1. Traditional food: functional properties and perspectives Diana Di Gioia 1 Department of Agroenvironmental Science and Technology Alma Mater Studiorum-University of Bologna - Italy

2. 2 The Agriculture Faculty of UNIBO Microbiology Agronomy- crop physiology D. Di Gioia, B. Biavati, G. Dinelli, I. Marotti, Department of Agroenvironmental Science and Technology

3. What is a “traditional food”? 3 For the European consumers: “food products that are well-known, that one can eat often and that were eaten already by our grandparents ”. The European legal framework: Protected Designation of Origin (PDO), along with Protected Geographical Indication (PGI) and Traditional Speciality Guaranteed (TSG), are systems created by the European Union in 1992 to promote and protect regional food products within the EU. Different countries have differences in the definition and characterization of Traditional Food Products (TFPs) depending on cultural heritage, regional specificities and many other factors Food produced, processed and prepared in a given geographical area using recognised know-how The geographical link must occur in at least one stage of the production It does not refer to the origin but highlights traditional character either in the composition or means of production

4. Unique definition for TFPs? 4 ("The Ricotta eaters") Vincenzo Campi, ~1585 traditional vs modern Time immutable? Mortadella: sausage (PGI) The Romans called the sausage "farcimen mirtatum" (myrtle sausage), because the sausage was flavored with myrtle berries. No history? Hamburger Mongols (1200) Tartare stick WimpyWimpy (1930) Immigrants (1850)

5. Content 5 The aim is to give an overview of the functional properties of TFPs The health image of TFPs: the role of plant ingredients The health image of TFPs: the role of microorganisms Challenges and perspectives of the TFP sector The role of TFPs against obesity

6. The health image of TPFs: the role of plant ingredients 6 Recent surveys have evidenced that EU consumers associate several attributes to TFPs : are the presumed “health benefits” of TFPs confirmed by scientific data? Traditional ingredients (for example old vs modern crop genotypes) Traditional production of ingredients (for example organic vs conventional cropping systems)

7. 7 Comparison of functional properties (bioactive compounds) of old and modern varieties of common and durum wheat Modern cultivar Introduced after the “green revolution” (1955-1970) Extraordinary yield and optimal rheological properties Adapted to “high input” farming (fertilizers, herbicides) Relatively low genetic variability Modern varieties Agronomic drawbacks (not dwarf, lodging, weed resistance) Adapted to “low input” farming Old cultivar Old varieties The health image of TPFs: the role of plant ingredients

8. 8 Lignans are important bioactive compounds in wheat Lignans pharmacological properties:  anti-inflammatory activity  immunosuppressive activity  anti-oxidant activity  antiviral actions  protective effect against estrogen-related disease such as osteoporosis  anti-cancer properties (reduced incidence rate of breast, prostate and colon cancer)

9. The health image of TPFs: the role of plant ingredients 9 Lignan content (µg/g dry weight) in 10 soft wheat varieties Mean content: 2.60 ± 0.21 Mean content: 5.00 ± 1.30 (tot 1 )= sum of SECO and ANYDROSECO Dinelli et al. (2007), Electrophoresis, 28, 4212-4219. 2.67 ± 0.041.09 ± 0.071.58 ± 0.15n.d. Eureka 2.29 ± 0.160.98 ± 0.081.30 ± 0.15n.d. Nobel 2.66 ± 0.121.02 ± 0.081.64 ± 0.08n.d. Mieti 2.77 ± 0.161.22 ± 0.101.55 ± 0.13n.d. Bolero TotalPIN+ISOSECO (tot 1 ) SYRHINARC 5.84 ± 0.111.28 ± 0.071.53 ± 0.121.34 ± 0.110.84 ± 0.060.85 ± 0.08Inallettabile 4.38 ± 0.101.09 ± 0.081.33 ± 0.08n.d.1.00 ± 0.100.96 ± 0.12Andriolo 3.36 ± 0.041.01 ± 0.101.34 ± 0.11n.d.1.00 ± 0.08n.d.Sieve 4.64 ± 0.141.22 ± 0.071.44 ± 0.12n.d.0.95 ± 0.101.02 ± 0.11 Verna 7.11 ± 0.051.86 ± 0.092.46 ± 0.141.35 ± 0.141.59 ± 0.101.20 ± 0.08 4.67 ± 0.111.42 ± 0.101.25 ± 0.13n.d.0.93 ± 0.071.08 ± 0.08 Gentil Rosso aristato Gentil Rosso mutico

