ABSTRACT DUNN, MARSHALL ROBERT. Viscosity and Gel

ABSTRACT

DUNN, MARSHALL ROBERT. Viscosity and Gel Formation of Micellar Casein Concentrates. (Under the direction of Dr. MaryAnne Drake).

Milk protein products have gained widespread use in the food and sports nutrition industries. Processing techniques, such as micro and ultrafiltration (UF), have led to the development of novel protein ingredients, particularly micellar casein concentrate (MCC).

Liquid MCC has unique rheological properties, specifically that it will form a thermo-reversible gel at colder temperatures. The mechanisms responsible for the rheological properties of MCC are not completely understood but are thought to be related to jamming of the micelles and to the dissociation of β-casein from the micelle at colder temperatures. This thesis determined the basis for viscosity increase and cold gelation of liquid MCC at protein concentrations from 6 to 20% during refrigerated storage. Skim milk (ca 350 kg) was pasteurized (72°C for 16 sec) and filtered through a ceramic MF system to make micellar casein concentrate (MCC). The liquid

MCC was immediately concentrated via a plate ultrafiltration (UF) system to 18% protein (w/w).

The MCC was then diluted to various concentrations (6 to 18%, w/w). Apparent viscosity readings were collected from liquid MCC samples (6, 8, 10, 12% protein w/w) at 4, 20, and

37°C. Instron compression force of MCC gels (14, 16, 18% protein w/w) was collected over a period of 2 weeks at 4°C. The maximum compressive load was compared at each time point to assess the changes in gel strength over time. Supernatants from MCC of 6.5 and 10.5% protein were collected after ultracentrifugation (100,605 x g for 2 h at 4, 20 and 37°C) and the nitrogen distribution (total, noncasein, casein, and nonprotein nitrogen) was determined. The entire experiment was replicated 3 times. The highest protein concentrations of MCC formed gels almost immediately on cooling to 4°C, while lower concentrations of MCC were viscous liquids.

The protein, casein, and casein as a percent of true protein in the liquid phase around casein micelles in MCC increased with increasing casein concentration of the MCC and with decreasing temperature. Casein as a percent of true protein at 4°C in the liquid phase around casein micelles increased from about 16% for skim milk to about 78% for an MCC containing 10.5% protein.

AV of MCC solutions in the range of 6 to 13% casein increased with increasing casein concentration and decreasing temperature. There was a strong temperature by protein concentration interaction for viscosity with AV increasing non-linearly with decreasing temperature at high protein concentration. MCC containing 16 and 18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels increased in strength over 10 days of storage at 4oC, likely due to migration of casein out of the micelles and interaction of the non-micellar casein to form a network that further strengthened the random loose particle jamming gel structure. With an increased understanding of the mechanism of cold thickening of MCC, there may be potential to replace hydrocolloids for thickening and stabilizing beverages with a clean label ingredient and also to provide a high level of protein with superior flavor and color.

Viscosity and Gel Formation of Micellar Casein Concentrates

Marshall Robert Dunn

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Food Science

Raleigh, North Carolina

2021

APPROVED BY:

______Dr. MaryAnne Drake Dr. Clint Stevenson Chair of Advisory Committee

______Dr. Dana Hanson

BIOGRAPHY

Marshall Robert Dunn was born on June 11, 1990 to his amazing parents, Holly and

Michael Dunn. Marshall spent most of his adolescence in Ithaca, NY and Salem, UT. Growing up, Marshall was exposed to food science through his father who worked as a food scientist and then taught the subject at Brigham Young University. In 2008 Marshall attended Brigham

Young University and followed in his father’s footsteps, receiving his Bachelor of Science in

Food Science, with a minor in Chemistry. Upon finishing his undergraduate, Marshall worked for a small, local, startup business for a year before accepting a job doing beverage R&D with

Bolthouse Farms in Bakersfield, CA. It was while working at Bolthouse that Marshall met his future wife, Lauren Gillespie, who helped to move his love of food science beyond the lab and into the kitchen. To further his education Marshall left Bolthouse and began his graduate studies at North Carolina State University with Dr. MaryAnne Drake in 2018. Towards the end of

Marshall’s master’s program, the world was hit with the COVID-19 pandemic, adding uncertainty to the future. It was during this time that Marshall and Lauren got engaged, married, and pregnant with their first child, all while finishing his thesis. Marshall hopes to be able to use his past experience and the knowledge gained during his master’s degree to positively impact the future direction of the dairy industry and to share his love of food and his passion of science with the rising generation.

DEDICATION

I would like to thank Dr. MaryAnne Drake and Dr. Dave Barbano for their immense help, direction, and support during my time as a master’s student, especially after the restrictions and changes from COVID-19. Your understanding, patience and help during these unprecedented times have been invaluable. I am eternally grateful for all the time you both spent helping me with my writing, interpreting data and helping me complete this thesis. I also want to thank Dr.

