Aim: The efficacy of probiotic Lactobacillus casei (Shirota strain) in reducing incidence of ventilator-associated pneumonia (VAP) in medical patients who received mechanical ventilation at Siriraj Hospital was evaluated.

Method: A prospective, randomized, open-label controlled trial was conducted on 150 adult hospitalized patients in medical wards who were on mechanical ventilation for 72 hours or longer: The patients were randomized to the probiotic group or the control group. The patients in the probiotic group received 80 ml of a probiotic fermented milk drink containing 8 billion Lactobacillus casei (Shirota strain) for oral care after having standard oral care once daily and additional 80 ml of the aforementioned fermented dairy product was given via enteral feeding once daily. The primary outcomes were incidence of VAP and incidence rate of VAP episodes per 1,000 ventilator-days. The secondary outcomes were length of hospital stay, mortality at day 28 and 90, incidence of diarrhea, and presence of resistant bacteria in oropharyngeal and rectal swab samples taken from the patients at baseline, day 7 and day 28 after enrollment.

Results: The baseline characteristics of the patients in the probiotic group (75) and the control group (75) were not significantly different. The patients in the probiotic group were less likely to develop VAP compared with the control group (24% vs. 29.3%, p = 0.46). The incidence rates of VAP in the probiotic and control groups were 22.64 and 30.22 episodes per 1,000 ventilator-days, respectively (p = 0.37). A trend of lower prevalence of resistant bacteria cultured from oropharyngeal swabs in the probiotic group than that in the control group was observed. Overall 28 and 90-day mortality and length of hospital stay of the patients in both groups was not significantly different.

Conclusions: Administration of probiotic containing Lactobacillus casei (Shirota strain) resulted in a tendency to reduce the incidence of VAP and colonization with resistant bacteria in oropharyngeal cavity without significant effects on mortality and length of hospital stay.

PURPOSE:There is growing evidence that probiotics confer health benefits to the host by modulating immune function, especially in older people, where immunosenescence is a feature even of healthy ageing. The aim of this study was to investigate the effect of a probiotic drink containing Lactobacillus casei Shirota (LcS) on immune function in a healthy non-immunocompromised older population.

METHODS: Thirty healthy old volunteers were recruited into a randomized placebo-controlled, single-blind crossover study. The volunteers were supplemented with the probiotic drink containing 1.3 × 10(10) CFU (LcS) or skimmed milk per day for 4 weeks, followed by 4 weeks of washout and were crossed over to the other treatment. Peripheral blood and saliva samples were collected at baseline and end of each treatment.

RESULTS: Probiotic consumption was associated with a significant increase in natural killer (NK) cell activity relative to baseline and a significant decrease in the mean fluorescence intensity of CD25 expression in the resting T cells compared with placebo. Additionally, there was a trend towards an increased ratio of IL-10 to IL-12 relative to baseline after (LcS) intake.

CONCLUSIONS: Consumption of a probiotic drink containing (LcS) improved NK cell activity and tended to produce a more anti-inflammatory cytokine profile in an older population.
Probiotics have already proven beneficial effects in the treatment of several intestinal infections, but the underlying mechanisms how the probiotics can affect responses of porcine IPEC-J2 enterocytes to oxidative stress remained to be elucidated. The immunomodulatory effect of five bacterial strains (Lactobacillus plantarum 2142, Lactobacillus casei Shirota, Bifidobacterium animalis subsp. lactis BB-12, Bacillus amyloliquefaciens CECT 5940 and Enterococcus faecium CECT 4515) on 1 mM peroxide-triggered upregulation of interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) level was screened by q RT-PCR. Our data revealed that spent culture supernatant (SCS) of L. plantarum 2142 had significant lowering effect on IL-8 and TNF-α level with concomitant promoting activity on protective Hsp70 gene expression. According to our results, lactic acid (racemic, D: - and L: -lactic acid) and acetic acid produced by lactobacilli had no protective effect in quenching upregulation of proinflammatory cytokines. Furthermore, L. plantarum 2142-specific supernatant peptides were detected by gel electrophoresis and capillary zone electrophoresis.
CONTEXT:It is of great importance to evaluate the safety of probiotics in dysregulated immune conditions, as probiotics can possibly modulate immune functions in the host.

OBJECTIVE:We tried to confirm the safety of using Lactobacillus casei strain Shirota (LcS) to help prevent autoimmunity in the central nervous system.

METHODS: We used two chronic experimental autoimmune encephalomyelitis (EAE) models, a relapse and remission type EAE model in SJL/J mice and a durable type model in C57BL/6 mice. (LcS) was administered from 1 week before antigen sensitization until the end of the experiments, and neurological symptoms and histopathological changes of the spinal cord were observed. Immunological parameters were also examined in the SJL/J mouse model.

