Survival of new probiotic strains with anti-inflammatory & anti-obesity effects used in non-fat yogurt and low-fat Cheddar cheese making

Transcription

1 Survival of new probiotic strains with anti-inflammatory & anti-obesity effects used in non-fat yogurt and low-fat Cheddar cheese making Veronique Demers-Mathieu, Ph.D. Department of Microbiology & Medicine Laval University

2 Presentation plan Introduction: - Obesity, inflammation, intestinal microbiota, probiotics and dairy products Hypothesis Methods & Results 1) Genetic & phenotypic identification and selection of probiotic isolates 2) Anti-inflammatory (in vitro) & anti-obesity (in vivo) effects of probiotics 3) Manufacturing of probiotic Cheddar cheese & yogurt Conclusion & Perspectives

3 Prevalence of obesity in adults (BMI >30 kg/m 2 ), billion adults worldwide are overweight (312 million of them are obese) 155 million children worldwide are overweight or obese Research is urgently needed to prevent and control obesity.

4 Obesity & Adipose tissue inflammation Number and size of adipocytes = secretion of pro-inflammatory cytokines > Adipose tissue macrophage infiltration > visceral fat (obesity) induced chronic inflammation (Catalàn et al., 2007)

5 Potential mechanisms of probiotic to prevent obesity Produce metabolites = intestinal permeability (inhibit the passage of LPS = metabolic endotoxemia). Stimulate immune cells by activation of anti-inflammatory pathways (cytokines, IgA). Create physical barrier against pathogen or produce bacteriocins that inhibits microbe invasion. Reinforce intestinal barrier integrity by increase production of junction proteins or mucus. Le Barz et al. (2005)

6 Viability of probiotics in dairy products Probiotics: Live microorganisms, when administered in adequate amounts confer a health on the host (FDA/WHO, 2001) Cheese Mesophilic strains Salt (water activity) ph Ripening time Viability depends : Strain and species Manufacturing steps Nutrients available Oxygen and organic acids Metabolism and adaptability Yogurt Thermophile starter (S. thermophilus & Lb. bulgaricus) ph Storage time The serving size of a product should contain at least one billion of probiotic (Health Canada & FDA)

7 Hypothesis New probiotics, with anti-inflammatory and anti-obesity properties, will survive during manufacture and storage of dairy products if their technological properties are well characterized and adapted to manufacturing processes. Their anti-obesity effect will be retained in dairy products since these ones protect them during gastrointestinal transit.

8 Identification and selection of new probiotic isolates 1) Isolate strains from dairy products and/or adult & baby feces (200) 2) Identify genus by sequencing the 16S rdna gene (>98%) - Lactobacillus (40) et Bifidobacterium (10) 3) Evaluate their resistance to acid and bile salts (30) 4) Identify species : - B. animalis ssp lactis identified by qpcr (Random primers ) (10) - Lactobacillus identified by rplb gene (20) 5) Differentiate strains with RAPD profiles (30) 6) Determine their technological properties (30) : - Compatibility of probiotic with lactic acid bacteria (Spectrophotometry) - Growth and proteolytic capacities (Pearce test) - Survival in dairy products (cheese slurry)

9 Anti-inflammatory and anti-obesity effects 1) Determination of anti-inflammatory effect (in vitro) (30 isolates) Macrophages cell line J774.1 treated with bacterial extracts (heat-killed bacteria). Nitric oxide (NO) and cytokines measured by Griess nitrite test and immunology multiplex assay. 5 candidates with anti-inflammatory effect were selected! 2) Determination of anti-obesity effect (in vivo) (5 isolates) Eight-week-old C57Bl/6J male mice (n=12, 108 mice). Mice were fed with a high-fat-high-sucrose (HFHS: induced obesity) diet. One group were fed with chow diet (healthy control) without probiotic in milk. Animals were treated by daily gavage with probiotic milk received daily doses of 1 x 10 9 cfu in 110 μl of milk. Mice fed with chow diet (healthy control) Mice fed with HFHS diet during 8 weeks Treated with milk WP Treated with milk WP Treated with Milk Bf26 Treated with Milk Bf141 Treated with Milk Lb38 Treated with Milk L79 Treated with Milk Lb102

10 Manufacturing steps of yogurt and Cheddar cheese A) Non-fat yogurt making Thermophilic starter + probiotic + yogurt milk B) Low-fat Cheddar cheese making Mesophilic starter + probiotic + cheese milk Coagulation Fermentation (41 C until ph 4.65) Cooking Stirred yogurt Wheying off ph 6.2 Cooling Storage (4 C) C) Bacterial count & incubation - MRS bile ( %) agar with bile extracts (0.15 % w/v) (37 C) - Anaerobic condition for 72 h Cheddarisation Salting (3.5% S/M) Pressing (275.9 kpa), ph 5.4 Ripening (4 C)

