Exploring encapsulation strategies as a protective mechanism to avoid amensalism in mixed populations of Pseudomonas taetrolens and Lactobacillus casei.

Pseudomonas taetrolens constitutes an efficient platform for the biosynthesis of lactobionic acid, a potentially prebiotic compound. Unfortunately, an amensalistic interaction has been demonstrated between P. taetrolens and probiotic lactic acid bacteria (LAB), characterized by the competitive exclusion of P. taetrolens, hindering the in situ production of fermented dairy products with synbiotic properties. In the present research, encapsulation was explored as a barrier to the diffusion of the antimicrobial metabolites generated by LAB. Mixed fermentations involving P. taetrolens LMG 2336 and Lactobacillus casei CECT 475 were cultivated, entrapping both microorganisms alternately. Alginate, alginate/starch and carboxymethyl cellulose/k-carrageenan were tested as encapsulating agents. The immobilization of L. casei in 2% alginate/2% starch beads was found to be the best strategy, improving the production of lactobionic acid by 182% with respect to co-cultures with free cells. This study proves the potential of LAB encapsulation for the protection of sensitive strains in mixed food fermentations.

Using Raman spectroscopy and chemometrics to identify the growth phase of Lactobacillus casei Zhang during batch culture at the single-cell level.

BACKGROUND: As microbial cultures are comprised of heterogeneous cells that differ according to their size and intracellular concentrations of DNA, proteins, and other constituents, the detailed identification and discrimination of the growth phases of bacterial populations in batch culture is challenging. Cell analysis is indispensable for quality control and cell enrichment. METHODS: In this paper, we report the results of our investigation on the use of single-cell Raman spectrometry (SCRS) for real-time analysis and prediction of cells in different growth phases during batch culture of Lactobacillus (L.) casei Zhang. A targeted analysis of defined cell growth phases at the level of the single cell, including lag phase, log phase, and stationary phase, was facilitated by SCRS. RESULTS: Spectral shifts were identified in different states of cell growth that reflect biochemical changes specific to each cell growth phase. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, whereas protein-specific and lipid-specific Raman vibrations increased at different rates. Furthermore, a supervised classification model (Random Forest) was used to specify the lag phase, log phase, and stationary phase of cells based on SCRS, and a mean sensitivity of 90.7% and mean specificity of 90.8% were achieved. In addition, the correct cell type was predicted at an accuracy of approximately 91.2%. CONCLUSIONS: To conclude, Raman spectroscopy allows label-free, continuous monitoring of cell growth, which may facilitate more accurate estimates of the growth states of lactic acid bacterial populations during fermented batch culture in industry.

Effect of salt on cell viability and membrane integrity of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum as observed by flow cytometry.

The aim of the current study was to investigate the effect of varying sodium chloride concentrations (0-5%) on viability and membrane integrity of three probiotic bacteria, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum, using conventional technique and flow cytometry. Double staining of cells by carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) enabled to evaluate the effect of NaCl on cell esterase activity and membrane integrity. Observations from conventional culture technique were compared with findings from flow cytometric analysis on the metabolic activities of the cells and a correlation was observed between culturability and dye extrusion ability of L. casei and B. longum. However, a certain population of L. acidophilus was viable as per the plate count method but its efflux activity was compromised. Esterase activity of most bacteria reduced significantly (P < 0.05) during one week storage at NaCl concentrations greater than 3.5%. The study revealed that L. casei was least affected by higher NaCl concentrations among the three probiotic bacteria, as opposed to B. longum where the cF extrusion performance was greatly reduced during 1 wk storage. The metabolic activity and salt resistance of L. casei was found to be highest among the bacteria studied.

Microencapsulation of Lactobacillus casei by spray drying.

This study evaluates the use of spray drying to produce microparticles of Lactobacillus casei. Microorganism was cultivated in shaken flasks and the microencapsulation process was performed using a laboratory-scale spray dryer. A rotational central composite design was employed to optimise the drying conditions. High cell viability (1.1 × 10(10) CFU/g) was achieved using an inlet air temperature of 70 °C and 25% (w/v) of maltodextrin. Microparticles presented values of solubility, wettability, water activity, hygroscopicity and humidity corresponding to 97.03 ± 0.04%, 100% (in 1.16 min), 0.14 ± 0.0, 35.20 g H2O/100 g and 4.80 ± 0.43%, respectively. The microparticles were spherical with a smooth surface and thermally stable. Encapsulation improved the survival of L. casei during storage. After 60 days, the samples stored at -8 °C showed viable cell concentrations of 1.0 × 10(9) CFU/g.