RESULTS:(LcS) administration did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model but instead tended to improve neurological symptoms in the SJL/J mouse EAE model. (LcS) administration transiently upregulated IL-17 production by antigen-stimulated lymphocytes of draining lymph nodes 7 days after sensitization. Enhanced production of IL-10 and an increase in the percentage of CD4(+)CD25(+) T regulatory cells were also observed at the same sites. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by (LcS) administration.

CONCLUSION:These results demonstrate that (LcS) does not exacerbate, but instead may improve EAE depending on the immunization conditions, and that IL-17 responses at peripheral sites may not always result in a worsening of autoimmune diseases.
There is considerable interest in the strain specificity of immune modulation by probiotics. The present study compared the immunomodulatory properties of six probiotic strains of different species and two genera in a human peripheral blood mononuclear cell (PBMC) model in vitro. Live cells of lactobacilli ( Lactobacillus casei Shirota, L. rhamnosus GG, L. plantarum NCIMB 8826 and L. reuteri NCIMB 11951) and bifidobacteria (Bifidobacterium longum SP 07/3 and B. bifidum MF 20/5) were individually incubated with PBMC from seven healthy subjects for 24 h. Probiotic strains increased the proportion of CD69+ on lymphocytes, T cells, T cell subsets and natural killer (NK) cells, and increased the proportion of CD25+, mainly on lymphocytes and NK cells. The effects on activation marker expression did not appear to be strain specific. NK cell activity was significantly increased by all six strains, without any significant difference between strains. Probiotic strains increased production of IL-1β, IL-6, IL-10, TNF-α, granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein 1α to different extents, but had no effect on the production of IL-2, IL-4, IL-5 or TNF-β. The cytokines that showed strain-specific modulation included IL-10, interferon-γ, TNF-α, IL-12p70, IL-6 and monocyte chemotactic protein-1. The Lactobacillus strains tended to promote T helper 1 cytokines, whereas bifidobacterial strains tended to produce a more anti-inflammatory profile. The results suggest that there was limited evidence of strain-specific effects of probiotics with respect to T cell and NK cell activation or NK cell activity, whereas production of some cytokines was differentially influenced by probiotic strains.
Dietary probiotics supplementation exerts beneficial health effects. Since cigarette smoking reduces natural killer (NK) activity, we evaluated the effect of Lactobacillus casei Shirota LcS intake on NK cytotoxic activity in male smokers. The double-blind, placebo-controlled, randomised study was conducted on seventy-two healthy Italian blue-collar male smokers randomly divided for daily intake of LcS powder or placebo. Before and after 3 weeks of intake, peripheral blood mononuclear cells were isolated and NK activity and CD16 cells' number were assessed. Daily LcS intake for 3 weeks significantly increased NK activity (P < 0.001). The increase in NK activity was paralleled by an increase in CD16 cells (P < 0.001). Before intake, NK cytotoxic activity inversely correlated with the number of cigarettes smoked (R - 0.064). LcS intake prevented the smoke-dependent expected NK activity reduction. The analysis of the distribution of changes in smoke-adjusted NK activity demonstrated that the positive variations were significantly associated with LcS intake, while the negative variations were associated with placebo intake (median value of distributions of differences, 20.98 lytic unit (LU)/10 cells for LcS v. - 4.38 LU/10 cells for placebo, P = 0.039). In conclusion, 3 weeks of daily LcS intake in Italian male smokers was associated with a higher increase in cytotoxic activity and CD16 cells' number in comparison to the placebo intake group.
Probiotics have been reported to be efficacious against cancers, infections, allergies, inflammatory bowel diseases and autoimmune diseases, and it is important to explain how such multifunctional activities are realized. Lactobacillus casei Shirota (LcS) is one of these multifunctional probiotics, and its ability to augment the host immune system has been extensively examined. We have shown that the cell wall structure of this probiotic strain is responsible for potently inducing IL-12 production. In addition, we have recently found that LcS differentially controls the inflammatory cytokine responses of macrophages and T cells in either Peyer's patches or the spleen. Other studies revealed that LcS-induced IL-12 production by macrophages is modified when other bacteria or their cell components are simultaneously present. These findings can provide a theoretical basis for understanding the multifunctional activities of specific probiotics.
Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota LcS, on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS-induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8(+) and CD56(+) subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL-1beta production but inhibited LPS-induced IL-10 and IL-6 production, and had no further effect on TNF-alpha and IL-12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti-inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS-induced cytokine production. Monocytes play an important role in LcS-induced immunological responses.
The purpose of this study was to examine the effects of a probiotic supplement during 4 mo of winter training in men and women engaged in endurance-based physical activities on incidence of upper respiratory-tract infections (URTIs) and immune markers. Eighty-four highly active individuals were randomized to probiotic (n = 42) or placebo (n = 42) groups and, under double-blind procedures, received probiotic (PRO: Lactobacillus casei Shirota [LcS]) or placebo (PLA) daily for 16 wk. Resting blood and saliva samples were collected at baseline and after 8 and 16 wk. Weekly training and illness logs were kept. Fifty-eight subjects completed the study (n = 32 PRO, n = 26 PLA). The proportion of subjects on PLA who experienced 1 or more weeks with URTI symptoms was 36% higher than those on PRO (PLA 0.90, PRO 0.66; p = .021). The number of URTI episodes was significantly higher (p < .01) in the PLA group (2.1 ± 1.2) than in the PRO group (1.2 ± 1.0). Severity and duration of symptoms were not significantly different between treatments. Saliva IgA concentration was higher on PRO than PLA, significant treatment effect F(1, 54) = 5.1, p = .03; this difference was not evident at baseline but was significant after 8 and 16 wk of supplementation. Regular ingestion of LcS appears to be beneficial in reducing the frequency of URTI in an athletic cohort, which may be related to better maintenance of saliva IgA levels during a winter period of training and competition.
BACKGROUND: Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements.

