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microbiologically stable foods and it has been archived by controlling the growth of spoiling and pathogenic food-related microorganisms.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, oxidereduction 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.

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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 yearold 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).

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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. -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 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 -lactamase was observed in 17 % of the resistant isolates in Japan (Marumo et al., 1999).

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1.4 Preservatives of Natural Origin Recently, there has been increasing interest in discovering new natural antimicrobials (Sagdi 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 nontoxic to humans and detrimental to food-borne pathogens (Simes et al., 1999).

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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; Sagdi 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 Kivan, 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.

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COMMON NAME Aloe barbadensis (Aloe)

COMPOUND Latex

ACTIVITY Corynebacterium spp., Salmonella, S. aureus and Streptococcus.

Matricaria chamomilla (Chamomile) Capsicum annuum (Chilli peppers) Onion Allium cepa Purple prairie clover Petalostemum purpureum Japanese Herb Rabdosia trichocarpa

Anthemic acid (Phenolic acid)

Salmonella typhimurium, S. aureus and Helminths.

Capsaicin (Terpene) Allicin (Sulfoxide) Petalostemumol (Flavanol) Trichorabdal A (Diterpene)

Bacteria

Bacteria and Candida

Bacteria and Fungi

Helicobacter pylori

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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

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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

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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

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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 hazards10

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

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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

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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-Prez 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-Prez 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 (low13

pH), H2O2 and bacteriocins produced by starter cultures are responsible for preventing the growth of food poisoning and spoilage bacteria in meat (Krckel, 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 the14

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

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(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 Kivan, 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 antimicrobial 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)

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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 after18

treatment at 100 C 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 Kivan et al 1999) 2.4.4 Ocimum basilicum (Thiruneetrupacchai Tulsi)

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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 zones21

(MIC) of the hexane, methanol, and ethanol extracts ranged from 125 to 250 l/ ml, 62.50 to 500 l/ ml, and 125 to 250 l/ 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--cadinol (8.6-11.4%), bergamotene (7.4-9.2%) and -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 radicalscavenging ability, bleaching -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 SchottDuran) 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 121C/15 mins. 3.2 Media, Antibiotics and ReagentsTable: 2 Bacteriological media

INGREDIENTS MEDIA Peptic digest of animal tissue Beef Extract Nutrient broth Yeast Extract Sodium chloridepH (at 25C) 7.40.2

GM/LT. 10.00 01.50 01.50 05.00 300.00 17.50 01.50 17.00

Beef infusion Mueller-Hinton agar Caesin acid hydrolysate Starch AgarpH (at 25C) 7.30.1

Table: 3 Antibiotics employed for the assay

Gram ve organism A Ce Co AMPICILLIN Cephotaxime Co Trimoxazole

Gram +ve organism AC E P Ox 25 Ce Cd Tb Tobramycin AMOXYCLAV Ac Amoxyclav Erythromycin G Gentamicin Penicillin-G Oxacillin Cephalothin Clindamycin

Table: 4 TLC ( Spray reagents )

ANISALDE HYDE H2SO4

1 ML OF CONC. H2SO4 DETECTION CONTAINING 0.5 ML TERPENES,

OF

MANY SUGARS,

IN 50 ML ACETIC ACID COMPOUNDS, ESPECIALLY ANISALDEHYDE. PHENOLS AND STEROIDS. Few grams of iodine Detection of many compounds crystals were added to a especially flavonoids and beaker and saturated. terpenoids.

Iodine

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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).

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.

e d c

f

g

h b c a j k

i

l

Fig: 1 List of Spices & Herbs used for the studya. 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. 2. 3. 4. 5. 6. 7. 8. 9. 10. ESCHERICHIA COLI Klebsiella pneumonia Proteus vulgaris Pseudomonas aeruginosa Salmonella paratyphi-A Salmonella paratyphi-B Shigella boydii Staphylococcus aureus Staphylococcus epidermidis 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 (Scholl29

and Schultz). The filtrate was condensed in a rotary evaporator (Buchi R200 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) 67 68 64 61 65 61 60 60 60 65 60 60

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Allium cepa (Onion) Allium cepa (Scallion) Allium sativum (Garlic) Azadirachta indica (Neem) Citrus limon (Lemon) Curcuma longa (Turmeric) Syzygium aromaticum (Clove) Zingiber officinale (Ginger) Ocimum basilicum (Thiruneetrupacchai) Ocimum tenuiflorum (Black Thulasi) Ocimum sanctum (Rama Thulasi) Ocimum canum (Nai Thulasi)

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 6 2 12

7 8 C

5 4

3

11 10

9

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 37C 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 37C 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 complete34

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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. E1 + E2 + E3 + E4 E1 + E2 E2 + E3 E3 + E4 E2 + E4 E2 + E3 + E4 E1 + E2 + E3 E1 + E3 + E4 E1 + E2 + E4 E1 + E3 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, 230400mesh) 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 l 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. NO. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

MOBILE PHASE CHCl3: MeOH: EtOAc: H2O EtOAc: MeOH: HCOOH: H2O EtOAc: HCOOH: HOAc: H2O DCM: MeOH: H2O BuOH: HOAc: H2O THF: H2O: H3PO4 CH2Cl2: HOAc: H2O CHCl3: MeOH: EtOAc CHCl3: MeOH: EtOAc CHCl3: MeOH: EtOAc AcOH: CHCl3: H2O CHCl3: MeOH: H2O EtOAc: MeOH: H2O EtOAc: HCOOH: H2O CHCl3: MeOH: HOAc MeOH: Water EtOAc: HOAc DCM: AcOH DCM: AcOH DCM: AcOH

RATIO 2.5 : 3 : 1.5 : 2 5 : 0.2 : 0.3 : 6 2.5 : 0.2 : 0.2 : 0.4 4.5 : 1 : 0.1 7 : 1.5 : 2 6 : 6 : 6.1 2:1:1 4 : 0.75 : 0.5 4 : 0.45 : 0.5 4 : 0.35 : 0.5 8:2:1 6.1 : 3.2 : 0.7 5 : 0.3 : 1 6:1:1 9 : 0.5 : 0.5 4:1 5:1 8.5 : 1.5 9:1 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 l 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 C. 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: 5a). 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 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)

l

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 A. sativum C. limon S. aromaticum O. basilicum Amoxyclav Erythromycin Penicillin-G Oxacillin Cephalothin Clindamycin Solvent Cont.

