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antimicrobial and antioxidant activity of selected turkish spices

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master programme final project

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antimicrobial and antioxidant activity of selected turkish spices

  1. 1. BAZI TÜRK BAHARATI ÇESİTLERİNİNBAZI TÜRK BAHARATI ÇESİTLERİNİN ANTİMİKROBİYAL VE ANTİOKSİDANANTİMİKROBİYAL VE ANTİOKSİDAN AKTİVİTELERİNİN DEĞERLENDİRİLMESİAKTİVİTELERİNİN DEĞERLENDİRİLMESİ Gioacchino dell’AQUILADanışman: Yard. Doç. Dr. Duygu ALTIOK Yüksek Lisan Tezi İstanbul, 2013
  2. 2. EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANTEVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SELECTED TURKISH SPICESACTIVITIES OF SELECTED TURKISH SPICES Gioacchino dell’AQUILAAdvisor: Ass. Prof. Dr. Duygu ALTIOK İstanbul, 2013 Master Thesis
  3. 3. Introduction and Objectives
  4. 4. What are spices? FOOD AND DRUG ADMINISTRATIONFOOD AND DRUG ADMINISTRATION Any aromatic vegetable in whole, broken or ground formAny aromatic vegetable in whole, broken or ground form whose significant function in food is seasoning ratherwhose significant function in food is seasoning rather than nutritionalthan nutritional INTERNATIONAL ORGANISATION FOR STANDARDISATIONINTERNATIONAL ORGANISATION FOR STANDARDISATION The termThe term spices and condimentsspices and condiments refers to natural plantrefers to natural plant or vegetable product or mixtures that are used toor vegetable product or mixtures that are used to impart flavor, aroma and pungency to the foodimpart flavor, aroma and pungency to the food 4
  5. 5. Spice Market in Turkey FAOSTAT 2013 Turkey is the third world biggest spices producer and its market is increasing in terms of Harvested area (Ha), Yield (Hg/Ha) and Production (Ton) Turkey 79.097 *1000 $ 113.783 *1000 Kg 5
  6. 6. Definition of drug Adapted from Vaidya et al., (2008) Botanical nameBotanical name SpiceSpice DrugDrug Cinnamomum aromaticum Cinnamon Bark Myristica fragrans Mace Aril Myristica fragrans Nutmeg Endosperm Eugenia caryophyllata Cloves Flower bud Curcuma longa Turmeric Root Zingiber officinale Ginger Rhizome Laurus nobilis Bayleaf Leaves Sesamum indicum Sesam Seed, seed oil 6
  7. 7. Properties **Flavoring agentsFlavoring agents in meals, curries, bakery, pickles, processedin meals, curries, bakery, pickles, processed meats, beverages, liquors.meats, beverages, liquors. ** Enhance or vary the flavors of foods.Enhance or vary the flavors of foods. ** AAntioxidantntioxidant -- antimicrobialantimicrobial propertiesproperties againstagainst food spoilagefood spoilage ** Used asUsed as preservativespreservatives in pickle and chutney.in pickle and chutney. ** Possess importantPossess important nutritionalnutritional andand medicinal propertiesmedicinal properties.. 7
  8. 8. Basic Uses of Spices Adapted from Ravindran et al., (2002) FlavoringFlavoring Cinnamon, Allspice, Dill, Mint, Cumin, Marjoram, Basil, Anise, Mace, Nutmeg, Fennel, Sesame, Vanilla, Fenugreek, Cardamom, Celery OOdodouur maskingr masking Garlic, Savory, Bay leaves, Clove, Leek, Thyme, Rosemary, Caraway, Oregano, Onion, Coriander PungencyPungency Garlic, Bay leaves, Clove, Leek, Thyme, Rosemary, Caraway, Oregano, Onion, Coriander, Mustard, Ginger, Red pepper, Pepper ColoringColoring Paprika, Turmeric, Saffron 8
