Impact of probiotic Lactobacillus sp. on autochthonous lactobacilli in weaned piglets.

AIMS: This study aimed to determine whether host-adapted lactobacilli exhibit superior survival during intestinal transit relative to nomadic and free-living organisms, and to characterize the impact of probiotic lactobacilli on autochthonous lactobacilli. METHODS AND RESULTS: Mixed cultures of Lactobacillus casei K9-1 and Lactobacillus fermentum K9-2, or reutericyclin producing Lactobacillus reuteri and its isogenic mutant were fed to piglets as freeze-dried culture, or as part of fermented feed. Lactobacilli in digesta and faecal samples were quantified by strain-specific quantitative PCR (qPCR), high-resolution-melting curve qPCR, and high-throughput sequencing of 16S rRNA gene sequence tags. The abundance of the host adapted L. reuteri in digesta and faeces was higher (P < 0·05) when compared to L. casei or L. fermentum. Feed fermentation or chemical acidification of feed reduced (P < 0·05) cell counts of Lactobacillus salivarius in colonic digesta. The reutericyclin producing L. reuteri TMW1.656 transiently reduced (P < 0·05) the faecal abundance of lactobacilli. However, the overall impact of probiotic intervention on autochthonous lactobacilli was minor. CONCLUSIONS: The vertebrate host-adapted L. reuteri survives better during intestinal transit of piglets compared to L. casei and L. fermentum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ecology and lifestyle of Lactobacillus strains may be suitable criteria for selection of probiotic strains for use in swine production.

Antifungal activity of secondary plant metabolites from potatoes (Solanum tuberosum L.): Glycoalkaloids and phenolic acids show synergistic effects.

AIMS: To study the antifungal effects of the potato secondary metabolites α-solanine, α-chaconine, solanidine and caffeic acid, alone or combined. METHODS AND RESULTS: Resistance to glycoalkaloids varied among the fungal species tested, as derived from minimum inhibitory concentrations assays. Synergistic antifungal activity between glycoalkaloids and phenolic compounds was found. Changes in the fluidity of fungal membranes caused by potato secondary plant metabolites were determined by calculation of the generalized polarization values. The results partially explained the synergistic effect between caffeic acid and α-chaconine and supported findings on membrane disruption mechanisms from previous studies on artificial membranes. LC/MS analysis was used to determine variability and relative amounts of sterols in the different fungal species. Results suggested that the sterol pattern of fungi is related to their resistance to potato glycoalkaloids and to their taxonomy. CONCLUSION: Fungal resistance to α-chaconine and possibly other glycoalkaloids is species dependent. α-Chaconine and caffeic acid show synergistic antifungal activity. The taxonomic classification and the sterol pattern play a role in fungal resistance to glycoalkaloids. SIGNIFICANCE AND IMPACT OF THE STUDY: Results improve the understanding of the antifungal mode of action of potato secondary metabolites, which is essential for their potential utilization as antifungal agents in nonfood systems.

Biochemical analysis of respiratory metabolism in the heterofermentative Lactobacillus spicheri and Lactobacillus reuteri.

AIMS: This study evaluated the aerobic and respiratory metabolism in Lactobacillus reuteri and Lactobacillus spicheri, two heterofermentative species used in sourdough fermentation. METHODS AND RESULTS: In silico genome analysis, production of metabolites and gene expression of pyruvate oxidase, pyruvate dehydrogenase and cytochrome oxidase were assessed in anaerobic and aerobic cultures of Lact. reuteri and Lact. spicheri. Respiring homofermentative Lactobacillus casei N87 and Lact. rhamnosus N132 were used for comparison. Aerobiosis and respiration increased the biomass production of heterofermentative strains compared to anaerobic cultivation. Respiration led to acetoin production by Lact. rhamnosus and Lact. casei, but not in heterofermentative strains, in which lactate and acetate were the major end-products. Lactobacillus spicheri LP38 showed the highest oxygen uptake. Pyruvate oxidase, respiratory cytochromes, NADH oxidase and NADH peroxidase were present in the genome of Lact. spicheri LP38. Both Lact. spicheri LP38 and Lact. rhamnosus N132 overexpressed pox in aerobic cultures, while cydA was up-regulated only when haeme was supplied; pdh was repressed during aerobic growth. CONCLUSIONS: Aerobic and respiratory growth provided physiological and metabolic advantages also in heterofermentative lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The exploitation of oxygen-tolerant phenotypes of Lact. spicheri may be useful for the development of improved starter cultures.

