Understanding the role of pH in cheese manufacturing: general aspects of cheese quality and safety

Abstract

Cheese production has emerged as science and technology in the past few years, which was considered an artisan craft in the earlier period. However, despite intensive research work from many decades, the complex changes that occur during preparation and ripening of cheese are not apparent, affecting the quality and safety of cheese. Over time, several factors are studied and reviewed that affect cheese quality. The pH of the cheese curd matrix from manufacturing till ripening is one of the most crucial parameters that governs several aspects of cheese quality. Therefore, this paper aims to highlight the effect of pH on various processes (such as rennet coagulation, whey syneresis, salt absorption and ripening), microstructure and dynamic rheology, and microbiological changes that regulate the overall quality and safety aspects of cheeses. Understanding the role of pH on cheese quality parameters will aid to make better and more consistent cheeses that will satisfy both the consumers and cheese-makers.

Introduction

Cheese is categorized under fermented milk products and is one the most ancient dairy-based fermented food. It is acknowledged that cheese evolved accidentally about 8000 years back in a province of 'Fertile Crescent' from the Tigris and Euphrates rivers (Fox and McSweeney 2017). Yang et al. (2014) suggested cheese was made in Xinjiang, China, as early as 1980–1450 BC. However, in the present era, cheese is one of the best crowd-pleasing fermented milk products globally, all age group savors. Cheese production has evolved from farm-based to industrial-scale representing about 40% of the total milk production in the world. It is amongst the most dynamic food segment, growing at an impressive average annual rate of 2.6% from 2013 to 2019. In 2019, the world cheese production has nearly to 26 million tonnes (MT) where US (6.3 MT), Germany (3.5 MT) and France (1.9 MT) are the leading cheese producing countries with a combined 46% share of global production (Dairy Industries International 2020).

As the cheese market is growing worldwide, there is an increase in demand for consistently high-quality cheeses with desired characteristics. However, it is not easy to produce the same quality of cheese throughout the year owing to variability in one or more of the critical parameters such as microflora of the milk, rennet activity, starter and non-starter lactic acid bacteria (NSLAB) which affects the cheese composition and ripening (Fox et al. 2017a). Nevertheless, the past 100 years' extensive research work in the field of the chemistry, biochemistry, and microbiology of cheese has uplifted the production of consistently high-quality cheese. Among various parameters, pH is one of the most crucial to regulate cheeses' quality and safety aspects.

The shift in pH either by direct acidification or starter lactic acid bacteria (SLAB) governs various aspects of chemical, biochemical and microbiological processes which occur during cheese production leading to end-product quality. Bansal and Mishra (2020) reported that pH affects salt absorption and diffusion, enzymes' activity during ripening, growth of cheese microflora, and calcium distribution in micelle and serum phase, which are essential factors to decide the final product quality (Fig. 1). Indeed, cheese's pH is a defining parameter that shapes all cheeses' identity and quality. Every cheese variety has a specific pH, and a change in pH (either decrease or increase) may alter the quality of cheese. For instance, the difference in pH of well-made Cheddar cheese (pH 5.2) and poorly made Cheddar cheese (pH 5.4) is only 0.2 units. However, a difference of 1 pH unit is equivalent to a tenfold difference in the hydrogen ions concentration (H⁺) which means the difference in H⁺ between 5.2 and 5.4 is twofold.

Fig. 1.

Influence of pH on cheese manufacturing process

The influence of pH on quality and safety aspects of cheese is widely reported in many literatures (Fox et al. 2017a; Picon 2018; Ong et al. 2020; Wolfschoon-Pombo and Andlinger 2013). However, concise information on the role pH in cheese manufacturing has not been presented in literature. Therefore, this article perspective is to provide conceptual and applied information for the impact of pH on the quality and safety aspects. Under this review, the effect of pH on rennet coagulation, whey syneresis, salt absorption, enzymatic activity during ripening, microstructure, rheological properties, and microbiology has been presented in detail to understand the relationship of pH with sensory, textural, and rheological properties of cheeses.

Effect of pH on cheese quality and safety

Johnson (2017), in his extensive review on "100 years of cheese production and quality", mentioned that during the early period, the acidity of cheese was measured by the feel of the curd, which was relied on the senses of the cheese-makers. Recognizing the role of acidity on cheese quality, technologies were developed to measure the change in curd texture during coagulation. The first qualitative method to measure the acidity of cheese curd was the hot iron test, which measures the strechability of curd, and it was perceived that with fermentation, the longer the stretch, the more acid produced in the curd (Johnson 2017). During the 1920s, quantitative titratable acidity was the most common test to evaluate the progress of fermentation during coagulation and replaced the qualitative hot iron method (Johnson 2017). Brown and Price (1934) advocated the use of pH measurements in place of titratable acidity, but pH measurement during cheese making did not become common until the 1980s. Nowadays both pH and titratable acidity are used to measure the quality assurance during cheese making because it is easy to use and less expensive.

