Bile acids (BA) have emerged as signalling molecules regulating intestinal physiology. The importance of intestinal microbiota in production of secondary BA, for example, lithocholic acid (LCA) which impairs enterocyte proliferation and permeability, triggered us to determine the effects of oral probiotics on intestinal BA metabolism. Piglets were weaned at 28 d of age and allocated into control (CON, n 14) or probiotic (PRO, n 14) group fed 50 mg of Lactobacillus plantarum daily, and gut microbiota and BA profile were determined. To test the potential interaction of LCA with bacteria endotoxins in inducing damage of enterocytes, IPEC-J2 cells were treated with LCA, lipopolysaccharide (LPS) and LCA + LPS and expressions of genes related to inflammation, antioxidant capacity and nutrient transport were determined. Compared with the CON group, the PRO group showed lower total LCA level in the ileum and higher relative abundance of the Lactobacillus genus in faeces. In contrast, the relative abundances of Bacteroides, Clostridium_sensu_stricto_1, Parabacteroides and Ruminococcus_1, important bacteria genera in BA biotransformation, were all lower in the PRO than in the CON group. Moreover, PRO piglets had lower postprandial glucagon-like peptide-1 level, while higher glucose level than CON piglets. Co-administration of LPS and LCA led to down-regulated expression of glucose and peptide transporter genes in IPEC-J2 cells. Altogether, oral L. plantarum altered BA profile probably by modulating relative abundances of gut microbial genera that play key roles in BA metabolism and might consequently impact glucose homoeostasis. The detrimental effect of LCA on nutrient transport in enterocytes might be aggravated under LPS challenge.

Lactic acid bacteria (LAB) are usually employed as alfalfa silage inoculants to obtain high-quality feed for animal husbandry. However, the effects of these inoculants are still unclear and need to be studied extensively. Therefore, the objective of this meta-analysis was to quantitatively summarize published research studies that assess the effects of homofermentative (HoLAB) and heterofermentative lactic acid bacteria (HeLAB) on fermentation parameters, nutritive value, microbiological composition and aerobic stability of alfalfa silage. PubMed, ScienceDirect and Scopus have been screened for articles published from 1980 to 2018. The criteria for inclusion were: randomized and controlled trials using alfalfa silage and published in peer-reviewed journals. It was found that inoculation with LAB decreased silage pH, neutral detergent fibre, acid detergent fibre and ammoniacal nitrogen, while it increased dry matter and crude protein compared to control in the pooled raw mean difference random-effect model. Additionally, LAB inoculation decreased acetate, propionate, ethanol and butyrate concentrations, whereas it increased lactate. In addition, inoculants reduced the counts of yeasts and moulds. Lastly, LAB inoculation improved aerobic stability. To the best of our knowledge, this is the first meta-analysis that aims at comparing the application of HoLAB and HeLAB for alfalfa silage. In the pool estimate, positive effects attributable to the application of microbial silage inoculants were found in most of the evaluated parameters; supporting the importance of applying both types of inoculants to improve forage preservation for the livestock industry.

The current study aimed to select the strains of lactic acid bacteria (LAB) isolated from forage cactus plants and silage and assess their effects on silage fermentation and aerobic stability. Forty wild isolates from plant and cactus silage, classified as LAB, were evaluated for metabolite production and identified by 16S ribosomal DNA sequencing. These wild isolates were identified as Lactobacillus plantarum, Weissella cibaria, Weissella confusa and Weissella paramesenteroides and the LAB populations differed among the silage. The use of microbial inoculants did not influence gas or effluent losses in forage cactus silage. The silage inoculated with the microbial strain GP15 showed the highest number of LAB populations. The amounts of water-soluble carbohydrates (WSC) and ammonia nitrogen differed among the silage. The silage inoculated with the GP1 strain presented the highest WSC. Populations of enterobacteria and yeasts and moulds were below the minimum detection limit (<2.0 log cfu/g silage) in all the silage studied. The predominant action of inoculants was to maximize dry matter recovery of the silage, which could be the criterion adopted to select the strains of LAB for use as inoculants in Opuntia silage.

