RESUMO
Spray drying was applied for the production of kefir powder. The survival of microorganisms after drying, storage and simulated gastrointestinal (GI) conditions was investigated. Kefir was obtained by fermentation of milk and whey permeate, and was dehydrated directly (traditional kefir) or using different carriers (skim milk, whey permeate and maltodextrin). Low survival (5.5 log and <2 log CFU/g for lactic acid bacteria and yeast respectively) of microorganisms was achieved when kefir was dehydrated without thermoprotectants (carriers). In contrast, survival of the microorganisms was significantly improved in the presence of different carriers. When skim milk (SM) was used as the carrier medium, lactic acid bacteria (LAB) survival was above 9 log CFU/g. In contrast, viability of yeast was dramatically reduced after spray drying in these conditions. When whey permeate was used as the carrier medium, LAB survival was 8 log CFU/g and yeast survival was above 4 log CFU/g. LAB in the kefir powder survived better the simulated GI conditions when spray drying was conducted in SM. LAB in kefir powder sample dehydrated in SM and SM plus maltodextrin remained stable for at least 60â¯days at 4⯰C. Our results demonstrated that spray drying of kefir is a suitable approach to obtain a concentrated kefir-derived product.
Assuntos
Digestão , Manipulação de Alimentos/métodos , Microbiologia de Alimentos/métodos , Kefir/microbiologia , Lactobacillales/fisiologia , Leveduras/fisiologia , Aerossóis , Animais , Contagem de Colônia Microbiana , Dessecação , Fermentação , Suco Gástrico/química , Viabilidade Microbiana , Leite/metabolismo , Polissacarídeos/metabolismo , Proteínas do Soro do Leite/metabolismoRESUMO
BACKGROUND: Fungal contamination of poultry feed causes economic losses to industry and represents a potential risk to animal health. The aim of the present study was to analyze the effectiveness of whey fermented with kefir grains as additive to reduce fungal incidence, thus improving feed safety. RESULTS: Whey fermented for 24 h at 20 °C with kefir grains (100 g L(-1) ) reduced conidial germination of Aspergillus flavus, Aspergillus parasiticus, Aspergillus terreus, Aspergillus fumigatus, Penicillium crustosum, Trichoderma longibrachiatum and Rhizopus sp. Poultry feed supplemented with fermented whey (1 L kg(-1) ) was two to four times more resistant to fungal contamination than control feed depending on the fungal species. Additionally, it contained kefir microorganisms at levels of 1 × 10(8) colony-forming units (CFU) kg(-1) of lactic acid bacteria and 6 × 10(7) CFU kg(-1) of yeasts even after 30 days of storage. CONCLUSION: Fermented whey added to poultry feed acted as a biopreservative, improving its resistance to fungal contamination and increasing its shelf life.
Assuntos
Ração Animal/microbiologia , Fermentação , Microbiologia de Alimentos/métodos , Fungos , Proteínas do Leite/metabolismo , Aves Domésticas , Animais , Aspergillus/efeitos dos fármacos , Aspergillus/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Produtos Fermentados do Leite/microbiologia , Aditivos Alimentares , Contaminação de Alimentos/prevenção & controle , Conservação de Alimentos/métodos , Leite/microbiologia , Penicillium/efeitos dos fármacos , Penicillium/crescimento & desenvolvimento , Rhizopus/efeitos dos fármacos , Rhizopus/crescimento & desenvolvimento , Trichoderma/efeitos dos fármacos , Trichoderma/crescimento & desenvolvimento , Proteínas do Soro do LeiteRESUMO
We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.