OBJECTIVE: We sought to investigate the role that Lactobacillus casei Shirota LcS may play in modulating seasonal allergic rhinitis (SAR).

METHODS:The study format was double-blinded, placebo-controlled with 10 SAR sufferers in each group. We have documented and compared changes in immune status arising through the daily ingestion of a milk drink with or without live LcS, over a period of 5 months. Pre-, peak- and post-grass pollen season blood samples were collected for determination of plasma total IgE and grass pollen-specific IgG and IgE levels by an enzyme immunoassay. At the same time, cytokine levels were determined by flow cytometric bead array technology following culture of peripheral blood mononuclear cells for 6 days in the presence or absence of specific grass pollen antigens.

RESULTS:Volunteers treated with LcS showed a significant reduction in levels of antigen-induced IL-5, IL-6 and IFN-gamma production compared with volunteers supplemented with placebo. Meanwhile, levels of specific IgG increased and IgE decreased in the probiotic group.

CONCLUSION: Changes in antigen-induced production of cytokines were observed in patients treated with probiotics. These data show that probiotic supplementation modulates immune responses in allergic rhinitis and may have the potential to alleviate the severity of symptoms.
Nine healthy middle-aged and 10 elderly volunteers drank fermented milk containing 4 x 10(10) live cells of Lactobacillus casei strain Shirota daily for 3 wk, and their natural killer (NK) activity and other immunological functions were examined. In the experiments with middle-aged volunteers, NK activity significantly increased (P < 0.01) 3 wk after the start of intake, elevated NK cell activity remained for the next 3 wk, and this effect was particularly prominent in the low-NK-activity individuals. In the experiments with elderly volunteers, NK activity significantly decreased (P < 0.01) in the control group 3 wk after the start of intake; however, the intake of Lactobacillus casei strain Shirota maintained the NK activity. These results suggest that daily intake of Lactobacillus casei strain Shirota provides a positive effect on NK-cell activity.
BACKGROUND: Lactobacillus casei strain Shirota LcS has been found to exert antiallergic effects in animal experiments, but there is little information about its clinical effects in human patients with allergy.

METHODS:We performed a randomized double-blind, placebo-controlled study to investigate the effects of LcS in patients with allergic rhinitis triggered by Japanese cedar pollen (JCP). Participants were asked to drink fermented milk containing LcS group or placebo (control group) for 8 weeks. Clinical symptoms and immunological parameters were compared between the two groups.

RESULTS:Symptom-medication scores (SMS) worsened in accordance with the increase in the amount of scattered JCP. In terms of the nasal and ocular SMS, there was no significant difference between the LcS group and the placebo group during the ingestion period. In the subgroup of patients with moderate-to-severe nasal symptom scores before starting the ingestion of test samples, supplementation with LcS tended to reduce nasal SMS.

