Antimicrobial Active Packaging Systems to Extend the Shelf Life of Lamb Meat

1	Introduction	1
2	Irish Lamb Meat Production	3
3	Shelf Life characteristics of Lamb meat	3
3.1	Microbial characteristics of Lamb Meat	3
3.2	Biochemical characteristics of Lamb Meat	4
3.2.1	Changes in Lamb pH	4
3.2.2	Changes in Lamb Colour	5
3.2.3	Lipid Oxidation in Lamb Meat	6
3.3	Sensory characteristics of Lamb Meat	6
4	Packaging Systems for Lamb meat	7
4.1	Modified Atmospheric Packaging	7
4.2	Vacuum Packaging/ Vacuum Skin Packaging	9
5	Smart Packaging	10
5.1	Antioxidant active Packaging /Oxygen Scavenging Packaging	10
5.2	Carbon dioxide Emitting Packaging	11
5.3	Antimicrobial active Packaging	11
6	Bioactive Edible Films and Coatings	12
6.1	Protein based Edible Films	13
6.1.1	Gelatin Films	13
6.2	Gelatin Nano solubilisates Films	15
7	Rosemary as an Antimicrobial	16
7.1	Antioxidant and Antimicrobial properties of Rosemary oils	17
7.2	Antimicrobial Mode of Action of Rosemary Nano solubilisates	18
7.3	Factors affecting the antimicrobial activity of Rosemary oil	19
8	Conclusion	19
9	References	20

1. Introduction

Food and Agricultural organisation of the United nations (2017) reported that over 1.3 billion tonnes of food products gets wasted every year accounting for about one third of the total food production in the world. The meat food contributes over 20% loss of the total food produced and this loss occurred mainly at the later stages of supply chain i.e. retail losses. Gunders (2012) reported that retail losses of meat products are largely influenced by the short shelf life of the product and the reports from European Commission (2010) showed that the retail food waste contributes to 58% of the total food wastage in the EU countries, therefore signifying the importance to extend the shelf life of the meat products. Lamb meat is a very perishable food product with a shelf life of nine to ten days under MAP or VP (Williams, 1991) making it hard for commercialisation and exporting over distant countries. Therefore, the lamb meat industries are looking for technologies to extend the shelf life of raw and processed lamb meat products which would provide them huge economic benefits and help them in managing the food waste related problems.  Extending the shelf life of the lamb meat would benefit the lamb meat industries in establishing their product among the competitors and helps them in effective transport to long distant export markets and satisfy consumer requirements. This can solve major food wastage issues related to the short shelf life of the product in the supply chain.

The shelf life of packaged lamb meat is affected by several factors such as microbial, biochemical and sensory characteristics, of which, the microbial metabolic activity is the most predominant factor affecting the shelf life of these products as it is hard to produce a meat product without some degree of microbial contamination (Mills, 2012) that arises during slaughtering techniques. Both aerobic and anaerobic bacteria can grow on the surface of the meat, therefore, by Controlling the microbial growth using antimicrobial materials, the shelf life of lamb meat product can be extended. Recent Studies have reported the extension of shelf life of lamb by using the antimicrobial agents such as whey protein isolate, TiO­₂ nanoparticles, rosemary essential oil, food by-products, grape pomace and so on (Andres et al., 2017; Alizadeh Sani et al., 2017; Guerra -Rivas et al., 2016; Ortuno et al., 2015; Karabagias et al., 2011). The shelf life of the meat can also be affected by changes on the biochemical characteristics such as colour of the meat, lipid oxidation to a certain extent as these factors are responsible for the sensory changes in the meat thereby reducing its retail quality and shelf life. Currently techniques like chilling, addition of preservatives and packaging techniques are used to preserve meat products and extend their shelf life (Ortuno, Serrano and Banon, 2015).

The primary purpose of packaging is to contain, protect, preserve and provide entire information of the food product right from the manufacturing point to the consumer usage point (Cruz Romero and Kerry, 2017). Packaging systems used to package fresh and processed meat products, control microbial contamination thereby delaying the meat spoilage. In other words, they are simply responsible for the maintenance of the sensory qualities and other important parameters within the estimated product shelf life. Active packaging, according to the European Union Guidance to the commission Regulation No 450/2009, is a packaging system with an additional function apart from acting as a protective barrier from the external factors. Antimicrobial active packaging is the form of active packaging that are used to control the microbial growth using antimicrobial materials in the packaging system and are used in the form of coatings, edible films, sachets/ pads, polymer coated. The edible films or coatings are generally impregnated with the naturally occurring antimicrobial nano solubilisates and then surface coated on to the conventional polymers to control the growth of the microbes. Gelatin films containing antimicrobial materials Sodium octonoate and Auranta FV are used as carrier coatings on the commercial polymer surface to control the microbial growth on the meat surface and control its spoilage effects (Clarke et al., 2016). Rosemary essential oil known to have antioxidant and antimicrobial activity due to the presence of carnosic acid and used for the development of antimicrobial active packaging to extend the shelf life of lamb meat products (camo et al., 2008; Ortuno et al., 2015; Pineros- Hernandez et al., 2016).