Intravaginally administered lactic acid bacteria expedited uterine involution and modulated hormonal profiles of transition dairy cows.

The objective of this investigation was to evaluate whether intravaginal infusion of lactic acid bacteria (LAB) around parturition could expedite involution rate of the uterus and improve reproductive performance of postpartum dairy cows. One hundred pregnant Holstein dairy cows were assigned to 1 of 3 experimental groups: (1) 1 dose of LAB in wk -2 and -1 and 1 dose of carrier in wk 1 relative to the expected day of parturition (TRT1); (2) 1 dose of LAB in wk -2, -1, and 1 (TRT2); and (3) 1 dose of carrier in wk -2, -1, and 1 (CTR). The LAB treatment was a lyophilized mixture of Lactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 with a cell count of 10(8) to 10(9) cfu/dose. Uterine involution and ovarian activity was evaluated by transrectal ultrasonography weekly from d 7 to 49 postpartum. Blood samples were collected from a subset of cows to quantify prostaglandin (PG) F2α metabolite (PGFM), PGE2, and progesterone. Cows treated with LAB had smaller cross-sectional areas of gravid horn and uterine body on d 14 postpartum. Cows in TRT2 resumed ovarian cyclicity earlier, as indicated by increased concentrations of serum progesterone. Cows in TRT1 had fewer days open than those in the CTR (110 vs. 150 d), whereas cows in TRT2 and CTR did not differ in days open. In addition, both TRT1 and TRT2 increased the concentrations of PGFM at calving week, and cows in TRT2 also had greater concentrations of PGE2 on d 14 and d 21 postpartum relative to CTR. Overall, cows treated intravaginally with LAB had smaller gravid horn and uterine body on d 14 postpartum than those in the CTR group. Treatment with LAB also increased concentrations of serum PGFM (3,533±328pg/mL in TRT1, 4,470±372pg/mL in TRT2, and 2,000±328pg/mL in CTR on d 0, respectively), with the TRT1 group having fewer cows that resumed ovarian cyclicity but fewer days open compared with both TRT2 and CTR groups. More research is warranted to better understand the mechanism(s) by which intravaginal LAB expedited uterine involution and affected hormonal profiles.

Evaluation of exopolysaccharide producing Weissella cibaria MG1 strain for the production of sourdough from various flours.

This study determined exopolysaccharide (EPS) production by Weissella cibaria MG1 in sourdoughs prepared from gluten-free flours (buckwheat, oat, quinoa and teff), as well as wheat flour. Sourdoughs (SD) were fermented without sucrose, or by replacing 10% flour with sucrose to support EPS production. The amount of EPS depended on the substrate: high amounts of EPS corresponding to low amounts of oligosaccharides were found in buckwheat (4.2 g EPS/kg SD) and quinoa sourdoughs (3.2 g EPS/kg SD); in contrast, no EPS but panose-series oligosaccharides (PSO) were detected in wheat sourdoughs. Organic acid production, carbohydrates and rheological changes during fermentation were compared to the EPS negative control without added sucrose. Corresponding to the higher mineral content of the flours, sourdoughs from quinoa, teff and buckwheat had higher buffering capacity than wheat. Fermentable carbohydrates in buckwheat, teff and quinoa flours promoted W. cibaria growth; indicating why W. cibaria failed to grow in oat sourdoughs. Endogenous proteolytic activity was highest in quinoa flour; α-amylase activity was highest in wheat and teff flours. Protein degradation during fermentation was most extensive in quinoa and teff SD reducing protein peaks 18-29, 30-41 and 43-55 kDa extensively. Rheological analyses revealed decreased dough strength (AF) after fermentation, especially in sucrose-supplemented buckwheat sourdoughs correlating with amounts of EPS. High EPS production correlated with high protein, fermentable sugars (glucose, maltose, fructose), and mineral contents in quinoa flour. In conclusion, W. cibaria MG1 is a suitable starter culture for sourdough fermentation of buckwheat, quinoa and teff flour.