  9. 9. Chemistry of flavour FlavoFlavouurr is combination of taste,is combination of taste, aroma and texturearoma and texture derived from anderived from an overall combination of volatileoverall combination of volatile components (components (AAromaroma) and nonvolatile) and nonvolatile components (components (TTasteaste) in a spice.) in a spice. 9
  10. 10. Sensory characteristics Adapted from Farrel, (1985) FlavorFlavor SpicesSpices FlavorFlavor SpicesSpices Sulfurous Onion, Garlic Sweet Anise Cardamom Fragrant Basil Warm, fruity and camphrous Anise Bay leaf Pungent and hot Capsicum Mustard Warm, spicy, and very aromatic Coriander Ginger Pungent and sweet Cloves Cinnamon Woody Cinnamon Cloves 10
  11. 11. Colour ComponentsSource Agriculture Research Institute, United States Department Agriculture PigmentPigment TintTint SpiceSpice β- Carotene Reddish Orange Red pepper, mustard, saffron Cryptoxanthin Red Paprika, red pepper Lutein Dark red Paprika Zeaxanthin Yellow Paprika Capsanthin Dark red Paprika, red pepper Crocetin Dark red Saffron Crocin Yellowish orange Saffron Flavonoids Yellow Ginger Curcumin Orange- yellow Turmeric Chlorophyll Green Coriander, Bay leaf 11
  12. 12. SpiceSpice 100 g /KCal100 g /KCal SpiceSpice 100 g /KCal100 g /KCal Asafoetida 297 Mint 48 Cardamom 229 Mustard 541541 Coriander Seed 288 Nutmeg Fruit 472472 Cumin Seeds 356356 Nutmeg Rind 52 Garlic (Dry) 145 Bishops weed 363363 Ginger (Fresh) 67 Parsley 87 Poppy Seeds 408408 Turmeric 349349 Nutritional factsSource National Institute of Nutrition, ICMR, India 12
  13. 13. SpiceSpice Medicinal propertiesMedicinal properties Basil Anti-helminthic, antipyretic, carminative, diuretic Coriander Carminative, tonic, analgesic, antinflammatory Fennel Stimulant, carminative, stomachic Mint Stimulant, stomachic, carminative, antiseptic Oregano Stimulant, carminative, stomachic, diuretic Thyme Carminative, anti-helminthic, stomachic, tonic Medicinal properties Adapted from Mazza et al. (2010) 13
  14. 14. Thymbra spicata: ZaaterZaater Distribution: Mediterranean area Turkey: Karakekik, Zahter, Zaater Traditional uses: sorethroat, stomachache, headache, toothache and herbal teas Modern uses: food flavouring, liqueur production, perfumery, antiseptic Drug: leaf and flowering tops Constituents: carvacrol, terpinene, cymene, myrcene, thymol 14
  15. 15. Rhus coriaria: SumakSumak Distribution: Middle East – Mediterranean Turkey: Sumak Traditional uses: wound healing, flavour, antimicrobial and antiviral, antiseptic, blood flow regularator Modern uses: food flavouring, drinks, colorant, Drug: fruits Constituents: gallotannins, flavonoids, minerals 15
  16. 16. Ocimum basilicum: ReyhanReyhan Distribution: Worldwide Turkey: Reyhan, (Fesleğen) Traditional uses: rithuals, refreshing drinks, kidneys malfunctions, headache, cough Modern uses: condiment, dentistry, perfumery, antiseptic, insect repellent Drug: Stems and leaves Constituents: related to the variety, camphor, cineole, linalool, chavicol 16
  17. 17. Mentha spicata: NaneNane Distribution: Worldwide Turkey: Nane Traditional uses: chest pain, stomachache, various type of traditonal foods and salads (mercimek çorbası, taboouleh, cacık, Modern uses: aromatherapy, liqueur, confectionery, food flavouring, cosmetics Drug: stems and leaves Constituents: essential oil, menthol 17
  18. 18. Origanum vulgare: KekikKekik Distribution: Mediterranean Turkey: Kekik Traditional uses: colds, indigestion, aromatherapy, cooking Modern uses: carminative, antiseptic, cholagogue, diaphoretic, expectorant, tonic Drug: leaves and flowering stems Constituents: carvacrol, thymol, pinene, caryophyllene, cimene 18