However, to the best of our knowledge, rosemary nano solubilisates have not been used to develop antimicrobial active packaging and also no work have been reported on the use of gelatin film as a carrier of antimicrobial rosemary nano solubilisates, coated on the inner surface of conventional vacuum packaging pouches, to extend the shelf life of lamb meat. Therefore, the objectives of this study are

1. To develop conventional vacuum packaging pouches coated with gelatin as a carrier of antimicrobial substances and to determine its mechanical and barrier properties.
2. To assess the performances of the developed antimicrobial active packaging in extending the shelf life of the vacuum packaged lamb meat.

 

2. Irish Lamb Meat Production

Lamb meat industries are the indigenous meat industry in Ireland contributing for about 3% of total food exports in the year 2016. Over 50,000 tonnes of sheep meat valuing approximately around € 240 million was exported in the year 2016 (Boardbia) with France (45%) and UK (25%) forming the major export market contributing 70% of the total Irish lamb exports. Irish lambs also account for about 7% of total meat product consumed in Ireland. Due to the small domestic market and huge demand in European Union market, lamb meats produced are mainly for the export purpose. This signifies the importance of shelf life extension of the lamb meat products for the effective export qualities.

 3. Shelf Life Characteristics of Lamb Meat

The shelf life of the lamb meat is characterised by the microbial, biochemical and sensory changes that occur during storage. These characteristic changes are dependent on the intrinsic and extrinsic factors of the lamb meat. The Intrinsic factors include pH, its oxidation- reduction potential, water activity (a_w), available sources of water, carbon sources, vitamins, minerals and other important constituent of the meat. Extrinsic factors include availability of oxygen, relative humidity, temperature and constituent of the storage atmosphere (Cutter, 2002). The proximate composition study of lean lamb meat indicated the level of moisture (72.9%), fat (4.7%), protein (21.9%) and other essential vitamins and minerals present which are suitable for the above characteristic changes in the meat (Williams et al., 2002).

3.1 Microbial Characteristics of Lamb Meat

Growth of microbes in lamb meat is influenced by its intrinsic, extrinsic and processing factors. Availability of enough nutrient content  and the suitable environmental factors makes the lamb meat as an excellent medium for the growth and survival of  many spoilage and pathogenic microbes on the meat (Cutter, 2002). These microbes utilises the sugar in the meat and breaks down the meat protein producing slime and off-odour, thereby reducing the meat quality and causes food borne disease on consumption. Salmonella, Pseudomonas, E. coli, Staphylococcus, Bacillus are some of the common bacteria that grow on the meat. The main microorganisms found in packed lamb meat are:  Entereobacteriaceae, Pseudomonas spp., Brochothrix thermosphacta, Lactic acid bacteria are the major bacteria that are found to grow on the lamb meat (Rubio et al., 2016).

Table 1 Main Spoilage Bacteria found in Chilled Packed Lamb meat (adapted from Mills et al., 2014).

Organism	Mode of Survival	Threshold level	Spoilage Potential
Pseudomonas	Aerobe	107/ cm2 at Packing	High
LAB	Anaerobe	~ 108 cfu/g	Low
Brochothrix themosphacta	Facultative anaerobe	~ 106 cfu/g	High
Enterobacteriaceae	Facultative anaerobe	~ 108 cfu/g	High

In MAP lamb products, the composition of gaseous atmosphere, water activity, storage temperature and pressure inside the package determines the growth of the microbes (Cutter, 2002).

