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.

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.

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.

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.

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.

Intravaginal administration of lactic acid bacteria modulated the incidence of purulent vaginal discharges, plasma haptoglobin concentrations, and milk production in dairy cows.

This investigation studied the effects of intravaginal administration of a mixture of lactic acid bacteria (LAB) on the incidence of purulent vaginal discharges (PVD), plasma haptoglobin concentrations, and milk production in dairy cows. A total of 82 pregnant primiparous and multiparous Holstein dairy cows were used in this study. Half of the cows received intravaginally 1mL of LAB at 10(10)-10(12)cfu/mL and the other half 1mL of reconstituted skim milk (i.e., carrier) (controls). Administration of LAB was conducted once per wk during 2 and 1wk before the expected day of calving and at 1, 2, 3, and 4wk postpartum. Data demonstrated that intravaginal administration of LAB decreased the occurrence of PVD at 3wk postpartum (P<0.05). Concentrations of plasma haptoglobin, an acute phase protein often associated with uterine infections, was lower in cows treated with the LAB mixture at 2wk (P<0.001) and 3wk (P<0.05) postpartum. Treatment with LAB did not improve overall pregnancy rate, but the treated multiparous cows produced more milk than their control counterparts (P<0.05), whereas no difference was observed in primiparous cows regarding milk yield (P>0.05). Overall, this is the first study demonstrating that intravaginal LAB administration lowers the incidence of PVD and enhances milk production in dairy cows. Further research is warranted to evaluate the effects of LAB on reproductive performance in a larger cohort of cows.

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.

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.

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.

The effects of pure nucleotides on performance, humoral immunity, gut structure and numbers of intestinal bacteria of newly weaned pigs.

Weaning is often stressful for piglets and accompanied by morphological, histological, microbial, and immunological changes along the digestive tract. Dietary nucleotides are bioactive compounds which have the potential to diminish weaning-associated challenges. The experiment was carried out with 5 litters each of 7 pigs (mixed sex), weaned at 20 d of age. One baseline pig per litter was slaughtered at d 0. The remaining 30 pigs were housed individually and randomly allocated to 2 dietary treatments: the control diet or the control diet supplemented with a mixture of nucleotides. Measurements of growth performance traits included ADFI, ADG, G:F, and BW. At d 17, fresh fecal samples were taken to determine bacterial numbers. On d 19 and 20, pigs were slaughtered and blood samples were analyzed for plasma immunoglobulins and intestinal samples were assessed for morphological traits. Digesta from the jejunum and cecum were collected for analysis of the microbiome. The ADFI was greater in the nucleotide treatment compared with the control treatment (P < 0.05), but ADG, G:F, and BW did not differ between treatments. Plasma IgA concentrations increased with age and were greater in the nucleotide (P < 0.05) compared with the control group. There were no treatment differences in plasma IgG and IgM, gut morphology, or intestinal and fecal bacterial counts. Supplemental nucleotides may increase ADFI but without having any impact on growth performance of the pigs. Greater plasma IgA concentrations indicate that adding nucleotides in the weaning diet supported humoral immunity. However, there was no effect of dietary nucleotide supplementation on the composition of the bacterial community in parts of the small and large intestine. Further research is warranted before the use of nucleotide as a feed additive in pig diet can be recommended.

Slowly digestible starch influences mRNA abundance of glucose and short-chain fatty acid transporters in the porcine distal intestinal tract.