Due to the number of variants, cheeses are grouped based on type of coagulation (acid, rennet, heat/ acid coagulation) and moisture content (very soft, soft, semi-hard, hard, and extra hard) which can be further categorized into sub-groups based on the source of milk, texture and ripening agents (Gobbetti and Cagno 2022). However, no single classification categorizes all the variants of cheeses. Trmčić et al (2017) categorized cheeses manufactured from raw milk based on pH and water activity and suggested that this categorization is the most optimistic approach for food safety assessment purposes. Table 1 represents the group of cheeses (raw and pasteurized milk) on the basis of pH (Trmčić et al. 2017).

Table 1.

Grouping of cheese on the basis of pH

pH range	Type of cheeses	Characteristics
< 5.0	Feta, Gammelost cheese, Cottage, Cream, Quarg, Queso blanco	• Fresh acid or acid/heat coagulated cheeses
5.01–5.4	Asiago medium, Brick, Cheddar, Canestrato, Castelmagno, Fiore, Fossa, Idiazabal, Kefalotyri, Mahón, Manchego, Montasio, Mozzarella, Parmigiano Reggiano, Pecorino cheese, Pecorino Romano, Pecorino Sardo, Pecorino Siciliano, Provolone, Roncal, Sbrinz	• Ripened hard or extra-hard cheeses • High scalding temperature • Saturated brine and/or dry salting
5.41–5.80	Asiago old cheese, Edam, Gouda, Panela, Swiss and Emmentaler cheese	• Long ripened hard or semisoft (washed curd) cheeses • Eye formation in the cheese
5.81–6.20	Tilsit, Comte, Trappist, Havarti, Taleggio, Muster, Gorgonzola, Roquefort, Camembert, Panela	• Soft-ripened varieties • Mold-ripened or surface ripened (washed rind) cheeses • Brine salted
> 6.2	Camembert, Queso blanco, Queso fresco, Stilton, Gruyere, Limburger, Reblochon	• Blue cheese varieties • Soft or semisoft (washed rind/bloomy rind) cheeses • Low scalding temperature • Saturated brine and/or dry salting

Source: Gobbetti and Cagno 2017; Trmčić et al. 2017

Measuring pH during cheese making is crucial to ensure quality and safety aspects of cheese. Cheese pH affects the texture and flavor of curd by influencing the degree of casein hydration, enzymes activity during ripening (e.g. plasmin and chymosin), metabolic activity of SLAB and NSLAB, amino acid decarboxylase activity and bacteriocin production. In general, too low pH (excessive acidity) produces grainy texture and might results in mold growth during storage depending upon the varieties of cheese (pasty body in soft cheeses while short and brittle body in dry hard cheeses) (Johnson and Sommer 2020). In contrast, cheese with high pH values (> 5.6) may not knit very well and leads to crumbly body and curdy cheese texture which can result in poor functional, stretching and melting characteristics. This might be because of lesser solubilization of colloidal calcium in cheese curd. Moreover, high pH might also lead to food safety concern (Johnson and Sommer 2020). Figure 2 depicts the relationship between pH and the quality of cheese. In the following sections, the influence of pH on various processes (such as rennet coagulation, whey syneresis, salt absorption and enzymatic activity during ripening), microstructure, rheological properties, and cheese ecology is presented.

Fig. 2.

Relationship between pH and cheese quality

Rennet coagulation

The pH of milk has a profound impact on coagulation, and the pH dependency varies with the source of milk clotting enzymes (i.e., animal, plant and microbial origin). Harboe et al. (2010) reported that coagulation characteristics of destabilized Rhizomucor meihi (XL type) are exceedingly sensitive to change in pH in comparison to fermentation produced chymosin (FPC, Camelus) or Cryphonectria parasitica. The authors also reported that in a typical pH range of 6.4–6.6, gel strength properties of different sources of rennet increases in the order of C. parasitica < FPC (Camelus) < calf chymosin < FPC (bovine) < R. miehei (L type) < R. miehei (XL type) < bovine pepsin.

