Fucoidan as a marine-origin prebiotic modulates the growth and antibacterial ability of Lactobacillus rhamnosus.

Fucoidan has received much attention in healthy food and biomedicine owing to their unique (bio)physicochemical properties, particularly antibacterial and antiviral. Pathogenic microorganisms and probiotics are coexisting in many tissues (e.g., gut, oral, and vagina). However, the effect of fucoidan on probiotics has not been examined. Herein, fucoidan sterilized by different methods (i.e., 0.22 µm filter and high-temperature autoclave) is applied to explore its effect on the responses of Lactobacillus rhamnosus. It is found that high-temperature autoclave treatment causes the depolymerization of fucoidan. Further, the proliferation, morphology, and metabolism of probiotics are greatly dependent on the concentrations of fucoidan. The formation of probiotic biofilm is reduced with an increased concentration of fucoidan. Moreover, the antibacterial ability of probiotics initially increases and then decreases with an increased concentration of fucoidan. Thus, fucoidan could serve as a new marine-origin prebiotic, offering new insight into probiotic modulation and its application in inhibiting bacterial infections.

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Alginate-based double-network hydrogel improves the viability of encapsulated probiotics during simulated sequential gastrointestinal digestion: Effect of biopolymer type and concentrations.

The impact of secondary polysaccharide, i.e., low methoxyl pectin (LMP) or κ-carrageenan (KC), and its concentration (0.2, 0.4, and 0.6%) on particle size, shape, morphological, textural properties and swelling behavior of sodium alginate (ALG)- based double-network hydrogel particles, as well as the viability of encapsulated probiotics Lactobacillus rhamnosus GG (LGG) in simulated sequential gastrointestinal (GI) digestion was investigated. We found the addition of LMP impaired the sphericity of double-network hydrogel particles, while the incorporation of KC increased the particle size. The FT-IR results indicated the miscibility and cross-linking capacity of the two polysaccharides in forming double-network hydrogel particles. With respect to the swelling behavior in simulated GI digestion, all hydrogel particles shrank in simulated gastric fluid (SGF) but swelled in simulated intestinal fluid (SIF). Among the two types of double-networking, ALG-KC hydrogel particles showed noticeable shrank in SGF in conjunction with the reduced swelling in SIF, which was unfavorable for protection and the controlled release of probiotics. In the case of death rate of encapsulated LGG, the presence of LMP at a lower level (0.2 or 0.4%) exhibited protective effect against LGG death during the sequential GI digestion, while addition of KC demonstrated an opposite role.

Preparation and characterization of alginate and gelatin microcapsules containing Lactobacillus rhamnosus.

This paper describes the preparation and characterization of alginate beads coated with gelatin and containing Lactobacillus rhamnosus. Capsules were obtained by extrusion method using CaCl2 as cross linker. An experimental design was performed using alginate and gelatin concentrations as the variables investigated, while the response variable was the concentration of viable cells. Beads were characterized in terms of size, morphology, scanning electron microscopy (SEM), moisture content, Fourier Transform Infrared Spectrometry (FTIR), thermal behavior and cell viability during storage. The results showed that the highest concentration of viable cells (4.2 x 109 CFU/g) was obtained for 1 % w/v of alginate and 0.1 % w/v of gelatin. Capsules were predominantly spherical with a rough surface, a narrow size distribution ranging from 1.53 to 1.90 mm and a moisture content of 97.70 ± 0.03 %. Furthermore, FTIR and thermogravimetric analysis indicated an interaction between alginate-gelatin. Cell concentration of alginate/gelatin microcapsules was 105 CFU/g after 4 months of storage at 8 oC.

Preparation and characterization of alginate and gelatin microcapsules containing Lactobacillus rhamnosus

ABSTRACT This paper describes the preparation and characterization of alginate beads coated with gelatin and containing Lactobacillus rhamnosus. Capsules were obtained by extrusion method using CaCl2 as cross linker. An experimental design was performed using alginate and gelatin concentrations as the variables investigated, while the response variable was the concentration of viable cells. Beads were characterized in terms of size, morphology, scanning electron microscopy (SEM), moisture content, Fourier Transform Infrared Spectrometry (FTIR), thermal behavior and cell viability during storage. The results showed that the highest concentration of viable cells (4.2 x 109 CFU/g) was obtained for 1 % w/v of alginate and 0.1 % w/v of gelatin. Capsules were predominantly spherical with a rough surface, a narrow size distribution ranging from 1.53 to 1.90 mm and a moisture content of 97.70 ± 0.03 %. Furthermore, FTIR and thermogravimetric analysis indicated an interaction between alginate-gelatin. Cell concentration of alginate/gelatin microcapsules was 105 CFU/g after 4 months of storage at 8 oC.

