1.

1 Food Preservation

Food preservation has been characterized for nutritious and

microbiologically stable foods and it has been archived by controlling
the growth of spoiling and pathogenic food-related microorganisms.
Microbial control in foods could be assured by suppressing one or more
essential factors for microbial survival (Horace, 1982). It could be
possible by adding suitable substances (weak organic acids, hydrogen
peroxide, chelators, and organic biomolecules) and applying physical
(temperature, packaging) and/ or chemical procedures (pH, oxide-
reduction potential, osmotic pressure) would make some microorganism
unviable (Ray, 1996; Brull and Coote, 1999).

1.2 Synthetic Chemical Preservatives

There has been increasing concern of the consumers about foods free or with
lower level of chemical preservatives because these could be toxic for
humans (Bedin et al., 1999). Concomitantly, consumers have also
demanded for foods with long shelf-life and absence of residual
chemicals. This perspective has put pressure on the food industry for
progressive removal of chemical preservatives and adoption of natural
alternatives to obtain its goals concerning microbial safety.

1
Uncontrolled use of synthetic chemical preservatives has been inducing
factor for appearance of microbial strains more and more resistant to
classic antimicrobial agents. Fifty years of increasing use of chemicals
antimicrobials have created a situation leading to an ecological
imbalance and enrichment of multiple resistant pathogenic
microorganisms (Levy, 1997). The successful story of microbial chemo
control lies in the continuous search for new antimicrobial substances to
control the challenge posed by resistant strains (Notermans and
Verdegaal, 1992).

Case Study: Great Britain did a 4 week study involving 277 normal three year-
old children, to find the effects chemical food dyes and the preservative sodium
benzoate, would have on preschool children. Much to their surprise, the
researchers found that even a modest amount of food additives had a profound
effect on the children's behavior. Although none of these children were
considered to be hyperactive, ADD, ADHD, or PDD before the study, during the
test period when they consumed drinks with these food dyes and sodium
benzoate, nearly one child in four clearly showed disturbed behavior.

For two weeks the children drank fruit juice that did not contain additives, and
during the other two weeks their juice looked the same, but contained a blend of
four food dyes and the preservative sodium benzoate. The parents were not aware
of when the children received the plain juice and when their juice was laced with
additives. During this "challenge period" parents reported these reactions:
disturbing others, difficulty settling down to sleep, poor concentration and temper
tantrums (www.bantpractitioners.com).

2
1.3 Antimicrobial Resistance

Antibiotic resistance in food borne pathogens is a reality, though substantial

qualitative and quantitative differences have been observed (Teuber,
1999). Strains of resistant food borne pathogens to a variety of
antimicrobials have become a major health concern (Kiessling et al.,
2002) and it could decrease the successful application of control
measures on spoilage and pathogenic microorganisms, many times
leading for use of less safe, ineffective or expensive alternatives (Levy,
1997). Changes in the antimicrobial target, inactivation by enzymes,
changes in cellular permeability, antimicrobial active efflux and
overproduction of target enzymes and bypass of the antimicrobial have
been common mechanisms of antimicrobial resistance (McKeegan et al.,
2002).

Brull and Coote (1999) have reported microbial resistance for some
antimicrobials used in food conservation as weak-organic acids, hydrogen
peroxide, chelators and some small organic biomolecules. β-lactams falls
under the most important class of antibiotics, accounting for the two-thirds
of the drug arsenal against bacteria. E. coli produces ‘Extended-spectrum β-
lactamase’ (ESBL), which destroys a large number of widely used
antibiotics. A new class of ESBL (CTX-M) was detected in this strain. The
ESBL producing E. coli resist penicillins and cephalosporins (Novais et al.,
2006). The spread of multi-drug resistant strains of S. aureus (MRSA) have
made those infections more difficult to treat (Cosgrove et al., 2003). P.
aeruginosa producing OXA-4 β-lactamase was observed in 17 % of the
resistant isolates in Japan (Marumo et al., 1999).

3
1.4 Preservatives of Natural Origin

Recently, there has been increasing interest in discovering new natural

antimicrobials (Sagdiç et al., 2003a); this is also has been true in food
microbiology. Plant products with antimicrobial properties notably have
obtained emphasis for a possible application in food production in order to
prevent bacterial and fungal growth (Lanciotti et al., 2004). Plant products
are characterized for a wide range of volatile compounds, some of which are
important flavor quality factors (Utama et al., 2002). Moreover, plant
volatiles have been generally recognized as safe (GRAS) (Newberne et al.,
2000).Systematic screening for biological interactions between
microorganisms and plant products has been valuable source of new and
effective antimicrobial substances, which could have different action ways
on the microbial cell, when compared to other conventional antimicrobials.
Plants synthesize, by a secondary metabolism, many compounds with
complex molecular structures and some of them have been related with
antimicrobial properties found in plant and their derivatives. Among these
secondary metabolites, flavonoids, isoflavonoids, tanins, coumarins,
glycosides, terpenes and phenolic compounds are generally found to be non-
toxic to humans and detrimental to food-borne pathogens (Simões et al.,
1999).

4
1.4.1 Spices and Herbs

Being natural food products, spices appeal to consumers who tend to

question the safety of synthetic additives (Farag et al., 1989; Sagdiç
2003b). Spices have been defined as plant substances from indigenous or
exotic origin, aromatic or with strong taste, used to enhance the taste of
foods (Germano and Germano, 1998). Spices include leaves (bay, mint,
rosemary, coriander, laurel, and oregano), flowers (clove), bulbs (garlic,
onion), fruits (cumin, red chilli, and black pepper), stems (coriander,
cinnamon), rhizomes (ginger) and other plant parts (Shelef, 1983).
Spices are well known for their medicinal, preservative and antioxidant
properties, they have been currently used with primary purpose of
enhancing the flavor of foods, indirectly extending shelf-life (Aktug and
Karapinar 1986, Ristori et al., 2002).

Antimicrobial properties of spices have been documented in recent years and

the interest continues to be pursued (El Shami et al., 1985; Akgul and
Kivanç, 1988; Cosentino et al., 1999; Domans and Deans, 2000; Ristori
et al., 2002; Sridhar and Rajaopal, 2003). Iranian garlic exhibited a dose
dependant activity on Staphylococcus aureus 8327 (Shokrzadeh and
Ebadi, 2006). Still, little information is available emphasizing the
preservative and antimicrobial role of spices in the prevention of foods
of the microbial action (Arora and Kaur, 1999).

Table: 1 List of certain spices & herbs and its chemicals with proven antimicrobial
activity.

5
COMMON NAME COMPOUND ACTIVITY
Aloe barbadensis Corynebacterium spp.,
(Aloe) Latex Salmonella, S. aureus and
Streptococcus.

Matricaria
Anthemic acid Salmonella typhimurium,
chamomilla
(Phenolic acid) S. aureus and Helminths.
(Chamomile)

Capsicum annuum
Capsaicin
(Chilli peppers) Bacteria
(Terpene)

Onion Allicin
Allium cepa (Sulfoxide) Bacteria and Candida
Purple prairie clover
Petalostemum Petalostemumol
Bacteria and Fungi
purpureum (Flavanol)
Japanese Herb
Rabdosia Trichorabdal A
Helicobacter pylori
trichocarpa (Diterpene)

6
Spices are recognized to stabilize the foods from the microbial deterioration.
This could be observed when spices show initially high microbial charge
and as time progresses, the microbial growth become progressively
slower or it is eventually totally suppressed (Kizil and Sogut, 2003).
Antimicrobial activity of spices depend on several factors, which
includes: (i) kind of spice, (ii) composition and concentration of spice,
(iii) microbial species and its occurrence level, (iv) substrate
composition and (v) processing conditions and storage (Shelef, 1983;
Farag et al., 1989). Spices are also used for medicinal purposes
(Ekweney and Elegalam, 2005).

Many plant extracts have shown the presence of antimicrobial properties.

Anti-infective agents like emetine, quinine and berberine remain highly
effective instruments in the fight against microbial infections. There are
wide ranges of antibiotics that are used for treatment of bacterial
infections but there are still some challenges to be met in microbial
chemotherapeutic agent is due to abuse of these drugs (Reuter, 2005).

1.5 Scope of the present study

7
Owing to the side effects and the resistance that pathogens build against
antibiotics, much attention has been paid to extracts and biologically
active compounds isolated from plant species. Antimicrobial activity of
various spices has been well documented. Nevertheless, their synergistic
activity (enhancement/ suppression) against bacterial contaminants has
not been studied, particularly in the food-borne pathogens. Essential oils
from the genus Ocimum has been reported to exhibit antimicrobial
activity. But their synergistic activity with spice extracts has never been
attempted. Thus, our study was focused on the aforementioned avenue
for deriving formulations that could be applied as an additive for food
preservation.