Optimization of the formulation for preparing Lactobacillus casei loaded whey protein-Ca-alginate microparticles using full-factorial design.

CONTEXT: This article presents specific approach for microencapsulation of Lactobacillus casei using emulsion method followed by additional coating with whey protein. METHODS: Experimental design was employed using polynomial regression model at 2nd level with three independent variables, concentrations of alginate, whey protein and CaCl2. Physicochemical, biopharmaceutical and biological properties were investigated. RESULTS: In 11 series generated, negatively charged microparticles were obtained, with size 6.99-9.88 µm, Ca-content 0.29-0.47 mg per 10 mg microparticles, and viability of the probiotic 9.30-10.87 log10CFU/g. The viability after 24 hours in simulated gastrointestinal conditions was between 3.60 and 8.32 log10CFU/g. DISCUSSION: Optimal formulation of the microparticles that ensures survival of the probiotic and achieves controlled delivery was determined: 2.5% (w/w) alginate, 3% (w/w) CaCl2 and 3% (w/w) whey protein. CONCLUSION: The advantageous properties of the L. casei-loaded microparticles make them suitable for incorporation in functional food and/or pharmaceutical products.

Osmotic stress adaptation in Lactobacillus casei BL23 leads to structural changes in the cell wall polymer lipoteichoic acid.

The probiotic Gram-positive bacterium Lactobacillus casei BL23 is naturally confronted with salt-stress habitats. It has been previously reported that growth in high-salt medium, containing 0.8 M NaCl, leads to modifications in the cell envelope of this bacterium. In this study, we report that L. casei BL23 has an increased ability to form biofilms and to bind cations in high-salt conditions. This behaviour correlated with modifications of surface properties involving teichoic acids, which are important cell wall components. We also showed that, in these high-salt conditions, L. casei BL23 produces less of the cell wall polymer lipoteichoic acid (LTA), and that this anionic polymer has a shorter mean chain length and a lower level of d-alanyl-substitution. Analysis of the transcript levels of the dltABCD operon, encoding the enzymes required for the incorporation of d-alanine into anionic polymers, showed a 16-fold reduction in mRNA levels, which is consistent with a decrease in d-alanine substitutions on LTA. Furthermore, a 13-fold reduction in the transcript levels was observed for the gene LCABL_09330 coding for a putative LTA synthase. To provide further experimental evidence that LCABL_09330 is a true LTA synthase (LtaS) in L. casei BL23, the enzymic domain was cloned and expressed in E. coli. The purified protein was able to hydrolyse the membrane lipid phosphatidylglycerol as expected for an LTA synthase enzyme, and hence LCABL_09330 was renamed LtaS. The purified enzyme showed Mn(2+)-ion dependent activity, and its activity was modulated by differences in NaCl concentration. The decrease in both ltaS transcript levels and enzyme activity observed in high-salt conditions might influence the length of the LTA backbone chain. A putative function of the modified LTA structure is discussed that is compatible with the growth under salt-stress conditions and with the overall envelope modifications taking place during this stress condition.

Strategies to decolorize high concentrations of methyl orange using growing cells of Lactobacillus casei TISTR 1500.

Batch, fed-batch, and continuous fermentation was used in the processing of methyl orange decolorization using growing cells of Lactobacillus casei TISTR 1500. This report presents the optimal conditions for methyl orange decolorization by the strain TISTR 1500 in modified MRS via a central composite design (CCD) experiment. In particular, the highest decolorization efficiencies were obtained with 13.41 g/L of meat extract, and with 10.89 g/L of yeast extract at pH 6.88 at 35 °C. Under the optimal conditions, the rate of decolorization increased to 322% of that obtained for un-optimized MRS medium. The high concentration of methyl orange (5 g/L) was completely degraded within 9 h in batch fermentation. The total methyl orange load with 8.075 g/L was also decolorized in fed-batch fermentation within 13 h, and the biomass of the strain dramatically decreased after an incubation time of 8 h due to a shortage of sucrose. In the continuous system with a dye-loading rate of 600 mg/L/h and a total of loaded azo dye of 7.2 g/L, high efficiency of methyl orange removal was significantly high, at 98%.

Lactobacillus casei combats acid stress by maintaining cell membrane functionality.

Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress.

Antiproliferative and anticytotoxic effects of cell fractions and exopolysaccharides from Lactobacillus casei 01.

