antimicrobial and antioxidant activity of selected turkish spices
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Transcript of antimicrobial and antioxidant activity of selected turkish spices
BAZI TÜRK BAHARATI ÇESİTLERİNİN BAZI TÜRK BAHARATI ÇESİTLERİNİN ANTİMİKROBİYAL VE ANTİOKSİDAN ANTİMİKROBİYAL VE ANTİOKSİDAN
AKTİVİTELERİNİN DEĞERLENDİRİLMESİAKTİVİTELERİNİN DEĞERLENDİRİLMESİ
Gioacchino dell’AQUILA Danışman: Yard. Doç. Dr. Duygu ALTIOK
Yüksek Lisan Tezi
İstanbul, 2013
EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF SELECTED TURKISH SPICESACTIVITIES OF SELECTED TURKISH SPICES
Gioacchino dell’AQUILA Advisor: Ass. Prof. Dr. Duygu ALTIOK
İstanbul, 2013
Master Thesis
Introduction and Objectives
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 rather whose significant function in food is seasoning rather
than nutritionalthan nutritional
INTERNATIONAL ORGANISATION FOR STANDARDISATIONINTERNATIONAL ORGANISATION FOR STANDARDISATION
The term The term spices and condiments spices 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
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Spice Market in TurkeyFAOSTAT 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)
Turkey79.097
*1000 $ 113.783
*1000 Kg
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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
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Properties
**Flavoring agentsFlavoring agents in meals, curries, bakery, pickles, processed in 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 properties properties againstagainst food spoilagefood spoilage
* * Used as Used as preservativespreservatives in pickle and chutney. in pickle and chutney.
** Possess important Possess important nutritionalnutritional and and medicinal propertiesmedicinal properties..
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Basic Uses of SpicesAdapted from Ravindran et al., (2002)
FlavoringFlavoring Cinnamon, Allspice, Dill, Mint, Cumin, Marjoram,
Basil, Anise, Mace, Nutmeg, Fennel, Sesame,
Vanilla, Fenugreek, Cardamom, Celery
OOdodouur maskingr maskingGarlic, 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
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Chemistry of flavour
FlavoFlavouurr is combination of taste, is combination of taste,
aroma and texture aroma and texture derived from an derived from an
overall combination of volatile overall combination of volatile
components (components (AAromaroma) and nonvolatile ) and nonvolatile
components (components (TTasteaste) in a spice.) in a spice.
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Sensory characteristics Adapted from Farrel, (1985)
FlavorFlavor SpicesSpices FlavorFlavor SpicesSpices
Sulfurous Onion, Garlic Sweet Anise Cardamom
Fragrant BasilWarm, fruity and
camphrousAnise
Bay leaf
Pungent and hotCapsicum Mustard
Warm, spicy, and very aromatic
Coriander Ginger
Pungent and sweetCloves
Cinnamon WoodyCinnamon
Cloves
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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
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Spice Spice 100 g /KCal 100 g /KCal SpiceSpice 100 g /KCal 100 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 facts Source National Institute of Nutrition, ICMR, India
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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 propertiesAdapted from Mazza et al. (2010)
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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
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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
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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
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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
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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
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mg GAE /100 gr dw TEAC (µM Trolox/100 g dw) Literature ReferenceOriganum 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)
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
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Materials and Methods
Materials
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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
*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
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Methods
Preliminary operationsPreliminary operations before the experiments before 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
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Total Microbial Count
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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
Agar Disc Diffusion: Spice Extracts
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Antioxidant Activity Assessments
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Antioxidant Activity Assessments
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Results and Discussion
Total Microbial Count by Agar Pour Plate in number of CFU
AGAR POUR
PLATE10-2 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 - - - - -
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Total Microbial Count by Agar Spread Plate in number of CFU
AGAR SPREAD
PLATE10-2 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
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
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
Agar Disc Diffusion: Spice TabletsOriganum vulgare (Kekik)
35
Agar Disc Diffusion: Spice TabletsThymbra spicata (Zahter)
36
Agar Disc Diffusion: Spice TabletsRhus coriaria (Sumak)
37
Agar Disc Diffusion: Spice TabletsMentha spicata (Nane)
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Agar Disc Diffusion: Spice TabletsOcimum basilicum (Reyhan)
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Agar Disc Diffusion: Spice TabletsSpice mixture (
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The experiment had the same results on the spice mixture.
Presence of colonies might be related to:
Different composition on fito-complex.Antimicrobial concentrationPresence of resistant coloniesObserved in Zahter samplesAssay on selective Culture media demonstrated no contamination
Agar Disc Diffusion: Spice TabletsSpice Mixture
41
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
Agar Disc Diffusion: EthanolOriganum vulgare (Kekik)
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Agar Disc Diffusion: EthanolRhus coriaria (Sumak)
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Agar Disc Diffusion: EthanolThymbra spicata (Zahter)
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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
Agar Disc Diffusion: DMSO
S. aureus E. coli
% DMSO
extract100 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
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
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 AssessmentsTrolox Equivalent Antioxidant Capacity
49
Values are expressed in µM Trolox /100 g dw
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 AssessmentsGallic Acid Equivalent Total Phenolic Content
50
Values are expressed in mg GAE/100 g dw
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 AssessmentsCatechin Equivalent Total Flavonoid Content
51
Values are expressed in mg CE/100 g dw
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
Conclusions
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
Designing new food spiceExamine the Examine the efficiencyefficiency and and functionalityfunctionality of spices. of spices.
Need to study Need to study toxicologytoxicology and and safetysafety..
InteractionsInteractions with food components. with food components.
Mechanisms of action Mechanisms of action against microorganisms.against microorganisms.
Influence on Influence on nutritionalnutritional and and sensory sensory qualities.qualities.
Method of applications in Method of applications in marketable foodmarketable food..
Extraction, isolation, and Extraction, isolation, and economic productioneconomic production55
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 cuisines cultural cuisines
*Consumers seeking fresh and preservatives free food*Consumers seeking fresh and preservatives free food
*Green consumerism, ecofriendly lifestyles*Green consumerism, ecofriendly lifestyles
56
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Recommendations**Increase of plant species to investigate from antimicrobial Increase of plant species to investigate from antimicrobial and antioxidant point of viewand antioxidant point of view
**Differentiation of sample accessions in Turkey to underline Differentiation of sample accessions in Turkey to underline regional /endemic plant characteristics regional /endemic plant characteristics
**Differentiation of method to compare plant propertiesDifferentiation of method to compare plant properties..
**Evaluation of synergistic antimicrobial and antioxidant Evaluation of synergistic antimicrobial and antioxidant capacitiescapacities
**Consideration about the biological effects in food systemsConsideration about the biological effects in food systems
Thank you for your time!Thank you for your time!