Hanson and Dr. Stevenson for being on my committee and helping me through this process.

I want to express my sincere gratitude to all my lab and classmates who helped me out along the way, I could not have done this without all of your help. I would especially like to thank the pilot plant crew- Brandon for being our fearless leader and a mentor, Daniel for being our resident ceramic MF operator, Alex for being a jack of all trades, and Hayden for being side by side with me every step of the way. I will forever feel bonded to you all.

My family was instrumental in helping me get through this master’s program; thank you for all your prayers and support. There were multiple times where I felt God’s guidance and I attribute it directly to all your prayers and faith on my behalf. Dad, I wanted to especially thank you for all your advice and help, it truly helped me to keep moving forward.

Most importantly I want to thank my amazing wife Lauren. The idea of finishing my degree and immediately marrying you was the dream that kept me sane throughout all the craziness. Without that light at the end of the tunnel I do not think that I would have been able to make it. And lastly, to my soon to be baby girl Kendall, ultimately this degree is so that you can have the best life I can give you. I love you and your mom more than I could ever express. This thesis is dedicated to you both.

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TABLE OF CONTENTS

LIST OF TABLES ...... vi LIST OF FIGURES ...... vii CHAPTER 1: LITERATURE REVIEW: THE PRODUCTION, PHYSICAL PROPERTIES, AND RHEOLOGICAL TESTING METHODS OF MICELLAR CASEIN CONCENTRATE ....1 Abstract ...... 2 Introduction ...... 3 Milk Fundamentals ...... 4 Structure of Casein Micelle Models ...... 5 Hydrophobic Properties of β-casein...... 8 MCC Production ...... 10 Liquid vs Spray Dried MCC ...... 15 Uses and Functionality of MCC ...... 19 Functional Properties of MCC ...... 25 Protein Concentration and Steric Effects ...... 26 Types of Fluids ...... 27 Gels ...... 29 Jammed Systems ...... 30 Cold Gelation of MCC ...... 32 Cold Gelation of HC-MCC ...... 33 Rheological Measurements ...... 37 MCC Protein Fraction Analysis ...... 47 References ...... 53 CHAPTER 2: VISCOSITY AND GEL FORMATION OF MICELLAR CASEIN CONCENTRATES ...... 74 Abstract ...... 75 Introduction ...... 77 Materials and Methods ...... 82 Experimental Design ...... 82 Processing of Micellar Casein Concentrate (MCC) ...... 83 Preparation of MCC Dilutions for Analysis ...... 85 Ultracentrifugation ...... 86 Instron Analysis ...... 88 Brookfield Analysis ...... 89

Chemical Analysis Methods ...... 90 Statistical Analysis ...... 90 Results and Discussion ...... 91 Protein Content of Micellar Casein Concentrates ...... 91 Protein Composition of the Aqueous Phase Around the Casein Micelles ...... 91 Impact of Protein Concentration and Temperature on Apparent Viscosity ...... 93 Impact of Protein Concentration and Temperature on MCC Gel Strength ...... 95 Conclusions ...... 96 Acknowledgments...... 97 References ...... 98

LIST OF TABLES

Table 1. Brookfield viscometer shear rates (1/s) used for micellar casein concentrates

(MCC) at 4, 20, and 37°C over the range of 6 to 12% protein...... 119

Table 2. Protein content of MCC and skim milk from each replicate (Rep)...... 120

Table 3. Casein as a percentage of true protein (relative %) in the ultracentrifugation (UC)

supernatants for skim milk (3.43% protein) and micellar casein concentrates at

6.93 and 11.51% protein as measured by Kjeldahl analysis...... 121

Table 4. True protein (%), casein as a % of true protein, casein (%), and estimated phase

volume of the MCC used for Instron analysis of gel strength...... 122

LIST OF FIGURES

Figure 1. Micellar casein concentrate (MCC) ultracentrifuged at 100,000 x g at 4°C for 2 h. .123

Figure 2. True protein concentration (%) in ultracentrifugation supernatants from skim

milk (3.43%) protein and micellar casein concentrates at 6.93 and 11.51%

protein ultracentrifuged at 4, 20, 37°C...... 124

Figure 3. Casein (CN) concentration (%) in ultracentrifugation supernatants from skim

milk (3.43% protein and micellar casein concentrates at 6.93 and 11.51% protein

ultracentrifuged at 4, 20, and 37°C...... 125

Figure 4. Apparent viscosity (AV) from Brookfield for MCC 6.54, 8.75, 10.66, and 13.21%

micellar casein concentrates at different temperatures of 4, 20, and 37°C...... 126

Figure 5. Log of apparent viscosity (AV) from Brookfield for MCC 6.54, 8.75, 10.66, and

