Abstract
Somatic cells are an important component naturally present in milk, and somatic cell count is used as an indicator of udder health and milk quality. The role of somatic cells in dairy processes and products is ill-defined in most studies because the role of these cells combines also the concomitance of physicochemical modifications of milk, bacterial count, and the udder inflammation in the presence of high somatic cell count. The aim of this review is to focus on the role of somatic cells themselves and of endogenous enzymes from somatic cells in milk, in dairy transformation processes, and in characteristics of final products overcoming biases due to other factors. The immune function of somatic cells in the udder defense and their protective role in milk will be primarily considered. Different characteristics of milk induced by various somatic cell counts, types, and their endogenous enzymes influencing directly the technological properties of milk and the final quality of dairy products will be discussed as well. By comparing methods used in other studies and eliminating biases due to other factors not considered in these studies, a new approach has been suggested to evaluate the effective role of somatic cells on dairy processes and products. In addition, this new approach allows the characterization of somatic cells and their endogenous enzymes and, in future research, will allow the clarification of mechanisms involved in the release of these components from somatic cells during dairy processes, particularly in cheese technologies.

Keywords: Milk, Somatic cells, Enzymes, Cathepsin D, Cheese

Introduction
Milk is known to be a high-value nutritional biological fluid composed of water, proteins, fat, sugars, minerals, etc. Other important components existing naturally in raw milk are somatic cells (SCs), and the predominant cell type, besides shed epithelial cells, in most species is leucocytes, including macrophages, polymorphonuclear neutrophils cells (PMNs), and lymphocytes (Boutinaud and Jammes 2002).

The amount of SCs, usually called somatic cell count (SCC), in milk is used as an important indicator of udder health since SCs are involved in protecting the mammary gland from infection as part of the innate immune system. SCC in milk is influenced by many factors, such as animal species, milk production level, lactation stage, and also the individual and environmental factors as well as management practices (Rupp et al. 2000). Though SCC is subjected to variation, it is still used as an indicator of milk quality in several species, especially in ruminant and human (Hunt et al. 2013; Sharma et al. 2011). Taking cow milk as an example, when SCC >2 × 105 cells.mL−1, the udder is considered to be infected, and when SCC >4 × 105 cells.mL−1, the milk is deemed unfit for human consumption in the European Union (EU). The legal SCC threshold for milk acceptance in dairy industries varies in different countries, e.g., the values for bovine milk in Germany, Canada, and the USA are 1 × 105, 5 × 105, and 7.5 × 105 cells.mL−1, respectively (Olechnowicz and Jaskowski 2012; Schwarz et al. 2011). For caprine and ovine milk, the cutoff value is 1 × 106 in the USA but is not yet defined in the EU (Council Directive 92/46/EEC 1992).

Whether SC is “a friend or a foe” in the dairy field remains a question (Souza et al. 2012). Generally, SCs until now have been considered as negative. High SCC is associated with udder inflammation, which leads to bacteriological problems in milk, an alteration of milk composition, and finally, the major modifications of dairy product characteristics compared to the normal values (Le Maréchal et al. 2011; Lindmark-Mansson et al. 2006; Raynal-Ljutovac et al. 2007; Sharma et al. 2011). However, besides their immune function in the udder and protective functions in milk, SCs have recently been shown to influence, in a positive way, the composition and technological properties of dairy products, thus participating in the final quality of dairy products through their endogenous enzymes (Sanchez-Macias et al. 2013; Souza et al. 2012).

The role of SCs is generally assessed as a global effect, although the influence of the other factors has not been considered separately, and then, includes intrinsic characteristics of milk modified by the inflammation of the mammary gland, consequences on milk biosynthesis and secretion, and bacterial count (Albenzio et al. 2004; O’Farrell et al. 2002; Raynal-Ljutovac et al. 2007). Few research studies until now have given a clear view of the role of SCs without the combination of the other causative factors. This review focuses on the state of our knowledge of SCs in the dairy field and on their effective role in dairy processes and products.

