Konstantinos C. Mountzouris

Effects of probiotic inclusion levels in broiler nutrition on growth performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora composition

The aim of this work was to investigate the effect of inclusion levels of a 5-bacterial species probiotic in broiler nutrition. Five hundred twenty-five 1-d-old male Cobb broilers were allocated in 5 experimental treatments for 6 wk. The experimental treatments received a corn-soybean coccidiostat-free basal diet and depending on the addition were labeled as follows: no addition (C), 10(8) cfu probiotic/kg of diet (P1), 10(9) cfu probiotic/kg of diet (P2), 10(10) cfu probiotic/kg of diet (P3), and 2.5 mg of avilamycin/kg of diet (A). Each treatment had 3 replicates of 35 broilers each. Treatment effects on broiler growth performance and biomarkers such as ileal and total tract nutrient digestibility, plasma Ig concentration, and cecal microflora composition were determined. Differences among treatments were considered significant when P < or = 0.05. Overall BW gain was significantly higher in treatment P1 (2,293 g) compared with P2 (2,163 g), C (2,165 g), and P3 (2,167 g), with A (2,230 g) being intermediate and not different from P1. Overall feed conversion ratio values were similar and significantly better for P1 (1.80) and A (1.80) compared with P2 (1.87), C (1.89), and P3 (1.92). Ileal apparent digestibility coefficients (ADC) of CP and ether extract were higher in A. Generally, treatments A and P1 showed an improved total tract ADC for DM, organic matter, ash, ether extract, and AME(n) values. The total tract ADC of CP was higher in P1, C, and P2. There were no differences between treatments regarding plasma Ig in 14- and 42-d-old broilers. Treatments P2 and P3 were effective at beneficially modulating cecal microflora composition. In particular, the lower cecal coliform concentration (log cfu/g of wet digesta) was seen in P2 (6.12) and P3 (4.90) in 14- and 42-d-old broilers, respectively, whereas at 42 d, P3 and P2 had the highest Bifidobacterium (8.31; 8.08) and Lactobacillus concentrations (8.20; 7.86), respectively. It is concluded that probiotic inclusion level had a significant effect on broiler growth responses, nutrient ADC, AME(n), and cecal microflora composition.

Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis

In this study 635 lactic acid bacteria of food origin were evaluated for their potential application as protective cultures in foods. A stepwise selection method was used to obtain the most appropriate strains for application as protective cultures in chicken meat. Specifically, all strains were examined for antimicrobial activity against various Gram positive and Gram negative pathogenic and spoilage bacteria. Strains exhibiting anti-bacterial activity were subsequently examined for survival in simulated food processing and gastrointestinal tract conditions, such as high temperatures, low pH, starvation and the presence of NaCl and bile salts. Selected strains where then examined for basic safety properties such as antibiotic resistance and haemolytic potential, while their antimicrobial activity was further investigated by PCR screening for possession of known bacteriocin genes. Two chosen strains were then applied on raw chicken meat to evaluate their protective ability against two common food pathogens, Listeria monocytogenes and Salmonella enteritidis, but also to identify potential spoilage effects by the application of the protective cultures on the food matrix. Antimicrobial activity in vitro was evident against Gram positive indicators, mainly Listeria and Brochothrix spp., while no antibacterial activity was obtained against any of the Gram negative bacteria tested. The antimicrobial activity was of a proteinaceous nature while strains with anti-listerial activity were found to possess one or more bacteriocin genes, mainly enterocins. Strains generally exhibited sensitivity to pH 2.0, but good survival at 45 degrees C, in the presence of bile salts and NaCl as well as during starvation, while variable survival rates were obtained at 55 degrees C. None of the strains was found to be haemolytic while variable antibiotic resistance profiles were obtained. Finally, when the selected strains Enterococcus faecium PCD71 and Lactobacillus fermentum ACA-DC179 were applied as protective cultures in chicken meat against L. monocytogenes and S. enteritidis respectively, a significantly reduced growth of these pathogenic bacteria was observed. In addition, these two strains did not appear to have any detrimental effect on biochemical parameters related to spoilage of the chicken meat.

Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella enteritidis

1. Probiotics are beneficial microbes that are currently considered as alternatives to antimicrobial growth promoters (AGP) in animal nutrition. In addition, they are purported to suppress pathogens in the gut via the concept of competitive exclusion (CE). The aim of this work was to evaluate the efficacy of a multistrain probiotic against Salmonella enteritidis (SE) in broilers. 2. Three hundred and four, one-day-old, male Cobb broilers were fed a corn–soybean basal diet and were allocated to four experimental treatments for 6 weeks. Three of the four treatments were challenged with SE. Treatments were: C− (Negative Control, no SE challenge), C+ (Positive control, SE challenge, no other additives), AB (SE challenge + Avilamycin used as AGP) and PFW (SE challenge + probiotic in feed and water). Morbidity, mortality, incidence of Salmonella positive broilers, cecal microflora composition and antibody response (IgA and IgG) in the blood and intestine were determined as biomarkers of probiotic CE efficacy against SE. 3. The three SE challenged treatments had a significantly higher morbidity compared to C− during the first 2 weeks of age, while there were no differences between treatments regarding mortality. Salmonella shedding was evidenced in 75 and 50% of the birds at 5 and 21 d post-challenge, respectively. Microbiological analysis of cecal digesta in 42-d-old broilers revealed that while broilers in treatment C+ were 100% Salmonella positive, the broilers in treatments PFW and AB were 50% positive and in addition they had lower Salmonella levels (CFU/g digesta) by 2⋅7 logs compared to C+. At the age of 42 d significantly higher IgA and IgG specific immune responses against SE were detected at systemic and at intestinal level only for the positive control treatment (C+). 4. In conclusion, the lack of significant levels of specific IgA and IgG against SE at systemic and intestinal level combined with the lower prevalence of SE positive broilers and the lower cecal SE levels in treatments AB and PFW compared to C+, suggest that treatments PFW and AB were efficacious at reducing SE.

Dietary probiotic inclusion level modulates intestinal mucin composition and mucosal morphology in broilers

The aim of this study was to investigate the effect of dietary probiotic inclusion level on mucin composition (monosaccharide ratio), mucosal morphometry, mucus layer staining intensity, and mucus layer thickness along the broiler intestinal tract. One-day-old male Cobb broilers were administered maize-soybean meal basal (BD) diets for 42 d and depending on the feed additive used, broilers were allocated into the following 5 experimental treatments: control C (BD, no additive), treatment P1 (10(8) colony forming units of probiotic/kg of BD), treatment P2 (10(9) cfu of probiotic/kg of BD), treatment P3 (10(10) cfu of probiotic/kg of BD), and treatment A (2.5 mg avilamycin/kg of BD). Intestinal samples from duodenum, ileum, and cecum of 14- and 42-d-old broilers were collected and analyzed. Mannose (Man) decreased linearly with increasing probiotic level in duodenum (P=0.015) and ileum (P=0.042) of 14-d-old broilers. N-Acetyl-glucosamine and galactose decreased linearly (P=0.012 and P=0.001, respectively), while fucose increased linearly (P<0.001) with increasing probiotic feed inclusion level in 42-d-old broiler cecum, with treatment A not differing from treatment C (P≥0.05). Cecal villus height and crypt depth increased linearly (P=0.016 and P=0.003, respectively) with probiotic inclusion level, with treatment A having higher (P≤0.05) values only from treatment C. Mucus layer thickness increased linearly with probiotic inclusion level in duodenum at 14 d and 42 d (P=0.007 and P=0.030, respectively). Finally, mucus layer staining intensity was influenced (P<0.001) by villus fragment (i.e., tip, midsection, and base) but not from the treatment, age, and intestinal segment examined. As a conclusion, this study provides evidence that probiotic inclusion level affects intestinal mucin monosaccharide composition, mucus layer thickness, and intestinal morphology in broilers.

Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi - Assessment of their effect on the final product and spent substrate properties

Nine agro-industrial and forestry by-products were subjected to solid-state fermentation by Agrocybe cylindracea and Pleurotus ostreatus, and the process and end-products were comparatively evaluated. Grape marc waste plus cotton gin trash was the best performing medium for both fungi, while substrate composition had a marked effect on most cultivation parameters. Biological efficiency was positively correlated with nitrogen, lignin and ash, and negatively with hemicelluloses and carbohydrate content of substrates. Spent substrates demonstrated high reductions in hemicelluloses and cellulose in contrast to lignin; fibre fractions were correlated with nitrogen, fat and ash content of initial materials, while residual mycelial biomass was affected by mushroom productivity. Mushroom proximate analysis revealed significant variations of constituents depending on the substrate. Crude protein and fat were correlated with substrates nitrogen for both species. Alternative cultivation substrates of high potential are proposed, while spent material could be exploited as animal feed due to its upgraded properties.