  19. 19. 19 mg GAE /100 gr dw TEAC (µM Trolox/100 g dw) Literature Reference Origanum vulgare 0.15 ± 0.01 19.9 ± 1.00 Wojdyło et al. (2007) 0.05 ± 0.01 Lan Su (2007) 0.178 ± 0.007 Samushenkova (2012) 0.2 ± 0.01 Po-Jung (2007) 0.420 ± 0.24 11.23 ± 0.02 Ünver at al. (2009) 0.18 ± 0.09 Hossain et al. (2008) 20 ± 1.00 USDA ORAC (2006) Ocimum basilicum 0.14 ± 0.01 7.8 ± 1.00 Hinneburg et al. (2006) 0.054 ± 0.006 Samushenkova (2012) 0.054 ± 0.01 1.2 ± 0.09 Alzoreki et al. (2001) 0.09 ± 0.01 Po-Jung et al. (2007) 6.7 ± 1.00 USDA ORAC (2006) 0.028 ± 0.02 Hossain et al. (2008) Rhus coriaria 0.05 ± 0.05 8.2 ± 0.01 Ünver at al. (2009) 0.34 ± 0.03 Kosar et al. (2007) 0.5 ± 0.01 17.32 Yizhang et al. (2004) Thymbra spicata 0.04 4.9 ± 0.06 Kosar et al. (2007) 0.04 ± 0.06 7.08 ± 0.01 Ünver at al. (2009) Mentha spicata 0.03 ± 0.06 6.2 ± 0.02 Ünver at al. (2009) 1.8 ± 0.02 Phansawamtl (2008) 0.039 ± 0.03 Charanjit (2002) 0.033 ± 0.002 4.3 ± 0.03 Tawaha et al. (2007)
  20. 20. Objectives *Evaluation of the potential efficacy of spices as bio-preservatives *Establishment of investigation protocol for raw spice material *Determination of the antimicrobial activity  Agar Disc Diffusion Assay with Spice Tablets and Spice Extracts *Quantification of the antioxidant activity  Trolox Equivalent Antioxidant Capacity  Total Phenol Gallic Acid Equivalent  Total Flavonoid Catechin Equivalent 20
  21. 21. Materials and Methods
  22. 22. Materials 22 Spice samplesSpice samples Collected from Konya organic farmer's market in October 2012 >Rhus coriaria and Thymbra spicata were wild samples >Ocimum basilicum, Origanum vulgare, Mentha spicata were produced and handled following organic procedures Botanical identification Department of Pharmacognosy at Eskisehir University Preparation *cleaning from foreign bodies *separately grinding *stored in dark glass jars
  23. 23. *Total Microbial Count <Agar Spread Plate Technique <Agar Pour Plate Technique * Antimicrobial Tests <Agar Disc Diffusion Assay With Spice Tablets <Agar Disc Diffusion Assay with Spice Extracts *Antioxidant Activity Assessments <Trolox Equivalent Antioxidant Capacity (TEAC) <Total Phenolic Content (Gallic Acid Equivalent) <Total Flavonoid Content (Catechin Equivalent) Methods 23
  24. 24. Methods Preliminary operationsPreliminary operations before the experimentsbefore the experiments :: *Sterilization by autoclave of equipment and materials needed -Nutrient agar; Nutrient Broth; Phosphate Buffered Pepton Water; *Activation of culture media (E. coli; S. aureus ) and incubation *Cleaning surfaces and operator hands with 70% ETOH *Performing in sterile conditions by Bunsen flame 24
  25. 25. Total Microbial Count 25
  26. 26. Agar Disc Diffusion: Spice Tablets *100 μL of Microorganism culture is inoculated and swabbed to cover all the agar surface *By ensuring sterility each tablet is placed in the middle of Petri dishes *All test were performed in duplicate for each microorganism *Petri dishes were incubated for 24 h at 37°C before reading results 26
  27. 27. Agar Disc Diffusion: Spice Extracts 27
  28. 28. Antioxidant Activity Assessments 28
  29. 29. Antioxidant Activity Assessments 29
  30. 30. Results and Discussion
  31. 31. Total Microbial Count by Agar Pour Plate in number of CFU AGAR POUR PLATE 10-2 10-3 10-5 10-7 10-9 10-11 10-13 K 50 4 - - - - - K1 2 - - - - - - N + 40 - - - - - N1 + 35 - - - - - Z 10 - - - - - - Z1 20 - - - - - - R + + 70 35 20 10 9 R1 + + 80 40 26 12 10 S - - - - - - - S1 20 1 - - - - - 31