Inorganic salts and intracellular polyphosphate inclusions play a role in the thermotolerance of the immunobiotic Lactobacillus rhamnosus CRL 1505.

In this work, the thermotolerance of Lactobacillus rhamnosus CRL1505, an immunobiotic strain, was studied as a way to improve the tolerance of the strain to industrial processes involving heat stress. The strain displayed a high intrinsic thermotolerance (55°C, 20 min); however, after 5 min at 60°C in phosphate buffer a two log units decrease in cell viability was observed. Different heat shock media were tested to improve the cell survival. Best results were obtained in the mediumcontaining inorganic salts (KH2PO4, Na2HPO4, MnSO4, and MgSO4) likely as using 10% skim milk. Flow cytometry analysis evinced 25.0% live cells and a large number of injured cells (59.7%) in the inorganic salts medium after heat stress. The morphological changes caused by temperature were visualized by transmission electronic microscopy (TEM). In addition, TEM observations revealed the presence of polyphosphate (polyP) granules in the cells under no-stress conditions. A DAPI-based fluorescence technique, adjusted to Gram-positive bacteria for the first time, was used to determine intracellular polyP levels. Results obtained suggest that the high initial polyP content in L. rhamnosus CRL 1505 together with the presence of inorganic salts in the heat shock medium improve the tolerance of the cells to heat shock. To our knowledge, this is the first report giving evidence of the relationship between polyP and inorganic salts in thermotolerance of lactic acid bacteria.

Therapeutic effect of tyndallized Lactobacillus rhamnosus IDCC 3201 on atopic dermatitis mediated by down-regulation of immunoglobulin E in NC/Nga mice.

The therapeutic effect of oral administration of Lactobacillus rhamnosus IDCC 3201 tyndallizate (RHT3201) on atopic dermatitis (AD)-like skin lesions in NC/Nga mice were investigated. After induction of dermatitis in NC/Nga mice with house-dust mite extract, each group was fed RHT3201 with 1 × 10(8) , 1 × 10(9) , or 1 × 10(10) cells orally once a day for 8 weeks. Dermatitis scores and frequency of scratching were improved by oral feeding with RHT3201. In contrast to the control group, RHT3201-fed mice showed significantly down-regulated mast cell numbers and serum immunoglobulin E (IgE) concentrations had significantly less IL4 in their axillary lymph node cells. The therapeutic effect of RHT3201 was found to be dose-dependent. These findings indicate that RHT3201 has potential for treating AD.

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus.

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42-114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal-host interactions and adaptation to this ecological niche.

Functional identification of conserved residues involved in Lactobacillus rhamnosus strain GG sortase specificity and pilus biogenesis.

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.

Adhesion and nanomechanics of pili from the probiotic Lactobacillus rhamnosus GG.

Knowledge of the mechanisms by which bacterial pili adhere to host cells and withstand external forces is critical to our understanding of their functional roles and offers exciting avenues in biomedicine for controlling the adhesion of bacterial pathogens and probiotics. While much progress has been made in the nanoscale characterization of pili from Gram-negative bacteria, the adhesive and mechanical properties of Gram-positive bacterial pili remain largely unknown. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of pili from the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG (LGG). First, we show that SpaC, the key adhesion protein of the LGG pilus, is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Homophilic and heterophilic interactions display similar binding strengths and dissociation rates. Next, pulling experiments on living bacteria demonstrate that LGG pili exhibit two unique mechanical responses, that is, zipper-like adhesion involving multiple SpaC molecules distributed along the pilus length and nanospring properties enabling pili to resist high force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stresses in the natural environment. The single-molecule experiments presented here may help us to design molecules capable of promoting or inhibiting bacterial-host interactions.

Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG.

Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.

Cell enumeration and visualisation by transmission electron microscopy of Lactobacillus rhamnosus treated with cinnamon (Cinnamomum zeylanicum B.) essential oil.

