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1.
J Biosci Bioeng ; 132(6): 606-612, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34563462

RESUMO

The transition of the bacterial biota of Kishu saba-narezushi (mackerel-narezushi) in the Hidaka region of Wakayama prefecture, Japan, was analyzed using amplicon sequencing based on the V3-V4 variable region of the 16S rRNA gene. In the non-fermented sample (0 day), the major genus with the highest abundance ratio was Staphylococcus. In the early stage (fermentation for 2 days), however, the genus Lactococcus became a dominant species, and in the later stage (fermentation for 5 days), the abundance ratio of the genus Lactobacillus increased significantly. Lactococcus lactis strains isolated from the narezushi samples had the ability to suppress the growth of not only Staphylococcus genera but also Lactobacillus. Moreover, the isolates produced a bacteriocin, which was identified as nisin Z. On the basis of these results, it is concluded that L. lactis plays an important role in preparing the fermentation conditions of Kishu saba-narezushi in the early stage by suppressing unwanted microorganisms using lactic acid and nisin Z.


Assuntos
Lactococcus lactis , Nisina , Perciformes , Animais , Bactérias/metabolismo , Biota , Fermentação , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/metabolismo , Perciformes/metabolismo , RNA Ribossômico 16S/genética
2.
Curr Microbiol ; 78(9): 3430-3438, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255153

RESUMO

An attempt was made, to characterize natural antibiotics or lantibiotics from unconventional sources and its antibacterial spectrum against food borne pathogens and drug resistant bacteria. Six different traditional fermented foods i.e., fermented fish, fermented soybeans, Soibum (fermented bamboo shoots), milk, idly and dosa batter were used for the isolation of bacteriocin producing Lactic acid bacteria (LAB). Among all bacterial cultures isolated from the various sources, 129 cultures have found to produce antimicrobial compounds. Nisin specific reporter bacteria was utilized as biosensor to identify the Nisin like bacteriocin, where 10 cultures found to be positive Nisin producer. Identified Nisin like bacteriocin was partially concentrated by using ammonium sulphate followed by butanol extraction. Minimum inhibitory concentration (MIC) was analyzed against food borne pathogen and drug resistant bacteria. MIC of partially purified Nisin (pp-Nisin) of all the LAB isolates against food-borne pathogens are ranged between 0.5 and 92 µg/ml respected to various Gram-positive bacteria. Similarly, the drug resistant bacteria were also inhibited by pp-Nisin (MIC ranged between 15 and 175 µg/ml). All samples of ppnisin exhibited auto induction ability. Taxonomic identification of the nisin producers was done by whole genome sequencing which reveals that cultures belongs to Lactococcus lactis ssp. lactis. Also it was found that Lactococcus lactis ssp. lactis C2d and Lactococcus lactis ssp. lactis SP2C4 harbor nisA gene and Lactococcus lactis ssp. lactis FS2 (L. lactis FS2) harbor nisQ gene. The finding of this study highlights the first case of L. lactis FS2 isolated from fermented fish harbor nisQ gene. Antibacterial activity of pp-Nisin against drug resistant LAB is also reported.


Assuntos
Bacteriocinas , Alimentos e Bebidas Fermentados , Lactobacillales , Lactococcus lactis , Nisina , Animais , Bacteriocinas/genética , Fermentação , Lactobacillales/genética , Lactobacillales/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/metabolismo
3.
Front Immunol ; 12: 647987, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248935

RESUMO

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.


Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Chaperonina 60/administração & dosagem , Chaperonina 60/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Mycobacterium leprae/enzimologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia
4.
Food Microbiol ; 99: 103813, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119100

RESUMO

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L-1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) - even suppressing it completely in some cases - in four of the six tested tyramine producing strains.


