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

4

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

5

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

16

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

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

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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

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Agar Disc Diffusion: Spice Extracts

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Antioxidant Activity Assessments

28

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 - - - - - - -

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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 - - - - -

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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.

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Agar Disc Diffusion: Spice TabletsOriganum vulgare (Kekik)

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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

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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)

44

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

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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

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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

57

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!