Viability of human-derived probiotic lactobacilli in ice cream produced with sucrose and aspartame

A mixture of human-derived probiotic strains of Lactobacillus acidophilus, L. agilis and L. rhamnosus was used as a probiotic culture in ice cream manufacture. Viability and survival of these probiotic cultures were investigated in two different ice cream formulations. Ice cream with sucrose and ice cream with aspartame were prepared and each of these was divided into two subgroups: one with direct addition of the probiotic culture and one with milk fermented by the same probiotic culture. Ice cream samples were stored at −20°C for 6 months and the survival rate of cultures were determined monthly. Probiotic cultures underwent tests for resistance to bile salts, antibiotics, acidic conditions; they were found to be highly resistant to such challenges. Chemical analysis of ice cream samples, such as determination of acidity, pH and solid matter, was also performed. The probiotic cultures remained unchanged in ice cream stored for up to 6 months regardless of the sweeteners used. Using probiotic cultures in ice cream mixes did not alter the characteristics of the product.     

Effect of some pharmaceutical excipients on the survival of probiotic vaginal lactobacilli

Lactobacilli are the predominant microorganisms of the vaginal bacterial microbiota, and they play a major role in the maintenance of a healthy urogenital tract. In consequence, the interest in their potential use as probiotics has significantly increased during the last decade. In the present study we assessed the influence of different excipients on the survival of 4 probiotic vaginal lactobacilli incorporated into glycerinated gelatin ovules and stored at 5 degrees C for 60 d. Results showed that viability after storage was a strain-dependent characteristic, but inclusion of ascorbic acid significantly increased survival in 3 of the 4 strains tested. The best survival was observed for Lactobacillus salivarius CRL 1328 in ovules containing skimmed milk. No significant differences in viability were observed between control ovules (glycerogelatin base without excipients) and those containing lactose or Tween 80 for any of the strains tested. Lactobacillus acidophilus CRL 1259 and Lactobacillus crispatus CRL 1266 were, respectively, the most resistant and sensitive strains to the storage with the different substances. In conclusion, these results provide a basis for selecting excipients to improve the survival of lactobacilli in a probiotic product, in an attempt to ensure the delivery of an adequate number of viable cells to the urogenital tract.     

Influence of probiotic vaginal lactobacilli on in vitro adhesion of urogenital pathogens to vaginal epithelial cells

AIMS: Lactobacilli, the predominant micro-organisms of the vaginal microbiota, play a major role in the maintenance of a healthy urogenital tract by preventing the colonization of pathogenic bacteria. The aim of the present study was to assess the ability of four vaginal Lactobacillus strains, previously selected for their probiotic features, to block in vitro the adherence of three human urogenital pathogens to vaginal epithelial cells (VEC). METHODS AND RESULTS: Three types of assays were performed in order to determine the inhibitory effect of lactobacilli on adhesion of urogenital pathogens to VEC: blockage by exclusion (lactobacilli and VEC followed by pathogens), competition (lactobacilli, VEC and pathogens together) and displacement (pathogens and VEC followed by the addition of lactobacilli). Bacterial adhesion to VEC was quantified by microscopy (x1000) after Gram's stain. All the strains were able to inhibit by exclusion and competition the adhesion of Staphylococcus aureus to VEC but none was able to decrease the attachment of Escherichia coli by neither of the mechanisms assayed. Only Lactobacillus acidophillus CRL 1259 and Lactobacillus paracasei CRL 1289 inhibited the attachment of Group B streptococci (GBS) to VEC by exclusion and competition respectively. CONCLUSIONS: Lactobacillus of vaginal origin were able to inhibit the attachment of genitouropathogenic Staph. aureus and GBS to the vaginal epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the probiotic potential of these Lactobacillus strains as anti-infective agents in the vagina and encourage further studies about their capacity to prevent and manage urogenital tract infections in females.     

Prior frozen storage enhances the effect of edible coatings against Listeria monocytogenes on cold-smoked salmon during subsequent refrigerated storage

