antimicrobial and antioxidant activity of selected turkish spices english version

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INDEX:

TEZ ONAYI ………………………………………………………………..…….i

BEYAN ……………………………………………………………………..…ii

ABSTRACT......………………………………………………........................iii

ÖZET.…..…………………………………………..…………………………...iv

ABBREVIATIONS...………..……………………………………………….….v

INDEX OF FIGURES………………………………………………………..vi

INDEX OF TABLES...…… ..……………………………........................vii

1. INTRODUCTION AND OBJECTIVES ............................................................... 1

2. LITERATURE REVIEW ....................................................................................... 5

2.1 SPICES CHARACTERISTICS ............................................................................... 5

2.1.1 Thymbra spicata ..................................................................................... 7

2.1.1.1 Taxonomy and distribution .............................................................. 7

2.1.1.2 Ethnobotany and history .................................................................. 8

2.1.1.3 Bioactive constituents ...................................................................... 8

2.1.2 Rhus coriaria .......................................................................................... 9

2.1.2.1 Taxonomy and distribution .............................................................. 9

2.1.2.2 Ethnobotany and history ................................................................ 10

2.1.2.3 Bioactive constituents .................................................................... 11

2.1.3 Ocimum basilicum ................................................................................ 11

2.1.3.1Taxonomy and distribution ............................................................. 12



2.1.4 Mentha spicata ...................................................................................... 15

2.1.4.1 Taxonomy and distribution ............................................................ 15



2.1.5 Origanum vulgare ................................................................................. 17

2.1.5.1 Taxonomy and distribution ............................................................ 17



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2.2 BIOPRESERVATION ......................................................................................... 20

2.2.1 History .................................................................................................. 20

2.2.2 Today's methods .................................................................................... 21

2.2.3 Consumers' green perspective on food safety ....................................... 23

3. MATERIALS AND METHODS ..................................................................... 27

3.1 MATERIALS ................................................................................................... 27

3.1.1 Samples collection ................................................................................ 27

3.1.1.1 Sample preparation ........................................................................ 27

3.2 METHODS ...................................................................................................... 28

3.2.1 Total Microbial Count ........................................................................... 28

3.2.1.1 Plate Count Agar preparation ........................................................ 28

3.2.1.2 Phosphate-buffered peptone water ................................................ 28

3.2.1.3 Agar Pour plate and Spread Plate techniques ................................ 28

3.2.2 Agar Disc Diffusion Method ................................................................. 30

3.2.2.1 Preparation of Nutrient Agar plates ............................................... 30

3.2.2.2 Culture media activation ................................................................ 30

3.2.2.3 Agar disc diffusion method ............................................................ 31

3.2.2.3.1 Agar disc diffusion assay with spice tablets ........................... 39

3.2.2.3.2 Agar disc diffusion assay with spice extracts ......................... 32

3.2.3 Antioxidant activity assessments ........................................................... 33

3.2.3.1 Preparation of the plant extracts .................................................... 33

3.2.3.2 Trolox Equivalent Antioxidant Capacity Assay ............................ 33

3.2.3.3 Total phenolic content (Folin-Ciocalteu) ....................................... 37

3.2.3.4 Total flavonoid content .................................................................. 37

4. RESULTS .......................................................................................................... 36

4.1 TOTAL MICROBIAL COUNT ............................................................................. 36

4.2 AGAR DISC DIFFUSION ASSAY ........................................................................ 38

4.3 TROLOX EQUIVALENT ANTIOXIDANT CAPACITY ASSAY .................................. 52

5. DISCUSSION ................................................................................................... 57

6. REFERENCES ................................................................................................. 62

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ABBREVIATIONS

A : Spice mixture

APP : Agar Pour Plate

ASP : Agar Spread Plate

BPW : Buffered Peptone Water

CFU : Colony Forming Unıt

EOs : Essential oils

GRAS : Generally Recognized As Safe

K : Kekik (Origanum vulgare)

mM : milliMoles

N : Nane (Mentha spicata)

NA : Nutrient Agar

PCA : Plate Count Agar

R : Reyhan (Ocimum basilicum)

S : Sumac (Rhus coriaria)

TEAC : Trolox Equivalent Antioxidant Capacity

Z : Zaater ( Thymbra spicata)

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INDEX OF FIGURES

FIGURE 1: THYMBRA SPICATA FLOWERING TOP ............................................................ 7

FIGURE 2: RHUS CORIARIA FRUITS CLUSTER ............................................................... 9

FIGURE 3: OCIMUM BASILICUM EDIBLE TOP STEM AND LEAVES ................................. 11

FIGURE 4: MENTHA SPICATA FLOWERING STEM AND LEAVES .................................... 15

FIGURE 5: ORIGANUM VULGARE SUBS. HIRTUM FLOWERING TOP ............................... 17

FIGURE 6: AGAR PLATE SWABBING TECHNIQUE ....................................................... 31

FIGURE 7: SPICE MIXTURE TABLETS INHIBITION ZONES ON E. COLI AND S. AUREUS .. 41

FIGURE 8: OREGANO TABLETS INHIBITION ZONES ON E. COLI AND S. AUREUS ......... 41

FIGURE 9: THYMBRA TABLETS INHIBITION ZONES ON E. COLI AND S. AUREUS ......... 42

FIGURE 10: SUMAC TABLETS INHIBITION ZONES ON E. COLI AND S. AUREUS ............ 42

FIGURE 11: OREGANO ETHANOL EXTRACT AGAINST E. COLI AND S. AUREUS ........... 49

FIGURE 12: EFFECTS OF THYMBRA ETHANOL EXTRACT ON

E. COLI AND S. AUREUS COLTURES ................................................................... 49

FIGURE 13: SUMAC ETHANOL EXTRACTS INHIBITION ZONES

AGAINST E. COLI AND S. AUREUS ..................................................................... 50

FIGURE 14: SUMAC EXTRACT ABSORBANCE LOSS ..................................... 46

FIGURE 15: THYMBRA EXTRACT ABSORBANCE

LOSS…………..…………..46

FIGURE 16: BASIL EXTRACT ABSORBANCE LOSS…… …… …..….……..47

FIGURE 17: MINT EXTRACT ABSORBANCE LOSS…………… …….……..47

FIGURE 18: MINT EXTRACT ABSORBANCE LOSS………………… .……..47

FIGURE 19: TROLOX ABSORBANCE STANDARD SLOPE ………… .……..48

FIGURE 20: TEAC VALUES FOR PLANT EXTRACTS……………… .……..48

FIGURE 21: TOTAL PHENOLS VALUES FOR PLANT EXTRACTS …. .……..48

FIGURE 22: TOTAL FLAVONOIDS VALUES FOR PLANT EXTRACTS.……..48

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INDEX OF TABLES

TABLE 1: RESULTS OF TOTAL MICROBIAL COUNT BY AGAR POUR

PLATE EXPRESSED IN NUMBER OF CFU .............................................. 38

TABLE 2: RESULTS OF TOTAL MICROBIAL COUNT BY AGAR SPREAD

PLATE EXPRESSED IN NUMBER OF CFU .............................................. 38

TABLE 3: RESULTS OF TOTAL MICROBIAL COUNT EXPRESSED IN CFU

AVERAGE ........................................................................................................ 39

TABLE 4: RESULTS OF AGAR DISC DIFFUSION METHOD EXPRESSED IN

MM OF CLEAR ZONE AROUND THE TABLET ...................................... 40

TABLE 5: RESULTS OF AGAR DISC DIFFUSION METHOD WİTH DMSO

SPICE EXTRACTS EXPRESSED IN MM OF CLEAR ZONE .................... 42

TABLE 6: RESULTS OF AGAR DISC DIFFUSION METHOD WİTH ETOH


TABLE 7: RESULTS OF AGAR DISC DIFFUSION METHOD WİTH WATER


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1. INTRODUCTION AND OBJECTIVES

Nowadays despite the availability of novel technologies, there is still a

remarkable outbreak of food borne infections resulting from the consumption of

food contaminated by microorganisms. The research then is directing its interest

towards the discovery of new powerful antimicrobial agents that could enable

marketed food to be safe and not harmful for consumers.

Since the customers current perception has turned negative against synthetic

preservatives the research effort was shifted towards the development of

alternatives that end users perceive as 'green'. The antimicrobial properties of

herbs and spices have then been recognized as a focus of interest due to the

evidence of their traditional use both for food preservation and medicinal

purposes.

Natural preservatives have risen in popularity both in pharmaceutical as

well as in food industry due to their ability to extend food shelf-life through their

antioxidant and antimicrobial activities. There are many herbs and spices that are

known to possess antimicrobial activity and are used from time immemorial to

keep food from spoilage and to prevent its contamination by pathogen

microorganisms for humans. Food preservation issues in recent years became

more complex with increasing concern over the presence of chemical residues in

food. Besides the demand for non-toxic natural preservatives is increasing

everyday owing to the absence of studies on chemicals long-term side effects.

A totally renewed interest in ‘natural preservation’ or 'bio preservation'

seems to be stimulated by present food safety concerns, difficulties related to

microbial resistance and production of minimal processed food according to ‘eco-

friendly’ image or vision policies of food companies. Nature is a flourishing

source of biological active molecules that need to be investigated as food

preserving agents. Those substances could be found in different parts of the plant

and commonly, the plant portion containing the highest concentration of

metabolites is considered as 'drug'. For example: cardamom seeds, bay leaves,

clove from flower bud, pepper from fruit, cinnamon from bark or ginger from

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rhizome.

Spices are desirable food ingredients in order to create and explore new

tasty products. Understanding and analysing their properties while developing

new methods and instruments to study them are critical factors for food product

manufacturing [1Madsen at al. 2000]. There is no particular definition of spices

because it is very difficult to define what a spice is compared to a herb. Many

spices require tropical or subtropical climates to grow. Herbs are soft-stemmed

plants and both in fresh or dried forms, their leaves and flowering tops are used

for seasoning foods.

The smell and the taste of spices are dependent on their chemical

compositions. In many cases not a single component creates the characteristic and

original smell, a complex mixture influences the overall odour quality. Mostly the

volatile oil fraction of spices is lost during processing. On the other hand, different

compounds are entrapped by fat and proteins in the food matter. In addition to

their aroma and pungency factors, spices contain many different compounds such

as fat and resin that contribute to the natural flavour of spices. Some spices like

paprika, turmeric and saffron have the advantage of not only giving a flavour but

also giving attractive colours to food.