CONCLUSION:These results indicate that fermented milk containing LcS does not prevent allergic symptoms in patients sensitive to JCP, but may delay the occurrence of allergic symptoms in patients with moderate-to-severe nasal symptom scores.
We conducted a placebo-controlled, cross-over trial to examine the effect of Lactobacillus casei Shirota LcS on natural killer (NK) cell activity in humans. NK cell activity exhibited a declining trend during the period of placebo ingestion, but NK cell activity increased after intake for 3 weeks of fermented milk containing 4 x 10(10) live LcS. When human peripheral blood mononuclear cells were cultured in the presence of heat-killed LcS, NK cell activity was enhanced. The ability of LcS to enhance NK cell activity and induce interleukin (IL)-12 production was correlated, and the addition of anti-IL-12 monoclonal antibody reduced the enhancement of NK cell activity triggered by LcS. In addition, separation of NK cells from LcS-stimulated monocytes with membrane filter reduced NK cell activity to the intermediate level and almost deprived monocytes of the ability to produce IL-12. These results demonstrate that LcS can enhance NK cell activity in vivo and in vitro in humans, and IL-12 may be responsible for enhancement of NK cell activity triggered by LcS.
BACKGROUND:Habitual smoking significantly reduces natural killer (NK) cell activity. To clarify whether the intake of fermented milk containing lactic acid bacteria restores NK cell activity in habitual smokers, we conducted a placebo-controlled double-blind test.

RESULTS:NK cell activity in individuals was inversely correlated to numbers of cigarettes smoked. Averaged NK cell activity adjusted by the numbers of cigarettes was significantly higher in individuals drinking fermented milk containing L. casei than those drinking a placebo. However, the proportion of NK cells was not different between individuals drinking either fermented milk containing L. casei or the placebo.

CONCLUSIONS:Intake of fermented milk containing lactic acid bacteria was considered effective for restoring the NK cell activity of habitual smokers.
Nine healthy volunteers drank fermented milk containing 4 x 10(10) live cells of Lactobacillus casei strain Shirota daily for 3 weeks, and their NK activity and other immunological functions were measured. NK activity significantly increased (p < 0.01) 3 weeks after the start of intake and remained elevated for the next 3 weeks. The effect was particularly prominent in low-NK-individuals.

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1. Probiotic effects on intestinal fermentation patterns in patients with irritable bowel syndrome.

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Objective: To demonstrate the recovery of Lactobacillus casei strain Shirota LcS from feces of Thai subjects who regularly took LcS containing milk product for 1 week and demonstrate the disappearance of LcS after stopped taking milk product.

Material and Method: First fecal samples were collected from 20 healthy adults at 10 days after they abstained from all lactobacillus containing milk products. Second specimens taken after the subjects ingested LcS containing milk product for 7 days and third specimens at 7 days after they stopped taking LcS containing milk product. All the fecal specimens were culture for LcS using LLV-FOS culture medium and enumeration of LcS was calculated. All stool samples were also tested for the presence of LcS by using nested PCR to confirm the presence of LcS obtained from culture method.

Results: Both culture and nested PCR method showed that all the stools samples obtained from subjects prior to the administration of LcS containing milk product were devoid of LcS, except for 3 specimens which showed weakly positive test for PCR. At 7 days after ingesting LcS containing milk product, all stool specimens were positive for LcS on both culture and PCR method. At 7 days after stopped taking LcS containing milk product, 1/19 specimens were positive from culture and 6/ 19 specimens were positive for PCR method.

Conclusion: LcS could survive in the gastrointestinal tract of Thai subjects and could be recovered from the feces after ingestion.
Aim: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers.

Methods and Results: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10) Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated.

Conclusions: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period.

Significance and Impact of the Study: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.
Aims: This study aimed at determining whether oral administration of a probiotic strain, Lactobacillus casei strain Shirota LcS, can improve insulin resistance, which is the underlying cause of obesity-associated metabolic abnormalities, in diet-induced obesity (DIO) mice.

Methods and Results: DIO mice were fed a high-fat diet without or with 0•05% LcS for 4 weeks and then subjected to an insulin tolerance test (ITT) or oral glucose tolerance test (OGTT). Oral administration of LcS not only accelerated the reduction in plasma glucose levels during the ITT, but also reduced the elevation of plasma glucose levels during the OGTT. In addition, plasma levels of lipopolysaccharide-binding protein (LBP), which is a marker of endotoxaemia, were augmented in the murine models of obese DIO, ob/ob, db/db and KK-A(y) and compared to those of lean mice. LcS treatment suppressed the elevation of plasma LBP levels in DIO mice, but did not affect intra-abdominal fat weight.

Conclusions: LcS improves insulin resistance and glucose intolerance in DIO mice. The reduction in endotoxaemia, but not intra-abdominal fat, may contribute to the beneficial effects of LcS.

Significance and Impact of the Study: This study suggests that LcS has the potential to prevent obesity-associated metabolic abnormalities by improving insulin resistance.

