SPOILAGE ASSOCIATED WITH MEAT AND MEAT PRODUCTS BY MICROBES

SPOILAGE ASSOCIATED WITH MEAT AND MEAT PRODUCTS BY MICROBES

Department of Meat Hygiene and Technology

Faculty of Veterinary Hygiene and Ecology

University of Veterinary and Pharmaceutical Sciences

Caristas Enugu Nigeria.

INTRODUCTION

             Meat is excellent food for microorganism because of its composition and quality. Meat is also nutritious, protein rich food which is highly perishable and has a short shelf-life unless preservation methods are used.  Shelf life and maintenance of meat quality are influenced by a number of interrelated factors including holding temperature, which can result in detrimental changes in the quality attributes of meat (Gill 2007). Spoilage by microbial growth is the most important factor in relation to the keeping quality of meat. Muscle tissue that is in good health at the time of slaughtering are derived from domestic bird or animals, which can be consumed for food. Examples are goats, antelope, cow, bill etc and birds such as chicken, turkey, geese and ducks. This also includes the offal’s such as tongue, kidney, liver, intestine of animals etc. The type of animal used for meat is dependent on its environment and the society of attractive with minimum risk to the health of consumers. To produce a palatable, nutritive and whole some meat and meat product, consideration will be given to the complicated processes and accomplishment added to the cost of processes involved and subsequent maintenance of standard which have to be maintained (Ayres 1999). People, that is their culture and religious belief as well as their choice. Food produced with respect to meat and meat product should be capable of providing .animals and birds used as food excluding fish and fowl which consist of muscular tissues with the joint but also with age e.g cow, goats. Though meat is one of the principal body building food, various parts of meat are eaten in different parts of the world according to their availability or because of local custom. Meat is composed of bean meat which contains protein such as myosin and globulin is high in biological value because of all the important amino acid it contains. Meat(liver)is an excellent source of protein, vitamin A, B and Iron. Meat also contains fermentable carbohydrate (Glycogen), therefore the importance of meat in hospitality industry cannot be over emphasized.  

Meat preservation written as an integrated and all encompassing text that includes historical aspects and treads, discussion of background information, the evaluation and status of techniques and procedure and treatment of potential future basic on consumers desires, there is a definite trend developing to produce and market meat and meat products that have been subjected to a loss degree of preservation, yet appear to be fresh and more healthful. Today there is an intense interest to produce the safest meat possible(Currier et al 2001). The overriding theme of meat preservation provides the understanding of the science of meat and discussion for using known technologies to achieve the goal of safe meat of high quality. Proper preservation of meat is important to prevent economic loss due to spoilage and to prevent the transmission of food borne illness (Clarence et al 2009).

1.2 STATEMENT OF PROBLEM

In general, the metabolic activity of the ephemeral microbial association which prevails in a meat ecosystem under certain in aerobic conditions or generally introduced during processing, leads to the manifestation of changes or spoilage of meat. These changes or spoilage are related to the

i.                    Type, composition and population of the microbial association and

ii.                  The type and the availability of energy substrates in meat.

Indeed the type and the extent of spoilage is governed by the availability of low-molecular weight compounds (e.g glucose, lactate) existing in meat. A wide range of microorganisms coming from different sources are introduced onto carcass surfaces, which contain abundant nutrients and which have high water availability. Only a few of the contaminants will be able to initiate growth and only some of these will eventually spoil the meat by means of their biochemical attributes. Predominance of different groups of micro organisms on meat depends on the characteristics of the meat, the environment in which meat is stored as well as the processing that meat may undergo. As earlier noted, a vast number of studies in meat microbiology have established that spoilage is caused by only a fraction of the initial microbial association that comes to dominate which are yeast, bacteria and mold.

YEASTS

         Are a subset of a large group of organisms called fungi that also includes molds and mushrooms. They are generally single-celled organisms that are adapted for life in specialized, usually liquid, environments and unlike some molds and mushrooms, do not produce toxic secondary metabolites. Yeasts can grow with or without oxygen (facultative) and are well known for their beneficial fermentations that produce bread and alcoholic drinks and  there are four main group that affects food which are: Zygosaccharomyces, Saccharomyces spp, Candida and related genera, Dekkera/ Brettanomyces

MOLDS

Are filamentous fungi that do not produce large fruiting bodies like mushrooms. Molds are very important for recycling dead plant and animal remains in nature but also attack a wide variety of foods and other materials useful to humans.

They are well adapted for growth on and through solid substrates, generally produce airborne spores and require oxygen for their metabolic processes. Most molds grow at a pH range of 3 to 8 and some can grow at  very low water activity levels (0.7-0.8) on dried foods. And there are four main group that causes spoilage of food, which are: Zygomycetes, Penicillium spp, Aspergillus and Fusarium  spp.

