Improvement of texture and structure of reduced-fat Cheddar cheese by exopolysaccharide-producing lactococci

The objective of this study was to evaluate the effect of capsular and ropy exopolysaccharide (EPS)-producing strains of Lactococcus lactis ssp. cremoris on textural and microstructural attributes during ripening of 50%-reduced-fat Cheddar cheese. Cheeses were manufactured with added capsule- or ropy-forming strains individually or in combination. For comparison, reduced-fat cheese with or without lecithin added at 0.2% (wt/vol) to cheese milk and full-fat cheeses were made using EPS-nonproducing starter, and all cheeses were ripened at 7°C for 6 mo. Exopolysaccharide-producing strains increased cheese moisture retention by 3.6 to 4.8% and cheese yield by 0.28 to 1.19 kg/100 kg compared with control cheese, whereas lecithin-containing cheese retained 1.4% higher moisture and had 0.37 kg/100 kg higher yield over the control cheese. Texture profile analyses for 0-d-old cheeses revealed that cheeses with EPS-producing strains had less firm, springy, and cohesive texture but were more brittle than control cheeses. However, these effects became less pronounced after 6 mo of ripening. Using transmission electron microscopy, fresh and aged cheeses with added EPS-producing strains showed a less compact protein matrix through which larger whey pockets were dispersed compared with control cheese. The numerical analysis of transmission electron microscopy images showed that the area in the cheese matrix occupied by protein was smaller in cheeses with added EPS-producing strains than in control cheese. On the other hand, lecithin had little impact on both cheese texture and microstructure; after 6 mo, cheese containing lecithin showed a texture profile very close to that of control reduced-fat cheese. The protein-occupied area in the cheese matrix did not appear to be significantly affected by lecithin addition. Exopolysaccharide-producing strains could contribute to the modification of cheese texture and microstructure and thus modify the functional properties of reduced-fat Cheddar cheese.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: composition and proteolysis

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar cheese, EPS-producing cultures should be used in conjunction with debittering strains.     

Effects of exopolysaccharide-producing cultures on the viscoelastic properties of reduced-fat Cheddar cheese

The objective was to study the influence of different exopolysaccharide (EPS)-producing and nonproducing lactic cultures on the viscoelastic properties of reduced-fat Cheddar cheese. Changes in the viscoelastic properties were followed over a ripening period of 6 mo. Results showed that the elastic, viscous, and complex moduli were higher in reduced-fat cheeses made with EPS-nonproducing cultures than in full-fat cheese. No differences in the viscoelastic properties were found between young reduced-fat cheese made with a ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and its full-fat counterpart. Interestingly, the changes in viscoelastic moduli in both full-fat cheese and reduced-fat cheese made with JFR1 during ripening followed the same pattern. Whereas the moduli increased during the first month of ripening in those 2 cheeses, a dramatic decrease was observed in all other cheeses. Slopes of the viscoelastic moduli as a function of frequency were lower in the full-fat than in reduced-fat cheeses. The creep test showed that fresh reduced-fat cheese made with JFR1 was less rigid and more deformable than that made with EPS-nonproducing cultures. The creep and recovery properties of young reduced-fat cheese made with JFR1 and the full-fat type were similar. No differences were found in the viscoelastic properties between reduced-fat cheese made with no EPS and those made with EPS-producing adjunct cultures of Streptococcus thermophilus. After 6 mo of ripening, cheeses made with EPS-producing cultures maintained lower elastic and viscous moduli than did those made with no EPS.     

Ultrafiltered milk reduces bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide-producing culture

The objectives were to reduce bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide (EPS)-producing culture and study relationships among ultra-filtration (UF), residual chymosin activity (RCA), and cheese bitterness. In previous studies, EPS-producing cultures improved the textural, melting, and viscoelastic properties of reduced-fat Cheddar cheese. However, the EPS-positive cheese developed bitterness after 2 to 3 mo of ripening due to increased RCA. We hypothesized that the reduced amount of chymosin needed to coagulate UF milk might result in reduced RCA and bitterness in cheese. Reduced-fat Cheddar cheeses were manufactured with EPS-producing and nonproducing cultures using skim milk or UF milk (1.2×) adjusted to a casein:fat ratio of 1.35. The EPS-producing culture increased moisture and RCA in reduced-fat Cheddar cheese. Lower RCA was found in cheese made from UF milk compared with that in cheese made from control milk. Ultrafiltration at a low concentration rate (1.2×) produced EPS-positive, reduced-fat cheese with similar RCA to that in the EPS-negative cheese. Slower proteolysis was observed in UF cheeses compared with non-UF cheeses. Panelists reported that UF EPS-positive cheese was less bitter than EPS-positive cheese made from control milk. This study showed that UF at a low concentration factor (1.2×) could successfully reduce bitterness in cheese containing a high moisture level. Because this technology reduced the RCA level (per g of protein) to a level similar to that in the control cheeses, the contribution of chymosin to cheese proteolysis would be similar in both cheeses.     

