Animicrobial Effect Of Polyethylene Film Biology Essay

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A number of nontraditional preservation techniques are being developed to satisfy consumer demand with regard to nutritional and sensory aspects of foods. Generally, foods are thermally processed by subjecting them to temperatures varying from 60 to 100 ËšC for the duration of a few seconds to a minute in order to destroy vegetative microorganisms. During this period of treatment, a large amount of energy is transferred to the food. However, this energy can trigger unwanted reactions, leading to undesirable organoleptic and nutritional effects. Ensuring food safety and at the same time meeting such demands for retention of nutrition and quality attributes has resulted in increased interest in alternative preservation techniques for inactivating microorganisms and enzymes in foods.

Quality attributes of importance include flavor, odor, color, texture, and nutritional value. This increasing demand has opened new dimensions for the use of natural preservatives derived from plants, animals, ormicroflora. In bio preservation, storage life is extended, and safety of food products is enhanced by using natural or controlled microflora, mainly lactic acid bacteria (LAB) and their antibacterial products such as lactic acid, bacteriocins, and others. Typical examples of investigated compounds are lactoperoxidase (milk), lysozyme (egg white, figs), saponins and flavonoids (herbs and spices), bacteriocins (LAB), and chitosan (shrimp shells). Antimicrobial compounds present in foods can extend the shelf life of unprocessed or processed foods by reducing the microbial growth rate or viability.

Originally, spices and herbs were added to change or to improve taste. Some of these substances are also known to contribute to the self defence of plants against infectious organisms. Extensive research has investigated the potential application of natural antimicrobial agents in food preservation. Natural antimicrobials in food preservation can be used alone or in combination with other non thermal technologies. Edible, medicinal, and herbal plants and their derived essential oils and isolated compounds contain a large number of secondary metabolites that are known to retard or inhibit the growth of bacteria, yeast, and molds. Many of these compounds are under investigation and are not yet exploited commercially.

The antimicrobial compounds in plant materials are commonly found in the essential oil fraction of leaves (rosemary, sage, basil, oregano, thyme, and marjoram), flowers or buds (clove), bulbs (garlic and onion), seeds (caraway, fennel, nutgem, and parsley), rhizomes (asafetida), fruits (pepper and cardamom), or other parts of plants. Plant essential oils and their constituents have been widely used as flavouring agents in foods since the earliest recorded history, and it is well established that many have a wide spectra of antimicrobial action. These compounds may be lethal to microbial cells or they might inhibit the production of secondary metabolites.

Plant essential oils are generally more inhibitory against Gram-positive than Gram-negative bacteria. While this is true for many essential oils, there are some agents that are effective against both groups, such as oregano, clove, cinnamon, and citral. The major essential oils components with antimicrobial effects found in plants, herbs, and spices are phenolic compounds, terpenes, aliphatic alcohols, aldehydes, ketones, acids, and isoflavonoids. Chemical analysis of a range of Eos revealed that the principal constituents of many include carvacrol, thymol, citral, eugenol (see Scheme 1 for their chemical structure), and their precursors. It has been reported that some nonphenolic constituents of essential oils are more effective or quite effective against Gram-negative bacteria such as allyl isothiocyanate (AIT) and garlic oil, respectively. In addition, AIT is also effective against many fungi.

1.2 Problem Statements

Meat and meat products being primary causes of food borne disease are also prone to spoilage during storage. As many refrigerated food, microbial growth is generally responsible for the spoilage in meat and meat product is normally ready to eat product (Isabelle, Philippe, Andre & Jean-Dominique, 2005).

In order to prolong the storage stability of foods, synthetic antimicrobials are mainly used in industrial processing. However according to toxicologist and nutritionists there are side effect to use some synthetic antimicrobials in food packaging. For this reason the search for antimicrobial from natural sources such as spices and essential oils to replaces synthetic ones has received much attention. Following is the problem statement for this study:

What is the antimicrobial activity of E. coli in linear low density polyethylene (LLDPE) incorporated with 8% w/w of garlic oil on surface of beef slice?

What is the antimicrobial activity of L. Monocytogenes in linear low density polyethylene (LLDPE) incorporated with 8% w/w of garlic oil on surface of beef slice?

1.3 Aim and Objectives

The aim of this study is mainly to determine the antimicrobial activity of LLDPE incorporated with 8% w/w of garlic oil.

To determine the antimicrobial activity of LLDPE toward E. coli incorporated with 8% w/w of garlic oil on the surface of the beef slice.

