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1.
J Appl Microbiol ; 125(2): 564-574, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29654625

RESUMO

AIM: The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS: Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and ß-caseins, α-lactalbumin and ß-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION: Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.


Assuntos
Queijo/microbiologia , Lactobacillales , Hipersensibilidade a Leite , Animais , Brasil , Bovinos , Lactobacillales/isolamento & purificação , Lactobacillales/metabolismo , Leite/microbiologia , Proteínas do Leite/química , Proteínas do Leite/metabolismo
2.
Benef Microbes ; 8(4): 635-643, 2017 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-28726509

RESUMO

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the ß-subunit from ß-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.


Assuntos
Antígenos de Plantas/imunologia , Enterococcus faecalis/metabolismo , Globulinas/imunologia , Proteínas de Armazenamento de Sementes/imunologia , Leite de Soja/metabolismo , Proteínas de Soja/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fermentação , Globulinas/química , Globulinas/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/metabolismo , Leite de Soja/química , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Glycine max/química , Glycine max/imunologia , Glycine max/metabolismo , Glycine max/microbiologia
3.
J Dairy Sci ; 99(7): 5144-5154, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27179865

RESUMO

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and ß-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Leite/imunologia , Leite/microbiologia , Peptídeo Hidrolases/metabolismo , Proteólise , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Caseínas/metabolismo , Proteínas do Leite/imunologia , Proteínas do Leite/metabolismo , Proteínas do Soro do Leite/metabolismo
4.
Benef Microbes ; 6(5): 679-86, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26192744

RESUMO

The allergenicity of ß-lactoglobulin (ß-Lg) was studied by using Ussing chamber in a murine model of ß-Lg allergy supplemented with hydrolysates obtained after fermentation of milk for 48 h at 37 (°)C with Enterococcus faecalis DAPTO 512, isolated from cow milk and identified by 16S rDNA sequence analysis. Balb/c mice were sensitised intraperitoneally with ß-Lg. Three groups of mice were formed: group 1, composed of naive mice used as control received only NaCl; group 2, positive control composed of mice sensitised intraperitoneally with ß-Lg; group 3, formed by mice which were given hydrolysates of 48 h then sensitised with ß-Lg. After 48 h of fermentation ß-casein and ß-Lg were degraded by E. faecalis DAPTO 512. ß-Lg immunisation was associated with strong IgG and IgE production in case of positive controls and a significant increase in short current circuit (Isc) and high conductance (G) responses were observed. The control and the hydrolysate groups showed a significant decrease in the production of IgG and IgE anti ß-Lg compared to the positive control. The allergenic potential of ß-Lg was markedly reduced in the group that received hydrolysates (Isc and G remained unchanged after intestine challenge with ß-Lg). The histological scrutiny showed villi atrophy, lymphocyte hyperplasia and a significant chorion detachment in the positive control group. In the group administered with hydrolysates of fermented milk, inflammatory signs were lower, the villi were long and thin and lymphocytes were less dense. The results showed that feeding of milk fermented with E. faecalis DAPTO 512 during 18 days prior to ß-Lg allergy induction exerts a protecting effect on the murine intestine and induces a significant decrease in the ß-Lg allergenicity.


Assuntos
Alérgenos/metabolismo , Caseínas/metabolismo , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Mucosa Intestinal/patologia , Lactoglobulinas/metabolismo , Hipersensibilidade a Leite/prevenção & controle , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Modelos Animais de Doenças , Enterococcus faecalis/classificação , Enterococcus faecalis/isolamento & purificação , Histocitoquímica , Camundongos Endogâmicos BALB C , Microscopia , Hipersensibilidade a Leite/patologia , Proteólise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
5.
J Appl Microbiol ; 116(5): 1195-208, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24506656

RESUMO

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactobacillus/metabolismo , Produtos da Carne/microbiologia , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/química , Bacteriocinas/biossíntese , Bacteriocinas/química , Brasil , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos
6.
Food Microbiol ; 36(2): 296-304, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24010610

RESUMO

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactobacillus/metabolismo , Probióticos/metabolismo , Animais , Galinhas , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Humanos , Lactobacillus/genética , Lactobacillus/isolamento & purificação
7.
Protein Pept Lett ; 20(5): 607-12, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23116059