13.21% samples at different temperatures of 4, 20, and 37°C ...... 127

Figure 6. Maximum compressive force (N) from Instron for MCC 15.6, 17.9, and 20.3%

protein over a 10-d of storage at 4°C...... 128

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CHAPTER 1: LITERATURE REVIEW: THE PRODUCTION, PHYSICAL PROPERTIES, AND RHEOLOGICAL TESTING METHODS OF MICELLAR CASEIN CONCENTRATE

M.R. Dunn, and M.A. Drake

Department of Food, Bioprocessing and Nutrition Sciences, Southeast Dairy Foods Research

Center, North Carolina State University, Raleigh, NC 27695

*Corresponding Author: MaryAnne Drake Box 7624, Department of Food, Bioprocessing and Nutritional Sciences North Carolina State University Raleigh, NC 27695-7624 Phone: 919-513-4598 Fax: 919-513-0014 E-mail: [email protected]

Abstract

Milk protein products have gained widespread use in the food and sports nutrition industries. Processing techniques, such as micro and ultrafiltration, have led to the development of novel dairy protein ingredients such as micellar casein concentrate (MCC). MCC has many benefits including a mild flavor, white appearance, heat stability, and maintaining the functional properties of the native casein micelle. One serious challenge in using spray dried MCC is rehydrating the product, as it takes several hours to fully rehydrate, which has limited its widespread adoption. This obstacle is avoided when MCC is left in its native liquid state and can subsequently be highly concentrated (HC-MCC) by ultrafiltration or vacuum evaporation to allow for better shipping costs. HC-MCC has been shown to have unique rheological properties, specifically that it will form a thermo-reversible gel at colder temperatures. The mechanisms responsible for the rheological properties of MCC and HC-MCC are not completely understood but are thought to be related to jamming of the micelles and to the dissociation of β-casein from the micelle at colder temperatures. Understanding and exploring the properties that MCC and

HC-MCC have will help formulators and manufactures to better produce and utilize this product.

Key Words: Milk, Micellar Casein Concentrate, Viscosity, Gelation

Introduction

Bovine fluid milk has long been a mainstay in the American and European diets over the past century and beyond (Barbano, 2017). Milk has been well established in having a positive effect on bone density, muscle mass, and vitamin A and D consumption and body weight control.

However, the consumption of fluid milk in the U.S. has been in steady decline over the past 50 years (Nielsen and Popkin, 2004; Stewart et al., 2012; Barbano, 2017). This is in part due to milk being present at less eating occasions, as well as the rise in alternative dairy products.

Throughout the last several years, although the fluid milk category remains down, the consumption of dairy products, as measured on a milk-fat basis, continues to increase. This increase is being primarily driven by consumption of butter, cheese, and whey protein powders

(USDA, 2020). These consumer consumption patterns in the U.S. have caused a surplus of skim milk; additionally, there are seasonal variations in milk production which lead to supply and demand issues throughout the year (Weldon et al., 2003). These issues have led to a need for a viable opportunity to process, use, and store the valuable components of skim milk.

Amelia and Barbano (2013) proposed a method using membrane technology of isolating and concentrating the valuable parts of skim milk, namely the casein and whey, for various uses and storage of up to 3 months. This process produces a high-quality whey stream, which has immense value in the sports nutrition and supplement industry, and a high purity casein stream known as micellar casein concentrate (MCC). The MCC produced could be used in beverage, cheese, yogurt, ice cream and other dairy applications where a high protein content would be wanted. Additionally, the MCC showed potential for storage in refrigerated conditions for up to

3 months.

While the MCC that Amelia and Barbano (2013) produced showed great promise, it did show some problems. Namely that upon concentration to 18% protein (w/w), the product formed a strong gel upon cooling to < 22°C. Adams and Barbano (2016) noted that If the MCC forms a gel upon cooling, it may be difficult to pump from a holding tank to another point in a manufacturing facility or to pump through plate heat exchange unit. This has created a need for exploration and research into the rheological behavior and the mechanism of the observed cold gelation in order to make this process more viable and provide the dairy industry with another tool for controlling milk supply and meeting customer needs.

Unfortunately, very little has been published in the way of looking at this gelation behavior. Lu et al. (2015, 2017), has looked at this phenomenon in the context of using a highly concentrated MCC (HC-MCC) created with spiral wound polymeric membranes. They analyzed the rheological behavior of the MCC via the use of electron microscopy and a rheometer.

However, the MCC that was used in their studies was not to the same purity level of that produced in Amelia and Barbano (2013), who used a ceramic membrane, in order to achieve serum protein removal rates of > 95%, compared with 70% by Lu et al. (2015, 2017). The ability to produce and control the rheological behavior of a liquid MCC concentrate is very valuable to the dairy industry, and the exact mechanisms are still not clearly understood. This literature review will summarize the current status of the literature in regards to MCC production, uses, rheological behaviors, and methods of analysis.