To determine the antimicrobial activity of LLDPE toward L. Monocytogenes incorporated with 8% w/w of garlic oil on the surface of the beef slice.

1.4 Scope

This study describes the antimicrobial activity of LLDPE incorporated with 8% w/w of garlic oil toward beef slice.

1.4.1 Antimicrobial Activity on the Surface of Beef Slice

The beef slice will be cut into small piece and then inoculate it with microbes then wrap with the LLDPE incorporated with 8% w/w of garlic oil. The beef slice is then enumerated using peptone water. At specific time interval numbers of visible cells remain in the agar plate by doing serial dilution with the enumerated peptone water.

CHAPTER 2

LITERATURE REVIEW

2.1 Background of essential oils as antimicrobial agent in polymer films

Food quality and safety is a major concern of consumer and food industry since consumers prefer fresher and minimal processed product. In particular, bacterial contamination in ready to eat products because it is concern to human health (Pranoto, Rakshit & Salokhe, 2005). Most contaminant occurs after processing. Natural antimicrobials such as essential oils can minimize the post packaging pathogens contamination. Post processing antimicrobial packaging system able to control the growth and re-contamination of food borne pathogens (Sivarooban, Hettiarachchy & Johnson, 2008).

Application of antimicrobial dips or sprays on the surface of the food is one of the traditional ways of controlling microbial growth in packed food product. It also helps in improving the safety and delaying spoilage. However the efficiency of the antimicrobial substance is restricted. This is due to the uncontrolled migration into the food and partial inactivation of the active compounds and the interaction with the food components. One of the new approaches to overcome this limitation is the use of antimicrobial packaging technique as a promising active packaging (Zehra, Gokce, Betul & Kezban, 2010).

Antimicrobial packaging is a form of active packaging that could extend the lifespan of product and provides microbial safety for consumers. It acts to reduce, inhibit, or retard the growth of pathogen microorganisms in packed foods and packaging material. Antimicrobials can be incorporated into packaging system through various methods such as sprayed or coated on food surface, volatilized from an insert or a sachet placed in the package into the head space, sprayed or chemically bound to the surface of packaging film, formulated homogeneously with the film polymers and designed to occupy pores or channels within the film (Nadarajah, Han & Holley, 2005).

Several compounds have been used for antimicrobial activity such as lysozyme and fungicides such as benomyl, imazalil and natural antimicrobial compounds such as spices. Spices carry mostly antimicrobial and some antioxidant properties. Natural compound such as nisin and lysozyme also has been used to incorporate in food packaging at the same time safe for human consumption. Besides, all this essential oils possess the strongest antibacterial properties against food borne pathogens contain higher concentration of the phenolic compound such as carvacrol, eugenol and thymol. This compound exhibits the wide range of effect including antioxidant and antimicrobial properties (Seydim & Sarikus, 2006).

Since food packaging brings significant effects to human health, therefore food packaging is highly regulated around the world including active and microbial packaging. In US since no specific regulations exist for active packaging it will follow food addictive standard. Until now only Zeomic, a silver substituted Zeolite and chlorine dioxide have been approved by Food and Drug Administration (FDA). This can be avoided when we use essential oil as antimicrobial since essential oils are classified as generally recognized as safe (GRAS). Thus, is good scope to study on the essential oils as food packaging antimicrobial (Paola & Joseph, 2002).

2.2 Type of essential oil as antimicrobial in polymer film

Essential oils comprise more than sixty individual components. Major components can constitute up to 85% of the essential oils, whereas other components are present only as a trace. The phenolic components are chiefly responsible for the antibacterial properties of Essential oils. There is some evidence that minor components have a critical part to play in antibacterial activity, possibly by producing a synergistic effect between other components. This has been found to be the case for sage, certain species of Thymus and oregano.

The composition of essential oils from a particular species of plant can differ between harvesting seasons and between geographical sources. Generally, essential oils produced from herbs harvested during or immediately after flowering possesses have the strongest antimicrobial activity. The composition of essential oils from different parts of the same plant can also differ widely. For example, essential oil obtained from the seeds of coriander has a quite different composition to essential oils of cilantro, which is obtained from the immature leaves of the same plant.

