Psychrotrophic microorganisms in raw milk and the cheese quality

Psychrotrophic microorganisms, especially Pseudomonas spp., are present in the microbiota of refrigerated milk as they can grow at refrigeration temperatures irrespective of their optimal growth temperature. Psychrotrophic counts ranging from 105 to 108 CFU/mL in refrigerated raw milk effect cheese quality, since the synthesized thermoresistant enzymes affect the nutritional value, sensory properties, and texture. Cheese is the dairy product with the highest growth rate in the food industry in Brazil in recent years and meets the current consumption trends of nutritious and practical foods. The objective of this review was to address the importance and influence of the psychrotrophic raw milk microbiota on the quality and sensory properties of cheese. The enzymes produced by psychrotrophic microorganisms lead to taste changes, undesirable clotting times, increased concentrations of free fatty acids and free amino acids, and a reduced shelf-life, in addition to negatively affecting cheese yields. Proteases from psychrotrophs are also associated with slicing problems and progressive loss of the elasticity of cheese, a bitter taste, and increased clotting times of cheese produced with pasteurized milk. On the other hand, their lipases increase the clotting time and have a negative effect on the sensory properties by providing a rancid, soap, metallic, or oxidized smell and taste. The control of the psychrotrophic population found in refrigerated raw milk contributes to better cheese production yields and desirable texture and sensory properties, which extends the shelf-life of cheese and improves consumer acceptance.

Psychrotrophic microorganisms predominantly have a lipoproteolytic metabolism at temperatures below 10ºC by producing thermoresistant extracellular enzymes that influence the shelf-life and quality of milk and its dairy products (Arslan et al., 2011). Therefore, the storage of milk in refrigeration tanks (4ºC) and the collection of raw material every 48 hours (Brasil, 2018) promote the multiplication of psychrotrophic bacteria and the synthesis of enzymes (Nornberg et al., 2009).
Gram-positive bacteria, such as Bacillus, Clostridium, Corynebacterium, Streptococcus, Lactobacillus, and Microbacterium, are also present but to a lesser extent (Mcphee & Griffiths, 2011). As these microorganisms originate from the milking environment and equipment (Fagundes et al., 2006;Molineri et al., 2012), good practices can minimize the growth of psychrotrophic agents and the action of enzymes, thus, extending the shelf-life of milk and its dairy products by maintaining the integrity of nutritional components and, consequently, improving industrial performance (Ribeiro Júnior et al., 2018).
Among psychrotrophic bacteria, Pseudomonas spp. predominate in refrigerated raw milk since they have great genetic diversity, metabolic versatility, and can efficiently adapt to cold temperatures due to the large amount of unsaturated lipids present in the cell membranes (Cleto et al., 2012;Samarzija et al., 2012;Decimo et al., 2014;Hammad et al, 2015;Neubeck et al., 2015;Oliveira et al, 2015;Al-Rodhan & Nasear, 2016;Xin et al., 2017 a). The enzymes synthesized by Pseudomonas spp. in raw milk have 100% hydrolytic activity and can maintain this activity after pasteurization and sterilization, respectively (Sorhaug & Stepaniak, 1997;Bagliniere et al., 2013). The Pseudomonas spp. most isolated from milk, the milking environment, and industrial plants are P. fluorescens, P. putida, P. fragi, and P. aeruginosa (Wiedmann et al., 2000;Dogan & Boor, 2003;Decimo et al., 2014;Scatamburlo et al., 2015). Research, Society and Development, v. 9, n. 9, e127997217, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7217 8 Brazilian legislation does not establish a standard of identity and quality for the enumeration of psychrotrophic microorganisms (Brasil, 2018). However, the synthesis of enzymes that cause changes in milk and its dairy products is associated with a high cell density with psychrotrophic bacterial populations ranging from 10 5 to 10 7 CFU/mL (Mahieu, 1991;Muir, 1996;Walstra et al., 2006). It is important to note that differences in extracellular enzymatic activities of different strains are associated with the genetic characteristics of each group (Haryani et al., 2003). In addition to good milking practices, genetic studies that evaluate the strains of Pseudomonas spp. present in the milking environment and the origin of these isolates should be considered as tools for better quality and shelf-life of dairy products (Ercolini et al., 2009). Researchers detected an increase in proteolysis after the storage of milk at 2ºC, 4ºC, and 8ºC, even with low counts of psychrotrophic bacteria (Haryani et al., 2003;Wiking et al., 2002). According to Ribeiro et al. (2018), reducing the temperature and storage times of milk with a high initial count of Pseudomonas spp. does not affect this population of psychrotrophs over time.
The proteases from psychrotrophs are associated with sensory defects and technological problems during cheese production, such as changes in clotting times, reduced yields, slicing problems, and progressive loss of elasticity (Kindstedt & Fox, 1993;Samarzija et al., 2012). The first change caused by the presence of psychrotrophs is the destabilization of the milk plasmin system, and this stimulates the release of plasmin and plasminogen from the casein micelle, which are lost in the serum. Subsequently, this affects the development of cheese flavor and texture since plasmin is an important enzyme in these processes (Samarzija et al., 2012).
The clotting time is reduced in cheese produced from raw milk but the use of pasteurized milk increases the time of this manufacturing step. Clotting time is reduced due to higher concentrations of free amino acids resulting from enzymatic action, which stimulates the growth of starter cultures (Champagne et al., 1994;Samarzija et al., 2012). The clotting time can increase due to protein changes in raw milk by the action of proteases, which makes this component more sensitive to heat treatment. For example, the formation of different complexes, such as β-lactoglobulin and casein, increase the clotting time of milk (Cousin, 1982). Problems with clotting times are also associated with the partial solubilization of ßcasein in raw milk, which subsequently tends to leave the casein micelle. Consequently, the casein diameter decreases and the hydration capacity increases, which promotes a more fragile and less compact curd (Samarzija et al. , 2012).
The cheese yield can decrease due to the action of bacterial proteases that partially degrade β-and қ-casein and, thus, promote the release of soluble components, such as polypeptides and amino acids, which are lost in the serum (Mcphee & Griffiths, 2011). A bitter taste is an important sensorial defect and occurs due to the presence of low molecular weight peptides (Fox & Mcsweeney,1998), more pronounced in aged cheeses. Researchers evaluate the influence of the milk's psychrotrophic population in raw milk on smoked provolone cheese and related that the greatest psychrotrophic population of raw milk induced a 3% reduction in cheese yield (Gasparini et al., 2020). The texture is an important characteristic regarding cheese quality and, consequently, consumer acceptance and is directly associated with the intact casein-to-moisture ratio and the pH (Lawrence, 1987). Bacterial proteases reduce cheese elasticity (rate at which the product returns to its original shape when it is compressed between teeth) and hardness (force necessary to compress a solid food between teeth (Szczesniak, 2002). According to Gasparini et al. (2020) an increase in hardness and chewiness parameters in smoked provolone cheese produced with raw milk containing psychrotrophic populations of 7 log cfu/mL presented. These technological defects Development, v. 9, n. 9, e127997217, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7217 11 are associated with a reduction in the amount of intact αs1-casein, which no longer contributes to the formation of the cheese protein matrix following hydrolysis by bacterial proteases (Ayyash & Shah, 2011).