Spices consumed in small quantities contain little macro-nutrient values

compared to vegetables that contain high amounts of protein, carbohydrates, fat,

starch, fibre, minerals and different vitamins. However, spices supply secondary

compounds that have medicinal, antioxidant and antimicrobial effects.

In particular, spices contain variable amounts of protein, fat, carbohydrate,

small quantities of vitamins (e.g., carotene, thiamine, riboflavin and niacin) and

inorganic elements (calcium, magnesium, manganese, phosphorous, potassium,

chlorine, copper, iron, sodium and zinc). Some spices also contain fatty acids,

starch, sugars, cholesterol and fibre.

Logically the proportion of the use of spices is higher in countries where

spices grow. Extracts of plants, spices and herbs play an important role in

promoting human health by their anticancer, antioxidant and anti-inflammatory

properties. Flavonoids from tea beverages act as free radical-scavengers and

antioxidants. Anthocyanins and flavonoids from teas and cherries possess anti-

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allergic, antiviral, anticancer and anti-carcinogenic properties and prevent

cardiovascular diseases and aging [2Balentine at al. 1999]. Some components in

spices also possess colorant, bioactive (i.e. antioxidant and antimicrobial),

acidulant and sweetener effects [3Wang et al. 2000].

Essential oils (EOs) are distilled parts of spices by mostly steam and also

by cold, dry and vacuum distillation methods. As far as it has been recognised that

many EOs have strong antimicrobial properties, spices have just recently gained

the scientific interest due to the current enhancement in popularity for 'eco-

friendly consumerism'. Nonetheless the researches carried on this topic are still

not considering the innumerable opportunities for improving products features in

term of energy efficiency, safety and shelf-life on the other hand we are

experiencing a totally novel generation of meals with high technologic value, new

taste and whose production is completely sustainable both from the manufacturers

and consumers point of view.

The application of spices has in the food industry a wide range of possible

targets. For instance a study carried out on unpasteurised apple juice shows that

the treatment of the juice with 1.25 mM carvacrol or p-cymene reduced the

numbers of E. coli O157:H7 significantly within 1–2 days at 25° and 4°C storage

temperatures. The effective concentrations of carvacrol at 0.5 mM could be

reduced even further by combining it with cymene at 0.25 mM. The phenolic

compounds were biocides against both spoilage yeasts and E. coli O157:H7

thereby increasing the shelf-life and improving the safety of un-pasteurised apple

juice, particularly when stored at chill temperatures [4Kiskò et al. 2005].

The effectiveness of spices in preventing both spoilage and foodborne

outbreaks should be well investigated because, as previously shown in well tested

procedures, they could be efficient also at chill storage temperatures. In addition,

their efficacy has been proved also on unpasteurised and unsterilized food stuffs

demonstrating once and for all that thermal processing with its serious implication

in retaining the nourishing quality of food is not at all necessary. Besides the lack

in the scientific literature of spices antimicrobial and antioxidant activities

characterizing assay protocols there is an evidence that the plants derived

components, in their whole forms or as extracts, have been used both in the

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ancient medicine and in the traditional gastronomy to avoid decay of food, to

extend its shelf-life and to threat the most common pathogens related illness.

Moreover, those ingredients belongs to nature so they have none or little toxic

effect on the human organism whilst the potential use in the food industry as bio-

preservative and foodaceutics should be assumed by food scientists as a

perspective opportunity to investigate on novel preserving methods that have less

impact on human metabolism and environment.

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2. LITERATURE REVIEW

2.1 Spices characteristics

Medicinal and edible plants are used by 80% of the world population as

the only available therapy especially in developing countries [5Hashim et al. 2010]

while in developed ones the research on natural products as a source of new

powerful drugs is a lively scenario for strategic investments. Current research on

natural molecules and products primarily focuses on plants since they can be

sourced more easily and be selected based on their ethno-medicinal uses [6Arora

et al. 2007].

Nowadays spices are primarily condiments used in cooking in everyday

life but in ancient times they were used as basic ingredients for incense,

embalming preservatives, ointments, perfumes, antidotes against poisons,

cosmetics and medicines but they were food condiments for cooking to a limited

extent

Medieval Europeans used spices to flavour the drab and partially

decomposed food, to provide fragrance and to mask noxious odours [7Erdogan et

al. 2007]. The demand for spices played an important role in world history; it

stimulated the exploration of the globe, the discovery of continental America

starting trade and cultural interaction between the countries of East and West.

The first recorded use of spices was dated from the Pyramid Age in Egypt

(2600 BC). Onions were fed to labourers as medicinal herbs to preserve their

health during construction of pyramids. The spices and herbs used today as

condiments such as Anise, Caraway, Cassia, Coriander, Fennel, Cardamom,

Onions, Garlic, Thyme, Mustard, Sesame, Fenugreek, Saffron, and Poppy seed

were used in medicine, cosmetics, cooking and embalming.

In China, the first authentic record of the use of Cassia was found in the

Ch’u Ssu (Elegies of Chu) in the fourth century BC. The great philosopher

Confucius (551-479 BC) mentioned the use of Ginger in his Analects. Excavations

in the Indus Valley showed that spices and herbs have been used since the first

millennium BC.

In the ancient Greece, spices and herbs played an important role in medical

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science and as condiments in food. They imported some Eastern spices such as

Pepper, Cassia, Cinnamon and Ginger and also used spices and herbs grown in the

Mediterranean area such as Anise, Caraway, Poppy seeds, Parsley and Marjoram.

Hippocrates (460-377 BC), known as the “Father of Medicine”, wrote

many treatises on medicinal plants and their use. The Greek philosopher and

scientist Theophrastus (327-287 BC), sometimes called the “Father of Botany”

wrote two books, named On Odours and An Enquiry into Plants that gathered the

botanical information of spices and herbs.

The transportation of spices and other goods from East to West created

several ancient trade routes: the Incense Route and the Silk Route. High demand

and cost of spices in the Middle Age encouraged the Europeans to find the new

routes to primarily spice growing regions in the Orient. Marco Polo, Pedro

Cabral, Vasco da Gama, Ferdinando Magellano, Cristoforo Colombo and

Hernando Cortes were the pioneers who established new routes for spice trading.

The crucial role of spices in the countries’ economy resulted in the

discovery of new lands, wars between countries and raids of spice growing

countries. Although most of the spices came from the East, some popular spices

were introduced to Europe and Asia after discovering the ‘New World’. Chili

peppers, Sweet peppers, Allspice, Annatto, Cocoa, Epazote, Sassafras and Vanilla

were used by Aztecs, Mayans and Incas both to enhance the flavour of their food

or drinks and for medicinal purposes.

A wide range of dietary an medicinal plants parts is used to be extracted as

raw drugs that possess several biological activities. While some of these drugs are

collected in smaller quantities by the local communities and folk healers for local

uses, many other raw drugs are collected in larger quantities and traded in the

market as the raw materials for many herbal industries [8Uniyal et al. 2006].

Plants used for traditional medicine contain a wide range of substances

that can be used to treat chronic as well as infectious diseases. Clinical

microbiologists have great interest in screening of medicinal plants for new

therapeutics intended as dietary supplements or nutraceutics [9Periyasami et al.

2010]. The active principles of many drugs found in plants are derived from the

secondary metabolism thus the antimicrobial activities of those extracts may

7

reside in a variety of different components.

The development of drug resistance in human pathogens against

commonly used antibiotics has necessitated a search for new antimicrobial

substances from other sources including plants. Screening of plants for

antimicrobial activities is important for finding potential new compounds for both

food and pharmaceutical industry.

2.1.1 Thymbra spicata

Figure 1: Thymbra spicata flowering top

2.1.1.1 Taxonomy and distribution

Species of the genus Thymbra (Lamiaceae) are widely distributed in the

Mediterranean area, Asia and Northern America regularly found in sunny, dry,

rocky habitats. Thymbra consists of about 200 species, usually aromatic herbs and

shrubs. The leafy parts of plants such as Thymbra species are used in traditional

medicine in the treatment of various diseases. In Turkey, [10

Baydar et al. 2004]

8

Thymbra is represented by two species. The most common is Thymbra spicata

(black thyme) growing wild in some Eastern Mediterranean countries and the

dried leaves are used as spice and herbal tea. The essential oils of this plant have

wide industrial applications, from the flavouring of foods, liqueur production,

perfumery and antiseptic to being used as antimicrobial agents [11

Ozkan et al

2003].

2.1.1.2 Ethnobotany and history

In Mediterranean folk medicine infusion of this plant is used to soothe a

sore throat, treat mouth ulcer, stomachache, headache and toothache. The plant is

a woody shrublet with leaves that are lanceolate-elliptic with slightly revolute

margins. The inflorescence is a loose, often elongated head with a lilac corolla.

The essential oil of this species is used in folk medicine as an antiseptic, tonic,

gastric sedative and diuretic [12

Capone et al. 2009].

Furthermore, the aerial parts of some Thymbra plants have been widely

used in traditional medicine, to treat many ailments, for instance muscle pains,

indigestion, cramps, nausea, diarrhoea and infectious diseases. In addition, they

are commonly applied as an antibacterial for the treatment of cold and bronchitis.

The leaves have a thyme-like flavour and are used as a seasoning for

pulses, savoury breads, brine-cured olives and vegetables. The leaves and young

shoots are used as a tea substitute. It is said that this make one of the best-tasting

of all herbal teas.

2.1.1.3 Bioactive constituents

The essential oil of T. spicata is characterized by high content of carvacrol,

γ-terpinene and p-cymene, respectively. Moreover, its essential oil contains a low

percentage of myrcene, α- terpinene, bornylacetate, borneol and thymol [13

Baydar

et al. 2004]. It is clear as for other aromatic plants and spices that the composition

of the essential oil is strongly realted to the production conditions and the

positioning of the lots if cultivated, moreover the harvesting time and the

9

processing equipment has great influence on the essential oil composition and

yield.

The two varieties of T. spicata are known by different local names and

have traditional uses in various regions of Turkey. In South Anatolia T. spicata

var. spicata is known as “Saater” or “Zaater”. In Southwest Anatolia, T. spicata

var. intricata is called “Karaba” or “Karakekik” [14

Baytop et al. 1999]. The T.

spicata var. intricata, which is endemic in Turkey, comprises 10-40 cm shrubs that

grow at altitudes of 150 to 1520 m in dry stony places, rocks and limestone cliffs.