Aim: Although several studies have demonstrated the efficacy of probiotics for preventing upper respiratory tract infections (URTIs) in at-risk populations, including children and the elderly. Few studies have investigated the efficacy of probiotics in healthy adults living normal, everyday lives. Thus, this study evaluated the effect of Lactobacillus casei strain Shirota-fermented milk (LcS-FM) on the incidence of URTIs in healthy middle-aged office workers.

Method: In a randomized controlled trial, 96 male workers aged 30–49 years consumed LcS-FM containing 1.0 × 1011 LcS bacteria or control milk (CM) once daily for 12 weeks during the winter season. URTI episodes were evaluated by a physician via a questionnaire of URTI symptoms.

Results: The incidence of URTIs during the intervention period was significantly lower in the LcS-FM group as compared to the CM group (22.4 vs. 53.2 %, P = 0.002). The time to-event analysis showed that the LcS-FM group had a significantly higher URTI-free rate than the CM group over the test period (log-rank test: χ2 11.25, P = 0.0008). The cumulative number of URTI episodes and cumulative days with URTI symptoms per person was lower in the LcS-FM group, and the duration per episode was shorter. Inhibition of both reductions in NK cell activity in peripheral blood mononuclear cells and increase in salivary cortisol levels was observed in the LcS-FM group.

Conclusions: The results suggest that the daily intake of fermented milk with LcS may reduce the risk of URTIs in healthy middle-aged office workers, probably through modulation of the immune system.

Aim: There is insufficient evidence of preventive effect of probiotics on upper respiratory tract infections (URTIs) in an elderly population.

Method: A multicenter, double-blinded, randomized, placebo-controlled parallel group study was conducted. Elderly persons had participated who used day care at 4 facilities in Tokyo. We used fermented milks containing Lactobacillus casei strain Shirota (LcS) and placebo drinks as test drinks.

Results: A total of 154 subjects was analyzed. The number of persons diagnosed with an acute URTIs was almost identical in both groups (LcS: 31, placebo: 32), whereas the number of acute URTIs events (LcS: 68, placebo: 51) and the symptom score (LcS: 425, placebo: 396) were both higher in the LcS group. Permutation tests performed using the total number of acute URTIs infection events/total days of observation and the total symptom score/total days of observation found no statistically significant difference respectively (P values of .89 and .64, respectively). Comparing the mean duration of infection per infection event found a shorter mean duration in the LcS group (LcS: 3.71 days, placebo: 5.40 days), and the difference was statistically significant.

Conclusions: The results suggest that fermented milk containing LcS probably reduces the duration of acute URTIs.

Aim: This pilot study investigated the effects of the probiotic Lactobacillus casei strain Shirota (LcS) on psychological and physical stress responses in medical students undertaking an authorized Nationwide examination for promotion.

Method: In a double-blind, placebo-controlled trial, 24 and 23 healthy medical students consumed a fermented milk containing LcS and a placebo milk, respectively, once a day for 8 weeks until the day before the examination. Psychological state, salivary cortisol, faecal serotonin and plasma L-tryptophan were analysed on 5 different sampling days (8 weeks before examination, 2 weeks before examination, 1 day before examination, immediately after examination and 2 weeks after the examination). Physical symptoms were also recorded in a dairy by subjects during the intervention period for 8 weeks.

Results: In association with a significant elevation of anxiety at 1 day before the examination, salivary cortisol and plasma L-tryptophan levels were significantly increased in only the placebo group (P < 0.05). Two weeks after the examination, the LcS group had significantly higher faecal serotonin levels (P < 0.05) than the placebo group. Moreover, the rate of subjects experiencing common abdominal and cold symptoms and total number of days experiencing these physical symptoms per subject were significantly lower in the LcS group than in the placebo group during the pre-examination period at 5-6 weeks (each P < 0.05) and 7-8 weeks (each P < 0.01) during the intervention period.

Conclusions: The results suggest that daily consumption of fermented milk containing LcS may exert beneficial effect by preventing the onset of physical symptoms in healthy subjects exposed to stressful situations.