Manufacture of low-fat Cheddar cheese by exopolysaccharide-producing Lactobacillus plantarum JLK0142 and its functional properties

This study aimed to evaluate the effect of exopolysaccharide (EPS)-producing Lactobacillus plantarum JLK0142 on the ripening characteristics and in vitro health-promoting benefits of low-fat Cheddar cheese. Three batches of cheese were made by employing a non-EPS–producing cheese starter (control), in combination with Lb. plantarum JLK0142 as an adjunct and the purified EPS as an ingredient. Lactobacillus plantarum JLK0142 survived well in cheese, with counts of 7.99 log cfu/g after 90 d of ripening. All experimental cheeses (with adjunct culture or EPS ingredient) had higher moisture, proteolysis, and sensory scores, and lower hardness and cohesiveness compared with the control cheese. Water-soluble extracts from the experimental cheeses outperformed that of the control in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl radicals, and inhibiting α-amylase, angiotensin-converting enzyme, and HT-29 tumor cell growth. Therefore, incorporation of the EPS-producing culture of Lb. plantarum JLK0142 is promising for improvement of low-fat cheese quality and bioactivities.     

Effect of anthocyanin-absorbed whey protein microgels on physicochemical and textural properties of reduced-fat Cheddar cheese

Reduced-fat foods have become more popular due to their health benefits; however, reducing the fat content of food affects the sensory experience. Therefore, it is necessary to improve the sensory acceptance of reduced-fat foods to that of full-fat equivalents. The aim of this study was to evaluate the effect of adding whey protein microgels (WPM) with an average diameter of 4 μm, or WPM with adsorbed anthocyanins [WPM (Ant)] on the textural and sensory properties of reduced-fat Cheddar cheese (RFC). Reduced-fat Cheddar cheese was prepared in 2 ways: (1) by adding WPM, designated as RFC+M, or (2) by adding WPM (Ant), designated as RFC+M (Ant). For comparison, RFC without fat substitutes and full-fat Cheddar cheese were also prepared. We discovered that the addition of WPM and WPM (Ant) increased the moisture content, fluidity, and meltability of RFC, and reduced its hardness, springiness, and chewiness. The textural and sensory characteristics of RFC were markedly inferior to those of full-fat Cheddar cheese, whereas addition of WPM and WPM (Ant) significantly improved the sensory characteristics of RFC. The WPM and WPM (Ant) showed a high potential as fat substitutes and anthocyanin carriers to effectively improve the acceptance of reduced-fat foods.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: cryo-scanning electron microscopy observations

The microstructure of reduced- and full-fat Cheddar cheeses made with exopolysaccharide (EPS)-producing and nonproducing cultures was observed using cryo-scanning electron microscopy. Fully hydrated cheese samples were rapidly frozen in liquid nitrogen slush (−207°C) and observed in their frozen hydrated state without the need for fat extraction. Different EPS-producing cultures were used in making reduced-fat Cheddar cheese. Full-fat cheese was made with a commercial EPS-nonproducing starter culture. The cryo-scanning electron micrographs showed that fat globules in the fully hydrated cheese were surrounded by cavities. Serum channels and pores in the protein network were clearly observed. Young (1-wk-old) full-fat cheese contained wide and long fat serum channels, which were formed because of fat coalescence. Such channels were not observed in the reduced-fat cheese. Young reduced-fat cheese made with EPS-nonproducing cultures contained fewer and larger pores than did reduced-fat cheese made with a ropy strain of Lactococcus lactis ssp. cremoris (JFR1), which had higher moisture levels. A 3-dimensional network of EPS was observed in large pores in cheese made with JFR1. Major changes in the size and distribution of pores within the structure of the protein network were observed in all reduced-fat cheeses, except that made with JFR1, as they aged. Changes in porosity were less pronounced in both the full-fat and the reduced-fat cheeses made with JFR1.     