2.2.1 Major components of selected essential oils

The major essential oils components with antimicrobial effects found in plants, herbs, and spices are phenolic compounds, terpenes, aliphatic alcohols, aldehydes, ketones, acids, and isoflavonoids. Table 2.1 shows the major component of selected essential oil. Chemical analysis of a range of essential oils revealed that the principal constituents of many include carvacrol, thymol, citral, eugenol, and their precursors. It has been reported that some non phenolic constituents of essential oils are more effective or quite effective against Gram-negative bacteria such as allyl isothiocyanate and garlic oil. In garlic oil, diallyl disulphide, diallyl trisulfide and allyl propyl disulfide are the main compounds that inhibit the act as antiagent (Yudi, Vilas & Sudip, 2005).

Table 2.1: Major Component of Selected Essential Oil (Sara, 2004)

Common name of essential oil

Latin name of plant source

Major components

Approximate composition (%)

Cilantro

Coriandrum sativum

Linalool

26

(immature leaves)

E-2-decanal

20

Coriander

Coriandrum sativum

Linalool

70

(seeds)

E-2-decanal

-

Cinnamon

Cinnamomum zeylandicum

Trans-cinnamaldehyde

65

Oregano

Origanum vulgare

Carvacrol

Trace

Thymol

Trace

γ-Terpinene

2 -25

p-Cymene

Trace

Rosemary

Rosmarinus officinalis

α-pinene

2 -25

Bornyl acetate

0 -17

Camphor

2 -14

1,8-cineole

3 -89

Sage

Salvia officinalis L.

Camphor

6 -15

α-Pinene

4 - 5

β-pinene

2 -10

1,8-cineole

6 -14

α-tujone

20-42

Clove (bud)

Syzygium aromaticum

Eugenol

75-85

Eugenyl acetate

8 -15

Thyme

Thymus vulgaris

Thymol

10-64

Carvacrol

2 - 11

γ-Terpinene

2 -31

 

 

p-Cymene

10-56

Generally, the antimicrobial efficacy of essential oils is dependent on the chemical structure of their components as well as the concentration. Many of the antimicrobial compounds present in plants can be part of their pre- or post infection defense mechanisms for combating infectious or parasitic agents. Consequently, plants that manifest relatively high levels of antimicrobial action may be sources of compounds that inhibit the growth of food borne pathogens. Compounds are also generated in response to stress from inactive precursors, which may be activated by enzymes, hydrolases or oxidases, usually present in plant tissues. In mustard and horse radish, precursor glucosinolates are converted by enzyme myrosinase to yield a variety of isothiocynates including the allyl form, which is a strong antimicrobial agent (Sara, 2004).

The application of plant essential oils for controlling the growth of food borne pathogens and food spoilage bacteria requires evaluation of the range of activity against the organisms of concern to a particular product, as well as effects on a food's organoleptic properties. Plant essential oils are usually mixtures of several components. Oils with high levels of eugenol (allspice, clove bud and leaf, bay, and cinnamon leaf), cinnamamic aldehyde (cinnamon bark and cassia oil), and citral (lemon myrtle, Litsea cubeba, and lime) are usually strong antimicrobials. The essential oils from Thymus possess significant quantities of phenolic monoterpenes and have reported antiviral, antibacterial, and antifungal properties. The volatile terpenes carvacrol, p-cymene, γ-terpinene, and thymol contribute to the antimicrobial activity of oregano, thyme, and savory. The antimicrobial activity of sage and rosemary can be attributed to borneol and other phenolic compounds in the terpene fraction. The terpene thejone was responsible for the antimicrobial activity of sage, whereas in rosemary, a group of terpenes (borneol, camphor, 1, 8 cineole, a-pinene, camphone, verbenonone, and bornyl acetate) was responsible (Sara, 2004).

2.2.2 Mechanisms of antimicrobial action

Mechanism of antimicrobial action has not been discussed in great detail this is due to the large number of different group of chemical compounds present is essential oils. Not all mechanisms works individually, some work in consequence of other mechanisms targeted (Sara, 2004). Essential oils with high percentage of phenolic compounds such as carvacrol, eugenol and thymol have strongest antimicrobial properties against food borne pathogens. The chemical structure of the individual essential oil components affects their precise mode of action and antimicrobial activity (Sara, 2004).

The main groups are composed of terpenes and aromatic constituents. Terpenes made from combinations of several 5-carbon-base units called isoprene. A terpene containing oxygen is called a terpenoid. For terpenes when the molecules are optically active, two enantiomers are very often presents in different plants such as α-pinene from Pinus palustris, β-pinene from Pinus caribaea. Whereas for aromatic compounds which is derived from phenylpropane occurs less frequently compared to terpenes. The mostly available palnt with aromatic compound essential oil is anise, cinnamon, clove, nutmeg, parsley, and some botanical families (Bakkali, Averbeck, Averbeck & Idaomar, 2008). Non-phenolic component of essential oil which gives antibacterial effect is influenced by alkyl group. As example, limonene is more active than p-cymene (Sara, 2004).