10. The health image of TPFs: the role of plant ingredients 10 45.5 ± 3.0 Modern cv. (6) 60.5 ± 3.5 313.6 ± 15.9 Old cv (17) Free polyphenols (mg/100g) Bound polyphenols (mg/100g) Total polyphenols (mg/100g) 263.1 ± 15.7 178.0 ± 12.4 223.5 ± 12.1 0.41 ± 0.12 Modern cv. (6) 0.51 ± 0.13 1.92 ± 0.22 Old cv (17) Free flavonoids (  mol/g) Bound flavonoids (  mol/g) Total flavonoids  mol/g) 1.41 ± 0.25 0.84 ± 0.20 1.25 ± 0.21 Modern cv. (6) Old cv (17) 149.9 ± 26.5 232.9 ± 14.0 Total carotenoids (  g/100g) 4.09 ± 0.83 Resistant starch (%) 2.83 ± 0.49 Lutein (  g/100g) 91.9 ± 9.5 70.0 ± 8.5 Dinelli et al. (2009), submitted to Food chem

11. The health image of TPFs: the role of plant ingredients 11 They did not undergo strict breeding programs and thus they are characterized by a wide genetic variability; General considerations on old crop genotypes: Modern crop breeding programs are often aimed to reduce the crop carbon investment in secondary metabolites (=functional compounds); Old genotypes are an important biodiversity source to be used in organic farming and to increase the content of health- promoting compounds. Plant breeding: the art and science of changing the genetics of plants for the benefit of humankind (Poehlman & Sleeper, Breeding Field Crops, 1995)

12. The health image of TPFs: the role of plant ingredients 12 Wounding cholorogenic acid coumarins lignin Dixon & Paiva, The plant cell, 1995, 7; 1085-1097. Low Nitrogen flavonoids isoflavonoids UV Light anthocyanidins flavonols/ flavones psoralens Pathogen Attack isoflavones coumarins flavonols stilbenes Low Phosphate anthocyanidins Low Iron phenolic acids It is recognized that highly-intensive agricultural practices (conventional farming) may reduce the natural production of secondary metabolites involved in plant defense; Under organic farming conditions, plants are easily subject to stress conditions  elicitation effects on secondary metabolites  increment in functional compounds.

13. 13 Beneficial bacteria in the food chain can have: a protective role (protective cultures), to reduce the growth of pathogenic and/or spoilage microrganisms in food  BIOPRESERVATION a probiotic role (probiotic cultures), i.e. conferring a beneficial effect upon the host, either on a farm animal through animal feed, or on humans, through different food products. The health image of TPFs: the role of microorganisms BIOPRESERVATION: exhended storage life and enhanced safety of foods using their natural or controlled microflora and (or) their antibacterial products

14. 14 LAB and bifidobacteria are the best candidates for use as protective & probiotic cultures:  Have been used since the beginning of history as starter cultures  Present in almost all fermented foods-vegetables, meat products, dairy products  Are part of the natural microbiota of both farm animals and humans  Have a long history of consumption and safe use Lactobacillus LactococcusStreptococcus Pediococcus Oenococcus Bifidobacterium The health image of TFPs: the role of microorganisms

15. 15 Relevance of LAB in different fermented food products Wine Beer Cider Sauerkraut Fermented milk Cheese Fermented sausages The health image of TFPs: the role of microorganisms

16. 16 The health image of TFPs: food preparation Protective cultures: applications in traditional dairy products Yogurt  The starter cultures used can be considered protective cultures  Starter cultures can also have a role as probiotic cultures Brie Mascarpone Soft Cheese Soft cheeses  Control of pathogens such as Listeria spp. Cheddar Pecorino Semi hard & hard cheeses  Growth control of undesirable spoilage bacteria Parmiggiano Reggiano

17. 17 Protective cultures: applications in traditional meat products Beef jerkyBresaola Semi dry sausages Cured meat, dry or semi dry  Control of pathogens such as Campylobacter spp. Cooked or ground meat  Control of pathogens such as Campylobacter spp. Cured meatsGround meatPoultry The health image of TFPs: the role of microorganisms

18. 18 The health image of TFPs: the role of microorganisms Protective/probiotic cultures: applications in traditional meat products the use of antibiotics in feed to prevent colonization of Campylobacter spp. has been prohibited in Western Europe alternative strategies to contrast the infection from this bacterium in animals have to be identified and used there is an increasing interest in the development of alternative therapies based on the use of probiotics derived from the natural intestinal flora to contrast the infection of this bacterium “Control and Prevention of Emerging and Future Pathogens at Cellular and Molecular Level throughout the Food Chain” Campylobacteriosis is the most frequently reported zoonotic disease in Europe