Temperature-induced moisture migration in reduced-fat Cheddar cheese

Moisture migration during cooling of 290-kg Cheddar cheese blocks is a problem. The problem is of greater magnitude in reduced and low fat varieties. The objective of this study was to design and evaluate the performance of a laboratory-scale apparatus for simulation of temperature induced moisture migration in 290-kg blocks of Cheddar cheese. Two apparati were designed to produce a systematic temperature gradient in small cheese slabs over a 36-h period to simulate the temperature gradient that develops during cooling of a 290-kg block. One of the apparati was designed to induce a moisture migration downwards with gravity and the other against gravity. The apparati produced moisture migration ranges of 9.7 and 6.4%, for the apparatus to induce moisture migration downwards and upwards, respectively. The moisture moved from areas of warm cheese to areas of cold cheese during cooling, as occurs in 290-kg blocks. These ranges were comparable to those obtained with 290-kg reduced-fat Cheddar blocks. In addition, small but significant differences in pH were created within slabs. The direct effect of the temperature gradient on moisture migration within cheese slabs appeared to be more important than the possible impact of the small pH gradient produced within the cheese by the temperature gradient.     

Reduced fat process cheese made from young reduced fat Cheddar cheese manufactured with exopolysaccharide-producing cultures

In a previous study, exopolysaccharide (EPS)-producing cultures improved textural and functional properties of reduced fat Cheddar cheese. Because base cheese has an impact on the characteristics of process cheese, we hypothesized that the use of EPS-producing cultures in making base reduced fat Cheddar cheese (BRFCC) would allow utilization of more young cheeses in making reduced fat process cheese. The objective of this study was to evaluate characteristics of reduced fat process cheese made from young BRFCC containing EPS as compared with those in cheese made from a 50/50 blend of young and aged EPS-negative cheeses. Reduced fat process cheeses were manufactured using young (2 d) or 1-mo-old EPS-positive or negative BRFCC. Moisture and fat of reduced fat process cheese were standardized to 49 and 21%, respectively. Enzyme modified cheese was incorporated to provide flavor of aged cheese. Exopolysaccharide-positive reduced fat process cheese was softer, less chewy and gummy, and exhibited lower viscoelastic moduli than the EPS-negative cheeses. The hardness, chewiness, and viscoelastic moduli were lower in reduced fat process cheeses made from 1-mo-old BRFCC than in the corresponding cheeses made from 2-d-old BRFCC. This could be because of more extensive proteolysis and lower pH in the former cheeses. Sensory scores for texture of EPS-positive reduced fat process cheeses were higher than those of the EPS-negative cheeses. Panelists did not detect differences in flavor between cheeses made with enzyme modified cheese and aged cheese. No correlations were found between the physical and melting properties of base cheese and process cheese.     

Effect of various high-pressure treatments on the properties of reduced-fat Cheddar cheese

A major problem with reduced-fat cheese is the difficulty in attaining the characteristic flavor and texture of typical full-fat versions. Some previous studies have suggested that high hydrostatic pressure (HHP) can accelerate the ripening of full-fat cheeses. Our objective was to investigate the effect of HHP on reduced-fat (~7.3% fat) Cheddar cheese, with the goal of improving its flavor and texture. We used a central composite rotatable design with response surface methodology to study the effect of pressure and holding time on the rheological, physical, chemical, and microbial characteristics of reduced-fat Cheddar cheese. A 2-level factorial experimental design was chosen to study the effects of the independent variables (pressure and holding time). Pressures were varied from around 50 to 400 MPa and holding times ranged from 2.5 to 19.5 min. High pressure was applied 1 wk after cheese manufacture, and analyses were performed at 2 wk, and 1, 3, and 6 mo. The insoluble calcium content as a percentage of total Ca in cheeses were not affected by pressure treatment. Pressure applications ≥225 MPa resulted in softer cheese texture during ripening. Pressures ≥225 MPa increased melt, and resulted in higher maximum loss tangent values at 2 wk. Pressure treatment had a greater effect on cheese microbial and textural properties than holding time. High-pressure-treated cheeses also had higher pH values than the control. We did not observe any significant difference in rates of proteolysis between treatments. In conclusion, holding times of around 5 min and pressures of ≥225 MPa could potentially be used to improve the excessively firm texture of reduced-fat cheese.     