In situ determination of Clostridium endospore membrane fluidity during pressure-assisted thermal processing in combination with nisin or reutericyclin.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

Metabolism of isomalto-oligosaccharides by Lactobacillus reuteri and bifidobacteria.

UNLABELLED: Commercial isomalto-oligosaccharides (IMO) are functional food ingredients. They are composed of α(1→6)- and α(1→4)-linked oligosaccharides. IMO are partially indigestible, and dietary IMO stimulate beneficial members of intestinal microbiota, including lactobacilli and bifidobacteria. However, data on IMO metabolism by lactobacilli are not available. It was the aim of this study to identify metabolic pathways of IMO metabolism in lactobacilli. This study focused on the host-adapted species Lactobacillus reuteri. Metabolism of bifidobacteria was analysed for comparison. Commercial IMO contained IMO with a degree of polymerization (DP) of up to four and panose-series oligosaccharides (POS) with a DP of up to 5. Lactobacilli metabolized isomaltose preferentially over oligosaccharides with higher DP. Bifidobacteria preferentially metabolized oligosaccharides with higher DP and accumulated glucose. Metabolism of IMO and POS by L. reuteri was attributed to α(1→6)-specific glucanase DexB and maltose phosphorylase. Contribution of maltose phosphorylase was verified by quantification of IMO and POS phosphorolysis in crude cellular extracts of L. reuteri 100-23. In conclusion, metabolism of IMO by lactobacilli is limited to short-chain oligosaccharides, while bifidobacteria preferentially metabolize oligosaccharides with higher DP. The functionality of commercial IMO can thus be modified by degree of polymerization. SIGNIFICANCE AND IMPACT OF THE STUDY: Isomalto-oligosaccharides (IMO) are applied as functional food ingredients, but the composition and biological functionality of current commercial products are poorly documented. This study is the first to analyse IMO metabolism by Lactobacillus reuteri. Bifidobacteria were used for comparison. Commercial IMO contained IMO with degree of polymerization (DP) of up to four and panose-series oligosaccharides with DP of up to 5. L. reuteri preferentially metabolized short-chain oligosaccharides, whereas bifidobacteria preferentially metabolized higher oligosaccharides. Results of this study allow the modification of the biological and technological functionality of commercial IMO by adjustment of the degree of polymerization and will thus facilitate the application development for IMO.

Influence of isomalto-oligosaccharides on intestinal microbiota in rats.

AIMS: Isomalto-oligosaccharides (IMO) with α(1 --> 6) and α(1 --> 4) glucosidic linkages are produced by enzymatic conversion of starch. IMO are only partially digestible but data on their influence on intestinal microbiota are limited. It was the aim of this study to investigate the effect of IMO diet on intestinal microbiota and short-chain fatty acids production (SCFA) in rats. METHODS AND RESULTS: Three groups of F344 rats, each consisting of six animals, were fed IMO, inulin or a control diets for six weeks. A qualitative assessment of the intestinal microbiota was achieved by PCR-denaturing gradient gel electrophoresis (DGGE). Major bacterial taxa were quantified by quantitative PCR (qPCR), and SCFA were measured using gas chromatography. Quantitative PCR demonstrated that lactobacilli were one of the dominant bacterial taxa in faecal samples from rats. IMO increased the number of lactobacilli and the total number of intestinal bacteria in rats fed IMO compared with animals receiving control and inulin diets. Furthermore, PCR-DGGE with lactobacilli-specific primers showed an altered biodiversity of lactobacilli in rats fed IMO compared with control diet. CONCLUSIONS: IMO selectively stimulates lactobacilli and increases their diversity in rats. SIGNIFICANCE AND IMPACT OF STUDY: Isomalto-oligosaccharides specifically stimulate growth of intestinal lactobacilli in a rat model system.