19. 19 MAIN OBJECTIVES OF THE WORK : To select protective and probiotic cultures of LAB and bifidobacteria able to: - inhibit pathogenic organisms common in the food industry (Campylobacter jejuni) - survive food processing conditions (presence of salt, high temperature, lack of nutrients) - survive the passage in the gastrointestinal tract of animals or humans (low pH, presence of bile salts) Study of the role of protective and probiotic cultures in food The health image of TFPs: the role of microorganisms To evaluate the capacity of orally administered probiotics (selected as active against Campylobacter jejuni ) to colonise the intestinal tract of broiler chickens and assess their effect on the Campylobacter jejuni population

20. 20 92 strains (LAB and bifidobacteria) were tested against: C. jejuni CIP 70.2 C jejuni LMG 8842 C. jejuni 221/05 The health image of TFPs: the role of microorganisms Selection of protective and probiotic cultures of LAB and bifidobacteria able to inhibit Campylobacter jejuni strains

21. 21 Screening methods: agar spot assay with LAB and bifidobacteria cultures well diffusion agar assay using neutralized cell-free cultures supernatants of the strains showing inhibitory activity Nutrient soft agar (0,5 %) containing about 10 7 cells per ml of C. jejuni Spot of culture The health image of TFPs: the role of microorganisms Lactobacillus delbrueckii ssp delbrueckii PCK 103 L. plantarum PCK 73 B. Longum PCB 133 Antimicrobial activity against Campylobacter spp.

22. 22 Inhibitory activity: well diffusion agar assay with neutralized cell-free surnatants The health image of TFPs: the role of microorganisms Campylobacter jejuni CIP 70.1 Campylobacter jejuni LMG 8842 Campylobacter jejuni 221/05 L. pentosus PCA 227-+- L. plantarum PCA 236-+- L. plantarum PCA 263-++ L. plantarum PCA 275-++ L. plantarum PCS 20-++ B. longum PCD 733B--+ Enterococcus durans PCD 103 -+- Leuconostoc mesenteroides PCK 18 --+ L. plantarum PCK 73+-+ L. delbrueckii PCK 103-++ B. longum PCB 133-++

23. 23 Survival to food processing conditions: 6% NaCl, thermal stress (50°C and 55°C), starvation stress The health image of TFPs: the role of microorganisms Heat Stress all strains survive well after 1h of treatment to 55 °C Osmotic stress all strains retain their viability after 1h in presence of 6% NaCl Starvation stress three strains after 6 hours loose more than 50% of their initial population. The others strains, after 24 hours, didn’t show a significant vitality decrease

24. 24 Survival the passage in the GIT of animals or humans pH 2.5, bile salts 1 and 2% The health image of TFPs: the role of microorganisms pH 2,5 all strains rapidly loose their viability after 1 h of treatment Bile salts 1% and 2% w/v more than 50% of the initial population survive after 4 hours of treatment LAB and Bifidobacteria strains Resistance to 2% bile salts Survival % Microencapsulation of freeze-dried cells to improve survival

25. Administration in poultry of probiotic cultures selected as active against Campylobacter jejuni Probiotic administration ( frozen cultures 10 8 -10 9 by oral gavage ) Bifidobacterium longum PCB133 Lactobacillus plantarum PCS20 Chickens hygienic condition 5 days naturalization In vivo conditions for all the trials 15 chickens each group 10 chickens chosen for faeces collection The health image of TFPs: the role of microorganisms

26. Molecular analysis Daily probiotic administration for two weeks Samples collection: T0-T1 (15 days)-T2 (20 days) DNA extraction Microbiota analysis Culture-dependent techniques Culture- independent technique Analysis Workflow to assess microorganisms vitality in faeces The health image of TFPs: the role of microorganisms Real Time-PCR

27. B. longum colonization L. Plantarum PCS20 did not Only the probiotic strain B. longum PCB133 colonised the intestinal tract of the broiler chickens and was detected in the faeces of the treatment group End of administration Culture-dependent vs culture-indipendent technique ~ 1 log difference Sensible reduction of C. jejuni (preliminary results to be confirmed) The health image of TFPs: the role of microorganisms

28. 28 The health image of TFPs: the role of microorganisms  Impact of two prebiotic additives on the intestinal microbiota of poultry  Detection of pathogens in animal feed by RealTime PCR and evaluation of the survival of the microencapsulated probiotics  Application of a new synbiotic formula in the feeding of poultry with relevance to microbiological aspects and growth performance Work in progress.....