The effect of aging on low-fat, reduced-fat, and full-fat Cheddar cheese texture

This study investigated the effects of aging and fat content on the texture of Cheddar cheese, both mechanical and sensory aspects, over a 9-mo aging period. Cheeses of 6, 16, and 33% fat were tested at 0.5, 3, 6, and 9 mo of aging. Cheeses were evaluated by a trained sensory panel using an established texture lexicon as well as instrumental methods, which were used to probe cheese structure. Sensory analysis showed that low-fat cheeses were differentiated from full-fat cheeses by being more springy and firm and this difference widened as the cheeses aged. In addition, full-fat cheeses broke down more during chewing than the lower fat cheeses and the degree of breakdown increased with aging. Mechanical properties were divided by magnitude of deformation during the test and separated into 3 ranges: the linear viscoelastic region, the nonlinear region, and fracture point. These regions represent a stress/strain response from low to high magnitude, respectively. Strong relationships between sensory terms and rheological properties determined in the linear (maximum compliance) and nonlinear (critical stress and strain and a nonlinear shape factor) regions were revealed. Some correlations were seen with fracture values, but these were not as high as terms related to the nonlinear region of the cheeses. The correlations pointed to strain-weakening behavior being the critical mechanical property. This was associated with higher fat content cheeses breaking down more as strain increased up to fracture. Increased strain weakening associated with an increase in fat content was attributed to fat producing weak points in the protein network, which became initiation sites for fracture within the structure. This suggests that fat replacers need to serve this functional role.     

Sensory properties of Cheddar cheese: changes during maturation

The aroma, flavour and texture of 16 samples of commercial Cheddar cheese have been profiled after ripening at 10 °C for 3, 4, 6, 8, 10 and 12 months. Systematic changes in sensory character have been studied and the main changes during maturation identified. Although sensory character changed slowly during ripening, assessment early in the maturation period was an unreliable estimate of ultimate sensory character. Progressive changes in Cheddar aroma and flavour, creamy flavour, acid flavour and mouth-coating character were noted during ripening. Changes in minor components of aroma and flavour were also observed but, on average, were small. Two samples eventually developed marked rancid character and another became excessively bitter. The relation between gross composition of the cheese and sensory properties was investigated. In the early stages of ripening, the ratings for Cheddar flavour and mouth-coating character were associated with the salt content of the cheese and with the concentration of fat in dry matter. However, as the cheese matured these associations weakened.     

原料乳体细胞数不同对契达干酪产出量、质构及感官的影响

用体细胞数(SCC)分别是5.6×104,48.8×104,476.1×104 mL-1的原料乳制作契达干酪,得到LSCC,MSCC,HSCC组干酪。从干酪真正产出量来看:LSCC组>MSCC组>HSCC组(P<0.05)。在干酪成熟过程中,质构与SCC在P<0.01的水平下负相关,其中硬度、剪切力相关系数分别为0.5482和1.3977。感官评定结果表明,HSCC组干酪有酸味,且组织状态软而粘。同时对干酪成熟过程中的水溶性氮和脂解进行了测定,其结果是:WSN/TN与SCC在P<0.01水平下线性相关,相关系数为0.4261;HSCC组干酪的FFA在P<0.05的水平下显著高于LSCC和MSCC组干酪,且FFA与SCC在P<0.0001的水平下正相关。     

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities ≤90%, significant differences in the cheeses microstructure, and a significant improvement in both textural and cooking properties, without negatively affecting the flavor profiles of the cheeses.     

ADSA Foundation Scholar Award: Possibilities and challenges of exopolysaccharide-producing lactic cultures in dairy foods

Exopolysaccharides (EPS) from lactic acid bacteria are a diverse group of polysaccharides exhibiting various functional properties. Two forms of EPS are produced by lactic acid bacteria: capsular and unattached. Capsular EPS does not cause ropiness nor does production of unattached EPS ensure ropiness. The functions of EPS in dairy products are not completely understood. This is for 2 main reasons: the major variations among exopolysaccharides even from the same group of micro-organisms, which makes it difficult to apply information from one EPS to others, and the lack of availability of techniques with the ability to observe the microstructure and distribution of the highly hydrated EPS in fermented dairy products. The introduction of relatively new microscopic techniques such as confocal scanning laser microscopy and cryo-scanning electron microscopy made it possible to directly observe the distribution of fully hydrated EPS in dairy products. Recently, EPS produced by nonropy strains have drawn the attention of the dairy industry. This is because of the ability of some nonropy strains to produce large capsular and unattached EPS that would improve the texture of dairy products without causing the undesirable slippery mouthfeel produced by the ropy strains. Factors affecting functions of EPS are their molecular characteristics and ability to interact with milk proteins. Studying the interaction between EPS and milk proteins is complex because EPS are gradually produced during fermentation, unlike polysaccharides added directly to milk to stabilize the fermented product. The concentration and possibly molecular characteristics of EPS and protein characteristics such as charge and hydrophobicity change during fermentation. Consequently, the interaction of EPS with proteins might also change during fermentation. Exopolysaccharides provide functions that benefit reduced-fat cheeses. They bind water and increase the moisture in the nonfat portion, interfere with protein-protein interactions and reduce the rigidity of the protein network, and increase viscosity of the serum phase. This review discusses the production of capsular EPS and their role in structure formation in fermented milk, the mechanism of ropiness formation, and applications of EPS-producing cultures in reduced-fat cheeses.     