Cell fractions including heat-treated cells, crude cell walls, intracellular extracts and exopolysaccharides (EPSs) obtained from Lactobacillus casei 01 were first studied for their effects on the proliferation of human intestinal epithelial cells, intestine 407 and the human colon cancer cell, HT-29. Their effects on the cytotoxicity of 4-nitroquinoline 1-oxide (4-NQO) against intestine 407 were further investigated. The results revealed that EPS exhibited the highest antiproliferation activity on HT-29 cells while the viability of intestine 407 cells was not affected by EPS at a concentration of 5-50µg/mL. It was also noted that all the cell fractions and EPS from L. casei 01 reduced the cytotoxicity of 4-NQO against intestine 407 with EPS showing the highest anticytotoxic activity. Additionally, it was found that EPS might exert blocking and bioanticytotoxic effects by both adjusting the function of intestine 407 and repairing the 4-NQO-damaged cells, thus reducing cytotoxicity of 4-NQO.

Probiotic Lactobacillus casei expressing human lactoferrin elevates antibacterial activity in the gastrointestinal tract.

In this study, Lactobacillus casei was used to deliver and express human lactoferrin (hLF) to protect the host against bacterial infection. Full-length hLF cDNA was cloned into a Lactobacillus-specific plasmid to produce the L. casei transformants (rhLF/L. casei). Antimicrobial activity of recombinant hLF was examined in inhibition of bacteria growth in vitro. A mouse model was established to test in vivo antibacterial activity and protective effect of orally-administered probiotic L. casei transformant in the gastrointestinal tract. Trials were conducted in which animals were challenged with E. coli ATCC25922. E. coli colony numbers in duodenal fluid from the group fed with rhLF/L. casei were significantly lower than those of the group fed with wild-type L. casei or placebo (P < 0.01). Histopathological analyses of the small intestine, showed both decreased intestinal injury and increased villi length were observed in the mice fed with rhLF/L. casei as compared with the control groups (P < 0.01). Our results demonstrate that L. casei expressing hLF exhibited antibacterial activity both in in vitro and in vivo. It also provides a potentially large-scale production of hLF as applications for treatment of infections caused by clinically relevant pathogens.

Protective effect of sucrose on the membrane properties of Lactobacillus casei Zhang subjected to freeze-drying.

The purpose of this research was to investigate the influence of sucrose at 2.0, 4.0, and 8.0% as a protectant during freeze-drying on the viability and membrane properties of Lactobacillus casei Zhang. Membrane properties were determined using zeta potential, hydrophobicity, fluidity, and integrity before and after freeze-drying. Exposing L. casei Zhang to sucrose protected it from drastic changes in cell surface electrophoretic mobility and hydrophobicity in contrast with the untreated condition, and the effect was dose related. Sucrose caused an increase in membrane fluidity compared with the control sample. Moreover, 2.0% sucrose decreased the general polarization values less than 4.0 or 8.0% sucrose, while 4.0% sucrose and 8.0% sucrose had no significant difference in decreasing general polarization values (P < 0.05). L. casei Zhang freeze-dried in the presence of 2.0% sucrose retained up to 23.7% membrane integrity, whereas cells freeze-dried with 4.0 and 8.0% sucrose had 32.4 and 37.6% membrane integrity compared with that of L. casei Zhang before freeze-drying. Correspondingly, the number of survivors of L. casei Zhang, determined by the plate count method, decreased from 8.02 to 0.63 log CFU/ml after freeze-drying in the absence of sucrose. However, in the presence of 2.0, 4.0, and 8.0% sucrose, the numbers of survivors were 2.01, 2.87, and 3.20 log CFU/ml after freeze-drying, respectively. The present work suggested that sucrose was an effective membrane protectant at 2.0, 4.0, or 8.0% on the surface zeta potential, hydrophobicity, fluidity, and integrity of L. casei Zhang.

Inactivation of Escherichia coli in broth and sausage by combined high pressure and Lactobacillus casei cell extract.

