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Study Of Microorganisms Found In Raw Milk From Dairy Farms

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Introduction

Milk’s characteristics as a highly nutritional product containing proteins, fats, carbohydrates, vitamins,minerals and essential amino acids, with its neutral pH and high water activity make it not only an excellent food source for human consumption, but a great medium for microorganisms to grow (Frank, 1997), particularly bacterial pathogens (Chyeet al. , 2004). Computer models of lactase persistence in adults, estimate that humans have using milk from other species as a food source since 5000-6000 BC (Itan et al, 2009). Milk proteins taken from preserved ceramic vessels indicate evidence of dairy farming in areas occupied by present day Hungary and Romania 7,900–7,450 years ago(Craig et al. , 2005).

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Despite approximately 7500 years of advancement in dairy technology, it wasn’t until around 150 years ago that Louis Pasteur discovered that microorganisms were not only utilizing dairy products, but also causing spoilage of raw untreated milk (Schwatrz, 2001). His work helped determine Mycobacterium tuberculosis as a zoonotic agent in raw milk (Holsinger et al 1997).

Prior to 1977 when the first generation of DNA sequencing (the Sanger method) was developed (Heather & Chain 2015) microorganisms were studied through microbiological culturing. Carried out by matching the characteristics of the growth to know morphological, physiological and biochemical traits. (Quigley et al. , 2011).

Over 40 years advancements in DNA sequencing are resulting in faster and cheaper sequencing techniques. Creating huge reference libraries of know genome sequences, which are used with modern bioinformatic tools to improve our understanding of the taxonomy of microorganisms in raw milk and its derived products (Loman et al. , 2012).

As our we improve our technology to understand and manipulate raw milk with its subsequent products, to suit market demands, we are discovering that microorganisms are also evolving to match the new constraints and opportunities we are directly/indirectly providing for them, e. g. through genomic decay and adaptation to the milk environment (Quigley et al. 2013). Increased knowledge of microorganisms in milk, that were difficult to culture in the past, gives a better indication of the microbiome of raw milk, the interactions between spp. and the circumstances that allow for beneficial and harmful microorganisms to grow.

Milk from a healthy udder was thought to be almost sterile, with environmental contamination by microorganisms occurs during milking and milk handling, from water and milk equipment (Cousins & Bramley, 1981). There are now challenges to that theory as a result of molecular methods, our understanding of microbiota, may indicate that there may be commensal microbial communities in the mammary gland, previously undetected by culturing. This novel concept is opening questions about dysbiosis and its interactions with mastitis causing bacteria (Oikonomou, 2012).

The bacterial load in milk directly influences the quality and safety of dairy products (Arcuriet al. , 2006). Inadequate dairy products can be attributed to the poor microbial quality of the milk and heat-resistant enzymes (Marshall, 1982; Muir et al. , 1986). Despite growth in production using the modern technology, some milk producers still use non-specialized methods, resulting in raw milk of poor quality (Correa, et al. , 2009).

Globalization of dairy markets, resulting in a variety of imports, have made consumers highly demanding regarding price, quality and safety of the products offered (Nada et al. , 2012). Hungary’s accession to the EU resulted in exposure to increased competition, reducing domestic market share (Kőnig, 2006). As the trend in Hungary’s main dairy export is raw milk (Vőneki, 2014, pp. 6), in order to compete in open markets the production of high quality raw milk should be priority both for small holdings and larger dairy plants.

The objective of this study were to determine the microbiological quality of raw milk produced in dairy farms across Hungary.

Milk Characteristics As A Medium For Microbial Growth

It was thought normal physiological conditions, milk is almost sterile at the point it is secreted from the udder (Tolle,1980). Our increasing understanding of microbiota, may indicate that there may be commensal microbial communities in the mammary gland, previously undetected by non selective methods. (Oikonomou, 2012). The high level of water, neutral pH and high nutrient content, make milk an ideal environment for bacteria growth, making it highly perishable and conducive to proliferation of bacterial pathogens (Maldaneret al. , 2012; Chyeet al. , 2004). Raw milk is an representation of a medium containing a diverse and complex microbial population (Quigley et al. , 2011; Vacheyrou et al. , 2011). Some of these microorganisms proliferate by directly using the nutrients available and some require other specific microbial populations to metabolize major components and metabolites from the primary nutrients (Frank, 1997).

