Calcium Phosphate Formation in the Dairy Industry

Calcium phosphate (Ca₃(PO₄)₂) is a common compound in the dairy manufacturing industry, where it naturally occurs due to the high concentrations of calcium and phosphate in dairy products. It is especially prevalent in the cheese manufacturing industry which will be a primary focus of this article but it also has especial relevance in all types of diary product manufacture. The formation of calcium phosphate is a highly complex phenomenon and one that has been the subject of much research.

While essential for the nutritional value of cheese, calcium phosphate can pose significant challenges when it precipitates and forms films on surfaces such as membranes and heated surfaces. It also includes analytical equipment such as Near Infrared (NIR) Reflectance probes. These probes are vital for real-time monitoring of moisture, fat, and protein content during cheese production. However, calcium phosphate buildup on their surfaces can lead to inaccurate readings, reduced efficiency, and increased maintenance costs.

Mitigating calcium phosphate formation and film development on surfaces, especially on sensitive analytical equipment, requires a multi-faceted approach that includes preventive strategies, cleaning protocols, and equipment design considerations.

Calcium phosphate formation is highly problematic in health. In medicine it is associated with the formation of kidney stones (Nancollas, 1990), in the calcification and decalcification of bone and tooth enamel (Nancollas et al., 1989).

Calcium phosphate formation is also an important step coating step and is needed as a protecting agent for example in the formation of bone and teeth. It can be found in many different forms including hydroxyapatite. In other industrial applications, calcium is added to encourage phosphate removal from phosphate rich waste streams.

Understanding Calcium Phosphate Formation

The formation of calcium phosphate is highly complex and produces many phases:-

• brushite or dicalcium phosphate dehydrate (DCPD) (Ferreira et al., 2003)
• octaclacium phosphate (OCP)
• hydroxyapatite (HAP)
• calcium-deficient apatite (CDA)

Their formation depends on a number of different conditions.  Before discussing mitigation strategies, it’s essential to understand how such calcium phosphate forms and adheres to surfaces in the dairy manufacturing environment.

(1) Chemical Composition of Milk

Milk contains high levels of calcium and inorganic phosphate, both of which are prone to precipitate together when conditions such as pH, ionic strength, temperature, concentration of both calcium and phosphate ions, presence of other ions and time for precipitation favour crystallization.

The other  salts in milk besides calcium and phosphate (Pi) are Mg, K, Na, Cl, and citrate. These are distributed between the micellar phases and serum. Virtually all the ions including two-thirds of the magnesium, one third of the calcium and half of the phosphate reside in the aqueous phase. The remainder are bound to phosphorylated residues on the protein casein where they form micellar calcium phosphate (MCP) or colloidal calcium phosphate (CCP) as it is sometimes called (Gaucheron, 2005). The degree of equilibrium between aqueous and MCP phases is constantly dynamic. It is the main part of a system which dictates not just coagulation of proteins in cheese manufacture but also fouling by calcium phosphate too (Lewis, 2011).

Calcium Phosphate Solubility

Most salts in milk are well below their solubility limit but calcium phosphate especially once formed will often exceed its level of solubility and start to precipitate. Bovine milk contains about 1200 mg Ca/L (approx. 30 mM). It is in the form of colloidal inorganic calcium ( approx. 12.5 mM), caseinate calcium (8.5 mM), soluble unionised calcium (6.5 mM) and serum ionic calcium (2.5 nM). Roughly 30% remain soluble and usually occurs as an unionized salt with citrate. Another 30% is in the cation form which implies that a total calcium content of 10% exists (approx. 2 -3 mM). This level of calcium has profound effects on rennet activity.  The remaining calcium is present in the form of colloidal calcium which we referenced earlier. Its precise structure is still trying to be understood.