The production of viable functional probiotics presupposes stability of strain features in the final product. We evaluated the impact of acquisition of heat-tolerance and subsequent freeze-drying on the adhesion properties of Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Bifidobacterium lactis Bb-12 and Bifidobacterium animalis IF20/1 and on their ability to inhibit the adhesion of pathogens in a mucus model. Both fresh and freeze-dried cultures were evaluated. Significant differences were observed between fresh, freeze dried, fresh heat-tolerant and freeze dried heat-tolerant strains, especially in the ability of the freeze dried probiotics to exclude, displace or outcompete pathogens. Based on our study characterizing probiotic properties such as adhesion and competitive exclusion, it seems possible to adapt probiotics to processing stresses, such as heat, without significantly changing the probiotic properties of the strains assessed. This may provide new options for future probiotic production technology. However, our results also emphasize that the properties of the stress-adapted strains, as well as the effect of the production processes should always be assessed as these are strain-specific
The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082Mb and a G+C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L.casei LcA and LcY harboured a 59.6kb plasmid that shared high similarities with plasmids found in other L.casei strains, such as W56 and BD-II. Further analysis revealed that the L.casei plasmids constitute a good evolution marker within the L.casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L.casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.
AIMS: Aflatoxin B1 (AFB1 ) is considered as the most toxic food contaminant, and microorganisms, especially bacteria, have been studied for their potential to reduce the bioavailability of mycotoxins including aflatoxins. Therefore, this research investigated the efficacy of oral administration of Lactobacillus casei Shirota LcS in aflatoxin-induced rats.

METHODS AND RESULTS:Sprague Dawley rats were divided into three groups of untreated control, the group induced with AFB1 only, and the group given probiotic in addition to AFB1 . In the group induced with AFB1 only, food intake and body weight were reduced significantly. The liver and kidney enzymes were significantly enhanced in both groups induced with AFB1, but they were lower in the group given LcS. AFB1 was detected from all serum samples except for untreated control group's samples. Blood serum level of AFB1 in the group induced with AFB1 only was significantly higher than the group which received probiotic as a treatment (P < 0•05), and there was no significant difference between the control group and the group treated with probiotic.

CONCLUSIONS:LcS supplementation could improve the adverse effect of AFB1 induction on rats' body weight, plasma biochemical parameters and also could reduce the level of AFB1 in blood serum.

SIGNIFICANCE AND IMPACT OF THE STUDY:This study's outcomes contribute to better understanding of the potential of probiotic to reduce the bioavailability of AFB1. Moreover, it can open an opportunity for future investigations to study the efficacy of oral supplementation of probiotic LcS in reducing aflatoxin level in human.
BACKGROUND: There is insufficient evidence of preventive effect of probiotics on upper respiratory tract infections (URTIs) in an elderly population.

METHODS:We conducted a multicenter, double-blinded, randomized, placebo-controlled parallel group study. Elderly persons had participated who used day care at 4 facilities in Tokyo. We used fermented milks containing Lactobacillus casei strain Shirota (LcS) and placebo drinks as test drinks.

RESULTS:A total of 154 subjects was analyzed. The number of persons diagnosed with an acute URTIs was almost identical in both groups (LcS: 31, placebo: 32), whereas the number of acute URTIs events (LcS: 68, placebo: 51) and the symptom score (LcS: 425, placebo: 396) were both higher in the LcS group. Permutation tests performed using the total number of acute URTIs infection events/total days of observation and the total symptom score/total days of observation found no statistically significant difference respectively (P values of .89 and .64, respectively). Comparing the mean duration of infection per infection event found a shorter mean duration in the LcS group (LcS: 3.71 days, placebo: 5.40 days), and the difference was statistically significant.

CONCLUSION:The results suggest that fermented milk containing LcS probably reduces the duration of acute URTIs.
INTRODUCTION: During exercise, ammonia is generated as a natural metabolic waste product, normally excreted via the kidneys after conversion into urea in the liver. If the exercise is strenuous, blood ammonia levels can build up and exceed the liver's capacity to remove it. This can cause ammonia levels to increase in the brain, and if this happens the ammonia is removed through the astrocytes through the enzymatic activity of glutamine synthetase, which combines ammonia with glutamate to produce glutamine. Osmotic stress may be induced by the subsequent increase in intracellular glutamine, however, leading to water accumulation, potentially mild encephalopathy and impaired cognitive function. Successful treatment with a phenylacetate precursor has been used in patients suffering urea cycle disorders and raised levels of ammonia in the blood. Phenylacetate forms a stable conjugate with glutamine to make phenylglutamine (PAG) which can be readily excreted via the kidneys.

RATIONALE: As lactobacilli can metabolise phenylalanine into phenylacetate, the study was based on the hypothesis that supplementation with L. casei Shirota could help to regulate ammonia metabolism in healthy people undertaking strenuous exercise via the natural generation of phenylacetate by the probiotic. This would enable ammonia, trapped as glutamine, to be excreted as phenylacetylglutamine via the kidneys.