3.2 Biochemical Characteristics of Lamb Meat

During chilled storage of lamb meat biochemical changes such as pH, Colour and lipid oxidation can occur. These changes can effectively alter the eating quality of the lamb meat and contribute to the shelf life of these products

3.2.1 Changes in Lamb pH

pH is defined as the numerical measure of acidity or alkalinity of the solution on a logarithmic scale which in other words, is the measure of hydrogen ion concentration of the solution. The pH of lamb meat is an important parameter that can affect the eating quality of the product. The pH of the living sheep muscle is almost at neutral pH around the range 7.0-7.2. After slaughtering, the glycogen in the meat converts into lactic acid which brings down the pH around 5.4-6.0. The ultimate pH_u of the lamb determines the quality of the meat which is around 5.4-5.7. The variation in the ultimate pH of the meat has influence on the tenderness and water retaining capacity of the meat. Lower ultimate pH results in dry meat with reduced water holding capacity, less tender and pale coloured lamb product (Ronald Klont, 2005) Higher the ultimate pH, increased water holding capacity with the dark coloured and highly tender (softer texture) meat product. Also, the higher pH of meat above 5.8 increases the chances of microbial growth on the product surface declining the quality and shelf life of the product which is the major concern for the meat industries (Villarroel et al., 2003).

Table 2 Change in quality of Lamb with ultimate pH change. (Adapted from Lomiwes et al.)

Ultimate pH	Quality of Meat
5.4-5.7	Tender Meat
5.7-6.2	Inconsistent Tenderness
> 6.2	DFD, Tender, soft texture, Dark

 

 

 

3.2.2 Changes in Lamb Colour

Meat colour is one of the main sensory attributes that determines the freshness of the meat from the customer’s point of view (Brewer et al., 2002). In case of red meats like lamb, beef, the freshness is determined by the bright red colour of the meat. Studies report that consumers prefer to buy the bright red coloured meat than the meat that turns into brown colour upon storage (Calnan et al., 2013). The colour appearance of the meat product is due to the presence of the heme protein pigment named Myoglobin. Myoglobin on reaction with oxygen gives oxymyoglobin that is responsible for the bright red colour of the meat. Oxymyoglobin on oxidation turns out into metmyoglobin that results in the brown colour of the meat.

 oxygenation

         Purple Red

deoxygenation

Reduction Oxidation

 

Brown

Fig.1 Colour Formation in Meat adopted from O’ Sullivan and Kerry, 2012.

The brown colour formation of the lamb meat on storage i.e. the formation of the metmyoglobin is favoured by various factors such as rate of auto- oxidation of myoglobin, rate of oxygen utilisation by the meat, lipid oxidation, partial pressure of the oxygen present, muscle oxidative capacity pH, temperature and microbial population in some cases (Mancini and Hunt, 2005). After slaughtering, the iron molecules present in the lamb meat becomes free ion which forms metal chelates. Thus, upon storage, these free ferrous ions promote the oxidation of oxymyoglobin resulting in the discolouration of the lamb meat.

Population of aerobic microbes like Pseudomonas spp. which grows on the surface of the lamb meat creates oxygen demand in the logarithmic phase of their growth, causing the oxidation of metmyoglobin (Walker, 1980). When lamb meat is vacuum packaged, the unavailability of oxygen may result in the rapid discolouration of the lamb meat, however, unpacking the meat resulted in the blooming of the colour of the meat because of its exposure to the atmospheric oxygen.

3.2.3 Lipid Oxidation in Lamb meat

The flavour of packaged lamb meat is can be affected by the oxidation of lipids or fatty acids present. Compared to beef or pork, the high content of ω-3 polyunsaturated fatty acids in the lamb leads to the easy oxidation of fats, resulting in the reduced shelf life of the product (Banon et al., 2011). Modified Atmospheric packaging (MAP) containing high concentrations of O₂ increased lipid oxidation and developed off-flavours in the lamb meat packs (Kerry et al., 2000). The oxidation of lipid in fresh meat produces off- odours which is significantly due to the rapid increase in the development of rancidity of meat. This have a minor role in the discolouration and reduction of other quality aspects of the meat. The oxidation occurs when the meat is exposed to the excess concentration of oxygen which is catalysed in the presence of enough light and catalysts like free iron molecules (Campo et al, 2006). Meat products packaged with high O₂ and the conditions favouring the above situations will result in the development of rancidity of meat product producing off-odours on unpackaging.