BACTERIA

Spore- forming bacteria are usually associated with spoilage of heat-treated foods because their spores can survive high processing temperatures. Bacterial genera commonly infecting meat while it is being processed, cut, packaged, transported, sold and handled include Salmonella spp, Shigella spp, E. coli, B. proteus, S. epidermidis and Staph.aureus, Cl. Weldi, B. cereus and faecal Streptococci.

As these microorganisms colonize a piece of meat, they begin to break it down, leaving behind toxins that can cause enteritis or food poisoning, potentially lethal in the rare case of botulism. The microorganisms do not survive a thorough cooking of the meat, but several of their toxins and microbial spores do. The microbes may also infect the person eating the meat, although against this the microflora of the human gut is normally an effective barrier.

1.3 AIMS AND OBJECTIVES OF THE STUDY

1.      To attain the keeping quality of raw meat.

2.      To determine the spoilage associated with meat and meat products by microbes.

3.      To determine the effect of preservation of raw meat

4.      To know the effect of temperature on raw meat.

5.      To find out the various preservation method used in the preservation of meat.

6.      To determine the effectiveness of the method of preservation in use.

7.      To help reduce wastage of meat resulting from poor method of preservation.

8.      To help prevent the incidence of food poisoning and infections.

9.      To identify some of the problem that could be encountered in preservation meat.

10.  To determine the microorganism that causes meat spoilage.

11.  To determine that good glycogen reserve can keep meat quality for 24 hours without spoilage.

1.4  IMPORTANT OF THE STUDY

The importance of meat preservation cannot be over emphasized in the present day activities of meat for proper consumption. In order words, meat have to be preserved so that they can be fit for consumption, the various meats consumed also have to be free from microbes in order to avoid disease condition. Meats have to be kept properly to prolong the shelf life and also to make it fit for healthy consumption. During drought, food kept in storage condition will serve as immediate food for consumption where there is scarcity.

1.5 SCOPE OF THE STUDY

Since of food, the nutrients and other substances there in their action infraction and balance in relation to health and disease  and the processes by which the organism ingests, absorbs, transports, utilizes and excretes food substances. The national library  of medicine (NLM), the national agricultural library (NAL) and the library of congress (LC) acquires publications devoted to human nutrition and food and provide document and information services. Together NAL, NLM and LC attempt to collect, retain and preserve all significant information on human nutrition food.

1.6  LIMITATIONS OF THE STUDY

The factor that operates against the study is finance, much money is involved carrying out this experiment, the time this project work was carried out alongside with our academic studies, instruments used are only available in the laboratory in AAmorji Polytechnic, instability of electricity, inadequate material like the current journals, textbook and related literature in the school library and inadequate equipment in the school laboratory.

1.7 DEFINITION OF TERMS

a.       Spoilage- Is a process in which food deteriorates to the point in which it is not edible to human or its quality of edibility becomes reduced. Various external forces are responsible for the spoilage of food. Food that is capable of spoiling is referred to as perishable food.

b.      Culture- A batch of cells, which can be microorganism or of animal, or of plant origin that are grown under specific condition of nutrient levels, temperature, pH, oxygen level, osmotic factor, light, pressure and water content. Culture cells are prepared in the laboratory for a wide spectrum of scientific research. A culture medium provides the appropriate conditions for growth.

c.       Glycogen- A polysaccharide consisting of a highly branched polymer of glucose occurring in animals tissue, especially in liver and muscle cells. It is the major store of carbohydrate emerging in animal cells.

d.      Rigor mortis- The stiffening  of the body of an animal after death due to a temporary rigidity of the muscle. This condition arises because ATP, which is no longer synthesized after death is required to breakdown the bridges that form between acting  and myosin filaments in muscle tissue during contraction .

e.       Glycolysis-(Embden-megerhof pathway) the series of biochemical reactions in which glucose is broken down to pyruvate with the release of usable energy in the form of ATP. On molecule of glucose undergoes two phosphorylation reaction reaction and is then split to form two triose phosphate molecules. Each of these is converted to pyruvate. The net energy yield is two ATP molecules per glucose molecule. In aerobic respiration, pyruvate then enters the krebs cycle. Alternatively, when oxygen is in short supply or absent, the pyruvate is converted to various products by anaerobic respiration. Other simple sugars e.g fructose and galactose and pathway at intermediate stages.

f.       Zoonosis- An infectious disease of non human vertebrates that can be transmitted to humans. Rabies and anthrase are well known examples and certain midges and the tsetse fly act as carriers for a variety of nematodes- worm zoonosis.

g.      Microorganisms- Are tiny organisms that are not visible within the naked eye except with the use of   microscope that have economic importance.

h.      Preservation of Meat-As of most perishable  foods, usually is accomplished by combination of preservative methods. The fact that most meat are very good culture media high in moisture, nearly neutral in pH. And high in nutrients coupled with the fact that some organisms maybe in the lymph nodes, bone and muscle and contamination with spoilage organisms are almost unavoidable, makes the preservation of meat more difficult than that of most kinds of food.

