master programme final project

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. 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. Introduction and Objectives

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
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. 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. Materials and Methods

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. *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. 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. Total Microbial Count
25

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. Agar Disc Diffusion: Spice Extracts
27

28. Antioxidant Activity Assessments
28

29. Antioxidant Activity Assessments
29

30. Results and Discussion

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. 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. 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. 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. Agar Disc Diffusion: Spice Tablets
Origanum vulgare (Kekik)
35

36. Agar Disc Diffusion: Spice Tablets
Thymbra spicata (Zahter)
36

37. Agar Disc Diffusion: Spice Tablets
Rhus coriaria (Sumak)
37

38. Agar Disc Diffusion: Spice Tablets
Mentha spicata (Nane)
38

39. Agar Disc Diffusion: Spice Tablets
Ocimum basilicum (Reyhan)
39

40. Agar Disc Diffusion: Spice Tablets
Spice mixture (
40

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. 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. Agar Disc Diffusion: Ethanol
Origanum vulgare (Kekik)
43

44. Agar Disc Diffusion: Ethanol
Rhus coriaria (Sumak)
44

45. Agar Disc Diffusion: Ethanol
Thymbra spicata (Zahter)
45

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. 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. 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. 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. 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. 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. 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. Conclusions

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. 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. 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
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. Thank you for your time!Thank you for your time!