Effects of subclinical mastitis on physicochemical, nutritional, and technological properties of bovine milk: A systematic review

Introduction

The global dairy sector is a vital component of agricultural economies and human nutrition, providing high-quality proteins, bioactive peptides, essential lipids, and highly bioavailable minerals (Narváez-Semanate, 2022) [42] . The dairy industry depends on the biochemical quality and physical stability of raw bovine milk to ensure efficient processing, product consistency, and desirable sensory characteristics (Darbaz et al., 2023; Bobbo et al., 2017) [16,13] . However, milk quality is continually challenged by bovine mastitis, an inflammatory disease of the mammary gland commonly caused by bacterial pathogens (Bobbo et al., 2017) [13] . While clinical mastitis presents with visible udder inflammation and obvious milk abnormalities, subclinical mastitis (SCM) remains clinically silent (Contreras, 2007; Olechnowicz, 2014) [14,45] . Cows affected by SCM produce apparently normal milk from udders without visible lesions, yet experience reduced milk production, altered milk composition, and elevated somatic cell counts (SCC) (Samad, 2022) [60] .

Subclinical mastitis is one of the most prevalent diseases in dairy herds, occurring 15-40 times more frequently than clinical mastitis (Darbaz, 2023) [16] . Because infected animals often remain undetected for extended periods, SCM serves as a persistent source of infection within herds and contributes substantially to economic losses in the dairy sector (Samad, 2022; Ndahetuye, 2023) [60,43] . These losses arise from decreased milk yield, increased veterinary and replacement costs, higher culling rates, and the loss of milk quality incentives offered by processors (Martins, 2020; Antre et al., 2019) [39,2] . Beyond farm-level losses, SCM significantly affects dairy processing. Milk from affected cows introduces elevated SCC, altered mineral balance, and increased concentrations of heat-stable proteolytic and lipolytic enzymes into the bulk milk supply. These changes impair industrial performance by reducing cheese yield and coagulation efficiency (Politis & Ng-Kwai-Hang, 1988; Auldist et al., 1996; Leitner et al., 2004) [52,4,37] , delaying yogurt fermentation and increasing syneresis (Ma et al., 2000; Barbano et al., 2006) [38,7] , and decreasing the heat stability of milk intended for powder manufacture (Kelly et al., 2006; Santos et al., 2003) [30,61] . Although numerous studies have investigated the effects of SCM on milk composition and processing characteristics, inconsistencies remain. Some reports describe significant reductions in milk fat and protein concentrations, whereas others report little change or apparent increases due to concentration effects associated with reduced milk volume (Le Maréchal et al., 2011; Bobbo et al., 2017) [34,13] . In addition, the pathogen-specific effects of SCM on milk proteins, peptides, and cheese-making performance require further clarification (Leitner et al., 2011; Cremonesi et al., 2015) [36,15] . Therefore, this systematic review synthesizes current peerreviewed evidence to provide a comprehensive evaluation of the effects of bovine subclinical mastitis on the physicochemical, nutritional, and technological properties of milk, while highlighting key research gaps and future priorities for the dairy industry.

Search Strategy and Review Protocol

This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines and the PRISMA-S search reporting framework to ensure transparency, reproducibility, and methodological rigor (Page et al., 2021; Rethlefsen et al., 2021) [50,57] .

Search Strategy

A comprehensive literature search was performed using five major databases: PubMed, Scopus, Web of Science, CAB Abstracts, and AGRICOLA. Additional searches were conducted through ScienceDirect, SpringerLink, Wiley Online Library, Taylor & Francis Online, MDPI, and Google Scholar. Literature published from January 2000 to the present was considered, with particular emphasis on highquality peer-reviewed studies published after 2015. Databasespecific search strategies were developed using controlled vocabulary and relevant keywords combined with Boolean operators (Higgins et al., 2023) [25] .

Search string

("Subclinical mastitis" OR "SCM" OR "intramammary infection" OR "udder health") AND ("milk composition" OR "milk quality" OR "physicochemical properties" OR "nutritional composition" OR "technological properties" OR "cheese yield" OR "coagulation properties" OR "rennet coagulation time" OR "curd firmness" OR "heat stability" OR "yogurt fermentation" OR "somatic cell count" OR "SCC") AND ("bovine" OR "cow" OR "dairy cattle")

Eligibility Criteria

Predefined eligibility criteria were established to ensure the inclusion of scientifically robust studies (Higgins et al., 2023; Page et al., 2021) [25,50] .