Short communication: comparison of covered and uncovered Schreiber test for cheese meltability evaluation

Schreiber meltability tests were performed on glass Petri dishes, with and without the Petri dish cover placed over the cheese samples, at 100, 150, and 232 degrees C. Meltability of different process cheese and Cheddar cheese samples was determined based on the melt spread distance and area. At the test temperature of 232 degrees C, the covered Schreiber was significantly superior to the uncovered test because of no crust formation, no browning, and a circular melting pattern, which were attributed to the barrier effect of covering the cheese samples (which inhibits moisture loss during the test). The covered Schreiber test data were statistically more robust as measured by the lower average coefficient of variation than the data from the traditional uncovered Schreiber test.     

藏灵菇胞外多糖的理化性质及其在切达干酪中的应用

藏灵菇KW1在SDM培养基、37 ℃条件下发酵产胞外多糖（exopolysaccharide,EPS）达624.82?mg/L,经分离纯化及单糖分析测定,明确此多糖由鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖组成,相对物质的量比1∶3.02∶2.12∶1.59∶3.04。红外光谱结果显示该EPS表现出典型的多糖吸收峰模式;扫描电镜显示,藏灵菇KW1?EPS微观结构中分布着许多球形结构和片状结构,且表面比较光滑;原子力显微观察表明,EPS具有一定聚集现象,呈现出膜状、簇状结构。将藏灵菇KW1?EPS应用于发酵剂菌株培养以及切达干酪制作中,结果表明EPS对发酵剂菌株生长有促进作用,并且随着添加量的增加,这种作用先增强后减弱。同时EPS的加入能提高干酪得率、持水能力以及成熟期间的活菌数。采用气相色谱-质谱联用从干酪中检测出69?种挥发性物质,香气活性值显示共有17?种风味物质对EPS干酪整体风味有贡献,其中丁酸乙酯、己酸乙酯、辛酸乙酯是关键性风味物质。本研究可为藏灵菇EPS在发酵乳制品中的应用提供一定技术参考。     

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: pH buffering properties of cheese

The pH buffering capacity of cheese is an important determinant of cheese pH. However, the effects of different constituents of cheese on its pH buffering capacity have not been fully clarified. The objective of this study was to characterize the chemical species and chemical equilibria that are responsible for the pH buffering properties of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), residual lactose (2.4 vs. 0.78%), and salt-to-moisture ratio (6.4 vs. 4.8%) were manufactured. The pH-titration curves for these cheeses were obtained by titrating cheese:water (1:39 wt/wt) dispersions with 1 N HCl, and backtitrating with 1 N NaOH. To understand the role of different chemical equilibria and the respective chemical species in controlling the pH of cheese, pH buffering was modeled mathematically. The 36 chemical species that were found to be relevant for modeling can be classified as cations (Na⁺, Ca²⁺, Mg²⁺), anions (phosphate, citrate, lactate), protein-bound amino acids with a side-chain pKa in the range of 3 to 9 (glutamate, histidine, serine phosphate, aspartate), metal ion complexes (phosphate, citrate, and lactate complexes of Na⁺, Ca²⁺, and Mg²⁺), and calcium phosphate precipitates. A set of 36 corresponding equations was solved to give the concentrations of all chemical species as a function of pH, allowing the prediction of buffering curves. Changes in the calculated species concentrations allowed the identification of the chemical species and chemical equilibria that dominate the pH buffering properties of cheese in different pH ranges. The model indicates that pH buffering in the pH range from 4.5 to 5.5 is predominantly due to a precipitate of Ca and phosphate, and the protonation equilibrium involving the side chains of protein-bound glutamate. In the literature, the precipitate is often referred to as amorphous colloidal calcium phosphate. A comparison of experimental data and model predictions shows that the buffering properties of the precipitate can be explained, assuming that it consists of hydroxyapatite [Ca₅(OH)(PO₄)₃] or Ca₃(PO₄)₂. The pH buffering in the region from pH 3.5 to 4.5 is due to protonation of side-chain carboxylates of protein-bound glutamate, aspartate, and lactate, in order of decreasing significance. In addition, pH buffering between pH 5 to 8 in the backtitration results from the reprecipitation of calcium and phosphate either as CaHPO₄ or Ca₄H(PO₄)₃.