2.1.2 Rhus coriaria

Figure 2: Rhus coriaria fruits cluster


Sumac is the common name for a genus (Rhus) of the Anacardiaceae that

contains over 250 individual species of flowering plants. In Turkey it is

represented by three genera, one of these is R. Coriaria. It is found in temperate-

10

tropical regions worldwide but generally, Sumac can grow in non-agriculturally

viable regions. The various species have been used by indigenous cultures for

medicinal and other purposes, suggesting potential for exploiting the bioactivity

of these plants without competing for food production land uses.

Rhus coriaria is a 1-3 m high shrub or small tree. The leaves are

imparipinnate with 9-15 leaflets. The inflorescence is a compact and erect panicle,

the flowers are small and greenish white and the fruit is a villose, reddish, 1-

seeded drupe. Sumac is a very popular condiment in Turkey and Iran, where the

ground fruits are liberally sprinkled over rice. Mixed with freshly cut onions it is

frequently eaten as an appetizer. The well-known Turkish fast food specialty

döner kebab is sometimes flavoured with Sumac powder.


Rhus glabra (smooth Sumac) is traditionally used by native peoples of

North America in the treatment of bacterial diseases such as syphilis, gonorrhea,

dysentery and gangrene. R. coriaria (tanner’s Sumac), which grows wild in the

region from the Canary Islands through the Mediterranean region to Turkey, Iran

and Afghanistan, is commonly used as a spice by grinding the dried fruits with

salt, and is also widely used as a medicinal herb in the Mediterranean and Middle

East, particularly for wound healing [15

Sezik at al. 1991].

The leaves of this plant contain tannins, sugars, waxes and flavone

derivatives (myricetine) which are yellow of color. They are used for the

protection of leather against microorganisms. Its wood, the so called “yellow root”

and “yellow wood”, has been used for the painting of leather and textile for long

times. Moreover, R. Coriaria fruit extracts can be used in the form of an internal

infusion (5%) as an antiseptic, protector of constipation, regulator of blood flow,

and temperature reducer.

In the Kahramanmaras region of Turkey, aqueous extracts obtained from

the fruits of R. coriaria were used to produce a sour taste in food. Moreover,

aqueous extracts of the plant have been used against viruses (Stomatitis aphthosa

epizootica) that result in a typical disease (Aphthae epizootica) in the nail of sheep

[16

Digrak et al. 2001].

11


This plant is reported to posses hydrolysable tannins, gallotannins, volatile

oil, flavonoids, anthocyanin, gallic acid, flavones, such as, myricetin, quercetin

and kaempferol, nitrate and nitrite contents, moisture, oil, protein, fiber, and ash.

Malic, palmitic, stearic, oleic, and linoleic acids are found as the major

components of Sumac oil. Minerals present in plant are K, P, Si, Br, Al, Cu, S, Cl,

Pb, Ti, Ca, Mn, Fe, Zn, Sr, Mg, Ba, Cr, Li, N [17

Shabbir, 2012].

Sumac is documented to possess antibacterial, hepatoprotective [18

Pourahmad et

al. 2010], antifungal, antioxidant, anti-inflammatory/chondroprotective, DNA

protective [19

Chakraborty et al. 2009], anti-ischemic, vasorelaxant, vascular

smooth muscle cell migration inhibition, hypoglycaemic, xanthine oxidase

inhibition and non-mutagenic properties.

2.1.3 Ocimum basilicum

Figure 3: Ocimum basilicum edible top stem and leaves

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2.1.3.1Taxonomy and distribution

Ocimum genus contains between 50 to 150 species of herbs and shrubs

from the tropical and subtropical regions of Asia, Africa, and Central and South

America. However, the major place of diversity appears to be in Africa [20

Paton,

1992]. Plants have square stems, fragrant opposite leaves and whorled flower on

spiked inflorescence.

Ocimum basilicum is an aromatic, annual herb, 0.3-0.5 metres tall, but

some cultivars can reach up to 1 m. The plant is almost hairless. Some cultivars,

such as the 'Dark Opal', have leaves and stems deep purple in colour. The leaves

are ovate, often puckered, flowers white or pink, and fruits have four small

nutlets, which are mucilaginous when wet.

Ocimum basilicum is closely related to and frequently confused with

Ocimum africanum and Ocimum americanum, but they can be identified on the

basis of indumentum (hair distribution) and flower size. Lemon-scented cultivars

are usually the result of crosses between O. basilicum and O. africanum.

Basil is one of the oldest spices belonging to the Ocimum genus and to the

Lamiaceae (Labiatae) family. The botanical nomenclature of the Ocimum

basilicum L. varieties from which the different types of basil oil are distilled is

complicated. The reason for this complexity stems from the fact that botanists

have assigned several designations to the same varieties and in some instances,

have confused some varieties with forms of other species [21

Guenther, 1975].

This genus is characterized by a great variability in its morphology and

chemiotypes [22

Lawrence, 1988]. The ease of its cross-pollination contributes to a

myriad of subspecies, varieties and forms.


The essential oil of basil extracted via steam distillation from the leaves

and flavouring tops are used to flavour foods, dental and oral products, in

fragrances and in traditional rituals and medicines.

Basil is a popular culinary herb, and its essential oils have been used

13

extensively for many years in the flavouring of confectionary and baked goods,

condiments (e.g., ketchups, tomato pastes, chili sauces, pickles, and vinegars),

sausages and meats, salad dressings, non alcoholic beverages, ice cream and ices.

Basil oil has also found a wide application in perfumery, as well as in dental and

oral products.

Basil is used to flavour soups and sauces and is the main ingredient of

‘pesto sauce’. The leaves can be eaten as a salad. Basil is also used in perfumery,

soap-making, and to flavour liqueurs. The seeds are edible, and when soaked in

water become mucilaginous. In parts of the Mediterranean they are made into a

refreshing drink known as cherbet tokhum [23

Bremmess, 2002].

Basil is widely used in systems of traditional medicine, including

Ayurveda and traditional Chinese medicine. It is used for treating digestive system

disorders, such as stomachache and diarrhoea, kidney complaints and infections.

In Africa, for example, it is used for treating whooping cough and various types of

fever. The leaves are pulped in water to make ear- and eye-drops in parts of West

Africa and a leaf decoction is used for treating coughs.

The leaves are used to make an insecticide that can protect stored crops

from beetle damage.

Extracted essential oils have also been shown to contain biologically active

constituents that are insecticidal, nematicidal and fungistatic. These properties can

be frequently attributed to predominate essential oil constitutes such as methyl

chavicol, eugenol linalool, camphor and methyl cinnamate. Traditionally, basil has

been used as a medicinal plant in treatment of headaches, coughs, diarrhea,

constipation, warts, worms, and kidney malfunctions. It is also thought to be an

antispasmodic, carminative, stimulant and insect repellent.


The oils of basil, especially the camphor-containing oil, have antibacterial

properties. Volatile compounds produced by sweet basil have been shown to

influence the composition, distribution, and spore germination of some fungal

populations. The volatile terpenes camphor and 1,8-cineole present in basil and

14

other members of the Lamiaceae have been suggested as agents in allopathic

reactions [24

Simon et al. 1999].

Sweet basil (Ocimum basilicum L.) a common garden herb is cultivated in

the United States for culinary purposes as a fresh herb and as a dried spice. There

are several types of basil oil in international commerce, each derived principally

from different cultivars and chemiotypes of sweet basil. The oils of commerce are

known as European, French, Egyptian, Reunion or Comoro and to a lesser extent

Bulgarian and Java basil oils.

The perfume, pharmacy and food industries use aromatic essential oil

extracted from the leaves and flowers of basil. Since O. basilicum and O. sanctum

oils have shown strong anti microbial effects may be used as a potential

preservative in food preparations. The European type, a sweet basil is considered

to have the highest quality aroma, containing linalool and methyl chavicol as

major constituents.

15

2.1.4 Mentha spicata

Figure 4: Mentha spicata flowering stem and leaves


Mints are aromatic, almost exclusively perennial, rarely annual herbs.

They have wide-spreading underground and over ground stolon [25

Aflatuni et al.

2005] and erect, square, branched stems. The leaves are arranged in opposite

pairs, from oblong to lanceolate, often downy, and with aserrate margin. Leaf

colours range from dark green and grey-green to purple, blue, and sometimes pale

yellow. The flowers are white to purple and produced in false whorls called

verticillasters. The corolla is two-lipped with four subequal lobes, the upper lobe

usually the largest. The fruit is a small, dry capsule containing one to four seeds

[26

Tucker et al 2007].

16


Mint leaves are used to make mint sauce and jelly, which are commonly

served alongside lamb, tzatziki and tabbouleh. Leaves can be candied and also

used for flavouring in herb teas and iced drinks. Apple mint leaves are hairy and

hence considered less suitable for use as a garnish than those of Mentha spicata

(spearmint).

Commonly cultivated as a pot plant and culinary herb, mint is an invasive

plant and must be kept in check if grown in open ground.

The leaf, fresh or dried, is the culinary source of mint. Fresh mint is

usually preferred over dried one when storage is not a problem. The leaves have a

pleasant warm, fresh, aromatic, sweet flavour with a cool aftertaste. Mint leaves

are used in teas, beverages, jellies, syrups, candies, and ice creams. In Middle

Eastern cuisine as well as in Turkey, mint is used on lamb dishes, in cold and hot

traditional soups (Mercimek çorbası), the leaves fresh are a common ingredient

for salads. Mint is a necessary ingredient in Touareg tea, a popular tea in northern

African and Arab countries [27

Ortiz et al. 1992].


Mint was originally used as a medicinal herb to treat stomachache and

chest pains, and it is commonly used in the form of tea as a home remedy to help

alleviate stomach pain. Menthol from mint essential oil (40–90%) is an ingredient

of many cosmetics and some perfumes. Menthol and mint essential oil are also

much used in medicine as a component of many drugs, and are very popular in

aromatherapy. Menthol is also used in cigarettes as an additive, because it blocks

out the bitter taste of tobacco and soothes the throat.

17

2.1.5 Origanum vulgare

Figure 5: Origanum vulgare subs. hirtum flowering top


Origanum vulgare is an aromatic, woody-based perennial, which grows to

20-90 cm in height. Its leaves are ovate, 10-40 mm long and 5-25 mm wide and

opposite on the stem. The edges of the leaves are smooth or very shallowly

toothed and the leaf tips vary from acute to obtuse. The inflorescence has flowers

grouped into short dense lateral or terminal spikes. The corolla is white to

purplish, 4-8 mm long and has two lips. The calyx is five-toothed. Each fruit has

four small nutlets (single-seeded units)

Oregano has particular requirements for growing since it prefers range of

pH between 6.0 (mildly acid) and 9.0 (strongly alkaline) with a preferred range

between 6.0 and 8.0. It prefers a hot, relatively dry climate, but will do well in

18

other environments [28

Peter, 2011].