Assuntos
Aderência Bacteriana , Mucosa Gástrica/microbiologia , Mucosa Intestinal/microbiologia , Lactobacillus/fisiologia , Muco/microbiologia , Probióticos , Animais , Aderência Bacteriana/efeitos dos fármacos , Colo , Produtos Fermentados do Leite/microbiologia , Hexoses/farmacologia , Intestino Delgado , Proteínas de Membrana/farmacologia , SuínosRESUMO
Giardiasis, caused by the protozoan Giardia intestinalis, is one of the most common intestinal diseases worldwide and constitutes an important problem for the public health systems of various countries. Kefir is a probiotic drink obtained by fermenting milk with 'kefir grains', which consist mainly of bacteria and yeasts that coexist in a complex symbiotic association. In this work, we studied the ability of kefir to protect mice from G. intestinalis infection, and characterized the host immune response to this probiotic in the context of the intestinal infection. Six- to 8-week-old C75BL/6 mice were separated into four groups: controls, kefir mice (receiving 1â:â100 dilution of kefir in drinking water for 14 days), Giardia mice (infected orally with 4×10(7) trophozoites of G. intestinalis at day 7) and Giardia-kefir mice (kefir-treated G. intestinalis-infected mice), and killed at 2 or 7 days post-infection. Kefir administration was able to significantly reduce the intensity of Giardia infection at 7 days post-infection. An increase in the percentage of CD4(+) T cells at 2 days post-infection was observed in the Peyer's patches (PP) of mice belonging to the Giardia group compared with the control and kefir groups, while the percentage of CD4(+) T cells in PP in the Giardia-kefir group was similar to that of controls. At 2 days post-infection, a reduction in the percentage of B220-positive major histocompatibility complex class II medium cells in PP was observed in infected mice compared with the other groups. At 7 days post-infection, Giardia-infected mice showed a reduction in RcFcε-positive cells compared with the control group, suggesting a downregulation of the inflammatory response. However, the percentages of RcFcε-positive cells did not differ from controls in the kefir and Giardia-kefir groups. An increase in IgA-positive cells was observed in the lamina propria of the kefir group compared with controls at 2 days post-infection. Interestingly, the diminished number of IgA-positive cells registered in the Giardia group at 7 days post-infection was restored by kefir feeding, although the increase in IgA-positive cells was no longer observed in the kefir group at that time. No significant differences in CXCL10 expression were registered between groups, in concordance with the absence of inflammation in small-intestinal tissue. Interestingly, a slight reduction in CCL20 expression was observed in the Giardia group, suggesting that G. intestinalis might downregulate its expression as a way of evading the inflammatory immune response. On the other hand, a trend towards an increase in TNF-α expression was observed in the kefir group, while the Giardia-kefir group showed a significant increase in TNF-α expression. Moreover, kefir-receiving mice (kefir and Giardia-kefir groups) showed an increase in the expression of IFN-γ, the most relevant Th1 cytokine, at 2 days post-infection. Our results demonstrate that feeding mice with kefir reduces G. intestinalis infection and promotes the activation of different mechanisms of humoral and cellular immunity that are downregulated by parasitic infection, thus contributing to protection.
Assuntos
Produtos Fermentados do Leite/imunologia , Fermentação/imunologia , Giardia lamblia/imunologia , Giardíase/imunologia , Giardíase/prevenção & controle , Leite/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Produtos Fermentados do Leite/metabolismo , Regulação para Baixo/imunologia , Feminino , Genes MHC da Classe II/imunologia , Giardia lamblia/metabolismo , Giardíase/metabolismo , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interferon gama/imunologia , Interferon gama/metabolismo , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Leite/metabolismo , Mucosa/imunologia , Mucosa/metabolismo , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The objective of this work was to test the protective effect of a mixture (MM) constituted by kefir-isolated microorganisms (Lactobacillus plantarum, Lactobacillus kefir, Lc. lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae) in a hamster model of infection with Clostridium difficile, an anaerobic Gram-positive bacterium that causes diarrhoea. Placebo or MM was administered ad libitum in drinking water from day 0 to the end of treatment. Hamsters received orally 200 µg of clyndamicin at day 7 and then were infected with 1 × 10(8) CFU of C. difficile by gavage. Development of diarrhoea and death was registered until the end of the protocol. Surviving animals were sacrificed at day 16, and a test for biological activity of clostridial toxins and histological stainings were performed in caecum samples. Six of seven infected animals developed diarrhoea and 5/7 died at the end of the experimental protocol. The histological sections showed oedema and inflammatory infiltrates with neutrophils and crypt abscesses. In the group of animals infected and treated with MM1/1000, only 1 of 7 hamsters showed diarrhoea and none of them died. The histological sections showed only a slight thickening of the mucosa with presence of lymphocytic infiltrate. These results demonstrate that an oral treatment with a mixture of kefir-isolated bacteria and yeasts was able to prevent diarrhoea and enterocolitis triggered by C. difficile.