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility.

AIMS: This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef-processing industry, and to determine their heat resistance. METHODS AND RESULTS: Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90-99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D(60) values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D(60) values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log(10) CFU g(-1) in ground beef patties cooked to an internal temperature of 71°C. CONCLUSIONS: Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef-processing facilities. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathogen interventions in current commercial beef slaughter may select for extremely heat-resistant strains of E. coli.

Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria.

AIMS: To determine structure-function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB). METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 6 hydroxybenzoic and 6 hydroxycinnamic acids were determined with Lactobacillus plantarum, Lactobacillus hammesii, Escherichia coli and Bacillus subtilis as indicator strains. The antibacterial activity of phenolic acids increased at lower pH. A decreasing number of hydroxyl groups enhanced the activity of hydroxybenzoic acids, but had minor effects on hydroxycinnamic acids. Substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic, but not of hydroxycinnamic, acid. Metabolism of chlorogenic, caffeic, p-coumaric, ferulic, protocatechuic or p-hydroxybenzoic acids by L. plantarum, L. hammesii, Lactobacillus fermentum and Lactobacillus reuteri was analysed by LC-DAD-MS. Furthermore, MICs of substrates and metabolites were compared. Decarboxylated and/or reduced metabolites of phenolic acids had a lower activity than the substrates. Strain-specific metabolism of phenolic acids generally corresponded to resistance. CONCLUSIONS: The influence of lipophilicity on the antibacterial activity of hydroxybenzoic acids is stronger than that of hydroxycinnamic acids. Metabolism of phenolic acids by LAB detoxifies phenolic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: Results allow the targeted selection of plant extracts for food preservation, and selection of starter cultures for fermented products.

Metabolic pathway of α-ketoglutarate in Lactobacillus sanfranciscensis and Lactobacillus reuteri during sourdough fermentation.

AIM: To identify metabolites of α-ketoglutarate (α-KG) in Lactobacillus sanfranciscensis and Lactobacillus reuteri in modified MRS and sourdough. METHODS AND RESULTS: Lactobacillus sanfranciscensis and L. reuteri were grown with additional α-KG in mMRS and in wheat sourdough. In mMRS, α-KG was used as an electron acceptor and converted to 2-hydroxyglutarate (2-OHG) by both organisms. Production of 2-OHG was identified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography (GC). Crude cell extracts of L. sanfranciscensis and L. reuteri grown with or without α-KG exhibited OHG dehydrogenase activity of 6.3 ± 0.3, 2.3 ± 0.9, 1.2 ± 0.2, and 1.1 ± 0.1 mmol l(-1) NADH (min x mg protein)(-1), respectively. The presence of phenylalanine and citrate in addition to α-KG partially redirected the use of α-KG from electron acceptor to amino group acceptor. In wheat sourdoughs, α-KG was predominantly used as electron acceptor and converted to 2-OHG. CONCLUSIONS: Lactobacillus sanfranciscensis and L. reuteri utilize α-KG as electron acceptor. Alternative use of α-KG as amino group acceptor occurs in the presence of abundant amino donors and citrate. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of α-KG as electron acceptor in heterofermentative lactobacilli impacts the formation of flavour volatiles through the transamination pathway.

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis.

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Effects of feeding fermented wheat with Lactobacillus reuteri on gut morphology, intestinal fermentation, nutrient digestibility, and growth performance in weaned pigs.