It is recommended that pre-acidification of milk by 0.1 to 0.2 pH units, either through use of LAB or by gluconic acid δ-lactone followed by pasteurization and results in superior and more uniform rennet coagulation characteristics and thus cheese quality (Fox 2011). Lowering the pH of milk has a significant effect on the rennet induced gel formation. At neutral pH, gelation needs more than 85% of κ-casein hydrolysis to occur whereas at lower pH, gelation occurs at a lower level of κ-casein hydrolysis (Dalgleish and Corredig 2012; Sandra et al. 2011). It is normally recognized that rennet coagulation time (RCT) is negatively correlated to milk's pH. Lucey (2022) reported that lowering of pH is exhibited by a decrease in RCT and an increase in gel firmness. This might be because of reduced electrostatic repulsion, solubilization of colloidal calcium phosphate (CCP), flocculation at a lesser degree of κ-casein degradation, and enhanced rennet and starter activity. Nevertheless, the authors noted the rise in gel firmness values with the decrease of pH up to 6.0–6.3 and a further decrease in pH (< 6.0) resulted in the decrease in gel strength. This could perhaps be due to excessive solubilization of CCP, a consequent decrease in number of bonds protecting the micellar integrity, an increased tendency for reorganizations of casein molecules within and between aggregated micelles and reduced enzyme activity. Choi et al. (2007) studied the effect of preacidification of milk prior to gelation. The highest storage modulus value was observed in gels made at pH 6.4 than at pH 6.7, probably due to decrease in electrostatic repulsion, whereas the CCP content only marginally decreased at pH 6.4. Further drop in pH from 6.4 to 5.4 exhibited lower storage modulus values because of the reduction in CCP crosslinking in the casein micelles. Thus, concentration of CCP associated with casein micelles had a significant role on the properties of rennet-induced gels. Recently, Radovanovic et al. (2021) noted significant decrease in RCT and setting time whilst an increase in aggregation rate and gel firmness was observed upon decreasing the pH from 6.5 to 6.3 in medium heat treated (65 °C/ 30 min) buffalo milk. The authors also concluded that rennet coagulation is not proper in high heat treated (95 °C/ 10 min) milk (if the pH is more than 6.5), however, it can be overcome by decreasing the pH to 6.3.

The impact of pH on rennet coagulation is very well studied and documented. It is usually recognized that lowering the pH of milk is accompanied by a decrease in RCT; however, reduction in RCT may also influence the quality of cheese due to its effect on gel strength, solubilization of the CCP, and interactions between particles. The quality and calcium content of pooled milk for cheese making varies for so many factors; altering the pH before rennet addition could be employed to apprehend the consistent cheese quality.

Whey syneresis

Whey drainage during cheese manufacturing influences the composition (mainly moisture content), sensory, textural, and rheological properties of cheese (Johnson and Sommer 2020). The degree and extent of whey expulsion is governed by many factors including, the composition of milk (especially the proportion of colloidal to soluble calcium and casein content), rennet coagulation, gel firmness, cutting of coagulum (small pieces promote syneresis), pH of the whey, size of curd particles, cooking temperature, speed of stirring and time, salting and pressing pressure. However, during the manufacturing of cheese, pH is one of the most critical parameters that regulate the expulsion of whey and thus the moisture content. The moisture content of cheese significantly affects the quality and safety aspects of cheese because of its effect on the hydration of casein and water activity (A_w). All cheese types have an optimum moisture content, which affects the sensory, microbiological, textural, and rheological properties of that specific type of cheese. Change in moisture content may modify the characteristics of that specific variety of cheese in negative or positive aspects.

Every cheese is characterizes with appropriate pH and increase in acidity enhances the whey syneresis. The increase in whey syneresis with lowering of pH may probably because of the increased rate of rearrangements. This is exhibited by an increase in permeability and syneresis pressure (pressure exerted by the gel on the whey phase; also called endogenous syneresis), assisting whey syneresis from cheese grains (Janhøj and Qvist 2010). However, Lucey (2022) testified that small adjustment of milk pH from 6.5 to 6.3 leads to a reduction in syneresis and is probably because of an increase in stiffness of the rennet gels and greater attractive interactions between casein particles. Further decrease in pH from 6.3 to 5.2 might increase whey syneresis which is probably due to increased lactic acid formation solubilizes the CCP and increased rate of rearrangements. Mineral content of cheese is greatly depends on the pH at whey drainage. The loss of calcium and phosphate from casein micelles determines the extent to which micelles are disrupted. This has a major effect on basic structure and texture of a cheese.

