RESUMO
Lactobacillus acidophilus were encapsulated by complex coacervation followed by transglutaminase crosslinking, aiming to improve the resistance of the microcapsules and improve the protection for probiotics. Subsequently, microcapsules were dried by freeze drying. The encapsulation efficiency, morphology, thermal resistance, gastrointestinal simulation and storage stability were analysed for wet and dry forms. The treatments offered high encapsulation efficiency (68.20-97.72%). Transglutaminase maintained the structure rounded, multinucleate and homogeneous distribution of probiotics in the microcapsules. In relation to the thermal resistance, in general, microencapsulation was effective in protecting and crosslinked microcapsules demonstrated greater protection for probiotics, obtaining viable cell counts of up to 10 log CFU g-1, approximately. On exposure to the simulated gastrointestinal tract, microencapsulation coupled to crosslinking demonstrated good results and the dry form was more efficient in the protection and the treatment with greater amount of transglutaminase was highlighted (9.07 log CFU g-1). As for storage, probiotic viability was maintained for up to 60â¯days in freezing temperature, with counts of up to 9.59 log CFU g-1. The results obtained in the present work are innovative and present a promising alternative for the protection of probiotics and their addition in food products.
Assuntos
Células Imobilizadas/microbiologia , Lactobacillus acidophilus/enzimologia , Viabilidade Microbiana , Probióticos , Cápsulas/química , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Armazenamento de Alimentos , Liofilização , Trato Gastrointestinal/metabolismo , Modelos Biológicos , Transglutaminases/metabolismoRESUMO
Background Lactic acid bacteria are able to reduce the immunoreactivity of proteins of cereal grains during wheat dough fermentation or may be a source of proteolytic preparations added during bread making. The key enzyme in prolamin degradation is prolyl endopeptidase. This study was aimed at optimizing the composition of a culture medium and culture conditions that would enhance the synthesis of intracellular prolyl endopeptidase (PEP) by Lactobacillus acidophilus 5e2. Results The application of Plackett-Burman screening plans enabled demonstrating that the concentration of a nitrogen source in the culture and the initial pH value of the culture medium were significant for PEP synthesis. Further optimization conducted with the method of central composite designs (CCD) confirmed both the linear and square impact of nitrogen concentration and initial pH value of the culture medium on PEP production. In turn, the response surface method (RSM) allowed determining the optimal nitrogen concentration and pH value at 26.88 g/l and pH 4.85, respectively. Conclusions Validation of the resultant model enabled over 3-fold increase in the quantity of the synthesized enzyme.
Assuntos
Prolil Oligopeptidases/biossíntese , Lactobacillus acidophilus/enzimologia , Meios de Cultura , Fermentação , Concentração de Íons de HidrogênioRESUMO
Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⻹ for L. acidophilus, with a cell mass yield (Y (X/S)) of 0.37 g g⻹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⻹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis-Menten constant (K(m)) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⻹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K(m) for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.
Assuntos
Biocombustíveis/análise , Glicerol/metabolismo , Lactobacillus acidophilus/metabolismo , Lactobacillus delbrueckii/metabolismo , Lactobacillus plantarum/metabolismo , Probióticos/metabolismo , Proteínas de Bactérias/metabolismo , Glicerol Quinase/metabolismo , Cinética , Lactobacillus acidophilus/química , Lactobacillus acidophilus/enzimologia , Lactobacillus acidophilus/crescimento & desenvolvimento , Lactobacillus delbrueckii/química , Lactobacillus delbrueckii/enzimologia , Lactobacillus delbrueckii/crescimento & desenvolvimento , Lactobacillus plantarum/química , Lactobacillus plantarum/enzimologia , Lactobacillus plantarum/crescimento & desenvolvimento , Desidrogenase do Álcool de Açúcar/metabolismoRESUMO
We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.
Assuntos
Antibacterianos/farmacologia , Conservantes de Alimentos/farmacologia , Lactobacillus acidophilus/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/farmacologia , Nisina/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Sinergismo Farmacológico , Microbiologia de Alimentos , Genes Bacterianos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Temperatura Alta , Testes de Sensibilidade Microbiana , Permeabilidade , Polilisina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Tensoativos/farmacologiaRESUMO
We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verification of the enzymatic activity.
Assuntos
Lactobacillus acidophilus/enzimologia , Glicoproteínas de Membrana/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Parede Celular/metabolismo , Clonagem Molecular , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.
Assuntos
Lactobacillus acidophilus/enzimologia , Amido/metabolismo , alfa-Amilases/genética , Adsorção , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Plasmídeos/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , alfa-Amilases/metabolismoRESUMO
Lactobacillus acidophilus NCFM-S, sensível à bacteriocina, L. acidophilus NCFM-R1, e L. acidophilus NCFM-R2, resistentes à bacteriocina, foram avaliadas "in vitro" quanto à resistência a suco gástrico artificial, sais biliares, lisozima e quimioterápicos. O objetivo do trabalho foi saber se a resistência à bacteriocina, uma substância inibitória secretada por L. acidophilus NCFM contra espécies homólogas, poderia estar relacionada com a habilidade das estirpes de sobreviver no trato gastrointestinal humano. As três culturas apresentaram sensibilidade após 24 horas em presença de suco gástrico artificial a 37ºC. Oxgall a 0.3 por cento pareceu ser inibitório para as três culturas, apesar de haver menor inibiçäo para a estirpe NCFM-R2. Após 30 minutos de contato com uma soluçäo de 100 µg/ml de lisozima, a NCFM-R2, mostrou menor percentagem de inibiçäo. Entre as estirpes estudadas, a NCFM-R2, mostrou-se a mais resistente aos quimioterápicos ampicilina, neomicina, penicilina G, estreptomicina, cloranfenicol, tetraciclina e sulfanilamida. De acordo com os resultados, observou-se que a estirpe NCFM-R2, resistente à bacteriocina, foi menos afetada pelas condiçöes estressantes do trato intestinal avalidadas neste trabalho, recomendado seu uso como adjunto dietético.