i.        Abattoir– A large or small place usually a house which involves the slaughtering, processing and distribution of cattle, sheep and hogs or is a place where animals are killed for their meat.

j.        Symptoms- Indication of illness or disease felt by patient especially one experienced by the patient or animal e.g pain, dizziness, or is a sign of the existing of something especially something undesirable.

k.      Glycogen reserve- Is a polysaccharide found in the liver and muscle of animals that is easily converted a glucose for energy or is a polysaccharide stored in the live or and muscle of an animal. When these reserves are filled, glucose is converted to fat and deposited in a dispose tissue.

CHAPTER TWO

LITERATURE REVIEW

2.1 SPOILAGE OF MEAT

The spoilage of meat occurs, if untreated in a matter of hours or days and results in the meat becoming unappetizing, poisonous or infectious. Spoilage is caused by the practically unavoidable infection and subsequent decomposition of meat by bacteria and fungi, which are borne by the animal itself, by the people handling the meat, and by their implements. Meat can be kept edible for a much longer time, though not indefinitely if proper hygiene is observed during production and processing, and if appropriate food safety, food preservation and food storage procedures are applied. Without the application of preservatives and stabilizers, the fats in meat may also begin to decompose rapidly after cooking or processing, leading to an objectionable taste known as warmed over flavor (Baumah 2002).

2.2 MEAT AND THEIR TYPES

        Meat is animal flesh that is eaten as food. Most often, this means the skeletal muscles and associated fat and other tissues, but it may also describe other edible tissues such as organs and offal. In commerce, meat is generally used by the meat packing industry in a more restrictive sense, the flesh of mammalian species (pigs, cattle, lambs etc) raised and prepared for human consumption, to the exclusion of fish, poultry and other animals. Usage varies worldwide by culture, and some countries such as India have large populations that avoid the consumption of all or most kinds of meat. Game or bush meat is also generally distinguished from that produced by agriculture.

 The consumption of meat has various traditions and rituals associated with it in different cultures such as kosher and halal and its production is generally regulated by state authorities as well. This article is mainly focused on that process from primary production to consumption (Tompkin 2010).

2.2.1 RED AND WHITE MEAT

Meat can be broadly classified as “red” or “white” depending on the concentration of myoglobin in muscle fibre. When myoglobin is exposed to oxygen, reddish oxymyoglobin develops, making myoglobin- rich meat  appear red. The redness of meat depends on species, animal age and fibre type. Red meat contains more narrow muscle fibres that tend to operate over long periods without rest, while meat contains more broad fibres that tend to work in short fast bursts. The meat of adult mammals such as cows, sheep, goats and horses are generally considered red, while domestic chicken   and turkey breast meat are generally considered white (USDA 1996).

2.3 COMPOSITION OF MEAT

      The animal carcass is composed of the muscular tissues, fatty tissues and bone, the chemical composition of meat varies with age of animals, breed, species, dietary, the cut (i.e the location) and other factors (Mc Meekin 2000).

       Muscular tissues: the muscular tissues including the connective tissues consist of approximately 75% water, 19% proteins, 3.5 fats, 2.5% of insoluble non protein materials. The water content varies with age and degree of fatness of the animal involved. The older  the animal, the lower the water content. The amount of water present also varies with the breed of the animal, thus veal and lamb contains more water than the beef and mutton. A considerably proportion of the water of the muscular tissue is replaced by fat during fattening in the form of marbling. The muscular tissue consists of approximately 25%  dry  matters. The dry matters consist of mainly coagulate proteins which make up 18-20% of muscular meat. Traces of glucose and animal starch (glycogen) are found in all muscle tissues and the liver. It also consists of 1-2% minerals matter mianly iron and phosphorus, important vitamins like thiamin, niacin, riboflavin, folic acid and vitamin B12. Liver, heart and kidney are good source of high quality protein. They are rich in iron, liver is very rich in vitamin A. tripe is a good source of calcium. The protein in muscle can broadly be divided into sarcoplasmic protein which are soluble in water or dilute salt solution, the myofibrillar protein which are soluble in concentrated salt solution and those which are insoluble in concentrated salt solution at lower temperature.

Fatty tissues: the fatty tissues is composed of mainly fat but very low amount of connective tissues chiefly collagen, together with some water and minerals. The composition depends upon the fatness of animal. The fatter the animal, the more fat and less water, protein and materials in the fatty tissues. Fatty tissues vary in composition from 76-93% pure fat, 5-20% water, 2-5% protein and 0.1-0.2% minerals. Vitamins content is highly negligible in fatty tissues.