Whilst the simplest possible tertiary structure of calcium phosphate is Ca2 (PO4)2. Law & Tamime, (2011) give a very sophisticated assessment of how calcium is dispersed in the colloidal system.  Colloidal calcium occurs in part bound to phosphate which is indirectly attached to the organic serine phosphate groups. The latter is said to be attached directly to casein via dissociated epsilon-carboxyl groups of acidic amino acids such as aspartic and glutamic acid.

One of the most useful descriptions from Law & Tamime (2011) is to view milk as a milk and salt system – a soup! That soup is composed of large colloidal anions of calcium phosphate bound loosely to casein which is dispersed in a serum of soluble salts and ionic species based on calcium citrate, sodium phosphate and ionic calcium and potassium. There is an equilibrium between the insoluble colloidal salts and the soluble or serum salts. The whole equilibrium is controlled by casein because soluble citrate and phosphate compete for calcium ions with casein.  tricalcium phosphate has a very low solubility product of 2.1 x 10E-33 mol/L at 25C whereas tricalcium citrate has a solubility product of 3.23 x 10E-3. This leads to the production of insoluble calcium phosphate  along with calcium citrate. It is a good description and I couldn’t put it better!

(2a) Processing Conditions In Dairy

Fresh milk at its natural pH can be heated at 140C for over 10 minutes before coagulation occurs. When the pH changes or the milk is concentrated, the degree of heat stability decreases. Whilst precipitation of salts can occur much of the chemistry of coagulation concerns protein precipitation.

Any heat-induced coagulation of milk is the product of aggregation of milk protein especially casein irrespective of whether rennet has been added or not. There is a heat-induced dissociation of kappa-casein from the micelles with an acid-induced collapse of the kappa-casein brush on the micelle surface due to heat-induced acidification. This destabilises the micelles which makes them more susceptible to aggregation.

There is also heat-induced denaturation of whey. When whey is denatured it associatesd with casein micelles stabilising them against heat-induced coagulation. Unfortunately in concentrated milk, heat-induced whey protein aggregation can be a strong destabilizing factor particular when the pH is high (Huppertz, 2016).

(2b) Processing Conditions In Cheese production

During cheese production, processes like heating, acidification, and concentration can increase the likelihood of calcium phosphate precipitation. For example, the pH reduction during cheese curdling can decrease the solubility of calcium phosphate, leading to its deposition on surfaces.

(3) Surface Interaction

Once calcium phosphate precipitates, it can adhere to various surfaces, including stainless steel equipment, sensors, and probes. The interaction between the surface and calcium phosphate crystals often leads to the formation of a tightly bound film that is difficult to remove.

The formation of DCPD is as follows:-

Ca²⁺ + HPO4²⁻ + 2H₂O = CaHPO4·2H2O (aq)

The calcium to phosphate ratio is 1:1 The reaction occurs between the pH of 4 to 6 at ambient and raised temperatures to 37ºC. At 60ºC, the pH of reaction is optimal at pH 4.

The formation of OCP is as follows:-

8Ca²⁺ + 2HPO4²⁻ + 4PO4³⁻ + 5H2O = Ca₈(HPO₄)₂(PO₄)4·5H2O (aq)

The ratio of Ca:P is 1.33. This tends to form when the pH of the dairy product is slightly closer to neutral such as 6.5 but the pH optimum becomes more acidic  as the temperature rises from 37 to 80ºC.

The formation of HAP is as follows:-

10Ca2+ + 6PO4³⁻ + 2OH− = Ca10(PO4)6(OH)2 (aq)

The ratio of Ca:P is 1.67. It forms when the pH of the dairy food is between 7 and 9 and usually when the temperature is high at 60 to 80C

The formation of CDA is as follows:-

(10−x)Ca2+ + xHPO42− + (6−x)PO43− + (2−x)OH− = (10−x/6)Ca10−x(HPO4)x(PO4)6−x(OH)2−x (aq)

The ratio of calcium to phosphorous is based on the value of x which is between 0 and 2. It also forms when the pH is between 7.5 and 9 but at 37 ºC.