Assuntos
Clostridioides difficile/fisiologia , Produtos Fermentados do Leite/microbiologia , Enterocolite Pseudomembranosa/prevenção & controle , Kluyveromyces/isolamento & purificação , Lactobacillus/isolamento & purificação , Saccharomyces cerevisiae/isolamento & purificação , Administração Oral , Animais , Toxinas Bacterianas/efeitos adversos , Ceco/microbiologia , Ceco/patologia , Cricetinae , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Feminino , Humanos , Kluyveromyces/fisiologia , Ácido Láctico/metabolismo , Lactobacillus/fisiologia , Mesocricetus , Saccharomyces cerevisiae/fisiologiaRESUMO
The aim of this study was to evaluate the ability of Bifidobacterium strains to prevent the effects associated with Clostridium difficile infection in a hamster model of enterocolitis. After clindamycin treatment (30 mg/kg), animals were infected intragastrically with C. difficile (5×108 CFU per animal). Seven days prior to antibiotic administration, probiotic treatment was started by administering bacterial suspensions of bifidobacteria in drinking water. Strains CIDCA 531, CIDCA 5310, CIDCA 5316, CIDCA 5320, CIDCA 5323 and CIDCA 5325 were used. Treatment was continued during all the experimental period. Development of diarrhoea, enterocolitis and mortality were evaluated. All the infected animals belonging to the placebo group developed enterocolitis (5/5) and only two dead (2/5) whereas in the group administered with Bifidobacterium bifidum strain CIDCA 5310 the ratio of animals with enterocolitis or dead decreased significantly (1/5 and 0/5 respectively). Biological activity of caecum contents was evaluated in vitro on Vero cells. Animals treated with strain CIDCA 5310 presented lower biological activity than those belonging to the placebo group. The present study shows the potential of selected strains of bifidobacteria to antagonise, in vivo, the virulence of C. difficile.
Assuntos
Bifidobacterium/metabolismo , Clostridioides difficile , Enterocolite Pseudomembranosa/veterinária , Probióticos/uso terapêutico , Animais , Ceco/microbiologia , Ceco/patologia , Chlorocebus aethiops , Cricetinae , Enterocolite Pseudomembranosa/patologia , Enterocolite Pseudomembranosa/prevenção & controle , Feminino , Conteúdo Gastrointestinal/microbiologia , Mesocricetus , Células Vero/efeitos dos fármacosRESUMO
Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide-cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.
Assuntos
Sobrevivência Celular , Produtos Fermentados do Leite/microbiologia , Escherichia coli O157 , Lactobacillus plantarum/fisiologia , Toxina Shiga II/toxicidade , Animais , Parede Celular/fisiologia , Chlorocebus aethiops , Lactobacillus plantarum/ultraestrutura , Células Vero/efeitos dos fármacosRESUMO
We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.
Assuntos
Grão Comestível/microbiologia , Fermentação , Leite/microbiologia , Polissacarídeos/metabolismo , Animais , Grão Comestível/metabolismo , Concentração de Íons de Hidrogênio , Kluyveromyces/isolamento & purificação , Lactobacillus/isolamento & purificação , Lactococcus lactis/isolamento & purificação , Leite/metabolismo , Polissacarídeos/química , Saccharomyces/isolamento & purificação , TemperaturaRESUMO
In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/metabolismo , Lactobacillus/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Antibiose , Toxinas Bacterianas/metabolismo , Linhagem Celular , Chlorocebus aethiops , Ligação ProteicaRESUMO
A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin-producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.