The relationship between starch chemistry and intestinal nutrient transporters is not well characterized. We hypothesized that inclusion of slowly instead of rapidly digestible starch in pig diets will decrease glucose and increase short-chain fatty acid (SCFA) transporter expression in the distal gut. Weaned barrows (n = 32) were fed 4 diets containing 70% starch [ranging from 0 to 63% amylose and from 1.06 (rapidly) to 0.22%/min (slowly) rate of in vitro digestion] at 3 × maintenance energy requirement in a complete randomized block design. Ileal and colon mucosa was collected on day 21 to quantify mRNA abundance of Na(+)-dependent glucose transporter 1 (SGLT1), monocarboxylic acid transporter 1 (MCT1), and Na(+)-coupled monocarboxylate transporter (SMCT). Messenger RNA was extracted and cDNA manufactured prior to relative quantitative reverse transcription PCR. Data were analyzed using the 2(-Δ ΔC)(T) method, with ß-actin and glyceraldehyde-3-phosphate dehydrogenase as reference genes, and regression analysis was performed. As in vitro rate of digestion decreased, SGLT1 linearly increased (P < 0.05) in the ileum. Contrary to SGLT1, MCT1 tended to linearly decrease (P = 0.08) in the ileum and increased quadratically (P < 0.001) in the colon with decreasing rate of digestion. Starch digestion rate did not affect SMCT in the ileum; however, colonic SMCT quadratically decreased (P < 0.01) with decreasing rate of digestion. In conclusion, in contrast to our hypothesis, slowly digestible starch increased ileal glucose and decreased ileal SCFA transporter mRNA abundance, possibly due to an increased glucose in the luminal ileum. Effects of starch on colonic SCFA transporter mRNA abundance were inconsistent.

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.

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.

Bacterial fermentation affects net mineral flux in the large intestine of pigs fed diets with viscous and fermentable nonstarch polysaccharides.

The impact of colonic fermentation on postileal absorption of Ca, Mg, P, Cu, Fe, Mn, and Zn was investigated in 8 ileally cannulated grower pigs (initial BW = 29.1 ± 1.6 kg) according to a double 4 × 4 Latin square. A semi-purified diet was supplemented with 5.20% low viscous, low fermentable cellulose (CEL), 6.25% high viscous, low fermentable carboxymethylcellulose (CMC), 8.95% low viscous, high fermentable oat beta-glucan (LG), or 9.25% high viscous, high fermentable oat beta-glucan (HG), resulting in 5% actual added nonstarch polysaccharides (NSP) in the diets. Because of the intrinsic mineral content in LG and HG, pigs receiving the LG and HG diets had a greater (P < 0.05) daily intake of Mg, P, Cu, Fe, Mn, and Zn, and also Ca for the HG diet compared with the CEL and CMC diets. Different amounts of minerals reached the large intestine for the 4 diets as indicated by the 60 to 86% less (P < 0.05) ileal flow of Ca, Mg, P, and Fe for CMC compared with CEL and HG. Apparent mineral retention was generally less (P < 0.05) for CEL compared with CMC. Regression analyses indicated that postileal flux of Ca, Cu, and Zn were related (R(2) = 0.24 to 0.99; P < 0.05) to short-chain fatty acid (SCFA) concentrations in feces. Postileal Ca absorption was negatively related (R(2) = 0.24; P < 0.05) to fecal total SCFA concentrations when SCFA concentrations were greater than 95.6 mmol/kg of DM. Furthermore, postileal Zn (R(2) = 0.99; P < 0.001) and Cu secretion (R(2) = 0.94; P < 0.001) decreased with increasing total SCFA concentrations in feces. Additionally, postileal secretion of Fe increased (R(2) = 0.20; P < 0.05) with increasing 16S rRNA gene copies of Enterobacteriaceae in feces, whereas the secretion of Cu decreased (R(2) = 0.25; P < 0.01) with increasing gene copies of Enterobacteriaceae. Overall, the apparent retention of Ca, Mg, and P was 27 to 85% less (P < 0.05) for CEL and HG than for CMC, whereas the apparent retention of Fe, Mn, and Zn was less (P < 0.05) for CEL than for CMC, LG, and HG. In conclusion, these data indicate that the stimulation of fermentation by dietary NSP affects net mineral flux in the large intestine that, in turn, can influence mineral excretion in feces. Additionally, negative effects of CEL on apparent retention may increase the daily requirement for minerals of grower pigs.

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.

Glutamine deamidation by cereal-associated lactic acid bacteria.

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.

Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave.

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment.

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.

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.