Making Wheat Technically Gluten Free: New Research in Sourdough Methods. Marco Gobbetti

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1 Making Wheat Technically Gluten Free: New Research in Sourdough Methods Marco Gobbetti Boston, November 9 11, 2014

2 A mixture of flour (wheat, rye, rice, etc ) and water, fermented by lactic acid bacteria and yeasts, which are responsible for its capacity to leaven a dough, while contemporarily and unavoidably acidifying it (Gobbetti, Trends Food Sci. Technol.)

3 How sourdough lactic acid bacteria and yeasts contribute to the organoleptic features of baked goods Acidification Proteolytic activity Moderate increase of the volume (heterofermentative lactic acid bacteria) Synthesis of volatile compounds Improvement of the dough gas retention Decrease of the rate of bread staling Synthesis of volatile compounds Increase of the volume

4 Biodiversity of the Italian sourdoughs Minervini et al., Applied Environ Microb, 78: L. plantarum, L. paralimentarius, Leuc. mesenteroides L. plantarum, L. paralimentarius L. sanfranciscensis, L. paralimentarius L. sanfranciscensis, L. gallinarum L. plantarum, L. casei, L. sanfranciscensis, P. inopinatus L. sanfranciscensis, L. plantarum, Lc. lactis, L. brevis, C. humilis L. sanfranciscensis, L. plantarum, C. humilis L. plantarum, P. pentosaceus W. cibaria, W. confusa P. pentosaceus L. plantarum, L. brevis, Lc. lactis, Carasau W. paramesenteroides, Leuc. mesenteroides L. sanfranciscensis L. sanfranciscensis L. sanfranciscensis, L. plantarum, L. brevis, L. rossiae, P. argentinicus L. plantarum, Leuc. citreum, L. sanfranciscensis, Leuc. citreum L. sanfranciscensis, E. durans, C. humilis L. sanfranciscensis, L. sakei, Leuc. mesenteroides L. spicheri, L. paralimentarius, K. exigua, K. barnetti L. sanfranciscensis, L. paralimentarius, L. namurensis

5 The first sourdough "library" (Saint-Vith, Belgium) in the world

6 Future therapeutic approaches for celiac disease treatment (Caputo et al., Enzyme Research doi /2010/174354; Gujral et al., World J Gastroenterol. 18: ) Therapeutic agent - Glutenenases and endoprotease Hydrolysis of toxic gliadin - Prolyl endopeptidase - Sourdough lactobacilli - Lyophilised bacteria (VSL#3) - Hexapeptide derived from zonula Prevention of gliadin absorbtion Larazotide occludens toxin ofvibrio cholera - Synthetic polymer poly - Anti-gliadin IgY - Dihydroisoxazoles ttg2 inhibitor - Cinnamoyltriazole - Aryl β-aminoethyl ketones - Three deamidated peptides derived Peptide vaccination from Wheat α-, ω-gliadin, β-hordein - Human hookworm inoculation Mechanism - HLA-DQ2 blocker Modulate immune response - Interleukin blocker - R-spondin-1 Restore intestinal architecture

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8 The struggle of our research team against the gluten 18,000 6,000 Residual gluten (ppm) 2,480 < Time (year)

9 Pool 1 (ca. log 9/ml) St 1 2 Lb. brevis 14G Lb. alimentarius 15M Lb. hilgardii 51B Lb. sanfranciscensis 7A Pool 2 + (ca. log 8/ml) ϖ -gliadins α, β, ϖ -gliadins Lb. sanfranciscensis E14 Lb. sanfranciscensis E21 Lb. sanfranciscensis 174 Lb. sanfranciscensis 13 Lb. sanfranciscensis A1 Lb. sanfranciscensis Fungal proteases (200 ppm) (CAD) Alb.-Glob. Glutenins Gliadins hydrolized gliadins (SLP) Glutenins Water salt soluble St, European gliadin standard; 1, chemically acidified dough (CAD); 2, fermented dough (20% wheat flour) with pool 1, 2 and proteases (SLP) Organic nitrogen concentration of spray-dried flour R5-ELISA SLP < 10 ppm

