Casei ATCC Research Articles

Two potential probiotic lactobacillus strains isolated from olive microbiota exhibit adhesion and anti-proliferative effects in cancer cell lines

The beneficial effects of two potential probiotic lactobacillus strains, namely Lactobacillus pentosus B281 and L. plantarum B282 were examined and the mechanisms of action were investigated. Quantitative analysis and confocal microscopy showed that both strains exhibited a significant higher adherence to Caco-2 cells in comparison to the reference strain L. casei ATCC 393. Treatment with conditioned media (CM) of the two strains caused significant reduction of cell proliferation, as demonstrated by Sulforhodamine B (SRB) and clonogenic assays. Moreover, the CM of the two strains induced a G1-phase arrest and down-regulation of specific cyclin genes, as indicated by flow cytometry and real-time PCR analysis. To begin elucidating the nature of the bacterial components conveying these responses, the anti-proliferative effect of heat-treated CM was analysed. The anti-proliferative activity of heat-treated CM was similar to the non-heated CM in a time- and dose-dependent manner, indicating the presence of thermostable bioactive compounds.

Survival of spray dried microencapsulated Lactobacillus casei ATCC 393 in simulated gastrointestinal conditions and fermented milk

In the present study, the survival of spray-dried microencapsulated Lactobacillus casei ATCC 393 cells was evaluated under simulated gastrointestinal conditions, as well as during production and storage of fermented milk. Under simulated gastric juice and bile salts solutions, microencapsulation provided significantly improved protection compared to free cells. Furthermore, it retained a significantly higher number of viable L. casei compared to free cell system during fermented milk production. No significant differences in the physicochemical and sensorial characteristics of produced fermented milk with spray-dried L. casei cells were observed compared to the control and commercial samples, which is an important feature for consumers' acceptability. Importantly, after all in vitro tests, the number of microencapsulated L. casei cells remained over 6 log cfu g−1, in contrast to free cells, which is the minimum required level for conferring a probiotic effect.

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 109 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

Effect of cooling rate, freeze-drying, and storage on survival of free and immobilized Lactobacillus casei ATCC 393

The optimum conditions for freeze-drying of free or immobilized Lactobacillus casei ATCC 393 on apple pieces and casein were investigated. Initially, the effect of cooling rate on cell viability of free cells was assessed. Application of 5 °C/min resulted in significant higher levels (10.8 log cfu/g) after 15 days of storage at 4 °C, compared to other rates tested. In a second step, the effect of lyoprotectants (solutions of sugars, starch, polyols, nitrogenous compounds and whey) on viable cell counts of both free and immobilized cells was evaluated. Lactose and monosodium glutamate were the most efficient agents for immobilized cells on apple pieces and casein, accounting for 9.2 log cfu/g and 9.7 log cfu/g, respectively. In all cases, concentrations above 6 log cfu/g (minimum required levels for conferring a probiotic effect) were monitored after storage at 4 °C for up to 12 months, suggesting the suitability of the technology for the production of probiotic foods.

Effect of curing salts and probiotic cultures on the evolution of flavor compounds in dry-fermented sausages during ripening

The effect of the concentration of curing salts, the nature of the probiotic culture (free or immobilized Lactobacillus casei ATCC 393 on wheat grains) and the ripening time on the generation of volatile compounds in probiotic dry-fermented sausages was investigated. Samples were collected after 1, 28 and 71days of ripening and subjected to SPME GC/MS analysis. All factors affected significantly the concentration of all volatile compounds. Noticeably, the content of esters, organic acids and total volatiles was significantly increased after 28 and 71days of ripening in almost all products. In most cases, reduction of curing salts content resulted in significant increase of esters and organic acids during ripening, whereas the opposite effect was observed in carbonyl compounds at days 28 and 71. Principal Component Analysis of the semi-quantitative data revealed that primarily the ripening process affected the volatile composition.

Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

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.

Evaluation of newly isolated probiotics in the protection against experimental intestinal trichinellosis

The potential use of probiotics in controlling enteric infections has generated tremendous interest in the last decade. The protective efficacy of seven oral doses of two newly isolated Egyptian probiotic strains; Lactobacillus acidophilus P110 (L. acidophilus) and Lactobacillus plantarum P164 (L. plantarum) versus Lactobacillus casei ATCC 7469 (L. casei) – against experimental intestinal trichinellosis – was assessed via parasitological, immunological and histopathological parameters, after verifying their in vivo safety and intestinal colonization.Parasitologically, the highest adult count reduction was observed in L. plantarum-fed infected sub-subgroup (56.98, 65.42 and 69.02%) – on the 5th, 12th and 17th days post infection (P.I.), respectively. Lesser percentage reductions were recorded in both the L. casei-fed infected sub-subgroup (36.19, 23.68 and 31.58%) and L. acidophilus-fed infected sub-subgroup (36.50, 11.8 and 7.61%) at the same intervals. On the 28th day post challenge, the highest larval count reduction was in L. plantarum-fed infected sub-subgroup (87.92%). While lower percentage yet still significant were observed in the L. casei-fed infected (74.88%) and L. acidophilus-fed infected sub-subgroups (60.98%).Immunologically, serum IFN-γ levels in the probiotic-fed non infected sub-subgroups were higher than those in the probiotic-fed infected sub-subgroups. Both showed higher levels of IFN-γ than the non probiotic-fed sub-subgroups. Histopathologically, intestinal sections of the probiotic-fed infected sub-subgroups showed amelioration of the inflammation and damage resulting from Trichinella spiralis (T. spiralis) infection. Results indicate that, through mechanical and immunological mechanisms, L. plantarum showed parasitological and histopathological protective superiority with respect to both L. casei and L. acidophilus against murineT. spiralis infection.