Oregano is an important herb in Greek and Italian cuisine, the dried form

having more flavour than the fresh leaves. Perhaps the dish most widely

associated with oregano is pizza. The flavour of oregano varies according to

cultivar, environmental conditions (such as climate and soil type) and time of year

when harvested.

Oregano is also used in traditional medicine for treating colds, indigestion

and stomach upsets. Its oil is used in aromatherapy, in perfumes and toiletries and

in the food industry as flavouring.

It is most frequently used with roasted, fried or grilled vegetables, meat

and fish. The herb is also widely used in Turkish, Middle Eastern, Greek,

Portuguese, Spanish, Philippine and Latin American cuisines. In Turkish cuisine,

oregano is mostly used for flavouring meat, especially for mutton and lamb. In

barbecue and kebab restaurants, it can be usually found on table, together with red

chilli pepper, salt and black pepper. The leaves are most often used in Greece to

add flavour to salad and it is usually added to the lemon-olive oil sauce that

accompanies many fish or meat barbecues and some casseroles. Hippocrates used

oregano as an antiseptic, as well as a cure for stomach and respiratory ailments.

The Cretan oregano (O. dictamnus) is still used today in Greece as a palliative for

sore throat. Oregano is high in antioxidant activity, due to a high content of

phenolic acids and flavonoids. It also has shown antimicrobial activity against

strains of the food-borne pathogen Listeria monocytogenes [29

Faleiro et al. 2005].


The main chemical constituents include carvacrol, thymol, limonene,

pinene, cimene, and caryophyllene. The leaves and flowering stems are strongly

antiseptic, antispasmodic, carminative, cholagogue, diaphoretic, emmenagogue,

expectorant, stimulant, stomachic and mildly tonic. Oregano is an important

culinary herb, used for the flavour of its leaves, which can be more flavourful

19

when dried than fresh. It has an aromatic, warm and slightly bitter taste, which

can vary in intensity.

Good quality oregano may be strong enough almost to numb the tongue,

but the cultivars adapted to colder climates often have a lesser flavour. Factors

such as climate, seasons and soil composition may affect the aromatic oils present

and this effect may be greater than the differences between the various species of

plants.

20

2.2 Biopreservation

2.2.1 History

Food preservation has long been a necessary pursuit of humans through

the ages. While short term food preservation methods are largely dominated by

today’s refrigerators and long term preservation is dominated by canning or

freezing, our ancient cultures thrived without such technology by collecting,

drying and storing grain in large ceramic pots. Hunter-gatherers preserved meat

and fish by air drying or smoking. Salt and sugar, when readily available, were

also used as a preservative.

Spices have always been used to enhance the flavour and palatability of

food. Several studies evaluated critical predictions in order to address the question

of why people use spices. Scientists evaluated the prediction of the use of 43

spices in 4,578 meat-based recipes from 36 countries. They concluded that in hot

climate countries the proportion of recipes with spices, number of spices used in

each recipe, total number of spices and the use of most antimicrobial spices

were higher [30

Billing et al. 1998].

Countries with high mean annual temperature use numerous spices

compared to countries with low mean annual temperature. In hot climate countries

spices are more frequently used at higher amounts than cool climate countries.

Spices with strong antimicrobial activity such as Garlic, Onion, Capsicum,

Cinnamon and Cumin are used more frequently in countries with hot climates

than countries with cooler climates.

Furthermore, hot country cuisines and spicier cuisines have more

antimicrobial potent against food-borne microorganisms. Billing and Sherman

(1998) had two hypotheses about how people started using spices. First, people

who used spices, especially in hot countries, suffered less from food-borne

illnesses and stored their food for longer periods of time. Second, adding spices

changed the taste and flavour of food and made it more palatable and safe for

consumption.

21

2.2.2 Today's methods

Many food products are perishable by nature and require protection from

spoilage during their preparation, storage and distribution to give them desired

shelf-life. Because food products are now often sold in areas of the world far

distant from their production sites, the need for extended safe shelf-life for these

products has also expanded. Bio-preservatives may constitute a wide range of

natural products from both plants and microorganisms which can be useful in

extending shelf-life of foods, reducing or eliminating survival of pathogenic

bacteria and increasing overall quality of food products.

Selected herbal extracts have been shown to have potent antimicrobial

properties both in bacterial cultures and in food applications such as marinades, in

edible films and in beverages. Bacteriocins are widely used in cheese making and

other food processes to increase safety and shelf-life of foods. As the popularity of

bio-preservatives continues to increase, consumers, regulatory agencies and food

processors require reliable information on the safety, standardization and efficacy

of these products.

The definition of a plant or botanical bio-preservative is somewhat vague.

These can include products made from the roots, leaves, stems, fruits or nuts from

a plant. Some of these products are GRAS (“spices”) and some are not. There are

fuzzy legal lines between what is considered a food and a dietary supplement,

particularly when botanical bio-preservatives are further processed.

Spices can be prepared by a variety of methods such as physical, aqueous,

solvent or supercritical gas extraction. Physical crushing followed by heating

and/or vacuum concentration is common. Concentrated extracts can be plated on

neutral dry carriers such as malt dextrin or diluted in vegetable oil.

Each of these methods of preparation will have an effect on the active

ingredients within the preparation. Standardization is the biggest challenge to the

food industry in the effort to use bio-preservatives since multiple varieties of

plants, widely varying growing conditions, time and method of harvest and

storage of plants or preparations may all affect the extract content [31

Draughton,

2003].

The development of food preservation processes has been driven by the

22

need to extend the shelf-life of foods. Food preservation is a continuous deal

against food spoilage microorganisms and food pathogens. Several food

preservation systems such as heating, refrigeration and addition of antimicrobial

compounds to food can be used to reduce the risk of outbreaks of food poisoning;

however, these techniques frequently have associated adverse changes in

organoleptic characteristics and loss of nutrients.

Within the disposable arsenal of preservation techniques, the food industry

investigates more and more the replacement of traditional food preservation

techniques by new preservation techniques due to the increased consumer demand

for tasty, nutritious, natural and easy-to-handle food products. Improvements in

the cold distribution chain have made international trade of perishable foods

possible but refrigeration alone cannot assure the quality and safety of all

perishable foods.

For instance, a study concerning food safety and food quality, tested bio-

preservative features and applications of plant-derived, animal-derived, microbial-

derived and enzyme for aquatic products basing on the characteristics of hurdle

technology. The feasibility of preservation combined with low temperature

preservation and modified atmosphere packaging was analysed and it was clear

that the combination of bio-preservatives and other technology effectively could

greatly prolong shelf life of aquatic foodstuff [32

Zhao and Xie, 2009].

The most common classical preservative agents are the weak organic

acids, for example acetic, lactic, benzoic and sorbic acid. These molecules inhibit

the outgrowth of both bacterial and fungal cells and sorbic acid is also reported to

inhibit the germination and outgrowth of bacterial spores.

Microbial growth in foods can be controlled by using natural

antimicrobials such as plant extracts, essential oils and protective cultures

(especially Lactic Acid Bacteria — LAB) and their metabolites. Plant extracts and

essential oils can also help to control undesirable microorganisms in minimally

processed foods. Recent antimicrobial essays showed that the addition of herbal

essential oils (Thyme and Oregano) in fish stored at 0 to 2°C during 33 days led to

a reduction in the numbers of spoilage micro-flora [33

Harpaz et al. 2003].

Many bacterial pathogens can survive and develop resistance when they

23

are exposed to a single antimicrobial factor thus to overcome this problem, the

application of multiple antimicrobial factors has been proposed for effective food

preservation because different antimicrobials may act by diverse mechanisms on

the same cellular target and enhance the intensity of damages to the

microorganisms [34

Galvez et al. 2007].

2.2.3 Consumers' green perspective on food safety

The term preservative is defined then as a natural or synthetic chemical

agent that prevents decomposition by microbial growth or any undesirable

chemical change in finished products. They are added to various products to retard

their spoilage, discoloration or contamination by micro-organisms. Instead they

help them retain their colour, texture, flavour and nutritional value.

In the production of food it is crucial that proper measures are taken to

ensure the safety and stability of the product during its whole shelf-life. In

particular, modern consumer trends and food legislation have made the successful

attainment of this objective much more of a challenge to the food industry.

It is becoming highly essential that operations particularly in the area of

fruits, meat, fish, and vegetables are cleaner and have less environmental impact.

Packaging, recycling, disposing, and waste treatment methods need to be

standardised so as to ascertain a stronger commitment towards the environment.

In such a context, it is also essential for the food industry to adopt greener

technologies for their processing lines.

How the 'green' way is going to get translated into the industry might

however vary depending upon the market requirement, the available infrastructure

and the investment profile of the companies. For instance, green technologies do

not comprise only those that promise less wastage and greener disposal; those that

is energy saving and renewable energy dependent are also valid candidates for

heralding the green trend in the food processing sector

Throughout the development of both Western and Eastern civilization,

plants, plant parts, and derived oils and extracts have functioned as sources of

food and medicine, symbolic articles in religious and social ceremonies, and

remedies to modify behaviour. Taste and aroma not only determine what we eat

24

but often allow us to evaluate the quality of food and, in some cases, identify

unwanted contaminants.

The principle of self-limitation taken together with the long history of use

of natural flavour complexes in food argues that these substances are safe under

intended conditions of use. Originally added to change or improve taste, spices

and herbs can also enhance shelf-life because of their antimicrobial nature. Some

of these same substances are also known to contribute to the self-defence of plants

against infectious organisms [35

Kim et al. 2001].

In spite of modern improvements in food production techniques, food

safety is an increasingly important public health issue. It has been estimated that

as many as 30% of people in industrialized countries suffer from a food borne

disease each year and in 2000 at least two million people died from diarrhoeal

disease worldwide [36

World Health Organization, 2002a].

There is therefore still a need for new methods of reducing or eliminating

food borne pathogens, possibly in combination with existing methods. At the

same time, Western society appears to be experiencing a trend of ‘green’

consumerism [37

Smid et al. 1999], desiring fewer synthetic food additives and

products with a smaller impact on the environment. Furthermore, the World

Health Organization has already called for a worldwide reduction in the

consumption of salt in order to reduce the incidence of cardio-vascular disease

[38

World Health Organization, 2002b].