Aims: Listeria monocytogenes is a major safety concern for ready‐to‐eat foods. The overall objective of this study was to investigate whether prior frozen storage could enhance the efficacy of edible coatings against L. monocytogenes on cold‐smoked salmon during subsequent refrigerated storage. Methods and Results: A formulation consisting of sodium lactate (SL, 1·2–2·4%) and sodium diacetate (SD, 0·125–0·25%) or 2·5% Opti.Form (a commercial formulation of SL and SD) was incorporated into each of five edible coatings: alginate, κ‐carrageenan, pectin, gelatin and starch. The coatings were applied onto the surface of cold‐smoked salmon slices inoculated with L. monocytogenes at a level of 500 CFU cm^?2. In the first phase, the slices were first frozen at ?18°C for 6 days and stored at 22°C for 6 days. Alginate, gelatin and starch appeared to be the most effective carriers. In the second phase, cold‐smoked salmon slices were inoculated with L. monocytogenes, coated with alginate, gelatin or starch with or without the antimicrobials and stored frozen at ?18°C for 12 months. Every 2 months, samples were removed from the freezer and kept at 4°C for 30 days. Prior frozen storage at ?18°C substantially enhanced the antilisterial efficacy of the edible coatings with or without antimicrobials during the subsequent refrigerated storage. Conclusions: Plain coatings with ≥2 months frozen storage and antimicrobial edible coatings represent an effective intervention to inhibit the growth of L. monocytogenes on cold‐smoked salmon. Significance and Impact of the Study: This study demonstrates the effectiveness of the conjunct application of frozen storage and edible coatings to control the growth of L. monocytogenes to enhance the microbiological safety of cold‐smoked salmon.     

Influence of chicory inulin on the survival of microbiota of a probiotic fermented milk during refrigerated storage

Inulin-supplemented and control probiotic fermented milks were produced using a dairy starter culture containingsStreptococcus thermophilus CHCC 742/2130,Lactobacillus acidophilus La-5 andBifidobacterium lactis Bb-12, and were then stored at 4°C for 6 weeks. Microbiological analyses were performed at weekly intervals. The results showed that the presence of inulin at 1 t o 5% (wt/v) did not influence significantly (P>0.05) the survival rates of eitherS. thermophilus orL. acidophilus. However, the addition of inulin at 5% (wt/v) had a significant beneficial effect (P<0.05) on the viability of bifidobacteria after 28 days of refrigerated storage.     

Estimation of vaginal probiotic lactobacilli growth parameters with the application of the Gompertz model

Lactobacilli are widely described as probiotic microorganisms used to restore the ecological balance of different animal or human tracts. For their use as probiotics, bacteria must show certain characteristics or properties related to the ability of adherence to mucosae or epithelia or show inhibition against pathogenic microorganisms. It is of primary interest to obtain the highest biomass and viability of the selected microorganisms. In this report, the growth of seven vaginal lactobacilli strains in four different growth media and at several inoculum percentages was compared, and the values of growth parameters (lag phase time, maximum growth rate, maximum optical density) were obtained by applying the Gompertz model to the experimental data. The application and estimation of this model is discussed, and the evaluation of the growth parameters is analyzed to compare the growth conditions of lactobacilli. Thus, these results in lab experiments provide a basis for testing different culture conditions to determine the best conditions in which to grow the probiotic lactobacilli for technological applications.     

Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains ( Salmonella enteritidis and Escherichia coli ). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3·0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo . It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Characterization of intestinal lactobacilli as putative probiotic candidates

AIMS: To use antioxidative activity and antagonistic properties of lactobacilli against selected pathogens and members of the normal microflora as a basis for screening probiotic candidates. METHODS AND RESULTS: Antagonistic activity of lactobacilli against target bacteria in both microaerobic and anaerobic environments was tested. Production of antagonistic metabolites (ethanol, hydrogen peroxide (H2O2), acetic, lactic and succinic acid) by lactobacilli as well as their total antioxidative activity were assessed. In general, the lactobacilli tested were most effective against Gram-negative bacteria and their antagonistic activity was strain-specific. However, obligately heterofermentative lactobacilli had the strongest activity when tested in a microaerobic environment. Additionally, facultatively heterofermentative lactobacilli were equally effective in either milieu and produced significant levels of acetic and lactic acid. Moreover, obligately homofermentative lactobacilli had high H2O2 production and total antioxidative activity but weak antagonistic activity. CONCLUSIONS: Antioxidative and antagonistic activity of intestinal lactobacilli is strain-specific but typically can be related to their fermentation type which may be used for rapidly screening large numbers of lactobacilli for probiotic candidates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the utilization of group characteristics to screen lactobacilli intended for specific probiotic use. Such uses include the targeting of particular gut niches and pathogens as well as allowing for long-term benefits to the host.     

Fate of Listeria monocytogenes in processed meat products during refrigerated storage

The fate of Listeria monocytogenes during refrigerated storage was determined on several processed meat products, including ham, bologna, wieners, sliced chicken, sliced turkey, fermented semidried sausage, bratwurst, and cooked roast beef. The meats were surface inoculated with a five-strain mixture of less than or equal to 200 or ca. 10(5) L. monocytogenes cells per package, vacuum packaged, and stored at 4.4 degrees C. Survival or growth of listeriae was determined for up to 12 weeks of storage or until the product was spoiled. The organism survived but did not grow on summer sausage, grew only slightly on cooked roast beef, grew well on some wiener products but not on others, grew well (10(3) to 10(5) CFU/g increase within 4 weeks) on ham, bologna, and bratwurst, and grew exceptionally well (10(3) to 10(5) CFU/g increase within 4 weeks) on sliced chicken and turkey. The rate of growth depended largely upon the type of product and the pH of the product. Growth was most prolific on processed poultry products. The organism generally grew well on meats near or above pH 6 and poorly or not at all on products near or below pH 5. These results indicate the importance of preventing postprocessing contamination of L. monocytogenes in a variety of ready-to-eat meat products.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