S. AUR 12.6 14.6 R R R R R R -

S. EPI 17.3 14.6 10.3 16.6 10.3 10.3 8.3 28.6 -

S. FAE 18.6 11.3 21 11 12.6 8.6 18.3 -

Note: 100 l 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 S A. sativum C. limon S. aromaticum O. basilicum Ampicillin Amoxyclav Cephotaxime Cotrimoxazole Gentamicin Tobramycin Solvent Cont.

E. COLI 11 13.3 22.6 20.3 16.6 -

K. PNE 9.6 10.6 8.6 -

P. VUL 11 14.6 36.3 -

S. BOY 10 11.3 15.6 11.6 17.3 23.6 18.3 14.3 -

S. PARAA 10.3 14.6 15 21.6 23 26 28.3 18.6 17 -

S. PARAB 8.6 11.3 13.6 13 21.6 23 26.6 31.3 23.3 15.3 -

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

43

a

b

c

d

e 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

f

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

i

Antibiotics (Gram ve ): Ampicillin (A), Cephotaxime (C), Cotrimoxazole (CO), Tobramycin (Tb), Amoxyclav (Ac), Gentamicin (G) Antibiotics (Gram +ve ): Amoxyclav (Ac), Erythromycin (E), PenicillinG (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-garlicclove-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 within46

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 6 2 C

7 8

5 4

3

11 10

9

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 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

l

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

Table: 11 Anti-bacterial activities of botanical combinations of promising extracts against Gram -ve bacteria

TREATMEN TS E1+E2+E3+E 4 E1+E2+E3 E1+E2+E4 E1+E3+E4 E2+E3+E4 E1+E2 E1+E3 E1+E4 E2+E3 E2+E4 E3+E4 Solvent Cont.

E. COLI 9.4 -

K. PNE 15.4 11 8.6 9.5 19.2 14 18.3 -

P.AER U 15.3 12.3 11.5 10.3 14.3 7.4 11.5 15.1 11.2 17.3 14.1 -

P. VUL 16.2 16.2 17.3 7.3 -

S. BOY 10.4 11.7 7.5 10.2 10.7 11.3 -

S .PARAA 10.2 10.5 12.3 11.3 16.2 17.4 12.3 -

S. PARAB 12.2 10.3 9.6 12.4 8.3 11.5 -

Note: E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum. 100 l 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 TS

S. EPI

S. FAE

51

E1+E2+E3+E4 E1+E2+E3 E1+E2+E4 E1+E3+E4 E1+E2 E1+E3 E1+E4 E2+E3 E2+E4 E3+E4 E2+E3+E4 Solvent Cont.

11.6 12 8.5 10.3 11.2 11.3 8.2 8.3 7.6 7.4 -

7.5 14.7 7.6 9 11 15.1 -

Note: E1- C. limon, E2- A. sativum, E3- S. aromaticum, E4- O. basilicum. 100 l 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 didnt undergo any change in its structure and fluorescence property along with any of the mixtures, except with clove extract. Corollary to this, the antibacterial of52

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. aeruginosa53

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 m

b n

c o

d

e

f

g

h

I

j

k

l

Rf

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 EXTRACTS 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) 1Ice blue Dark Violet Dark Violet Ice blue Dark Violet Dark Violet Ice blue Dark Violet Ice blue Ice blue Ice blue Dark Violet Pale Violet Dark Violet Ice blue

2Dark Violet Pale Violet Pale Violet Mild Violet Pale Violet Pale Violet Mild Violet Mild Violet Mild Violet Pale Violet Mild Violet Pale Violet Mild Violet Mild Violet Mild Iceblue

3Pale blue Pale Violet Mild Violet Pale Violet Ice blue Mild Violet Mild Iceblue Mild Violet Mild Violet Mild Violet Mild Violet Mild Violet Pale Violet Mild Iceblue

4Pale Violet Mild Violet Pale Violet Ice blue Mild Violet Dark Violet Mild Violet Mild Violet Pale Violet Pale Violet Mild Violet -

5Mild Violet Mild Violet Pale Violet Pale Violet Pale Violet Mild Iceblue -

6Dark Violet Dark Violet -

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 l 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 SAMPLE Coconut paste Control Raw Mushroom Control TREATM ENTS E1 + E2 E1 + E2 T AGAR (103) Nil TNTC 300 TNTC

MACCONK EY AGAR (103) Nil Nil Nil 119

Note: E1- C. limon & E2- A. sativum. 100 l 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 l) 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

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

NUTRIEN TREATME SAMPLE NTS E1 + E2 E2 + E3 E1 + E3 E1 + E2+E3 T AGAR (103) 119 Nil Nil 53 TNTC

MACCONK EY AGAR (103) Nil Nil Nil Nil Nil

Tomato Puree Control

Note: E1- C. limon, E2- A. sativum & E3- S. aromaticum. 100 l 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 of64

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 activity65

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 l 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.1. Abbas, N. S. M. and Halkman, A. K. (2004), Antimicrobial effect of

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