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Figure 2.1: Location and Mechanisms in the Bacterial Cell (Sara, 2004)

Essential oil and their component have hydrophobic characteristics which will disturb the structure and rendering bacteria cell membrane and mitochondria which can be seen in Figure 2.1. Essential oil components embedded in the cytoplasmic membrane and act on the cell protein. Lipophilic hydrocarbon molecules could accumulate in the lipid bilayer and distort the lipid-protein interaction. Excessive loss of cell content or the exit of critical molecules and ions will lead to death of bacteria. Generally, phenolic compound act as disturbance of cytoplasmic membrane, electron flow and coagulation of cell contents (Sara, 2004).

2.3 Type of antimicrobial packaging

Antimicrobial polymer can be used in several food related application including packaging. There are few important considerations being focused in food packaging. First is to extend the shelf life and promote safety by reducing the growth of specific microorganisms. Second will be self sterilization of packaging material. This means, antimicrobial packaging materials able to reduce the potential for processed product. Third one is self sterilization food. This method is used in liquid type of food especially high acid product. To make all this workable antimicrobial packaging is highly significant. Antimicrobial packaging can be clustered into few types which will be discussed further in following subtopics.

2.3.1 Incorporation of antimicrobial agent into polymer

Incorporation of bioactive agents including antimicrobial into polymer has been commercially applied in drug, pesticides and other medical devices. But only few food related topics has been widely discussed. Among all the available antimicrobial silver substitute zeolites is widely used in food application. Sodium ions present in zeolites are substituted by silver ions, which are antimicrobial against wide bacteria and mold. The substituted zeolites are incorporated into polymer like polyethylene at level of 1-3%. Silver ions will disruptive the cell enzymatic activity. There are few commercially available silver substitute zeolite are Zeomic®, Apacider®, AgIon, and Navaron. Antimicrobial enzymes such as lactoperoxidase and lactoferrin, antimicrobial peptides such as magainins, cecropins, defensins, natural phenol like hydoquinones, fatty acid ester and metal like copper also potential to incorporated in polymer (Paola & Joseph, 2002).

Preventing surface growth in foods was a large portion of spoilage and contamination main aim of incorporation of antimicrobial into packaging. Table 2.2 shows the antimicrobial incorporated directly into polymer used for food packaging. Sterilization of food product like meat able to reduces the addition of large quantity of antimicrobials. In latter process, antimicrobial activity may be rapidly lost due to dilution below active concentration due to migration into the bulk food matrix. Many antimicrobial are incorporated at 0.1-5% w/w of the packaging films. Extrusion and injection molding used with thermally stable antimicrobials like silver substitute into zeolite which able to withstand up to 800 ËšC. Whereas, for heat-sensitive antimicrobials like enzymes and volatile compound uses solvent compounding to incorporate it. For an example, Lysozyme incorporated into cellulose ester films by solvent compounding in order to prevent heat denaturation of the enzyme (Paola & Joseph, 2002).

Nisin shows that activity of the bacteriocin in the cast films is three times greater than that of heat-pressed films. Solvent compounding works by polymer and antimicrobial soluble in same solvent. Biopolymers based on lipids, protein and carbohydrates are normally are the one normally uses solvent compounding. These types of polymers and their combination are soluble in water, ethanol and many other solvent compatible with antimicrobials. There are many antimicrobials are not easily incorporated into homogeneously distributed in poly (olefic) and related hydrophobic polymer. Organic acids with LDPE by forming the anhydride of the acid prior to additional to the polymer melt. In moisture condition anhydride hydrolyzed to the acid from the film's surface to the food which is good in retarding the growth of mold (Paola & Joseph, 2002).

Antimicrobial packaging material normally can be divided into two broad categories which is volatile and non-volatile packaging material. For volatile packaging the antibacterial able to penetrate the bulk matrix of the food and the polymer not necessarily need to be in direct contact with the food product. This type of packaging material is good for application where there is no contact between required portions of food such as packaging of ground beef. In volatile packaging material precursor molecules are incorporated directly into the polymer. As an example, Allylisothiocyanate has been entrapped in cyclodextrins that are coated to package. Besides all the factors discussed above, control of vapor pressure and stability of the gases are essential to sustain their release and antimicrobial properties through self-life (Paola & Joseph, 2002).