Assuntos
Antibacterianos/farmacologia , Cerveja/microbiologia , Queijo/microbiologia , Meios de Cultura/farmacologia , Lactobacillaceae/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Lactococcus lactis/química , Tiramina/farmacologia , Antibacterianos/análise , Antibacterianos/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Meios de Cultura/metabolismo , Lactobacillaceae/crescimento & desenvolvimento , Lactobacillaceae/isolamento & purificação , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/isolamento & purificação , Lactococcus lactis/metabolismo , Tiramina/análise , Tiramina/metabolismo
5.
Food Microbiol ; 99: 103835, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119119

RESUMO

In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75-7.5 µM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 µM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1-2 log10 CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Lactococcus lactis/metabolismo , Listeria/efeitos dos fármacos , Nisina/metabolismo , Nisina/farmacologia , Antibacterianos/química , Bioengenharia , Lactococcus lactis/genética , Listeria/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Nisina/química , Nisina/genética
6.
J Dairy Sci ; 104(9): 9556-9569, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34147226

RESUMO

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield.


Assuntos
Lactococcus lactis , Nisina , Ácidos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/genética
7.
Appl Environ Microbiol ; 87(16): e0077921, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34105983

RESUMO

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIAmtl, and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.


Assuntos
Lactococcus lactis/metabolismo , Manitol/metabolismo , Óperon , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial , Lactococcus lactis/genética
8.
Fish Shellfish Immunol ; 114: 253-262, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33979691

RESUMO

Vibriosis, an illness caused by the Vibrio bacteria species, results in significant economic loss in olive flounder farms. Here we present a novel anti-Vibrio feed vaccine protecting multiple strains of Vibrio pathogens, a universal vaccine effect. The vaccine was generated by engineering Lactococcus lactis BFE920 to express the fusion antigens of Vibrio outer membrane protein K (OmpK) and flagellin B subunit (FlaB). These antigen genes are highly conserved among Vibrio species. Olive flounder (7.1 ± 0.8 g and 140 ± 10 g) were fed the vaccine adsorbed to a regular feed (1 × 107 CFU/g) for one week with a 1-week interval, repeating three times (a triple boost). The vaccinated fish increased the significant levels of antigen-specific antibodies, T cell numbers (CD4-1, CD4-2, and CD8α), cytokine production (T-bet and IFN-γ), and innate immune responses (TLR5M, IL-1ß, and IL-12p40). Also, the survival rates of adult and juvenile fish fed the vaccine were significantly elevated when challenged with V. anguillarum, V. alginolyticus, and V. harveyi. In addition, weight gain rate and feed conversion ratio were improved in vaccinated fish. The feed vaccine protected multiple Vibrio pathogens, a universal vaccine effect, by activating innate and adaptive immune responses. This oral vaccine may be developed as an anti-Vibrio vaccine to protect against a broad spectrum of Vibrio pathogens.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Linguado , Lactococcus lactis/metabolismo , Vibrioses/veterinária , Vibrio/metabolismo , Imunidade Adaptativa , Animais , Vacinas Bacterianas/administração & dosagem , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Imunidade Inata , Probióticos , Vibrio/imunologia , Vibrioses/prevenção & controle
9.
Mol Syst Biol ; 17(4): e10093, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33821549

RESUMO

Cells adapt to different conditions via gene expression that tunes metabolism for maximal fitness. Constraints on cellular proteome may limit such expression strategies and introduce trade-offs. Resource allocation under proteome constraints has explained regulatory strategies in bacteria. It is unclear, however, to what extent these constraints can predict evolutionary changes, especially for microorganisms that evolved under nutrient-rich conditions, i.e., multiple available nitrogen sources, such as Lactococcus lactis. Here, we present a proteome-constrained genome-scale metabolic model of L. lactis (pcLactis) to interpret growth on multiple nutrients. Through integration of proteomics and flux data, in glucose-limited chemostats, the model predicted glucose and arginine uptake as dominant constraints at low growth rates. Indeed, glucose and arginine catabolism were found upregulated in evolved mutants. At high growth rates, pcLactis correctly predicted the observed shutdown of arginine catabolism because limited proteome availability favored lactate for ATP production. Thus, our model-based analysis is able to identify and explain the proteome constraints that limit growth rate in nutrient-rich environments and thus form targets of fitness improvement.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Aptidão Genética , Glucose/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteoma/metabolismo , Trifosfato de Adenosina/metabolismo , Evolução Biológica , Modelos Biológicos , Mutação/genética , Reprodutibilidade dos Testes
10.
mBio ; 12(2)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832972

RESUMO

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic ß-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in ß-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.