METHOD: An open-label, pilot, proof of principle study was conducted in 20 male football players assigned to consume either probiotic (L. casei Shirota; minimum 6.5x109 twice per day) or no supplementation. The players undertook an exhaustive routine designed to exercise all the major muscle blocks (two cycles of a 9-station static exercise program with a one minute rest between the two cycles; water supplied ad libertum). The players provided a four hour timed urine sample after the exercise program. Both the exercise program and urine sampling were repeated after one month. Urine samples were measured for phenyacetylglutamine and ammonia and corrected by creatinine levels.

RESULTS: The results (expressed as the difference in urinary levels for each volunteer between the two sampling points) showed that phenylacetylglutamine significantly increased in the probiotic group (2.98 ±1.04 vs -0.911 ±0.477; P less than 0.01) and, while not reaching statistical significance, their ammonia levels were also lower compared to the control group (0.953 ± 0.868 vs 1.486 ± 0.865; P=0.064).

CONCLUSIONS: The researchers concluded that probiotic supplementation with a probiotic Lactobacillus strain appeared to help regulate exercise-generated ammonia in young health sportsmen.
This study involved 678 hospital patients (mean age 71) who were on a range of single or multiple antibiotic regimes. One group (n=340) consumed a daily fermented milk drink containing L. casei Shirota during the antibiotic treatment and for three days after this ceased. (At the time of probiotic intervention, all patients and staff on the ward received probiotic). A control group of patients also on antibiotics (n=338), did not receive the probiotic; these patients were matched to the probiotic group in terms of age, sex, antibiotics, disease severity, length of hospital stay, use of proton pump inhibitors.

There was no exclusion of any particular antibiotic. Antibiotics taken included penicillins, cephalosporins, quinolones, clindamycin, vancomycin, and were multiple as well as single antibiotic regimes.

During probiotic intake, all patients on the ward were offered probiotic, as well as the staff. During periods on the wards of no probiotic intake, neither staff nor patients took probiotic.

AAD:* 17/340 (5%) in LcS group vs 63/338 (18.6%) control group (P less than 0.001)
CDI*: 1/340 (0.3%) in LcS group vs 21/338 (6.2%) control group (P less than 0.001)

Faecal analysis showed that antibiotic therapy decreased abundance of total bacteria, Bifidobacterium spp, Clostridium clusters IV and XI whereas Enterobacteriaceae increased. LcS intervention reduced the observed antibiotic-induced decrease in abundance of total bacteria and Bifidobacterium. LcS intervention also increased Lactobacillus abundance.

*Antibiotic-associated diarrhoea = AAD; C. difficile infection = CDI
With more than 1.4 million of the 9 million child deaths being attributed to diarrhoea in 2008 and 49% of them occurring in five countries namely, India, Nigeria, Democratic Republic of the Congo, Pakistan and China, there is an urgent need for intervention to prevent and control diarrhoeal diseases. Of the various interventions, probiotics offer immense potential. The past decade has witnessed the validation of their utility for the prevention, treatment and management of a variety of infective and non infective disorders. The most investigated field continues to remain infectious diarrhoea and compelling evidence comes from randomized placebo controlled trials. While results from these studies are encouraging most of them reflect the outcomes of the developed world. Developing countries like India continue to struggle with nutritional and health challenges and bear the greatest burden of diarrhoea. A paucity of data from the developing countries limits the definite recommendation of probiotics. In these countries curd, often confused for a probiotic, is practiced as an integral part of the culture. While the nutritional benefits of these products cannot be understated, it is still uncertain whether these products can be classified as a probiotic. The emergence of probiotic foods which are scientifically validated for their efficacy and impart defined health benefits offer an excellent opportunity to improve public health. A recent randomized controlled trial conducted by the National Institute of Cholera and Enteric Diseases in Kolkata, India demonstrated a protective efficacy of 14% in preventing diarrhoea among children who received a probiotic. For the developing world however the vision for probiotics would mean a fundamental change in perception and developing a well planned strategy to allow interventions like probiotics to permeate to impoverished settings, where the assault of micro organisms is on a daily basis. This would mean that probiotics are ingrained into the public health system without being seen as a medicine.
Background: para-Cresol, which is present in the blood mainly as p-cresyl sulphate, is a protein-bound uraemic toxin that is produced in the intestine by certain intestinal bacteria, and its production is affected by various intestinal environmental factors. Patients with end-stage renal disease who are undergoing haemodialysis (HD) often have defective bowel function leading to abnormal defecation. Since treatment with synbiotics (SYN), which are a combination of probiotics and prebiotics, is reported to improve bowel habit, we examined the effects of SYN on the serum p-cresol level in HD patients.