2.4 POST MORTEM CHANGES IN MEAT

       In the living animal, one of the important reactions in the muscle resulting to generation of energy required for the muscle concentration in glycolysis (anaerobic breakdown of glycogen).

Glycogen           enzymes         glucose +O₂  enzymes energy + H₂ 0 + CO₂

         When the animal is killed, it can no longer make use of oxygen and hence this oxygen becomes unavailable to the tissues. This result in the breakdown of glycogen present to lactic acid which has a substantial affect on the drop in pH of the muscle.

         In living animals, this lactic acid is used along another biochemistry pathway, thereby maintaining the pH of the muscle above 6.0 but after the killing of the animal, this alternative pathway can no longer be utilized and so a pH drop to about 5.4 or lower result. This acidity gives the fresh meat some level of protection against microbial spoilage (Jay 2011).

      Postmortem glycolysis usually results in many other changes apart from production of lactic acid. The most important of these being the denotation of protein at the iso-electric point (pH 5.3-5.5). The meat texture is also affected. The production and accumulation of lactic acid in the muscle of dead animal also causes a contraction of muscle fibers and increased toughness. This result in a high decrease in extensibility giving rise to the phenomenon referred to as rigor mortis. At the completion of the rigor mortis, the muscle will recover its extensibility and become money tenderized ( Ozlem 2005).

           Meat from an animal of low glycogen in the muscle will have poor tenderness quality and will be sticky and flabby. The meat colour will be impaired. An animal that been greatly exercised before slaughter will have low glycogen content. Glycogen in the muscle can be depleted through walking, running or fighting and it may take some time for a reasonable amount of glycogen to be restored. Therefore, an animal should be killed after prolong good feeding, when it’s muscle should contain a high concentration of glycogen in order that its meat may have good organoleptic qualities and keep well storage (Ismail et al  1995).

2.5 MEAT COLOUR

         Meat colour is the single greatest appearance factor that determines whether or not a meat cut will be purchased (Kraft, 2008). The colour of muscle tissue is determined by the concentration of oxygen and the oxidation state of the muscle pigment myoglobin.

         The display life of meat of limited by the time required for oxidation of oxymyoglobin to metmyoglobin, initially on the surface layers of muscle tissues, and reaches proportions of total pigment concentrations that the meat appears dull and eventually brown(Gill, 2007). During normal distribution of meat products, the primal and sub-primals are marketed to the retailers vacuum packaged and the retailer fabricate these into smaller retail cuts and display them in over-wrapped packages.

         The myoglobin normally will be in deoxy-form under vacuum and is converted to oxy-form during fabrication and display. The oxymyoglobin is gradually oxidized to form metmyyoglobin and the kinetics of the process is dictated by several factors such as the muscle type, rate of postmortem pH decline, packaging film, Oxygen consumption, display lighting and temperature, and the intrinsic metmyoglobin reducing activity of the muscle. Exposure of pork and pork products to nitrogen will be delayed by incorporation of 20 to 30% carbon dioxide in the gas mixtures (Gill, 2007).

          Incorporation of carbon monoxide in the gas mixture can provide a stable, cherry red colour to the meat by formation of carboxymyoglobin, which is more resistant to oxidation compared to the oxymyoglobin (Silliker 1992.) As discussed earlier, combination of carbon monoxide with other gases can provide the advantages of colour stability in addition to microbial control (Currier et al 2001).

2.6 PRESERVATION OF MEAT

Meat preservation, whether done at home or in the industries are by asepsis, use of heat,  use of low temperature, drying, use of ozone, use of irradiation, use of antibiotics, smoking and addition of preservatives. Some are discussed below.

2.6.1 ASEPSIS

           Is the act of keeping micro-organisms away from meats as much as practicable during slaughtering and handling. It permit easier preservation by any method. Storage time under chilling conditions may be lengthened, aging for tenderizing becomes less of a risk, curing  and smoking methods are more certain and heat processes are more successful (Turtura 2009).

2.6.2 USE OF HEAT

         The rate of heat penetration on beef range  from fairly in meat soup to very slow in tightly packaged meats or in pastes. Chemical added to meats, such as   spices, salt and nitrites in curing processes, also affect the heat processing usually making it more effective.

        Heat may be applied to meat in other ways than canning, treatment of meat surfaces with hot water to lengthen the keeping time has suggested, although this may lessen nutrients and damage colour. The proper cooking of meats for direct consumption greatly reduces the microbial content and hence lengthens the keeping time (Adak et al 2005).

2.6.3 USE OF LOW TEMPERATURE

       More meat is preserved by the use of low temperature than by any other method, and much more by chilling than by freezing.

       Chilling is a modern packaging house involving chilling meat promptly and rapidly to temperature near freezing and chilling storage at only slightly above the freezing point with the temperature range from -1.4 to 2.2⁰C, the bacteria that gives trouble here are Acinetobacter, Moraxella, Alcaligenes, Micrococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Flavobacterium and Protoeus and even yeasts and molds can grow at  low temperature.