The in vivo effects of administering free and microencapsulated Lactobacillus plantarum LIP-1 cells (2·0×109 colony-forming units/d) were evaluated in high-fat-diet-induced hyperlipidaemic rats. Results from real-time quantitative PCR targeting to LIP-1 cells showed a higher colon colonisation count of LIP-1 in the rats receiving microencapsulated cells compared with free cells (P<0·05). Moreover, the microencapsulated LIP-1 treatment resulted in a more obvious lipid-lowering effect (P<0·05). Meanwhile, their faecal samples had significantly less lipopolysaccharide-producing bacteria (especially Bilophila, Sutterella and Oscillibacter) and mucosa-damaging bacteria (Bilophila and Akkermansia muciniphila), whereas significantly more SCFA-producing bacteria (P<0·05) (namely Lactobacillus, Alloprevotella, Coprococcus, Eubacterium and Ruminococcus) and bacteria that potentially possessed bile salt hydrolase activity (Bacteroides, Clostridium, Eubacterium and Lactobacillus), and other beneficial bacteria (Alistipes and Turicibacter). Further, Spearman’s correlation analysis showed significant correlations between some of the modulated gut bacteria and the serum lipid levels. These results together confirm that microcapsulation enhanced the colon colonisation of LIP-1 cells, which subsequently exhibited more pronounced effects in improving the gut microbiota composition of hyperlipidaemic rats and lipid reduction.

The enzymatic hydrolysis of cheese whey was optimised using the enzymes iZyme, Alcalase or Flavourzyme under different conditions. Hydrolysates supplemented with commercial nutrients were evaluated as fermentation broths to produce DL-3-Phenyllactic acid (PLA) from phenylalanine (Phe) by Lactobacillus plantarum CECT-221. Optimised hydrolysates were obtained using Flavourzyme at 50 °C and 100 rpm during 12 h, and assayed in 250 ml Erlenemyer flasks using different proportions of vinasses as economic nutrient. The process was then scaled up using a 2 litres Bioreactor working under the continuous modality. Under the intermediate dilution rate of 0·0207 h−1 0·81 ± 0·026 mM of PLA and 38·8 ± 3·253 g/l of lactic acid were produced. A final evaluation revealed that lactic acid, and bacteriocins exerted the highest inhibitory effect among the extracted components of cell-free supernatants.

The immunomodulatory effects of live or non-viable lactic acid bacteria have been extensively investigated. We reported that oral intake of heat-killed Lactobacillus plantarum L-137 (HK L-137) augmented innate and acquired immunity in mice and human subjects. To examine the effects of HK L-137 intake on upper respiratory tract infection (URTI) symptoms and immune functions in human subjects, a randomised, double-blind, placebo-controlled, parallel study was conducted in subjects with high psychological stress levels. A total of seventy-eight healthy subjects (thirty-three men and forty-five women; mean age 50·6 years) with scores of >41 on eighteen-item subscales of psychological distress in the Brief Job Stress Questionnaire were randomly assigned to receive a tablet containing HK L-137 (10 mg) or a placebo tablet daily for 12 weeks. The URTI symptoms were rated once daily on the validated twenty-one-item Wisconsin Upper Respiratory Symptom Survey-21. Immune functions, such as concanavalin A-induced proliferation and percentages of interferon (IFN)-γ- and IL-4-producing CD4 T cells of peripheral blood mononuclear cells (PBMC), and serum IFN-β concentrations were measured every 4 weeks. URTI incidence was significantly lower in the HK L-137 group than in the control group. URTI incidence, duration and severity, and duration of medication showed significant negative correlations with duration of HK L-137 intake. The percentage change from baseline of concanavalin A-induced proliferation of PBMC was significantly greater in the HK L-137 group than in the control group. These findings suggest that daily HK L-137 intake can decrease URTI incidence in healthy subjects, possibly through augmentation of immune functions.

Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide–cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

Previous studies have indicated that supplementation with probiotic bacteria may improve lipid metabolism. The present study was aimed at investigating the effects of a mixture of three strains of Lactobacillus plantarum (CECT 7527, CECT 7528 and CECT 7529) on cholesterol-lowering efficacy in hypercholesterolaemic patients. A total of sixty volunteers (thirty participants in the placebo group and thirty counterparts in the L. plantarum group), aged 18–65 years old, participated in a controlled, randomised, double-blind trial. The study group received one capsule daily containing 1·2 × 109 colony-forming units of Lactobacillus strains in a unique dose; the placebo group consumed the same product without bacteria for 12 weeks. A significant reduction of 13·6 % in plasma total cholesterol (TC) levels was observed after 12 weeks of consumption in the L. plantarum group when compared with the placebo group. The lipidic outcomes were also analysed based on TC values at baseline: low initial values (LIV, 2000–2500 mg/l) v. high initial values (HIV, 2510–3000 mg/l). In the HIV group, the L. plantarum treatment showed a reduction after 12 weeks of consumption compared with the placebo group in TC, LDL-cholesterol (LDL-C) and oxidised LDL-C (17·4, 17·6 and 15·6 %, respectively). In the LIV, the L. plantarum treatment only showed a reduction after 12 weeks of consumption when compared with the placebo group in TC (9·4 %). The present results showed that the biofunctionality of L. plantarum (CECT 7527, CECT 7528 and CECT 7529) is proportional to the cardiovascular risk of the patient, having a better effect in patients with higher levels of cholesterol.

