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ABSTRACT

The present study was carried out to determine the bacteriological quality of pasteurized liquid milk samples sold at different shops in students market located at Imo state University front gate. A total of four samples were collected. The samples were examined to determine the total heterophilic bacterial count and total coliform count. Results revealed that two of the samples (1 and 2) yielded no growth on both Nutrient agar and MacConkey agar hence, were tagged satisfactory. Sample 2 and 3 yielded growth that ranges from 7.0X104 – 9.0 X104, 1.3 X105 – 1.3 X105 respectively. Further identification test carried out showed the presence of Escherichia coli and Enterobacter spp. in sample 3 and 4 hence they were tagged unsatisfactory. The presence of coliform bacteria shows the possibility of pathogenic bacteria and could cause disease.

 CHAPTER ONE

1.0 INTRODUCTION/LITERATURE REVIEW

1.1 INTRODUCTION

Milk is an important food of diet of vast population on earth, due to its high nutritional value for human beings. Milk is an excellent growth medium of microorganism when suitable temperature exists. If it is produced unhygenically and handled carelessly, it gets contaminated very easily leading to its early spoilage (Oliver et al., 2005). Many milk-borne epidemics of human diseases have been spread by contamination of milk by spoiled hands of dairy workers, unsanitary utensils, flies and polluted water supplies. The same thing can be said for improper handling of foods in the home, restaurants, hospitals and other institutions.

The quality of milk is determined by aspects of composition and hygiene. Due to its complex biochemical composition and high water activity milk serves as an excellent culture medium for the growth and multiplication of many kinds of microorganisms.

The contamination of milk and milk products is largely due to human factor and unhygienic conditions. Usually milk gets contaminated with different kinds of microorganisms at milk collecting places. Milk is a major part of human food and plays a prominent role in the diet. Approximately 50% of the milk produced is consumed as fresh or boiled, one sixth as yoghurt or curd and remaining is utilized for manufacturing of indigenous varieties of milk products such as Ice cream, Butter, Khoa, Paneer, Rabri, Kheer, Burfi and Gulabjaman. The manufacture of these products is based on traditional method without any regard to the quality of raw material used and/or the hygienic quality of the products. Under such conditions many microorganisms can find access to the milk products.

Coliforms are considered as normal flora of intestinal tract of human and animals. They have been used as indicator organisms for bacteriological quality of milk and its products (Chatterjee et al., 2006).

Coliform count is always being taken as a definite index of fecal contamination of milk and its products, that besides the possible presence of enteric pathogens which may constitute health hazards to the consumers. The most important index of microbiological quality is total bacterial count, coliforms, yeast and moulds count and detection of specific pathogens and their toxins (Szita et al., 2008).

Among all micro-organisms Escherichia coli is frequently contaminating organism in food and is reliable indicator of fecal contamination and generally present due to insanitary conditions of water, food, milk and other dairy products (Jayarao and Henning, 2001). Recovery of E. coli from food is an indicative of possible presence of enteropathogenic or toxigenic micro-organism which could constitute a public health hazard. Enteropathogenic E. coli (EEC) can cause severe diarrhea and vomiting in infants and young children. Coliforms particularly Escherichia coli are frequently used in the microbiological analysis of food as an indicator of poor hygienic condition.

Microbiological examination of milk is essential to find the degree of contamination and enumeration of indicator organisms. The coliform bacteria are able to grow well in a variety of substrates and to utilize a number of carbohydrates and some other organic compounds as food for energy and a number of fairly simple nitrogenous compounds as a source of nitrogen. The coliform group of bacteria is defined as the indicator (faecal coliform) of suitability of milk for drinking (Wells et al., 1991). The present study has been designed to assess the milk quality of different milch animals with special reference to coliforms.

1.2 AIMS AND OBJECTIVES

The aims of this study is to determine the microbial quality of liquid milk sold in Owerri metropolis in Imo State..

 Objective

The objectives of this study is as follows;

  • Determination of the microbial load on the milk sample
  • To isolate organisms associated with liquid milk contamination
  • Identification of organism associated with liquid milk.