      Freezing is a popular technique used for preservation of meat because it is easy and yield good result with the meat subjected to a -12.2 to -28.9^OC, this method kills about half of the microorganism. But in Nigeria, the use of refrigerator is not an affordable way of preservation of meat by most citizens because of unavailable electricity power supply (Polster et al 2004).

2.6.4 DRYING

        This method is done naturally in the sun, oven and in dehydrators. In Nigeria, the northern part uses this method in preserving their food especially meat. Drying of meat is appealing because the finished products can be stored in tightly closed plastic containers at room temperature.

2.6.5 SMOKING

         Is the process of flavouring, cooking  or preserving meat by exposing it to the smoke from burning or smoldering plant materials, most often wood. Meat is the most common smoked food.

2.6.6 USE OF OZONE

         This is an active oxidizing agent that gives an oxidized or tallow flavour to meat. The level of ozone cited will inhibit microorganism, but much higher concentration are needed to stop growth of that microorganism that has already begun.

2.6.7 IRRADIATION OF MEAT

         The use of ultraviolet ray has been employed chiefly on large, hung piece of meat in plant storage room to lengthen the keeping time. The ray serve at reduce numbers of microorganism in the air and to inhibit or kill them on surface of the meat reached directly by the rays.

2.7 PULSED ELECTRIC FIELDS OF MEAT

       Processing meat may include compounds that alter the water activity or pH of foods, thereby limiting   growth of many organisms.

      Antimicrobial compounds may be added to meat or packaging to inhibit growth of many spoilage organisms.

1.      Organic acids can help control bacteria, molds and yeasts in meat.

2.      Bacteriocins, including nisin, can help control spoilage bacteria in meat.

3.      Chitosan incorporated into meat or used as a coating for fruits and vegetables inhibits growth of some spoilage bacteria and yeasts.

4.Many herbs, essential oils and spices have demonstrated some inhibitory activity against spoilage microbes in a variety of foods. Thyme, oregano, vanillin and cinnamon are the most commonly mentioned substances in recent papers.

2.8 EFFECTS OF SPOILAGE OF MEAT

        Some spoiled foods are harmless to eat and may simply be diminished in quality. But foods exhibiting certain types of spoilage maybe harmful to consume. Uncooked or undercooked animal flesh that spoils is typically quite toxic, and consumption can result in serious illness or death. The toxic effects from consuming spoiled food are known colloquially as “food poisoning” and more properly as “food borne”. As living organisms go, bacteria lead fairly simply lives. They don’t walk or crawl, so the only time they go anywhere is when someone moves them. Otherwise they pretty much stay put, content to spend their time eating and making more of themselves.

       Unfortunately, what they are eating is our food- especially foods that are high in protein, like meats, poultry, fish, eggs and dairy products. To be sure, some of them will go for low protein foods like fruits and vegetables, but those ones are a lot slower. Which is why an onion or a peach left on your kitchen counter for a couple of days would still be safe to eat, while a steak clearly would not. It is important to note that spoiled food is not necessarily dangerous food. For one thing, most people won’t eat food that smells bad, or looks slimy. And one can not get food poisoning from something you did not eat (Jay 2011).

     Moreover, the microorganisms that cause ordinary  food spoilage are not necessarily harmful to us. In fact, centuries before refrigerators, the earliest sauces and seasonings were used to mask the off tastes and smells of food that had begun to spoil. This continues to be true in parts of the world where people don’t have home refrigeration units. The bacteria we are concerned with from a food safety standpoint are so called pathogens that cause food poisoning. And these pathogens like Salmonella or Escherichia coli, don’t produce any smells, off-tastes or changes in the food appearance e.g a slimy surface or some sort of discoloration.

2.9 MEAT NUTRITIONAL INFORMATION

	Typical meat	Calories	Nutritional content
			Protein	CHO	Fat
1	Fish	110-140	20-25g	Og	1-5g
2.	Chicken breast	160	28g	Og	7g
3.	Lamb	250	30g	Og	14g
4.	Steak (beef top round)	210	36g	Og	7g
5.	Steak (beef T-bone)	450	25g	Og	35g

    All muscle tissue is very high in protein, containing all of the essential amino acids and in most cases is a good source of zinc, vitamin B₁₂, selenium, phosphorus, Niacin, vitamin B6, chlorine, riboflavin and iron. Several forms of meat are high in vitamin K₂ which is only otherwise known to be found in fermented foods, with natto having the highest concentration (Ayres 1999).