Inclusion Criteria

Studies were included if they: [58,27] . • Reported quantitative data on physicochemical, nutritional, or technological properties of milk.

Exclusion Criteria

Studies were excluded if they:

• Focused exclusively on clinical mastitis.

• Investigated non-bovine dairy species.

• Were review articles, editorials, conference abstracts, book chapters, patents, or case reports without original data. • Contained duplicate datasets or lacked adequate scientific validation (Higgins et al., 2023) [25] .

Study Selection Process

After duplicate removal, titles and abstracts were independently screened by two reviewers. Potentially relevant articles underwent full-text evaluation against the predefined eligibility criteria. Any disagreements were resolved through discussion and consensus (Page et al., 2021; Higgins et al., 2023) [50,25] .

Data Extraction

Data were extracted using a standardized template that recorded author, publication year, geographic region, breed, diagnostic methods, SCC thresholds, sample size, and quantitative outcomes related to physicochemical, nutritional, and technological milk properties (Higgins et al., 2023) [25] .

Quality Assessment

Methodological quality and risk of bias were assessed using criteria adapted from the Cochrane Handbook for Systematic Reviews and the SANRA guidelines (Higgins et al., 2023; Baethge et al., 2019) [25,5] . Studies were evaluated based on sample size adequacy, accuracy of SCM diagnosis, use of microbiological confirmation, clarity of analytical procedures, and appropriateness of statistical analyses, including adjustment for potential confounding factors such as parity, stage of lactation, and herd effects.

PRISMA Flow Diagram

Effects of Subclinical Mastitis on Physicochemical Properties of Milk

The inflammatory processes associated with subclinical mastitis (SCM) induce measurable alterations in the physicochemical properties of bovine milk. These changes primarily result from disruption of tight junctions between mammary epithelial cells, allowing the non-selective transfer of extracellular fluid components and altering milk ionic composition (Leitner et al., 2004; Silanikove et al., 2014) [37,65] .

pH and Titratable Acidity

Healthy bovine milk is slightly acidic, with a normal pH ranging from 6.59 to 6.68. This acidity is maintained by natural buffering components, including caseins, dihydrogen phosphate (H₂PO₄⁻), and citric acid (Fox et al., 2015; Walstra et al., 2006) [20,70] . Numerous studies have reported a significant increase in milk pH during SCM, often reaching values between 6.69 and 7.07. This increase results from impaired secretion of acidifying constituents due to mammary epithelial damage and the influx of blood-derived sodium bicarbonate (NaHCO₃) and chloride (Cl⁻) ions into the alveolar lumen (Kitchen, 1981; Leitner et al., 2004; Sharma et al., 2011) [31,37,64] . Conversely, titratable acidity decreases significantly during SCM. Normal bovine milk typically exhibits a titratable acidity of approximately 0.14% lactic acid, which may decline to about 0.105% in milk from affected quarters. This reduction reflects losses of caseins and phosphate ions that contribute to the natural buffering capacity of milk (Batavani et al., 2007) [9] .

Density and Specific Gravity

The specific gravity and density of normal bovine milk generally range from 1.029 to 1.034 g/cm³ and are largely determined by solids-not-fat (SNF) and fat content (Walstra et al., 2006; Fox et al., 2015) [70,20] . During SCM, specific gravity decreases significantly, often approaching 1.024 g/cm³. This reduction is mainly attributed to decreased lactose and casein synthesis, resulting in lower SNF content and reduced milk density (Batavani et al., 2007; Bansal et al., 2005) [9,6] .

Electrical Conductivity

Electrical conductivity (EC) reflects the concentration and mobility of free ions in milk. In healthy milk, EC typically ranges from 4.0 to 5.5 mS/cm at 20°C because most calcium and magnesium remain bound within casein micelles, leaving relatively low concentrations of free conductive ions in the serum phase (Norberg et al., 2004; Walstra et al., 2006) [44,70] . Subclinical mastitis causes a marked increase in EC, often exceeding 6.0 mS/cm. Inflammatory damage elevates sodium (Na⁺) and chloride (Cl⁻) concentrations while reducing potassium (K⁺) levels. The increased abundance of highly mobile Na⁺ and Cl⁻ ions enhances the electrical conductivity of milk (Kitchen, 1981; Norberg et al., 2004; Pyörälä, 2003) [ 31, 44, 54] .