  32. 32. Total Microbial Count by Agar Spread Plate in number of CFU AGAR SPREAD PLATE 10-2 10-3 10-5 10-7 10-9 10-11 10-13 K 5 - - - - - - K1 - - - - - - - N - - - - - - - N1 10 - - - - - - Z 15 - - - - - - Z1 25 - - - - - - R + + 70 37 15 7 - R1 + + 80 40 12 10 - S - - - - - - - S1 - - - - - - - 32
  33. 33. Results of Total Microbial Count expressed in CFU average Pour Plate Method Spread Plate Method Dilution (1mL) CFU Dilution (0.1 mL) CFU K N Z R S K N Z R S 10-2 2.6*103 + 1.5*103 + 1*103 10-2 5*103 1*104 2*104 + - 10-3 2*103 3.7*104 - + 5*102 10-3 - - - + - 10-5 - - - 7.5*106 - 10-5 - - - 7.5*109 - 10-7 - - - 3.7*108 - 10-7 - - - 3.8*109 - 10-9 - - - 2.3*1010 - 10-9 - - - 1.3*1011 - 10-11 - - - 1.1*1012 - 10-11 - - - 8.5*1012 - 10-13 - - - 0.9*1014 - 10-13 - - - - - 33
  34. 34. Antimicrobial Activity ►Kind of spice.Kind of spice. ►Composition and concentration of spice.Composition and concentration of spice. ►Microbial species and its occurrence level.Microbial species and its occurrence level. ►Substrate composition.Substrate composition. ►Processing conditions and storage.Processing conditions and storage. 34
  35. 35. Agar Disc Diffusion: Spice Tablets Origanum vulgare (Kekik) 35
  36. 36. Agar Disc Diffusion: Spice Tablets Thymbra spicata (Zahter) 36
  37. 37. Agar Disc Diffusion: Spice Tablets Rhus coriaria (Sumak) 37
  38. 38. Agar Disc Diffusion: Spice Tablets Mentha spicata (Nane) 38
  39. 39. Agar Disc Diffusion: Spice Tablets Ocimum basilicum (Reyhan) 39
  40. 40. Agar Disc Diffusion: Spice Tablets Spice mixture ( 40
  41. 41. The experiment had the same results on the spice mixture. Presence of colonies might be related to: Different composition on fito- complex. Antimicrobial concentration Presence of resistant colonies Observed in Zahtersamples Assay on selective Culture media demonstrated no contamination Agar Disc Diffusion: Spice Tablets Spice Mixture 41
  42. 42. Spice Tablets Inhibition Zones (diameter clear zone – tablet diameter) mm S. aureus E. coli Positive Control + + A1 10 4 A2 12 6 R1 10 8 R2 8 10 Z1 17 16 Z2 14 13 N1 13 11 N2 13 11 S1 18 15 S2 20 18 K1 28 36 K2 28 35 42
  43. 43. Agar Disc Diffusion: Ethanol Origanum vulgare (Kekik) 43
  44. 44. Agar Disc Diffusion: Ethanol Rhus coriaria (Sumak) 44
  45. 45. Agar Disc Diffusion: Ethanol Thymbra spicata (Zahter) 45
  46. 46. Agar Disc Diffusion: Ethanol   S. aureus E. coli % ethanol extract 100 75 50 25 100 75 50 25 Sterility - - - - - - - - Positive 10 - - - - - - - K 17* 12 11 9 17 10.5 10 9 R 10 - - - - - - - Z 12* - - - 11.5 9 - - N 10 - - - - - - - S 15* 10 10 7 15 14 13 12 (*=values have been recorded by detracting 10 mm ethanol control inhibition zone at 100%) 46
  47. 47. Agar Disc Diffusion: DMSO S. aureus E.  coli % DMSO extract 100 75 50 25 100 75 50 25 Sterility - - - - - - - - Positive - - - - - - - - K 13 13 10 9 15 14 8 8 R 12 11 11 10 12 12 11 11 Z 14 13 12 12 13 12 9 9 N 13 12 11 11 12.5 12 12 12 S 16 15 12 11 15 14 13 12 47
  48. 48. Discussion *All the sample were active against E. coli and S. aureus * Rhus coriaria, Thymbra spicata, Origanum vulgare were the most active *Wider Inhibition Zones populated by colonies would be absent increasing the concentration *Two different inhibition zones shows that more than one compounds are charged of antimicrobial activity *Qualitative studies on the samples would clarify which components are responsible for the antimicrobial activity 48