Assuntos
Produtos Fermentados do Leite/química , Produtos Fermentados do Leite/microbiologia , Probióticos/metabolismo , Animais , Fenômenos Químicos , Chlorocebus aethiops , Escherichia coli Êntero-Hemorrágica/metabolismo , Fermentação , Humanos , Kluyveromyces/metabolismo , Lactobacillus plantarum/metabolismo , Toxinas Shiga/antagonistas & inibidores , Olfato , Streptococcus thermophilus/metabolismo , Paladar , Células VeroRESUMO
In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700-1500 cm(-1), 1500-1185 cm(-1) and 1800-400 (whole spectrum) cm(-1) Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700-1500 cm(-1), 1500-1185 cm(-1) and 1185-1020 cm(-1), i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.
Assuntos
Lactobacillus/química , Lactobacillus/classificação , Análise Espectral Raman , Técnicas Bacteriológicas , Análise de Componente Principal , Especificidade da EspécieRESUMO
The effect of freeze-drying on viability and probiotic properties of a microbial mixture containing selected bacterial and yeast strains isolated from kefir grains (Lactobacillus kefir, Lactobacillus plantarum, Lactococcus lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) was studied. The microorganisms were selected according to their potentially probiotic properties in vitro already reported. Two types of formulations were performed, a microbial mixture (MM) suspended in milk and a milk product fermented with MM (FMM). To test the effect of storage on viability of microorganisms, MM and FMM were freeze-dried and maintained at 4°C for six months. After 180 days of storage at 4°C, freeze-dried MM showed better survival rates for each strain than freeze-dried FMM. The addition of sugars (trehalose or sucrose) did not improve the survival rates of any of the microorganisms after freeze-drying. Freeze-drying did not affect the capacity of MM to inhibit growth of Shigella sonnei in vitro, since the co-incubation of this pathogen with freeze-dried MM produced a decrease of 2 log in Shigella viability. The safety of freeze-dried MM was tested in mice and non-translocation of microorganisms to liver or spleen was observed in BALB/c mice feed ad libitum during 7 or 20 days. To our knowledge, this is the first report about the effect of freeze-drying on viability, in vitro probiotic properties and microbial translocation of a mixture containing different strains of both bacteria and yeasts isolated from kefir.
Assuntos
Produtos Fermentados do Leite/microbiologia , Liofilização , Lactobacillaceae/fisiologia , Probióticos , Saccharomyces cerevisiae/fisiologia , Animais , Translocação Bacteriana , Feminino , Fermentação , Kluyveromyces/fisiologia , Lactobacillus/fisiologia , Lactobacillus plantarum/fisiologia , Lactococcus lactis/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Leite/microbiologia , Shigella/fisiologiaRESUMO
The injuries caused by spray drying (SD) of three potential probiotic lactobacilli isolated from kefir grains and the impact on some probiotic properties, were evaluated. Results demonstrated that Lactobacillus plantarum 83114 and L. kefir 8321 showed a slight reduction of viability (0.11 and 0.29 log CFU/ml respectively) after SD process, and L. kefir 8348 was found to be more sensitive to the process with a reduction in viability of 0.70 log CFU/ml. Neither membrane damage, evaluated by increased sensitivity to NaCl, lysozyme, bile salt and penicillin G, nor changes in acidifying activity in MRS and milk by lactobacilli were detected after SD. L. plantarum 83114 and L. kefir 8321 after SD did not lose their capacity to adhere to intestinal cells. Nevertheless, L. kefir 8348 showed a significant loss of adhesion capacity after SD. In addition, rehydrated spray-dried L. kefir 8321 retained the ability to protect against Salmonella invasion of intestinal cells. This effect was observed when L. kefir is co-incubated with Salmonella before invasion assay. This work shows that the membrane integrity evaluated by indirect methods and some probiotic properties of lactobacilli isolated from kefir did not change significantly after SD, and these powders could be used in functional foods applications.