Because of its rapid response to mammary inflammation, EC is widely used as a real-time indicator in automated and robotic milking systems for early detection of subclinical mastitis (Norberg et al., 2004; Hovinen & Pyörälä, 2011) [44,26] .

Freezing Point

The freezing point depression (FPD) of bovine milk is a highly stable physical parameter, normally ranging from -0.520°C to -0.540°C. It is primarily regulated by lactose and dissolved minerals to maintain osmotic equilibrium with blood plasma (Fox et al., 2015; Walstra et al., 2006) [20,70] . During SCM, lactose synthesis declines markedly, reducing its osmotic contribution. To maintain osmotic balance, sodium and chloride ions move from blood into milk. Although total osmotic pressure remains relatively constant, this shift in osmolyte composition alters the freezing point, which may move closer to zero under severe inflammatory conditions (Auldist & Hubble, 1998; Leitner et al., 2004) [3,37] .

Viscosity

Milk viscosity is influenced by the concentration and size of fat globules, casein micelles, and somatic cells. Under normal conditions, bovine milk behaves largely as a Newtonian fluid; however, SCM can alter its rheological characteristics (Walstra et al., 2006) [70] . The elevated somatic cell count associated with SCM introduces increased quantities of cellular proteins and highmolecular-weight DNA into milk, particularly following leukocyte lysis. Together with protein aggregation, this cellular debris can modify milk flow properties and increase viscosity (Silanikove et al., 2014; Leitner et al., 2004) [65,37] . This phenomenon forms the basis of the California Mastitis Test (CMT), in which detergent-induced lysis of somatic cells releases DNA that produces a characteristic gel. The degree of gel formation is directly proportional to leukocyte concentration and serves as a practical indicator of subclinical inflammation (Schalm & Noorlander, 1957; Sharma et al., 2011) [62,64] .

Milk Yield

Subclinical mastitis is consistently associated with reduced milk yield despite the absence of visible clinical signs. Production losses result from damage to secretory alveolar tissue caused by invading pathogens and their toxins, which reduces mammary secretory capacity. Additionally, the inflammatory response increases the demand for energy and glucose, thereby competing with lactose synthesis, the principal osmotic regulator of milk volume (Seegers et al., 2003; Halasa et al., 2007) [63,22] . The extent of milk loss depends on the causative pathogen and duration of infection. [21] demonstrated that chronic SCM substantially reduced both milk production and total solids yield at the cow level. These findings highlight the considerable economic impact of persistent intramammary infections on dairy herd productivity (Gonçalves et al., 2020) [21] .

Fat, Lipid, and Oxidative Alterations

The effects of subclinical mastitis (SCM) on milk fat concentration are inconsistent. Several studies have reported reductions in milk fat percentage due to impaired fatty acid synthesis by mammary epithelial cells and decreased uptake of blood-derived triglycerides (Auldist et al., 1995; Bobbo et al., 2017). In contrast, other studies have observed little change or slight increases in fat percentage, largely as a concentration effect resulting from reduced milk volume rather than increased fat synthesis (Safak et al., 2023; Leitner et al., 2004) [59,37] . Beyond changes in fat concentration, SCM substantially alters lipid composition and stability. Inflammatory processes stimulate the release of lipolytic enzymes, particularly lipoprotein lipases derived from somatic cells and pathogenic bacteria, which hydrolyze triglycerides into free fatty acids (FFAs) and diacylglycerols. This enhanced lipolysis increases susceptibility to rancidity, off-flavors, and deterioration during storage (Ma et [38,30,34] . Subclinical mastitis is also associated with increased oxidative stress within the mammary gland. Activated polymorphonuclear neutrophils generate reactive oxygen species (ROS) as part of the immune response against invading pathogens. During persistent intramammary infections, ROS production may exceed the antioxidant capacity of mammary tissues, resulting in oxidative damage (Sordillo & Aitken, 2009; Turk et al., 2017) [66,69] .

Oxidative stress promotes lipid peroxidation of milk fat globule membranes, accelerates fatty acid deterioration, and contributes to rancid flavors and reduced shelf life of dairy products. In addition, oxidative modifications of milk proteins, particularly caseins, can impair protein functionality, stability, and processing characteristics, further reducing milk quality (Bernabucci et [10,66,64] . Evidence also suggests that improving antioxidant status through dietary supplementation, including antioxidant nutrients and rumen-protected methionine, may help alleviate oxidative stress, support mammary tissue integrity, and improve lactational performance in dairy cows exposed to inflammatory challenges (Osorio et al., 2013; Zhou et al., 2016) [48,72] .