10 Mechanisms of gluten proteolysis Fungal Prt Peptides Gluten Free amino acids Opp Dpp Dtp T Peptides H + Cell wall Membrane Cytoplasm LAB Peptidases (PepQ, PepX, PepO, PepN, PepP)

11 33 mer L-Q-L-Q-P-F-P-Q-P-Q-L-P-Y-P-Q-P-Q-L-P-Y-P-Q-P-Q-L-P-Y-P-Q-P-Q-P-F PepN/PepO/PEP L Q L-Q-P F-P-Q-P-Q L-P-Y-P-Q-P-Q L-P-Y-P-Q-P-Q L-P-Y-P-Q-P-Q-P-F PepN/PepX L Q P F-P Q-P-Q L-P Y-P Q-P-Q L-P Y-P Q-P-Q L-P Y-P Q-P Q-P-F PepQ/PepX F P Q-P Q L P Y P Q-P Q L P Y P Q-P Q L P Y P Q P Q-P F PepQ Q P Q P Q P Q P De Angelis et al. 2010, Appl Environ Microbiol 76:508 H + Amino acids

12 Other cereals Durum wheat cultivars Ciccio Colosseo Duilio Simeto Gluten < 10 ppm Other species Barley Rye Oat Gluten > 10 ppm Svevo

13 Viability of Caco-2/TC7 cells (De Angelis et al. 2006a) A 540 nm ctr PT rye LAB PT rye Intracellular F-actin reorganization (IEC-6) ** * Time (hours) ** * Zonulin Release (IEC-6) zonulin(ng/mg protein) 4 3,5 3 2,5 2 1,5 1 0,5 0 Control GliadinA Gliadin C Time 0 time 120 Tissue epithelial electrical resistance Delta Tissue Resist ance (Om hs/cm Ti me (m in ) CD3 + intraepithelial lymphocytes infiltration con tr ol Glut en A gluten C (De Angelis et al. 2006b) CD3+ cells/mm epithelium Agglutination test (K562) 0 VSL#3 CAD Medium (De Angelis et al. 2006b) Synthesis of NO (Caco-2) 10 nanomoles/mg prot/ml * ** (Di Cagno et al., 2004; De Angelis et al. 2006a, 2006b) Fas expression (biopsies) Caspase 3 activity (Caco-2) Caspase-3 activity (micromoles/min/ml) * ** ctr ctr LAB PT rye LAB PT rye 0 ctr ctr LAB PT rye LAB PT rye (De Angelis et al. 2006) (De Angelis et al. 2006a)

14 Clinical Challenge 1: Department of Pediatric, Sapienza - University of Roma Sweet baked goods (200 g) corresponding to 100 g of processed wheat flour which contained 10 g of hydrolyzed gluten Clinical challenge - Eight CD patients (age median 13, range 8-17 years), - Daily consumption of ca. 200 g of Sweet baked goods, - The challenge lasted 60 days (A) Total serum IgA IgA -AGA Serology analyses (A) and intestinal permeability (B) at 0 (t0), 30 (t1), and 60 (t2) days of the clinical challenge (B) IgG -AGA IgA -ttg