RESUMO

Regular consumption of natural antioxidants reduces the risk of developing diseases. Aloin is one of the main active phenolic components of Aloe vera. The main disadvantage of aloin is its concentration limit of use that causes cell damage. One of the aims of this study was to investigate the antioxidant activity of aloin in the presence and absence of camel ß-casein ( ß-CN) and its peptide fractions. The mixture of aloin, ß-CN and peptides showed a very high antioxidant activity in a synergistic manner as compared to each component alone. The alpha ( α)-glucosidase inhibitory activity of aloin was also investigated in the presence and absence of ß-CN and its peptides. Aloin alone is a potent inhibitor of α-glucosidase. The α-glucosidase inhibitory activity of aloin is reduced in the presence of ß-CN or its peptides. The combination of aloin and ß-CN or its peptides makes a high antioxidant functional ingredient.


Assuntos
Antioxidantes/metabolismo , Caseínas/metabolismo , Emodina/análogos & derivados , Inibidores de Glicosídeo Hidrolases , Aloe/química , Animais , Camelus , Caseínas/química , Emodina/metabolismo , Emodina/farmacologia , Hidrólise , Modelos Lineares , Leite/química , Peptídeos/química , Peptídeos/metabolismo , alfa-Glucosidases/metabolismo
8.
Lett Appl Microbiol ; 56(4): 237-44, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23256522

RESUMO

The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l(-1) of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria.


Assuntos
Antifúngicos/farmacologia , Bacteriocinas/farmacologia , Debaryomyces/efeitos dos fármacos , Enterococcus , Fungos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Bacteriocinas/síntese química , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Queijo/microbiologia , Debaryomyces/ultraestrutura , Enterococcus/isolamento & purificação , Enterococcus/metabolismo , Listeria/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química
9.
Benef Microbes ; 2(1): 15-27, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21831786

RESUMO

Five bacterial cocci isolates were selected from a wide pool of 503 isolates collected from traditional Egyptian dairy products on the basis of their inhibitory activities against Lactobacillus brevis F145, Lactobacillus bulgaricus 340, Enterococcus faecium HKLHS, Listeria ivanovii ATCC, Listeria innocua CIP 80.11 and Listeria monocytogenes EGDe 107776. These 5 isolates were identified as E. faecium TX1330 and E. faecium E980 by 16S rDNA amplification and sequencing. The antibacterial activity of the two strains was not affected by treatment of the cell free culture supernatant with catalase but their activities disappeared completely when digested with protease K, α-chymotrypsin and trypsin. The antimicrobial substance was stable over a wide range of pH (2-10) and was active after heating at 100 °C for 10 min. Bacteriocin yield in two strains reached a maximum (1,600 AU/ml) at the end of the exponential phase (6 h) and remained stable until the end of 24 h-incubation period when the medium reached pH 5.5. Maximal production of bacteriocin was obtained when growing the bacterial cells at temperatures ranging between 30 and 37 °C. Bacteriocin production was unaffected when the bacterial cells grew under severe conditions of pH (9.6) and in high salt (6.5% NaCl). Thanks to PCR gene amplification the bacteriocins produced by E. faecium TX1330 could be identified as enterocins A and B structural genes, while the bacteriocins produced by E. faecium E980 could be identified as enterocins P and L50A structural genes, which can be classified into two enterocin subclasses (IIa and IIc), respectively. PCR amplification demonstrated that the two studied strains did not contain virulence factors asal, cyl A and B, ace, efaAfs and espfm. These two strains were sensitive to most of the tested antibiotics but were resistant to tetracycline. E. faecium E980 was also resistant to chloramphenicol.