    Muscle tissue is very low in carbohydrates and does not contain dietary fiber. The fat content of meat can vary widely depending on the species and breed of animal, the way in which the animal was raised, including what it was fed with, the anatomical part of the body and the methods of butchering and cooking. Wild animals such as deer are typically learner than farm animals, leading those concerned about fats content to choose game such as venison. Decades of breeding meat animals for fatness are being reversed by consumer demand for meat with less fat. Red meat, such as beef, pork and lamb, contains many essential nutrients necessary for healthy growth and development in children. Nutrients in red meat include iron, zinc, vitamin B₁₂ and protein.

The table in this section compares the nutritional content of several types of meat. While each kind of meat has about the same content of protein and carbohydrates, there is a very wide range of fat content. It is the additional fat that contributes most of the calorie content of meat and to concern about dietary health (Clarence et al 2004).

2.10 CONTROL OF SPOILAGE MICROORGANISMS

 Spoilage organisms are not originally an integral part of food but are widely present in water, soil, air and other animals. Healthy living plants and animals can ward off bacteria and fungi, but as soon as they are slaughtered or harvested, their defenses deteriorate and their tissues become susceptible to spoilage microbes.

 Good manufacturing practices with strict attention to sanitation and hygiene can prevent colonization by many but not all microbes and are the most important first step in delaying the spoilage processes.

Microbes requires certain conditions for growth and therefore management of the environment of foods can change these factors and delay spoilage.

1. Many, but not all, microbes  grow slowly or not at all, at low temperatures and refrigeration can prolong the lag phase and decrease growth rate of microbes.
2. Many microbes require a high water activity and therefore keeping foods such as grains and cereal products  dry, will help to preserve them.
3. Some microbes requires oxygen, others are killed by oxygen and still others are facultative.

  Managing the atmosphere during storage in packaging can retard or prevent the growth of some microbes. Several types of modified atmosphere packaging have been developed to retain growth of pathogenic and spoilage organisms (Eze et al 2012).

  However, microbes are endlessly innovative and eventually seem to circumvent the barriers we set up against them. Therefore further   strategies and multiple hurdles are utilized to extend shelf life. These procedures must be assessed for compatibility with different foods so that there are no significant organoleptic changes in the foods caused by the treatment or preservative.

CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1 MATERIALS

Nutrient Agar, Distilled Water, Measuring Cylinder, Beaker, Conical Flask, Stirrer, pH Meter, Cotton Wool, Aluminum Foil, Bunsen Burner, Weighing Balance, Microscope, Autoclave, and Sterile Petri Dishes, Acetone Alcohol, Crystal Violet, Lugos Iodine, Safranine, Plasma, Hydrogen Peroxide, Eosin Methylene Blue Agar, Potato Dextrose Agar, MacConkey Agar.

3.2 PREPARATION OF AGAR PLATES

3.2.1 Nutrient Agar

• Weigh 28g of nutrient agar using a weighing balance and pour in a beaker

• Using a measuring cylinder, measure 1000ml of distilled water, and mix with the nutrient agar in the beaker.

• Warm slightly and Stir with a spatula for ten minutes to dissolve completely

• Using a pH meter, check the pH and adjust appropriately

• Transfer the mixture to a conical flask.

• Cover the flask with cotton wool wrapped in aluminum foil.

• Sterilize by autoclaving at 121°C for 15 minutes;

 •Allow to cool to 45°C then aseptically pour into sterile Petri dishes.

3.2.2 Eosin Methylene Blue Agar (Modified) Levine

• Weigh 37.5g of Eosin Methylene Blue Agar and dissolve in 1 litre (l000ml) of distilled water.

• Bring to the boil to dissolve completely while you stir using a glass stirrer.

• Sterilize by autoclaving at 121°C for 15 minutes.

• Cool to 60°C and shake the medium in order to oxidize the methylene blue (i.e. restore its blue colour) and to suspend the precipitate which is an essential part of the medium.

• Aseptically pour into sterile Petri dishes.

3.2.3 MacConkey Agar

• Weigh 50g of MacConkey agar using a weighing balance and pour in a beaker

• Using a measuring cylinder, measure 1000ml of distilled water, and mix with the MacConkey agar in the beaker.

• Warm slightly and Stir with a spatula for ten minutes to dissolve completely

• Using a pH meter, check the pH and adjust appropriately

• Transfer the mixture to a conical flask.

• Cover the flask with cotton wool wrapped in aluminum foil.

• Sterilize by autoclaving at 12 1°C for 15 minutes;

• Allow to cool at   45°C then aseptically pour into sterile Petri dishes.

3.2.4 Potato Dextrose Agar

• Weigh 39g of Potato dextrose agar using a weighing balance and pour in a beaker

• Using a measuring cylinder, measure 1000ml of distilled water, and mix with the Potato dextrose agar in the beaker.

• Warm slightly and Stir with a spatula for ten minutes to dissolve completely

• Using a pH meter, check the pH and adjust appropriately

• Transfer the mixture to a conical flask.

• Cover the flask with cotton wool wrapped in aluminum foil.

