Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081.

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70-80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 109 to 1010 CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.

Protective effects of tyndallized Lactobacillus acidophilus IDCC 3302 against UVB­induced photodamage to epidermal keratinocytes cells.

Photoaging is a consequence of chronic exposure to ultraviolet (UV) radiation and results in skin damage. In this study, whether tyndallizate of the probiotic bacterium Lactobacillus acidophilus IDCC 3302 (ACT 3302) can protect against UVB­induced photodamage to the skin was investigated. For this, HaCaT keratinocytes were used as a model for skin photoaging. HaCaT cells were treated with ACT 3302 prior to UVB exposure and skin hydration factors and matrix metalloproteinase (MMP)­1, MMP­2, and MMP­9 levels in the culture supernatant were evaluated by ELISA. The protective effects of ACT 3302 against UVB­induced oxidative stress in HaCaT cells was also assessed by measuring superoxide dismutase and catalase activity and detecting the expression of pro­inflammatory cytokine­encoding genes and mitogen­activated protein kinase (MAPK) signaling components by reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. UVB exposure increased MMP expression and MAPK activation; these changes were attenuated by pretreatment with ACT 3302. Treatment with ACT 3302 prior to UVB exposure also attenuated inflammation. These results demonstrate that tyndallized ACT 3302 can mitigate photodamage to the skin induced by UVB radiation through the suppression of MMPs and could therefore be used clinically to prevent wrinkle formation.

Effects of polymerised whey protein-based microencapsulation on survivability of Lactobacillus acidophilus LA-5 and physiochemical properties of yoghurt.

Lactobacillus acidophilus LA-5 has poor survivability in yoghurts. This study aims to microencapsulate L. acidophilus LA-5 using polymerised whey protein as wall material and evaluate its survivability and effects on physiochemical properties of yoghurts. The microencapsulation yield was 92.90% and average beads size was 744 µm. Microencapsulated L. acidophilus LA-5 showed higher survival at simulated gastrointestinal conditions. Microencapsulated L. acidophilus LA-5 had improved survivability (∼106 cfu/mL) than free cells (∼104 cfu/mL) after digesting by simulated intestinal juice (6 h) in yoghurts during 10-week storage. Microencapsulated L. acidophilus LA-5 improved the firmness and protein content and decreased the spontaneous whey separation and synaeresis of yoghurts significantly (p < 0.01). Changes in pH and free microorganism population of yoghurts during storage were not affected by adding microencapsulated L. acidophilus LA-5. Microencapsulation of L. acidophilus LA-5 using polymerised whey protein as wall material improved its survivability and the physiochemical properties of the yoghurt.

Soy extract and maltodextrin as microencapsulating agents for Lactobacillus acidophilus: a model approach.

The present study aimed to optimise the microencapsulation of Lactobacillus acidophilus La-05 by spray drying, using soy extract and maltodextrin as encapsulants. Air inlet temperature, maltodextrin/soy extract ratio and feed flow rate were investigated through Central Composite Rotational Design (CCRD). Probiotic viability increased with increasing the proportion of soy extract. Temperature and feed flow rate had a negative effect. Particle diameter ranged from 4.97 to 8.82 µm, water activity from 0.25 to 0.52 and moisture from 2.30 to 7.01 g.100g-1 Particles produced following the optimised conditions (air temperature of 87 °C, maltodextrin/soy extract ratio of 2:3 w.w-1, feed flow rate of 0.54 L.h-1) reached Encapsulation yield (EY) of 83%. Thermogravimetry and FTIR analysis suggested that microcapsules could protect L. acidophilus cells against dehydration and heating. During storage, microencapsulated probiotic had high cell viability (reductions ranged between 0.12 and 1.72 log cycles). Soy extract/maltodextrin presented well-encapsulating properties of Lactobacillus acidophilus La-05.

Efficient synthesis and activity of beneficial intestinal flora of two lactulose-derived oligosaccharides.

Lactulose is considered as a prebiotic because it promotes the intestinal proliferation of Lactobacillus acidophilus which is added to various milk products. Moreover, lactulose is used in pharmaceuticals as a gentle laxative and to treat hyperammonemia. This study was aimed at the total synthesis of two Lactulose-derived oligosaccharides: one is 3-O-ß-d-galactopyranosyl-d-fructose, d-fructose and ß-d-galactose bounded together with ß-1,3-glycosidic bound, the other is 1-O-ß-d-galactopyranosyl-d-fructose, d-fructose and ß-d-galactose bounded together with ß-1,1-glycosidic bound, which were accomplished in seven steps from d-fructose and ß-d-galactose and every step of yield above 75%. This synthetic route provided a practical and effective synthetic strategy for galactooligosaccharides, starting from commercially available monosaccharides. Then we evaluated on their prebiotic properties in the search for potential agents of regulating and improving the intestinal flora of human. The result showed that the prebiotic properties of Lactulose-derived oligosaccharides was much better than Lactulose. Among them, 3-O-ß-d-galactopyranosyl-d-fructose displayed the most potent activity of proliferation of L. acidophilus.