Apart from cheese composition, the insufficient expulsion of whey can also cause certain defects in cheese owing to the formation of water pockets in the cheese network. Vliet and Walstra (1994) testified that maximum water in the cheese matrix is free and is mechanically entrapped within the cheese matrix. Residual lactose may percolate into these water pockets, and when fermented, it causes bleached or light-colored areas due to localized reduction in pH (Johnson and Law 2010). Moreover, the amount of chymosin retained in the curd is also influenced by the low pH during drainage, high moisture content and lack of curd cooking. Adjusting the pH can regulate the whey syneresis and thus moisture content of cheese, mainly to produce low/ reduced sodium and low-fat cheeses. In general, higher moisture content in cheese can result in weak body and pasty texture while lower moisture content in cheese can cause hard, firm and corky body and texture (Johnson and Sommer 2020). Optimizing the moisture content through reduced syneresis could be a feasible technical approach to improve the quality of low/ reduced-fat cheeses.

Salt absorption

The effect of pH on salt absorption is vital as salt plays a crucial part in the overall quality of cheese. However, salt absorption in cheese is governed by several factors, including curd moisture, temperature, dimension and geometry, salting time, pH of curd, brine concentration and concentration gradient, temperature and pH of brine solution (Giroux et al. 2022). Among different factors, the pH of curd prior salting and pH of brine solution also affects the rate of salt absorption. The rate of salt absorption in cheese curd differs for dry-salted and brine-salted cheeses. The salt absorption rate decreases with an increase in pH for brine-salted cheeses, whereas, for dry-salted cheeses (such as Cheddar), salt uptake increases with an increase in pH (Fox et al. 2017c). The increase in pH of brine salted cheeses (such as Gouda and other cheeses) from 4.7 to 5.7, the level of salt uptake decreases during brining even when cheeses with similar pre-brining moisture content. Uptake of high salt at lower cheese pH coincides with a lower water loss during brining (per unit weight of salt gained), which might be due to higher content of lactic acid in low pH cheeses (Guinee and Sutherland 2022). However, Cheddar curd dry salted at high pH retained more salt than low pH cheese curd. Since at higher pH values moisture being bound more tightly by the curd and lesser whey syneresis due to high water binding capacity of casein, thus lower losses of added dry salt and greater salt absorption by the curd chips.

In practice, pH of brine solution is adjusted nearly to 5.0–5.3, which is close that of most brine-salted cheeses before brine salting. This has preservative effect and also reduces the risk of surface defects (e.g., soft rind) associated with loss of hydrogen ion (Guinee and Sutherland 2022). Excessive lowering the pH (e.g., 4.6) of brine would lead to casein precipitation, greater loss of moisture from cheese surface, which in turn would reduce salt uptake.

Enzymatic activity during ripening of cheese

The biochemical changes occurring during the ripening of cheese are the fundamental bio-reactions (glycolysis, proteolysis and lipolysis) that govern the flavor, texture, microstructure, and functional properties. The change in pH may affect the activity of enzymes and thus the degradation of proteins and lipids, which in turn influence the quality of cheese. The pH during ripening also regulates the proliferation of cheese microflora and thus influences safety aspects of cheese. During the ripening of cheese, several factors can affect the rate of biochemical changes, viz., A_w, pH, enzymes activity, salt content, temperature and time of ripening, and the presence of any inhibitors or activators.

At the beginning of ripening the pH of the hard and semi-hard cheese is around 5.3 and may increase gradually to pH 5.5 during long ripening period. Conversely, the pH of mold- and surface-ripened cheeses, for example, Camembert, Brie, Blue, Limburger, Münster and Tilsit increase to around neutral (pH 7.0–7.5) during ripening.

Glycolysis

Metabolism of residual lactose and of lactate, citrate and related events are caused by both SLAB and NSLAB. The most essential step in the manufacture of cheese curd is production of lactic acid from lactose by SLAB. Fresh cheese curd contains only 1–2% lactose because most of lactose (~ 98%) in milk is removed in the whey. The pH of curd for Cheddar-type cheese is lower than that of many cheese varieties (pH ~ 5.2) at milling, the low pH and rapid penetration of salt in the curd prevents the lactose metabolism and lactose is metabolized slowly (by residual SLAB) to L(+) lactic acid. However, SLAB are inhibited, if salt concentration is too high, and residual lactose is metabolized by NSLAB to DL-lactic acid. Recemization of L(+)-lactate to D(−)-lactate by both Lactobacilli and Pediococci (NSLAB) is pH dependent (optima: 4.5–6.0 and 4.0–5.2, respectively), which has significance with respect to crystal formation in Cheddar cheese (Murtaza et al. 2014). These polycrystalline deposits are white precipitates composed of calcium lactate pentahydrate (CLP). Cheddar cheese with low pH is highly susceptible to CLP crystallization as the levels of both lactate and soluble calcium increases. Crystallization of CLP is more rapid and of greater severity as the post-salting pH drops, especially when the pH is allowed to drop to 5.0 and below (Swearingen et al. 2004).