• Sterilize by autoclaving at 12 1°C for 15 minutes;

• Allow to coo at 45°C then aseptically pour into sterile Petri dishes. (kanika, 2009).

3.3 COLLECTION OF SAMPLES

In this study, four butchers who had a supply of fresh beef at the time of visit (between the hours of 7:00am and 8:00am) to Amorji market were randomly sampled. Freshly cut beefsteaks, all arising from the same meat piece (flank abdominal wall, fore and hind limb areas) were consistently sampled. Eight samples (one from each butcher weighing 1.0 kg) were aseptically collected in sterile polythene pouches, sealed and transported in ice packs to the laboratory for microbiological analysis within one hour of collection. This exercise was repeated weekly for two weeks. A total of sixteen (16) fresh meat (beef) samples, were thus obtained from selected slaughterhouses / beef vendors in Amorji market.

1.0g of beef was minced aseptically and dissolved in 10ml of distilled water after which serial dilution was carried out.

A series of sterile test tubes are arranged on a test tube rack with 9mls of distilled water each. 1ml of the mixture of 1.0g minced meat dissolved in l0mls of distilled water is aseptically taken and transferred to the first tube, mix thoroughly, take lml from this first tube and transfer to the next; the series continues until you get to the last tube.

3.4 INOCULATION! ISOLATION OF CULTURES.

The first and third dilutions of each sample from the butchers were cultured. Total bacterial count was determined using the pour plate method and individual colonies were streaked on prepared sterile solid agar to isolate pure cultures (nutrient agar and potato dextrose agar). Streaking is by sterilizing a wire loop using the Bunsen burner until it is red hot; the loop is allowed to cool close to the flame and then used to take a loop-full of the inoculum; this is then used to make non-overlapping streaks on the solid agar. The inoculated plates were labeled appropriately and incubated at 37°C for 24hrs except the potato dextrose agar that was incubated at 25°C (room temperature) for 3-5days. The potato dextrose agar was treated with chloramphenicol to suppress bacteria growth while the nutrient agar was treated with Gruiseofulvin to suppress the growth of fungi. Growths were observed after which colonies were sub-cultured on fresh agar plates. The pure cultures were identified using different methods which include; Gram staining, sub culturing on selective/differential mediums, catalase test, coagulase test, indole test, and methyl red test. Cultural characteristics of the growth cultures were observed and recorded, while the morphological characteristics were also observed under the oil immersion objective of the microscope. Growth cultures of fungi were observed on the potato dextrose agar plates and were stained using methylene blue stain and observed under the microscope using the 10x and 40x objectives which were then compared with standards and identified.

3.4.1 GRAM STAINING:

• Make a smear of the growth organism on a clean grease-free slide using a sterile wire loop.

•      Flood the smear with crystal violet solution for 30- 60 seconds.

•      Wash with distilled water and blot.

•      Flood with iodine solution for 30- 60 seconds.

•      Wash with distilled water and blot.

•      Flood with. alcohol solution to decolourizes and wash immediately.

•      Wash with distilled water and blot.

•      Flood with safranin solution for 2 minutes.

•      Wash with distilled water and blot.

•      Place the slide on a slide rack to dry.

•      The dried gram stained slides were then viewed under the oil immersion objective of the microscope.

3.4.2 CATALASE TEST

·         Divide a clean grease-free slide into 2 sections using a grease pencil, label one part as “test’’ and the other as “control’’. Place a drop of normal saline on each area/part.

·         With a sterilized cooled inoculating wire loop, pick a small amount of culture from the nutrient agar slant or petri plate.

·         Emulsify one or two colony on each drop of saline to make a smooth suspension.

·         With a Pasteur pipette, place one drop of hydrogen peroxide over the test smear.(Do not place any drop of hydrogen peroxide on the smear that acts as control).

·         Observe the fluid over the smear (hydrogen peroxide over the test smear) for the appearance of gas bubbles.

·         When there is gas bubbles,it means it is catalase positive.

·         When there is no gas bubbles, it means it is catalase negative.

3.4.3 COAGULASE TEST(SLIDE METHOD)

•      Place three separate drops of saline on a clean slide.

•      Suspend a loopful of test colony in two of these, and a loopful of control organism (Staphylococcus aureus) in the third.

•      With a sterile loop, add a drop of plasma to one test and the control suspension.

•      Occurrence of clumping within 10 seconds indicates a positive result.

•      The saline control should remain evenly suspended.

•      This test detects the presence of Staphylococcus aureus in a growth culture.

CHAPTER FOUR

4.0 DISCUSSION OF RESULT

The study on the examination of spoilage associated with meat and meat products by microbes was conducted mainly to determine the state of hygiene (microbial load) and also to observe contamination organisms and ways through which we can reduce the incidences of cross contamination of beef in the market arena.