The precipitation phenomenon has been studies to some extent in real dairy systems and with models mimicking these conditions. A critical study by Van Kemenade and De Bruyn (1987) examined the kinetics of calcium phosphate precipitation with supersaturated solutions. They confirmed the Ostwald rule of stages.

The presence of calcium and phosphate salts in milk is vital for the stability of milk. Phosphate salts are added for example as a stabiliser when manufacturing sterilized evaporated milk (Huppertz, 2016).

The most comprehensive examination of the phenomenon has been by Mekmene et al., (2009) and then in relation to dairy processing by Nieuwenhuijse and Huppertz (2022).

Mitigation Strategies for Calcium Phosphate Formation

Mitigating calcium phosphate formation requires a combination of preventive and active measures to control its precipitation and accumulation on surfaces. Here are some key strategies:

1. Control of Processing Parameters

Regulating key processing parameters such as pH, temperature, and concentration of calcium and phosphate can significantly reduce the risk of calcium phosphate formation.

• pH Control: Maintaining the pH of the cheese-making environment within a range that keeps calcium phosphate soluble is crucial. Avoiding extreme pH conditions, especially during stages like curdling and whey separation, can minimize precipitation. For example, adding acid slowly during the curdling process can prevent rapid pH drops that could trigger calcium phosphate crystallization.
• Temperature Management: Controlling the temperature during processing is another critical factor. High temperatures can promote the formation of calcium phosphate crystals by increasing the concentration of calcium and phosphate ions in solution. Employing temperature control measures, such as gradual heating and cooling, can help reduce precipitation.
• Ion Concentration: Adjusting the concentration of calcium and phosphate ions through careful management of milk composition and the use of additives can also mitigate calcium phosphate formation. For instance, the use of chelating agents like EDTA (ethylenediaminetetraacetic acid) can bind free calcium ions, reducing the likelihood of precipitation.

2. Surface Treatment and Coatings

The interaction between calcium phosphate and equipment surfaces can be minimized through surface treatments and coatings that reduce adhesion or prevent crystallization.

• Anti-Adhesion Coatings: Applying anti-adhesion coatings to equipment surfaces, including NIR reflectance probes, can prevent calcium phosphate from sticking. These coatings are typically hydrophobic or oleophobic, creating a barrier that reduces the surface energy and inhibits the deposition of calcium phosphate.
• Surface Smoothing: Polishing and smoothing the surfaces of equipment can reduce the number of nucleation sites where calcium phosphate crystals can initiate and grow. Smooth surfaces are less likely to promote adhesion, making it easier to clean and maintain equipment.
• Electropolishing: Electropolishing, a process that smooths and passivates metal surfaces, can be particularly effective for stainless steel equipment. By removing microscopic peaks and valleys on the surface, electropolishing reduces the tendency of calcium phosphate to adhere.

2a. Anti-Adhesion Coatings

Anti-adhesion coatings are becoming available prevent all types of fouling. They function by having low wettability and a self-cleaning capability. Their application is hampered by the heavy usage of fluorinated compounds, having a low stability in various process conditions.

Anti-fouling performance is related to surface morphology and chemical composition. When discussing surface morphology, anti-fouling surfaces are divided into two types. The first type is the superhydrophobic or superoleophobic surface with micro- and nanostructures. These are made using organic solvents and fluorinated compounds but greener chemistry approaches are now being explored (Zhao et al., 2019).

An amphiphilic silicone coating was explored in a model dairy fluid using a pilot plant pasteuriser. The coating was modified RTV silicone with a  PEO-silane amphiphile comprised of a PEO segment and flexible siloxane tether ([(EtO)₃Si-(CH₂)₂-oligodimethylsiloxane_m–block-(OCH₂CH₂)_n-OCH₃]). This was applied to treated stainless steel. there was some loss of the coating after five cycles of pasteurization and then a standard cleaning-in-place process. The coating was also resistant to adhesion by foodborne pathogenic bacteria (Zouaghi et al., 2018).