1.6 Objectives of the study

8
To determine the efficacy of crude extracts from various common spices i.e.
Allium cepa (Onion & Scallion), Allium sativum (garlic), Curcuma longa
(Turmeric), Syzygium aromaticum (Clove), Citrus limon (Lemon),
Azadirachta indica (Neem) and 4 species of Ocimum (Thulasi) i.e.
Ocimum sanctum, O. basilicum, O. canum and O. tenuiflorum against
selected resistant isolates of bacterial food-borne human pathogens

To identify the most promising leads and testing the efficacy of various
possible combinations of these extracts against the susceptible pathogens

Standardization of thin layer chromatograms for the promising leads (pure

extracts & mixtures) to identify the number of compounds and its classes

Testing the efficacy of the most promising extract combinations in certain

food products, under in vitro conditions.

Foods are freshest and at optimum quality at the time of their harvest or
slaughter. To maintain this quality in foods that will be consumed later,
the foods can be preserved by cold, heat, chemical preservatives, or
combinations of these methods. Cold preservation literally means
refrigeration or freezing, while heating involves many processing methods,
such as pasteurization, commercial sterilization, and drying (Morris et al.
2004). Adding preservative ingredients and processing by means of
fermentation are also ways to preserve foods. The shelf-life of a product is

9
defined as the expected time duration that a product will remain
organoleptically acceptable.

2.1 Synthetic Chemical Preservatives

Prescott et al. (2002) defined preservatives as a group of chemical

compounds deliberately added to food or that appears in food as a result of
pre-processing treatment, processing or storage. These include simple
organic acids (such as propionic acid, sorbic acid, benzoic acid) p-hydroxyl
benzoate alkylester (parabens), ethylene/propylene oxides, sulfides, ethylene
oxide (gas sterilant), ethyl formate and sodium nitrates. The sulphites inhibit
yeasts, moulds and bacteria and are most effective as inhibitors of browning
in foods. Sulphur dioxide and sulphites are metabolized to sulphate and are
excreted in the urine without any obvious pathological result (Charles et al.
2000).

Benzoic acid has been widely employed as an antimicrobial agent in foods

and it occurs naturally in cranberries, prunes, cinnamon and cloves. It is well
suited for acid foods such as fruit juices, carbonated beverages, pickles and
sauerkraut (Park et al., 2001). Benzoic acid has been found to cause no
deleterious effect when used in small amounts. It is, however, readily
eliminated from the body after conjugation with glycine to form hippuric
acid (Ihekoronye and Ngoddy, 1995). This work was therefore aimed at
determining the effect of some chemical preservatives on the shelf-life of
various food stuffs.

2.2 Food hazards

10
A hazard can be defined as a potential danger which, if unchecked, can
cause harm. The emphasis is on the word potential, which implies that
although dangers are always present, their effects can be avoided if proper
preventive and corrective measures are taken in food handling. Food hazards
relate physical, chemical and biological contaminants that can render the
food unsafe for consumption. In case foods contaminated with these hazards
are consumed, the person will demonstrate a range of harmful effects. The
elimination of hazards or their reduction to minimum levels is the basis of
food safety. Food hazards can be classified into three categories: (i)
Physical, (ii) Chemical and (iii) Biological.

Physical hazards are commonly referred to as foreign objects and are

visually present in foods. These items are not intentionally added to food
and find themselves there by accident, poor inspection practices or bad
hygiene. Common foreign objects are dirt, grease, plant fragments and
chipped bones.
Chemical hazards are contaminants of chemical origin and occur in foods as
chemical residues. The use of chemicals is an indispensable part of modern
society. Food production and preparation are not exempt from this practice.
Cleaning agents, insecticides and food preservatives are examples of
chemical hazards. Some of these chemicals are inherently present in foods
and function as natural defence mechanisms to protect the species. If foods
that contain natural hazards are consumed in large amounts, the possibility
of harm arises. Solanine in green potatoes and Ciguatoxin in large reef fish
are examples of natural hazards (Schmidt and Rodrick, 2003). Chemical

11
poisoning will produce symptoms of vomiting, diarrhoea and become fatal,
depending on the strength and type of chemical.

Biological and more particularly microbiological hazards are the effects

from growth and toxins produced by micro-organisms. Food poisoning and
food infections are caused by harmful micro-organisms and cause extremely
serious problems. Salmonella typhi, S. paratyphi-A, B and Clostridium
botulinum are examples of hazardous micro-organisms. Food spoilage,
although less hazardous, is a symptom of the activity of micro-organisms.

2.3 Biopreservatives

About 30% of people in developed countries at least once a year experience

a food borne disease. Therefore, there is a need for new methods to prevent
the growth of food borne pathogens or decrease the number of them in food
(Basti et al., 2007). The food industry has tend to reduce the use of chemical
preservatives in their products due to increasing pressure from consumers or
legal authorities, to either completely remove or to adopt more herbal
alternatives for maintenance or extension of product shelf life (Dillon and
Board, 1994; Nychas, 1995).

One of the concerns in food industry is the contamination by pathogens,

which are frequent cause of food borne diseases. Over the past decade,
recurrent outbreaks of diarrhea, combined with the natural resistance of the
causative agents, contributed to its status of hazard. The problem of selection
of bacteria resistant to antibiotics (Parada, 1980; Chopra et al., 1997; Rao,
1998; Kapil, 2005) and the increasing demand for safe foods, with less

12
chemical additives, has increased the interest in replacing these compounds
by natural products, which do not injure the host or the environment.

Biotechnology in the food-processing sector targets the selection, production

and improvement of useful microorganisms and products from plants and
other natural sources, as well as their technical application in food quality.
The use of non-pathogenic microorganisms and/ or their metabolites, to
improve microbiological safety and extend the shelf life of foods is defined
as ‘Biopreservation’ (De Martinis et al., 2001). Another important aspect of
biopreservation is the use of secondary chemicals from plant extracts, which
strictly falls under the category of natural chemical preservatives.

2.3.1 Microbial food preservatives

Antagonistic properties of lactic acid bacteria (LAB) allied to their safe

history of use in traditional food fermented products make them very
attractive to be used as biopreservatives (Parada, 1984; Caplice and
Fitzgerald, 1999). LAB and their metabolites had been investigated by
several authors (Buncic et al. 1997; Sakhare and Narasimha Rao, 2003).
Considerable research has been done on the ability of LAB to inhibit growth
of pathogenic microorganisms (Winkowski et al.1993; Minor-Pérez et al.
2004). To be successful in biopreservation, a bacteriocinogenic LAB culture
must compete with the relatively high indigenous microbial loads of raw
meat, to actively inhibit pathogenic and spoilage bacteria (Sakhare and
Narasimha Rao, 2003; Minor-Pérez et al. 2004). It was reported that the
shelf-life of meat could be extended by low temperatures combined with a
treatment with LAB strain (Babji and Murthy, 2000). Acid formation (low

13
pH), H2O2 and bacteriocins produced by starter cultures are responsible for
preventing the growth of food poisoning and spoilage bacteria in meat
(Krõckel, 1995; Budde et al. 2003).

Several studies have been carried out on the Physical-chemical

characteristics, sensory proprieties and nutritive values of camel meat treated
with biopreservatives (El-Faher et al. 1991; Elgasim and Al-Kanhal, 1992).
Al-Sheddy et al. (1999) explained the use of organic acid salts combined
with Bifidobacterium for the preservation of camel meat.

2.3.2 Bacteriocins

As antibiotics are at present restricted only to foods and feeds, Bacteriocins,

an interesting group of biomolecules with antimicrobial properties represents
a good alternative (Jack et al., 1995). The increasing interest in these
compounds has stimulated the characterization of many novel peptides
(Deraz et al., 2005). The successful development of nisin from an initial
biological observation through regulatory approval for commercial
applications is a model that has stimulated new contributions in the field of
bacteriocin research (Deegan et al., 2006).

2.3.3 Secondary compounds as biopreservatives

Secondary compounds or Shunt metabolites are small molecules (< 1500

amu) that are synthesized by plants and microorganisms, in response to
stress and overproduction of primary metabolites (Cannell, 1999). Most of
these secondary compounds that are used in the food industry fall under the

14
class of flavonoids and phenolics. In addition to the studies on antimicrobial
activity of essential oils in spices, the effectiveness of their chemical
compounds (small molecules) have also been investigated in order to
improve the understanding on the cell targets of these molecules found in
spices (Karatzas et al., 2000; Vasquez et al., 2001). Carvacrol, (+) carvone,
thymol and trans-cinnamaldehyde assayed against E. coli O157:H7 and S.
typhimurium suggested that carvacrol and thymol decreased the intracellular
ATP content of E. coli cells, while simultaneously increasing the
extracellular ATP. This indicated the disruptive action of these compounds
toward cytoplasmic membrane Helander et al. (1998)

Delaquis and Mazza (1998) described antimicrobial properties of

isothiocyanate derived from Allium cepa (Onion) and A. sativum. It was
hypothesized that isothiocyanates inactivated extracellular enzymes through
the oxidative cleavage of disulphide bonds (Brul and Coote, 1999). Delaquis
and Mazza (1998) purposed that the formation of reactive thiocyanate
radical could mediate the antimicrobial property. Ramos-Nino et al. (1996)
found that benzoic acids, benzaldehydes and cinnamic acid were able to
inhibit the growth of Listeria monocytogenes. The lipophilic molecular
portion of these compounds was recognized as being responsible for this
antimicrobial property.
2.4 Spices and Herbs

Spices have been defined as plant substances from indigenous or exotic

origin, aromatic or with strong taste, used to enhance the taste of foods
(Germano and Germano, 1998). Spices include leaves (bay, mint, rosemary,
coriander, laurel, oregano), flowers (clove), bulbs (garlic, onion), fruits

15
(cumin, red chilli, black pepper), stems (coriander, cinnamon), rhizomes
(ginger) and other plant parts (Shelef, 1983). Although, spices have been
well known for their medicinal, preservative and antioxidant properties, they
have been currently used with primary purpose of enhancing the flavor of
foods rather than intending to extend the shelf-life (Aktug and Karapinar
1986, Ristori et al., 2002).