If the level of salt in processed foods is reduced, it is possible that other

additives will be needed to maintain the safety of foods. There is therefore scope

for new methods of making food safe which have a natural or ‘green’ image. One

such possibility is the use of essential oils (EOs) as antibacterial additives.

The dietary concerns of both today’s ageing population and people with

fast paced lifestyles have moved from foods that prevent nutritional deficiency

and associated diseases to foods that offer longer-term prevention of chronic

diseases. Countries are currently faced with health challenges arising from

changing population demographics and increases in lifestyle-related diseases.

Consumers are becoming more aware of the relationships between diet and

disease. Changing views and perceptions about the effects of dietary compounds

25

can profoundly influence the consumption of foods.

Modern food innovations are pushed either directly by consumer demand

or by advances in science and technology. A large body of research has been

conducted which aimed at the identification of the physiologically active

components in foods from all the edible sources that are believed to reduce the

risks of a variety of health problems. Most recently, consumer demand for foods

with health benefits beyond simple nutrition is increasing. Furthermore, to be

commercially successful, these new foods ultimately still must meet consumer

needs.

Physiologically active components with positive health outcomes in foods

may come from plant, animal or microbial sources. A significant proportion of

these components are derived from plants. Epidemiological studies suggest that

regular or increased consumption of fruits may reduce the risk of chronic and

infectious diseases [39

Vattem et al. 2005] and these health benefits are thought to

be mainly attributable to the plants' intrinsic antioxidants and dietary fibre content

[40

Scott et al. 2008].

Health benefits can be obtained through a balanced diet (e.g. 5+ fruit and

vegetable servings per day) or through foods containing fruit- and vegetable-

derived ingredients. Growing consumer demand exists for plant-based functional

foods that improve general health and well-being and fruits are one of the most

popular functional platforms because of their perceived ‘naturalness’ and their

diverse nutrient composition [41

Starling, 2005].

Firstly, consumers require more high quality, preservative-free, safe but

mildly processed foods with extended shelf-life. For example, this may mean that

foods have to be preserved at higher pH values and have to be treated at mild-

pasteurization rather than sterilization temperatures. As acidity and sterilization

treatments are two crucial factors in the control of outgrowth of pathogenic spore-

forming bacteria, such as Clostridium botulinum, addressing this consumer need

calls for innovative approaches to ensure preservation of products.

Secondly, legislation has restricted the use and permitted levels of some

currently accepted preservatives in different foods. This has created problems for

the industry because the susceptibility of some microorganisms to most currently

26

used preservatives is falling.

An increasing number of consumers prefer minimally processed foods, prepared

without chemical preservatives. Many of these ready-to-eat and novel food types

represent new food systems with respect to health risks and spoilage association.

Against this background, and relying on improved understanding and knowledge

of the complexity of microbial interactions, recent approaches are increasingly

directed towards possibilities offered by biological preservation.

Quite clearly, the most predominant trend in the industry today is that of

being environmental friendly and less waste producing. The "Clean and Green

trend," as it is popularly known, has thus become increasingly imperative in the

food processing sector as well. Thanks to consumer pressure and governmental

regulations, food processing and packaging are required to be eco-friendly in both

their production processes and disposal methods.

27

3. MATERIALS AND METHODS

3.1 Materials

3.1.1 Samples collection

Samples of the five plants analysed in this work (Thymbra, Rhus, Ocimum,

Mentha and Origanum) were collected from a farmers market in the province of

Konya in the Central Anatolian region in October 2012. The Origanum, Ocimum,

Mentha cultures are cultivated and harvested following organic procedures whilst

Rhus and Thymbra are collected from the nature.

Each plant material was identified and characterized by Anadolu

University, Faculty of Pharmacy, Department of Pharmacognosy (Eskisehir, TR)

as:

Fam. Labiatae - Thymbra spicata, Zaa'ter

Fam. Anacardiaceae – Rhus coriaria, Sumac

Fam. Labiatae – Ocimum basilicum, Basil

Fam. Labiatae – Mentha spicata, Mint

Fam. Labiatae – Origanum vulgare subsp. hirtum, Oregano

3.1.1.1 Sample preparation

Each plant material was separated from foreign bodies that naturally occur

due to organic production and harvesting methods. Subsequently all the five

spices were powdered using a blender (Waring, Two-speed blender 7011G) and

stored at 4-5 °C in dark glass to preserve their organoleptic and hygienic features.

With the purpose to prepare an ideal spice blend 2 g of each sample has

been weighted and then mixed in a grinding bowl; 0.25 g of this mixture has been

pressed with a 3 ton manual press to obtain round tablets 2 mm height and 10 mm

diameter.

28

3.2 Methods

3.2.1 Total Microbial Count

3.2.1.1 Plate Count Agar preparation

Plate Count Agar (PCA) (LabM Limited Topley House, Batch N˚

121302/103, GB), the microbiological growth media is used to assess the viable

bacterial growth of samples. PCA is not a selective medium and its composition

normally contains (w/v):

0.5% peptone

0.25% yeast extract

0.1% glucose

1.5% agar

The assay was performed in double parallel using both the Agar pour plate

and Agar spread plate techniques. 50 ml Agar have been capped and sterilized by

autoclave (Hirayama, Hmclave Hv-110 L) at 121 °C for 15 min.

3.2.1.2 Phosphate-buffered peptone water

Phosphate-buffered peptone water (Fluka Analytical, Batch N˚ 105450-

0500, ES) with a solubility of 25.5 g/L has been prepared using distilled water as a

solvent. After mixing the solution on a magnetic stirrer (Ika®, C-Mag Hs10) until

it becomes clear and with no residues, 18 ml of the solution has been transferred

to the 50 ml Falcon conical tubes (BD Falcon™, Batch N˚ 352098, US). The test

tubes have been sterilized by autoclave (Hirayama, Hmclave Hv-110 L) at 121 °C

for 15 min.

3.2.1.3 Agar Pour plate and Spread Plate techniques

The experiment has been performed in a previously cleaned with 70%

ethanol (Düzey Lab, C2H6O 96% Batch N˚ 64-175, TR) and UV sterilized

29

microbiological safety cabinet (Lab Culture, Esco Class II, Type A2). In this

experiment, 2 gr of each spice sample has been introduced to the falcon test tubes

containing 18 mL of Phosphate-buffered peptone water obtaining dilution of 10-1

.

All the spice samples have been placed in duplicate obtaining 2x5

specimens. The test tubes have been labelled and vigorously shaken for 1 min

using a manual vortex (Stuart, Vortex Mixer SA8). Then they have been

centrifuged for 5 min at 3000 rpm using a centrifuge (Hettich Zentrifugen,

Rotofix 32A) in order to obtain the supernatant that will be used for further

dilutions.

From each spice Ten-fold serial dilutions in the range of 10-1

to 10-13

were

achieved by taking 1 ml sample from the previous dilution and pouring it into

consecutive test tube including 9 ml sterile buffered peptone water.

In the Agar Spread Plate technique, 15 ml of 45-50 °C PCA has been

poured in sterile and previously numbered and labelled Petri dishes using a sterile

25 mL plastic pipette (LP Italiana, Batch N˚ Q1056D, IT). After solidification of

the agar, by using a sterile micropipette (Eppendorf Research, 200 μL), 100 μL of

the spice-buffered peptone mixture from each decimal dilution of each sample has

been transferred on the agar surface and swabbed uniformly using a triangular

glass swab that every has been washed in ethanol, flamed and cooled after every

application.

Agar Pour Plate technique has been adopted by pouring 1 mL of the

chosen dilution from each spice sample directly to the labelled and numbered

sterile Petri dishes. The PCA has been kept melt above 50 °C in water bath

(Stuart, Water Bath SWBD) and with sterile 25 mL pipette (LP Italiana, Batch N˚

Q1056D, IT) has been added in a measure of 15 mL in the dish avoiding direct

contact with the spice solution. The dish then has been briefly swung to mix the

two components avoiding bubbling and left to solidify. All the Petri dishes were

incubated upside-down (Nüve Incubator, EN 120) for 24 h at 37.5 °C before

collecting the results

.

30

3.2.2 Agar Disc Diffusion Method

3.2.2.1 Preparation of Nutrient Agar Plates

Nutrient Agar (Merck, Batch N˚ VM185150 036, DE) has a solubility of

20 g/L of distilled water. For our purpose we placed a Schott bottle on a magnetic

stirring plate and completely dissolved the agar. The bottle has been closed with

screw cap. It needed to be loosening to avoid blasting during sterilization process.

The nutrient agar terrain prepared according to the labelled procedure has been

then sterilized by autoclave (Hirayama, Hmclave Hv-110 L). Autoclave

sterilization of media was carried on 121 °C for 15 min, temperature time

necessary to sterilize a liquid (warm up, sterilization, cold up).

After autoclave sterilization 15 mL of Nutrient Agar is poured in sterile

Petri dishes using disposable plastic pipette (LP Italiana, Batch N˚ Q1056D, IT).

This procedure has been carried in a UV sterilized microbiological safety cabinet

(Lab Culture, Esco Class II, Type A2).

3.2.2.2 Culture media activation

Nutrient Broth (Merck, Batch N˚ VM229043 107, DE) has been prepared

to activate and refresh the laboratory microbial stock that will be used in the

experiment. Nutrient Broth has been dissolved on a magnetic stirring plate (Velp

Scientifica, Magnetic Micro-stirrer) in distilled water. After the solution is clear

with no residues, 8 ml of solution has been poured in glass test tubes and capped

with cotton.

Cultured microorganism was refreshed by taking 100 μL of 24 hours

culture and inoculating into 8 ml fresh and stable medium. In the disc diffusion

test, microorganisms in their exponential growth phase (6-7 h) were used.

31

3.2.2.3 Agar disc diffusion method

The agar disc diffusion experiment aimed to test the potential

antimicrobial activity of five different spices and a mixture of them. Agar

diffusion test was performed in sterile conditions inoculating G(+) and G(-)

microorgansisms, Escherichia coli (NRRL B-3008) and Staphylococcus aureus

(ATCC 29213), respectively. Using a sterile micropipette (Eppendorf Research,

200 μL), consequentially and carrying out duplicated and parallel experiments,

100 μL of the nutrient broth containing microorganisms was poured in the Petri

dishes and spread using a cotton swab (LP Italiana, Batch N˚ Q0020, IT) as in the

following figure (figure n. 6). It is necessary to underline that the agar surface is

covered by rotating the dish three times of 60 degrees.