Chemical compounds in milk are generally similar across species, the main constituents are water, lipids, lactose sugar and proteins. As well as numerous minor constituents, mostly found at trace levels, such minerals, vitamins, hormones and enzymes. (Fox 1997). Lactose is the only sugar naturally present is milk, the enzyme lactase is required to break it down to glucose and galactose. While glucose is utilized by microorganisms for energy, most cannot synthesize it from lactose as they lack the lactase enzyme. Lactic acid bacteria (LAB) do have lactase and proliferate in milk as a result. Some LAB can also break down galactose in to glucose also, futher improving their advantage over other microrganisms. (Fox 1997). LAB produce metabolic products that are can interfering with the growth of competing microorganisms, further improving their proliferation rates (Vandenbergh 1993).

The pH of milk is near the physiological pH of 6. 8 which most microorganisms have optimal growth, meaning that milk is a advantageous growth medium with respect to acidity (pH) (Fox, 1997)

Microorganisms vary greatly in their ability to survive and/or grow at reduced water activity. Most bacteria can survive between 0. 90 – 0. 91 and most yeast: 0. 87 – 0. 94, The high water activity of milk of 0. 97- 1, makes an ideal medium. (Fox, 1997)

Milk processing exploits many of the physicochemical properties of milk, it is practiced worldwide, particularly in Europe and North America. Milk is a very versatile raw material, from which a numerous different products, including about 1000 varieties of cheese, are produced. (Fox 2003)

The microorganisms, colonizing milk are normally harmless and low numbers, (up to a few hundred per ml). However, in cases of mastitis, the milk is heavily contaminated with bacteria and may even be unfit for consumption. (Arcuriet al. , 2006)

Ways To Determine Microorganism Populations In Milk

Tests to determine microbial populations in milk had traditionallly been phenotypically through the use of broths or agars targeting specific growth of the selected microbial population supplemented with morphological, biochemical or physiological characterisation. These testing methods are still the used in industrial settings as they are low-tech and inexpensive and typically involve determining total bacteria counts, to reflect general milk quality, or to detect specific pathogens/microorganisms, which indicate the presence of contamination. However these methods are relatively labour intensive and time-consuming, and in some cases the tests distinguishing power is insufficient (Quigely et al, 2011).

In the last 40 years, has seen development of more rapid genotypic analysis, with high-throughput tests that are DNA-based,. Usually relying on polymerase chain reaction (PCR) technology. DNA analysis initially used for confirmation of traditional tests is increasing seen as an alternative to culture-based analysis to the identity of the microorganisms that are present in raw milk, and resultant dairy products (Quigely et al 2013)

A key benefit to replacing the culturing, is due to aversion of many microorganisms to isolation using common culturing methods. This can lead to a significant underestimation of the microbial communities. Though DNA analysis has many advantages, it is far from straight forward, with numerous factors requiring consideration when applying these culture-independent methods. Protocols must be selected to ensure efficient extraction of nucleic acids from as many of the microorganisms present, using strategies, such as the use of DNA-binding agents or an alternative focus on RNA. Care must be taken to avoid to false positives resulting from the amplification of DNA from dead cells and contaminants (Quigley et al. ,2011).

Using DNA sequincing to determine microorganisms, requires genes to be targeted using oligonucleotides (probes) to detect target-specific genes or non-target-specific gene amplification to provide an overview of microorganism in a niche, this requires detection of conserved gene (those key genes that have been unaltered by evolution and remain a constant). such as the 16S or 23S rRNA genes. Further analysis by techniques such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE) or single-stranded conformation polymorphism (SSCP), are used to determine similarities or differences in the populations. These approaches may be used in conjunction with Sanger (first generation) DNA sequencing, to help specifically identify the populations present. (Quigley et al. ,2011).

Since 2005, there has been a rapid evolution in next-generation DNA sequencing (NGS) technologies. Resulting in the production of millions of sequence reads from a single run, allowing a much more detailed and accurate estimation of microbial diversity at a much lower cost (Kchouk et al, 2017). NGS are incapable of reading complete Genome DNA sequences, they are limited to sequencing small DNA fragments and generating millions of reads. This is somewhat limiting as it requires high computing resources. (Kchouk et al, 2017). However the ever-increasing number and length of the sequence reads, coupled with the availability of databases and bioinformatic tools are resulting in NGS becoming increasingly accessible (Loman et al. , 2012).

Although, to date, high-throughput sequencing approaches have not been extensively applied to assess the microbiota of dairy-based environments, there have been a number of recent publications, suggesting this will change dramatically in the coming years (Masoud et al. , 2011, 2012; Alegria et al. , 2012; Quigley et al. , 2012).

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