Somatic cells and udder defense mechanism
The immune function of somatic cells in the udder
SCs are known to be one of the major defense components of the mammary gland against disease or intramammary infections (Paape et al. 1979, 2002; Sharma et al. 2011). The four main cell types composing SC, namely, macrophages, PMNs, lymphocytes, and epithelial cells, are briefly presented in Table 1.

Macrophages are generally the predominant cell type in healthy cow milk. They can fight against bacterial invasion quickly by engulfing action. In the case of infection, macrophages release chemical messengers or chemoattractants that are detected by PMNs and direct PMNs in turn towards the infection site. Both macrophages and PMNs can ingest microbial cells by phagocytosis and have an essential role in the innate immune system. Moreover, macrophages participate in the specific immunity as do lymphocytes (Burvenich et al. 2003).

PMNs can be recruited and increase milk SCC when the infection continues. They can be present to a large extent in mastitic milk, even up to 92 % in bovine milk (Paape et al. 1979). When PMNs arrive at the site of infection, they phagocyte microorganisms and kill them by using a combination of oxidative and non-oxidative mechanisms (Pham 2006).

Lymphocytes have a determinant role in the specific immune system. They are the only cells able to recognize the antigens through specific membrane receptors for invading pathogens (Sordillo et al. 1997).

Mammary epithelial cells are the cells that produce milk. They are shed from the mammary epithelium during lactation (Boutinaud and Jammes 2002). Prior to 1980, they were confused with macrophages because of their similar morphological traits and finally discovered in milk by electron microscopy (Lee et al. 1980). The epithelial cells are the first defense line of the mammary glands, and they may participate in the immunity of neonates in different species (Boutinaud and Jammes 2002). This type of cell is often detected below 15 % (Table 1) as also confirmed in other studies (Ben Chedly et al. 2011; Boutinaud et al. 2008; Rankl 2004). The mammary epithelial feature of these cells has been further determined in cows and goats using transcriptomic and proteomic analyses, demonstrating that their content in mRNA and proteins, such as cytokeratin and enzymes involved in milk synthesis, is specific to mammary epithelial cells (Ben Chedly et al. 2011; Boutinaud et al. 2008; Janjanam et al. 2013).

The protective function of somatic cells in milk
Besides the immune defense role in the udder, SCs can continue their protective function in milk. Additionally, some components identified as being from SCs are present in milk and also help to enhance the host defense. For example, PMNs have bactericidal and respiratory burst activities and they can eliminate the invading bacteria by releasing reactive oxygen species (ROS) and granular enzymes (Paape et al. 2002). Some antibacterial proteins identified in bovine milk also arise from SC such as macrophage scavenger receptor types I and II, PMN peptidoglycan recognition protein and lymphocyte cytosolic protein 1 and cathelicidins. They can continue to exert their protective properties when they are in skim milk, whey, or milk fat globule membranes (Hettinga et al. 2011; Smolenski et al. 2006). Recently, information was obtained on the protein changes of the bovine and goat innate immune system following exposure to lipopolysaccharide (LPS) from the cell walls of gram-negative Escherichia coli, leading to an increase in SCC without having the contribution of bacteria themselves (Hinz et al. 2012; Olumee-Shabon et al. 2013). In both ruminant species, an increase in the antimicrobial proteins was observed and more precisely of cathelicidins, which are involved in the degranulation of PMNs. In caprine milk, acute-phase proteins, haptoglobin, and serum amyloid A were shown in milk collected after 18 h after infusion with LPS.

The enzymes initially from SCs can be released or secreted in milk and are therefore considered as endogenous SC enzymes and play an important protective role in milk. The role of the lysozyme, one SC endogenous enzyme, is well known for the ability to destroy bacteria (Paape et al. 2003). Some proteinases from PMNs, such as cathepsin G, elastase, and proteinase 3, have antimicrobial activities during phagocytosis of invading microorganisms. They could also contribute, after release, to the extracellular killing of microorganisms by cleaving their bacterial virulence factors as shown in mice (Pham 2006). Catalase, an endogenous enzyme from PMNs, is one of the main antioxidant enzymes in milk and is suspected of being responsible for changed redox potential of milk that limited the survival capability of microorganisms (Hamed et al. 2008).