Table 2.2: Antimicrobials Incorporated Directly into Polymers Used for Food Packaging (Paola & Joseph, 2002)

Antimicrobials

Polymer/carrier

Main target microorganisms

Organic acids/anhydrides:

Propionic, benziocsorbic, acetic, lactic, malic

Edible films, EVA, LLDPE

Molds

Inorganic gases:

Sulfur dioxide, chlorine dioxide

Various polyolefins

Molds, Bacteria, Yeasts

Metals:

Silver

Various polyolefins

Bacteria

Fungicide:

Benomyl, imazalil

LDPE

Molds

Bacteriocins:

Nisin, pediocins, lacticin

Edible films, cellulose, LDPE

Gram-positive bacteria

Enzyme:

Lysozyme, glucose oxidase

Cellulose acetate, PS Edible films

Gram-positive bacteria

Chelating agents:

EDTA

Edible films

Gram-negative bacteria

Spices:

Cinnamic, caffeic, p-coumaic acids

Nylon/PE, cellulose

Molds, yeast, bacteria

Essential oils:

Grapefruit seed extract, hinokitiol, bamboo powder, Rheum palmatum, coptis chinesis extracts

LDPE, cellulose

Molds, yeast, bacteria

Parabens:

Propylparaben, ethylparaben

Clay-coated cellulose LDPE

Molds

2.3.2 Polymer that inherently antimicrobial

Some polymers are inherently antimicrobial and have been used in films and coatings. Chitosan and poly-L-lysine are cationic polymer promote cell adhesion. Charged amines interact with negative charges on the cell membrane causing interruption in intracellular constituent. The antimicrobial effect is depending on the antifungal properties of chitosan. Chitosan act as barrier between the nutrient contained in product and microorganisms. Whereas, calcium alginate films reduces the growth of the natural flora and coliform inoculated on beef due to the presence of calcium chloride (Paola & Joseph, 2002).

Physical modification of polymer is one of the key elements to render surface antimicrobial. Antimicrobial potential of polyamide films treated with UV irradiation has been discussed in past. Positively charged amine group which present in polymer at times only able to enhance the adhesion of cell but not necessarily death of cell. UV-treated nylon film shows a good respond of surface amino group to bactericidal but bacterial cells were absorbed to the surface and diminished the effectiveness of the amine groups. Mostly the studies conducted in buffer. Addition of nutrient prevents cell damage due to interaction of salt and other cations with surface.

2.3.3 Polymer with addition of sachets and pads containing antimicrobial agents

In most of the commercially available packaging the antimicrobial agent is attach to the interior of a packaging. Oxygen absorbers, moisture absorber and ethanol vapour generation. Ethanol vapour generation consist of ethanol absorbed in carrier material and enclosed in polymer packets. Amount of ethanol generated is relatively small and effective only in product with reduced water activity. Commercial examples include Ethicap®, heat sealed packets containing microencapsulated ethanol in silicon dioxide powder, and Fretek®, a paper wafer in which center layer is impregnated with ethanol in acetic acid and sandwiched between layers of polyolefic films. Absorbing pads used in trays for packaged retail meats and poultry to soak up meat exudates (Paola & Joseph, 2002).

2.4 Effectiveness of essential oil tested in vitro against microbes

Essential oil has been incorporated in various types of films and tested its antimicrobial activity against microbes. Antimicrobial activity testing can be classified into three groups which are diffusion, dilution or bio autographic methods. At the same time there is no standardised test to evaluate antimicrobial activity of possible preservatives against food-related microorganism. The minimum inhibitory concentration (MIC) is cited by most researchers as a measure of the anti-bacterial performance of essential oils. Table 2.3 shows the selected MIC of essential oil tested in vitro against microbes. The strength of the antibacterial activity can be determined by dilution of essential oil in agar broth. Previous studies shows that, dilution in agar have been used different solvent to incorporate the essential oil in the medium, different volume of inoculation and sometimes streaked onto the agar surface. The rapidity of a bactericidal effect or the duration of a bacteriostatic effect can be determined by time-kill analysis. This can be observed from the graph viable cell remaining in broth after the addition of essential oil is plotted against time. In following subsection the type of film incorporated with various essential oil and its antimicrobial activity discussed in detail (Sara, 2004).