Milk lipids and the action of bacterial lipases
On average, milk contains approximately 33 g of fat per liter and triacylglycerols account for 95% of this lipid fraction. The other lipids in milk are diacylglycerols (2%), cholesterol (up to 0.5%), phospholipids (about 1%), and free fatty acids (up to 1%) (Walstra et al., 2006;Jensen & Newburg, 1995).
Triglycerides are composed of three fatty acids covalently linked to a glycerol molecule by ester bonds. This fat is secreted by mammary gland cells, is found in the form of globules that are surrounded by a lipid bilayer that promotes stabilization, and forms an emulsion in the milk aqueous phase (Walstra et al., 2006) The membrane that maintains the milk fat globule structure also prevents the lipolysis of triglycerides (Cousin, 1982;Sorhaug & Stepaniak, 1997). Mechanical processes, such as homogenization, high-speed pumping, and agitation, can disrupt the membranes of the milk fat globules. In addition, the synergistic action of enzymes can damage this membrane through the hydrolysis of its proteins and phospholipids. When the membrane of the milk fat globule is damaged, the triglycerides are exposed and subject to the activity of hydrolases, such as lipases (Muir, 1996).
Lipolysis of milk fat globules can be defined as a process of hydrolytic breakdown of these lipids, which is catalyzed by enzymes and results in the production of free fatty acids and partial glycerides. These enzymes are usually called lipases and can be either endogenous, wherein most are associated with the casein micelle structures, or exogenous, which are secreted as a result of bacterial metabolic action (Deeth & Fitz-Gerald, 2006;Ray et al., 2013).
Milk contains a potent endogenous lipase, lipoprotein lipase, which contributes to the metabolism of bovine plasma triglycerides (Olivecrona et al., 2003) and is associated with casein. Endogenous lipases exhibit maximum activity at 37ºC and pH 8 (Ordonez-Pereda et al., 2005), are thermosensitive at pasteurization temperatures, and do not cause any damage to the fat fraction of processed and properly handled milk (Aehle, 2007). The sensory defects in raw milk are not associated with endogenous lipases since these enzymes are associated with casein molecules (Fox et al., 2004) in addition to having the fat globule membrane as a barrier that prevents their action. Development, v. 9, n. 9, e127997217, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7217