3.3 Sensory Characteristics of Lamb meat

Sensory of the meat generally described as tenderness and juiciness of the meat which is partly associated with the water holding capacity and muscle structure of the meat. Studies showed the consumer’s interest on paying high price for tender meat products in the market thus making tenderness as one of the most important qualitative characteristic of the meat product (Miller et al., 2001) and was also reported as the main parameter determining the consumer’s satisfaction on eating (Pipek et al., 2008). It is associated with the connective tissues present in the meat and the strength of these connective tissues are dependent on the age, species of the animal slaughtered, muscle type and its location. Pre-slaughter handling, transportation stress, animal diet also plays the key role in meat tender quality. Other factors like lipid oxidation, pH, protein oxidation also have an influence over the meat tenderness and juiciness. Protein oxidation plays a major role in meat quality as they cause both physical and chemical alterations like loss of enzyme activity, breakdown of amino acids, reduction of protein solubility range, etc., all of which contributes to the reduction of meat tenderness and juiciness. Studies on beef packed inside MAP packaging system reveals that the high O₂ content in the packaging induced the protein oxidation in the meat thereby decreasing the meat tenderness and shelf life (Zakrys et al., 2008).

4.  Packaging for Lamb Meat

The commonly used meat packaging systems to preserve the lamb meat products are Modified Atmospheric Packaging(MAP), Vacuum packaging(VP) systems and overwrapping systems. Vacuum packaging and MAP are used for long term cold storage while overwrap packs used for retail displays and for short term cold holding (Kerry et al., 2006). Recent studies in the advancement of packaging system had resulted in the development of new packaging technologies namely active packaging, intelligent packaging, nanomaterial packaging and edible films packaging systems.

4.1 Modified Atmospheric Packaging

Modified Atmospheric packaging is an advanced system of packaging meat products with Nitrogen (N₂), Oxygen (O₂) and Carbon dioxide (CO₂) used as primary gases. . These Primary gases are either used alone or as mixtures; however, oxygen and Carbon dioxide are the predominantly used MAP gases for packaging fresh lamb meat. The gas combination commonly used is 70% O₂ and 30% CO₂ respectively (Cruz Romero and Kerry, 2017). High O₂ concentration in the packaging helps in prolonging the metmyoglobin formation which in turn ensures the maintenance of the bright red colour of the lamb meat, however, high concentration of O₂ produced lipid oxidation in meat. The high level of CO₂ is responsible for the inhibition of microbial growth on the meat surface which ensures the extension of shelf life of the product (McMillin, 2008).

Table 3. Properties of Primary gases used in MAP (adapted from Cruz-Romero and Kerry, 2017).

Gas	Properties	Use in MAP meat products
Oxygen	Inhibits anaerobic bacteria  Moderately soluble in water and fat Oxidises fat, myoglobin	Avoids anaerobic growth  Maintain raw meat colour
Carbon dioxide	Supress anaerobic bacteria  Highly soluble in water and fat Acid taste at high concentration.	Inhibits microbial contamination  Produce tight contact packaging
Nitrogen	Inert  Low solubility in water and fat.	Prevent collapse of packaging  Replaces oxygen.

There are other secondary gases like argon, Helium, nitrous oxide, carbon monoxide and hydrogen used along with the primary MAP gases. MAP packaging helps in extending the shelf life of the product by inhibiting the microbial growth and maintains the colour of the meat product when compared with the product packaged with air i.e. normal packaging (Penney & Bell, 1993).

Fig. 2 MAP packed Lamb Racks

The main disadvantage of the MAP packaging is that the presence of excess oxygen induces the lipid oxidation that results in the rancidity of the meat generating the undesirable off- odours declining the quality scale of the meat product (Renerre & Labadie, 1993). Reports show that the lamb meat packed with 70% of O₂ and 30% of CO₂ can be stored for 8-9 days without any perceived level of microbial spoilage; however, the quality is degraded enough because of the lipid oxidation and browning action on the meat surface thus making it not suitable for the retail display (Banon et al, 2012).

4.2 Vacuum Packaging/ Vacuum Skin Packaging

Vacuum packaging generally refers to removal of air constituents from the package before sealing with the food product inside. Vacuum packaging ensures the complete evacuation of air creating a void situation for the growth of the aerobic bacteria. Also, it eliminates the probability of fat oxidation of the meat product thus prolonging the product shelf life and balances sensory qualities of the meat (Strydom & Hope-Jones, 2014). The ability to see the meat content inside the vacuum packaging has made a visual appeal to the consumers buying the product.