29. 29 The health image of TFPs: the role of microorganisms Interaction between probiotics and plant derived functional componds Flavonoids are a large group of plant secondary metabolites, which belong to the class/category of polyphenols. They are omnipresent in the human diet and are found in fruits, vegetables and grains. to boost the immune sytem to protect body from free radicals To kill pathogenic germs To decrease the risk of cancer Flavonoids in food are generally considered to be beneficial to consumers' health and are one of the most important categories of bioactive food components. Epidemiological observations ascribed to flavonoids:

30. Dietary glycosides FlaGlu CO2 Phenolic acid Bacterial hydrolysis Aglycones Bacterial hydrolysis Glu Fla Glucosidase Absorption Bioactive form Absorption Flavonoid bioconversion by microorganisms The health image of TFPs: the role of microorganisms

31. Gutmicroflora Bioavailability of dietary flavonoids  ability of gut microflora to degrade these compounds  glucosidase activity) Several groups of bacteria possess  -glucosidase activity (Bacteroides, Lactobacillus, Bifidobacterium) The health image of TFPs: the role of microorganisms Bifidobacterium Widely used in pharmaceutical and dairy preparations for their probiotic effects (maintain improved intestinal bacteria composition, stimulate the immune response, possible anticarcinogenic activity and protection against infections). Lactobacillus Flavonoid bioconversion by the gut microflora

32. Flavonoid glycosides Authentic standards (kaempferol 3-O-glucoside, kaempferol 3-O- xylosylglucoside, daidzin, glycitin, genistin) of the Italian common bean ecotype “Zolfino”. Seed and seedling extracts 38 bifidobacteria strains were screened for  -glucosidase activity B. catenulatum B. pseudocatenulatum B. adolescentis B. longum B. infantis B. breve 1) Screening of Bifidobacterium strains for  -glucosidase activity 2) Capability of Bifidobacterium strains to convert the flavonoid glycosides detected in common bean seeds and seedlings The health image of TFPs: the role of microorganisms Aim of the work Flavonoid bioconversion by the gut microflora

33. Bacterial surnatants Selected bacteria (pure culture) Flavonoid glycosides Standards Bean seeds Incubation (37°C, anerobiosis, 24h) Flavonoid extraction (SPE) HPLC analysis Screening of  -glucosidase activity determination of p-nitrophenyl β-D-glucopiranoside conversion in p-nitrophenol. The health image of TFPs: the role of microorganisms

34. 0.0 10.0 20.0 30.0 40.0 50.0 B7377B7304B7003 B7178B7291 B7184B7001B7875B7039 B7824 B7239B7370 B7023B7696B7792 B7031B7716 B7819 B7740B7162B7284B7305B7314B7290B7310B7308B7296B7229 B7213B7262B7249B7825B7352B7348B7302 B7254B7356B7751 β-glucosidase activity (U/g dry cell weight) = B. catenulatum = B. adolescentis = B. pseudocatenulatum = B. infantis = B. breve = B. bifidum = B. longum The health image of TFPs: the role of microorganisms Screening of  -glucosidase activity: results

35. 0.0 10.0 20.0 30.0 40.0 50.0 B7377B7304B7003 B7178B7291 B7184B7001B7875B7039 B7824 B7239B7370 B7023B7696B7792 B7031B7716 B7819 B7740B7162B7284B7305B7314B7290B7310B7308B7296B7229 B7213B7262B7249B7825B7352B7348B7302 B7254B7356B7751 β-glucosidase activity (U/g dry cell weight) * B. catenulatum * B. infantis * B. breve * B. pseudocatenulatum * B. adolescentis The health image of TFPs: the role of microorganisms  -glucosidase activity: results

36. 04812 Time (min) kaempferol 3-O-xylosylglucoside kaempferol 3-O-glucoside kaempferol 7-O-glucoside Flavonoid metabolism: selected bifidobacteria strains incubated with flavonoids extracted from “Zolfino” bean seeds Kaempferol AFTER incubation with B7003 Kaempferol-3-0-xylosylglucoside The health image of TFPs: the role of microorganisms

37. Glycosidic forms of daidzein, genistein, glycitein, kaempferol Chromatogram Seedling extract Zolfino landrace 04812 Time (min) Chromatogram after 24 h incubation with B7003 Genistein Glycitein Daidzein Kaempferol Flavonoid metabolism: selected bifidobacteria strains incubated with flavonoids extracted from “Zolfino” bean seedlings The health image of TFPs: the role of microorganisms