Bioactive compounds in spices have been included in class of naturally

occurring food preservatives and have their inclusion in foods allowed by
food production regulatory authorities (Brull and Coote, 1999). Several
scientific reports describe the inhibitory effect of spices on a variety of
microorganisms, although considerable variation for resistance of different
microorganisms to a given spice and of the same microorganisms to
different spices has been observed (Akgul and Kivanç, 1988). Gould (1995)
has emphasized the possible use of spices and derivatives like alternatives
for inclusion in a new perspective of food conservation called “natural anti-
microbial system”.

The antibacterial activities of essential oils from spices have been known for
a long time and a number of researches on the antibacterial effect of these
volatile oils and their derivatives have been reported (Betts, 2000; Hseigh et
al., 2001; Sagdic and Ozcan, 2003; Delgado et al., 2004; Nasar- Abbas and
Kadir Halkman, 2004; Basti et al., 2007 and Fazeli et al., 2007). The
antimicrobial effect of several plants and essential oils has been studied on
E. coli (Dorman and Deans, 2000; Nostro et al., 2000; Skandamis and
Nychas, 2000; Marino et al., 2001; Ozcan and Erkmen, 2001; Salvat et al.,
2001; Sagdic et al., 2002; Dadalioglu and Evrendilek, 2004)

16
2.4.1 Allium sativum (Garlic)

Garlic has had an important dietary and medicinal role for centuries. There is
extensive literature on the antibacterial effects of fresh garlic juice, aqueous
and alcoholic extracts, lyophilized powders, steam distilled oil and other
commercial preparations of garlic. Garlic exhibits a broad antibiotic
spectrum against gram-positive and gram-negative bacteria like Aerobacter,
Aeromonas, Bacillus, Citrella, Citrobacter, Clostridium, Enterobacter,
Escherichia, Klebsiella, Lactobacillus, Leuconostoc, Micrococcus,
Mycobacterium, Proteus, Providencia, Pseudomonas, Salmonella, Serratia,
Shigella, Staphylococcus, Streptococcus and Vibrio species (Kabelik and
Uhrova, 1968) Enterotoxic E. coli strains and other pathogenic intestinal
bacteria, which are responsible for diarrhea in humans and animals, are
effectively inhibited by garlic than the normal intestinal flora. As a result the
toxin production by the bacteria is also prevented (Dewitt et al., 1979)

The antimicrobial effect in vitro of aqueous and ethanolic extracts of Allium

sativum (Garlic), Zingiber officinale (Ginger) and Citrus aurantifolia (Lime)
juice were assayed against S. aureus, Bacillus spp., E. coli and Salmonella
spp. All the test organisms were susceptible to undiluted lime-juice. The
aqueous and ethanolic extracts of garlic and ginger singly did not inhibit any
of the test organisms. However, the highest inhibitions (19 mm) were
observed with the combination on S. aureus and Salmonella spp.

2.4.2 Syzygium aromaticum (Clove)

17
S. aromaticum syn. Eugenia aromaticum or E. caryophillata are the
aromatic dried flower buds of a tree in the family Myrtaceae (Srivastava and
Malhotra 1991; Chaieb et al., 2007a). Cloves are used in ayurveda, chinese
and western herbalism. Cloves are used as a carminative, to increase
hydrochloric acid in stomach and to improve peristalis (Phyllis and James,
2000). In addition to this, the cloves are antimutagenic (Miyazawa and
Hisama, 2003), antiinflammatory (Kim et al., 1998), antioxidant (Chaieb et
al., 2007b), antiulcerogenic (Bae et al., 1998; Li et al., 2005), anti
thrombotic (Srivastava and Malhotra ,1991) and anti parasitic ( Yang et al.,
2003).

The essential oil extracted from the dried flower buds of the cloves is used
for acne, warts, scars and parasites. Research has shown that clove oil is an
effective mosquito repellent (Trongtokit et al., 2005). The clove oil is also
used as a topical application to relieve pain and to promote healing and also
finds use in the fragrance and flavoring industries (Chaieb et al., 2007a).
However clove oil is toxic to human cells. If ingested or injected in
sufficient quantity, it has been shown to cause life threatening
complications, including acute respiratory distress syndrome (ARDS),
fulminant hepatic failure and central nervous system (CNS) disorder
(Prashar et al., 2006).
Ethanol, aqueous extracts, and essential oils of S. aromaticum were analyzed
for determination of antibacterial activity against 21 food borne pathogens.
Screening of clove extract showed antibacterial activity against the tested
organisms. The MIC values for ethanol, aqueous extracts, and essential oil
from cloves range from 0.5 to 5.5 mg/ ml, 0.8 to 5.5 mg/ ml, and 1.25 to 5%
respectively. Essential oil of cloves showed anti bacterial activity after

18
treatment at 100 ºC for 30 min suggesting that the high temperature does not
affect the activity of EO. The highest anti bacterial activity was found at pH
5.0 (Hoque et al., 2007).

A study was carried out to investigate the potential of using aqueous

infusion, decoction and essential oil extracts of S. aromaticum as natural
antibacterial agent against 100 isolates belonging to 10 different species of
Gram -ve bacilli viz., E. coli (36), P. mirabilis (6), P. aeruginosa (5),
Klebsiella ozaenae (2), Klebsiella pneumoniae (24), Serratia marcescens
(4), Salmonella typhi (3), Shigella dysentriae (5) and Vibrio Cholerae (5) by
standard disc diffusion method.

The aqueous infusion and decoction of clove exhibited maximum activity

against P. aeruginosa (10.43 and 10.86 mm). Essential oil of clove exhibited
maximum activity against V. cholerae (23.75 mm). K. ozonae, K.
pneumoniae, S. marcescens, S. typhi, S. dysenteriae and V. cholerae where
found resistant to aqueous infusion and decoction while the essential oil
showed strong antibacterial activity against all bacterial isolates tested
(Saeed and Tariq, 2008).

2.4.3 Citrus limon (Lemon)

Citrus limon (Lemon) is a popular citrus fruit and a food ingredient for
flavouring and adding acidity. Lemon juices have been reported to exhibit
anti bacterial activity against Vibrio cholerae (de Castillo et al., 2000; Mata
et al., 1994).

19
The antimicrobial activity of lime juice against V. cholerae has been
reported (Rodrigues et al., 2000).. Lemon, lime and sudachi juices were
tested for antibacterial activity against seven strains of Vibrio species. All
juices were effective in inhibiting the growth of the Vibrio strains. Citric
acid, the major organic acid in these juices, was found to be responsible for
inhibiting the growth of Vibrio parahaemolyticus. Sauce prepared from
sudachi juice showed a strong bactericidal activity against V.
parahaemolyticus, whereas the sauce adjusted to higher pH values had no
bacterial activity. Diluted sudachi juice or citric acid solution also had
antibacterial activity independently. These results suggest that citrus fruit
juices are effective in preventing infection with Vibrio species (Tomotake et
al., 2005)

Citrus fruits, inclusing lemon are well known to possess terpenoids. 7 citrus
essential oils (EOs) were screened by disc diffusion assay for their
antibacterial activity against 11 serotypes/ strains of Salmonella. The 3 most
active oils, orange terpenes, single-folded d-limonene, and orange essence
terpenes were selected to determine the minimal inhibitory concentration
(MIC). EOs were stabilized in broth by the addition of 0.15% (w/v) agar for
performance of the MIC tests. All the 3 oils exhibited inhibitory activity
against Salmonellae. Orange terpenes and d-limonene both had MICs of 1%.
The most active compound, terpenes from orange essence, produced an MIC
that ranged from 0.13% to 0.5% against the all the strains. (Merih Kivanç et
al 1999)

2.4.4 Ocimum basilicum (Thiruneetrupacchai Tulsi)

20
Mediterranean countries, including Turkey (Tada et al., 1996), (Reuveni , et
al., 2002) Leaves and flowering parts of O. basilicum are traditionally used
as antispasmodic, aromatic, carminative, digestive, galactogogue, stomachic,
and tonic agents (Chiej et al., 1984) , (Duke et al., 1985). They have been
also used as a folk remedy to treat various ailments such as; feverish
illnesses, poor digestion, nausea, abdominal cramps, gastro-enteritis,
migraine, insomnia, depression, gonorrhea, dysentery, and chronic diarrhea
exhaustion (Chopra et al., 1986).