3.2.2.3.1 Agar disc diffusion assay with spice tablets

After two microorganisms in question are swabbed uniformly across the

agar plate performing a duplicated parallel experiment, the spice tablets are placed

in the centre of agar plates with sterile forceps that have been washed in ethanol,

flamed and cooled after every application; then gently pressed down onto the agar

to stick the tablet onto its surface. Diffusion of antimicrobials in the disk begins

Figure 6: Agar Plate swabbing technique

32

immediately; therefore, once a disk contacts the agar surface, the disk should not

be moved. The concentration of the compound will be highest next to the disk,

and will decrease as distance from the disk increases.

3.2.2.3.2 Agar disc diffusion assay with spice extracts

Each spice sample has been extracted using different solvents to show

differences in antimicrobial activity. The solvents used in this study are DMSO

100% (Merck, Dimethyl Sulfoxide Batch N˚ K42088843-120, DE), ETOH 70%

(Düzey Lab, Ethanol C2H6O 96% Batch N˚ 64-175, TR) and distilled hot water.

10 g of spice material has been weighted and mixed with each solvent in a 1:5

ratio (w/v) in 250 mL glass flasks properly sealed and labelled.

The extraction process has been carried by mixing the flasks content using

rotating orbital shaker (Dragon Lab, Orbital Shaker SK-330-PRO) at 240 rpm for

48 h. Then, the extracts were vacuum-filtered and stored in labelled capped dark

glass jars until use. 75%, 50%, 25% dilutions of each extract and a sterility control

for each solvent at the same dilution were prepared. Round filter paper disk of 4

mm diameter were obtained by perforating filter paper (Munktell, Paper Sheets

67N Batch N˚ A522417) with a paper puncher. They were sterilized at 120 °C for

2 h in glass Petri dishes in dry oven (Binder, S3 Model Stove) and stored in a dry

place before the experiment.

Each of four filter paper disks was placed on a quarter of the solidified

Nutrıent Agar surface in all the numbered and labelled Petri dishes. 15 µL of

100%, 75%, 50%, 25% spice extract dilution has been poured onto each disk in

each Petri dish with a sterile micropipette (Eppendorf Research, 200 μL). For

example: for spice X we have X-ETOH, X-DMSO, X-WATER. For each spice

solvent combination it has been performed a positive and a sterility control. The

experiment has been carried by double parallel for both tested microorganisms.

If the compound is effective against bacteria at a certain concentration, no

growth will be observed where the drug concentration is greater than Minimum

Inhibition Concentration for that microorganism. This is called the Zone of

Inhibition. Thus, the size of the zone of inhibition is a measure of the compound's

effectiveness: how larger the clear zone around the filter disk, the more effective

33

the compound is. All the labelled Petri dishes are incubated at 37 °C to enhance

the microbial growth. The results related to inhibition zone dimension have been

taken after 24 h.

3.2.3 Antioxidant Activities Assessments

3.2.3.1 Preparation of the plant extracts

10 mg of each spice sample has been poured in a dark glass flask and

mixed with 10 ml of 70 % (v/v) ethanol solution. The flask has been capped and

sealed using parafilm (Menasha Plastic Packaging, WI54952) to avoid

evaporation of the solvent. In order to obtain the hydro-alcoholic extracts, the

flasks’ content has been mixed using rotating orbital shaker (Dragon Lab, Orbital

Shaker SK-330-PRO) at 240 rpm for 30 min. After the maceration process, the

extracts were vacuum-filtered and stored in capped dark glass jars until utilization.

All the hydro-alcoholic spice extracts were prepared at that dilution in order to be

red by UV spectrophotometer (Jeanway, Spectrophotometer 6315).

3.2.3.2 Trolox Equivalent Antioxidant Capacity

ABTS+ assay has been performed by modified method of Wojdylo et al.

(2007) [42

]. ABTS

[Sigma Aldrich, (2,2-azino-bis (3-ethylbenzothiazoline-8-

sulfonic acid) Batch N˚ 30931-67-0, DE] was dissolved in water to produce 7mM

stock solution. ABTS+ was obtained by reacting the stock solution with 2.45 mM

(final concentration) K2S8O2 (Fluka Chemika, Potassium peroxidisulfate Batch N˚

446720/1 51403070, TR). Solution was kept in dark at room temperature for 12

hours prior to use. For this study the samples containing the ABTS+ solution were

diluted with distilled water to an absorbance of 0.700 (± 0.02) at 734 nm by UV

spectrophotometer (Jeanway, Spectrophotometer 6315) and equilibrated at 30 °C.

A reagent blank reading was taken (A0).

After addition of 3.0 mL of diluted ABTS+ solution (A734 nm = 0.7 ± 0.02)

in a clean cuvette, sequentially 30, 40 and 50 µL of ethanol spice extracts sample

34

was poured reading the absorbance loss exactly every minute until 6 min after

initial mixing (At). All determinations were performed in duplicate. Data are

expressed in terms of TEAC (Trolox Equivalent Antioxidant Capacity) and the

results expressed in mg Trolox per mg of tested sample.

Percentage inhibition was measured according to following formula:

TEAC =𝑺 𝒔𝒂𝒎𝒑𝒍𝒆 ∗ 𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓

𝑺 𝒕𝒓𝒐𝒍𝒐𝒙 ∗ 𝐌𝐰 ∗ 𝐂 ∗ 𝟏𝟎𝟒

3.2.3.3 Total phenolic content (Folin-Ciocalteu)

Total phenolic content of the plant extracts has been determined by Folin-

Ciocalteu colorimetric method. In the experiment, 0.5 ml of hydro-alcoholic spice

sample has been mixed with 1.25 ml of daily prepared 20% Na2CO3 (Balmuncu

Kymia, Sodium Carbonate Batch N˚ 207-838-8, TR) solution. Then, to the

obtained solution has been added 0.5 ml 1 N Folin-Ciocalteu reagent (Sigma

Aldrich, Folin-Ciocalteu Batch N˚ 026K0008, DE) and after gently shaking, the

mixture has been left to react in a dark place for 40 min. It has been taken a

reagent blank and the absorbance has been red at 725 nm. All determinations were

performed in duplicate. Data are expressed in terms of Gallic acid equivalents.

3.2.3.4 Total flavonoids content

Total flavonoids content method for spice ethanol extracts has been

performed mixing 0.25 ml of the spice sample with 1.25 ml of distilled water and

75 μl of daily prepared 5% NaNO2 solution (Merck, Sodium Nitrite Batch N˚

A0248344115, DE) and it has been left to react in a dark place for 6 min. After the

reaction is complete, 150 μ of 10% AlCl3 solution (Merck, Aluminum Chloride

Batch N˚ S6038181, DE) has been added and the reaction has been carried on for

5 min more in a dark place. After that time, 0.5 ml of 1 M NaOH (Sigma Aldrich,

Sodium Hydroxide pellets Batch N˚ SZE93150, DE) solution and 275 μl of

35

distilled water has been poured to obtain the final solution. It has been taken a

reagent blank and the absorbance has been red at 510 nm. All determinations were

performed in duplicate. Data are expressed in terms of Catechin equivalents.

36

4. RESULTS AND DISCUSSION

4.1 Total microbial count

Total microbial count experiment has been performed in parallel choosing

a range of the dilution obtained from the original sample in order to assess the

number of viable microorganism naturally occurring in the spice matter. The

results expressed in number of Colony Forming Unit have been shown in Table 1:

Table 1: Results of Total Microbial Count by Agar Pour plate expressed 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 - - - - -

(+ = CFU determination was not possible since the number of colonies was more than 300n)

The evaluation of the outcomes of the experiment showed that all the

tested samples were contaminated by microorganism at the origin likely natural

micro-flora due to organic manufacturing practices or contaminant microorganism

interfering with life-cycle of the product. The only sample that did not showed

micro-flora at significantly higher concentrations (S 10 -2

) was Sumac, probably

related to its higher antioxidant capacıty.

The CFU determinations by serial diluting the samples, demonstrate that at

37

concentration of 10-3

Thymbra, Sumac and Oregano CFU number was relevantly

smaller that Mınt and Basil one. By proceeding with lower dilutions (from 10-5

to

10-13

) all the spice sample did not present any contamination but Basil. This

contamination is probably related to no proper handling during harvesting and

storage.

In order to obtain a greater number of significant data at the same purpose

and since the Agar Pour Plate technique predominantly enhance anaerobic

microorganisms growth, at the same purpose it has been adopted the Agar Spread

Plate technique concerning the number of aerobic microorganism in the original

spice sample at the dilutions that have been chosen. The results expressed in

number of Colony Forming Unit have been listed in Table 2:

Table 2: Results of Total Microbial Count by Agar Spread plate expressed 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 - - - - - - -

(+ = CFU determination was not possible since the number of colonies was more than 300n)

By comparing the results of the two different methods it appears clear that

all the samples were contaminated mainly by anaerobic microorganisms since

starting from concentrations relevantly high (from 10-3

to 10-13

), all the samples

38

were clean. Basil samples also demonstrated a slight decrease in CFU but still the

highest amount among the five samples.

The results expressed in Colony Forming Unit Average for the two techniques

that have been used, are listed in Table 3:

Table 3: 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 - - - - -

+ = CFU determination was not possible since the number of colonies was more than 300n)

39

4.2 Agar disc diffusion assay

With the purpose of testing the antimicrobial activity of spices as crude

sample, 12 mm diameter (0.25 g) spice tablet has been placed on the surface of

agar plate previously inoculated by the two test microorganisms (E. coli – Gram

negative and S. aureus – Gram positive). The results of the Agar Disc Diffusion

method has been recorded in mm of Zone of Inhibition around the spice tablet

after 24 h of incubation at 37 ºC.

The outcomes of the experiment performed in parallel have been listed in

Table 4:

Table 4: Results of Agar Disc Diffusion method are expressed in mm of clear

zone around the tablet. Tablet diameter (12 mm) has been detracted from the total.

S. aureus E. coli

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

When a tablet is placed on agar plate, immediately water is absorbed into

the tablet from the agar. The antimicrobial compounds begin to diffuse into the

surrounding agar. The rate of diffusion through the agar is not uniform therefore

the concentration of antimicrobial is highest closest to the tablet and a logarithmic

reduction in concentration occurs as the distance from the disk increases

[43

Jorgensen and Turnridge, 2007].

