Effect of Lactobacillus paracasei on Intestinal Colonisation of Lactobacilli,Bifidobacteria andClostridium difficile in Elderly Persons

Probiotics have been suggested to improve gastrointestinal health in humans. The present study investigated the effect of a fermented product containing Lactobacillus paracasei ssp. paracasei F19 and inulin on the prevalence of lactobacilli, bifidobacteria and Clostridium difficile in the intestinal microflora of elderly participants, seropositive for Helicobacter pylori. Thirty-five participants were included in the study and were randomized into two groups, the treatment group and the placebo group. Fermented milk products were consumed twice daily for 12 weeks. Faecal samples were collected before, during (4 and 12 weeks) and 8 weeks after the administration of the products. There was statistically significant increase in the numbers of L. paracasei F19 and lactobacilli other than F19 in the treatment group (p<0.01, Wilcoxon's signed rank test). In two individuals, L. paracasei was isolated also 8 weeks after the end of the administration period. In the placebo group, no changes occurred. C. difficile was only detected once in one of the participants in the treatment group.     

Safety evaluation of Lactobacillus paracasei subsp. paracasei LC-01, a probiotic bacterium

The safety of Lactobacillus paracasei subsp. paracasei LC-01 was evaluated for its use as a potential probiotic. In our in vitro study, the antibiotic resistance and the ability to produce biogenic amine were determined. The results showed that the strain was sensitive to all tested antibiotics and did not produce biogenic amine except for tyramine. The oral toxicity of this strain was evaluated in Balb/C mice. One hundred mice were divided into 10 groups. Four groups were administered 0, 10⁸, 10⁹, or 10¹⁰ CFU/mouse per day dissolved in saline solution respectively, for 28 days. Three groups were injected intraperitoneally with 10⁹ CFU/mouse dissolved in saline solution, and were killed 2, 5, and 10 days after injection. The last 3 groups were injected with the vehicle as controls respectively. The results showed that oral administration of the strain had no adverse effects on mouse body weight and that there was no treatment-associated bacterial translocation. Intraperitoneal administration caused a significant translocation to liver, spleen and kidney. However, this translocation did not cause illness or death throughout the experiment. The results suggest that L. paracasei subsp. paracasei LC-01 is likely to be safe for human consumption.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

Probiotic Characterization of Lactobacillus paracasei subsp. paracasei KNI9 Inhibiting Adherence of Yersinia enterocolitica on Caco-2 Cells In Vitro

The study was aimed to characterize the probiotic properties of Lactobacillus paracasei subsp. paracasei strain KNI9 and its antagonistic activity against Yersinia enterocolitica subsp. enterocolitica. The strain KNI9 was susceptible to antibiotics such as chloramphenicol, tetracycline, erythromycin, and streptomycin recommended by European food safety authority (EFSA). Strain KNI9 exhibited tolerance to simulated oro-gastrointestinal (OGT) condition, adherence to Caco-2 cells, and antimicrobial activity against intestinal enteric pathogens such as Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Listeria monocytogenes. Furthermore, the strain KNI9 inhibited the adherence and invasiveness of Y. enterocolitica subsp. enterocolitica to Caco-2 cell line. These results indicate that the L. paracasei subsp. paracasei KNI9 could be further developed into a potential probiotic strain after appropriate in vivo studies.

     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Lepr^fa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean^+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Lepr^fa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Lepr^fa/fa rats that received probiotics than in rats fed the placebo. Zucker-Lepr^fa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean^+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Lepr^fa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean^+/fa rats. Serum TNF-α levels decreased in the Zucker-Lepr^fa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Lepr^fa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.     

Lactobacillus paracasei subsp. paracasei 8700:2 Degrades Inulin-Type Fructans Exhibiting Different Degrees of Polymerization

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.     

Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [³H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) α-1,2 linked to two, three, or four fructose (F) units (GF₂, GF₃, and GF₄, respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF₂ and GF₃ was rapid, whereas little GF₄ uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.     

Evaluation of Lactobacillus paracasei subsp. tolerans isolated from Jenyn's sprat (Ramnogaster arcuata) as probiotic for juvenile rainbow trout Oncorhynchus mykiss (Walbaum, 1792)

A lactic acid bacterial strain, Lactobacillus paracasei subsp. tolerans F2, isolated from the intestine of Ramnogaster arcuata, was evaluated as a growth promoter in juvenile rainbow trout Oncorhynchus mykiss (Walbaum, 1972) farming. In addition, the safety of the strain was assessed according to the FAO recommendations. Strain F2 was susceptible to most antibiotics tested and no evidence of hemolytic activity was found. When the strain of Lactobacillus paracasei subsp. tolerans F2 was administered with food, no adverse effects on health were observed and fish biomass increased 12% more in the treatment group than in the control group. Significant differences were detected in the specific growth rate and feed conversion ratio. In the group receiving Lactobacillus paracasei subsp. tolerans F2‐supplemented feed, quantitative differences in the microbial composition of fish feces with respect to the control group were observed. An important decrease in fungi and enterobacteria was observed in feces from the treatment group, coincident with an increase in lactic acid bacteria. This result would indicate a change in the composition of the intestinal microbiota of fish treated with the putative probiotic. These results suggest that the strain of Lactobacillus paracasei subsp. tolerans F2 has the application potential to improve the performance in rainbow trout farming.     

Quantitative Real-Time PCR Analysis of Fecal Lactobacillus Species in Infants Receiving a Prebiotic Infant Formula

The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5′ nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% ± 0.3% versus 4.1% ± 1.5%) and the OSF group (0.8% ± 0.3% versus 4.4% ± 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.     

