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Previous research has demonstrated promising outcomes regarding the advantageous impact of probiotics in both cancer prevention and treatment. Nevertheless, the precise molecular mechanisms underpinning these effects remain elusive. Recent investigations have proposed a potential involvement of micro ribonucleic acids (miRNAs) in mediating the favorable influence of probiotics on cancerous cells. This study was designed to evaluate the effect of Lactobacillus casei condition medium on miR-21 relative expression in HT-29 colorectal cancer cells. Lactobacillus casei condition medium mixed with RPMI in different proportions (1:1, 1:3, and 1:7) and utilized to treat HT-29 cells for 24 and 48 h. Subsequently, percentage of early and late apoptotic cells were identified using a flow cytometry instrument. A real-time polymerase chain reaction was carried out to determine the relative expression of miR-21. Our findings revealed that L. casei condition medium induces apoptosis in a time- and dose-dependent manner in HT-29 cells. Furthermore, we found a significantly downregulated miR-21 after treatment with high doses of L. casei condition medium after 48 h. Overall, our results provide valuable insights into a potential mechanism through which L. casei condition medium mediates its apoptotic effect in colorectal cancer cells through downregulation of miR-21. However, further investigations are required to unravel its therapeutic, diagnostic, and treatment monitoring potential. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate the probiotic and safety properties of lactic acid bacterial (LAB) strains isolated from the gut microbiota of honey bee Apis mellifera L. since this source remains a promising reservoir of microbial diversity. A total of five bacterial isolates were molecularly identified using 16S rRNA gene sequencing as Enterococcus faecalis -HBE1, Lactobacillus brevis -HBE2, Enterococcus faecalis -HBE3, Enterococcus faecalis -HBE4 and Lactobacillus casei -HBE5. Gut tolerance conditions (low pH and bile salt) were evaluated. Exopolysaccharides (EPS) production, hemolytic, antioxidant activity, resistance toward antibiotics and technological characteristics (starter activity, pH and proteolysis) were examined. The five isolates showed a high survival rate (>95%), under gastrointestinal tract conditions indicating excellent potential for application as probiotics. The isolates showed no hemolytic activities and good acidification rates in the range of pH 4.6–4.98 after incubation at 37°C for 24h. The isolates exhibited promising proteolytic activity as well as DPPH radical scavenging activity in the range of 16.52–59.39%. All the tested isolates had the capability to produce exopolysaccharides except Lactobacillus casei -HBE5. These results put forward that lactic acid bacterial strain isolated from honey bee workers can be considered as promising candidates for future applications as starter cultures and could constitute new potential probiotics for the production of functional dietary products promoting health benefits. [ABSTRACT FROM AUTHOR]

The viability of probiotics is affected by several factors during manufacturing, storage and gastrointestinal tract passage. Protecting the probiotics from harmful conditions is particularly critical for oxygen sensitive species like Akkermansia muciniphila, a bacterium which recently has been proposed as a next-generation probiotic candidate. Previously, we have developed a protocol for microencapsulating A. muciniphila in a xanthan/gellan gum matrix. Here, we report the enhanced survival during storage and in vitro gastric passage of microencapsulated A. muciniphila embedded in dark chocolate. Lactobacillus casei, as a representative species of traditional probiotics, was included in order to compare its behavior with that of A. muciniphila. For A. muciniphila we observed a 0.63 and 0.87 log CFU g−1 reduction during 60 days storage at 4°C or 15°C, respectively. The viability of L. casei remained stable during the same period. During simulated gastric transit (pH 3), microencapsulated A. muciniphila embedded in chocolate showed 1.80 log CFU mL−1 better survival than naked cells, while for L. casei survival was improved with 0.8 log CFU mL−1. In a hedonic sensory test, dark chocolate containing microcapsules were not significantly different from two commercially available chocolates. The developed protocol constitutes a promising approach for A. muciniphila dosage. [ABSTRACT FROM AUTHOR]

The ability to serve as a delivery vehicle for various interesting biomolecules makes lactic acid bacteria (LAB) very useful in several applications. In the medical field, recombinant LAB expressing pathogenic antigens at different cellular locations have been used to elicit both mucosal and systemic immune responses. Expression-secretion vectors (ESVs) with a signal peptide (SP) are pivotal for protein expression and secretion. In this study, the genome sequence of Lactobacillus casei ATCC334 was explored for new SPs using bioinformatics tools. Three new SPs of the proteins Cwh, SurA and SP6565 were identified and used to construct an ESV based on our Escherichia coli-L. casei shuttle vector, pRCEID-LC13.9. Functional testing of these constructs with the green fluorescence protein (GFP) gene showed that they could secrete the GFP. The construct with CwhSP showed the highest GFP secretion. Consequently, CwhSP was selected to develop an ESV construct carrying a synthetic gene encoding the extracellular domain of the matrix 2 protein fused with the hepatitis B core antigen (M2e:HBc). This ESV was shown to efficiently express and secrete the M2e:HBc fusion protein. The identified SPs and the developed ESVs can be exploited for expression and secretion of homologous and heterologous proteins in L. casei. [ABSTRACT FROM AUTHOR]