Assuntos
Antibacterianos/farmacologia , Cefuroxima/farmacologia , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Lactococcus lactis/metabolismo , Potássio/metabolismo , Sistemas do Segundo Mensageiro
11.
J Sci Food Agric ; 101(14): 5807-5812, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33792042

RESUMO

BACKGROUND: Lactococcus lactis strain pGSMT/MG1363 is a genetically modified microorganism (GMM) that constitutively expresses human metallothionein-I fusion protein to combine with intracellular lead. Unlike traditional probiotics, pGSMT/MG1363 lacks a history of safe use in food. Administration of microorganism could influence the gut microbial community and consequently confer health benefits or cause disadvantages to the host. To date, little has been done to assess the influence of recombinant strain pGSMT/MG1363 on the stability of gut microbiota. RESULTS: Liver, testis and kidney sections of male Sprague-Dawley rats orally administered pGSMT/MG1363 for 6 weeks showed normal structure and no pathological damage. There were no adverse effects on the analyzed serum biochemical parameters between the pGSMT/MG1363 group and the MG1363 group. Principal coordinate analysis showed that, compared with the MG1363 group, the 6-week-old fecal gut microbiota of rats fed with pGSMT/MG1363 was not significantly different (Adonis, P = 0.802). pGSMT/MG1363 treatment for 6 weeks did not significantly change the relative abundance of gut microbiota at the phylum and genus levels in comparison with MG1363 treatment. CONCLUSION: Compared to the non-GM strain MG1363 group, administration of the recombinant strain pGSMT/MG1363 for 6 weeks showed no adverse effects on the analyzed physiological parameters and gut microbial compositions of male Sprague-Dawley rats. The results suggested that, in terms of gut microbiota stability, pGSMT/MG1363 could be considered as safe as MG1363, at least for short-term intake. Further toxicological evaluations still need to be considered before drawing a definite conclusion concerning the safe use of pGSMT/MG1363. © 2021 Society of Chemical Industry.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Lactococcus lactis/genética , Probióticos/administração & dosagem , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Fezes/microbiologia , Rim/metabolismo , Rim/patologia , Lactococcus lactis/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Probióticos/efeitos adversos , Probióticos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Int J Food Microbiol ; 345: 109130, 2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-33735781

RESUMO

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.


Assuntos
Bactérias/classificação , Bactérias/metabolismo , Queijo/microbiologia , Fungos/classificação , Fungos/metabolismo , Leite/microbiologia , Animais , Bactérias/isolamento & purificação , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/metabolismo , Fungos/isolamento & purificação , Geotrichum/isolamento & purificação , Geotrichum/metabolismo , Cabras , Lactobacillus paracasei/isolamento & purificação , Lactobacillus paracasei/metabolismo , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/metabolismo , Microbiota , Odorantes/análise , Penicillium/isolamento & purificação , Penicillium/metabolismo , Scopulariopsis/isolamento & purificação , Scopulariopsis/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
J Agric Food Chem ; 69(9): 2826-2835, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33646779

RESUMO

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.


Assuntos
Indústria de Laticínios , Lactococcus lactis , Nisina/biossíntese , Fermentação , Lactato Desidrogenases , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Oxirredução
14.
Mol Biol Rep ; 48(2): 1725-1734, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33586053

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Fígado/efeitos dos fármacos , Probióticos/farmacologia , Resveratrol/farmacologia , Administração Oral , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Colesterol/sangue , Biologia Computacional , Feminino , Regulação da Expressão Gênica/genética , Ontologia Genética , Imuno-Histoquímica , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Resveratrol/administração & dosagem , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
15.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573129

RESUMO

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.