Dong et al., (2021)  recently explored the use of hydrolytic condensation of methyltrimethoxysilane in isopropanol. thee were coatings with good anti-fouling ability against water, various solvents and food materials such as red wine. They were claimed to have good stability to boiling and various chemical solutions.

Sun et al., (2023) generated an anti-fouling coating using stearic acid (SA) modified organic montmorillonite (SA@OMMT) and poly(dimethylsiloxane) (PDMS). Stearic acid has a natural hydrophobicity and so works well as a natural coating. they are claimed to have a good physical and chamical stability even with physical abrasion testing and an exposure of corrosive liquids. A new type of coating based on cellulose acetate

3. Regular Cleaning and Maintenance Protocols

Establishing rigorous cleaning and maintenance protocols is essential for preventing the buildup of calcium phosphate films on equipment.

• CIP (Clean-In-Place) Systems: Implementing automated CIP systems can ensure that equipment is regularly cleaned with the appropriate solutions, such as acidic or alkaline cleaners that dissolve calcium phosphate deposits. Acidic cleaners, like those containing citric acid or phosphoric acid, are particularly effective in dissolving calcium phosphate films.
• Routine Inspections: Regularly inspecting equipment for signs of calcium phosphate buildup can help detect and address issues before they become severe. Early intervention can prevent extensive deposits that are difficult to remove.
• Cleaning Frequency and Duration: Adjusting the frequency and duration of cleaning cycles based on the specific conditions of the cheese manufacturing process can optimize the removal of calcium phosphate deposits. For instance, more frequent short cleaning cycles may be more effective than less frequent long cycles in preventing buildup.

4. Design Considerations for Analytical Equipment

When designing or selecting analytical equipment like NIR reflectance probes, it’s important to consider features that minimize the risk of calcium phosphate buildup.

• Material Selection: Choosing materials that are less prone to calcium phosphate adhesion is crucial. For example, probes made from non-stick materials or those coated with anti-fouling agents can reduce the likelihood of deposit formation.
• Probe Geometry: The design of the probe should minimize crevices and sharp angles where calcium phosphate can accumulate. A streamlined, smooth geometry facilitates easier cleaning and reduces the risk of buildup.
• Temperature Control in Probes: Incorporating temperature control mechanisms within the probes can prevent local overheating, which could otherwise increase the rate of calcium phosphate precipitation on the probe surface.
• Replaceable or Coated Windows: NIR probes can be designed with replaceable or coated windows that resist calcium phosphate deposition. These windows can be swapped out or recoated as needed, ensuring consistent performance without the need for frequent deep cleaning.

5. Water Treatment and Filtration

Water used in the cheese manufacturing process can be a source of calcium and phosphate ions. Treating and filtering process water to reduce the concentration of these ions can help mitigate calcium phosphate formation.

• Water Softening: Implementing water softening systems can reduce the calcium content in water used for processing and cleaning. This reduces the overall calcium load in the production environment, lowering the risk of calcium phosphate precipitation.
• Reverse Osmosis: Reverse osmosis systems can effectively remove both calcium and phosphate ions from water, providing a highly purified water source that minimizes the risk of calcium phosphate formation.
• Ion Exchange Systems: Using ion exchange resins to remove calcium ions from water can also be an effective strategy, especially in environments where high water hardness is a problem.

Case Study: Application in the Cheese Manufacturing Industry

To illustrate how these mitigation strategies can be applied in the cheese manufacturing industry, consider the following case study:

Problem

A cheese manufacturing facility noticed that calcium phosphate buildup on their NIR reflectance probes was leading to inaccurate moisture content readings, resulting in inconsistent product quality. The probes required frequent cleaning, leading to increased downtime and maintenance costs.

Solution

1. Process Parameter Optimization: The facility implemented tighter control over the pH during cheese curdling by using automated pH sensors and acid dosing systems. This reduced the frequency of rapid pH drops that previously triggered calcium phosphate precipitation.