Externally, they have been applied for the treatment of acne, loss of smell,
insect stings, snake bites, and skin infections (Martin et al., 2004). However,
O. basilicum, like many other Ocimum species, has not been investigated
very well in terms of antimicrobial activities. In recent years, multiple drug
resistance in both human and plant pathogenic microorganisms have been
developed due to the indiscriminate use of commercial. antimicrobial drugs
commonly used in the treatment of infectious diseases (Davis et al., 1994;
Service et al., 1995) .

Out of hexane, ethanol and methanol extracts from Ocimum basilicum

(sweet basil) 146 microbial organisms belonging to 55 bacteria, 4 fungi and
different strains of C. albicans that were assayed, hexane and methanol
extracts exhibited anticandidal and bactericidal activities. Ethanol extracts
inhibited 3 out of 23 strains of C. albicans studied. All three extract of O.
basilicum were different in terms of their antibacterial activities. The hexane
extract showed a stronger and broader spectrum of antibacterial activity,
followed by the methanol and ethanol extracts, which inhibited 10, 9 and 6%
of the 146 bacterial strains tested, respectively. The minimal inhibition zones

21
(MIC) of the hexane, methanol, and ethanol extracts ranged from 125 to 250
µl/ ml, 62.50 to 500 µl/ ml, and 125 to 250 µl/ ml, respectively (Ahmet et
al., 2005)

The antimicrobial activities of chloroform, acetone and two different

concentrations of methanol extracts of O. basilicum were studied. These
extracts were tested in vitro against 10 bacteria and 4 yeasts strains by the
disc diffusion method. The results indicated that the methanol extracts of O.
basilucum exhibited the antimicrobial activity against tested
microorganisms. Methanol extracts showed inhibition zones against strains
of P. aeruginosa, Listeria, S. dysenteriae and Staphylococcus and two
different strains of E. coli (Kaya et al., 2008)

The essential oils of O. basilicum consisted of linalool as the most abundant

component (56.7-60.6%), followed by epi-α-cadinol (8.6-11.4%), α-
bergamotene (7.4-9.2%) and γ-cadinene (3.2-5.4%). S. aureus, E. coli,
Bacillus subtilis, Pasteurella multocida and pathogenic fungi Aspergillus
niger, Mucor mucedo, Fusarium solani, Botryodiplodia theobromae,
Rhizopus solani were affected by the treatment with these oils.

Samples collected in winter were found to be richer in oxygenated

monoterpenes (68.9%), while those of summer were higher in sesquiterpene
hydrocarbons (24.3%). The contents of most of the chemical constituents
varied significantly with different seasons. The essential oils investigated,
exhibited good antioxidant activity as measurements by DPPH free radical-
scavenging ability, bleaching β-carotene in linoleic acid system and

22
inhibition of linoleic acid oxidation. Both the activities were seasonal
(Abdulla et al., 2008)

2.5 Antimicrobial property

The main factors that determine the antimicrobial activity are the type and
composition of the spice, amount used, type of microorganism, composition
of the food, pH value, temperature of the environment, and proteins, lipids,
salts, and phenolic substances present in the food environment. Spices such
as S. aromaticum were found to inhibit both fungal growth and toxin
production in food stuffs (Hitokoto et al., 1980).

A. sativum has been found to possess antibacterial activity on ground camel

meat (Al-Delaimy and Ali, 1970). Prasad and Sharma (1981) also reported
the antifungal properties of garlic in poultry feed substrate. Adekalu and
Fajemisin (unpublished data) used garlic to extend the shelf life of tomatoes.
The two spices are flavoring agents in cooking and in drug formulations in
the United States of America where 1 billion pounds of dehydrated garlic are
used annually. Garlic has also been used as preservatives in food such as
tomatoes and meat sausages (Al Dehylaimy and Barakat, 1971). The use of
chemical preservatives and antimicrobial extracts from plants to prolong the
shelf life of food crops during storage are actively being investigated.

Al-Delaimy and Barakat (1971) reported the prolonged storage of fresh

Carmel meat by the use of fresh garlic as an antimicrobial and preservative
agent. Plants and their constituents have proved successful as potent fungi
toxicant that appear harmless to humans (Fawcett and Spencer, 1970; Beye,

23
1978). Udo et al. (2001) reported the possibility of utilizing alcoholic extract
of garlic to protect potato and yam against rots during storage.

3.1 Chemicals and Glassware

Glassware, microtips and eppendorf tubes (Borosil®, Tarson® and Schott-

Duran®) were initially cleaned with detergent (Protasan® DS) and immersed
in chromic acid cleaning solution overnight, washed thoroughly with tap
water, rinsed with distilled water and dried in hot air oven. Analytical grade
(AR) grade solvents were used for extraction (Merck®, Qualigens®,

24
HiMedia®). Bacteriological media, spreaders and petri dishes were
autoclaved at 121°C/15 mins.

3.2 Media, Antibiotics and Reagents

Table: 2 Bacteriological media

INGREDIENTS GM/LT.
MEDIA
Peptic digest of animal tissue 10.00
Beef Extract 01.50
Nutrient broth Yeast Extract 01.50
Sodium chloride 05.00
pH (at 25°C) 7.4±0.2
Beef infusion 300.00
Caesin acid hydrolysate 17.50
Mueller-Hinton
Starch 01.50
agar
Agar 17.00
pH (at 25°C) 7.3±0.1

Table: 3 Antibiotics employed for the assay

Gram –ve organism Gram +ve organism

A AMPICILLIN Tb Tobramycin
AC AMOXYCLAV
Ce Cephotaxime Ac Amoxyclav
E Erythromycin
Co Co Trimoxazole G Gentamicin
P Penicillin-G
Ox Oxacillin
Ce Cephalothin
25
Cd Clindamycin
Table: 4 TLC ( Spray reagents )

1 ML OF CONC. H2SO4 DETECTION OF MANY

ANISALDE
IN 50 ML ACETIC ACID COMPOUNDS, ESPECIALLY
HYDE
CONTAINING 0.5 ML TERPENES, SUGARS,
H2SO4
ANISALDEHYDE. PHENOLS AND STEROIDS.
Few grams of iodine Detection of many compounds
Iodine crystals were added to a especially flavonoids and
beaker and saturated. terpenoids.

26
3.3 Plant source

The spices and fruits used in the study i.e. turmeric, clove, ginger, garlic,
onion, scallion and lemon were bought from the local vegetable market.
Neem was collected from a fully grown tree in a residential area. The four
species of herbs i. e. Ocimum sanctum (Rama Thulasi), O. tenuiflorum
(Karum Thulasi), O. canum (Nai Thulasi) and O. basilicum were purchased
from Anna Botanical Farm, Arumbakkam, Chennai (fig: 1). They were
washed thoroughly with tap water and ground in a mixer with minimum
water after which they were distributed in conical flasks for extraction
(percolation method at room temperature).

27
 .

e
f
d

c g

b c a

j k

i l

Fig: 1 List of Spices & Herbs used for the study

a. Allium cepa (Scallion), b. Allium cepa (Onion),

c. Zingiber officinale (Ginger), d. Allium sativum
(Garlic), e. Curcuma longa (Turmeric), f. Syzygium
aromaticum (Clove), g. Citrus limon (Lemon),
h. Azadirachta indica (Neem), i. Ocimum sanctum
(Rama Thulasi), j. Ocimum canum (Nai Thulasi),
k. Ocimum basilicum (Thiruneetrupacchai), l. Ocimum
tenuiflorum (Karunthulasi)

28
3.4 Microorganism source

3.4.1 Human pathogens

Clinical strains of bacterial human pathogens (Table: 5) were obtained from
the Department of Microbiology, A. L. Mudaliar Post Graduate Institute of
Basic Medical Sciences (A.L.M.PGIBMS), Velachery, Chennai. The
organisms were sub-cultured in sterile nutrient agar slants that were used as
a source for further assays. Routine sub-culturing was done every fortnight
to prevent contamination.