Antiproliferative effects of Lactobacillus casei ATCC 393 against colon carcinoma

Antiproliferative effects of Lactobacillus casei ATCC 393 against colon carcinoma

Anti-proliferative effect on a colon adenocarcinoma cell line exerted by a membrane disrupting antimicrobial peptide KL15

The antimicrobial and anticancer activities of an antimicrobial peptide (AMP) KL15 obtained through in silico modification on the sequences of 2 previously identified bacteriocins m2163 and m2386 from Lactobacillus casei ATCC 334 by us have been studied. While significant bactericidal effect on the pathogenic bacteria Listeria, Escherichia, Bacillus, Staphylococcus, Enterococcus is exerted by KL15, the AMP can also kill 2 human adenocarcinoma cells SW480 and Caco-2 with measured IC50 as 50 μg/ml or 26.3 μM. However, the IC50 determined for KL15 on killing the normal human mammary epithelial cell H184B5F5/M10 is 150 μg/ml. The conformation of KL15 dissolved in 50% 2,2,2-trifluroroethanol or in 2 large unilamellar vesicle systems determined by circular dichroism spectroscopy appears to be helical. Further, the cell membrane permeability of treated SW480 cells by KL15 appears to be significantly enhanced as studied by both flow cytometry and confocal microscopy. As observed under a scanning electron microscope, the morphology of treated SW480 cells is also significantly changed as treating time by 80 μg/ml KL15 is increased. KL15 appears to be able to pierce the cell membrane of treated SW480 cells so that numerous porous structures are generated and observable. Therefore, KL15 is likely to kill the treated SW480 cells through the necrotic pathway similar to some recently identified AMPs by others.

Effect of immobilized Lactobacillus casei on volatile compounds of heat treated probiotic dry-fermented sausages

The effect of the amount of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds during the production of heat treated probiotic dry-fermented sausages was investigated. For comparison reasons, sausages containing free L. casei cells or no starter culture as well as a similar commercial product were also included in the study. Samples ripened for 8days and heat treated to 70–72°C for 8–10min were subjected to Solid Phase Microextraction (SPME) Gas Chromatography/Mass Spectrometry (GC/MS) analysis. The starter culture affected significantly the production of volatile compounds. The highest content of esters and alcohols was observed in the sample containing 30g of immobilized cells/kg of stuffing mixture, while the highest concentration of organic acids was observed in the sausages with no starter culture. In contrast, the commercial product contained the lowest concentration of volatiles. Principal component analysis of the semi-quantitative data revealed that the volatile composition was affected primarily by the nature and concentration of the starter culture.

Antimicrobial peptide m2163 or m2386 identified from Lactobacillus casei ATCC 334 can trigger apoptosis in the human colorectal cancer cell line SW480.

Ribosomal synthesized antimicrobial peptides (AMPs) are widely distributed in nature and are toxic to certain microorganisms. Some of these AMPs are found to exhibit cytotoxic activity against the growth of cancer cells and thus have obvious anticancer potential. Here, we have studied the antiproliferation on the human colorectal cancer cell line SW480 of two AMPs, namely m2163 and m2386, identified by us from a lactic acid bacterium Lactobacillus casei ATCC 334 previously. A half maximal inhibitory concentration (IC50) of 40 μg/ml is determined first using the MTT (3-(4, 5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for either peptide m2163 or m2386. The apoptosis in treated SW480 cells by either peptide m2163 or m2386 is analyzed using flow cytometry with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide double staining. These analyses show that a substantial population of treated SW480 cells can undergo apoptosis by either peptide m2163 or m2386. The real-time quantitative polymerase chain reaction (qPCR) and Western blot analyses are subsequently used to study how the apoptosis is induced in the treated SW480 cells by either peptide m2163 or m2386. While m2163 is found to induce the expression of Fas and TRAILR1, the expression of Fas, TNFR1, and TRAILR1 death receptors on the cell surface of treated SW480 cells is found to be induced by m2386. Further, the expression of some mitochondria-related apoptosis proteins such as Smac is found to be also induced, suggesting that either peptide m2163 or m2386 can trigger both the extrinsic and intrinsic apoptosis pathways. The cell membrane permeability is greatly enhanced upon treatment with either peptide m2163 or m2386 as analyzed by the flow cytometry using both FITC-labeled peptides. The flow cytometry is also used to analyze the fluorescence intensity given by FITC-m2163 in either the mitochondria or cytoplasm fraction of the treated and fractionated SW480 cells. It is found that the detected fluorescence intensity of the mitochondria fraction is much weaker than that of the cytoplasm one, suggesting that most of the FITC-m2163 peptides are located in the cytoplasm rather than the mitochondria. This is further confirmed by a confocal microscopy study that either peptide m2163 or m2386 can localize on the cell membrane for a substantial length of time and then penetrate into the cell cytoplasm to induce the apoptosis.