Adherence capacities of oral lactobacilli for potential probiotic purposes

The most abundantly used probiotic strains come from the genus Lactobacillus and only a few studies have investigated their role in oral health. Even if a positive correlation has been established between the saliva Lactobacillus count and dental caries, this genus is generally recognized as safe (GRAS). Moreover, lactobacilli could in some cases play a beneficial role by inhibiting the growth of some oral pathogenic bacteria. This activity could justify their use as probiotic. To establish the potential health benefit of probiotic candidates, appropriate in?vitro tests are required, particularly on their adhesive capacity. The aim of this work was to investigate the adhesive properties and surface characteristics of 70 oral lactobacilli that could be used as probiotics for oral health. For this, three methods were used: biofilm formation on a glass surface and on saliva-coated hydroxyapatite discs and the microbial adhesion to solvent method. The results of the biofilm formed on glass surface showed 13 strains with an adhesion score equal to or higher than 3. 57/70 (81%) of the tested lactobacilli did not form any biofilm on glass surfaces. All of the 13 strains formed biofilms on HA discs. Among these 13 strains, 10/13 (77%) showed low surface hydrophobicity (0-35%) and 3/13 (23%) showed medium hydrophobicity (36-70%). Some of the selected strains showed potentially useful adhesive capacity. This work paves the way for the selection of probiotics that could be used for oral health purposes with the aim to reduce carious risk.     

Adhesion forces and coaggregation between vaginal staphylococci and lactobacilli

Urogenital infections are the most common ailments afflicting women. They are treated with dated antimicrobials whose efficacy is diminishing. The process of infection involves pathogen adhesion and displacement of indigenous Lactobacillus crispatus and Lactobacillus jensenii. An alternative therapeutic approach to antimicrobial therapy is to reestablish lactobacilli in this microbiome through probiotic administration. We hypothesized that lactobacilli displaying strong adhesion forces with pathogens would facilitate coaggregation between the two strains, ultimately explaining the elimination of pathogens seen in vivo. Using atomic force microscopy, we found that adhesion forces between lactobacilli and three virulent toxic shock syndrome toxin 1-producing Staphylococcus aureus strains, were significantly stronger (2.2-6.4 nN) than between staphylococcal pairs (2.2-3.4 nN), especially for the probiotic Lactobacillus reuteri RC-14 (4.0-6.4 nN) after 120 s of bond-strengthening. Moreover, stronger adhesion forces resulted in significantly larger coaggregates. Adhesion between the bacteria occurred instantly upon contact and matured within one to two minutes, demonstrating the potential for rapid anti-pathogen effects using a probiotic. Coaggregation is one of the recognized mechanisms through which lactobacilli can exert their probiotic effects to create a hostile micro-environment around a pathogen. With antimicrobial options fading, it therewith becomes increasingly important to identify lactobacilli that bind strongly with pathogens.     

Viability of frozen rat granulocytes and granulocyte precursors

Mature rat polymorphonuclear leukocytes (PMNs) were frozen to ?196 °C, thawed, and tested for functional viability using a variety of criteria. The assays for functional viability included: qualitative and quantitative nitroblue tetrazolium tests for phagocytic activity, fluorometric tests for membrane integrity, chemotaxis, and bactericidal activity. Maximal survival was obtained when mature PMNs were frozen in the presence of 10% dimethyl sulfoxide (Me₂SO) and 5% hydroxyethyl-starch (HES) for cells cooled at ~10 °C per minute, followed by rapid warming. Maximal survival was obtained for granulocyte precursor cells (as measured by CFU-c) after freezing in the presence of 10% Me₂SO and cooling at ~10 °C per minute. The principal new findings for mature PMNs were: (i) there was a synergistic effect between intra- and extracellular protective additives; (ii) the optimal cooling rate increases from approximately 0.3 to 10 °C per minute when an extracellular protective agent, such as HES is included in the freezing media; (iii) the zwitterion buffer Hepes has a small but consistently beneficial effect on survival; (iv) granulocytes obtained from peripheral blood consistently show a higher functional survival after freezing (95%) than do PMNs obtained from a glycogen-induced peritoneal exudate (70%); (v) neither serum, plasma, nor other macromolecules are needed in the postt-haw dilution media to obtain high survival; and (vi) cells frozen using an optimized two-step protocol survived as well as those frozen using a continuous cooling protocol.     

Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.