Assuntos
Bacteriocinas/biossíntese , Laticínios/microbiologia , Enterococcus/metabolismo , Bacteriocinas/isolamento & purificação , Sequência de Bases , DNA Ribossômico/genética , Egito , Enterococcus/genética , Enterococcus/isolamento & purificação , Microbiologia de Alimentos , Inocuidade dos Alimentos , Variação Genética , Lactobacillus , Listeria/efeitos dos fármacos , Listeria monocytogenes , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular
10.
J Environ Public Health ; 2010: 986460, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20811593

RESUMO

The aim of this research was to purify and characterize the mode of action of enterocin S37, a bacteriocin produced by Enterococcus faecalis S37, a strain recently isolated from the chicken feces. Enterocin S37 has a molecular weight comprised between 4 and 5 kDa. It remained active after 1 h at 80(o)C and at pH values ranging from 4.0 to 9.0. Furthermore, cell-free supernatant of Enterococcus faecalis S37 and purified enterocin S37 were active against Gram-positive bacteria including Listeria monocytogenes EGDe, L. innocua F, Enterococcus faecalis JH2-2, and Lactobacillus brevis F145. The purification of enterocin S37 was performed by ammonium sulfate precipitation followed up by hydrophobic-interaction chromatography procedures. Treatment of enterocin S37 with proteinase K, alpha-chymotrypsin, and papain confirmed its proteinaceous nature, while its treatment with lysozyme and lipase resulted in no alteration of activity. Enterocin S37 is hydrophobic, anti-Listeria and likely acting by depletion of intracellular K(+) ions upon action on K(ATP) channels. This study contributed to gain more insights into the mode of action of enterocins.


Assuntos
Antibacterianos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Enterococcus faecalis/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/farmacologia , Galinhas/microbiologia , Farmacorresistência Bacteriana , Enterococcus faecalis/isolamento & purificação , Fezes/microbiologia , Bactérias Gram-Positivas/efeitos dos fármacos
11.
Benef Microbes ; 1(1): 43-51, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21831749

RESUMO

Different Enterococci, isolated from starters for the production of the traditional Bulgarian yellow cheese 'Kashkaval' were screened for bacteriocin production, and one of them, Enterococcus faecalis 3915 demonstrated bacteriocin activity. In this study, we investigated the growth parameters of the producer strain as well as the production kinetics and preliminary characterisation of the produced bacteriocin named enterocin 3915. For the growth modelling, the logistic model was used, while bacteriocin production was monitored. Experiments on inducibility were conducted, and strain was checked for the presence of plasmids. The peptide was crudely purified by ammonium sulphate precipitation followed by preparative PAGE. The approximate molecular mass was determined electrophoretically, and the activity was visualised by electrophoresis and agar overlay technique. It was found that E. faecalis 3915 produces a bacteriocin with constitutive synthesis and chromosomal localisation of its genetic determinants. The peptide revealed to be relatively heat-stable with a molecular mass of about 6.5 kDa. As E. faecalis 3915 originates from cheese starter it can be classified as generally recognised as safe (GRAS). The inhibitory activity of enterocin 3915 comprises commensals or pathogens, so properties generally accepted as probiotic could be attributed to the producer, and potential application in the dairy industry is not to be excluded.


Assuntos
Bacteriocinas/biossíntese , Enterococcus faecalis/metabolismo , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Queijo/microbiologia , Enterococcus faecalis/química , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Peso Molecular
12.
Oral Microbiol Immunol ; 23(3): 254-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18402613

RESUMO

INTRODUCTION: Lactobacillus salivarius BGHO1, a human oral isolate with antagonistic activity against growth of Streptococcus mutans, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, and Salmonella enteritidis, probably produces more than one proteinaceous antimicrobial substance. The objective of this study was the purification of a bacteriocin, named LS1, produced by L. salivarius BGHO1. METHODS: A simple and fast procedure for bacteriocin purification was developed, consisting of reverse-phase chromatography of the ammonium sulfate precipitate of cell-free culture supernatant by fast protein liquid chromatography and high-performance liquid chromatography, followed by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the subsequent extraction of bacteriocin from the gel. RESULTS: The supernatant of L. salivarius BGHO1 culture retained its antimicrobial activity after boiling in a water bath for 15 min. Its antimicrobial activity was also maintained even after treatment for 20 min at 121 degrees C in an autoclave. Bacteriocin LS1 was purified to homogeneity. The molecular mass of bacteriocin LS1 was estimated to be approximately 10 kDa, based on tricine SDS-PAGE. During purification, another compound with antimicrobial activity, produced by L. salivarius BGHO1, was detected. The molecular mass of this compound was estimated to be approximately 5 kDa, based on tricine SDS-PAGE. CONCLUSION: Our results imply that LS1 is most probably a new bacteriocin, different from previously described bacteriocins produced by L. salivarius strains. The purification of bacteriocin LS1 enabled the further characterization of LS1 on both the molecular and genetic levels.