Selection of Escherichia coli-inhibiting strains of Lactobacillus paracasei subsp. paracasei

The aim of this study was to select Escherichia coli-inhibiting strains among lactic acid bacteria. On the basis of phenotypical and technological characteristics, 20 strains of lactic acid bacteria were screened from a total of 225 isolates, obtained from nine samples of artisanal Caprino d'Aspromonte cheese, made from raw goats' milk. The antagonistic activity of these 20 strains was detected in plates against three different strains of E. coli. Two strains of Lactobacillus paracasei subsp. paracasei showed a marked anti-E. coli activity against all three strains tested; the other lactic acid bacteria did not exhibit inhibiting activity. The E. coli inhibition can be ascribed to production of bacteriocin-like compounds. The use of L. paracasei subsp. paracasei strains to increase the safety of the cheeses made from raw milk is recommended because these cultures strongly inhibit E. coli, without foreseeable adverse sensory changes. Received 27 December 2001/ Accepted in revised form 28 June 2002     

Milk Fermented by Lactobacillus paracasei NCC 2461 (ST11) Modulates the Immune Response and Microbiota to Exert its Protective Effects Against Salmonella typhimurium Infection in Mice

Probiotics form a promising strategy to maintain intestinal health. Milks fermented with probiotic strains, such as the Lactobacillus paracasei ST11, are largely commercialized in Brazil and form a low-cost alternative to probiotic pharmaceutical formulations. In this study, we assessed the probiotic effects of milk fermented by L. paracasei ST11 (administered through fermented milk) in a Salmonella typhimurium infection model in BALB/c mice. We observed in this murine model that the applied probiotic conferred protective effects against S. typhimurium infection, since its administration reduced mortality, weight loss, translocation to target organs (liver and spleen) and ileum injury. Moreover, a reduction in the mRNA expression of pro-inflammatory cytokines such as IFN-γ, IL-6, TNF-α and IL-17 in animals that received the probiotic before challenge was observed. Additionally, the ileum microbiota was better preserved in these animals. The present study highlights a multifactorial protective aspect of this commercial probiotic strain against a common gastrointestinal pathogen.

     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Improved Stress Tolerance of GroESL-Overproducing Lactococcus lactis and Probiotic Lactobacillus paracasei NFBC 338

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52°C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54°C) and lactobacilli (at 60°C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.     

1H NMR investigation on the role of sorbitol for the survival of Lactobacillus paracasei ssp. paracasei in vacuum‐dried preparations

Aims: The aim of this work was to study the effect of sorbitol as protective agent on the survival of the probiotic strain Lactobacillus paracasei ssp. paracasei (F19) after vacuum drying. Methods and Results: The survival was studied after different drying times and for various concentrations of sorbitol by plate count method. Furthermore, time domain ¹H NMR studies on dehydrated suspensions of Lact. paracasei ssp. paracasei were performed to study the proton mobility in the dried samples. From the obtained signal, T2 relaxation times of single components and fractions with different proton mobility were determined. It was found out that the survival is increased by the presence of a minimum amount of sorbitol that is dependent on drying time. Furthermore, it is shown that the protective effect can only be observed below a critical water content of c. 20%. Nuclear magnetic resonance (NMR) results indicate a transition of sorbitol from liquid to solid like behaviour during drying. The onset of the transition coincides with the critical water content found for a protective effect. Conclusions: The data suggest that sorbitol protons are incorporated into the dried cells of Lact. paracasei ssp. paracasei (F19) below the critical water content and therefore leading to an enhanced survival. Significance and Impact of the Study: The results help to better understand the underlying mechanism of protection of Lact. paracasei ssp. paracasei using sorbitol and to establish vacuum drying as potential alternative drying technique to standard freeze drying.     

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.     

Enhanced Survival of GroESL-Overproducing Lactobacillus paracasei NFBC 338 under Stressful Conditions Induced by Drying

GroESL-overproducing Lactobacillus paracasei NFBC 338 was dried, and its viability was compared with that of controls. Spray- and freeze-dried cultures overproducing GroESL exhibited ~10-fold and 2-fold better survival, respectively, demonstrating the importance of GroESL in stress tolerance, which can be exploited to enhance the technological performance of sensitive probiotic cultures.     

Influence of a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® on bowel habits of healthy adults

Aims: This study aims to investigate the effect of different kinds of food products enriched with a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501^® and Lactobacillus paracasei IMC 502^®, on bowel habits of healthy adults. Methods and Results: Fifty healthy volunteers took part in a double‐blind placebo probiotic feeding study (25 fed probiotics, 25 fed placebo) for 12 weeks. Each volunteer ingested daily one or more food products enriched with a combination of the two potential probiotic strains (probiotic group) or the same food products without the probiotics (control group). Faecal samples were collected before, at the end and 2 weeks later the intervention period, and some of the main groups of faecal bacteria were enumerated by plate count and real‐time PCR. Questionnaires on bowel habits were submitted to volunteers. After the intervention, a significant increase in faecal lactobacilli and bifidobacteria were observed in the probiotic group, and stool frequency and stool volume were higher in the probiotic group than in the placebo group. Conclusions: Daily consumption of food products enriched with the two potential probiotic strains, Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^®, contributes to improve intestinal microbiota with beneficial properties and enhances bowel habits of healthy adults. Significance and Impact of the Study: The study revealed that Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^® exert a positive effect, in terms of improved bowel habits, on healthy adults.