Lactobacillus acidophilus-Rutin Interplay Investigated by Proteomics.

Dietary polyphenols are bioactive molecules that beneficially affect human health, due to their anti-oxidant, anti-inflammatory, cardio-protective and chemopreventive properties. They are absorbed in a very low percentage in the small intestine and reach intact the colon, where they are metabolized by the gut microbiota. Although it is well documented a key role of microbial metabolism in the absorption of polyphenols and modulation of their biological activity, molecular mechanisms at the basis of the bacteria-polyphenols interplay are still poorly understood. In this context, differential proteomics was applied to reveal adaptive response mechanisms that enabled a potential probiotic Lactobacillus acidophilus strain to survive in the presence of the dietary polyphenol rutin. The response to rutin mainly modulated the expression level of proteins involved in general stress response mechanisms and, in particular, induced the activation of protein quality control systems, and affected carbohydrate and amino acid metabolism, protein synthesis and cell wall integrity. Moreover, rutin triggered the expression of proteins involved in oxidation-reduction processes.This study provides a first general view of the impact of dietary polyphenols on metabolic and biological processes of L. acidophilus.

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.

Improved viability of Lactobacillus acidophilus NRRL-B 4495 during freeze-drying in whey protein-pullulan microcapsules.

In this research, pullulan was incorporated in protein-based encapsulation matrix in order to assess its cryoprotective effect on the viability of freeze-dried (FD) probiotic Lactobacillus acidophilus NRRL-B 4495. This study demonstrated that pullulan in encapsulation matrix resulted in a 90.4% survival rate as compared to 88.1% for whey protein (WPI) encapsulated cells. The protective effects of pullulan on the survival of FD-encapsulated cells in gastrointestinal conditions were compared. FD WPI-pullulan capsules retained higher survived cell numbers (7.10 log CFU/g) than those of FD WPI capsules (6.03 log CFU/g) after simulated gastric juice exposure. Additionally, use of pullulan resulted in an increased viability after bile exposure. FD-free bacteria exhibited 2.18 log CFU/g reduction, while FD WPI and FD WPI-pullulan encapsulated bacteria showed 0.95 and 0.49 log CFU/g reduction after 24 h exposure to bile solution, respectively. Morphology of the FD microcapsules was visualized by scanning electron microscopy.

Control of cell morphology of probiotic Lactobacillus acidophilus for enhanced cell stability during industrial processing.

The viability of bacteria during industrial processing is an essential quality criterion for bacterial preparations, such as probiotics and starter cultures. Therefore, producing stable microbial cultures during proliferation is of great interest. A strong correlation between the culture medium and cellular morphology was observed for the lactic acid bacterium Lactobacillus acidophilus NCFM, which is commonly used in the dairy industry as a probiotic supplement and as a starter culture. The cell shapes ranged from single short rods to long filamentous rods. The culture medium composition could control this phenomenon of pleomorphism, especially the use of peptone in combination with an adequate heating of the medium during preparation. Furthermore, we observed a correlation between the cell size and stability of the microorganisms during industrial processing steps, such as freeze-drying, extrusion encapsulation and storage following dried preparations. The results revealed that short cells are more stable than long cells during each of the industrially relevant processing steps. As demonstrated for L. acidophilus NCFM, the adaptation of the medium composition and optimized medium preparation offer the possibility to increase the concentration of viable cells during up- and survival rate during down-stream processing.

Interaction evaluation of bacteria and protoplasts with single-stranded deoxyribonucleic acid library based on capillary electrophoresis.

For whole-cell aptamers selection, cells surface situation has great impact on single-stranded (ssDNA) binding and aptamers selection. In this work, both Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were as cells targets, their interaction with ssDNA library were evaluated based on capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) with UV and LIF detection. Our results demonstrated that protoplasts without cells wall had apparently stronger interaction with ssDNA library than bacteria, the protoplasts-ssDNA complex could be observed clearly with CZE-LIF. Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol, formaldehyde and glutaraldehyde) showed binding difference with ssDNA library, which could be identified with ACE-UV. Binding constants indicated the interaction of E. coli with ssDNA library were in the order of E. coli protoplasts>methanol (ethanol) treated E. coli>formaldehyde (glutaraldehyde) treated E. coli≈E. coli. Above results suggest that cells surface situation determines their binding affinity with ssDNA, which should be considered in whole-cell aptamers selection and aptamers further application. Capillary electrophoresis is a preferable technique for interaction evaluation of composite targets binding with ssDNA library.

Effects of salt concentration and pH on structural and functional properties of Lactobacillus acidophilus: FT-IR spectroscopic analysis.

The effects of sodium chloride concentration and varying pH levels on the structural and functional properties of Lactobacillus acidophilus were investigated. Reconstituted skim milk was inoculated with Lb. acidophilus at varying salt concentrations (0, 1, 2, 5 and 10% NaCl) and pH levels (4.0, 5.0 and 6.0) and ACE-inhibitory activity and proteolytic activity were determined and the viable cell count was enumerated after 24h of fermentation at 37 °C. The degree of proteolysis exhibited an increase with higher salt concentration at pH 5.0 and 6.0. ACE-inhibitory activity was found to be the highest at pH 5.0 at all salt concentrations. Fourier transform infrared spectroscopy results demonstrated significant changes occurring beyond 2% NaCl particularly at low pH (4.0). The findings revealed that significant changes occurred in amide I and amide III regions when Lb. acidophilus was subjected to varying salt concentrations.