Vacuum skin packaging system is the recent alternative system where the meat is shrink-wrapped with the polymer of low oxygen transmission rate. This system has managed to overcome some of the disadvantages of vacuum packaging systems like appearance of purges in the crevices of the meat and the formation of typical purple colour of the meat surface due to the deoxy-myoglobin formation.  When unpackaged, both the VP and VSP packaged meat product bloomed on re-exposure to air, however, Vacuum skin packaged meat products has proved to be more colour stable than the meat product packaged with conventional vacuum packaging system (Li et al, 2012). VSP had made a huge visual appeal on consumers because of its shrinkage and reduced air pockets (Diz et al., 2005). Aaslyng et al. (2010) showed increased consumer preferences for the VSP over the MAP packaged meat in three Scandinavian countries. Consumers perceived that VSP packaged meat were tenderer and juicier than MAP packaged meat. Similarly, VSP packaged lamb steaks possessed better eating qualities than lamb steaks MAP packaged containing high oxygen concentration of 70% oxygen and 30% CO₂ (Lagerstedt et al., 2011). It also improved the shelf life of the freshly packed lamb meat to at least ten days with an excellent sensory quality (Frank et al.,2016).

Fig. 3 Vacuum Packed Lamb meat                           Fig. 4 Vacuum Skin Packaged Lamb meat

5. Smart Packaging System

Smart Packaging can be defined as the packaging that serve beyond its use as simple packaging material with traditional printed features. This new packaging has been classified in many ways namely active, intelligent, functional, enhanced and diagnostic packaging. Active and the intelligent packaging systems are widely-used packaging systems for packaging meat and meat products (Fang et al., 2017). The active packaging serve either by absorbing food chemicals from the food or releasing those chemicals into the food material that can act as preservatives, flavourings, etc., (EU, 2009). They also satisfy the customer requirements of being natural and biodegradable material by means of bioactive packaging (Lopez-Rubio et al., 2004). The active packaging finds its major applications on meat and meat products through antimicrobial, antioxidant and CO₂ emitting packaging. This packaging system can extend the product shelf life by protecting the product from microbial contamination and degradation (Ettinger, 2002) and maintain the quality of the packaged food product (Suppakul et al., 2003).

5.1 Antioxidant active packaging/ Oxygen- Scavenging Packaging

In case of packed meat products, the presence of excess range of oxygen in the packaging can result in many undesirable reactions such as lipid oxidation, colour change, microbial growth which in turn result in the overall deterioration of the nutritional and sensory qualities of the meat (Gomez- Estaca et al., 2014). Antioxidant active packaging is developed with a purpose of controlling the oxygen content of the package thereby maintaining the shelf life and quality of the meat product. This Packaging systems has two important classifications namely, Independent antioxidant devices and antioxidant packaging materials (Gomez-Estaca et al., 2014).

1. Independent antioxidant devices:  oxygen scavengers contained in the sachets, pads or labels which are added passively in the conventional packages. The scavenging reaction is controlled by specialised mechanisms to control the premature action of oxygen scavenger. Under suitable conditions these oxygen scavengers gets activated and removes the excess oxygen, for example, presence of humidity to initiate the oxygen removal in case of iron-based scavengers (Lopez Rubio et al., 2004).
2. Antioxidant Packaging: Incorporation of the antioxidant substance in to the walls of the packaging material or within the packaging systems. These application removes the undesirable substances within the package or releases the antioxidant substance to the product surface or into the packaging system thereby preventing the undesirable effects of oxidation reactions. The selection of antioxidant compound is of prime importance as it should be compatible to the packaging film and apt to the food product used (Decker, 1998).

5.2 Carbon dioxide Emitting Packaging

CO₂ has a desirable inhibitory effect on the growth of aerobic bacteria through antimicrobial effects, resulting in the increase of lag phase and generation time during log phase of the bacterial growth. CO₂ emitters are generally used in the meat product packaging in sachets/ pads. These emitters help in maintaining the carbon dioxide concentration which generally decreases due to the dissolution into the food particles or due to the permeation via the packaging material (Coma, 2008).

5.3 Antimicrobial active packaging

The purpose of the antimicrobial active packaging is to enhance the safety of meat products that are highly susceptible to microbial contamination. Basically, antimicrobial active packaging can be classified into four different categories (Cooksey, 2001). They are,

1. Antimicrobial agents being incorporated through sachets/pads that are kept inside the package. Subsequently, the antimicrobial agents are released from the sachets/pads and provides protection against microbial growth (Otoni et al., 2016).
2. Incorporating the antimicrobial agents directly into the packaging films through conventional heat treatment method where the antimicrobial agents are gradually released from them to the meat surface or to the package head space thus helping in controlling the microbial growth.
3. Coating the packaging material with a carrier matrix that possess antimicrobial compounds which are being released into the headspace through evaporation or migrated into the food material through diffusion process.
4. Use of Polymers such as chitosan and poly-L-lysine with an inherent antimicrobial activity to kill the bacterial cells are used as an active packaging materials. The charged amines of these polymers can interact with the microbes and can degenerate their cell membrane and kills them (Goldberg et al., 1990).