We previously reported that the β-1,4-Mannanase (manB) gene fromBacillus pumilus functions as a good reporter gene in Lactobacillus casei. Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SPUsp45) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SPSlpA), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SPUsp45 is functionally recognized and processed by the L. casei secretion machinery. The SPUsp45-mediated secretion efficiency was ∼87%, and SPSlpA drove the export of secreted ManB with ∼80% efficiency. SPSlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA. [ABSTRACT FROM AUTHOR]

In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We investigated whether protocols allowing high efficiency electrotransformation of other lactic acid bacteria were applicable to five strains of Lactobacillus casei (12A, 32G, A2-362, ATCC 334 and BL23). Addition of 1% glycine or 0.9 M NaCl during cell growth, limitation of the growth of the cell cultures to OD600 0.6-0.8, pre-electroporation treatment of cells with water or with a lithium acetate (100 mM)/dithiothreitol (10 mM) solution and optimization of electroporation conditions all improved transformation efficiencies. However, the five strains varied in their responses to these treatments. Transformation efficiencies of 106 colony forming units μg−1 pTRKH2 DNA and higher were obtained with three strains which is sufficient for construction of chromosomal gene knock-outs and gene replacements. [ABSTRACT FROM AUTHOR]

Concentrations of Na+, K+ and Ca2+ in the growth medium were varied within limits normally found in vivo to determine how cation concentrations affect the sensitivity of ruminal bacteria to the ionophores, monensin (a Na+/ H+ and K+/ H+ exchanger) and tetronasin ( Ca2+/ H+). High [ Na+] (172 mM cf. 137 mM in control medium) enhanced the efficacy of monensin towards Eubacterium ruminantium 2388, Streptococcus bovis C277, Lactobacillus casei LB17 and Prevotella albensis M384. High [ K+] (35 mM cf. 19 mM) alone caused a decreased potency of both ionophores, except with L. casei. Added Ca2+ (7.4 cf. 2.8 mM) increased the potency of tetronasin when [ Na+] was low. High [ Na+] alone also potentiated the efficacy of tetronasin. Monensin caused intracellular [ Na+] and [ K+] to be decreased in the most sensitive of these organisms, E. ruminantium, whereas only intracellular [Ca2+] fell with tetronasin. The changes were small; however, Δ p fell by only 20 mV after 2 h when ionophores caused immediate cessation of growth. ATP concentrations fell by 77% and 75% with monensin and tetronasin, respectively. Thus, altering cation concentrations might be used to potentiate the efficacy of ionophores, by increasing the rate of energy expenditure to maintain ionic homoeostasis in sensitive bacteria. [ABSTRACT FROM AUTHOR]

To study the mechanism of action of the lactobacilli, splenocytes were incubated with lactobacilli. We compared the ability of live and lyophilized Lactobacillus casei, Lactobacillus rhamnosus GG and Lactobacillus delbrueckii ssp. bulgaricus to modulate the production of interleukin 12p40 (IL-12p40), tumor necrosis factor α and IL-10 by splenocytes from C57BL/6 and BALB/c mice. Blocking contact between lactobacilli and immune cells abrogated all cytokine production. Toll-like receptor 2 (TLR2) was partially responsible, but not TLR4 or TLR9, for the induction of cytokine production in splenocytes. All cytokine production declined to basal levels when bacterial phagocytosis was inhibited. This shows that lactobacilli stimulation of cytokine production in splenocytes requires the process of phagocytosis and engagement of TLR2, but not TLR4 or TLR9. [ABSTRACT FROM AUTHOR]

The trans-sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans-sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans-sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei, which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli. The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds. [ABSTRACT FROM AUTHOR]

Twenty-seven Lactobacillus plantarum ssp. plantarum, 11 Lactobacillus paraplantarum and five Lactobacillus casei-related strains, isolated from various autochthonous Serbian and Montenegro-fermented foods, were identified using phenotypical characterization and current PCR methods based on PCR of the recA gene or the 23S–5S rRNA gene intragenic spacer (IS) region. The strains were genotypically characterized by a new method based on the insertion sequence element IS Lpl11 that grouped these lactobacilli into 10 IS-fingerprinting groups. Between six and 23 copies of the IS Lpl1 were found in each strain and the IS Lpl1-fingerprint groups correlated well with the origin of the strains. The method proved suitable for strain typing of lactic acid bacteria at the infraspecies level. [ABSTRACT FROM AUTHOR]