1.3 LITERATURE REVIEW

1.3.1 History

Humans first learned to regularly consume the milk of other mammals following the domestication of animals during the Neolithic Revolution or the development of agriculture. This development occurred independently in several places around the world from as early as 9000–7000 BC in Southwest Asia to 3500–3000 BC in the Americas (Bellwood and Peter, 2005). The most important dairy animals—cattle, sheep and goats—were first domesticated in Southwest Asia, although domestic cattle had been independently derived from wild aurochs populations several times since. Initially animals were kept for meat, and archaeologist Andrew Sherratt has suggested that dairying, along with the exploitation of domestic animals for hair and labor, began much later in a separate secondary products revolution in the fourth millennium BC (Sherratt and Andrew, 1981). Sherratt’s model is not supported by recent findings, based on the analysis of lipid residue in prehistoric pottery, that shows that dairying was practiced in the early phases of agriculture in Southwest Asia, by at least the seventh millennium BC (Vigue and Helmel, 2007).

From Southwest Asia domestic dairy animals spread to Europe (beginning around 7000 BC but not reaching Britain and Scandinavia until after 4000 BC), and South Asia (7000–5500 BC). The first farmers in central Europe and Britain milked their animals. Pastoral and pastoral nomadic economies, which rely predominantly or exclusively on domestic animals and their products rather than crop farming, were developed as European farmers moved into the Pontic-Caspian steppe in the fourth millennium BC, and subsequently spread across much of the Eurasian steppe. Sheep and goats were introduced to Africa from Southwest Asia, but African cattle may have been independently domesticated around 7000–6000 BC. Camels, domesticated in central Arabia in the fourth millennium BC, have also been used as dairy animals in North Africa and the Arabian Peninsula (Peters, J., 1997). The earliest Egyptian records of burn treatments describe burn dressings using milk from mothers of male babies. In the rest of the world (i.e., East and Southeast Asia, the Americas and Australia) milk and dairy products were historically not a large part of the diet, either because they remained populated by hunter-gatherers who did not keep animals or the local agricultural economies did not include domesticated dairy species. Milk consumption became common in these regions comparatively recently, as a consequence of European colonialism and political domination over much of the world in the last 500 years.

In the Middle Ages, milk was called the “virtuous white liquor” because alcoholic beverages were more safe to consume than water (Valenze, D.M., 2011).

1.3.2 Sources of milk

The females of all mammal species can by definition produce milk, but cow’s milk dominates commercial production. In 2011, FAO estimates 85% of all milk worldwide was produced from cows.

Human milk is not produced or distributed industrially or commercially; however, human milk banks collect donated human breast milk and redistribute it to infants who may benefit from human milk for various reasons (premature neonates, babies with allergies, metabolic diseases, etc.) but who cannot breastfeed.

In the Western world, cow’s milk is produced on an industrial scale and is by far the most commonly consumed form of milk. Commercial dairy farming using automated milking equipment produces the vast majority of milk in developed countries. Dairy cattle such as the Holstein have been bred selectively for increased milk production. About 90% of the dairy cows in the United States and 85% in Great Britain are Holsteins (McGee and Harold, 2004). Other dairy cows in the United States include Ayrshire, Brown Swiss, Guernsey, Jersey and Milking Shorthorn (Dairy Shorthorn).

Sources aside from cows

Aside from cattle, many kinds of livestock provide milk used by humans for dairy products. These animals include buffalo, goat, sheep, camel, donkey, horse, reindeer and yak. The first four respectively produced about 11%, 2%, 1.4% and 0.2% of all milk worldwide in 2011.

According to the US National Bison Association, American bison (also called American buffalo) are not milked commercially; however, various sources report cows resulting from cross-breeding bison and domestic cattle are good milk producers, and have been used both during the European settlement of North America and during the development of commercial Beefalo in the 1970s and 1980s (O’Connor and George).

1.3.3 Physical and chemical properties of milk

Milk is an emulsion or colloid of butterfat globules within a water-based fluid that contains dissolved carbohydrates and protein aggregates with minerals. Because it is produced as a food source for the young, all of its contents provide benefits for growth. The principal requirements are energy (lipids, lactose, and protein), biosynthesis of non-essential amino acids supplied by proteins (essential amino acids and amino groups), essential fatty acids, vitamins and inorganic elements, and water (Fox PF., 1995).

pH

The pH of milk ranges from 6.4 to 6.8 and it changes over time. Milk from other bovines and non-bovine mammals varies in composition, but has a similar pH (Fox PF., 1995).