Total Protein, Casein, and Whey Fractions

Total crude protein concentration often remains unchanged or may increase slightly during SCM; however, this apparent stability masks substantial deterioration in protein quality and functionality (Auldist et al., 1995; Bobbo et al., 2017). Milk proteins are broadly classified into caseins and whey proteins. Caseins (αs1-, αs2-, β-, and κ-casein) are synthesized within the mammary gland and play a critical role in dairy processing, whereas whey proteins include α-lactalbumin, βlactoglobulin, immunoglobulins, and bovine serum albumin (Fox et al., 2015) [20] . Concentrations of bovine serum albumin and immunoglobulins increase markedly because of enhanced vascular permeability and leakage of serum proteins into mammary secretions (Leitner et al., 2004; Pyörälä, 2003) [37,54] . In contrast, α-lactalbumin and β-lactoglobulin concentrations decline, reflecting reduced synthetic activity of damaged mammary epithelial cells (Leitner et al., 2004; Auldist et al., 1995).

Lactose

Lactose is the principal carbohydrate of milk and the primary osmotic regulator of milk secretion. Under normal conditions, its concentration remains relatively stable because of continuous synthesis within the Golgi apparatus of mammary epithelial cells (Fox et al., 2015; Walstra et al., 2006) [20,70] . A decline in lactose concentration is one of the most consistent compositional changes associated with SCM. This reduction results from impaired lactose synthesis, damage to mammary epithelial cells, leakage of lactose into the bloodstream through disrupted tight junctions, and bacterial utilization of lactose within the infected mammary gland (Kitchen, 1981; Leitner et al., 2004; Silanikove et al., 2014) [31, 37, 65] .

Total Solids and Solids-Not-Fat (SNF) Total solids (TS) and solids-not-fat (SNF) constitute the major dry matter components of milk. Because SCM reduces lactose synthesis, casein production, and overall mammary secretory activity, both TS and SNF generally decline during infection (Auldist et al., 1995; Bansal et al., 2005; Bobbo et al., 2017). These reductions diminish the nutritional value of milk and adversely affect processing efficiency and product yield during the manufacture of cheese, yogurt, and milk powder (Le Maréchal et al., 2011) [34] .

Ash and Mineral Profile

The mineral composition of milk is highly sensitive to mammary gland health. Subclinical mastitis alters both macro-and micro-mineral concentrations through changes in epithelial permeability and mammary secretory function (Kitchen, 1981; Leitner et al., 2004) [31,37] .

Calcium, Phosphorus, and Magnesium

In healthy milk, calcium, phosphorus, and magnesium are predominantly associated with casein micelles. Reduced casein synthesis and increased proteolytic degradation during SCM decrease the concentrations of these minerals (Auldist et al., 1995; Leitner et al., 2004; Fox et al., 2015). The resulting mineral depletion lowers the nutritional value of milk and destabilizes its colloidal structure, thereby impairing coagulation characteristics and overall processing performance (Politis & Ng-Kwai-Hang, 1988; Le Maréchal et al., 2011) [52,34] .

Sodium, Potassium, and Chloride

The concentrations of sodium, potassium, and chloride are normally maintained by active transport mechanisms within mammary epithelial cells (Kitchen, 1981) [31] . Disruption of the blood-milk barrier during SCM permits passive exchange between blood plasma and milk, leading to increased sodium and chloride concentrations and decreased potassium levels (Kitchen, 1981; Pyörälä, 2003; Leitner et al., 2004) [31,54,37] . This characteristic ionic shift contributes to the salty taste of mastitic milk and is largely responsible for the elevated electrical conductivity observed during intramammary infections (Norberg et al., 2004; Hovinen & Pyörälä, 2011) [44, 26] .

Trace Minerals and Total Ash

Trace minerals, including zinc, iron, and copper, are also influenced by mammary inflammation. During infection, host defense mechanisms alter the distribution of these micronutrients as part of nutritional immunity, thereby limiting their availability to invading pathogens (Moyes et al., 2009; Kehrli & Shuster, 1994) [41,28] . Experimental E. coli intramammary infections have demonstrated significant alterations in zinc, iron, and copper metabolism associated with the acute-phase inflammatory response (Moyes et al., 2009) [41] .