Feeding fermented feed to weaned pigs may improve nutrient digestibility and gut health and thereby reduce diarrhea incidence. Effects of feeding wheat grain fermented for 24 h with were evaluated with 36 weaned pigs (7.3 kg BW). Fermented wheat grain contained (DM basis) 14.2% CP, 0.45% chemically available Lys, and 7.8% NDF, whereas unfermented wheat grain contained 16.4% CP, 0.45% chemically available Lys, and 9.9% NDF. Pigs were fed 6 mash wheat-based diets balanced for water content during 2 phases: Phase 1 diets for 1 wk (d 0-7) with 20% unfermented or fermented wheat and, subsequently, Phase 2 diets for 2 wk (d 8-21) with 50% unfermented or fermented wheat. The 6 diets were unfermented wheat (CTRL), unfermented and chemically acidified wheat (ACD), fermented wheat with TMW1.656 and 10% sucrose, fermented wheat with TMW1.656 and 5% glucose + 5% fructose, fermented wheat with LTH5794 and 10% sucrose, and fermented wheat with LTH5794 and 5% glucose + 5% fructose. Diets were formulated to provide 2.5 and 2.4 Mcal NE/kg and 5.3 and 5.0 g standardized ileal digestible Lys/Mcal NE for Phase 1 and 2 diets, respectively. Feeding fermented wheat reduced ( < 0.05) apparent total tract digestibility (ATTD) of diet DM (84.7 vs. 85.4%), GE (84.4 vs. 85.3%), and CP (81.8 vs. 83.6%) for d 15 through 21 compared with the CTRL and ACD diets. Weaned pigs fed fermented wheat diets had lower ( < 0.05) ADFI than pigs fed the CTRL and ACD diets for d 0 through 7. The ADFI, ADG, and G:F did not differ between pigs fed fermented and unfermented diets. Concentrations of acetic, propionic, and branched-chain fatty acids and total VFA in feces increased ( < 0.05) for pigs fed fermented wheat diets containing exopolysaccharides (EPS). However, VFA did not differ in ileal digesta. Villus height in the duodenum and jejunum increased in pigs fed fermented wheat without EPS ( < 0.05) compared with pigs fed fermented wheat with EPS. However, pigs fed the CTRL and ACD diets had longer ( < 0.05) villi and deeper crypts in the ileum than pigs fed fermented wheat. The ratio of villus height to crypt depth did not differ in the 3 segments of small intestine of weaned pigs. In conclusion, feeding fermented wheat grain diets to weaned pigs did not affect gut morphology, intestinal fermentation, growth performance, and ATTD of nutrients; however, EPS stimulated hindgut fermentation and may promote health benefits.

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments.

The inactivation by 200-400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pHin), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pHin during pressure application but improved the ability to maintain a high pHin after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pHin and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane deltapH correlates to cell death and glu stabilizes the pHin of E. coli during post-pressure incubation.

A fuzzy logic-based model for the multistage high-pressure inactivation of Lactococcus lactis ssp. cremoris MG 1363.

The high-pressure inactivation (200 to 600 MPa) of Lactococcus lactis ssp. cremoris MG 1363 suspended in milk buffer was investigated with both experimental and theoretical methods. The inactivation kinetics were characterised by the determination of the viable cell counts, cell counts of undamaged cells, LmrP activity, membrane integrity, and metabolic activity. Pressures between 200 and 600 MPa were applied, and pressure holding times were varied between 0 and 120 min. Experiments were carried out in milk buffer at pH values ranging between 4.0 and 6.5, and the effect of the addition of molar concentrations of NaCl and sucrose was furthermore determined. The inactivation curves of L. lactis, as characterised by viable cell counts, exhibited typical sigmoid asymmetric shapes. Generally, inactivation of the membrane transport system LmrP was the most sensitive indicator of pressure-induced sublethal injury. Furthermore, the metabolic activity was inactivated concomitant with or prior to the loss of viability. Membrane integrity was lost concomitant with or later than cell death. For example, treatments at 200 MPa for 60 min in milk buffer did not inactivate L. lactis, but fully inactivated LmrP activity and reduced the metabolic activity by 50%. The membrane integrity was unaffected. Thus, the assay systems chosen are suitable to dissect the multistep high-pressure inactivation of L. lactis ssp. cremoris MG 1363. A fuzzy logic model accounting for the specific knowledge on the multistep pressure inactivation and allowing the prediction of the quantities of sublethally damaged cells was formulated. Furthermore, the fuzzy model could be used to accurately predict pressure inactivation of L. lactis using conditions not taken into account in model generation. It consists of 160 rules accounting for several dependent and independent variables. The rules were generated automatically with fuzzy clustering methods and rule-oriented statistical analysis. The set is open for the integration of further knowledge-based rules. A very good overall agreement between measured and predicted values was obtained. Single, deviating results have been identified and can be explained to be measurement errors or model intrinsic deficiencies.