Table 2.3: Selected MIC of Essential Oil Tested in vitro Against Microbes (Sara, 2004)

Plant from which EO is derived

Species of bacteria

MIC, approximate range

(µl ml-1)

Rosemary

Escherichia coli

4.5-˃10

Solmonella typhimurium

˃20

Bacillus cereus

0.2

Staphyloccus aureus

0.4-10

Oregano

Listeria monocytogenes

0.2

E. Coli

0.5-1.2

S. Typhimurium

1.2

Lemongrass

Staph. Aureus

0.5-1.2

E. Coli

0.6

S. Typhimurium

2.5

Sage

Staph. Aureus

0.6

E. Coli

3.5-5

S. Typhimurium

10-20

Staph. Aureus

0.75-10

Clove

L. Monocytogenes

0.2

E. Coli

0.4-2.5

S. Typhimurium

˃20

Staph. Aureus

0.4-2.5

Thyme

L. Monocytogenes

0.3

E. Coli

0.45-1.25

S. Typhimurium

0.450-˃20

Staph. Aureus

0.2-2.5

Turmeric

L. Monocytogenes

0.156-0.45

E. Coli

˃0.2

Tea bush (Lippia Spp.)

B.Cereus

0.2

E. Coli

2.5-˃80

Shigella dysenteria

5-˃80

Staph. Aureus

0.6-40

 

B.Cereus

5-10

2.4.1 Grape seed extract incorporated into soy protein edible films

Grape seed extract reduced L. monocytogenes population by 1 logCFU/ml after 1 h incubation at 25 ËšC. Escherichia coli and Salmonella typhimurium showed only 0.1 and 0.2 logCFU/ml reductions, respectively. Inactivation of L. monocytogenes will up to 3 logCFU/ml, when Grade seed extract and nisin used. The phenolic believed to be responsible for the antimicrobial activities. Phenolic act upon the cytoplasmic membrane of the bacteria, the addictive or synergistic effect of these compounds could enhance the inhibitory activity in combinations against L. monocytogenes. The grape seed extract incorporated soy protein edible film is effective to variable degrees in habiting the growth of L. monocytogenes, Escherichia coli and Salmonella typhimurium (Sivarooban, Hettiarachchy & Johnson, 2008).

2.4.2 Kiam wood extract incorporated into hydroxypropyl methylcellulose films

Kiam wood extract exhibited different inhibition level against Escherichia coli, Salmonella typhimurium and L. monocytogenes. Inhibition zone increased with increasing concentration of kiam wood extract. Edible film containing kiam wood extract at 1and 2 fold of minimum bacterial concentration was not effective against any tested microorganisms. Two fold shows little effect against L. monocytogenes and demonstrate a meagre inhibitory zone. Higher concentration such as 2 fold of minimum bacterial concentration, then antimicrobial activity also higher. When compare the effectiveness among the three bacteria Escherichia coli less sensitive to the inhibitory compared to other two microbes. The study on the kiam extract shows that it works well with gram-positive compared to gram-negative bacteria (Jutaporn, Suphitchaya & Thawien, 2011).

2.4.3 Clove bud oil, Cinnamon oil, Star anise oil and garlic oil incorporated into chitosan films

The star anise oil was proved to have poor antimicrobial activity. Besides that star anise oil did not form homogeneous films when star anise oil was added therefore star anise was ruled out from the discussion. When we come to the chitosan film forming the oil quantity incorporated in the chitosan film-forming solution was no more than 10% make the film forming solution too sticky. Inhibition zones were not significant when the incorporation oil quantities were less than about 2.5% for A. oryae and P. digitaum and about 5% for Escherichia coli and S. aureus (Lina, Fei , Yangfeng, Zhi, Pinglan, Yongqiang, Hao & Xiaojing, 2011).

When the clove bud oil concentration is higher than minimum inhibition concentration then, zone of inhibition increase rapidly with microorganism observed. The inhibition of the microbes for chitosan embedded with clove oil can be arranged in descending order based on its effectiveness with highest inhibition by A. oryze, P. digitatum, S. aureus and the lowest with E. coli. Chitason embedded with cinnamon have minimum inhibition zone near 2.5% for A. oryae and P. digitaum and 5% for E. coli and S. aureus. The inhibition effect of the chitason embedded with cinnamon on the fungi were about 2 to 3 fold stronger those with clove bud oil but for bacteria both the cinnamon and clove bud shows same level of inhibition (Lina, Fei , Yangfeng, Zhi, Pinglan, Yongqiang, Hao & Xiaojing, 2011).

In another study when chitosen is incorporated with garlic oil also reveal antimicrobial activity toward S. aureus, L. monocytogenes, B. cereus, E.coli and S. typhimurium. The most sensitive to the garlic oil is S. aureus and B. cereus. It clearly shows that the antimicrobial activity shows a positive result for the gram-positive bacteria instead of gram-negative bacteria (Pranoto, Rakshit & Salokhe, 2005).