Even if the release mechanism of these protective components from SCs is not fully understood yet, the release of these SC endogenous proteins including enzymes has already been mentioned in the last decade, as described in Section 3.1. The contribution of these SC endogenous enzymes to dairy transformation processing and final characteristics of products has been considered previously by several authors, and this issue will be discussed in Section 4.1.

Identification of somatic cell types
Several methods have been developed to differentiate SCs in milk, and among them, cytology methods are mainly used to visually identify the main cell types by optical microscopy (Baumert et al. 2009; Lindmark-Mansson et al. 2006; Sarikaya et al. 2004). Enzyme-linked immunosorbent assay (ELISA) was also applied for detecting and quantifying SCs, for example, by using the specific antibodies of PMNs. O’Sullivan et al. (1992) suggested to use a direct capture ELISA to diagnose bovine mastitis. In order to separate each type of SCs, immunomagnetic separation has been used for labeling the cell subpopulation. This method was successfully applied to isolate epithelial cells from bovine milk (Boutinaud et al. 2008), macrophages, and PMNs from sheep milk (Albenzio et al. 2009).

Recent advances of SC studies by flow cytometry in medicine diagnostic field give opportunities to study SCs in dairy field at the subpopulation level (Albenzio and Caroprese 2011; Leitner et al. 2000a; Reuter et al. 2014). The combination of immunofluorescence and flow cytometry allows the differentiation of cell types using specific antibodies (Kelly et al. 2000; Park et al. 1991; Riollet et al. 2001). Flow cytometry-cell sorting technologies distinguish and separate differential cells with more precise detection (Dosogne et al. 2003; Piepers et al. 2009). Compared with classical microscopy observation, flow cytometry allows counting SCs and identifying SC types with fewer samples and less time, thus allowing characterizing SCs and determining the roles of SCs in the milk.

Various somatic cell count and composition
The SC composition (SC types and their percentages in total SCC) in milk varies depending on many factors: animal species, breed, stage of lactation, genetics, parity, day-to-day variation, diurnal variation, milking interval, time of sampling, sampling procedures, stress and trauma, management factors, and seasonal and storage procedures (IDF 466/2013). The macrophage and PMN percentages showed an opposite trend during different stages of lactation in ewes, the highest macrophage level observed in early lactation and the highest PMN level in late lactation (Albenzio et al. 2009). In healthy bovine and ovine milk, macrophages are generally the predominant cells, while in both healthy and unhealthy goat milks, PMNs are predominant (Dulin et al. 1983; Ostensson 1993). Nevertheless, there are several contradictory examples concerning the predominant cell type in the case of healthy bovine milk, macrophages, lymphocytes, or epithelial cells becoming the main cell type according to the authors (Table 1).

Both SC count and composition are related to milk quality, but their relationship is not necessarily associated, except in the case of high SCC corresponding to a high amount of PMNs in bovine milk. SCC gives only the total amount of cells present in milk, but not the distribution of each cell type, making it difficult to have a relevant view of cell composition. Hence, milk with various SCCs and various SC compositions has a particular fingerprint that can lead to different final characteristics of dairy products, and this will be discussed in Section 4.

Endogenous enzymes from somatic cells
Besides protective functions in milk, SCs also provide numerous enzymes mentioned as SC endogenous enzymes in this review. These SCs as well as their endogenous enzymes have different profiles in terms of type, specificity, and activity and can be released into milk and further influence dairy processes and the quality of products. It is worthy to note that these SC endogenous enzymes are only part of all enzymes in milk, usually called milk indigenous enzymes. Actually, these indigenous enzymes in milk are well studied (Kelly et al. 2006; Kelly and Fox 2006; Kelly and McSweeney 2003), in particular the plasmin system (Ismail and Nielsen 2010). In this review, we will focus on SC endogenous enzymes.

Continued Atricle:
https://pmc.ncbi.nlm.nih.gov/articles/PMC4180028/