice were divided into 4 groups: Standard (ST), Lactococcus lactis (LL), Resveratrol (RSV), and Lactococcus lactis plus resveratrol (LL + RSV). Lactococcus lactis and resveratrol were administered by orogastric gavage. Blood parameters were assessed (total cholesterol, triglycerides, ALT and AST). IL-6 mRNA expression was evaluated by Real-time PCR and TNF-α protein expression was assessed by immunohistochemistry. The main findings showed that resveratrol and Lactococcus lactis association decreased body weight, aspartate aminotransferase and total cholesterol levels. LL and LL + RSV decreased triglycerides levels and IL-6 and TNF-α expression. These results open a perspective of using resveratrol and Lactococcus lactis to improve metabolic parameters and Lactococcus lactis in preventing inflammation and the hepatic diseases development.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Lactococcus lactis/metabolismo , Hígado/efectos de los fármacos , Probióticos/farmacología , Resveratrol/farmacología , Administración Oral , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Colesterol/sangre , Biología Computacional , Femenino , Regulación de la Expresión Génica/genética , Ontología de Genes , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/microbiología , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Resveratrol/administración & dosificación , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA+ (pValac::IL-10) resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.
Asunto(s)
Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética , Vectores Genéticos , Interleucina-10/genética , Interleucina-4/genética , Lactococcus lactis/genética , Animales , Glucemia/metabolismo , Colon/inmunología , Colon/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Inmunoglobulina A Secretora/metabolismo , Insulina/sangre , Interleucina-10/biosíntesis , Interleucina-10/sangre , Interleucina-4/biosíntesis , Interleucina-4/sangre , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Lactococcus lactis/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NODRESUMEN
AIMS: Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. METHODS AND RESULTS: In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. CONCLUSIONS: Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.
Asunto(s)
Asma/tratamiento farmacológico , Proteínas Bacterianas/farmacología , Chaperonina 60/farmacología , Inflamación/tratamiento farmacológico , Lactococcus lactis/inmunología , Administración Oral , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipersensibilidad/tratamiento farmacológico , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoterapia , Lactococcus lactis/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina , Linfocitos T Reguladores/inmunologíaRESUMEN
Oral tolerance is the physiological process that enables the immune system to differentiate between harmless dietary and microbiota antigens from pathogen derived antigens. It develops at the mucosal surfaces and can result in local and systemic regulatory and anti-inflammatory effects. Translation of these benefits to the clinical practice faces limitations involving specificity and doses of antigen as well as regimens of feeding. To circumvent these problems, we developed a recombinant Hsp65 delivered by the acid lactic bacteria Lactococcus lactis NCDO 2118 directy in the intestinal mucosa. Hsp65 is a ubiquitous protein overexpressed in inflamed tissues and capable of inducing immunoregulatory mechanisms. L. lactis has probiotic properties and is commonly and safely used in dairy products. In this study, we showed that continuous delivery of HSP65 in the gut mucosa by L. lactis is a potent tolerogenic stimulus inducing regulatory CD4+LAP+ T cells that prevented collagen-induced and methylated bovine serum albumin-induced arthritis in mice. Clinical and histological signs of arthritis were inhibited as well as levels of inflammatory cytokines such as IL-17 and IFN-γ, serum titers of anti-collagen antibodies and rheumatoid factor. Oral administration of L. lactis induced alterations in microbiota composition toward an increased abundance of anaerobic bacteria such as Bifidobacterium and Lactobacillus. Tolerance to HSP65 and arthritis prevention induced by the recombinant L. lactis was associated with increase in IL-10 production by B cells and it was dependent on LAP+ T cells, IL-10 and TLR2 signaling. Therefore, HSP65-producing treatment induced effective tolerance and prevented arthritis development suggesting it can be used as a therapeutic tool for autoimmune diseases.
Asunto(s)
Artritis/inducido químicamente , Artritis/prevención & control , Proteínas Bacterianas/metabolismo , Colágeno/efectos adversos , Proteínas de Choque Térmico/metabolismo , Lactococcus lactis/metabolismo , Albúmina Sérica Bovina/efectos adversos , Administración Oral , Animales , Artritis/inmunología , Enfermedades Autoinmunes/prevención & control , Proteínas Bacterianas/genética , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Microbioma Gastrointestinal , Proteínas de Choque Térmico/genética , Tolerancia Inmunológica , Mucosa Intestinal/inmunología , Lactococcus lactis/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Probióticos/administración & dosificación , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract (GIT) and to date, no efficient treatments exist. Interleukin-10 (IL-10), one of the most important anti-inflammatory cytokines of the immune response, has been under study due to its potential for IBD therapy; however, systemic treatments lead to undesirable side effects and oral administration is limited due to its quick degradation. To avoid these bottlenecks, we previously engineered an invasive Lactococcus lactis (L. lactis) strain capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) that diminished inflammation in two induced mouse models of intestinal inflammation. Thus, the aim of this study was to analyze its therapeutic effect in the IL-10-deficient mouse model (IL-10-/-) that spontaneously and gradually develops an inflammation that modifies the immune system and resembles Crohn's disease (CD) in humans, and evaluate if it would also diminish and/or prevent the onset of this disease. RESULTS: Oral administration of L. lactis MG1363 FnBPA+ (pValac:il-10) to IL-10-/- mice not only led to IL-10 production by these, but consequently also diminished the severe development of the disease, with animals showing lower macroscopic scores and histological damages, increased IL-10 levels and tendency to lower pro-inflammatory cytokine levels. CONCLUSIONS: The results of this study, together with the previously published ones using this DNA delivery-based strategy, show that it is capable of creating and maintaining an anti-inflammatory environment in the GIT and thus effectively diminish the onset of inflammation in various mouse models.