Recent advancement in the active packaging has resulted in the coating or incorporation of antimicrobial agents into the polymers that are normally used as the packaging material. These polymeric matrices can release active substances or retain undesirable food compounds that are threat to the food safety (Flores et al., 2007). The choice of antimicrobial agent for a particular meat should be made after a careful experimentation of the effect of that antimicrobial agent on the visual and sensory properties of the processed meat product and its delivery into the product packaging.  Many compounds that are supposed to possess antimicrobial activity such as ethanol, spices, organic acids, antibiotics, essential oils, silver ions have been examined for the reduction of microbial population of the food (meat) product (Zhao et al., 2013). Compounds like peptides, plant extracts like rosemary extracts can be used for the development of antimicrobial active packaging materials. Oregano and rosemary extracts showed antioxidant and antimicrobial effect and increased the shelf and display life of lamb meat without any significant effect on the colour and flavour for 8- 13 days  compared to control samples (Camo et al., 2008). More detailed study of rosemary as an antimicrobial agent can been studied below in section 6.1

6.  Bioactive Edible Films and Coatings

An innovative packaging system developed aiming at the consumer’s health with application of biopolymers that are degradable or edible and can transfer health benefits. Bioactive packaging system includes the use of active biodegradable materials as a functional component in the packaging system. These packaging materials have the ability to degrade by natural means. Bioactive packaging development has created a revolution in the packaging technologies that led to development of many ideas using different bioactive components (Lagaron, 2005) in the food packaging systems. This advancement has led to the development of edible films and coatings that can substitute the conventional packaging systems.

Edible films and coatings are the recent advanced packaging technology that utilises the biopolymers made from the bioactive ingredients like hydrocolloids derived from proteins, polysaccharides, pectin and lipids which may be of animal or plant origin. These ingredients are solvent casted to form a thin coating or a film that are meant to deliver the desired function apart from acting as barrier against the moisture, oxygen migration into the packaging. Although they have many advantages over the conventional packaging systems, their inability of certain mechanical characteristics of printability, stretch ability and other properties such as transparency, solubility, etc., eliminates them from the concept of replacing the conventional traditional packaging systems in industrial scale. Hence, these coating and films can be used alongside of these conventional packaging systems which are normally made up of polyethylene, polystyrene and polyamides.  The use of polymeric conventional packages with the bioactive gelatin films as a carrier of antimicrobial substance has been well demonstrated by Clarke et al, 2016 on the vacuum packaged bee.

6.1 Protein based Edible Films

Among the different sources used for edible films, protein based films are widely used since they possess acceptable mechanical properties than the others. Also, the gas barrier properties and casting properties of the protein based films makes them as the suitable choice over the others (Ou et al., 2004). Proteins derived from plant or animal source must be soluble so that they can be casted as film coatings. Proteins like casein, egg white, gelatin, wheat gluten, soy protein, collagen and myofibrillar proteins are generally used as films (Hanani et al., 2014). Reports of Hanani et al., (2013) proved the ability of the gelatin to be casted as a polymeric edible film and its cost effectiveness, transparency and mechanical properties. Therefore, gelatin with unique association characteristics, is widely used as a source for developing protein based edible films. The film is usually developed using solvent casting, thermoplasticization and extrusion process. Of these processes, solvent casting process generally used to cast the protein based films since the other process of film development has imperfections with the spatial arrangement of the molecules (Mensitieri et al., 2011).

6.1.1 Gelatin Films

Gelatin is a protein polymer derived from the hydrolysed insoluble fibrous collagen protein which is the major constituent of the skin, bones and connective tissues of animals. It consists of carbon, hydrogen, nitrogen and oxygen with 50.5%, 6.8%, 17% and 25.2% respectively (Hanani et al., 2014). The melting point of the gelatin is relatively low when compared to the other protein and thermo-reversible in nature. It is known for its usage as gelling agent, thickening agent, stabiliser, emulsifier, etc., Sequencing of gelatin for its amino acid reveals its uniqueness with high amount of proline, glycine and hydroxyproline which are responsible for its gelling properties. Gelatin, based on its pre-treatment, is classified into two types namely Type A and Type B. Type A gelatin is derived from collagen by treating it with the acid whereas Type B is obtained by treating the collagen with an alkali. Generally, pigskin gelatin and bovine gelatin are referred to as Type A and Type B gelatin respectively.