On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli.

The outer membrane (OM) of Gram-negative bacteria provides a protective barrier for natural occurring inhibitors. Pressure mediated OM permeabilisation therefore contributes to the elimination of Escherichia coli and Salmonella by pressure preservation processes. Pressure mediated inactivation, sublethal injury, and membrane permeabilisation of E. coli were determined using two strains differing in their barotolerance. Pressure treatment of E. coli TMW 2.427 at 300, 500 and 600 MPa for 40 min resulted in a 0, 1, and greater 6 log decrease of viable cell counts, respectively. The kinetics of OM and cytoplasmic membrane permeabilisation after pressure treatment were determined by staining of pressure treated cells with the fluorescent dyes propidium iodide (PI) and 1-N-phenylnaphtylamine (NPN), respectively. A slight increase of PI fluorescence was observed at conditions resulting in a greater 6 log decrease of viable cell counts only. In contrast, increased NPN fluorescence indicating OM permeabilisation was observed prior to cell death and sublethal injury. An on-line assay for determination of pressure mediated OM damage based on NPN fluorescence was established to distinguish between reversible and irreversible OM damage. Generally, the same degree of outer membrane damage was observed by either on line or off line determinations. However, whereas reversible membrane damage occurred fast and in thermodynamic equilibrium with pressure conditions, irreversible outer membrane damage was a time dependent process.

Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin.

Metabolic and functional properties of probiotic lactic acid bacteria (LAB) in the human gastro-intestinal ecosystem may be related to certain beneficial health effects. In this study, lactobacilli of either intestinal or fermented food origin were compared in their capability to survive low pH and bile, in their metabolic activity in the presence of bile salts and mucins, as well as in their potential to attach to enterocyte-like CaCO-2 cells. Food fermenting bacteria especially strains of the species Lactobacillus plantarum showed high tolerance to the consecutive exposure to hydrochloric acid (pH 1.5-2.5) and cholic acid (10 mM). Growth in and deconjugation of glycocholic (5 mM) and taurocholic acids (5 mM), as demonstrated for all lactobacilli of intestinal origin, was detected for food fermenting strains of the species L. plantarum, but not L. paracasei and L. sakei. Degradation of mucins was not observed for lactobacilli. Adhesion to the intestinal epithelial cell line CaCO-2 was demonstrated for several food fermenting bacterial strains in vitro. Soluble factors in the spent culture supernatants from intestinal and fermented food lactobacilli but not staphylococci cross reacted and synergized with cell wall components to promote adhesion to CaCO-2 cells. A competitive role of fecal bacteria on the adhesion of lactobacilli to CaCO-2 cells was demonstrated. In conclusion we have shown that metabolic and functional properties of intestinal lactobacilli are also found in certain bacteria of fermented food origin.

Resistance of Escherichia coli and Salmonella against nisin and curvacin A.