15 Clinical Challenge 2: Pediatrics and European Laboratory for the Study of Food Induced Diseases University of Naples, Federico II, Italy Biscuits (200 g) corresponding to 100 g of processed wheat flour which contained 8 g of hydrolyzed gluten Clinical challenge - Sixteen CD patients (age median 19, range years), - Daily consumption of ca. 200 g of biscuits, - The challenge lasted 60 days Greco et al. 2011, Clin Gastroenterol Hepatol 78:1087 Density of σδ + intraepithelial lymphocytes in jejunal biopsy from patient 4 F.I. at the beginning (panel A) and after 60 day of challenge (panel B) (A) (B) TIME ttg EMA MARSH ttg GRADE MARSH CD3 GRADE γδ CD25 CD3 γδ CD (pool S2 + proteasi fungine) F.I. t 0 NFBG 1.6 (controllo) NFBG T0 (controllo) A.An t 60 0 Negative 1.0 T T I.C. A.Am t 0 30 t 60 0 Light 1.9 positivity Negative 1.1 T T T1 0.9 T R.R. S.R. t 0 30 t 60 0 Positive 0.3 Negative 0.3 T T T T I.I. F.I. t 0 60 t 60 0 Positive 0.5 Light 0.3 positivity T0 >200 T T3b 8.4 T C.C. G.S. t 0 60 t 60 0 Positive 0.4 Negative 0.7 T T T3b 21 T ttg, anti-tissue t 60 Positive transglutaminase >200antibodies; T3bT0, normal L.R. duodenal t 0 mucosa; Negative CD3, T1, 0.5 architecturally T1 normal duodenal t 60 mucosa Positive with increased intraepithelial T3b S1BG (pool S1) lymphocytes infiltration; and CD3, γδ CD25 are markers S.A. for intraepithelial t 0 Negative lymphocytes in 0.5duodenal mucosa T t 60 Negative 0.5 T3a P.M. t 0 Negative 1.7 T t 60 Light positivity 9.2 T S2 BG (pool S2 + proteasi fungine)

16 Clinical Challenge 3: Pediatrics and European Laboratory for the Study of Food Induced Diseases University of Naples, Federico II, Italy Hydrolized wheat flour CLINICAL CHALLENGE IN PROGRESS 10 CD patients (range years) - Serology analyses - Morphometric analysis - Immunohistochemistry Time: t 0 t 1 t 2 t 3 - t 4 t 5 - t 6 MONTHS CLINICAL CHALLENGE IN PROGRESS 30 CD patients (range years) Gene Expression Profile of Peripheral Blood Monocytes

17 Water, wheat flour, fungal proteases, selected sourdough lactic acid bacteria 1 Addition of glutenfree ingredients (rice, mais, starch, gums) (short-time baker s yeast fermentation) Wheat gluten-free baked goods 12 Spray dryer (short-time baker s yeast fermentation) Wheat gluten-free flour - Long-time - Semi-liquid sourdough fermentation (structuring agents) Patent number(s) WO A1, US A1. Giuliani et al. 2014

18 Illusion Escher, 1974

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20 The advantages Wheat gluten-free bread Naturally gluten-free bread Versus Better sensory properties Better rheology properties and extended shelf life Higher mineral, vitamin, amino acid and fiber bioavailability Lower costs Improved social life Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti Università di Bari

21 Sourdough lactic acid bacteria and functional features Modification/increase of the bioavailability of dietary fibers, minerals and vitamins Decrease/elimination of wheat and rye allergies, and intolerance/sensitivity to gluten Enhancement of antioxidant activity Gluten-free productcs and celiac disease Pre-treatment of raw materials (i.e. wheat germ and bran) and use of byproducts of milling Decrease of glycaemic index / response Synthesis of exopolysaccharides Synthesis of bioactive peptides, amino acid derivatives and biogenic compounds (Katina et al., Trends Food Sci Technol. 2005; Katina et al., Food Microbiol. 2007; Gobbetti et al. Food Microbiol. 2014)

22 Sourdough biotechnology with lactic acid bacteria and enzymes to produce safe wheat foods for people suffering from irritable bowel syndrome (IBS) and non-celiac gluten sensitivity (NCGS) WHEATFORALL Phase I (Biotechnology) Phase II (Food processing) Phase III (Clinical) A Selection and use of enzymes and sourdough lactic acid bacteria (LAB) B Fermentation by fructophilic lactic acid bacteria (FLAB) C E F Reconstitution of Bread and Faecal and urine the sourdough pasta Clinical food microbiome and microbiota and processing challenges metabolome use/propagation D Drying In vivo clinical food challenges with IBS and NCGS people Fructose fermentation Full hydrolysis of fructans Full hydrolysis of ATIs Partial hydrolysis of gluten

23 - High throughput sequencing of sourdough microbiota - - Panis erat primis virides mortalibus herbae - (Annibale Carracci, 1646)

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