Bovine mastitis is the most important infectious disease on dairy farms. Conventional antibiotic therapy is often unsatisfactory and alternative treatments are continually under investigation. Lactobacillus (Lb.) perolens CRL 1724 and Lactobacillus plantarum CRL 1716 were previously isolated from milk of dairy cows and selected according to their potential probiotic properties. In the present work the in-vitro capacity of Lactobacillus strains to adhere to bovine teat canal epithelial cells (BTCEC) and to inhibit and co-aggregate 14 mastitis-causing pathogens (MCPs) was investigated. The effect of Lb. perolens CRL 1724 after intramammary inoculation in lactating cows was evaluated through determination of clinical signs of mastitis, milk appearance, somatic cell counts and Lb. perolens CRL 1724 recovery from milk. Lb. perolens CRL 1724 was able to inhibit 12 of 14 MCPs (85·7%) in vitro, especially those considered to be major pathogens. In addition, Lb. perolens CRL 1724 co-aggregated with all of them. Lb. plantarum CRL 1716 was able to inhibit 7 of 14 MCPs (50%) in vitro and showed co-aggregation ability similar to Lb. perolens CRL 1724. Lb. perolens CRL 1724 showed a higher efficacy of adhesion to BTCEC (values of percentage of adhesion and adhesion index of 75% and 14·4, respectively) than Lb. plantarum CRL 1716 (37% and 7·4, respectively). Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after the inoculation of 106 cfu/ml. The udders presented a normal aspect and there were no changes in the appearance of the milk. The results obtained will serve as the basis for further trials to evaluate the potential of Lb. perolens CRL 1724 to be included in a non-antibiotic formulation for the prevention of bovine mastitis.

A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin–producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.

Although a large number of in vitro and in vivo tests have confirmed that taking probiotics can improve the intestinal barrier, few studies have focused on the relationship between probiotics and the intestinal epithelial barrier in hyperbilirubinaemia. To investigate the effects of and mechanisms associated with probiotic bacteria (Lactobacillus plantarum; LP) and unconjugated bilirubin (UCB) on the intestinal epithelial barrier, we measured the viability, apoptotic ratio and protein kinase C (PKC) activity of Caco-2 cells. We also determined the distribution and expression of tight junction proteins such as occludin, zonula occludens (ZO)-1, claudin-1, claudin-4, junctional adhesion molecule (JAM)-1 and F-actin using confocal laser scanning microscopy, immunohistochemistry, Western blotting and real-time quantitative PCR. The present study demonstrated that high concentrations of UCB caused obvious cytotoxicity and decreased the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer. Low concentrations of UCB inhibited the expression of tight junction proteins and PKC but could induce UDP-glucuronosyltransferases 1 family-polypeptide A1 (UGT1A1) expression. UCB alone caused decreased PKC activity, serine phosphorylated occludin and ZO-1 levels. After treatment with LP, the effects of UCB on TER and apoptosis were mitigated; LP also prevented aberrant expression and rearrangement of tight junction proteins. Moreover, PKC activity and serine phosphorylated tight junction protein levels were partially restored after treatment with LP, LP exerted a protective effect against UCB damage to Caco-2 monolayer cells, and it restored the structure and distribution of tight junction proteins by activating the PKC pathway. In addition, UGT1A1 expression induced by UCB in Caco-2 cells could ameliorate the cytotoxicity of UCB.

Two greatly related Lactobacillus plantarum bacteriophages (named FAGK1 and FAGK2) were isolated from Kefir grains of different origins. Both phages belonged to the Siphoviridae family (morphotype B1) and showed similar dimensions for head and tail sizes. The host range of the two phages, using 36 strains as potential host strains, differed only in the phage reactivity against one of them. The phages showed latent periods of 30 min, burst periods of 80±10 min and burst size values of 11·0±1·0 PFU per infected cell as mean value. Identical DNA restriction patterns were obtained for both phages with PvuI, SalI, HindIII and MluI. The viral DNA apparently did not present extremes cos and the structural protein patterns presented four major bands (32·9, 35·7, 43·0 and 66·2 kDa). This study reports the first isolation of bacteriophages of Lb. plantarum from Kefir grains and adds further knowledge regarding the complex microbial community of this fermented milk.