In surface mold-ripened cheeses such as Camembert cheese, a pH gradient occurs during maturity from surface (pH ~ 7.5; lactic acid in the surface catabolized to water and carbon dioxide and production of ammonia through deamination of amino acids) to center (pH ~ 6.5; diffusion of lactic acid from center to surface) with a concentration gradient of lactic acid from center to the surface. The rise in pH and the related migration of phosphate and calcium from core of the cheese toward surface where it precipitates, contribute to softening of the texture of surface mold-ripened cheese. Batty et al. (2019) reported the pH range of Camembert cheese made with different recipes after salting (zero day) was pH 4.3–5.2 and may increase to the pH range of 7.0–7.4 and 7.5–8.0 in the rind after 10 and 50 day of ripening, respectively. Likewise, the rise in pH at the surface of smear-ripened cheese supports the growth of Coryneform bacteria.

Proteolysis

Proteolysis is the principal biochemical reaction occurs during cheese ripening which is catalyzed by residual rennet, indigenous milk proteinases (especially in cheeses made from raw milk and microfiltered milk) and peptidases, endogenous enzymes from SLAB and NSLAB.

The type of rennet used in cheese production determines how much is retained in the curd and in turn measures the levels of proteolysis of caseins. Only ~ 10% of chymosin and 2–3% of microbial rennet added to cheese milk is retained in the cheese curd whereas extensive denaturation of porcine pepsin occurs during cheese manufacturing. The low pH in cheese curd favors the retention of chymosin but not of microbial rennet or pepsin. Rennet retention in curd is also affected by the cooking temperature and extensive denaturation of rennet occurs in curds cooked at high temperature (~ 55 °C) such as Emmental cheese and during the later cooking–stretching step of the manufacture of Mozzarella. Nevertheless, Nega and Moatsou (2012) found considerable chymosin activity in Pasta-Filata cheeses (Kasseri and Metsovone) compared to Gruyere (Graviera Kritis and Graviera Naxou) and hard type (Ladotyri Mytilinis and San Michali) cheeses. The chymosin activity should be low in these type of cheeses as the processing temperature (80ºC) employed is quite high that are expected to inactivate residual chymosin. The high activity of chymosin (optimum pH 5.5) might be due to the low pH (~ 5.2) of the acidified cheese curd during kneading and increased interaction between casein micelles and coagulant. Authors also concluded that considering the cheese types with related pH and moisture content, the activity of residual chymosin depends on cooking temperature of cheese curd.

Moreover, chymosin activity also depends on the pH of the cheese during ripening (Vélez et al. 2016). pH and ionic strength influences the primary proteolysis of α_S1- and β-CNs but significantly lesser extent in α_S2-CN by chymosin and responsible for formation of peptides soluble in water or at pH 4.6. Cleavage of Phe₂₃–Phe₂₄ (principal site) of α_S1-casein in solution (pH 6.5) by chymosin produces a peptide α_S1-CN (f1-23), which is readily metabolized by proteinases of SLAB in cheeses such as Cheddar and similar cheeses within 4 months.

Among indigenous milk proteinases, plasmin (optimum pH ~ 7.5) is the most significant enzyme in regard to cheese ripening. Among the caseins, β-CN is the most susceptible to plasmin action, hydrolysis yields proteose peptone (PP) (PP5, PP8 slow and PP8 fast) and γ-casein (i.e. γ¹, γ² and γ³) followed by α_S2-CN and α_S1-CN. Although α_S1-CN less susceptible to plasmin action, it is readily hydrolyzed the solution to yield λ-CN. The activity of plasmin in cheese ripening differs with the type of cheeses. Ardo et al. (2017) reported that plasmin plays an important role in proteolysis of high-temperature cooked cheeses, mold-ripened and smear ripened cheeses whereas its activity is low in internal bacterially ripened cheeses (i.e., Cheddar). Larsson et al. (2006) reported decrease in plasmin activity with decrease in pH and hardly found any activity at pH 5.0 to 5.2. Also, plasmin retention is more when whey is drained at higher pH because of less dissociation of plasmin from casein micelle at higher pH (Voigt et al. 2011). Plasmin is responsible for formation of some γ-casein and hydrolysis of α_S2-casein during cheese ripening.