The purpose of this study was to investigate the effect of combined high pressure and Lactobacillus casei cell extract (CE) on Escherichia coli O157 strains with variation in pressure resistance in broth and sausage. Pressure-resistant (O157:H7 and O157:H12) and -sensitive (O157-M1 and O157-M2) E. coli strains were used. Pressure treatment at 350 MPa for 20 min in broth caused 1.1-1.2 logs reduction in O157:H12 and O157:H7 and 4.1-5.5 logs reduction in the O157-M1 and O157-M2. When high pressure was treated in the presence of CE (32 CEAU/mL), the combination treatment caused a significant inactivation in the pressure-resistant O157:H7 strains resulting in the viability loss of 4.3-4.6 logs and the synergistic effect increased with increase in treatment time (p < 0.05). Similar result was observed in sausage. Differential scanning calorimetry thermogram showed that the presence of Lb. casei CE may cause considerable damage to cellular components of E. coli during the high pressure treatment. The synergy between high pressure processing and Lb. casei OSY-LB6A CE against pressure-resistant E. coli O157 strains suggests the feasibility of using this combination to minimize the risk of transmission of E. coli O157 by food.

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system.

AIMS: To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied. METHODS AND RESULTS: An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and beta-galactosidase activities of the EPS(+) strains were higher than those of the EPS(-). Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected. CONCLUSIONS: The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.

Microencapsulation of a probiotic bacteria with alginate-gelatin and its properties.

Lactobacillus casei ATCC 393-loaded microcapsules based on alginate and gelatin had been prepared by extrusion method and the product could increase the cell numbers of L. casei ATCC 393 to be 10(7) CFU g(-1) in the dry state of microcapsules. The microparticles homogeneously distributed with size of 1.1 ± 0.2 mm. Four kinds of microcapsules (S(1), S(2), S(3) and S(4)) exhibited swelling in simulated gastric fluid (SGF) while the beads eroded and disintegrated rapidly in simulated intestinal fluid (SIF). Cells of L. casei ATCC 393 could be continuously released from the microcapsules during simulated gastrointestinal tract (GIT) and the release amounts and speeds in SIF were much higher and faster than that in SGF. Encapsulation in alginate-gelatin microcapsules successfully improved the survival of L. casei ATCC 393 and this approach might be useful in delivery of probiotic cultures as a functional food.

Lactic acid bacteria production from whey.

The main purpose of this work was to isolate and characterize lactic acid bacteria (LAB) strains to be used for biomass production using a whey-based medium supplemented with an ammonium salt and with very low levels of yeast extract (0.25 g/L). Five strains of LAB were isolated from naturally soured milk after enrichment in whey-based medium. One bacterial isolate, designated MNM2, exhibited a remarkable capability to utilize whey lactose and give a high biomass yield on lactose. This strain was identified as Lactobacillus casei by its 16S rDNA sequence. A kinetic study of cell growth, lactose consumption, and titratable acidity production of this bacterial strain was performed in a bioreactor. The biomass yield on lactose, the percentage of lactose consumption, and the maximum increase in cell mass obtained in the bioreactor were 0.165 g of biomass/g of lactose, 100%, and 2.0 g/L, respectively, which were 1.44, 1.11, and 2.35 times higher than those found in flask cultures. The results suggest that it is possible to produce LAB biomass from a whey-based medium supplemented with minimal amounts of yeast extract.

Low intensity ultrasound increases the fermentation efficiency of Lactobacillus casei subsp.casei ATTC 39392.

L-Lactic acid (L-LA) is one of the microbial products with several applications and its production efficiency is so important. In the present study, we have been exploring application of low intensity ultrasound technology to improve the metabolic activity for L-lactic acid production by Lactobacillus casei in different mediums. L-LA, biomass production and substrate (protein) consumption were measured as parameters of fermentation yield. L-LA and protein contents were determined using the titratable acidity and the biuret method respectively. Spectrophotometry (OD600nm) was used for measuring cell growths. L-LA, biomass production and protein consumption considered as dependent variables, but the amplitude of waves (20%, 40% and 60%), waves duration (15, 30, 45 s) and add of peptone (2, 6 and 10 g/l) as independent variables. The results showed that L-LA, biomass production and substrate consumption significantly increased (≈25%). Optimum conditions for biomass production was amplitude of 60%, 15s exposure time and 10 g/l peptone, while for acid lactic production and substrate consumption was 40%, 30s and 6g/l peptone, respectively. Flowcytometry analysis also showed that sonication led to increasing cell membrane permeability. This observation shows low intensity ultrasound as a potential parameter in the improvement of metabolic activity of L. casei.

Structural studies of the rhamnose-rich cell wall polysaccharide of Lactobacillus casei BL23.