Table: 5 List of bacterial food-borne human pathogens used for the assay

1. ESCHERICHIA COLI
2. Klebsiella pneumonia
3. Proteus vulgaris
4. Pseudomonas aeruginosa
5. Salmonella paratyphi-A
6. Salmonella paratyphi-B
7. Shigella boydii
8. Staphylococcus aureus
9. Staphylococcus epidermidis
10. Streptococcus faecalis

3.5 Extraction and preliminary bioassay with the crude extracts

Fresh spices and herbs were chosen for the study to specifically extract the
phytochemicals in its native form, devoid of the influence of any
temperature gradient. The plant material was extracted with methanol (6.8):
water (9.0) in the ratio 1:1, by percolation process at room temperature. This
procedure was particularly adopted to extract secondary chemicals, of both
high and low polar classes, in one step. The materials were soaked for 3
days, after which they were decanted and filtered using filter papers (Scholl

29
and Schultz®). The filtrate was condensed in a rotary evaporator (Buchi® R-
200 rotavapor) and dried under air draft for three days to completely remove
methanol, to obtain the crude water extract. The extract was stored at 5 °C
until usage (fig: 2).

a b c d e f g h

i j k l

Fig: 2 Aqueous methanolic extracts of the spices chosen for the study

a. A. cepa (Onion), b. A. cepa (Scallion), c. Z. officinale (Ginger),

d. A. sativum (Garlic), e. C. longa (Turmeric), f. S. aromaticum
(Clove), g. C. limon (Lemon), h. A. indica (Neem), i. O. sanctum
(Rama Thulasi), j. O. canum (Nai Thulasi), k. O. tenuiflorum
(Karuthulasi), l. O. basilicum (Thiruneetrupacchai)

Table: 6 Yield of Solutes

30
S. NO. SPICES & HERBS CONC.
(MG)

1. Allium cepa (Onion) 67

2. Allium cepa (Scallion) 68

3. Allium sativum (Garlic) 64

4. Azadirachta indica (Neem) 61

5. Citrus limon (Lemon) 65

6. Curcuma longa (Turmeric) 61

7. Syzygium aromaticum (Clove) 60

8. Zingiber officinale (Ginger) 60

9. Ocimum basilicum (Thiruneetrupacchai) 60

10. Ocimum tenuiflorum (Black Thulasi) 65

11. Ocimum sanctum (Rama Thulasi) 60

12. Ocimum canum (Nai Thulasi) 60

31
3.6 Antibacterial Disc diffusion assay (Kirby and Bauer) against
selected food-borne human pathogens

3.6.1 Preparation of extracts discs

2 ml of the crude extracts (6.5%) were taken in separate glass vials and
sterile whatman filter paper discs (7 mm diameter, HiMedia®) were soaked
in it for overnight (16 hrs). Then they were aseptically removed using sterile
forceps and thoroughly dried for 2 hrs under air draft to remove minute
traces of the solvent. The fortified discs were segregated according to the
concentrations (Fig: 3). Separate positive controls- Antibiotic discs
(HiMedia®) and negative controls- plain solvent were compared with the
activity of the extracts.

32
Fig: 3 Sterile filter paper discs (Whatman®) fortified with extracts

Plain discs were soaked in the respective extracts and dried in a sterile
glass plate

1 7

6 2 12 8

5 3 11 9

4 10

Fig: 4 Template of Bioassay- I

1. A. cepa (Onion), 2. A. cepa (Scallion), 3. Z. officinale (Ginger),

4. A. sativum (Garlic), 5. O. canum (Nai Thulasi), 6. S. aromaticum
(Clove), 7. C. limon (Lemon), 8. A. indica (Neem), 9. C. longa
(Turmeric), 10. O. sanctum (Rama Thulasi), 11. O. tenuiflorum
(Karunthulasi), 12. O. basilicum (Thiruneetrupacchai)

33
3.6.2 Preparation of starter culture and bioassay

A loopful of culture from different pathogens was derived from the slant
culture and inoculated in 2 ml of nutrient broth and incubated at 37°C
overnight. They were vortexed before the assay. Sterile Mueller-Hinton agar
(MHA) plates were seeded as a ‘lawn’ with the organisms using a sterile
cotton swab. The discs were placed equidistant using a template such that 6
discs were accommodated in a single plate. Triplicates were made and the
plates were incubated at 37°C for 24 hrs. The zone of inhibition around the
discs was measured using ruler and the average of the triplicates was
tabulated. Bacteriostasis was also observed by checking the growth of
resistant colonies within the zone of inhibition after 24 hrs of incubation
(fig: 4).

3.7 Assay with botanical combinations

Out of 9 extracts employed in the preliminary assay, 4 extracts turned out to

be promising. Hence, they were subjected to further assay in which several
possible combinations (Table: 7) were tested for its efficacy against the same
set of pathogens. This was specifically done to understand the effect of
combinatorial chemistry on the extracts. The protocol is as follows:

3.7.1 Bioassay

2 ml of equal concentrations of the different combinations (Table: 9) of the

most promising crude extracts short listed in the preliminary assay were
taken in separate glass vials. They were vortexed thoroughly for complete

34
homogenization and observed for consistency, before the fortification .
process (Section 3.6.1). Separate positive controls- commercial antibiotic
discs (HiMedia®) and negative controls- plain solvent were prepared along
with the extract combinations and tested for its detrimental activity. The
assay was carried out at room temperature in a biosafety level -II laminar
airflow unit. The process was performed as mentioned earlier (section 3.6.2)

Table: 7 Combinations of promising extracts used for the assay.

EXTRACT
INGREDIENTS
CODE
1. E1 + E2 + E3 + E4
2. E1 + E2
3. E2 + E3
4. E3 + E4
5. E2 + E4
6. E2 + E3 + E4
7. E1 + E2 + E3
8. E1 + E3 + E4
9. E1 + E2 + E4
10. E1 + E3
11. E1 + E4

E1- Citrus limon (Lemon), E2- Allium sativum (Garlic), E3- Syzygium aromaticum
(Clove), E4- Ocimum basilicum (Thiruneetrupacchai)

3.8 TLC Standardization of promising extracts and combinations

35
The promising pure extracts and botanical combinations were analyzed by
Thin Layer Chromatography (TLC). Ready made plates (SiO2 F-254, 230-
400mesh) of dimension (20 x 10 cm) were used as the sorbent. Various
combinations of hexane: EtOAc and Pet. ether: EtOAc were employed to get
the best separation of compounds. Finally, the mobile phase was
standardized as petroleum ether: EtOAc (7:3) that gave good resolution. 20
µl from each working standard solution was spotted and eluted with various
mobile phases mentioned (Table: 11). The dried plates were detected for
fluorescence under 254 nm. The chromogens were marked for its Rf values.

Table: 8 List of mobile phases used for standardization of TLC

S. MOBILE PHASE RATIO

NO.
1. CHCl3: MeOH: EtOAc: H2O 2.5 : 3 : 1.5 : 2
2. EtOAc: MeOH: HCOOH: H2O 5 : 0.2 : 0.3 : 6
3. EtOAc: HCOOH: HOAc: H2O 2.5 : 0.2 : 0.2 : 0.4
4. DCM: MeOH: H2O 4.5 : 1 : 0.1
5. BuOH: HOAc: H2O 7 : 1.5 : 2
6. THF: H2O: H3PO4 6 : 6 : 6.1
7. CH2Cl2: HOAc: H2O 2:1:1
8. CHCl3: MeOH: EtOAc 4 : 0.75 : 0.5
9. CHCl3: MeOH: EtOAc 4 : 0.45 : 0.5
10. CHCl3: MeOH: EtOAc 4 : 0.35 : 0.5
11. AcOH: CHCl3: H2O 8:2:1
12. CHCl3: MeOH: H2O 6.1 : 3.2 : 0.7
13. EtOAc: MeOH: H2O 5 : 0.3 : 1
14. EtOAc: HCOOH: H2O 6:1:1
15. CHCl3: MeOH: HOAc 9 : 0.5 : 0.5
16. MeOH: Water 4:1
17. EtOAc: HOAc 5:1
18. DCM: AcOH 8.5 : 1.5
19. DCM: AcOH 9:1
20. DCM: AcOH 9.5 : 0.5

36
3.9 In vitro testing models

Three models were chosen for the in vitro evaluation of the most promising
botanical combinations from the bioassays. Coconut paste (Chutney),
uncooked mushroom (Fresh) and tomato puree (fresh) were chosen for the
evaluation. The chutney was chosen specifically because it is freshly made
and served in various parts of food industries, as a supplementary. This
makes them prone to bacterial and fungal contamination, specifically by
food handlers, vessels and water with which it is made. Hence, freshly
prepared sample were bought from a restaurants. Uncooked mushroom is
bought as a packed material and tested for its purity and for the inhibition of
the spoilage after treatment with one of the most promising botanical
combination.

3.9.1 Organoleptic evaluation

Spoilage was determined by olfactory assessment and color change.

Expression of slight spoilage indicated moderate odor or color change while
complete spoilage indicated potent and objectionable deterioration.

3.9.2 Tomato Puree

37
Adekalu et al. (2009) has reported the preservative activities of A. sativum
and Eugenia aromatica on fresh tomato puree. This beckoned us to select
this sample for further testing with 4 different kinds of promising extract
combinations. Fresh tomatoes (Lycopersicon esculentum) were milled in a
blender and placed in covered glass beakers (Schott duran®, 50 ml). The
extract combinations (1 ml) were added to the tomato puree sample and
mixed thoroughly. Untreated samples (Control) were parallely maintained to
check the effect of atmospheric contamination. The samples were kept for
24 hrs, after which, they were subjected to plate count in nutrient agar.