Other proteinase enzymes of SLAB and NSLAB play an imperative role in cheese ripening mainly by hydrolyzing peptides produced from caseins and their activity is also pH dependent. Cell envelop proteinase (CEP or lactocepin) (pH optima of 5.5–6.5) of several strains of Lactococcus or Lactobacillus starter culture hydrolyzes the caseins in solution and in milk but it appears to have little effect on casein in cheese during ripening. However, CEP is mainly accountable for the hydrolysis of oligopeptides of β- and α_S1-CNs produced from plasmin and chymosin, respectively, to smaller peptides. A metalloprotease (optimum pH 4.5–8.5 and optimum pH for casein hydrolysis is 5.5) and an aspartic protease (optimum pH 3.5–6.0) are the two extracellular proteases expressed by Pencillium roqueforti during ripening of blue-mold cheese, especially when mold has become visible in the cheese (i.e., 2–5 weeks of maturation). The metalloprotease has broad specificity and mainly hydrolyzes the β- and α_S1-CNs while aspartic protease hydrolyzes casein to high-molecular weight peptides and like chymosin primary specificity on α_S1-CN. Several exopeptidases released by P. roqueforti further hydrolyze the peptides to acidic, basic, hydrophobic amino acids (by serine carboxypeptidase, stable at pH 5.0–5.5) and apolar amino acids (by metallo-aminopeptidase, pH 8.0) (Cantor et al. 2017).

NSLAB contains peptidases are the most important in ripening of cheese since they are responsible for the production of smaller peptides and free amino acids through secondary proteolysis, which are greatly converted into components that contribute cheese flavor. Gobbeti et al. (1999) studied the interactive effects of pH (5.5–7.0), temperature and NaCl on peptidase activities of several strains of NSLAB. While the peptidases of Lb. casei ssp. casei and Lb. plantarum were less sensitive to variation in pH than Lb. casei ssp. pseudoplantarum and Lb. curvatus. Significant variation in the concentration of free amino acids was observed during ripening of hard and extra-hard varieties and concentration of amino acids decreases during the end of ripening. However, some mesophilic lactobacilli produce γ-aminobutyric acid through decarboxylation of glutamic acid in later stages of maturation of extra-hard cheese (Fiore Sardo) (Piras et al. 2013). Microbial decarboxylase activity (by NSLAB) is responsible for production of biogenic amines in extra-hard varieties (Pecorino cheeses) during ripening.

Lipolysis

Lipolysis is important for flavor formation of most varieties of ripened cheeses which is catalyzed mainly by lipolytic enzymes from milk, SLAB and NSLAB. Lipolytic enzymes from LAB have pH optima in the range of 7.0–8.5 whereas milk lipoprotein lipase is 7.0 and their activity varies with cheese types. Moreover, the composition of cheese and pH influences the aroma threshold of free fatty acids which in turn affect the taste. Especially at high pH, dissociated fatty acids anions are less volatile which gives soapy flavor (Kilcawley and O’Sullivan, 2018). Significantly high amount of free fatty acids are released during ripening of blue mold cheeses and extra-hard cheeses compared to other cheese varieties. P. roqueforti performs the metabolism of lipids in blue-mold cheeses using two extracellular lipases (one acidic (pH optima at 6.0) and one alkaline (pH optima at 9.0)) and one intracellular lipase and releases large amount long-chain fatty acids (C_12:0–C_18:3) than of short-chain fatty acids (C_4:0–C_10:0) from triglycerides, which is opposite to the action of milk lipoprotein lipase (Cantor et al. 2017). Rennet paste is more commonly used in several extra-hard Italian varieties (Pecorino cheese and Fiore Sardo), which characterizes with piquant flavor due to action of pre-gastric esterase in rennet paste, which serves as a both coagulant and lipolytic agent in cheese production. These esterase releases large amount of short-chain fatty acids (especially butanoic, hexanoic, and octanoic acids) esterified at Sn-3 position of triglycerides (Gobbetti and Cagno 2022).

Microstructure and rheological properties

The microstructure of cheese is a crucial element that governs the rheological and functional attributes of cheese. Cheese microstructure is a three-dimensional particulate gel matrix of calcium phosphate-para-casein. Other substances like fat, moisture and minerals are present within the pores of these matrices. The microstructure of cheese is influenced by numerous factors, and it differs from the type of cheese. Changes in pH during manufacturing and ripening can affect the microstructure of cheese and thus textural and functional characteristics in positive or negative aspects. As discussed above, pH profoundly affects the proportion of phosphate and calcium ions in cheese curd, retention of chymosin and pepsin, syneresis, and composition. All these parameters determine the basic cheese structure. Cheese structure is influenced by interaction forces such as electrostatic, hydrophobic, hydrogen, and disulfide bonding that depends on the pH.