40

The experiment parallel results exhibit the strong antimicrobial and

bacteriostatic activity of crude spices with the highest zone of inhibition given by

Oregano (28 mm), Sumac (18-20 mm) and Thymbra (14-17 mm) on S. aureus that

appeared to be the most susceptible between the two tested microorganisms. Basil

and Mint samples validate the results with average zones of inhibition of 9 mm

and 13 mm respectively.

The tablets applied on nutrient agar inoculated by E.coli manifested strong

antimicrobial evidence against the bacteria, mainly Oregano (35-36 mm), Sumac

(15-18 mm) and Thymbra (13-16 mm).

The spice tablets exerted varying levels of antimicrobial effects against

microorganisms. As confirmed in this study concerning spice hydrosols [44

Sagdic

and Ozcan, 2004] Sumac, Oregano and Thymbra extracts were active against all

the tested microorganisms including S. aureus and E. coli.

The rate of diffusion of the antimicrobial through the agar is dependent on

the diffusion and solubility properties of used agar and the molecular weight of

the antimicrobial compound [45

Hudzicki, 2009]. Larger molecules will diffuse at a

slower rate than lower molecular weight compounds.

The diameters of the zones of microbial inhibition on the agar surface

originated by the antimicrobial activity of the spice tablets have been recorded and

they are shown in the following pictures:

41

Figure 7: Spice mixture tablets inhibition zones on E. coli and S. aureus

Figure 8: Oregano tablets inhibition zones on E. coli and S. aureus

42

Figure 9: Thymbra tablets inhibition zones on E. coli and S. aureus

Figure 10: Sumac tablets inhibition zones on E. coli and S. aureus

43

In this study the antimicrobial activity of spices such as Sumac, Oregano,

Thymbra, Basil and Mint, has been investigated by testing their Ethanol,

Dymethylsulfoxide and water extracts. In order emphasize the outcomes of the

antimicrobial assay on spice tablet, antimicrobial activity has been tested using the

agar diffusion method on paper disc at different extract dilution on nutrient agar

plates inoculated with the two test microorganisms (E. coli – Gram negative and S.

aureus – Gram positive).

The results showed a prominent antimicrobial activity with the largest

inhibition zones on both tested microorganism (E. coli and S. aureus) by Sumac,

Oregano and Thymbra Ethanol and Dymethylsulfoxide extracts while water

extracts were generally ineffective.

The results of Dymethysulfoxyde spices extract have been shown in Table

5:

Table 5: Results of Agar Disc Diffusion method with DMSO spice extracts

expressed in mm of clear zone around the paper disc.

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

44

The control taken for DMSO diluted as the spice extracts dilution (positive

control) did not showed any inhibition referring to the solvent antibiotic,

bacteriostatic or toxic activity against the microorganisms; for this reason all the

values recorded related to the inhibition zone created by the spice antimicrobial

constituents.

All the tested spice extract at all dilutions (from 100% to 25% of spice

extract) were active against the tested microorganism. However, the most active

extract was Sumac showing a 5 mm difference in zone of inhibition on S. aureus

at 100%. The inhibition zones diameters of Oregano extract were recorded as the

biggest with a marked difference (7 mm) between the 100% extract and the 25%

extract inhibition zones.

Thymbra extracts were more efficient at higher concentration (100% and

75%) than at smaller ones on both tested microorganisms while as long as they

were active against the microorganism there was no significant difference related

to the concentration of Basil and Mint diluted extracts.

The five spice sample has been extracted also in 70% Ethanol solution

performing in parallel. A control plate with ethanol at the same concentration of

the diluted spice extract has been taken as a positive control. The results are

expressed in mm of zone of inhibition around the paper disc and recorded in Table

6:

45

Table 6: Results of Agar Disc Diffusion method with ethanol spice extracts


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%)

The control taken for Ethanol diluted as the spice extracts dilution

(positive control) showed an inhibition zone of 10 mm on S. aureus plates at

100% (70% v/v) suggesting that the solvent has antibiotic, bacteriostatic or toxic

activity against Gram positive bacteria; for this reason the values of 100% Ethanol

extract in S. aureus inoculated nutrient agar plates have been recorded by

detracting 10 mm from the final inhibition zones.

All the tested spice extract at all dilutions (from 100% to 25% of spice

extract) were active against the tested microorganism but Mint and Basil extracts

at all dilutions. The most active extracts were Sumac and Oregano showing a 8

mm difference in zone of inhibition on S. aureus from 100% to 25% of spice

extract dilution.

At the same time, Thymbra extract was efficient at 100% and 75% original

extract dilutions on E. coli (11.5 and 9 mm, respectively) and at 100% original

extract on S. aureus (12 mm) both tested microorganism, but decreasing the

concentration of the solution it has not been showing any antimicrobial activity.

Recent studies in food preservation have been taken into consideration

while evaluating the outcomes showing prominent results in enhancing food shelf-

46

life by plant extracts. Biological properties of plant extracts and their

antimicrobial activity have been attributed to phenolic compounds, such as the

carvacrol and thymol [46

Seydim and Saricus, 2006]. These compounds have

hydrophobic characteristics and interact with different sites of microbial cell (e.g.,

cell wall and cytoplasmic membrane), causing loss of cellular constituents,

collapse of membrane structure, and cell death [47

Burt, 2004].

Scientific literature lacks of studies concerning crude spices antimicrobial

evaluations and due to the variability of plants in essential oil and active

components content, mainly evaluations have been carried on molecules. For

example, Carvacrol and Thymol are the most widely investigated molecules and

they have been found to be the most abundant constituents of the plants that have

been tested in this study as well as in the most commonly used herbs and spices.

Those molecules are the main constituents of Oregano and Thymbra and they

have been involved in further researches aimed to investigate their antimicrobial

effect at 0.03 or 0.06% on Escherichia coli in Tryptic Soy Broth. At 0.03 and

0.06% they showed an inhibitory activity against E. coli O157:H7 during storage

at 25 ºC [48

Hammou et al. 2011].

In further studies, the essential oil of Thymbra (containing mainly Carvacrol

75.5%) at 1/50 and 1/100 concentration was inhibitory against several bacteria

including E. coli and S. aureus. Synergism of Thymol and Carvacrol and other

powerful antioxidant has been investigated and it exhibited greater inhibition than

sorbic acid alone at same concentrations [49

Akgul and Kivanc, 1988].

For instance, in this study the content in Carvacrol and Thymol had shown

antagonistic activity against food-borne pathogenic bacteria [50

Baydar et al.

2004]. The essential oils characterised by high contents of cavracrol, c-terpinene

and p-cymene, respectively from the aerial parts of wild Oregano (Origanum

Minutiflorum, endemic in Turkey), Oregano (Origanum vulgare), Black thyme

(Thymbra spicata) and wild Savory (Satureja cuneifolia Ten.) were inhibitory to

the growth of all the bacteria under test including E. coli and S. aureus.

It was previously determined that the decoction, extract and hydrosol of black

thyme inhibited moulds, yeasts and bacteria [51

Sagdic and Ozcan, 2004] and some

other researchers reported that Origanum inhibited yeasts and moulds [52

Sokovic

47

et al. 2002] prospecting a wider range of activities of plants extracts in food

systems.

Alcohol extracts from Turkish Sumac fruit have been reported to show

different inhibitory capabilities towards the tested bacterial strains, with Gram-

positive bacteria being more sensitive than Gram-negative bacteria

[53

NasarAbbas, et al., 2004]. Results obtained from the present study revealed a

similar trend for Syrian sumac fruit extract. The antimicrobial activity

demonstrated by Sumac extract against the microorganisms tested in this study is

comparable with those of other spices reported in the literature which inhibited the

growth of Gram-positive and Gram-negative microoorganisms [54

Ahmad I. et al.,

2001].

The zones of inhibition of diluted spices ethanol extracts on Nutrient Agar

inoculated with the two test microorganisms (E. coli – Gram negative and S.

positive – Gram negative) after 24 h of incubation at 37 ºC have been shown in

the following figures:

48

49

Figure 11: Oregano ethanol extract against E. coli and S. aureus

Figure 12: Effects of Thymbra ethanol extract on E. coli and S. aureus coltures

50

Figure 13: Sumac ethanol extracts inhibition zones against E. coli and S. aureus

Spices hot water extracts have been tested using the same procedure placing

sterile water paper discs as a sterility control. The results have been recorded and

listed in Table 7:

51

Table 7: Results of Agar Disc Diffusion method with hot water spice extracts


S. aureus E. coli

% water

extract 100 75 50 25 100 75 50 25

Sterility - - - - - - - -

K - - - - - - - -

R - - - - - - - -

Z - - - - - - - -

N - - - - - - - -

S - - - - - - - -

52

4.3 Antioxidant Activity Assessment

4.3.1 Trolox equivalent antioxidant capacity assay

The antioxidant activity of spices ethanol extracts has been measured by

sequentially recording the loss of absorbance of the ABTS+ solution in a range of

6 min by a spectrophotometer. All experiments have been taken in parallel at

different extract concentrations and the results have been expressed in average by

slopes as shown in the following figures:

Figure 14: Sumac absorbance loss

Figure 15: Thymbra absorbance loss

y = 74650x + 3,1102 R² = 0,9487

0

10

20

30

40

50

60

70

0 0,0002 0,0004 0,0006 0,0008 0,001

% i

nh

ibit

ion

concentration

Rhus coriaria

y = 50222x + 1,6621 R² = 0,9717

05

1015202530354045

0 0,0002 0,0004 0,0006 0,0008 0,001

% i

nh

ibit

ion

concentration

Thymbra spicata

53

Figure 16: Basil absorbance loss

Figure 17: Oregano absorbance loss

y = 27868x - 0,137 R² = 0,9914

-5

0

5

10

15

20

25

0 0,0002 0,0004 0,0006 0,0008 0,001

Ocimum basilicum

% i

nh

ibit

ion

concentration

y = 64464x + 0,231 R² = 0,9997

0

10

20

30

40

50

60

0 0,0002 0,0004 0,0006 0,0008 0,001

Origanum vulgare

concentration

% i

nh

ibit

ion

54

Figure 18: Mint absorbance loss

Trolox equivalent antioxidant capacity (TEAC) values have been obtained

by correlating the rates of Trolox inhibition (a strong antioxidant analogue of Vit.