2.4.4 Oregano oil, rosemary oil and garlic oil incorporated into whey protein based edible films

Film containing 1% of oregano essential oil was not effective against any test microorganisms. The minimum inhibition needed for oregano is 2% for all microorganisms tested which are E. coli, S. aureus, S. enteritidis, L. monocytogenes, and L. plantarum. The greater zone of inhibition can be seen in 4% level for S. aureus, S. enteritidis and L. monocytogenes whereas it not effective for L. plantarum. Whey protein film containing 1% and 2% of garlic oil essential oil were not inhibitory against any of the test microorganisms. When increased to 3% significant inhibition can be seen and when make it to 4% greater inhibition can be seen onto S. aureus and L. monocytogenes. Rosemary essential oil incorporation did not show any inhibitory effect against the microorganisms tested (Seydim & Sarikus, 2006).

2.5 Antibacterial activity of essential oil in meat product

Limited number of food preservative from essential oil commercially available. In early 90s few journals had been published regarding the antibacterial activity in food product. Greater concentration of essential oil is needed when tested with food to achieve the same effect as in vitro. There is some exceptional case to this phenomena where no greater portion of essential oil is need to inhibit this species on cooked pork when compared to test in vitro (Sara, 2004). Foods have great nutrient content, therefore the bacteria able to recover by itself faster compared to laboratory scale (Bakkali, Averbeck, Averbeck & Idaomar, 2008). Besides that, pH also plays an important role in the antimicrobial effect of essential oil. Essential oils effect increases with the decrease in the pH of the food. This is due to the hydrophobicity of an essential oil increases at low pH which enable it to more easily dissolve in the lipids of the cell membrane. High level of fat and protein protects the food from the action of essential oil (Pandit & Shelef, 1994). Water content on the food also influences the antibacterial activity of essential oil. Low water content slows down the progress of antibacterial agent in bacterial cell (Sara, 2004).

2.5.1 Clove oil as a preservative in minced mutton

The antilisteric activity of clove oil in culture media has been reported by several earlier workers. The present study revealed its action in the food substrates minced mutton. The microorganism cannot be eliminated completely or inhibited but, clove oil was able to restrict the proliferation of L. monocytogenes in both food products. The effect was more pronounced with a 1% concentration of clove oil as compared to 0.5% at both 30ËšC and 7ËšC. Such delay in growth of the microorganism is particularly useful in terms of food safety for short storage of products because on prolonged storage Listeria may reach high levels in foods despite the presence of clove oil (Vrinda Menon & Garg, 2001).

2.5.2 Eugenol and pimento oil as preservatives in cooked chicken

Eugenol and pimento extracts are not only bacteriostatic but also bacteriocidic to both A. hydrophila and L. monocytogenes. The effect of plant extracts visible on the progress of bacterial growth, and it also has a significant reduction in the initial viable bacterial population. After pathogens were applied to chicken samples pretreated with plant extracts, samples were air-dried for 30min before they were packaged in bags and the day 0 samples were collected. Results revealed that eugenol and pimento were lethal to both A. hydrophila and L. monocytogenes. Application of these extracts to inoculated chicken resulted in an immediate and significant reduction nearly 3 log in populations of pathogens in the treated chicken. Eugenol and pimento extracts were the most effective extracts against both bacteria tested, and that eugenol was better than pimento extract. This is due to better quality of raw material. Good quality of raw material would theoretically maintain high microbiological quality longer (Hao, Brackett & Doyle, 1998).

2.5.3 Rosemary oil as preservatives in pork liver sausage

Rosemary oil was tested with pork liver sausage and the Listeria population in the untreated product increases by the 5 log cycle during the 21 days of testing period. Ground rosemary (0.5%) and its oil (1%) had limited antilisterial effect. Whereas the encapsulated oil (5%) and the antioxidant extract (0.3%) exhibit antilisterial activities and Listeria population increases by less than one log cycle during the first 21 days of storage. For the encapsulated higher concentration of oil needed to show a significant outcome (Pandit & Shelef, 1994).

2.5.4 Biodegradable gelatin-chitosan films incorporated with Thyme oil and clove oil as preservative in fish

Study on the fish shows a positive result toward clove and thyme by total inhibition followed by rosemary with partial inhibition. When a film placed in food surface, its solubility largely determine the release of antimicrobial compound. Gelatin film shows a low solubility value meanwhile when the clove essential oil is added the film shows a increase in the water solubility and when essential oil is added with gelatin-chitosan the solubility of the film maintained. The films protein-polyphenol is important in weaken the interaction that stabilized the protein net (Gomez-Estaca, Lopez de Lecey, Lopez-Caballero, Gomez-Guillen & Montero, 2010).