2. Surface Treatment: The probes were coated with a hydrophobic anti-adhesion coating, significantly reducing the adhesion of calcium phosphate. Additionally, the probes were redesigned with a smoother geometry to further minimize deposition.

3. Enhanced Cleaning Protocol: The facility upgraded its CIP system to include more frequent and targeted cleaning cycles using a citric acid-based solution specifically designed to dissolve calcium phosphate deposits. They also implemented routine manual inspections to ensure early detection of any buildup.

4. Water Treatment: A water softening system was installed to reduce the calcium content in the water used for cleaning and processing, which contributed to a reduction in calcium phosphate formation throughout the facility.

5. Probe Design Improvements: The NIR reflectance probes were replaced with models featuring replaceable, anti-fouling coated windows. This allowed for easy maintenance and ensured consistent performance without frequent deep cleaning.

Outcome

The combined approach led to a significant reduction in calcium phosphate buildup on the NIR reflectance probes. This resulted in more accurate readings, improved product quality, and reduced maintenance costs. The facility also experienced less downtime, enhancing overall production efficiency.

Calcium phosphate formation and its film development on surfaces, including analytical equipment like NIR reflectance probes, present a significant challenge in the cheese manufacturing industry. However, by implementing a comprehensive mitigation strategy that includes control of processing parameters, surface treatments, regular cleaning protocols, equipment design considerations, and water treatment, it is possible to effectively manage and reduce the impact of calcium phosphate buildup. This approach not only ensures the accuracy and reliability of analytical equipment but also contributes to consistent product quality and operational efficiency in cheese manufacturing.

References

Ferreira A., Oliveira C., Rocha F. (2003) The different phases in the precipitation of dicalcium phosphate dehydrate. J. Cryst. Growth 252 pp. 599–611

Gaucheron, F. (2011) Milk Salts: Distribution and Analysis. In: Encyclopedia of Dairy Sciences (2nd Edt).  Academic Press pp 908-916

Huppertz, T. (2016). Heat stability of milk. In: Advanced Dairy Chemistry: Volume 1B: proteins: applied aspects, pp. 179-196.

Nancollas, G.H. (1990) Physical chemistry of crystal nucleation, growth and dissolution of stones, in: Wickham J.E.A., Buck A.C. (Eds.), Renal Tract Stone, Metabolic Basis and Clinical Practice, Churchill Livingstone, Edinburgh. pp. 71–85  .

Nancollas G.H., Lore M., Perez L., Richardson C., Zawacki S.J., (1989) Mineral phases of calcium phosphate.  Anat. Rec. 224  pp. 234–241.

Nieuwenhuijse, H. and Huppertz, T. (2022) heat-Induced Changes in Milk Salts: A Review. Int. Dairy J. 126 (March) 105220 (Article)

Orme C., Giocondi J. (2007) Model systems for formation and dissolution of calcium phosphate minerals, in: Behrens P., Baeuerlein E. (Eds.), Handbook of Biomineralization: Biomimetic and Bio-inspired Materials Chemistry, Wiley-VCH, Weinheim. pp. 135–157

Sun, Y., Liu, R., Xu, J., Sun, Y., Gong, J., Long, L. (2023) A durable and environmental friendly superhydrophobic coatings with self-cleaning, anti-flouling performance for liquid-food residue reduction. Polymer Engineeering & Sci 63(4) pp. 1274-1288

Van Kemenade M.J.J.M., De Bruyn P.L., (1987)  A kinetic study of precipitation from supersaturated calcium-phosphate solutions. J. Col. Interface Sci. 118 pp. 564–585

Zouaghi, S., Barry, M. E., Bellayer, S., Lyskawa, J., Andre, C., Delaplace, G., … & Jimenez, M. (2018). Antifouling amphiphilic silicone coatings for dairy fouling mitigation on stainless steel. Biofouling, 34(7), pp. 769-783.