Transcriptional response of Lactobacillus acidophilus L-92 after attachment to epithelial Caco-2 cells.

Transcriptome analysis showed that Lactobacillus acidophilus L-92 cells having anti-allergy effects on human up-regulated 41 genes involved permease, ABC transporter, proteinase and transcriptional regulator after attached to epithelial Caco-2 cells. Inversely, 37 genes were down-regulated, including ATP synthases, ABC transporters and transcriptional regulators.

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758.

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.

Effects of gram-positive microorganisms and their products on in vivo survival of hemopoietic clonogenic cells.

The effects of gram-positive bacterial strains (Lactobacillus acidophilus and Lactobacillus rhamnosus) and their subcellular components on the survival of hemopoietic clonogenic cells were evaluated by the formation of endogenous splenic colonies. The effects of these preparations on NO production were studied by the spin-trap paramagnetic resonance spectroscopy. Bacterial preparations from gram-positive bacteria stimulated survival of hemopoietic clonogenic cells, but did not induce NO production in contrast to E. coli LPS.

Selection of aptamers against live bacterial cells.

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.

The role of oxygen in the viability of probiotic bacteria with reference to L. acidophilus and Bifidobacterium spp.

The various therapeutic benefits of Lactobacillus acidophilus and Bifidobacterium spp. have resulted in their increased incorporation into dairy foods such as yoghurts. Currently however, the efficacy of these probiotic bacteria is limited by their poor survival during the shelf life of yoghurt. Oxygen toxicity is widely considered to be responsible for the cell deaths of these bacteria. The intestinal origins and the microaerophilic and anaerobic characteristics of L. acidophilus and Bifidobacterium spp. respectively, can render them susceptible to oxygen contained in the food products. This review discusses the influence of the dissolved oxygen in yogurt on the viability of these bacteria. Suggested techniques to protect these probiotic bacteria from oxygen toxicity are evaluated. Although the problem of oxygen toxicity in probiotic bacteria is regarded as significant, little is known however about the cellular interaction of these bacteria with oxygen. This review summarizes what is known about the biochemistry of oxygen toxicity in these bacteria. The various metabolic and biochemical responses of L. acidophilus and Bifidobacterium to oxygen are examined. Additionally, the importance of NADH oxidase and NADH peroxidase in the oxygen tolerance of these bacteria is evaluated and assays used to measure their cellular concentrations are discussed.

Determination of cell viability in single or mixed samples using capillary electrophoresis laser-induced fluorescence microfluidic systems.

The advent of high-efficiency microbial separations will have a profound effect on both chemistry and microbiology. For the first time, it appears that it may be possible to obtain qualitative and quantitative information on microbial systems with the accuracy, precision, speed, and throughput that currently is found for chemical systems. Recently it was suggested that an analytical separations-based approach for determining the viability of cells would be advantageous. The feasibility of such an approach is demonstrated using CE-LIF of two bacteria and yeast. The analytical procedures and figures of merit are outlined. High-throughput analyses and evaluation of microorganisms now appear to be possible.

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli.

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding.

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.

Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.

The present paper provides an overview on the use of probiotic organisms as live supplements, with particular emphasis on Lactobacillus acidophilus and Bifidobacterium spp. The therapeutic potential of these bacteria in fermented dairy products is dependent on their survival during manufacture and storage. Probiotic bacteria are increasingly used in food and pharmaceutical applications to balance disturbed intestinal microflora and related dysfunction of the human gastrointestinal tract. Lactobacillus acidophilus and Bifidobacterium spp. have been reported to be beneficial probiotic organisms that provide excellent therapeutic benefits. The biological activity of probiotic bacteria is due in part to their ability to attach to enterocytes. This inhibits the binding of enteric pathogens by a process of competitive exclusion. Attachment of probiotic bacteria to cell surface receptors of enterocytes also initiates signalling events that result in the synthesis of cytokines. Probiotic bacteria also exert an influence on commensal micro-organisms by the production of lactic acid and bacteriocins. These substances inhibit growth of pathogens and also alter the ecological balance of enteric commensals. Production of butyric acid by some probiotic bacteria affects the turnover of enterocytes and neutralizes the activity of dietary carcinogens, such as nitrosamines, that are generated by the metabolic activity of commensal bacteria in subjects consuming a high-protein diet. Therefore, inclusion of probiotic bacteria in fermented dairy products enhances their value as better therapeutic functional foods. However, insufficient viability and survival of these bacteria remain a problem in commercial food products. By selecting better functional probiotic strains and adopting improved methods to enhance survival, including the use of appropriate prebiotics and the optimal combination of probiotics and prebiotics (synbiotics), an increased delivery of viable bacteria in fermented products to the consumers can be achieved.