3.9.3 Total and Coliform plate count

1 g of the sample is homogenized in 10 ml of sterile distilled water. This

forms the master stock. 1ml of the solution from the stock is diluted in 9 ml
of distilled water. The process is continued until the 4-fold dilution (104).
100 µl of 103 dilution was plated in Nutrient agar and MacConkey Agar
medium in triplicates using sterile glass spreaders. The plates were
incubated for 24 hrs at 37 ºC. Colony count was done using a colony counter
and results were tabulated .
4.1 Preliminary antimicrobial assay with pure (single) extracts of spices
and herbs

Out of 12 extracts of the spices and herbs tested against 11 isolates of

bacterial human pathogens, A. sativum, C. limon, S. aromaticum and O.
basilicum exhibited conspicuous inhibitory activity against 10 pathogens. C.
limon demonstrated remarkable broad-spectrum activity against all the 10
isolates with pronounced inhibition against S. aureus, S. epidermidis (17.3

38
mm), P. vulgaris (14.6 mm) and E. coli (13.3 mm) (Fig: 5-c, g, k & j) with
most promising activity against S. epidermidis and S. faecalis (18.6 mm)
(Fig: 5-f) compared to all the antibiotic controls, except Clindamycin (28.6
mm) (Table: 9). C. limon was also effective against S. paratyphi-A (Fig: 5-
a). Solvent controls showed no effect on the organisms tested.

S. aromaticum effectively exhibited bactericidal activity against S. boydii

(15.6 mm) and S. paratyphi-B (13.6 mm) (Fig: 5-h & d), the best among all
the extracts tested. Nevertheless, it did not show any inhibition against the
Gram +ve isolates. Similarly A. sativum was found to be ineffective at the
given concentration against the Gram +ve bacterial isolates. Nevertheless,
they exhibited notable activity against the Gram –ve organisms, with
maximum efficacy against E. coli and P. vulgaris (11 mm).

O. basilicum was the only herb extract that showed effective inhibition
against S. aureus (14.6 mm) compared to other extracts. It is also worthy to
note that none of the broad spectrum antibiotics were inhibitory to S. aureus
(Fig. not shown). This indicates the resistance developed in the isolate that
has rendered a supreme competence to the organism.

39
 a b

c d

e f

Fig: 5 Anti-bacterial assay with methanolic extracts (pure) of herbs

chosen for the study
a. S. paratyphi-A (C), b. S. paratyphi-A (A & S), c. S. aureus (C & O),
d. S. paratyphi-B (C & O), e. S. paratyphi-B (A & S), f. S. faecalis (C &
O)
Key: C- C. limon, A- A. sativum, S- S. aromaticum, O- O. basilicum

40
 g h

i j

k l

g. S. epidermidis (C & O), h. S. boydii (C), i. E. coli (C), j. E. coli (A),

k. P. vulgaris (C), l. K. pneumoniae (C & S)

Key: C- C. limon, A- A. sativum, O- O. basilicum, S- S. aromaticum

41
 Table: 9 Anti-bacterial activity of promising pure extracts of spices and herbs
against Gram +ve bacteria

TREATMENT S. S. EPI S. FAE

S AUR
A. sativum - - -
C. limon 12.6 17.3 18.6
S. aromaticum - - -
O. basilicum 14.6 14.6 11.3
Amoxyclav® R 10.3 21
Erythromycin® R 16.6 11
Penicillin-G® R 10.3 12.6
Oxacillin® R 10.3 8.6
Cephalothin® R 8.3 -
Clindamycin® R 28.6 18.3
Solvent Cont. - - -

Note: 100 µl of the sample is plated in each test. The average of the triplicates is tabulated.

4.2 Effect of commercial antibiotics on the tested pathogens

Out of the antibiotics tested, cephotaxime, gentamicin, tobramycin were

effective against Gram –ve bacteria (Fig: 6-a-e), except K. pneumonia (Fig:
6-g) that showed complete resistance to all the antibiotics tested. P. vulgaris
resisted all the antibiotics except Cephotaxime (Fig: 6-i). Gram +ve

42
pathogens, S. epidermidis resisted amoxyclav, penicillin-G, oxacillin and
cephalothin (Fig: 6-f) whereas S. faecalis was resistant to all the antibiotics
except clindamycin (Fig: 6-h). The results are mentioned in Table: 9 & 10.

Table: 10 Anti-bacterial activities of promising pure extracts of spices and herbs

against Gram -ve bacteria

TREATMENT E. K. P. S. S. PARA- S. PARA-

S COLI PNE VUL BOY A B
A. sativum 11 - 11 10 10.3 8.6
C. limon 13.3 9.6 14.6 11.3 14.6 11.3
S. aromaticum - - - 15.6 15 13.6
O. basilicum - - - - - 13
Ampicillin® - - - 11.6 21.6 21.6
Amoxyclav® - - - 17.3 23 23
Cephotaxime® 22.6 - 36.3 23.6 26 26.6
Cotrimoxazole® - - - - 28.3 31.3
Gentamicin® 20.3 10.6 - 18.3 18.6 23.3
Tobramycin® 16.6 8.6 - 14.3 17 15.3
Solvent Cont. - - - - - -

Note: 100 µl of the sample is plated in each test. The average of the triplicates is tabulated.

43
 a b

c d

e f

Fig: 6 Anti-bacterial assay with widely used antibiotics

a. S. boydii, b. S. paratyphi-B, c. E. coli, d. P. aeruginosa, e. S. paratyphi-A,

f. S. epidermidis

Antibiotics (Gram –ve ): Ampicillin (A), Cephotaxime (C), Cotrimoxazole

(CO), Tobramycin (Tb), Amoxyclav (Ac), Gentamicin (G)
Antibiotics (Gram +ve ): Amoxyclav (Ac), Erythromycin (E), Penicillin-G
(P), Oxacillin (Ox), Cephalothin (Ce), Clindamycin (Cd)

44
 g

h i

g. K. pneumoniae, h. S. faecalis, i. P. vulgaris

Antibiotics (Gram –ve ): Ampicillin (A), Cephotaxime (C),

Cotrimoxazole (CO), Tobramycin (Tb), Amoxyclav (Ac), Gentamicin
(G)
Antibiotics (Gram +ve ): Amoxyclav (Ac), Erythromycin (E), Penicillin-
G (P), Oxacillin (Ox), Cephalothin (Ce), Clindamycin (Cd)

45
4.3 Combinatorial activity of promising extracts of spices and herbs

Our attempt to study the bactericidal activity of the promising crude extracts
of the spices and herb species belonging to the genus Ocimum, revealed
many interesting and intriguing observations. From time immemorial, it has
been observed and thoroughly demonstrated that phytochemicals from
various plants, when treated as a mixtures, exhibits augmented/ suppressed
biological activities, under in vitro conditions. This observation was
strengthened by our experiments where the combined activities of the spice
and herbal extract either pronounced the antibacterial activity against the
tested pathogens and in some cases, suppressed the notable efficacy.

As we have observed that 3 spice extracts, i.e. C. limon, A. sativum, S.

aromaticum and 1 herbal extract, O. basilicum, the subsequent assay for
‘synergy’ evaluation was proceeded only with these leads. C. limon
independently inhibited the growth of E. coli in the screening assay,
whereas, the activity was totally nullified by the presence of other extracts.
On the contrary, the lemon extract in combination with other extracts
suppressed the growth of Gram +ve organisms, S. epidermidis and S.
faecalis (Table: 12). The triple combination of A. sativum, S. aromaticum
and O. basilicum was the only effective cocktail against E. coli.

All of the 11 combinations inhibited P. aeruginosa, where the ‘lemon-garlic-

clove-tnp Tulsi’ combination proved to be the one of the most efficacious
among all the combinations (15.3 mm). Nevertheless, the mixture of garlic
and tnp tulsi outweighed the activity tetra combination (17.3 mm). The
secondary growth of resistant organisms is predominantly observed within

46
the zone of inhibition in most of the plates (fig: 8) It is also evident that
lemon and clove extracts, at equal combinations, suppresses the activity of
the garlic.

Garlic-clove-tnp tulsi combination was the best among all the herbal
cocktails tested against K. pneumonia (19.2 mm), followed by clove-tnp
tulsi and the mixture of all the 4 extracts. Unusually, lemon in combination
with garlic, clove and tnp showed no evident inhibition (Table: 11). This
observation very clearly shows that phytochemicals precisely gets enhanced
and suppressed, revealing the activity of the ever-changing functionalities,
through assay systems. Further, it is quite convincing to observe that extracts
of pure compounds that were totally resisted by the pathogens, gains the
capacity to inhibit the same set of organisms when administered as cocktails,
where the ‘synergy’ plays a vital role in creation of new small molecular
entities that acquires functional groups to influence the growth of organisms,
under the in vitro conditions. This strengthens the practice of Ayurveda
where the physicians rely on botanical combinations for treatments.

Similar results were seen in the case of S. paratyphi-A, B and S. faecalis

when assayed with garlic-clove, garlic-tnp tulsi and clove-tnp-tulsi, where
there was no evident inhibition. Ironically, extract of lemon in combination
with other 3 extracts showed demonstrable inhibition (Table: 11). Among
the Gram +ve organisms tested, the behavior of S. aureus to the extracts
suggested a new dimension of thought on antimicrobial botanicals. Lemon
and tnp Tulsi inhibited the growth of S. aureus independently. Whereas,
none of the 11 cocktails tested, demonstrated inhibition. This total
suppression is quite interesting and beckoning to note.