Assuntos
Produtos Fermentados do Leite/microbiologia , Dessecação , Microbiologia de Alimentos , Lactobacillus/citologia , Lactobacillus/fisiologia , Probióticos , Animais , Células CACO-2/microbiologia , Membrana Celular/patologia , Humanos , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Viabilidade Microbiana , Leite/microbiologia , Salmonella/fisiologia , Infecções por Salmonella/patologiaRESUMO
Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0-11%), and monosodium glutamate and FOS proved to be the best protectants.
Assuntos
Aditivos Alimentares/farmacologia , Manipulação de Alimentos , Microbiologia de Alimentos , Lactobacillus/efeitos dos fármacos , Lactobacillus/fisiologia , Viabilidade Microbiana , Leite/microbiologia , Animais , Dessecação , Conservação de Alimentos , Temperatura Alta , Viabilidade Microbiana/efeitos dos fármacos , Pós , TemperaturaRESUMO
Toxigenic strains of Clostridium difficile were co-cultured with different strains of bifidobacteria and lactobacilli. Spent culture supernatants were tested for biological activity on cultured Vero cells. Co-culture of C. difficile with some potentially probiotic strains lead to a reduction of the biological activity of spent culture supernatants. The observed effects cannot be ascribed either to secreted factors from the probiotic strains or to toxin adsorption by bacterial cells. Immunological assays showed that there was significant diminution of both clostridial toxins (TcdA and TcdB) in spent culture supernatants of co-cultures as compared with pure clostridial cultures. Even though co-cultured clostridial cells showed a slight increase of intracellular toxins, this increase did not completely explains the reduction of toxin concentration in culture supernatants. The evidence suggests that the antagonism could be due to the diminution of the synthesis and/or secretion of both clostridial toxins. Our findings provide new insights into the possible mechanisms involved in the protective effect of probiotics in the context of C. difficile infection.
Assuntos
Antibiose , Clostridioides difficile/metabolismo , Probióticos/farmacologia , Fatores de Virulência/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/crescimento & desenvolvimento , Técnicas de Cocultura , Enterotoxinas/antagonistas & inibidoresRESUMO
The effect of several biocides, thermal treatments, and photocatalysis on the viability of four Lactobacillus plantarum phages was investigated. Times to achieve 99% inactivation (T99) of phages at 63, 72, and 90 degrees C were evaluated in four suspension media: deMan Rogosa Sharpe broth, reconstituted skim milk, a commercial EM-glucose medium, and Tris magnesium gelatin buffer. The four phages studied were highly resistant to 63 degrees C (T99 > 45 min); however, counts < 10 PFU/ml were achieved by heating at 90 degrees C for 5 min. Higher thermal resistance at 72 degrees C was observed when reconstituted skim milk and EM-glucose medium were assayed. Peracetic acid (0.15%, vol/vol) was an effective biocide for the complete inactivation of all phages studied within 5 min of exposure. Sodium hypochlorite (800 ppm) inactivated the phages completely within 30 min. Ethanol (100%) did not destroy phage particles even after 45 min. Isopropanol did not have any effect on phage viability. Phage counts < 50 PFU/ml were obtained within 180 min of photocatalytic treatment. The results obtained in this work are important for establishing adequate methods for inactivating phages in industrial plants and laboratory environments.
Assuntos
Fagos Bacilares , Desinfetantes/farmacologia , Irradiação de Alimentos , Temperatura Alta , Lactobacillus plantarum/virologia , Fagos Bacilares/efeitos dos fármacos , Fagos Bacilares/crescimento & desenvolvimento , Fagos Bacilares/efeitos da radiação , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Meios de Cultura , Relação Dose-Resposta a Droga , Cinética , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/efeitos da radiação , Ácido Peracético/farmacologia , Hipoclorito de Sódio/farmacologia , Fatores de Tempo , Raios UltravioletaRESUMO
Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.