The use of essential oils (EOs) in functional foods containing probiotic microorganisms must consider the antimicrobial activity of these oils against beneficial bacteria such as Lactobacillus rhamnosus. This study aimed to evaluate the sensitivity of L. rhamnosus cultures treated with cinnamon EO through viable cell counts and visualisation by transmission electron microscopy. Cinnamon EO at a concentration of 0.04% had a bacteriostatic activity after 2 h of incubation. Although slight alterations were detected in the cell structure, this concentration was considered to be bactericidal, since it led to a significant reduction in cell numbers after 24 h. On the other hand, cinnamon EO at a 1.00% concentration decreased cell counts by 3 log units after 2 h incubation and no viable cell count was detected after 24 h. Transmission electron microscopy indicated that cells treated with 1.00% cinnamon EO were severely damaged and presented cell membrane disruption and cytoplasmic leakage.

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG).

AIMS: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. METHODS AND RESULTS: Lactobacillus rhamnosus GG was frozen (-22 or -43 degrees C), freeze-dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze-concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold-stage microscopy and scanning electron microscopy. Trehalose and lactose-trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was -43 degrees C. CONCLUSIONS: State transitions of protective media affect ice formation and cell viability in freeze-drying and storage. Formation of a maximally freeze-concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze-drying. Freeze-drying must retain a solid amorphous state of protectant matrices. Freeze-dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.

Evaluation of the response of Lactobacillus rhamnosus VTT E-97800 to sucrose-induced osmotic stress.

Environmental osmotic changes are one of the stresses live probiotics may encounter either in their natural habitats or as a result of usage in food formulations and processing. Response to osmotic stress, induced by sucrose, of the probiotic strain Lactobacillus rhamnosus VTT E-97800 (E800) was investigated. The fluorescence-based approach used, by combined staining with caboxyfluorescein (cFDA) and propidium iodide (PI) could give insights on the osmotic-induced changes of microbial esterase activity and membrane integrity; also the extrusion of intracellular accumulated carboxyfluorescein (cF) upon energizing with glucose. Comparison of the flowcytometric viability assessment with the conventional culture techniques revealed that sucrose-stressed cells had a slight loss of culturability (logN/N(0) approximately -0.3) at 1.2 and 1.5M sucrose concentration though they could perform an enzymatic conversion of cFDA into cF. The presence of such metabolically active bacteria in food might be critical as they may excrete toxic or food spoilage metabolites. Moreover, the perturbation of cF extrusion activities became a limiting factor for reproductive capacities. There was no change in the cell morphology. These results proved the ability of the strain of study to tolerate sucrose, even at extreme concentrations and these must be taken into consideration for its usage in the formulation/processing of sugar-based foods, e.g. jams, candies, etc.

Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria.

The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.

Microentrapment of probiotic bacteria in a Ca(2+)-induced whey protein gel and effects on their viability in a dynamic gastro-intestinal model.

Entrapping probiotic bacteria in gels with ionic cross-linking is typically achieved with polysaccharides (alginate, pectin, carraghenan). In this study, whey proteins were used for this purpose by carrying out the Ca(2+)-induced gelation of pre-heated whey protein isolate (WPI). A Lactobacillus rhamnosus cell suspension was added in a denatured WPI solution in a 30 : 70 volume ratio. Gelation was carried out by extrusion of the cell suspension in a CaCl(2) solution. Beads of approximately 3 mm diameter were formed. The population in the beads was 8.0 x 10(8) cells g(-1). Entrapment efficiency in gel beads was 96%, with a survival level of 23%. Scanning electron microscopy of beads before freeze-drying showed a tight protein network containing encapsulated Lb. rhamnosus cells homogeneously distributed throughout the matrix. The survival to freeze-drying of the bead-entrapped cells was 41%. Viability of microentrapped cells in a dynamic gastro-intestinal (GI) model was studied and the results were compared to free cells freeze-dried in a milk-based cryoprotective solution, as well as in a pre-denatured WPI solution. Results showed that protein gelation provided protection against acidic conditions in the stomach after 90 min, as well as against bile after 30, 60 and 90 min in the duodenum. Moreover, the milk-based cryoprotective solution was equally effective after 90 min in the duodenum. It is concluded that the gelation of whey proteins induced by Ca(2+) ions can protect the cells against adverse conditions of the GI system. However, certain stages in the entrapment process, particularly extrusion in the solution of CaCl(2), still need to be optimized in order to reduce the mortality of the cells during gelation.