Lactobacillus strains inhibit biofilm formation of Salmonella sp. isolates from poultry.

Lactic acid bacteria (LAB) exert a strong antagonistic activity against many microorganisms including food spoilage organisms and may be used as an alternative to control biofilm formation of pathogens in food industries. The objective of this work was to investigate the ability of fifteen Salmonella strains isolated from poultry environment to form biofilms on different surfaces. In addition, the effect of Lactobacillus kefiri strains 8321 and 83113 and Lactobacillus plantarum 83114 and their surface proteins on biofilm development of Salmonella Enteritidis 115 was studied. The relationship between surface properties of bacteria (hydrophobicity, autoaggregation and coaggregation with lactobacilli) and biofilm formation was also investigated. Most of Salmonella strains were hydrophilic and five strains were moderately hydrophobic. In general, Salmonella strains showed high aggregation abilities (27-54%). S. Enteritidis 106 and S. Typhimurium 102 and 108 showed the highest percentages of autoaggregation. All Salmonella strains tested showed aggregation abilities with the three lactobacilli studied, but the percentage of coaggregation proved to be strain-specific. When comparing stainless steel, glass and polystyrene surfaces, higher levels of biofilm formation occurred on polystyrene plate than on glass surfaces or stainless steel. S. Enteritidis 115 exhibited the greatest attachment to polyestyrene surface. The preincubation or coincubation with the three lactobacilli strains significantly reduced (about 1 log CFU/ml of reduction) the ability of S. Enteritidis 115 to form biofilm compared to the control without lactobacilli. These results were confirmed by confocal microscopy. In the same way, when surface proteins extracted from lactobacilli strains were preincubated or coincubated with S. Enteritidis 115, biofilm formation of this strain was significantly decreased compared to the control. The results obtained showed that these Lactobacillus strains and their surface proteins can be used as alternatives for control of biofilm formation by Salmonella in the poultry industry.

Biofilm formation by Salmonella sp. in the poultry industry: Detection, control and eradication strategies.

Salmonella represents an important global public health problem and it is an emerging zoonotic bacterial threat in the poultry industry. Diverse registered human cases of salmonellosis shown poultry origins. Various control measures have been employed both at the farming and processing levels to address it. This review focuses on traditional and new detection techniques of biofilm formation by Salmonella spp. and different approaches that can be used to prevent and/or control biofilm formation by these bacteria. A number of methodologies based on different approximations have been recently employed to detect and evaluate bacteria attached to surfaces, including real-time polymerase chain reaction (PCR), confocal laser scanning microscopy and Optical Coherence Tomography. Due to persistence of Salmonella biofilm in food processing environments after cleaning and sanitation, control and eradication strategies in poultry industry should be constantly studied. In this sense, the use of several alternatives to control Salmonella biofilm formation, such as lactic acid bacteria, phagetherapy, extracts from aromatic plants, quorum sensing inhibitors, bacteriocins and nanomaterials, have been successfully tested and will be reviewed.

Suitability of kefir powder production using spray drying.

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 60 days at 4 °C. Our results demonstrated that spray drying of kefir is a suitable approach to obtain a concentrated kefir-derived product.

Incorporation of Lactobacillus plantarum and zeolites in poultry feed can reduce aflatoxin B1 levels.

The aim of this work was to evaluate the incorporation of a freeze-dried probiotic strain (Lactobacillus plantarum CIDCA 83114) into zeolites. The bacteria-zeolite mixture was added to poultry feed together with thyme, and the obtained product was stored for 60 days at 25 °C and 60-70% relative humidity. The ability of the obtained product to remove aflatoxin B1 (AFB1) was studied. The highest bacterial viability was observed when 50% w/w bacteria were added to zeolites. The bacterial:zeolite mixtures were then incorporated into poultry feed containing or not thyme. Initial counts of L. plantarum were in the range of 1-2 × 108 CFU/g for all samples. In all cases, bacterial viability decreased one logarithmic order after 20 days of storage, and three logarithmic orders after 60 days. No significant viability loss was observed after exposure of the poultry feed to gastro-intestinal conditions. Freeze-dried L. plantarum and zeolite were able to remove AFB1, with an average reduction of 20 and 80%, respectively. Moreover, the freeze-dried bacteria-zeolite mixture was capable to remove up to 90% AFB1. This work contributes to enhance the nutritional quality of poultry feed, with a strong impact in production.

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying.

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20-30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS.

Administration of kefir-fermented milk protects mice against Giardia intestinalis infection.

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 1 : 100 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.

Survival of spray-dried Lactobacillus kefir is affected by different protectants and storage conditions.

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.

Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties.

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.

Characterization of homofermentative lactobacilli isolated from kefir grains: potential use as probiotic.

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S-23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2.5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

Rapid discrimination of lactobacilli isolated from kefir grains by FT-IR spectroscopy.

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.