The major findings have been summarized and illustrated in Tables 1, 2 and 3. Meat samples were collected weekly for eight weeks and a total of six (6) pure culture species were isolated from the mixed population and identified using various methods as explained in chapter three.

The high microbial count enumerated from fresh beef samples indicated that the meat samples were contaminated. Microorganisms can easily be introduced either in the pre or post processing stages of meat processing. The high coliform count observed from meat is assumed to be an indicator of faecal contamination. It is likely that the observed increase of faecel bacteria is due to problem associated with removal of the fleece and its coming into contact with the surface of carcass (Ozlem, 2005) and (Chaubey et al. 2004) enumerated the coliform in the majority of the meat samples and suggested that raw meat and meat products should be handled under strict hygienic condition and stored in cool places to avoid contamination and safe guard the health of consumers. The high microbial load could be from the fleece of cattle to the carcass surfaces during hide removal. The main sources of microorganisms are the exterior of the animal which includes the hide, hooves and hair and the numbers and many kinds of microorganisms from the soil, wash water, feed and manure, as well as its natural surface flora and the intestinal contents contain the intestinal organisms. Knives, cloths, air environment of the abattoir, slaughter-slabs, hands and clothing of the workers and the physical facilities can serve as intermediate sources of contaminants, It has also been shown that during handling, contamination comes from carts, boxes or containers used in transporting the meat from where they are slaughtered to where they are sold. These resulted in the increase in the microbial load of the fresh beef samples. Retail cut could also result in greater microbial load because of the large exposed surface area, unavailability of potable water, nutrient and greater oxygen penetration. Hence smaller retail cuts displayed are conducive for microbial growth and proliferation which leads to spoilage of the meat. (Eze, and Nwosu, 2012). The fresh meat (beef) sold to the public in open markets is grossly contaminated with coliform bacteria as well as other bacteria and fungi. This work has revealed that the fresh beef sold in Amorji market is contaminated by both Gram positive and Gram negative bacteria.

The bacteria isolated were Pseudornonas species, Staphylococcus aureus, and, Escherichia coli. The organisms isolated are in line with the work of (Turtura 2009). They reported that Gram negative bacteria account for approximately 69% of the cases of bacterial food-borne diseases. The presence of these organisms in the meat is indicative of public health hazard and gives a signal of the possible occurrence of food borne intoxication and infection. This also implies that these meats are viable source of various diseases. Some of these diseases could spread and acquire epidemic status which poses serious health hazards(Adak et al 2005).

Staphylococcus aureus, which is a normal flora of the body, indicates contamination from handlers. The organism can pass onto food during harvesting, processing or even storage. It is the major cause of food poisoning known as staphylococcal food poisoning.(Clarence et al 2009). The poisoning is caused by the ingestion of an enterotoxin produced, which is characterized by diarrhea and Escherichia coli is an enteric organism and its presence is an indication of faecal contamination of the samples. This may be attributed to improper sanitary condition during processing of the meat from the water supply, unsterilized utensils and contamination by flies. The presence of other organisms is as a result of improper handling by butchers, when the efficiency of conventional methods to which meat is transported to markets as well as from the environment since some are ubiquitous in nature. (Pseudomonas species) (Eze, and Nwosu, 2012).

The determination of the level of fungal contaminants in the samples analyzed revealed a high microbial load in the fresh meat. This may not be unconnected with the fact that during slaughtering and selling of meat, it is opened to contamination.

Table 1

Total viable bacterial count (CFU/0.5m)

Sample	Count
A B C D E F G	10x10-1 15 x 10-3 7 x 10-3 12 x 10-3 19 x 10-3 27 x 10-3 21 x 10-3

Table 3

Showing the fungi isolated

Isolates	Colour when viewed	Suspected organism
1	Green	Aspergillus
2	White	Rhizopus
3	Gray	Yeast

CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 CONCLUSIONS

This project work revealed that the bacteria load present in the analyzed meat sample will not show a negative effect on the structure, appearance and odour of the meat; rather it has effect on the nutritional value and the health of individuals consuming these products.

The analyzed samples revealed the presence of Staphylococcus aureus, Pseudornonas species, Escherichia coli, and molds. These organisms are responsible for most acute food poisoning, infections and intoxication. This may be attributed to improper sanitary condition during processing of the meat from the water supplier, unsterilized utensils, and contamination by flies and poor handling.

5.2 RECOMMENDATIONS

Based on the results of this research, the following are therefore recommended to improve the standard of living/health of people who consume meat sold in Amorji market.

Local government health workers should routinely inspect meats that are sold in the market to ensure that necessary sanitary measures are observed.

Meat vendors should be educated on the importance of hygiene and treating sick persons before coming back to sell meat.

Consumers should also be educated on the danger of consuming contaminated meat, emphasizing the proper methods of preparing meat to halt possible incidence of food borne diseases of infections.

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