47
 1 7

6 2 C 8

5 3 11 9

4 10

Fig: 7 Template of Bioassay- II (Synergy testing)

1- E1+E2, 2- E2+E3, 3- E1+E3, 4- E1+E2+E3, 5- E1+E2+E3+E4,

6- E3+E4, 7- E2+E4, 8- E2+E3+E4, 9- E1+E3+E4, 10- E1+E2+E4, 11- E1+E4

E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4-Obasilicum,

48
 a b

c d

e f

Fig: 8 Anti-bacterial assay with mixture of methanolic extracts

of promising herbs

a. E. coli (i), b. P. vulgaris (iv), c. P. vulgaris (ii), d. S.faecalis (i),

e. S. faecalis (ii), f. S. epidermidis (iii)

Note
: i & iii- E1+E2, E2+E3, E1+E3, E1+E2+E3, E1+E2+E3+E4, E3+E4
ii & iv- E2+E4, E2+E3+E4, E1+E3+E4, E1+E2+E4, E1+E4

E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum

49
 g h

i j

k l

g. S. epidermidis (ii), h. S. boydii (iv), i. S. Paratyphi-A (ii),

j. S. paratyphi-A (i), k. P. aeruginosa (i), l. S. paratyphi-B (ii)

i - E1+E2, E2+E3, E1+E3, E1+E2+E3, E1+E2+E3+E4, E3+E4

ii & iv- E2+E4, E2+E3+E4, E1+E3+E4, E1+E2+E4, E1+E4

50 S. aromaticum, E4- O.basilicum

E1- C. limon, E2- A. sativum, E3-
 Table: 11 Anti-bacterial activities of botanical combinations of promising extracts
against Gram -ve bacteria

TREATMEN E. K. P.AER P. S. S .PARA- S. PARA-

TS COLI PNE U VUL BOY A B

E1+E2+E3+E
- 15.4 15.3 - - 10.2 12.2
4
E1+E2+E3 - 11 12.3 16.2 10.4 10.5 -
E1+E2+E4 - 8.6 11.5 - 11.7 12.3 10.3
E1+E3+E4 - 9.5 10.3 16.2 7.5 11.3 9.6
E2+E3+E4 9.4 19.2 14.3 - - - -
E1+E2 - - 7.4 - 10.2 16.2 12.4
E1+E3 - - 11.5 17.3 10.7 17.4 8.3
E1+E4 - - 15.1 - 11.3 12.3 11.5
E2+E3 - 14 11.2 - - - -
E2+E4 - - 17.3 - - - -
E3+E4 - 18.3 14.1 7.3 - - -
Solvent Cont. - - - - - - -

Note: E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum. 100 µl of the sample is plated in
each test. The average of the triplicates is tabulated.

Table: 12 Anti-bacterial activity of promising pure extracts of spices and herbs

against Gram +ve bacteria

TREATMEN S. EPI S. FAE

TS

51
 E1+E2+E3+E4 11.6 -
E1+E2+E3 12 7.5
E1+E2+E4 8.5 14.7
E1+E3+E4 10.3 7.6
E1+E2 11.2 9
E1+E3 11.3 11
E1+E4 8.2 15.1
E2+E3 8.3 -
E2+E4 - -
E3+E4 7.6 -
E2+E3+E4 7.4 -
Solvent Cont. - -

Note: E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum. 100 µl of the sample is plated in
each test. The average of the triplicates is tabulated.

Through this combinatorial study, it is quite convincing that there is a huge

scope of research in the area of phytochemical/ nutraceutical cocktails that
are short listed, by some unknown means, by our ancient medical practioners
to treat the patients. Validation of these crude medicines would clear the
mindset of the world that traditional remedies for curing ailments are based
on a deep understanding of the behavior of plants, where the plant is not
seen as a single molecule but as a ‘whole living being’.
4.4 TLC Fingerprinting and the influence of synergy of compounds

15 different formulations of the promising extracts and combinations that

were eluted through TLC suggested 6 major spots, in which the first 3 spots
were predominantly found in all the combinations. 2 ice blue chromogens at
the Rf 0.85 and 0.5 were typically found in the lemon extract and all of its
combinations, except E1+E3 mixture. These chromogens didn’t undergo any
change in its structure and fluorescence property along with any of the
mixtures, except with clove extract. Corollary to this, the antibacterial of

52
lemon (E1) was very much evident against E. coli and K. pneumoniae, while
in the presence of clove (E1+E3), the activity gets completely diminished.
Hence, these might be the active principles responsible for the antibiotic
activity against other species. Moreover, whether these 2 molecules are
singly acting on the bacterial system or they act together is still a question to
be addressed.

Extract of clove (E3) featured 3 predominant fluorescence spots (Dark

Violet) at Rf 0.75, 0.62 and 0.53 respectively (Fig: 9, Lanes D and I). All the
combinations showed the presence of these compounds. However,
E1+E2+E3 and E1+E3+E4 mixtures lacked the first major compound. It is
also important to note that the first ice blue chromogen in lemon shares the
same Rf (0.77) as that of the first spot of clove. But the spot found in lemon
has a very imminent fluorescence that either masked or quenched the latter,
to form a new compound in the same Rf. Another observation in both the
mixtures (E1+E2+E3 & E1+E3+E4) was the visibility of another compound
near the origin of spotting. This particular chromogen was not found in the
pure extracts of the three extracts, which might be attributed to the
observation that pure clove extracts which was inactive against K.
pneumoniae and P. aeruginosa, gained bactericidal activities (Table: 9 &
11).

Pure clove and tnp tulsi were not detrimental to E. coli, K. pneumoniae and
P. aeruginosa, but to S. boydii, S. paratyphi- A and B. But as a mixture
(E2+E3+E4), the activity reverts itself making the resistant organisms
susceptible. Garlic and thp tulsi independently inhibited S. paratyphi-B. But
the activity gets suppressed if they act together, where except P. aeruginosa

53
none other organism shows inhibition. The chromatogram of garlic was the
simplest of all the extracts with 2 dominant spots. Therefore, it would be
effortless for the future workers to purify the bioactive antibacterial
compounds.

54
 0.5

0.5

0.5

a b c d e f g h I j k l Rf
m n o
Fig: 9 Thin Layer Chromatographic fingerprinting of the bioactive extracts &
combinations

a- E1+E2, b- E2+E3, c- E1+E3, d- E1+E2+E3, e- E1+E2+E3+E4, f- E3+E4, g- E2+E4,

h- E2+E3+E4, i- E1+E3+E4, j- E1+E2+E4, k- E1+E4, l- E3, m- E2, n- E4, O- E1

E1- C. limon E2- A. sativum E3- S. aromaticum E4- O. basilicum

55
 Table: 13 Comparative TLC profiles of the promising extracts/ combinations

NUMBER OF SPOTS
1 2 3 4 5 6
EXTRACTS
A (E1 + E2) Ice blue Dark Violet Pale blue Pale Violet - -
B (E2 + E3) Dark Violet Pale Violet Pale Violet Mild Violet Mild Violet -
C (E1 + E3) Dark Violet Pale Violet Mild Violet Pale Violet - -
D (E1 + E2+ E3) Ice blue Mild Violet Pale Violet Ice blue Mild Violet Dark Violet
E (E1 + E2+ E3+E4) Dark Violet Pale Violet Ice blue Mild Violet Pale Violet Dark Violet
F (E3 + E4) Dark Violet Pale Violet Mild Violet Dark Violet - -
G (E2 + E4) Ice blue Mild Violet Mild Iceblue - - -
H (E2 + E3 + E4) Dark Violet Mild Violet Mild Violet Mild Violet Pale Violet -
I (E1 + E3+ E4) Ice blue Mild Violet Mild Violet Mild Violet Pale Violet -
J (E1 + E2+ E4) Ice blue Pale Violet Mild Violet Pale Violet Mild Iceblue -
K (E1 + E4) Ice blue Mild Violet Mild Violet Pale Violet - -
L (E3) Dark Violet Pale Violet Mild Violet Mild Violet - -
M (E2) Pale Violet Mild Violet - - - -
N (E4) Dark Violet Mild Violet Pale Violet - - -
O (E1) Ice blue Mild Iceblue Mild Iceblue - - -

Note: E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum

4.5 Application Studies

Based on the bench top assays, one of the most promising broad-spectrum
combination E1+E2 (Lemon and Garlic) was evaluated for their
preservative property in 2 different food products.

4.5.1 Study: 1 Effect of E1 and E2 on Coconut paste (Chutney) and Raw

Mushroom

56
The effect of E1+E2 was initially evaluated against 2 of the fast moving
food stuffs. Total plate count (TPC in 103) in nutrient agar for 5 g of coconut
paste (Fig: 10) was beyond measure, exhibiting a large amount of microbial
load. Hence, it was fixed as TNTC (too numerous to count) for the
unfortified control. Whereas, 5 g of the sample treated with 500 µl of E1+E2
exhibited ‘nil growth’, depicting complete control of the microorganism by
the formulation (Fig: 11).