The rheology of cheese is function of its composition and structure. In general, rennet cheeses with relatively low pH (4.9–5.3) are shorter and crumbly compared to cheeses with relatively high pH (5.3–5.6) (Fox et al 2017b). Guinee (2022) reported that a rise in pH in the range of 4.8–5.2 causes a decrease in elastic modulus and fracture stress in Cheddar and Gouda cheese. However, a further increase in pH from 5.2 to 5.6 is exhibited by a marked increase in fracture stress and slight increase in elastic modulus. The differences in rheological characteristics between cheese varieties is probably depends upon variation in pH, individual constituents (i.e., fat, protein and moisture content), salt-in-moisture, calcium content and hydrolysis of casein during ageing. Low pH cheeses (< 5.0) (e.g., Cheshire and Feta) tend to crumble into many pieces on fracturing and thus have lesser values of fracture stress and fracture strain. In contrast, cheeses (e.g., Emmental) with pH in the range of 5.35–5.50 exhibit more fracture stress and fracture strain values and tend to fracture in larger pieces. The effect of pH probably results from its effect on the proportion of colloidal to soluble calcium, casein hydration, and the type of inter-protein linkages.

Solubility of calcium and calcium content in cheese is mainly affected by changes in pH. During acidification of milk the calcium in colloidal phase solubilizes and becomes completely soluble in the aqueous phase at pH 3.5. The total calcium remaining in cheese therefore depends on the pH at which the curd is separated from the whey. Thus calcium content of natural cheeses (such as Cheddar ranges 20–40 mg/g protein) is greater than acid curd cheeses (such as Cottage and Cream cheese contains < 6 and 10 mg/g protein, respectively) (Wolfschoon-Pombo and Andlinger 2013). Any variation in acid gel pH before whey separation can change the microstructure and texture of resulting cheese by altering the incorporation of whey proteins and calcium content (Ong et al. 2020). The effect of variation in acid pH (pH 4.3, 4.5, 4.7 and 5.0) on Cream cheese microstructure and rheological properties was investigated by Ong et al. (2020). The microstructure of Cream cheese consists of corpuscular structure characterized by clusters of small fat globules coated extensively with protein aggregates and size of the structure appeared to decrease (from ~ 5 to ~ 4 μm) as the cheese pH was reduced from pH 5.0 to 4.7 or 4.3. Protein swelling, reduced interaction between whey proteins and casein and increased β-turn protein structure (observed by synchrotron-FTIR microspectroscopy) possibly contributed to the larger corpuscular structure and lower firmness of Cream cheese at pH 5.0 compared to pH 4.3–pH 4.7. Conversely, at lower pH, increased hydrophobic protein–protein interaction may cause the protein structure to contract, resulting in a smaller corpuscular structure, denser microstructure with increased aggregated β-sheet structure and a firmer product. In another study, pH of Cream cheese was adjusted to varying pH after manufacture by exposing the finished cheese to an atmosphere of volatile acid (acetic acid for 30, 60 and 90 min) and base (ammonia for 1, 3 and 5 min) and authors found that cheese firmness decreased and meltability increased with increase in pH from about 4.2 to 6.8 and a microstructure that appeared to be more continuous or swollen. Specifically, the volume of protein network surrounding the fat droplets increased markedly with increased pH, presumably due to extensive protein-to-water interaction and swelling of the protein network, thus producing a softer cheese (Monteiro et al., 2009).

Microbiology

The optimum pH for growth of most of the bacteria is towards neutral and growth is poor at pH values < 5.0 pH and acid content influence the growth and types of bacteria that will survive in cheese. In general, the pH of cheese curd varies from 4.5 to 5.3 which has significant role in controlling the microbial growth in cheese. Nevertheless, the cheese manufacturing process determines the composition of cheese and environmental conditions that further influence the fate of microflora during the process. Among various foodborne pathogens, Listeria monocytogenes, enteropathogenic Escherichia coli, Salmonella enterica, Staphylococcus aureus and Campylobacter spp. are the most common that are associated with cheese-borne illness outbreaks (Picon 2018). Generally, cheese related foodborne illnesses have been linked to soft cheese or cheeses produced from unpasteurized milk, but hardly from hard cheeses (Choi et al. 2016). In general, pasteurized milk is used for most of the cheese making process and should eliminate pathogenic microorganisms from milk. Yet, number of outbreaks associated with pasteurized milk cheeses have been reported in the literature (Centers for Disease Control and Prevention 2013; Gould et al. 2014; Schoder et al. 2013). In cheese, factors like pH, water activity, redox potential, organic acid content, time–temperature of cooking of curd, added salt and growth of SLAB can control the growth of pathogenic microorganisms. However, Kongo (2013) stated that pH of cheese from processing to ripening is a better parameter to gauge the actual product safety. Minimum pH values that have been reported for growth of Salmonella spp., L. monocytogenes and E. coli O157:H7 are 4.20, 4.39 and 4.40, respectively (Leong et al. 2014).