2.5.5 Soy edible films incorporated with thyme and oregano essential oil as preservative in ground beef patties

Greater antimicrobial activities of soy edible films incorporated with essential oils from Oregano and Thyme were demonstrated against S. aureus, E. coli and E. coli O157:H7 as compared to P. aureginosa and L. plantarum in growth media. The same inhibitory effect was not observed when the antimicrobial films were applied on ground beef patties against Staphylococcus spp., total viable bacteria and lactic acid bacteria. However, Oregano and Thyme essential oils incorporated films resulted in reductions in the counts of Pseudomonas spp. and coliform bacteria of ground beef patties during refrigerated storage. This limited antimicrobial activity of essential oils incorporated antimicrobial edible films on ground beef patties could be due to the complexity of ground beef matrix. It has been recently shown that antimicrobial activity of Oregano and Thyme essential oils was greater at acidic pH values and high concentrations of protein with moderate levels of simple sugars (Zehra, Gokce, Betul & Kezban, 2010).

2.5.6 Milk protein-based film containing oregano and pimento as preservative in whole beef muscle

The incorporation of oregano oil and pimento into milk protein-based edible film applied onto muscle meat helps to reduce microbial load and increase antioxidative activity, during 7 days of storage by inhibition of E. coli O157:H7 and Pseudomonas spp. Growth by essential oils depends on the nature of the phenolic compounds. The phenolic compound concentration could also play an important role in the antimicrobial and antioxidant activities that they confer to essential oils used in this study. Oregano-based films exhibited the most effective antimicrobial property, whereas pimento-based films presented the highest antioxidant activity. Also, the films allow a progressive release of phenolic compounds during storage. After 7 days, the availability of the phenolic compounds in films being always significant, the films remain effective. The use of edible films containing essential oils as a preservation method of meat is promising (Oussalah, Caillet, Salmieri, Saucier & Loicroix, 2004).

CHAPTER 3

METHODOLOGY

3.1 Experimental raw material

84 beef slice, E. coli and L. Monocytogenes

3.2 Formulation

LLDPE (3.6kg) + EVA (0.4kg) + Garlic oil (0.08kg)

LLDPE (3.6kg) + EVA (0.4kg) + Garlic oil (0.16kg)

LLDPE (3.6kg) + EVA (0.4kg) + Garlic oil (0.24kg)

LLDPE (3.6kg) + EVA (0.4kg) + Garlic oil (0.32kg)

Control: LLDPE (3.6kg) + EVA (0.4kg)

3.3 Sample preparation

E. coli prepared overnight into BHI broth. Then, 28 beef slice need to be prepared. UV-sterilization of beef slice will take place for 15min for all the beef slice. From 28 Beef slices, 21 Beef slices (5 cm x 5cm) which is sterilized with UV will be inoculated with E. coli which will be prepared by incubating overnight for 5min. The one inoculated with bacteria will be marked at set A and the one not inoculated marked as set B. Then all beef slice will be packed with LLDPE embedded with 8% w/w. Once the whole set of sample preparation and testing with E.coli finish then precede same process with L. Monocytogenes.

3.4 Sample testing

Any 3 slices from set A and any 1 slice from set B will be take out then each slice will be homogenized for 2 min in 100mL of sterile peptone water with 0.1% w/w. Tenfold serial dilution will be done using 0.1% peptone water. The serial dilutions will be spread plated on solidified agar plate and the plate will be incubated at 35 ËšC for 24 hr and the total bacterial population will be estimated. 40 agar plates that incubated in day 2 will be taken out and the total bacterial population will be estimated. Step 1,2,3 from the day 2 will be repeated again in day 3 and will be continuously do until day 7 and the day 3 step 1 will be do until day 8.

CHAPTER 4

EXPECTED RESULTS

4.1 Antimicrobial activity

Table 4.1 Expected result when tested with meat inoculated with E.coli

Sample

log CFU/cm2

Day 1

Day 4

Day 5

Day 6

Day 7

Meat without garlic oil

2.84

2.8

3.03

3.56

3.52

Control, film without garlic oil

ND

3.5

4.29

4.77

5.02

LLDPE with 8 w/w %

ND

2.4

2.44

2.4

2.4

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