11 Effect on murine macrophage nitric oxide and cytokine production NO production (%) a L79 Lb102 Lb38 Bf26 Bf141 Control LPS Basal IL-10 Concentartion (%) b L79 Lb102 Lb38 Bf26 Bf141 Control LPS Basal Effect of heat-killed probiotic strains on (a) NO (pro-inflammatory nitric oxide) and (b) IL-10 (antiinflammatory cytokine) production by macrophages in basal conditions (normal cell) and in LPS-stimulated macrophage cells (inflammatory condition). Heat-killed of all strains NO production in LPS-stimulated cells. IL-10 production in basal and in LPS-stimulated cells (except L79). Anti-inflammatory effect : Bf141, Bf26 & Lb38 > Lb102 > L79

12 Effect on the weight of mice fed with HFHS Bifidobacteria Ctr WP Lactobacillus Ctr WP Bf141 Lb102, Lb38 Bf26, Bf141: B. animalis; Lb38: Lb. plantarum; L79: Lb. paracasei/casei; Lb102: Lb. rhamnosus Body weight gain of mice after 8 weeks. Chow: mice fed with chow diet supplemented of milk WP; HFHS : mice fed with high-fat-high-sucrose (HFHS) diet supplemented of milk WP ; Bf26, Bf141, Lb38, Lb79 or Lb102 : mice fed with HFHS supplemented of each probiotic strain milk. Body weight gain of mice supplemented with probiotic milks < control mice (WP). Anti-obesity effect: Bf141, Lb102 and Lb38 > L79 and Bf26.

13 Effect on White Adipose Tissue (WAT) (a) ewat (b) iwat (c) rpwat White Adipose Tissue masses of (a) epididymal (ewat) (b) inguinal (c) retroperitoneal of mice treated with probiotic milk by daily gavage during 8 weeks. For mice fed with HFHS, ewat, iwat & rpwat weight of mice supplemented with probiotic milks < control mice (WP) Anti-obesity effect: Bf141, Lb38 & Lb102 > L79 & Bf26. For control mice, WATs weight of mice fed chow diet < mice fed HFHS diet.

14 Viability of probiotic strains in Cheddar cheese Population log (cfu g-1) Mi bere afc besa afsa d+1 d+2 d+14 d+30 d+60 d+120 Bf26, Bf14: B. animalis sp lactis Lb38: Lb. plantarum L79: Lb. paracasei/lb. casei Lb102: Lb. rhamnosus Period Bf26 Bf141 Lb38 L79 Lb102 Evolution of probiotic strain populations during production and ripening of low-fat cheeses (Mi: Initial milk; bere: before adding rennet; afc: after cooking; besa: before salting; afsa: after salting). Cheese composition being similar its effect on the viability of probiotics should be similar. The population of Lb38 and L79 was stable, while that of Bf26, Bf141 and Lb102 was reduced during cooking step and ripening time (60 d). The population of all bifidobacteria stayed stable, while that of all lactobacilli was decreased during salting step.

15 Viability of probiotic strains in yogurt 8.5 Population log (cfu g-1) Bf26, Bf141: B. animalis sp lactis Lb38: Lb. plantarum L79: Lb. paracasei/lb. casei Lb102: Lb. rhamnosus 6.0 im afferm afsm d+1 d+14 d+30 d+60 Time (days) Bf26N Bf141N Lb38N L79N Lb102N Evolution of probiotic strain populations during the production and storage of low-fat yogurts; im: Initial milk, afferm: after complete fermentation; afsm: after stirring, d: days. Yogurt composition being similar its effect on the viability of probiotics should be similar. The population of L79 and Lb102 was not affected, while that of Bf141, Bf26 and Lb38 was decreased during storage.

16 Conclusion & perspectives New probiotic strains had anti-inflammatory (in vitro) and anti-obesity (in vivo) effects. Their effects were higher with Bf141, Lb102 and Lb38. The heat-killed of probiotic strains induced a decrease in NO production and were able to increase cytokine IL-10 production (except L79) in macrophages. The weight gain and visceral fat masses in mice were more reduced with probiotic milks than control milk without probiotic. All probiotics were at the targeted count (10 9 per 50 g portion), but bifidobacteria counts dropped below 10 9 during ripening, while the lactobacilli counts remained stable or increased. Yogurts were formulated to contain 10 7 cfu g -1 of probiotic for at least 30 days of storage. After 60 days, this count was maintained in the case of lactobacilli, but not in the case of bifidobacteria. Studies are in process to evaluate the effects of these probiotics on the parameters associated with inflammation (cytokines and LPS levels) and on the composition of gut microbiota in mice.

17 Acknowledgments People involved in my project : Dr. Daniel St-Gelais Dr. Julie Audy People who helped me during my Ph.D.: Students: Mélanie Le Barz, Noémie Daniel, Sabrine Naïmi et Vincent Banville Research assistants: Annie Caron, Sophie Turcot Gaétan Bélanger, Denis Bélanger et Nancy Graveline, Stéphanie Bernard (Ultima). Dr. Ismail Fliss Dr. André Marette Dr. Geneviève Pilon Dr. Émilie Laurin Trainees: Laura Klein & Anne-Sophie Picard Dr. Denis Roy Granting agencies : NSERG Ultima Agropur