Biological Mechanisms Underlying Milk Composition Changes

The physicochemical and nutritional alterations observed during SCM are direct consequences of inflammatory processes and tissue damage within the mammary gland (Sordillo & Streicher, 2002; Pyörälä, 2003) [67,54] .

Inflammatory

Mechanisms, Blood-Milk Barrier Dysfunction, and Altered Lactose Synthesis The compositional changes observed during subclinical mastitis (SCM) originate from inflammatory processes within the mammary gland. The disease is initiated when pathogenic microorganisms enter through the teat canal and activate host immune responses through bacterial components such as lipopolysaccharides (LPS) and peptidoglycans, triggering the release of inflammatory mediators and cytokines (Rainard & Riollet, 2006; Wellnitz & Bruckmaier, 2012) [55,71] . These inflammatory signals recruit polymorphonuclear neutrophils (PMNs) from the bloodstream, resulting in a marked increase in somatic cell count (SCC). During mastitis, PMNs may account for more than 90% of somatic cells present in milk. Although essential for pathogen elimination, their activity contributes to tissue damage and impaired mammary secretory function, thereby exacerbating alterations in milk composition and quality (Paape et [49,55,67] . Inflammation also disrupts the integrity of mammary epithelial tight junctions, compromising the blood-milk barrier and increasing vascular permeability (Stelwagen et al., 1997; Wellnitz & Bruckmaier, 2012) [68,71] . As a result, sodium, chloride, serum albumin, immunoglobulins, haptoglobin, serum amyloid A, and other blood-derived constituents diffuse into milk, whereas lactose and potassium leak into the bloodstream. These exchanges are largely responsible for the physicochemical and nutritional alterations characteristic of SCM milk (Kitchen, 1981; Pyörälä, 2003; Leitner et al., 2004; Eckersall et al., 2006) [31,54,37,19] . The decline in lactose concentration, one of the most consistent changes associated with SCM, is closely linked to mammary epithelial damage and impaired secretory activity. Lactose is synthesized from glucose within mammary epithelial cells through the lactose synthetase complex, and disruption of this process, together with leakage of lactose through damaged tight junctions, contributes to reduced lactose levels and impaired milk secretion during inflammation ( [1,20,37] .

Proteolysis, Lipolysis, and Enzymatic Degradation

One of the most significant biochemical consequences of SCM is the acceleration of proteolytic activity within milk, leading to the degradation of functionally important proteins. This process is primarily mediated by activation of the endogenous plasminogen-plasmin system. Plasmin, a heatstable serine protease naturally present in milk as inactive plasminogen associated with casein micelles, becomes increasingly activated during SCM. Enhanced release of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) from somatic and damaged epithelial cells promotes the conversion of plasminogen into active plasmin, thereby increasing proteolytic activity (Politis, 1996 [51,29,35] . Active plasmin preferentially hydrolyzes β-casein and αs1casein, generating proteose-peptones and low-molecularweight peptides, whereas κ-casein is comparatively less susceptible. In parallel, lysosomal proteases such as cathepsins released from somatic cells further contribute to protein degradation. These processes destabilize casein micelles, impair coagulation properties, reduce cheese yield, and promote the formation of bitter peptides that adversely affect milk flavor and product quality ( [52,29,34] . Subclinical mastitis also enhances lipolytic activity within milk. Under normal conditions, the milk fat globule membrane (MFGM) protects triglycerides from enzymatic hydrolysis. However, inflammatory damage and microbial phospholipases compromise MFGM integrity, allowing endogenous lipoprotein lipase and bacterial lipases to access triglycerides and hydrolyze them into free fatty acids (FFAs) and glycerol (Deeth, 2006 [18,38,34] . The accumulation of FFAs accelerates rancidity, impairs sensory quality, and reduces the storage stability of milk and dairy products. Consequently, increased lipolysis is a major contributor to the reduced technological and commercial value of SCM-affected milk (Deeth, 2006; Santos et al., 2003) [18, 61] .