The aim was to investigate how blueberry husks and/or mixtures of probiotic strains (Lactobacillus crispatus DSM16743, L. gasseri DSM16737 and L. plantarum DSM15313 (LABmix), or Bifidobacterium infantis DSM15159 and DSM15161 (BIFmix)) affect colonic fermentation, caecal counts of lactobacilli, bifidobacteria and Enterobacteriaceae, body weight gain, and blood concentrations of carboxylic acids (CA) and ammonia in rats. Dietary fibres in blueberry husks were fermented to 61 % in colon, and the elevated faecal excretion of fibre and protein contributed to the high faecal bulking capacity (1·3). The caecal pool of CA was higher in rats fed blueberry husks than the fibre-free control (P < 0·05), and the propionic acid proportion was higher in the distal colon than in the control group (P < 0·05). Probiotics lowered the caecal amount of CA when added to blueberry husks (P < 0·001), while the propionic acid proportion was higher with LABmix (P < 0·01) than blueberry husks only. The propionic acid and butyric acid concentrations in blood were higher in rats fed blueberry husks and probiotics than those fed blueberry husks only (P < 0·01), implying that the absorption of these acids was facilitated by the bacteria. The caecal counts of lactobacilli, bifidobacteria and Enterobacteriaceae were lower in rats fed blueberry husks than the control diet (P < 0·05). The body weight gain was partly influenced by the caecal tissue and contents weights, and BIFmix decreased the ammonia concentration in blood (P < 0·05). We conclude that colonic fermentation is differentially affected by dietary fibre and probiotics, which may be of importance when developing foods with certain health effects.

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S–23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2·5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

Lactic acid bacteria have traditionally been thought to have immunomodulating effects. To verify this property, Lactobacillus plantarum was orally administered to mice (5 × 107 colony forming units (c.f.u.)), prior to infection with Listeria monocytogenes in order to evaluate the host resistance against an infectious micro-organism and to better define the influence of L. plantarum on such responses. Balb/c mice were treated daily with L. plantarum or received PBS (sham-treated mice as controls) for 4 weeks. Subsequently, mice were intravenously infected with a clinical isolate of L. monocytogenes. Our study revealed that the administration of L. plantarum did not significantly increase the survival (P = 0·13) of mice (fifteen in each group) after L. monocytogenes infection (106 c.f.u./ml), whereas a sub-lethal dose of L. monocytogenes (105 c.f.u./ml) was eliminated from liver and spleen 5 d after the challenge in both L. plantarum- and sham-treated mice (n 5). Nevertheless, the levels of IL-1β and IL-6 from sera of orally administered L. plantarum were drastically reduced at 0, 4 (P < 0·01) and 6 d after L. monocytogenes infection, whereas TNF-α production was unaltered. In conclusion, administration of L. plantarum reduced pro-inflammatory IL production after challenge with L. monocytogenes, although it did not significantly impact the survival of mice. We speculate that L. plantarum could exert anti-inflammatory effects, which may represent an important model to reduce inflammatory disorders. Therefore, further studies in human subjects should determine the role of L. plantarum as an immunomodulatory micro-organism and its relationship in the host protection to pathogens.

Herbage from the first regrowth of perennial ryegrass based swards was direct-ensiled following treatment with either an inoculant of Lactobacillus plantarum (Ecosyl, Imperial Chemical Industries pic) at 3·0 1/t, formic acid (850 g/kg) at 2·9 1/t, or no additive (control). During harvesting, alternate loads of inoculant material were treated with an absorbent polymer (ammonium polyacrylamide) at the rate of 1 kg/t herbage and ensiled in separate 100-t capacity silos. The mean dry matter (DM) and water soluble carbohydrate concentrations of herbage used for the four treatments was 157 and 120 g/kg respectively. Lactic acid levels post ensiling increased more rapidly in inoculant-treated herbage than with the other treatments. Formic acid and inoculant-treated silages were well preserved whereas control and inoculant-plus-polymer silages were only moderately well preserved. Losses of DM during ensilage were greater with the formic acid treatment with DM recovery values of 0·78, 0·72, 0·76 and 0·73 for the control, formic acid, inoculant and inoculant-plus-polymer silages respectively. Treatment of herbage with an absorbent polymer prior to ensiling resulted in a proportional reduction in effluent volume of 0·2 whereas formic acid treatment increased effluent flow by 0·28. The silages were evaluated in a changeover design experiment with two periods each of 4 weeks duration, using 24 British Friesian dairy cows. Animals were housed in individual stalls and in addition to the treatment silages, received 5 kg/day of supplement containing 193 g crude protein per kg DM. Silage intakes were increased by proportionately 0·10, 0·14 and 0·05 respectively with the formic acid, inoculant and inoculant-plus-polymer treatments compared with the control. The increased silage intakes with the inoculant treatment were reflected in an increased milk yield of 1·1 kg milk per day whereas formic acid and inoculant-plus-polymer treatments had no significant effect, although formic acid treatment did result in a significant increase in milk fat concentration. There were no major differences between treatments in energy or nitrogen digestibility, when determined on a complete ration basis. In conclusion, a large milk yield response was obtained as a result of treatment of herbage with inoculant prior to ensiling and this resulted from increased silage and hence energy intake. Treatment with formic acid increased silage and energy intake but had no effect on milk energy output.