Lactobacillus casei is a Gram positive lactic acid bacterium used in dairy fermentations and present in the normal human gut microbiota. Certain strains are recognized as probiotics with beneficial effects on human and animal health. L. casei BL23 is a potential probiotic strain endowed with anti-inflammatory properties and a model strain widely used in genetic, physiological and biochemical studies. A number of bacterial cell surface polysaccharides have been shown to play a role in the immune modulation activities observed for probiotic lactic acid bacteria. In the present work, we purified the most abundant carbohydrate polymer of L. casei BL23 cell wall, a neutral wall polysaccharide (WPS) and established its chemical structure by periodate oxidation, methylation analysis and 2D NMR spectroscopy. The WPS of L. casei BL23 was shown to contain α-Rha, α-Glc, ß-GlcNAc and ß-GalNAc forming a branched heptasaccharide repeating unit (variant 1) with an additional partial substitution with α-Glc (variant 2). A modified non-reducing end octasaccharide, corresponding to a terminal unit of the WPS (variant 3), was also identified and allowed to define the biological repeating unit of the WPS. To our knowledge, this is the first report of the identification of a biological repeating unit based on a chemical evidence, in a cell wall polysaccharide of a Gram positive bacterial species.

Effect of the association of maltodextrin and sucrose on the acidogenicity and adherence of cariogenic bacteria.

OBJECTIVE: The aim was to investigate the effect of maltodextrin and sucrose association on the acidogenic and adherence profiles of cariogenic bacteria. DESIGN: Streptococcus mutans (S. mutans) and Lactobacillus casei (L. casei) were cultivated in culture medium containing maltodextrin, sucrose, maltodextrin-sucrose mixture or glucose. Analyses of the acidogenicity and microbial adherence were conducted in triplicate for each microorganism and tested carbohydrate. RESULTS: For L. casei, maltodextrin, sucrose and maltodextrin-sucrose mixture showed lower acidogenic potential compared to glucose. When the microorganism was S. mutans, sucrose and maltodextrin-sucrose mixture presented higher acidogenic potential compared to maltodextrin and glucose. Microbial adherence analysis revealed higher adherence for S. mutans in presence of sucrose and maltodextrin-sucrose mixture compared to maltodextrin and glucose. For L. casei, all the carbohydrates showed similar adherence percentages. CONCLUSION: The addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria.

Change in cell surface properties of Lactobacillus casei under heat shock treatment.

We undertake this study in the aim to give new insight about the change in cellular physiological state under heat shock treatment and probiotic strain screening procedure. Different cell properties have been studied like adhesive ability to biotic and abiotic surfaces, the cell surface hydrophobicity and the fatty acids profiles. Compared to the normal cells, the heated cells increased their adhesive ability to biotic surface. However, the adhesion to abiotic surface was decreased. The cell surface hydrophobicity of the heated strains showed a significant decrease (P < 0.05). Our data revealed that high temperature change the fatty acids profiles of the treated cells, especially the proportions of unsaturated and saturated fatty acid. In fact, the ratio of saturated to unsaturated fatty acids of the heated Lactobacillus casei cells was significantly higher than that of the control cells (P < 0.05). The present finding could firstly add new insight about the response of probiotic to stressful conditions, such us the important role of cell membrane, considered as the first main structure to be damaged by physicochemical stress, in stress resistance because of their composition that can change in adaptation to harsh conditions. Secondly, there is no relationship between changes in membrane composition and fluidity induced by heat shock treatment and adhesion to biotic and abiotic surface.

Bacterial desorption in water-saturated porous media in the presence of rhamnolipid biosurfactant.

We investigated the effects of transients in elution chemistry on bacterial desorption in water-saturated porous media. Two typical Gram-positive bacterial strains of Lactobacillus casei and Streptococcus mitis were used as the model bacteria in this research. These two strains were first deposited in the porous medium, after which the medium with deposited bacteria was flushed with rhamnolipid biosurfactant solutions with a step increase in concentrations, and pulse-type bacterial releases were obtained. Bacterial desorption was quantified from bacterial breakthrough curves. It was found that bacterial retention in silica sand corresponded to bacterial interaction free energies with silica sand evaluated at the equilibrium distance, which were calculated based on independently determined bacterial, sediment and solution surface thermodynamic properties. With the increase in rhamnolipid biosurfactant concentrations, interactions between bacteria and silica sand decreased, and consequently less bacteria were retained. The decrease in interactions between bacteria and silica sand with increasing rhamnolipid biosurfactant concentrations was attributed to a decrease in the solution electron acceptor parameter of the Lewis acid/base component of surface tension, gamma3+. The increase in rhamnolipid biosurfactant concentrations favored the decrease in solution gamma3+, and consequently decreased the interactions between bacteria and silica sand.