38. 0.00 0.05 0.10 0.15 0.20 0.25 DaidzeinGenistein Glycitein Kaempferol Degradation rate k (1/h) Metabolism of flavonoid aglycones  degradation rates following incubation with selected strains Correlation between chemical structure and rate of microbial degradation: number and position of hydroxyl groups are important structural characteristics for flavonoid degradation by human bifidobacteria: genistein and kaempferol were degraded faster than daidzein and glycitein. Daidzein Glycitein Genistein Kaempferol Marotti I. et al. J. Agric. Food Chem. 2007, 55, 3913-3919 The health image of TFPs: the role of microorganisms

39. 0 2 4 6 8 10 = L. fermentum = L. buchneri = L. kefiri = L. helveticus = L. plantarum = L. bulgaricus β-glucosidase activity (U/g dry cell weight) = L. lactis L12L7 L8 L3L2L11L13 L9 L15L10L5 L1 L14 = L. acidophilus Studies in progress: wider screening of Lactobacillus strains Screening of β-glucosidase activity in Lactobacillus spp.: preliminary results The health image of TFPs: the role of microorganisms

40. PERSPECTIVES Potentiality of investigated Lactobacillus strains as functional starter cultures for new fermented products  facilitate bioavailability Mediterranean diet: bean-based dishes in the traditional cooking  common bean is a valuable ingredient to obtain products with health benefits Selected Bifidobacterium strains  probiotic dietary adjuncts to improve the organoleptic properties and health beneficial effects of flavonoid-based products, including hypothetical common bean food derivatives The health image of TFPs: the role of microorganisms

41. 41 Roles of TFPs against “obesity” dietary abundance sedentary lifestyle Obesity is associated with significant risks of ill health

42. Roles of TFPs against “obesity” 42 Overnutrition and energy density 400 800 1200 1600 0 Energy density (kJ 100 g -1 ) Desserts Chicken Wings Fries (chips) Crispy strips Sandwiches Vegetables Biscuit Popcorn Outlet A Burger King Desserts Fries (chips) Sandwiches Fried + side Condiments, etc Sandwiches Breakfast Outlet B Jack in the Box Outlet C Mc Donald’s Muffin/Danish Fries (chips) Chicken McNuggets Dressings Breakfast Sauces Dessert/Shakes Salads Sandwiches Traditional Gambian Meals Staples Stauces Breakfast Average 1167 1087 1054 439 British diet (670) Prentice & Jebb (2003), Obesity reviews, 4, 187-194.

43. Roles of TFPs against “obesity” 43 0400800120016002000 0 1000 2000 3000 4000 5000 Traditional Gambian Market (ready dishes) Fast foods Energy density (kJ 100 g -1 ) Weight of food eaten (g) Overnutrition and energy density For assuring 8500 kJ per day If a person made a 200 g error on a diet of 1200 kJ 100 g -1 when eating fast foods just twice a week, the cumulative effect would add up to 250000 kJ per year – equivalent to almost 8 kg of adipose tissue. Prentice & Jebb (2003), Obesity reviews, 4, 187-194.

44. Roles of TFPs against “obesity” 44 Obesity predisposes the individual to the development of non- transmissible diseases The role of traditional Mediterranean diet Trichopoulou et al., (2005), BMJ, 330, 991-997. high intake of vegetables, legumes, fruit, and cereals (in the past largely unrefined); moderate to high intake of fish; low intake of saturated lipids but high intake of unsaturated lipids (olive oil) moderate intake of dairy products (mostly cheese and yogurt) low intake of meat

45. A traditional food is not “innovative” but …… …..innovations are costantly looked for to guarantee the safety of the products (ensuring safety through innovation) improve the nutritional and health properties of the food (link between nutrition and health) preserve biodiversity and sustainability 45 Perspectives and challenges of TFP sector ….. this is what the modern consumer and the modern scientist always keep in mind……

46. 46 Perspectives and challenges of TFP sector Personalized Nutrigenetic Food Age 18th 19th 20th 21th Century Future Value (Technology +Iinformation) Survival food Convenience food Essential nutrients Engineered food Mass production Functional food Chemical analysis Nutraceutical Target group Health claim Nutrigenetic food Molecular nutrition Tailor-made personal food Home-test kits Harmonization of Eastern & Western Health knowledge

47. 47 Perspectives and challenges of TFP sector Comparison Personalized Nutrigenetic Food Age Pathologic status Health status TFPs Tailor made diet

48. 48 Thank you