Lipids

Initially milk fat is secreted in the form of a fat globule surrounded by a membrane. Each fat globule is composed almost entirely of triacylglycerols and is surrounded by a membrane consisting of complex lipids such as phospholipids, along with proteins. These act as emulsifiers which keep the individual globules from coalescing and protect the contents of these globules from various enzymes in the fluid portion of the milk. Although 97–98% of lipids are triacylglycrols, small amounts of di- and monoacylglycerols, free cholesterol and cholesterol esters, free fatty acids, and phospholipids are also present. Unlike protein and carbohydrates, fat composition in milk varies widely in the composition due to genetic, lactational, and nutritional factor difference between different species (Fox PF., 1995).

Like composition, fat globules vary in size from less than 0.2 to about 15 micrometers in diameter between different species. Diameter may also vary between animals within a species and at different times within a milking of a single animal. In unhomogenized cow’s milk, the fat globules have an average diameter of two to four micrometers and with homogenization, average around 0.4 micrometers. The fat-soluble vitamins A, D, E, and K along with essential fatty acids such as linoleic and linolenic acid are found within the milk fat portion of the milk (McGee and Harold, 2004).

Proteins

Normal bovine milk contains 30–35 grams of protein per liter of which about 80% is arranged in casein micelles.

Caseins

The largest structures in the fluid portion of the milk are “casein micelles”: aggregates of several thousand protein molecules with superficial resemblance to a surfactant micelle, bonded with the help of nanometer-scale particles of calcium phosphate. Each casein micelle is roughly spherical and about a tenth of a micrometer across. There are four different types of casein proteins: αs1-, αs2-, β-, and κ-caseins. Collectively, they make up around 76–86% of the protein in milk, by weight. Most of the casein proteins are bound into the micelles. There are several competing theories regarding the precise structure of the micelles, but they share one important feature: the outermost layer consists of strands of one type of protein, k-casein, reaching out from the body of the micelle into the surrounding fluid. These kappa-casein molecules all have a negative electrical charge and therefore repel each other, keeping the micelles separated under normal conditions and in a stable colloidal suspension in the water-based surrounding fluid (McGee and Harold, 2004).

Milk contains dozens of other types of proteins beside the caseins including enzymes. These other proteins are more water-soluble than the caseins and do not form larger structures. Because the proteins remain suspended in the whey left behind when the caseins coagulate into curds, they are collectively known as whey proteins. Whey proteins make up approximately 20% of the protein in milk, by weight. Lactoglobulin is the most common whey protein by a large margin (McGee and Harold, 2004).

Salts, minerals, and vitamins

Minerals or milk salts, are traditional names for a variety of cations and anions within bovine milk. Calcium, phosphate, magnesium, sodium, potassium, citrate, and chlorine are all included as minerals and they typically occur at concentration of 5–40 mM. The milk salts strongly interact with casein, most notably calcium phosphate. It is present in excess and often, much greater excess of solubility of solid calcium phosphate (Fox PF., 1995). In addition to calcium, milk is a good source of many other vitamins. Vitamins A, B6, B12, C, D, K, E, thiamine, niacin, biotin, riboflavin, folates, and pantothenic acid are all present in milk.

For many years the most accepted theory of the structure of a micelle was that it was composed of spherical casein aggregates, called submicelles, that were held together by calcium phosphate linkages. However, there are two recent models of the casein micelle that refute the distinct micellular structures within the micelle.

The first theory attributed to de Kruif and Holt, proposes that nanoclusters of calcium phosphate and the phosphopeptide fraction of beta-casein are the centerpiece to micellular structure. Specifically in this view, unstructured proteins organize around the calcium phosphate giving rise to their structure and thus no specific structure is formed.

The second theory proposed by Horne, the growth of calcium phosphate nanoclusters begins the process of micelle formation but is limited by binding phosphopeptide loop regions of the caseins. Once bound, protein-protein interactions are formed and polymerization occurs, in which K-casein is used as an end cap, to form micelles with trapped calcium phosphate nanoclusters.

Some sources indicate that the trapped calcium phosphate is in the form of Ca9(PO4)6; whereas, others say it is similar to the structure of the mineral brushite CaHPO4 -2H2O.

Carbohydrates and miscellaneous contents

Milk contains several different carbohydrate including lactose, glucose, galactose, and other oligosaccharides. The lactose gives milk its sweet taste and contributes approximately 40% of whole cow’s milk’s calories. Lactose is a disaccharide composite of two simple sugars, glucose and galactose. Bovine milk averages 4.8% anhydrous lactose, which amounts to about 50% of the total solids of skimmed milk. Levels of lactose are dependent upon the type of milk as other carbohydrates can be present at higher concentrations that lactose in milks (Fox PF., 1995).