Fig 5. Structure of Gelatin

Gelatin is soluble in water which makes them easier to undergo gelation after heating. The abundancy and relatively low cost makes gelatin a suitable choice for various applications. The notable functional properties of gelatin find its application in food, pharmaceutical, photographic, medical and cosmetic industries. Initially, the gelatin is used in the pharmaceutical area (Park et al., 2008). Recent studies shown that there is an increased utilisation of gelatin in developing the edible films or coatings as a food packaging. The biodegradable nature of the gelatin is suitable for the desired properties of a bioactive film (Wang et al., 2007).

Table 4 Studies made on different type of Gelatin based edible films. Adopted from Hanani et al., 2014.

Sources	Method	Plasticizer
Pork	Casting	Sorbitol
Bovine	Casting	Glycerol
Beef	Extrusion	–
Bovine Gelatin	Casting	Glycerol
Fish skin Gelatin	Casting	Glycerol
Tuna skin gelatin	Casting	Glycerol, Sorbitol

Usually, gelatin derived from pig, beef or fish is used for developing bioactive films that are used as packaging material in food industry. The films formed from the gelatin has shown good transparency, barrier, printing and other mechanical properties which most of the other bioactive packaging materials lacked. However, gelatin possess some weakness such as poor water resistant and moisture resistant properties which can be modified by blending with other ingredients that provide stability to the film. This type of film is referred as composite films where the gelatin is mixed with other biopolymer compound or with the supporting functional ingredients like corn oil, plant based oil, etc., to improve the desired characteristics of the film. Studies have reported that the composite films formed by combining chitosan with the gelatin improved the WVP of the beef gelatin films (Gomez- Estaca et al., 2010).

6.2 Gelatin Nano solubilisates Film

The concept of developing nano solubilisates film has been improved by blending nanoparticles with gelatin to form gelatin- based films with improved mechanical and physical barrier properties. The advantage of using nanoparticles is that their polymeric matrix arrangement makes it tough for the gases like oxygen to pass through them thereby delaying their passage from the package (Pereira de Abreu et al., 2007). Various studies to demonstrate the use of Nano solubiliates in the gelatin based films have proved their ability to form an effective film. Rabe et al in 2009, developed a pig skin gelatin film which is incorporated with ascorbic acid and sorbic acid. Their study revealed that the addition of nano solubilisates improved the structural strength of the gelatin films.

Table 5 Antimicrobial Nanocomposites used in Gelatin based Edible films. Adopted from O’ Callaghan and Kerry, 2014

Agents	Concentration	Dissolution Temperature	Solvent
Sorbic Acid Solubilisate	0.25 w/v	40°C	Water
Benzoic acid solubilisate	0.25 w/v	40℃	Water
Ascorbic acid	1 w/v	40℃	Water
Curcumin	0.2 w/v	40℃	Water
Rosemary	1 w/v	40℃	Water

This ability of the gelatin to form composite films with other natural and nano ingredients made the scientists to focus on the development of active gelatin based films incorporated with antimicrobial and antioxidant properties. Many studies have successfully developed active antimicrobial gelatin based films by using different antimicrobials such as nano solubilisates, Silver nanoparticles, for example, when incorporated with the gelatin based film, improved the tensile strength, hydrophobicity and water barrier potential of the film apart from imparting its own property of inhibiting the growth of the food pathogens (Kanmani, 2014). The study of bovine gelatin film coatings with the antimicrobial agents such as Sodium octanoate and Auranta FV on the vacuum packaged beef sub-primal cuts proved the effectiveness of the used antimicrobial agents in improving the film properties and controlling the microbial growth on the meat product. This study involved the gelatin films coated over the conventional polymeric packaging system through cold plasma treatment which improved the attachment of gelatin film over them. This system showed positive response in improving the shelf life of the product packaged (Clarke et al., 2016).

7. Rosemary as an Antimicrobial Agent

Rosmarinus officinalis, scientific name of the perennial herb Rosemary belonging to the family Lamiaceae is commonly found in the Mediterranean region. The leaves of the herb are traditionally used as an ingredient of many Mediterranean food and its extracts are generally used as a food additives to impart flavour, antioxidant and antimicrobial activity to the food. 

Fig. 6.  Leaves of Rosemary

Rosemary extract obtained from its leaves is rich in phenolic diterpenes, flavonoids and rosemary phenolic acids (Aguilar et al., 2008). It also contains many bioactive substances like monoterpene hydrocarbons such as borneol, verbenone, linalool, 1,8- cineole; triterpenes (Sirocchi et al., 2016).