Feed supplementation of Lactobacillus plantarum PCA 236 modulates gut microbiota and milk fatty acid composition in dairy goats--a preliminary study.

This study aimed to evaluate the potential of a promising Lactobacillus plantarum isolate (PCA 236) from cheese as a probiotic feed supplement in lactating goats. The ability of L. plantarum to survive transit through the goat gastrointestinal tract and to modulate selected constituents of the gut microbiota composition, monitored at faecal level was assessed. In addition, L. plantarum effects on plasma immunoglobulins and antioxidant capacity of the animals as well as on the milk fatty acid composition were determined. For the purpose of the experiment a field study was designed, involving 24 dairy goats of the Damascus breed, kept in a sheep and goat dairy farm. The goats were divided in terms of body weight in two treatments of 12 goats each, namely: control (CON) without addition of L. plantarum and probiotic (PRO) treatment with in feed administration of L. plantarum so that the goats would intake 12 log CFU/day. The experiment lasted 5 weeks and at weekly time intervals individual faecal, blood and milk samples were collected and analysed. All faecal samples were examined for the presence of L. plantarum PCA 236. In addition, the culturable population levels of mesophilic aerobes, coliforms lactic acid bacteria (LAB), Streptococcus, Enterococcus, mesophilic anaerobes, Clostridium and Bacteroides in faeces were also determined by enumeration on specific culture media. In parallel, plasma IgA, IgM and IgG and antioxidant capacity of plasma and milk were determined. No adverse effects were observed in the animals receiving the lactobacillus during the experiment. Lactobacillus plantarum PCA 236 was recovered in the faeces of all animals in the PRO treatment. In addition, PRO treatment resulted in a significant (P<or=0.05) increase in LAB coupled with a significant decrease in faecal clostridia populations compared to the CON treatment. The antioxidant capacity and the concentrations of immunoglobulins IgA, IgM and IgG in goat plasma did not differ between the treatments. In contrast, milk fat composition in the PRO treatment had a significantly higher content of polyunsaturated fatty acids such as linoleic, a-linolenic and rumenic acids compared to CON, while there were no differences in milk antioxidant capacity. The results obtained in this study, indicate that the L. plantarum PCA 236 strain has displayed an interesting probiotic potential, in terms of beneficially modulating the goat faecal microbiota and milk fatty acid composition that needs to be further researched.

Effect of banana consumption on faecal microbiota: A randomised, controlled trial

Banana is a widely consumed fruit, which contains considerable amounts of potential prebiotic indigestible carbohydrates. In our randomised, controlled trial we aimed to evaluate the in vivo prebiotic effect of banana consumption on faecal microbiota. Thirty-four healthy women participated in the study, having Body Mass Index (BMI) 24-30 kg/m(2), age 19-45 years, without history of gastrointestinal disease and no antibiotic and other medication use two months prior the initiation and during the study. All women were asked to maintain their usual dietary habits for 60 days and they were randomly assigned to consume twice a day a pre-meal snack, either one medium banana, or one cup of banana-flavoured drink or one cup of water (control group). Stool samples were collected at baseline, on days 30 and 60 of intervention for enumeration of total anaerobes, bifidobacteria and lactobacilli by plate count techniques, as well as for pH and short chain fatty acids (SCFAs) measurement. Gastrointestinal symptoms were also recorded. Mean bifidobacterial levels were increased only in the banana group both at 30 and 60 days of intervention, but this change did not reach a statistical significance. No significant overall differences in the total concentrations and molar ratios of SCFAs were detected according to dietary intervention. Analysis of the gastrointestinal symptoms records revealed significantly lower bloating levels in the banana group, compared to controls, at 26-35 days (p = 0.009) and 51-60 days (p = 0.010). Banana consumption had also no adverse effects on evacuation patterns. We concluded that daily consumption of bananas is a well-tolerated eating behaviour, which may induce bifidogenesis in healthy women experiencing body weight problems.

Effects of Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici on the nematode Caenorhabditis elegans include possible antitumor activity

This study examined the effects of three lactic acid bacteria (LAB) strains on the nematode Caenorhabditis elegans. Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici were found to inhibit the development and growth of the worm. Compared to Escherichia coli used as the control, L. reuteri and P. acidilactici reduced the lifespan of wild-type and short-lived daf-16 worms. On the contrary, L. salivarius extended the lifespan of daf-16 worms when used live, but reduced it as UV-killed bacteria. The three LAB induced the expression of genes involved in pathogen response and inhibited the growth of tumor-like germ cells, without affecting DAF16 localization or increasing corpse cells. Our results suggest the possible use of C. elegans as a model for studying the antitumor attributes of LAB. The negative effects of these LAB strains on the nematode also indicate their potential use against parasitic nematodes.