Assuntos
Antibacterianos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Lactobacillus/classificação , Antibiose , Bacteriocinas/classificação , Cromatografia , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Humanos , Lactobacillus/crescimento & desenvolvimento , Peso Molecular , Boca/microbiologia , Streptococcus mutans/crescimento & desenvolvimento , Água
13.
Clin Exp Allergy ; 38(4): 686-93, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18307527

RESUMO

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.


Assuntos
Caseínas/imunologia , Imunoglobulina E/sangue , Lactoferrina/imunologia , Hipersensibilidade a Leite/imunologia , Leite/imunologia , Análise Serial de Proteínas/métodos , Animais , Reações Antígeno-Anticorpo , Caseínas/química , Bovinos , Humanos , Imunoglobulina E/química , Lactoferrina/química , Leite/química , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho/métodos
14.
J Appl Microbiol ; 104(1): 243-50, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17887982

RESUMO

AIMS: To find different types of glucosyltransferases (GTFs) produced by Leuconostoc mesenteroides strain Lm 28 and its mutant forms, and to check the effectiveness of gluco-oligosaccharide synthesis using maltose as the acceptor. METHODS AND RESULTS: Constitutive mutants were obtained after chemical mutagenesis by ethyl methane sulfonate. Lm M281 produced more active GTFs than that obtained by the parental strain cultivated on sucrose. GTF from Lm M286 produced a resistant glucan, based on endo-dextranase and amyloglucosidase hydrolysis. The extracellular enzymes from Lm M286 catalyse acceptor reactions and transfer the glucose unit from sucrose to maltose to produce gluco-oligosaccharides (GOS). By increasing the sucrose/maltose ratio, it was possible to catalyse the synthesis of oligosaccharides of increasing degree of polymerization (DP). CONCLUSIONS: Different types of GTFs (dextransucrase, alternansucrase and levansucrase) were produced from new constitutive mutants of Leuc. mesenteroides. GTFs from Lm M286 can catalyse the acceptor reaction in the presence of maltose, leading to the synthesis of branched oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions were optimized to synthesize GOS by using GTFs from Lm M286, with the aim of producing maximum quantities of branched-chain oligosaccharides with DP 3-5. This would allow the use of the latter as prebiotics.


Assuntos
Reatores Biológicos/microbiologia , Glucosiltransferases/metabolismo , Leuconostoc/genética , Leuconostoc/metabolismo , Oligossacarídeos/biossíntese , Probióticos/metabolismo , Técnicas Bacteriológicas , Cromatografia Líquida de Alta Pressão , Dextranase/metabolismo , Eletroforese em Gel de Poliacrilamida , Metanossulfonato de Etila , Glicosiltransferases/metabolismo , Maltose/metabolismo , Mutação , Oligossacarídeos/análise , Sacarose/metabolismo
15.
J Appl Microbiol ; 103(3): 584-93, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17714391

RESUMO

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.


Assuntos
Microbiologia de Alimentos , Peróxido de Hidrogênio/metabolismo , Lactobacillus delbrueckii/isolamento & purificação , Iogurte/microbiologia , Antibacterianos/farmacocinética , Contagem de Colônia Microbiana , Meios de Cultura , Impressões Digitais de DNA/métodos , DNA Bacteriano/genética , DNA Ribossômico/genética , Escherichia coli/crescimento & desenvolvimento , Lactobacillus delbrueckii/genética , Lactobacillus delbrueckii/crescimento & desenvolvimento , Listeria/crescimento & desenvolvimento , Mongólia , Polimorfismo Genético , RNA Bacteriano/genética , RNA Ribossômico 16S/genética
16.
J Appl Microbiol ; 101(4): 837-48, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16968295

RESUMO

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).