Assuntos
Produtos Fermentados do Leite/microbiologia , Lactobacillus/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Glicosilação , Lactobacillus/crescimento & desenvolvimento , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The effects of kefir-fermented milk were tested against a toxigenic strain of Bacillus cereus. The incubation of milk with B. cereus spores plus 5% kefir grains prevented spore germination and growth of vegetative forms. In contrast, when 1% kefir grains was used, no effects were observed. The presence of metabolically active kefir grains diminished titers of nonhemolytic enterotoxin A, as assessed by enzyme-linked immunosorbent assay. During fermentation, kefir microorganisms produce extracellular metabolites such as organic acids, which could play a role in the inhibition of spore germination and growth of B. cereus, although the effect of other factors cannot be ruled out. Results of the present study show that kefir-fermented milk is able to antagonize key mechanisms involved in the growth of B. cereus as well as interfere with the biological activity of this microorganism.
Assuntos
Bacillus cereus/crescimento & desenvolvimento , Produtos Fermentados do Leite/microbiologia , Manipulação de Alimentos/métodos , Microbiologia de Alimentos , Contagem de Colônia Microbiana , Fermentação , Especificidade da Espécie , Esporos BacterianosRESUMO
Fourier transform infrared (FT-IR) spectroscopy was used in combination with multivariate statistical analysis for differentiation of lactic bacteria isolated from kefir grains. Twelve reference strains and 42 lactobacilli isolates from four local kefir grains, previously identified by biochemical traditional techniques at species level were included in this study. The spectra were analysed by hierarchical clustering analysis (HCA) using Pearson's product-moment correlation coefficient and Ward's algorithm. The differentiation between homo- and heterofermentative lactobacilli, proposed as a first level in the classification scheme, was performed with vector normalized first derivatives spectra in the windows 1789-1700, 1059-935, 3000-2927 and 896-833 cm(-1). For heterofermentative lactobacilli the windows 1780-1750, 1500-1200, 2950-2930 and 900-700 cm(-1) were found to contribute to the maximal separation among L. kefir, L. parakefir and Lactobacillus brevis. It was also demonstrated that although this model was robust against small variations in growth temperature (+/-5 degrees C) and growth time (+/-5 h), the make of culture medium used (Biokar or Difco) affected the separation of heterofermentative lactobacilli at species level. For homofermentative lactobacilli the spectral regions 1230-900, 1777-1690, 1357-1240 and 2960-2870 cm(-1), were selected for discrimination among 5 different species that are normally present in kefir grains: L. plantarum, L. acidophilus, L. kefirgranum, L. kefiranofaciens and L. cassei. The classification and discrimination schemes proposed in this work completely matched with the identification obtained by classical biochemical techniques at species level.
Assuntos
Produtos Fermentados do Leite/microbiologia , Microbiologia de Alimentos , Lactobacillus/classificação , Filogenia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise por Conglomerados , Meios de Cultura/química , Cinética , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/isolamento & purificação , Análise Multivariada , Especificidade da Espécie , TemperaturaRESUMO
The antimicrobial and anti-adhesive effects of extracellular factors from 27 strains of bifidobacteria isolated from healthy infants were tested against two reference strains of Clostridium difficile (ATCC 9689 and ATCC 43593). All bifidobacterial supernatants at pHs between 5.0 and 4.1 were able to produce strain-dependent growth inhibition of clostridia in the agar-diffusion assay. Six strains of Bifidobacterium produced during growth extracellular factors able to antagonize the adhesion of C. difficile ATCC 9689 and ATCC 43593 to cultured human enterocytes (Caco-2/TC7). Factors responsible for the anti-adhesive effect were thermolabile, active at neutral pH and unaffected by proteolytic cleavage (proteinase K and chymotrypsin). Results of the present paper show the potential of selected bifidobacteria to antagonize key mechanisms involved in the virulence of C. difficile.