Asunto(s)
Inflamación/terapia , Interleucina-10/deficiencia , Lactococcus lactis/genética , Plásmidos/metabolismo , Administración Oral , Animales , Modelos Animales de Enfermedad , Lactococcus lactis/metabolismo , Ratones , Ratones NoqueadosRESUMEN
AIMS: To investigate the anti-inflammatory activity of an invasive and Hp65-producing strain Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) in acute 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in mice as an innovative therapeutic strategy against Crohn's disease (CD). METHODS AND RESULTS: The pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, the anti-inflammatory capacity of the recombinant strain was evaluated in colitis induced with TNBS in BALB/c mice. Oral administration of the recombinant strain was able to attenuated the severity of colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and secretory immunoglobulin A levels. CONCLUSIONS: The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to a reduction in inflammatory damage in experimental CD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, which used L. lactis for the production and delivery of Hsp65, has scientific relevance because it shows the efficacy of this new strategy based on therapeutic protein delivery into mammalian enterocytes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperonina 60/metabolismo , Colitis/terapia , Inmunoglobulina A Secretora/metabolismo , Interleucina-10/metabolismo , Lactococcus lactis/fisiología , Administración Oral , Animales , Proteínas Bacterianas/genética , Células CACO-2 , Chaperonina 60/genética , Colitis/inducido químicamente , Colitis/inmunología , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inflamación/terapia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ratones , Ratones Endogámicos BALB C , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
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.
Asunto(s)
Antibacterianos/biosíntesis , Glutatión/metabolismo , Hemo/metabolismo , Lactococcus lactis/metabolismo , Nisina/biosíntesis , Ácido Pirúvico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Medios de Cultivo/análisis , Medios de Cultivo/metabolismo , Glutatión/análisis , Hemo/análisis , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrollo , Ácido Pirúvico/análisisRESUMEN
Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.
Asunto(s)
Industria Lechera/métodos , Lactococcus lactis/metabolismo , Nisina/metabolismo , Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Fermentación , Genes Bacterianos , Lactococcus lactis/genética , Nisina/análogos & derivados , Nisina/genéticaRESUMEN
Milk fermented with specific lactic acid bacteria (LAB) was reported to be a rich source of metabolites, such as peptides with different biological activities that may have a positive effect on cardiovascular health. Thus, in this study, the antithrombotic and hypocholesterolemic activities of fermented milk with specific strains of Lactococcus lactis were investigated before and after exposure to a simulated gastrointestinal digestion (SGD) model. The inhibition of thrombin-induced fibrin polymerization (IC50 peptide concentration necessary to inhibit thrombin activity by 50%), anticoagulant activity, inhibition of micellar solubility of cholesterol and bile acid binding capacity of water soluble fractions (WSF) <3 kDa from fermented milk were evaluated. Results indicated that the WSF from fermented milk with Lc-572 showed antithrombotic (IC50 = 0.049 mg/mL) and hypocholesterolemic (55% inhibition of micellar solubility of cholesterol and 27% bile acid binding capacity) activities. Meanwhile, fermented milk with Lc-571 showed mainly antithrombotic activity (IC50 = 0.045 mg/mL). On the other hand, fermented milk with Lc-600 presented mainly hypocholesterolemic activity (31.4% inhibition of micellar solubility of and 70% bile acid binding capacity). Moreover, biological activities were not lost after simulated gastrointestinal digestion conditions. Thus, fermented milk with these specific L. lactis strains show potential for the development of functional foods.
Asunto(s)
Anticolesterolemiantes/administración & dosificación , Fermentación , Fibrinolíticos/administración & dosificación , Lactococcus lactis/metabolismo , Leche/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Colesterol/metabolismo , Digestión , Humanos , Leche/química , Leche/microbiología , Unión ProteicaRESUMEN
The production of a highly concentrated probiotic preparation of Lactococcus lactis CECT 539 was studied in both batch and realkalized fed-batch fermentations in diluted whey (DW) media (DW25, DW50, DW75, DW100, and DW125) supplemented with MRS broth nutrients (except glucose and Tween 80) at 25, 50, 75, 100, and 125% of their standard concentrations in the complex medium. The fed-batch culture using DW100 medium provided the highest concentrations of probiotic biomass (5.98 g/L) and nisin (258.47 BU/mL), which were obtained at lower production costs than those estimated for the fed-batch culture in DW medium. The batch and fed-batch cultures reduced the initial chemical oxygen demand (COD) of the media by 29.1-41.7% and 31.2-54.2%, respectively. Graphical abstract.