Impact of beta-glucan on the faecal microbiota of polypectomized patients: A pilot study

Beta-glucans are polysaccharides present in the cell walls of higher plants, in the seeds of some cereals, and certain yeasts and fungi also produce them. It is suggested that they exhibit, among many other health benefits, protective effects against carcinogenesis in the colon, but there is not enough human data to support this. The aim of the study was to determine the effect of barley-derived beta-glucan in the gut microbiota of polypectomized patients. Subjects were randomly assigned to consume 125 g of bread per day with beta-glucan (3 g/d), or without (placebo group), for 3 months. Thirty-three polypectomized men and women (mean age 57.6 years) were recruited into the study, but only 20 completed. Subjects did not consume any probiotics, prebiotics or antibiotics 2 months prior the intervention, or during the study. Stool samples were collected at baseline, on days 30 and 90 of intervention, as well as 2 weeks after the intervention, for enumeration of total aerobes and anaerobes, coliforms, E. coli, enterococci, Bacteroides spp., Clostridium perfringens, bifidobacteria, lactobacilli and Candida spp. Faecal bacterial enzyme activity (beta-glucuronidase and beta-glucosidase), pH, faecal moisture and the concentration of volatile fatty acids in the faeces were measured. Gastrointestinal symptoms were also recorded. Overall, no significant differences were observed in bacterial viable counts between the two feeding groups. Group specific analysis for β-glucan group revealed significantly decreased total coliform counts on the 30th day of the trial compared to the baseline (p = 0.041). Clostridium perfringens concentration increased without reaching statistical significance, on the 30th day, while it decreased significantly on the 90th day of the intervention compared to the 30th day (p = 0.016). An increase was noted in the molar ratio of acetate on the 90th day of the trial compared to placebo (p = 0.018). The molar ratio of butyrate presented a trend to increase on the 30th day, which decreased (p = 0.013) on the 90th day and then increase 2 weeks after the intervention (p = 0.017) compared to placebo. A decrease was recorded in the β-glucan group in the bloating and abdominal pain score after the 30th day of the intervention (Day 30-37) compared to placebo. During β-glucan administration we did not observe any changes on beta-glucuronidase or beta-glucosidase activity, faecal pH, or on faecal moisture.

Effects of Lactobacillus acidophilus on gut microflora metabolic biomarkers in fed and fasted rats

BACKGROUND & AIMS Little is known about fasting effects on gut bacterial metabolism. As probiotics are purported to be beneficial for health, this study aimed to investigate the response of gut microbial metabolism on fasting with or without probiotic administration. METHODS Sixty male adult Wistar rats were allocated to six experimental treatments, for 6 days, arranged under three nutritional schemes namely: (a) ad libitum feeding (control), (b) fasting for 3 days and re-feeding for the remainder (re-fed) and (c) fasting for 6 days combined with parenteral liquid treatment during the last 3 days (starved). Each nutritional scheme had one non-probiotic and one probiotic treatment receiving orally Lactobacillus acidophilus. Rat caecal digesta were analyzed for bacterial enzyme activities and volatile fatty acids (VFA). RESULTS Fasted rats had significantly lower activities of alpha-galactosidase, alpha-glucosidase and beta-glucosidase and higher activities of beta-galactosidase and azoreductase compared to control and re-fed rats, irrespective of probiotic administration. Results were variable regarding cholylglycine hydrolase (CGH), while there were no differences between treatments regarding beta-glucuronidase and arylsulfatase activity. Fasted rats had significantly lower caecal VFA concentration and different fermentation patterns. L. acidophilus resulted in significantly reduced azoreductase activity and increased caecal acetate levels in fasted rats. Re-feeding appeared to restore most enzyme activities, fermentation intensity and to some extent fermentation patterns at control treatment levels. L. acidophilus resulted in significantly reduced CGH activity and increased butyrate levels in re-fed rats. CONCLUSION The results indicate a health beneficial potential of L. acidophilus in fasted and re-fed nutritional states via reduction of harmful azoreductase and CGH activities and promotion of useful VFA components for colonic function and health.