7.1 Antioxidant and Antimicrobial Properties of Rosemary oils

Carnosic acid, the phenolic diterpenes present in the rosemary extract is rich in higher antioxidant activity and antimicrobial activity. They can inhibit the oxidation of fats and oils thereby maintaining the flavour stability and shelf life of the product. They are also renowned for their very good sensory scores when applied to the meat products.

Fig. 7. Structure of Carnosic acid adopted from Xie et al., 2017.

Studies have reported that application of rosemary as dietary supplements and coating materials for active packaging systems and shown to possess strong antimicrobial and antioxidant activities. The assessment studies made on the antimicrobial activity of the rosemary nano solubilisates by O’ Callaghan and Kerry (2014) against the cheese derived microbes proved that rosemary exhibited strong antimicrobial action against S.aureus and B, cereus. This antimicrobial activity is highly due to the presence of structural composition of the functional carnosic acid present in the rosemary oil extract. Furthermore, rosemary nano solubilisates proved very effective against the growth of Gram positive bacteria even at lower concentrations. However, their action on Gram negative bacteria was found to be negligible (Holley and Patel, 2005).

Application of Rosemary Carnosic acid in lamb meat have shown results in controlling the microbial population. Studies of Camo et al., 2008; Banon et al., 2011; Serrano et al., 2013; Ortuno et al., 2013; Ortuno et al., 2015 has shown the promising results in controlling the microbial growth and extend the shelf life of the lamb meat proving the efficiency of antimicrobial activity of rosemary for lamb meat.

7.2 Antimicrobial mode of Action of Rosemary Nano solubilisates

The antimicrobial mode of action of rosemary essential oil is not clearly understood but it is believed to be the synergistic action of all the secondary metabolites such as carnosol, carnosic acid, rosemarinic acid, 1,8- cineol, α-pinene, camphene and common metabolites present in all essential oils such as thymol, carvacol, p-cymene and cinnamaldehydes. Reports of Hyldgaard et al., 2012 states that the application of rosemary essential oil makes the cell membrane rigid and affect the transport of ions through the membrane thereby killing the microbes.

1. Terpenoids such as thymol and carvacrol can affect the cellular membrane allowing for the passive transport of ions through the membrane. Carvacrol can disintegrate outer membranes of Gram-negative bacteria while in Gram-positive bacteria the membrane permeability is altered allowing permeation cations like H⁺ and K⁺ (Hyldgaard et al., 2012).
2. Phenylpropanoids such as eugenol and cinnamaldehyde can non-specifically permeabilise the cell membrane and can crosslink covalently with internal DNA and amino groups of proteins, respectively (Tongnuanchan et, 2014).

These are the primary metabolites that are responsible for the antimicrobial activity of essential oils while other minor antimicrobial constituents are discussed detail in Hyldgaard et al. (2012).

 

7.3 Factors affecting Antimicrobial activity of Rosemary oils

The release of bioactive compounds from the rosemary essential oil and its antimicrobial activity is characterised by several factors. The hydrophobic Rosemary essential oil is effective only against the gram-positive bacteria as they are easily penetrable through the cell membrane of gram-positive bacteria. The susceptibility of lipophilic end of rosemary oil on the lipoteichoic acid in the cell membrane helps in the easier penetration of essential oil into the cell whereas in case of gram- negative bacteria, there is a decrease in the susceptibility due to the presence of extrinsic membrane proteins and cell wall lipopolysaccharides (Tongnuanchan and Benjakul, 2014). Shape of the bacterial cell also determines the activity of the rosemary essential oil, where the rod-shaped cells are proved to be more sensitive to the essential oils than the bacteria with coccoid shape (Cui et al., 2015). pH also plays a major role in the release of bioactive compounds from the essential oil. If the pH of the lamb is too acidic, it may destabilise the nano solubilisates reducing their activity and losing their homogenous distribution (Fathi et al., 2012).

8. Conclusion

Studies have demonstrated the potential of rosemary essential oil to be used as an antimicrobial agent for the development of antimicrobial active packaging materials and extend the shelf life of lamb meat products. It has been reported that application of rosemary nano solubilisates have the potential to increase the antimicrobial properties of food packaging materials and this antimicrobial activity of Nanoparticles is predominantly determined by its shape, size, composition, crystallinity and morphology (Cruz-Romero et al. 2013). Reviews also highlights the lack of application of rosemary nano solubilisates on the development of antimicrobial active packaging system in extending the shelf life of the lamb meat. Also, Potential of gelatin to act as a carrier and coating polymer to develop conventional antimicrobial active packaging was successfully demonstrated and reviewed.

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