Methods: Nine HD patients received SYN ( Lactobacillus casei strain Shirota and Bifidobacterium breve strain Yakult as probiotics and galacto-oligosaccharides as prebiotics) three times a day for 2 weeks. The duration of the study was 4 weeks (2 weeks of pretreatment observation and 2 weeks of treatment). The subjects were asked to complete a questionnaire about their bowel habits (defecation frequency, stool quantity, stool form and ease of defecation) during the study period. Serum p-cresol levels before and after SYN treatment were determined.

Results: According to the questionnaire conducted during the pretreatment observation period, HD patients with a high serum p-cresol level tended to have hard stools with difficulty in defecation. With SYN treatment, stool quantity increased significantly and hard, muddy or soft stools tended to be replaced by normal ones. The serum p-cresol level also decreased significantly.

Conclusions: It was found that uraemic toxin, p-cresol, was associated with constipation and that SYN treatment resulted in normalization of bowel habits and a decrease of serum p-cresol levels in HD patients. Therefore, SYN treatment may be anticipated to reduce the toxic effect of p-cresol in HD patients.
Background: The efficacy of some probiotic strains for the management of lactose intolerance remains to be established.

Aim: To evaluate the effects of a 4-week consumption of a probiotic product containing Lactobacillus casei Shirota and Bifidobacterium breve Yakult (10(7)-10(9) CFU of each strain) on symptoms and breath hydrogen exhalation after a lactose load in lactose-intolerant patients and whether the beneficial results persisted after probiotic discontinuation.

Methods: Twenty-seven patients with lactose maldigestion and intolerance participated in this study, which comprised 4 hydrogen breath tests: baseline condition (20 g lactose), after lactase ingestion (9000 FCC units), at the end of 4-week probiotic supplementation, and a follow-up test performed 3 months after probiotic discontinuation. For each test, the area under the breath hydrogen concentration vs time curve (AUC (180 min)) was calculated, and symptom scores were recorded.

Results: The probiotic combination significantly reduced symptom scores (P < .01) and breath hydrogen AUC (P = .04) compared with the baseline condition. The comparison with the lactase test showed that symptom scores were similar (P > .05), despite the significantly higher (P = .01) AUC values after probiotic use. In the follow-up test, symptom scores and breath hydrogen AUC values remained similar to those found at the end of probiotic intervention.

Conclusion: Four-week consumption of a probiotic combination of L casei Shirota and B breve Yakult seems to improve symptoms and decrease hydrogen production intake in lactose-intolerant patients. These effects may persist for at least 3 months after suspension of probiotic consumption
Background / Objectives: Obesity and metabolic disorders are linked to inflammation via gut microbiota and/or gut permeability. Gut-derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. We intended to investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS.

Subjects / Methods: Patients with MetS were randomized to receive 3 × 6.5 × 10 CFU L. casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method and by determination of diaminooxidase serum levels, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14) levels.

Results: Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared with controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. High-sensitive C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups.

Conclusions: Gut permeability of MetS patients was increased significantly compared with healthy controls. L. casei Shirota administration in the MetS patients did not have any influence on any parameter tested possibly due to too-short study duration or underdosing of L. casei Shirota.
Objective: To determine inhibitory activity of fermented milk with Lactobacillus casei strain Shirota (FMLC) against common multi-drug-resistant (MDR) bacteria causing hospital-acquired infections.

Material and Method: Time-kill methods of FMLC and cell-free filtered fluid of FMLC (CF-FMLC) against Acinetobacter baumannii, Pseudomonas aeruginosa, ESBL-producing Escherichia coli & Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were conducted. The control solutions were Mueller Hinton broth (MHB) and distilled water. The mixtures of FMLC, CF-FMLC, MHB and DW with 10(5) to 10(6) CFU/ml of each bacterium were prepared and incubated at 35 degrees C. Each mixture was quantified for viable bacteria at 0, 1, 3, 6 and 24 hr after incubation onto brain heart infusion agar plates. The inoculated agar plates were incubated at 35 degrees C for 24-48 hr. Bacterial colonies on agar plates were counted and compared among the mixtures.

Results: Log CFUs of each organism in MHB and distilled water after incubation were increased from 5.1-6.3 at 0 h to 6.4- > 11 at 24 hr. Log CFUs of each organism in FMLC and CF-FMLC after incubation with study bacteria for 0, 1, 3, 6 and 24 hr were decreased to undetectable amounts at 24 hr.

Conclusion: FMLC and CF-FMLC exerted slow inhibitory activity against MDR bacteria resulting in eradication of all study bacteria at 24 hr. Such inhibitory effects were probably due to the products of the milk fermented by Lactobacillus casei strain Shirota. Clinical study is needed to determine if consumption of FMLC can prevent and treat colonization and infection with MDR bacteria in hospitalized patients.