Two principal mechanisms contribute to reduced lactose synthesis during SCM

Host-Pathogen Competition for Glucose: Intramammary pathogens utilize glucose as a major substrate for growth and replication. Simultaneously, activated immune cells require substantial amounts of glucose to support phagocytosis, respiratory burst activity, and cytokine production. This competition reduces glucose availability for lactose synthesis by mammary epithelial cells (Wellnitz & Bruckmaier, 2012; Sordillo & Streicher, 2002) [71,67] . Biosynthetic Failure: Direct epithelial injury caused by bacterial toxins and inflammatory mediators impairs the synthetic capacity of mammary epithelial cells. Reduced expression of α-lactalbumin, the regulatory component of the lactose synthetase complex, directly limits lactose production. Because lactose is the principal osmotic regulator of milk secretion, reduced lactose synthesis lowers osmotic pressure within the alveolar lumen, decreases water influx, and ultimately reduces milk yield (Akers & Nickerson, 2011; Leitner et al., 2004; Silanikove et al., 2014) [1,37,65] .

Enzyme Leakage and Inflammatory Biomarkers

Subclinical mastitis promotes the release of intracellular enzymes from damaged mammary epithelial cells and infiltrating leukocytes into milk. Among the most widely studied indicators of mammary tissue damage are N-acetyl-β-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) (Kitchen, 1981; Pyörälä, 2003) [31,54] . NAGase is a lysosomal enzyme present in high concentrations within mammary epithelial cells and neutrophils. During SCM, cellular injury and leukocyte activation result in elevated NAGase activity in milk. Numerous studies have demonstrated strong positive associations between NAGase activity, somatic cell count, and intramammary infection status, supporting its use as a sensitive indicator of subclinical mastitis (Kitchen et al., 1978; Pyörälä, 2003; Hovinen & Pyörälä, 2011) [32,54,26] . Similarly, lactate dehydrogenase (LDH), a cytoplasmic enzyme involved in glycolysis, is released into milk following epithelial cell damage and leukocyte disruption. Elevated LDH activity has consistently been associated with increased somatic cell counts, intramammary infection, and the severity of mammary inflammation (Batavani et al., 2010; Pyörälä, 2003) [8,54] . Because the activities of both NAGase and LDH correlate strongly with tissue damage, inflammatory intensity, and somatic cell count, these enzymes are widely recognized as sensitive and objective biomarkers for the detection of subclinical mastitis and for monitoring the recovery of mammary gland function following infection (Pyörälä, 2003; Hovinen & Pyörälä, 2011) [54,26] .

Effects of Subclinical Mastitis on Technological Properties of Milk

The compositional changes associated with subclinical mastitis (SCM) directly impair the technological performance of bovine milk, creating substantial challenges for dairy processing and product manufacture (Auldist & Hubble, 1998; Le Maréchal et al., 2011) [3,34] .

Cheese-Making Properties of SCM Milk

The manufacture of high-quality cheese depends on efficient rennet-induced coagulation of casein micelles, which is influenced by casein concentration, micellar integrity, and mineral balance. Subclinical mastitis (SCM) adversely affects all of these factors through reduced casein synthesis, plasminmediated degradation of αs1-and β-caseins, and disturbances in calcium and phosphorus balance, thereby compromising cheese-making performance ( [52,51,29,36,34] . As a result, SCM milk exhibits prolonged rennet coagulation time (RCT), slower curd-firming rate (k20), and reduced curd firmness (a30/a60). Structural disruption of casein micelles, elevated somatic cell counts, and increased concentrations of blood-derived whey proteins contribute to the formation of weak and fragile curd networks that are more susceptible to breakage during processing (Politis & Ng-Kwai-Hang, 1988; Bobbo et al., 2017; Leitner et al., 2011) [52,13,36] . These coagulation defects increase losses of fat and protein into the whey, leading to reduced cheese yield, altered composition, and lower processing efficiency. High-SCC milk has been shown to decrease the recovery of milk solids in cheese curd and increase moisture content, adversely affecting cheese texture, quality, and storage stability (Politis & Ng-Kwai-Hang, 1988; Auldist et al., 1996; Bobbo et al., 2017) [52,4,13] . Several studies have demonstrated that milk from SCMaffected quarters produces weaker gels and inferior clotting properties than milk from healthy cows. Furthermore, excessive proteolysis may alter cheese ripening and flavor development, with plasmin-derived peptides contributing to bitterness, texture defects, and reduced sensory acceptability of the final product (Bobbo et [13,36,29,34] .

Yogurt Production and Fermentation Performance

The manufacture of yogurt and other fermented dairy products is also adversely affected by subclinical mastitis (Ma et al., 2000; Le Maréchal et al., 2011) [38,34] .