  49. 49. Antioxidant Capacity (TEAC) R. coriaria 18,50 T. spicata 12,45 O. basilicum 6,90 O. vulgare 15,95 M. spicata 7,80 Antioxidant Activity Assessments Trolox Equivalent Antioxidant Capacity 49 Values are expressed in µM Trolox /100 g dw
  50. 50. Total Phenol Gallic Acid Equivalents (average) R. coriaria 0,409 T. spicata 0,337 O. basilicum 0,309 O. vulgare 0,3915 M. spicata 0,333 Antioxidant Activity Assessments Gallic Acid Equivalent Total Phenolic Content 50 Values are expressed in mg GAE/100 g dw
  51. 51. Catechin Equivalent  Total Flavonoid Content (average) R. coriaria 0,0775 T. spicata 0,058 O. basilicum 0,055 O. vulgare 0,067 M. spicata 0,055 Antioxidant Activity Assessments Catechin Equivalent Total Flavonoid Content 51 Values are expressed in mg CE/100 g dw
  52. 52. Antioxidant Mechanisms Free radical formation can be delayed by using chelating agents, singlet oxygen inhibitors and peroxide stabilizers. The propagation can be minimized by : Free radical scavenging (donate H) Quenching oxygen singlet (Carotenoids) Transition metal chelation (Ascorbic acid) Antioxidant complexing with radical molecule 52
  53. 53. Conclusions
  54. 54. Safety in Use of Spices ►Gaining popularity as bio-preservatives. ►GRAS (Generally Regarded As Safe). ►Natural and consumer friendly. ►Very small amount of extract is effective. ►Provides adequate shelf life and convenience. ►Sensory aspects of food are retained. 54
  55. 55. Designing new food spice Examine theExamine the efficiencyefficiency andand functionalityfunctionality of spices.of spices. Need to studyNeed to study toxicologytoxicology andand safetysafety.. InteractionsInteractions with food components.with food components. Mechanisms of actionMechanisms of action against microorganisms.against microorganisms. Influence onInfluence on nutritionalnutritional andand sensorysensory qualities.qualities. Method of applications inMethod of applications in marketable foodmarketable food.. Extraction, isolation, andExtraction, isolation, and economic productioneconomic production 55
  56. 56. Why not? *Limits use of doubtful safety artificial preservatives*Limits use of doubtful safety artificial preservatives (anti-microbial and anti-oxidants)(anti-microbial and anti-oxidants) *Prevents addition of artificial colorants.*Prevents addition of artificial colorants. *Food professionals always search for “new” spices*Food professionals always search for “new” spices *Growing demand for ethnic and cross*Growing demand for ethnic and cross--cultural cuisinescultural cuisines *Consumers seeking fresh and preservatives free food*Consumers seeking fresh and preservatives free food *Green consumerism, ecofriendly lifestyles*Green consumerism, ecofriendly lifestyles 56
  57. 57. 57 Recommendations **Increase of plant species to investigate from antimicrobialIncrease of plant species to investigate from antimicrobial and antioxidant point of viewand antioxidant point of view **Differentiation of sample accessions in Turkey to underlineDifferentiation of sample accessions in Turkey to underline regional /endemic plant characteristicsregional /endemic plant characteristics **Differentiation of method to compare plant propertiesDifferentiation of method to compare plant properties.. **Evaluation of synergistic antimicrobial and antioxidantEvaluation of synergistic antimicrobial and antioxidant capacitiescapacities **Consideration about the biological effects in food systemsConsideration about the biological effects in food systems
  58. 58. Thank you for your time!Thank you for your time!

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