Among different cheese-borne pathogens, L. monocytogenes can cause serious outbreaks even in pasteurized cheeses because it commonly occurs in processing environment and thus can contaminate cheese during the process of manufacturing as well as ripening (Centers for Disease Control and Prevention, 2013). Especially soft cheeses are more prone to outbreaks of Listeria as they can support the growth of this pathogen introduced after processing, irrespective of the heat treatment given to the milk (D’Amico et al. 2008). Furthermore, Falardeau et al. (2021) reported that L. monocytogenes can grow at pH levels as low as 4.9 to 5.0 in acid coagulated cheeses while growth of this pathogen occurs at pH above 5.5 in soft-ripened varieties. Moreover, L. monocytogenes can survive and grow even in ripened cheeses at refrigerated temperatures (D’Amico et al. 2008). Kapetanakou et al. (2017) evaluated the growth of L. monocytogenes in 11 types of (cream, soft and semi-hard) cheeses during storage at 7 °C. The authors found a correlation between pH and the survival of L. monocytogenes. Among the cheeses studied, Mascarpone (initial pH 6.45; A_w 0.988), Ricotta (pH 6.64; A_w 0.99), Camembert (pH 6.30; A_w 0.973), Halloumi (pH 6.60; A_w 0.965) showed high growth potential of L. monocytogenes compared to cottage cheese (pH 5.03; A_w 0.994), Edam (pH 5.58; A_w 0.953) and Gouda (pH 5.53; A_w 0.967). Although no significant difference in A_w of these cheeses, the lesser growth in latter cheese varieties might be due to low initial pH values which arrested the growth of Listeria. Similarly, Leong et al (2014) reported no growth of L. monocytogenes in semi-hard varieties like Gouda stored at 25 °C. The fate of L. monocytogenes at different temperatures is mainly depends on pH of the cheeses. An increase in pH favors the growth of L. monocytogenes and vice versa. Leong et al (2014) studied the growth of different pathogens (E. coli O157:H7, L. monocytogenes, Salmonella spp. and S. aureus) in 67 market cheeses stored at 25 °C. Out of 67 cheese samples, 53 did not show growth of all the pathogens studied while three samples supported the growth of E. coli, four of Listeria, six of Salmonella spp. and fourteen of S. aureus. The authors observed that pathogens growth were supported in cheeses having high initial pH values (5.41 to 6.50) in comparison with cheeses having low initial pH values (4.29 to 5.40).

It is important to highlight that synergistic effects of high number of LAB during cheese making (mainly SLAB) and ripening (mainly NSLAB), a decreased pH and bacteriocin production can negatively impact survival of pathogens in cheese made from raw or pasteurized milk. It also important to consider the rate of pH decrease in the curd while selecting the strains of SLAB. pH of curd decreases quickly and pathogens growth is arrested if the active starters is used in cheese making. Other factors which influence the growth of pathogens during ripening are moisture, pH, salt, nitrate and ripening temperature.

Conclusion

This review analyzed the scientific literature to comprehend the relationship between pH and cheese quality and safety. Various studies have reported the significant effect of pH on various key steps of cheese manufacturing affecting cheese quality and safety. However, there is a need to evident the effect of pH on individual cheese quality because the pH of different cheese varieties varies at different manufacturing stages. In literature, most of the studies on pH relate to change in calcium fraction of cheeses. Therefore, more focus must be given to gauge the impact of pH on quality parameters and specific cheese variety so that the accurate role of pH in cheese manufacturing can be recognized. Furthermore, the relationship between pH and microbiological safety needs to be validated further as the food poising outbreaks associated with cheese are most common.

The main aim of cheese-making is to produce consistently high-quality cheeses with desired characteristics. The critical steps in manufacturing that affect cheese's quality and safety include renneting, coagulation, whey syneresis, salting and ripening. It is evident from the above discussion that pH affects these crucial steps of cheese manufacturing. Altering the pH at different stages of cheese manufacturing can be employed to produce consistent cheese quality throughout the year. Moreover, accurate pH is also critical to ensure the safety of cheeses concerning the microbiological viewpoint. Cheese making is a holistic process where pH at different stages of manufacturing (rate and extent of acid development) is critical to achieve the desired cheese characteristics viz. flavor, structure and texture.

Abbreviations

CCP

Colloidal calcium phosphate

CN

Casein

CEP

Cell envelop protease

CLP

Calcium lactate pentahydrate

FPC

Fermentation produced chymosin

LAB

Lactic acid bacteria

MT

Million tonnes

NSLAB

Non-starter lactic acid bacteria

RCT

Rennet coagulation time

SLAB

Starter lactic acid bacteria

Authors' contributions

VB wrote the manuscript. NV edited the manuscript.

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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