E) at different concentration with the tested spices ethanol extracts rates at the

same concentration. Trolox standard inhibition slope is shown in Figure 19 while

final TEAC values have been shown in Figure 20:

Figure 19: Trolox absorbance standard slope

y = 31189x + 0,7104 R² = 0,9855

0

5

10

15

20

25

30

0 0,0002 0,0004 0,0006 0,0008 0,001

% i

nh

ibit

ion

concentration

Mentha spicata

y = 4,042x

R² = 0,9979

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25

% i

nh

ibit

ion

.

concentration

55

Figure 20: Teac values of tested spice extracts

4.3.2 Total phenols content

Figure 21: Total Phenols values for plant extracts

0

10

20

Rhuscoriaria

Thymbraspicata

Ocimumbasilicum

Origanumvulgare

Menthaspicata

TEAC

aox capacity

0

0,1

0,2

0,3

0,4

0,5

Rhuscoriaria

Thymbraspicata

Ocimumbasilicum

Origanumvulgare

Menthaspicata

Total Phenol

Total phenol average

Table 8: TEAC values

Antioxidant Capacity (TEAC)

R. coriaria 18,50

T. spicata 12,45

O. basilicum 6,90

O. vulgare 15,95

M. spicata 7,80

56

4.3.3 Total flavonoids content

Figure 22: Total flavonoids values for plant extracts

0

0,02

0,04

0,06

0,08

Rhuscoriaria

Thymbraspicata

Ocimumbasilicum

Origanumvulgare

Menthaspicata

Total Flavonoid

Total Flavonoid average

57

5. CONCLUSIONS

Food preservation dates back to very old times and the potential benefits of

aromatic plants have been used both for their preservative and medicinal attributes

as well as to impart flavour to food by improving its organoleptic features. Food

antimicrobials are considered as the compounds that hinder microbial growth or

kill microorganisms. Recently, there has been considerable interest in natural

extracts from medicinal and edible plants, herbs and spices for the development of

alternative food additives, in order to prevent the growth of food-borne pathogens

or to delay the onset of food spoilage [55

Oke et al. 2009].

It has long been recognized that plants have antimicrobial properties and

these have been reviewed in the past as have the antimicrobial properties of spices

[56

Shelef, 1983] but the relatively recent enhancement of interest in ‘green’

consumerism has led to a renewal of scientific interest in these substances.

Besides antibacterial properties [57

Rasooli and Owlia, 2005], plants or their

components have been shown to exhibit antiviral [58

Bishop, 1995], antimycotic

[59

Mari et al. 2003], antioxidant [60

Bektas et al. 2007], antitoxigenic [61

Ultee and

Smid, 2007], antiparasitic [62

Pessoa et al. 2002] and insecticidal [63

Karpouthsis et

al. 1998] properties. These characteristics are possibly related to the function of

these compounds in plant [64

Mahmoud and Croteau, 2002].

The antimicrobial activity of plants has been known for more than 60 years

and it is related mainly to the complex mixtures of organic compounds they

contain. However, only recently the biologically active compounds associated

with plant natural products were subjected to close investigations throughout

advanced analytical techniques that permitted the disclosure of their chemical

composition and the evaluation of their biological activities in vitro or in vivo.

Studies on this field are topical and could have reflexes of economic

importance considering that, in recent years increasing interest was directed

towards the discovery of new antimicrobial compounds, due to an alarming

increase of outbreaks and multiplying in the rate of infections with antibiotic-

resistant microorganism.

The scientific literature abounds with reports concerning the determination of

chemical compositions and antimicrobial properties of various herbs and spices,

58

as well as their applications in various commercial preparations, mainly as

antimicrobial and antioxidant agents [65

Baydar et al. 2004].

A growing awareness among consumers towards health and nutrition has

led the scientists’ interest on natural preservatives since, as an alternative to

certain disadvantages associated with chemical preservatives, industries are

paying more emphasis on the use of natural molecules. Utilization of natural

preservatives has rendered foods with high nutritional value, free from chemical

preservatives and adequate microbiological safety [66

Meena and Sethi, 1997].

Spices also stimulate appetite by increasing salivation, carminative action

and preserve the food by their antimicrobial and antioxidant properties. More than

400 spices are used in the different countries in the world. Since ancient times,

spices and herbs have been used for preventing food spoilage and deterioration,

and for extending shelf-life of food as well.

Until recently, plant extracts has been studied most from the viewpoint of

their flavour and fragrance only for flavouring foods, drinks and other goods.

However, plant extracts and their components are gaining increasing interest

because of their relatively safe status, their wide acceptance by consumers, and

their exploitation for potential multi-purpose functional use [67

Ormancey et al.

2001].

These molecules damage membrane integrity by increasing its

permeability followed by affecting pH homeostasis and equilibrium of inorganic

ions as well as dissipation of internal pH gradient [68

Lambert et al. 2011].

Moreover, p-cymene is reported to incorporate in the lipid bilayer of bacterial cell

to facilitate transport of carvacrol across the cytoplasmic membrane [69

Ultee et al.

1999].

Synergy is the interaction of the compounds and/or factors in such a way

that the activity of individual compounds or factors is increased when they are

applied together. Compounds in the mixtures of spices and herbs have shown to

have synergistic activity. Although the amount of spices in food systems may not

always be enough to produce antimicrobial effect, when combined with intrinsic

factors such as pH and extrinsic factors such as temperature they may exert

antimicrobial activity.

59

The use of spices with other food ingredients such as sodium chloride,

sugar and organic acids and also with thermal processing might provide a

synergistic effect in controlling microbial growth [70

Giese, 2004]

For example, heat stress may cause cellular membrane damage,

impairment to or lesions in the cytoplasmic membrane that allows essential oils to

move rapidly into the interior of the cells. The essential oils then impair the

metabolic functions and interfere with the recovery metabolism of injured cells by

their specific mode of actions. Essential oils moreover at low pH dissolve in

and/or attach to the lipid phase of the bacterial membrane [71

Skandamis and

Nychas, 2000] prospecting an interesting field of application in non thermal

processing.

Use of spice blends such as the chili powder (Red pepper, Onion, Paprika,

Garlic, Cumin and Oregano) and the oriental five (Pepper, Cinnamon, Anise,

Fennel and Coves) in food produces powerful antimicrobial effects. Spice extracts

are used to flavour and preserve many foods. Spices used in sausage making when

combined with organic acids (citric acid, acetic acid), salt and heating show

stronger antimicrobial effects [72

Ziauddin et al. 1996].

Furthermore, it is also known that the antimicrobial effects of essential oils

and the extracts of medicinal plants may be subjected to change based on the

variations in the chemical composition of an essential oil that may be observed

due to the origin, the locality, the climate conditions, and the harvest time of the

collected plant material.

Generally speaking, the functions of food antimicrobials are to inhibit or

inactivate spoilage and pathogenic microorganisms. These functions have

increased in importance in the past 10–15 years as food processors search for

more and better tools to improve food safety especially those depending on the

use of natural derivatives as antimicrobial agents [73

Davidson, 2001].

The main inducement to search for effective antimicrobials among naturally

occurring compounds is to expand the spectrum of antimicrobial activity over that

of the regulatory-approved substances; most of currently approved and traditional

food antimicrobials have limited application due to pH or food component

interactions [74

Davidson and Zivanovic, 2003].

60

Most antioxidants are phenolic substances, more rarely nitrogen heterocycles.

In the food industry, synthetic antioxidants are mostly used. However, modern

consumers are commonly afraid of any synthetic chemicals. They feel that natural

antimicrobials and antioxidants are safer and more acceptable to the human body.

Therefore, food producers try to add natural antioxidants when possible [75

Taiel

and El-tras, 2011].

At the same scope food industry is heading moreover non thermal

preservation techniques since they less affect food original characteristics. The

most recently studied are ionization, radiation, high hydrostatic pressure, pulsed

electric fields and active packaging. Among novel biopreservation techniques also

Lactic Acid Bacteria and other microbial cultures are used with the same purpose.

The noise about biopreservatives in the food supply chain could enable

manufacturers to guarantee convenient, shelf stable and safe products that might

easily reach the consumers target. At the same time the consumers raised need to a

preservative free food as well as a tasty and safe meal will be completely full-

filled.

Biological properties and antimicrobial activity are attributed to phenolic

compounds such as carvacrol, thymol whose hydrophobic features allows them to

interact with different sites of microbial cells causing collapse and death. Besides

antibacterial properties, plant constituents isolated or in the whole fraction have

been demonstrated to exhibit antiviral, antimycotic, anti-toxigenic, anti-parasitic

and insecticidal properties. These characteristics are possibly related to the

original function of these compounds in plants.

Antimicrobial and antioxidant activities of spices are recognized as an

important factor in providing their inclusion in food conservation systems when

proper measures are taken in order to assure their satisfactory microbiological

quality. These measures must include actions to control the water activity, good

sanitary conditions during processing, worker training, pertinent transport

conditions and properly storage. Microbial quality monitoring procedures should

be applied since harvest until their insertion in food systems. Use of spices as a

microbial growth inhibitor in food is often limited due to their flavour since in

most cases the effective antimicrobial dose exceed the organoleptic accepted

61

level.

Nonetheless, combination of spices and other antimicrobial barriers could

enhance food shelf-life stability and microbial safety even in moderated levels.

Due to this and due to the fact that spices are considered as G.R.A.S., the

antimicrobial properties of herbs and their constituents could be suitable

alternatives for inclusion in food conservation systems and could act sometimes as

main or adjuvant antimicrobial compounds. Before including spices or their

derivatives as preservative in food some opportune evaluations about

microbiological quality, economic feasibility, long-term antimicrobial or

antioxidant effects and toxicity should be carried out.

62

6. REFERENCES

1 Madsen, M.G., Grypa, R.D., 2000. Spices, Flavour Systems, The Electronic

Nose. Food Technology, 54(3):44-46.

2 Balentine, D.A., Albano, M.C., Nair, M.G., 1999. Role Of Medicinal Plants,

Herbs And Spices In Protecting Human Health. Nutrition Review, 57(9):541-

545.

3 Wang, M., Kikizaki, H., Zhu, N., Sang, S., Nakatani, N., HO, C., 2000.

Isolation And Structural Elucidation Of Two New Glycosides From Sage

(Salvia officinalis L.). Journal Agriculture Food Chemistry, 48(2):235-238

4 Kiskò, G., Roller, S., 2005. Carvacrol And P-Cymene Inactivate Escherichia

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