Assuntos
Reatores Biológicos/microbiologia , Vetores Genéticos/genética , Microbiologia Industrial/métodos , Lactococcus lactis/genética , Plasmídeos/genética , Administração através da Mucosa , Engenharia Celular/métodos , DNA Circular/biossíntese , DNA Circular/genética , DNA Circular/isolamento & purificação , Tecnologia de Alimentos/métodos , Engenharia Genética/métodos , Lactococcus lactis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Replicon/genética , Tecnologia Farmacêutica/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/biossíntese , Vacinas de DNA/genética , Vacinas de DNA/isolamento & purificação
16.
J Diabetes Res ; 2021: 6697319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604389

RESUMO

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.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Terapia Genética , Vetores Genéticos , Interleucina-10/genética , Interleucina-4/genética , Lactococcus lactis/genética , Animais , Glicemia/metabolismo , Colo/imunologia , Colo/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Imunoglobulina A Secretora/metabolismo , Insulina/sangue , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-4/biossíntese , Interleucina-4/sangue , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Lactococcus lactis/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD
17.
Biomolecules ; 11(1)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466817

RESUMO

As a multifunctional polypeptide, epidermal growth factor (EGF) increases growth performance or enhances resistance to diseases in commercial broilers under adverse conditions. In this study, a recombinant Lactococcus lactis was established to produce the secretary form of bioactive gEGF. The results of in vitro testing showed that gEGF promoted the proliferation of chicken embryo fibroblast cells. A total of 63 5-day-old broiler chickens were evenly divided into three groups and treated with either M17 medium (the control group), supernatant of LL-pNZ8149 fermentation product (the P-LL group), or supernatant of LL-pNZ8149-gEGF fermentation product (the gEGF group). In two weeks, many measurements of growth, immunity and the intestines were significantly higher in the gEGF group than those in the control and the P-LL groups. Our study showed that the bioactive gEGF could be expressed with Lactococcus lactis expression system with the potential to enhance growth performance, immune function, and intestinal development in broiler chickens.


Assuntos
Galinhas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Microbiologia de Alimentos , Lactococcus lactis/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Galinhas/imunologia , Intestinos/crescimento & desenvolvimento , Padrões de Referência
18.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33419737

RESUMO

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.


Assuntos
Caseínas/metabolismo , Lactobacillus rhamnosus , Lactococcus lactis , Lactose/metabolismo , Peptídeo Hidrolases/metabolismo , Probióticos , Animais , Conjugação Genética , Lactobacillus rhamnosus/genética , Lactobacillus rhamnosus/crescimento & desenvolvimento , Lactobacillus rhamnosus/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Leite/microbiologia , Plasmídeos , Proteólise
19.
J Microbiol Biotechnol ; 31(2): 298-303, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33397831

RESUMO

Comparative genomic analysis was performed on eight species of lactic acid bacteria (LAB)-Lactococcus (L.) lactis, Lactobacillus (Lb.) plantarum, Lb. casei, Lb. brevis, Leuconostoc (Leu.) mesenteroides, Lb. fermentum, Lb. buchneri, and Lb. curvatus-to assess their glutamic acid production pathways. Glutamic acid is important for umami taste in foods. The only genes for glutamic acid production identified in the eight LAB were for conversion from glutamine in L. lactis and Leu. mesenteroides, and from glucose via citrate in L. lactis. Thus, L. lactis was considered to be potentially the best of the species for glutamic acid production. By biochemical analyses, L. lactis HY7803 was selected for glutamic acid production from among 17 L. lactis strains. Strain HY7803 produced 83.16 pmol/µl glutamic acid from glucose, and exogenous supplementation of citrate increased this to 108.42 pmol/µl. Including glutamic acid, strain HY7803 produced more of 10 free amino acids than L. lactis reference strains IL1403 and ATCC 7962 in the presence of exogenous citrate. The differences in the amino acid profiles of the strains were illuminated by principal component analysis. Our results indicate that L. lactis HY7803 may be a good starter strain for glutamic acid production.


Assuntos
Ácido Glutâmico/biossíntese , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ácido Cítrico/metabolismo , Genoma Bacteriano , Genômica
20.
FEBS J ; 288(14): 4348-4363, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33482024

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Lactococcus lactis/metabolismo , Nisina/metabolismo , Fragmentos de Peptídeos/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Homologia de Sequência
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