Use of Pistacia terebinthus resin as immobilization support for Lactobacillus casei cells and application in selected dairy products.

Resin from Pistacia terebinthus tree was used for the immobilization of L. casei ATCC 393 cells. The encapsulated L. casei cells biocatalysts were added as adjuncts during yogurt production at 45°C and probiotic viability was assessed during storage at 4°C. For comparison reasons yogurt with free L. casei cells were prepared. The effect of encapsulated bacteria as adjuncts in yogurt on pH, lactic acid, lactose and other physicochemical parameters were studied for 60 storage days at 4°C. Samples were also tested for the microbiological and organoleptic characteristics during storage at 4°C. Encapsulation matrix seems to sustain the viability of embedded L. casei cells at levels more than 7 logcfug(-1) after 60days of storage at 4°C. Furthermore, the absence of pathogens such as Salmonella, Staphylococci, Enterobacteriaceae and coliforms in the produced yogurts is noteworthy where spoilage microorganisms such as yeasts and molds seem to affect yogurt quality only in absence of Pistacia terebinthus resin. The effect of the resin on production of aroma-related compounds responsible for yogurt flavor was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Alpha and beta- pinene were the major aroma compounds detected in produced yogurts (over 60% of total aromatic compounds detected). Yogurts with immobilized cells on P.terebintus resin had a fine aroma and taste characteristic of the resin.

Genome –Scale Reconstruction of Metabolic Networks of Lactobacillus casei ATCC 334 and 12A

Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications.

Effect of immobilized Lactobacillus casei on the evolution of flavor compounds in probiotic dry-fermented sausages during ripening

The effect of immobilized Lactobacillus casei ATCC 393 on wheat grains on the generation of volatile compounds in probiotic dry-fermented sausages during ripening was investigated. For comparison reasons, sausages containing free L. casei cells or no starter culture were also included in the study. Samples were collected after 1, 28 and 45days of ripening and subjected to SPME GC/MS analysis. Both the probiotic culture and the ripening process affected significantly the concentration of all volatile compounds. The significantly highest content of total volatiles, esters, alcohols and miscellaneous compounds was observed in sausages containing the highest amount of immobilized culture (300g/kg of stuffing mixture) ripened for 45days. Principal component analysis of the semi-quantitative data revealed that primarily the concentration of the immobilized probiotic culture affected the volatile composition.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

Free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter cultures for probiotic Feta-type cheese production

The use of free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter culture in probiotic Feta-type cheese production was evaluated. The probiotic cultures resulted in significantly higher acidity; lower pH; reduced counts of coliforms, enterobacteria, and staphylococci; and improved quality characteristics compared with cheese with no culture. Microbiological and strain-specific multiplex PCR analysis showed that both free and immobilized L. casei ATCC 393 were detected in the novel products at levels required for conferring a probiotic effect at the end of the ripening. The effect of starter culture on production of volatile compounds was investigated by the solid-phase microextraction gas chromatography-mass spectrometry analysis technique. The immobilized cells resulted in an improved profile of aroma-related compounds and the overall high quality of the novel products was ascertained by the preliminary sensory test. Finally, the high added value produced by exploitation of whey, which is an extremely polluting industrial waste, was highlighted and assessed.

Prebiotics Decrease the Antibacterial Effect of Nano Silver and Nano TiO<sub>2</sub> Particles Against Probiotic Bacteria of Food

Probiotics have been defined as “living microorganisms, which upon ingestion exert health benefits beyond inherent general nutrition”. Prebiotics are defined as short-chain carbohydrates, which are not digested by enzymes in the digestive system, which selectively stimulate the growth and activities of specific bacteria in the gut. Nano particles Ag and TiO2 have been shown to have wide applications in medicine and food industry. We investigated the antimicrobial activity of Ag and TiO2 nano particles on three species of Lactobacillus i.e. L. casei ATCC 39392, L. plantarum ATCC 8014 and L. fermentum ATCC 9338 in the presence and absence of Raffinose, Lactulose and Inulin, respectively. Result showed that nano silver particles decreased 85%, 85% and 71% of L. casei, L. plantarum and L. fermentum respectively after 48 hour and decreased percentages of L. casei, L. plantarum and L. fermentum that were 16%, 64% and 4% in the presence of the prebiotics and nano TiO2 particles decreased 59%, 85% and 61% number of L. casei, L. plantarum and L. fermentum, respectively after 48 h and decreased percentages of L. casei, L. plantarum and L. fermentum were 16% and 2% and 4% in the presence of these prebiotics.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier.

Beneficial Effects of Lactobacillus casei ATCC 334 on Halitosis Induced by Periodontopathogens

Beneficial Effects of Lactobacillus casei ATCC 334 on Halitosis Induced by Periodontopathogens

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.