Fermentation Kinetics

The growth and acidification activity of yogurt starter cultures (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) may be delayed in SCM milk. This inhibition is attributed to elevated concentrations of antimicrobial proteins, including lactoferrin, immunoglobulins, lysozyme, and lactoperoxidase, which increase during mammary inflammation and can interfere with starter culture activity (Leitner et al., 2011; Le Maréchal et al., 2011) [36,34] . Delayed acidification prolongs fermentation time, reducing processing efficiency and potentially affecting product consistency (Le Maréchal et al., 2011) [34] .

Product Quality, Texture, and Syneresis

The texture of yogurt depends on the formation of a stable acid-induced protein gel network capable of retaining water. Because SCM milk contains reduced casein concentrations and altered protein composition, the resulting gel network is often weaker and less stable (Auldist & Hubble, 1998; Le Maréchal et al., 2011) [3,34] . This weakened structure increases susceptibility to syneresis (whey separation), resulting in poorer visual appearance and reduced consumer acceptability. Although some studies have reported increased apparent viscosity in yogurt produced from high-SCC milk due to elevated whey protein concentrations and cellular material, overall texture quality and sensory acceptance are generally inferior to products manufactured from healthy milk (Le Maréchal et al., 2011) [34] .

Milk Powder Production and Heat Stability

The production of high-quality milk powders requires raw milk with excellent thermal stability to withstand concentration and spray-drying processes. Under normal conditions, bovine milk possesses sufficient heat stability to tolerate severe thermal treatments without coagulation (Fox et al., 2015) [20] . During subclinical mastitis, however, milk heat stability declines significantly. Elevated pH, altered mineral balance, increased proteolysis, and higher concentrations of serumderived proteins contribute to reduced thermal resistance and increased susceptibility to heat-induced coagulation (Kelly et al., 2006; Le Maréchal et al., 2011) [30,34] . These changes promote fouling of evaporators and heat exchangers, reduce processing efficiency, and negatively affect the solubility and functional quality of milk powders (Kelly et al., 2006; Auldist & Hubble, 1998) [30,3] .

Pathogen Transmission

A variety of bacterial pathogens associated with subclinical mastitis may be shed into milk, including Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus uberis, and Klebsiella pneumoniae. Because milk from subclinically infected quarters usually appears visually normal, contaminated milk may enter bulk tank systems if appropriate monitoring programs are not implemented (Oliver et al., 2005; Ruegg, 2017) [47,58] . The presence of pathogenic microorganisms in raw milk highlights the importance of effective mastitis control, hygienic milk production practices, and pasteurization for ensuring food safety (Oliver et al., 2009) [46] .

Heat-Stable Toxins

Although pasteurization effectively destroys viable bacterial cells, it does not necessarily eliminate all bacterial toxins. Certain strains of Staphylococcus aureus produce enterotoxins that are highly resistant to heat treatment and may remain biologically active after pasteurization and other processing operations (Hennekinne et al., 2012; Le Loir et al., 2003) [24,33] . These staphylococcal enterotoxins are recognized causes of foodborne intoxication and represent a potential food safety concern when milk from infected animals is used in dairy processing (Hennekinne et al., 2012) [24] . [cite: 25, 36, 45] Research Gaps and Future Directions While the immediate physicochemical, nutritional, and technological impacts of SCM on bovine milk have been extensively documented, several important knowledge gaps remain. Addressing these limitations will be essential for developing advanced diagnostic tools, effective control strategies, and innovative processing technologies capable of mitigating SCM-associated losses throughout the dairy value chain (Ruegg, 2017; Halasa et al., 2007) [58,22] . [53] .

Geographic and Environmental Gaps

Breed-Specific and Genetic Research Gaps

Most published studies investigating SCM-associated milk quality changes have focused on Holstein-Friesian cattle because of their global predominance in commercial dairy systems. However, increasing evidence suggests that breedspecific differences exist in udder health, immune response, milk composition, and technological characteristics (Reitz et al., 2024) [56] . Breeds such as Jersey and Brown Swiss produce milk with distinct protein, fat, and mineral profiles that may respond differently to inflammatory challenges. Furthermore, relatively little information is available regarding how genetic variants of milk proteins, particularly κ-casein and βlactoglobulin polymorphisms, influence coagulation properties and technological resilience during SCM (Reitz et al., 2024) [56] . Future research should focus on identifying genomic markers associated with both mastitis resistance and preservation of milk technological quality. Such information could support breeding programs aimed at improving udder health while simultaneously enhancing processing performance and dairy product quality (Bishop & Woolliams, 2014) [11] .