Assuntos
Antibacterianos , Bacteriocinas/isolamento & purificação , Produtos Fermentados do Leite/microbiologia , Microbiologia de Alimentos , Lactobacillaceae/metabolismo , Aminoácidos/análise , Bacteriocinas/química , Eletroforese em Gel de Poliacrilamida/métodos , Conservação de Alimentos , Temperatura Alta , Concentração de Íons de Hidrogênio , Espectrometria de Massas
17.
Biochemistry (Mosc) ; 68(8): 926-33, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12948394

RESUMO

Temperature dependences of kinetic constants (k(cat) and K(m)) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of cold-adapted fishes. The anionic and cationic trypsins were found to differ in organization of the S(1)-substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S(1)) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K(m) value) to the anionic and cationic trypsins and on the catalytic activity k(cat) should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.


Assuntos
Tripsina/metabolismo , Animais , Arginina/química , Sítios de Ligação , Bovinos , Compostos Cromogênicos/química , Ativação Enzimática/fisiologia , Concentração de Íons de Hidrogênio , Mucoproteínas/química , Mutação , Ratos , Especificidade por Substrato/fisiologia , Temperatura
18.
Int J Food Microbiol ; 87(1-2): 63-73, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12927708

RESUMO

New proteinaceous active substance produced by Lactobacillus paracasei subsp. paracasei strain M3 used as a starter for Bulgarian yellow cheese was identified and studied. It displayed bactericidal and fungistatic activities. Its activity was checked against over 60 bacterial and yeast strains. It was efficient against Bacillus subtilis ATCC 6633, several L. delbrueckii species, Helicobacter pylori NCIPD 230 and some yeast species, for example Candida albicans, C. pseudointermedia NBIMCC 1532, C. blankii NBIMCC 85 and Saccharomyces cerevisiae NBIMCC 1812. The synthesis of the substance by producing strain was detected in the late logarithmic growth phase during batch fermentation. Anion exchange chromatography, reversed phase chromatography (RPC) on C4 column and HPLC on C18 column were used for partial purification of this antimicrobial compound. The gene responsible for the synthesis of the active substance is located on the bacterial chromosome.


Assuntos
Bactérias/crescimento & desenvolvimento , Queijo/microbiologia , Lactobacillus/fisiologia , Leveduras/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/análise , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/análise , Fermentação , Microbiologia de Alimentos , Conservação de Alimentos/métodos , Lactobacillus/química , Leveduras/efeitos dos fármacos
19.
Int J Food Microbiol ; 80(2): 145-52, 2003 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-12381400

RESUMO

Lactic acid bacterium isolated from Bulgarian cheese and identified as Enterococcus faecium produces a small hydrophobic peptide substance (enterococcin A 2000) with broad spectrum of antimicrobial activity. The wide range of enterococcin antibacterial activity of this compound against Gram-positive, as well as against some Gram-negative bacteria, suggests a single mechanism of action. The mode of action of enterococcin A 2000 was studied in intact liver mitochondria and synthetic phospholipid liposomes used as model systems. Enterococcin A 2000 stimulated the ATPase activity in intact mitochondria. The kinetic curve of ATP hydrolysis differed from that obtained in presence of dinitrophenol (DNP) and showed a character similar to the ATP hydrolysis in the presence of classic ionophores. Enterococcin A 2000, when bound to synthetic phospholipid liposomes, permeabilized liposomes liberating the marker carboxyfluorescein (CF).


Assuntos
Adenosina Trifosfatases/metabolismo , Bacteriocinas/farmacologia , Queijo/microbiologia , Enterococcus faecium/metabolismo , Lipossomos/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Proteínas de Bactérias , Bacteriocinas/isolamento & purificação , Microbiologia de Alimentos , Hidrólise , Cinética , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos
20.
Folia Microbiol (Praha) ; 47(4): 391-400, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12422516

RESUMO

A strain of Enterococcus faecium isolated from Bulgarian yellow cheese "kashkaval" produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (< 2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti-Listeria motif YGNGV.


Assuntos
Antibacterianos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Enterococcus faecium/química , Sequência de Aminoácidos , Aminoácidos/análise , Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacologia , Sequência de Bases , Queijo/microbiologia , DNA Bacteriano/genética , Enterococcus faecium/genética , Escherichia coli/efeitos dos fármacos , Listeria/efeitos dos fármacos , Peso Molecular , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética
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