TPC (103) of raw mushroom (Fig: 12) in nutrient agar showed 300 colonies
compared to the control (TNTC). Whereas, the presence of coliforms (E.
coli, Salmonella, Shigella, Enterobacter and Citrobacter) was alarmingly
high (119 colonies) in the control. Treated sample showed ‘nil’ coliform
count, confirming the bactericidal preservative activity of E1+E2
combination (Fig: 13).

57
 a b

Fig: 10 Macroscopic changes observed in the samples studied

a. Layer of bacterial & fungal growth observed in coconut paste

(Chutney) exposed to open air for 2 days, b. Coconut paste treated
with 2 ml of E1-E2 combination. No spoilage was observed even
after 2 days rendering the product fit for human consumption.
Note: 1 ml of extract formulation is added to each sample.

a b

c d

Fig: 11 Spread plate enumeration of bacterial contamination

a. Control plate count in Nutrient agar showing bacterial

contamination, b. Total prevention of spoilage in treated (E1-E2)
sample combination. c. & d. Complete absence of coliform in
control and treated samples plated in Mac Conkey Agar media.
58
 Fig: 12 Macroscopic view of the mushroom sample
No physical changes were observed in the control and the treated samples
Note: 1 ml of extract formulation is added to the sample.

a b

c d

Fig: 13 Spread plate enumeration of bacteria contamination

a. Control plate count in Nutrient agar showing heavy bacterial

contamination, b. Notable reduction of spoilage in treated (E1-E2)
sample combination., c. Presence of Coliforms (lactose fermenting- pink
& non - lactose fermenting- colorless colonies) in the untreated control
puree sampleplated in Mac Conkey Agar media, d. Complete absence of
coliform in the treated sample

59
 Table: 14 Bacterial Total Plate Count (TPC) for Sample: 1 and 2

NUTRIEN MACCONK
SAMPLE TREATM T AGAR EY AGAR
(103) (103)
ENTS
Coconut paste E1 + E2 Nil Nil
Control - TNTC Nil
Raw Mushroom E1 + E2 300 Nil
Control - TNTC 119

Note: E1- C. limon & E2- A. sativum. 100 µl of the sample is plated in each
test. The average of the triplicates is tabulated. TNTC- Too Numerous to Count.

4.5.2 Study: 2 Effect of 4 promising combinations on Tomato Puree

Based on the assays, 4 different bioactive mixtures i. e. E1+E2, E2+E3,

E1+E3 & E1+E2+E3 were chosen for preservative efficiency testing in fresh
tomato puree sample. TPC (103) of fresh puree (control) in nutrient agar
showed numerous (TNTC) colonies, compared to the treatments with E1+E2
(119) and E1+E2+E3 (53). Whereas, samples treated with E2+E3 and
E1+E3 showed ‘nil’ growth of organisms. Coliforms were totally absent in
all the treatments and control (fig:15) This clearly showed that all the
combinations of garlic, lemon and clove tested possessed varying
preservative activities. Further studies on the active principles would give us
a better picture of the mode of action of these phytochemicals.

60
 a b

c d

e f

Fig: 14 Macroscopic changes observed in the Tomato Puree samples

a. Intact Tomato Puree sample obtained from a hotel, b. Control sample

exhibiting severe fungal & bacterial contamination after 24 hrs of
exposure to open air, c. Sample treated with E1+E2 and exposed for 24
hrs to open air, d. Sample treated with E2+E3, e. E1+E3, f. E1+E2+E3

Note: 1 ml of extract formulation is added to each sample. The color in

samples d., e. and f. were changed to brown due to the presence of clove
extract
61
 a

b c

d e

Fig: 15 Spread plate enumeration of bacterial contamination

a. Control sample (100 µl) exhibiting severe bacterial colonies after

overnight incubation, b. Sample treated with E1+E2 and exposed for 24
hrs to open air, c. Sample treated with E2+E3, d. E1+E3, e. E1+E2+E3

62
 Table: 15 Bacterial Total Plate Count (TPC) for Sample: 3

NUTRIEN MACCONK
TREATME T AGAR EY AGAR
SAMPLE
(103) (103)
NTS
E1 + E2 119 Nil
E2 + E3 Nil Nil
Tomato Puree
E1 + E3 Nil Nil
E1 + E2+E3 53 Nil
Control - TNTC Nil

Note: E1- C. limon, E2- A. sativum & E3- S. aromaticum. 100 µl of the sample
is plated in each test. The average of the triplicates is tabulated. TNTC- Too
Numerous to Count.

4.6 Synergy among spices

Even though the antibacterial activity of spices has been well demonstrated,
the synergistic study has not been attempted by many of the research groups.
The in vitro antibacterial effect of crude ethanol and aqueous extracts of A.
sativum, Z. officinale, C. longa and A. indica were assayed against S.
aureus, S. typhi and E. coli. The highest inhibition was observed with
synergistic combinations of ethanolic extracts of Garlic and Turmeric (70%)
extracts on S. aureus (15 mm) (Neogi et al., 2007).

Highest zone of inhibition observed with Garlic and Turmeric (Aqueous),

was 13 mm compared to standard antibiotics O-Trimoxazole, Ampicillin-A,
Cloxacilin, Chloramphenicol. Whereas, in our study, concentration ranges of
60-68 mg/ ml of spices were used for the assay. This might be a reason for
the absence of bioactivities in ginger, turmeric and neem that were below the

63
MICs mentioned. But in the present study, the detailed synergy effects of 3
spices were proved for further research. Results of these kinds herald an
interesting promise of designing a potentially active anti bacterial synergized
agent of plant origin.

The application of natural chemical preservatives in food industry is still in

its infancy. The discovery of biopreservatives is restricted specifically to
living organisms, especially beneficial bacteria that could be utilized for its
extracellular production of biomolecules. Novel lactobacillus species are
constantly being discovered around the world for large-scale application in
food preservation. Generally, many of the drug discovery projects are aimed
at these kinds of potent secondary compounds from spices for combating
various ailments like TB, duodenal ulcers, diabetes, cancer and AIDS.
Nevertheless, studies on ‘preservative small molecules’ of floral origin are
yet to be focused.

4.7 Pet food preservation

Chemical preservatives have been said to cause all sorts of different health
problems with household pets. Some of the common chemical preservatives
that you may find in pet foods are BHA (Butylated Hydroxyanisole), BHT
(Butylated Hydroxytoluene) and ethoxyquin. These three chemical
preservatives cause dry skin, dental disease and allergic reactions. They also
affect the functions of your pet's liver and kidneys. Ethoxyquin is regulated
by the FDA as a pesticide. (Petrozzella, 2008) Presently, Tocopherols
(vitamin E) and Ascorbic Acid (vitamin C) are two of the most common
natural preservatives that are used in pet foods. Hence, the application of

64
crude and purified phytochemicals will definitely redeem the strategy of
preservation among the pet food industries.

It is well known that except the food grown in your own garden all food
products have preservatives. Every manufacturer adds preservative to the
food during processing. The purpose is generally to avoid spoilage during
the transportation time. Because food is so important to survival, food
preservation is one of the oldest technologies used by human beings.
Different ways and means have been found and improved for the purpose.

Thus, the present study has exhibited the scope of employing

phytochemicals from spices as food preservatives in freshly made, quickly
perishable foods that are prone to bacterial and fungal infections.

Among the 12 spice and herbs extracts tested, A. sativum, C. limon, S.

aromaticum and O. basilicum exhibited conspicuous inhibitory activity

65
against 10 pathogens. C. limon demonstrated remarkable broad-spectrum
activity against all the isolates with pronounced inhibition against S. aureus,
S. epidermidis, P. vulgaris and E. coli. S. aromaticum exhibited effective
bactericidal activity against S. boydii and S. paratyphi-B, the best among all
the extracts tested against these 2 isolates. A. sativum exhibited notable
activity against the Gram –ve organisms, with maximum efficacy against E.
coli and P. vulgaris. O. basilicum was the only herb extract that showed
effective inhibition against S. aureus compared to other extracts. Most of the
extracts were better than the broad spectrum antibiotics used.

TLC suggested 6 major spots, in which the first 3 spots were predominantly
found in all the combinations. Extract of clove (E3) featured 3 predominant
fluorescence spots at Rf 0.75, 0.62 and 0.53. In the application studies, 5 g of
coconut paste and raw mushroom treated with 500 µl of Garlic and Lemon
(E1+E2) exhibited ‘nil growth’, depicting complete control of the
microorganisms, especially coliforms, by the formulation. The formulation
altered the color in tomato puree during treatment. Nevertheless, it
drastically reduced the microbial load.

Before including spices and/or their derivatives in food conservation

systems, some evaluations about microbiological quality, economic
feasibility, and antimicrobial effect for a long time and toxicity should be
carried out. If it is well established, spices and their derivatives could be
suitable alternatives for inclusion in food preservation systems.
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