Other components found in raw cow’s milk are living white blood cells, mammary gland cells, various bacteria, and a large number of active enzymes (McGee and Harold, 2004).

1.3.4 Appearance

Both the fat globules and the smaller casein micelles, which are just large enough to deflect light, contribute to the opaque white color of milk. The fat globules contain some yellow-orange carotene, enough in some breeds (such as Guernsey and Jersey cattle) to impart a golden or “creamy” hue to a glass of milk. The riboflavin in the whey portion of milk has a greenish color, which sometimes can be discerned in skimmed milk or whey products (McGee and Harold, 2004). Fat-free skimmed milk has only the casein micelles to scatter light, and they tend to scatter shorter-wavelength blue light more than they do red, giving skimmed milk a bluish tint (Goff and Douglas, 2010).

1.3.5 Processing of milk

In most Western countries, centralized dairy facilities process milk and products obtained from milk, such as cream, butter, and cheese. In the US, these dairies usually are local companies, while in the Southern Hemisphere facilities may be run by very large nationwide or trans-national corporations such as Fonterra.

Pasteurization

Pasteurization is used to kill harmful microorganisms by heating the milk for a short time and then immediately cooling it. The standard high temperature short time (HTST) process produces a 99.999% reduction in the number of bacteria in milk, rendering it safe to drink for up to three weeks if continually refrigerated. Dairies print expiration dates on each container, after which stores remove any unsold milk from their shelves.

A side effect of the heating of pasteurization is that some vitamin and mineral content is lost. Soluble calcium and phosphorus decrease by 5%, thiamin and vitamin B12 by 10%, and vitamin C by 20% (Wilson, G.S., 1943). Because losses are small in comparison to the large amount of the two B-vitamins present, milk continues to provide significant amounts of thiamin and vitamin B12. The loss of vitamin C is not nutritionally significant, as milk is not an important dietary source of vitamin C.

A newer process, ultrapasteurization or ultra-high temperature treatment (UHT), heats the milk to a higher temperature for a shorter amount of time. This extends its shelf life and allows the milk to be stored unrefrigerated because of the longer lasting sterilization effect.

Microfiltration

Microfiltration is a process that partially replaces pasteurization and produces milk with fewer microorganisms and longer shelf life without a change in the taste of the milk. In this process, cream is separated from the whey and is pasteurized in the usual way, but the whey is forced through ceramic microfilters that trap 99.9% of microorganisms in the milk (as compared to 99.999% killing of microorganisms in standard HTST pasteurization). The whey then is recombined with the pasteurized cream to reconstitute the original milk composition.

Creaming and homogenization

Upon standing for 12 to 24 hours, fresh milk has a tendency to separate into a high-fat cream layer on top of a larger, low-fat milk layer. The cream often is sold as a separate product with its own uses. Today the separation of the cream from the milk usually is accomplished rapidly in centrifugal cream separators. The fat globules rise to the top of a container of milk because fat is less dense than water. The smaller the globules, the more other molecular-level forces prevent this from happening. In fact, the cream rises in cow’s milk much more quickly than a simple model would predict: rather than isolated globules, the fat in the milk tends to form into clusters containing about a million globules, held together by a number of minor whey proteins (McGee and Harold, 2004). These clusters rise faster than individual globules can. The fat globules in milk from goats, sheep, and water buffalo do not form clusters as readily and are smaller to begin with, resulting in a slower separation of cream from these milks.

Milk often is homogenized, a treatment that prevents a cream layer from separating out of the milk. The milk is pumped at high pressures through very narrow tubes, breaking up the fat globules through turbulence and cavitation. A greater number of smaller particles possess more total surface area than a smaller number of larger ones, and the original fat globule membranes cannot completely cover them. Casein micelles are attracted to the newly exposed fat surfaces. Nearly one-third of the micelles in the milk end up participating in this new membrane structure. The casein weighs down the globules and interferes with the clustering that accelerated separation. The exposed fat globules are vulnerable to certain enzymes present in milk, which could break down the fats and produce rancid flavors. To prevent this, the enzymes are inactivated by pasteurizing the milk immediately before or during homogenization.

Homogenized milk tastes blander but feels creamier in the mouth than unhomogenized. It is whiter and more resistant to developing off flavors.[19] Creamline (or cream-top) milk is unhomogenized. It may or may not have been pasteurized. Milk that has undergone high-pressure homogenization, sometimes labeled as “ultra-homogenized,” has a longer shelf life than milk that has undergone ordinary homogenization at lower pressures (McGee and Harold, 2004).

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