Marina A. Golowczyc

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.

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.

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.

Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins

Several microbial interactions involving yeast and lactobacilli have been suggested in fermented products. Co-aggregation between Lactobacillus kefir and yeast Saccharomyces lipolytica isolated from kefir grains was studied by scanning electron microscopy and aggregation assays. Six out of twenty Lb. kefir strains were able to co-aggregate with Sacch. lipolytica CIDCA 812 and showed thermolabile non-covalently bound surface molecules involved in this interaction. Co-aggregation inhibition after Lb. kefir pre-treatment with 5 m-LiCl or 20 g SDS/l showed that bacterial S-layer proteins play an important role in this interaction. Presence of different sugar (mannose, sucrose and fructose) or yeast pre-treatment with sodium periodate inhibited co-aggregation between Lb. kefir and Sacch. lipolytica. Co-aggregating Lb. kefir strains were also able to agglutinate with human red blood cells and they lost this ability after treatment with 5 m-LiCl. These results and the capacity of purified S-layer proteins of Lb. kefir to haemagglutinate, strongly suggest that a lectin-like activity of bacterial surface proteins (S-layer) mediates the aggregation with yeast cells.

Use of whey permeate containing in situ synthesised galacto-oligosaccharides for the growth and preservation of Lactobacillus plantarum

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

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.

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.

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.