One csp-like gene, called cspA, from the lactic acid bacterium Lactobacillus casei was identified by an inverse polymerase chain reaction approach based on degenerate primers. cspA encodes a protein of 66 amino acid residues, which displays at least 74% identity with Csp proteins of Lactobacillus genera. Northern blot analysis revealed that cspA is transcribed monocistronically and that its expression is induced after a temperature downshift from 37°C to 20°C. The transcriptional start site has been determined and is situated 98 bp upstream of the initiation codon. A cspA mutant strain was constructed and it showed reduced growth rate compared with the wild type at both optimal and low temperatures, demonstrating that CspA plays an important role in the physiology of L. casei. [ABSTRACT FROM AUTHOR]

Abstract: Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. 13C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD+ regeneration. [Copyright &y& Elsevier]

Abstract: A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-ß-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361¿4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25°C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains. [Copyright &y& Elsevier]

A food-grade strain with nisRK stably integrated into the genome, was constructed in order to implement the nisin-controlled expression system (NICE) in Lactobacillus casei ATCC393. Expression of β-glucuronidase (gus) reporter gene was employed to optimize the system, which has been successfully used to produce the main antigenic protein from Norwalk virus, opening new perspectives for producing edible vaccines. [Copyright &y& Elsevier]

A number of recent research works in lactic acid bacteria aim towards the design of new strains that could be used as live vectors for the delivery of antigens for oral vaccination, or other therapeutic molecules. In this work, an inducible expression system based on the Lactobacillus casei lactose operon promoter was used to express three important surface antigens of Streptococcus pneumoniae in this lactic acid bacterium: a virulence-related pneumococcal surface antigen (PsaA) and two variants of the virulence factor PspA (pneumococcal surface protein A). Expression of the three proteins was induced upon growth on lactose and strongly repressed by glucose. These proteins were produced intracellularly. Also, secretion to the growth medium was achieved by means of a fusion to the secreting and processing signals from the L. casei surface proteinase. Interestingly, while secreted PspA proteins were found in the culture supernatants, PsaA remained trapped in the cell wall. Expression of pneumococcal antigens in a food-grade organism opens an alternative for mucosal vaccination against this important pathogen. [Copyright &y& Elsevier]

The use of ram horn hydrolysate (RHH) as a substrate for lactic acid production was investigated using Lactobacillus casei. For this purpose, first RHH was produced. Ram horns were hydrolyzed by treating with acids (6N-H2SO4 and 6N-HCl) and neutralizing the solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHH were determined. The effect of different concentrations (1–10% v/v) of RHH on the production of biomass, lactic acid concentration and sugar consumption was investigated, and a concentration of 6% RHH was found to be optimal. The content of lactic acid in the culture broth containing 6% RHH (44 g l−1) for 26 h was 22% higher than that of the control culture broth (36 g l−1). From this result, RHH was demonstrated to be a suitable supplement for lactic acid production, a use that would resolve a local environmental problem. [Copyright &y& Elsevier]

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFPUV) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFPUV expression, followed by aeration and a temperature of 30°C. Environmental factors favoring GFPUV accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 μg of GFP per mg of total protein, from which 55% was fluorescent. [Copyright &y& Elsevier]

The viability of probiotics is affected by several factors during manufacturing, storage and gastrointestinal tract passage. Protecting the probiotics from harmful conditions is particularly critical for oxygen sensitive species like Akkermansia muciniphila, a bacterium which recently has been proposed as a next-generation probiotic candidate. Previously, we have developed a protocol for microencapsulating A. muciniphila in a xanthan/gellan gum matrix. Here, we report the enhanced survival during storage and in vitro gastric passage of microencapsulated A. muciniphila embedded in dark chocolate. Lactobacillus casei, as a representative species of traditional probiotics, was included in order to compare its behavior with that of A. muciniphila. For A. muciniphila we observed a 0.63 and 0.87 log CFU g−1 reduction during 60 days storage at 4°C or 15°C, respectively. The viability of L. casei remained stable during the same period. During simulated gastric transit (pH 3), microencapsulated A. muciniphila embedded in chocolate showed 1.80 log CFU mL−1 better survival than naked cells, while for L. casei survival was improved with 0.8 log CFU mL−1. In a hedonic sensory test, dark chocolate containing microcapsules were not significantly different from two commercially available chocolates. The developed protocol constitutes a promising approach for A. muciniphila dosage. [ABSTRACT FROM AUTHOR]