We have determined the effects of the following factors on the resistance of Gram-negative bacteria against nisin and curvacin A: (i) chemotype of the lipopolysaccharide (LPS), (ii) addition of agents permeabilizing the outer membrane, (iii) the fatty acid supply of the growth medium, and (iv) the adaptation to acid and salt stress. Bacteriocin activity was determined against growing and resting cells as well as protoplasts. All smooth strains of Escherichia coli and Salmonella enterica serovar Typhimurium were highly resistant towards the bacteriocins, whereas mutants that possess the core of the LPS, but not the O antigen, as well as deep rough LPS mutants were sensitive. Antibiotics with outer membrane permeabilizing activity, polymyxin B and polymyxin B nonapeptide, increased the sensitivity of smooth E. coli towards nisin, but not that of deep rough mutants. Incorporation of 1 g l(-1) of either oleic acid or linoleic acid to the growth media greatly increased the susceptibility of E. coli LTH1600 and LTH4346 towards bacteriocins. Both strains of E. coli were sensitive to nisin and curvacin A at a pH of less than 5.5 and more than 3% (w/v) NaCl. Adaptation to sublethal pH or higher NaCl concentrations (pH 4.54 and 5.35 or 4.5% (w/v) NaCl) provided only limited protection against the bacteriocidal activity of nisin and curvacin A. Adaptation to 4.5% (w/v) NaCl did not result in cross protection to bacteriocin activity at pH 4.4, but rendered the cells more sensitive towards bacteriocins.

Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins.

The effect of food components and ecological factors on the activities of nisin, sakacin P and curvacin A was evaluated. Lactobacillus curvatus, Listeria innocua, Salmonella and Escherichia coli including E. coli O157:H7 were used as target organisms. Lecithin, casein, and divalent cations were antagonists of the bacteriocins at 0.1%, 0.1% and 10 mmol l(-1), respectively. A decrease in pH as well as the presence of EDTA, propyl-parabene or NaCl at concentrations of 0-1 mmol y(-1), 0-0.16 g l(-1), and 0-6% (w/w), respectively, increased the activity of all bacteriocins. These compounds as well as a pH < 5.5 rendered the Gram-negative target organisms sensitive against bacteriocins. Of practical importance is the respective effect of NaCl at concentrations > 5% which are achieved in fermentation and ripening processes, e.g. in production of fermented sausages. A characteristic response was observed for each of the bacteriocins. It is suggested that bacteriocins of lactic acid bacteria are effective against a wide range of microorganisms including E. coli O157:H7 if applied in combination with other preservative principles prevailing in foods.

Effect of bacteriocin-producing lactobacilli on the survival of Escherichia coli and Listeria in a dynamic model of the stomach and the small intestine.

The survival of Lactobacillus curvatus LTH 1174 (bac ) and (bac ) in combination with Escherichia coli LTH 1600 or Listeria innocua DSM20649 during transit through a dynamic model of the human stomach and small intestine (GIT model) was studied. Furthermore, we determined the digestion of curvacin A during gastro-intestinal transit and the effect of this bacteriocin on microbial survival. Lb. curvatus is rapidly killed in the gastric compartment at pH < 2.0, and less than 0.01% of the cells delivered to the small intestinal compartments were recovered from the ileal compartment of the model. Meat exerted a protective effect against the lethal action of bile against Lb. curvatus. The sensitivity of E. coli to acid depended on the aeration of the preculture and decreased in the order anaerobic > strongly agitated > agitated. Lactic acid and curvacin A enhanced the lethal effect of low pH on E. coli. Accordingly, cells from strongly agitated cultures were killed faster in the gastric compartment of the GIT model than those from agitated cultures, and inactivation was accelerated in the presence of curvacin A. E. coli tolerated the bile concentrations prevailing in the small intestinal compartments of the model. The survival of Listeria innocua in the GIT model was comparable to that of Lb. curvatus. The curvacin A produced by Lb. curvatus LTH1174 (bac+) killed > 90% of the L. innocua within 10 min after mixing of the cultures. Curvacin A was not degraded in the the gastric compartment, and could be detected in the ileal compartment during the first 180 min upon addition of the meal.