High-Throughput Peptidomics and Molecular Research Gaps

The molecular mechanisms underlying SCM-induced proteolysis remain incompletely understood. Although plasmin-mediated degradation of caseins has been widely investigated, the relative contributions of cathepsins, neutrophil-derived proteases, and pathogen-specific enzymes require further clarification (Kelly & McSweeney, 2003; Le Roux et al., 2003) [29,35] . Recent advances in proteomics, peptidomics, and mass spectrometry have revealed substantial alterations in the milk peptidome during mammary inflammation. However, pathogen-specific peptide signatures and their relationships with disease progression, technological defects, and product quality remain largely unexplored (Martins et al., 2020) [39] . Identification of these molecular fingerprints could facilitate the development of rapid diagnostic platforms capable of detecting pathogen-specific milk alterations before major quality deterioration occurs, thereby improving mastitis management and raw milk quality control (Martins et al., 2020) [39] .

Technological Gaps and Innovative Processing Solutions

Current industrial management of SCM-affected milk largely relies on dilution within bulk milk systems rather than targeted technological interventions. Consequently, relatively little research has evaluated processing approaches designed specifically to mitigate the adverse effects of elevated SCC and inflammatory enzymes on dairy product quality (Le Maréchal et al., 2011) [34] . Membrane-based technologies such as microfiltration and ultrafiltration offer promising opportunities to remove somatic cells, bacteria, and enzyme-containing cellular debris from raw milk. However, their effectiveness in restoring coagulation properties, improving heat stability, and recovering cheese-making performance under commercial processing conditions remains insufficiently investigated (Leitner, 2004; Le Maréchal et al., 2011) [37,34] . Future research should evaluate whether advanced separation technologies, alone or combined with enzymatic stabilization strategies, can reduce SCM-associated processing losses while maintaining nutritional and sensory quality.

Novel Non-Antibiotic Interventions

The growing global concern regarding antimicrobial resistance has increased interest in alternative approaches for mastitis prevention and treatment. Development of nonantibiotic interventions is now considered a major priority within dairy health management and One Health frameworks (Ruegg, 2017) [58] . Emerging technologies including vaccines, bacteriophage therapy, probiotics, immunomodulators, antimicrobial peptides, and physical therapies such as Acoustic Pulse Technology (APT) have shown potential for improving udder health while reducing reliance on conventional antimicrobials (Leitner et al., 2011; Blum et al., 2021) [36,12] . Although preliminary findings are encouraging, robust largescale longitudinal studies are required to determine whether these interventions can effectively eliminate intramammary infections, restore mammary tissue function, normalize milk synthesis, and reverse the physicochemical and technological defects associated with SCM. Future research should also evaluate the long-term economic feasibility and practical adoption of these technologies under commercial farming conditions (Leitner et al., 2011; Ruegg, 2017) [36,58] .

Conclusion

Subclinical mastitis (SCM) is a major constraint to sustainable dairy production, exerting profound effects on the physicochemical, nutritional, and technological quality of bovine milk despite the absence of visible clinical signs. The evidence synthesized in this review demonstrates that SCM disrupts mammary epithelial integrity and blood-milk barrier function, resulting in altered milk composition characterized by increased somatic cell count, elevated pH and electrical conductivity, reduced lactose concentration, and disturbed ionic balance. SCM also compromises the nutritional quality of milk through reduced casein synthesis, enhanced proteolysis, altered mineral composition, increased lipolysis, and oxidative stress. These changes significantly impair the technological functionality of milk, leading to prolonged coagulation time, weaker curd formation, reduced cheese yield, impaired fermentation performance, decreased heat stability, and shortened shelf life of dairy products. Consequently, SCM adversely affects the efficiency and profitability of dairy processing operations. Beyond its impact on milk quality, SCM imposes substantial economic losses through reduced milk yield, lower product recovery, increased management costs, and loss of qualitybased incentives. Furthermore, the potential presence of zoonotic pathogens, bacterial toxins, antimicrobial residues, and antimicrobial-resistant microorganisms highlights its importance as a critical One Health issue. Overall, SCM should be recognized not only as an udder health disorder but also as a significant milk quality and dairy processing challenge. Future progress will depend on the integration of precision monitoring technologies, early diagnostic biomarkers, genetic improvement strategies, and effective non-antibiotic interventions to enhance udder health, preserve milk quality, and ensure the long-term sustainability of the dairy industry.

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