Milk Fundamentals

Bovine milk has been and continues to be a major part of the food industry landscape due to its high nutritional content, diverse applications, and varied products that arise from the functionality of its proteins (Fox, 2001). Fluid milk has a protein content that ranges between 30

4 to 36g/L (or about 3.3%). The proteins are generally classified into two groups: serum proteins

(whey) and caseins, with caseins being the main protein fraction in fluid milk, making up around

80% of the protein content. Casein has several different protein sub-species: β-Caseins, κ-

Caseins, αS1-Caseins, and αS2-Caseins; each has their own specific and unique functional characteristics which are largely based on the net charge of the protein and the distribution of that charge across the molecule at the pH of milk (around 6.6) (Fox and Brodkorb, 2008;

Huppertz et al., 2018).

In raw milk, the casein sub-subspecies assemble into structures known as casein micelles.

These micelles are spherical colloidal particles which range from 50 to 600nm in diameter with an average around 150nm (Fox & Brodkorb, 2008; Lin & Morr, 1971; McMahon & Brown,

1984). Interestingly, the term micelle was applied to these structures before it was discovered that they do not exhibit a classical micellar configuration (McMahon & Oommen, 2008). The casein micelle includes all four casein species which encompass calcium ions, and are held together and stabilized by unique physicochemical properties. These properties include the ability of phosphorylated serine residues— as well as the carboxylate groups on glutamate and aspartate— to bind calcium; and the amphiphilic nature of partially glycosylated κ-casein, which acts as a stabilizing component on the micelle surface (Huppertz et al., 2018).

Structure of Casein Micelle Models

Casein micelles are in a group of proteins classified as phosphoproteins (Horne, 2002,

2006; Fox and Brodkorb, 2008; Thorn et al., 2015; Huppertz et al., 2018). Caseins have also been described as supramolecules, or a system consisting of multiple molecular entities held together and organized by means of noncovalent intermolecular binding interactions (McMahon

& Oommen, 2008). As a phosphoprotein, caseins belong to a rather rare class of proteins. It

5 contains 0.7–0.9% phosphorus, which is covalently bound to the casein by a serine ester linkage

(Southward, 2003). As previously mentioned, the casein micelle is a spherical colloidal structure comprised of the varying casein sub-components β-caseins, κ-caseins, and αS-caseins (αS1-casein and αS2-casein). In bovine skim milk, the ratio of αS1-casein, αS2-casein, β-casein and κ-casein is

4:1:4:1 (Ekken and Olsinger, 2000). Additionally, the casein micelle has other non-protein components which are critical to its formation and functionality. Protein material represents roughly 95% of the dry matter of casein micelles, with the remainder being minerals collectively referred to as micellar calcium phosphate, also sometimes referred to as colloidal calcium phosphate. Micellar calcium phosphate consists primarily of calcium and phosphate, with lesser amounts of magnesium, citrate, and other minerals.

Frustratingly, the casein micelle falls into a category of proteins that are non- crystallizable for the use of x-ray crystallography in determining the precise structure of the molecule (Kumosinski et al., 1991); because of this, the structure of native casein micelles is not fully known. However, a wide variety of other methods and experiments have been done to try to predict and model the native micelle structure. The models for the structure of the casein micelle have shifted and evolved over the years and there have been many papers published reviewing the current theories (Dalgleish, 2011; De Kruif, 2014; Fox, Uniacke-Lowe,

McSweeney, & O’Mahony, 2015; Horne, 2006; McMahon & Oommen, 2008; McMahon &

Brown, 1984; Walstra, 1999). Three main structural categories have been proposed: sub-micelle model, nanocluster model and dual-binding model. The sub-micelle model is characterized by the overall structure of the micelle being comprised of smaller micelle subunits which are made of spherical aggregates of various casein species. These submicelles are linked together via

6 calcium phosphate and also allow for salt bridging and hydrophobic interactions which in turn create the casein micelle (Slattery and Evard, 1973; Walstra, 1990, 1999).

The nanocluster model describes the casein micelle structure as a cross-linked protein gel that is connected via calcium phosphate nanoclusters (around 2 nm). These nanoclusters act as the base “seeds” or scaffolding from which the casein molecules can bind to and then branch out and grow the cross-linked protein gel (De Kruif, Huppertz, Urban, & Petukhov, 2012; Holt &

Horne, 1996). The dual-binding model is more so a modification of the nanocluster model. It also uses calcium phosphate nanoclusters as one of the key binding elements in the model, however it also gives equal consideration to the hydrophobic interactions between the various casein species in determining the structure of the casein micelle. In fact, these hydrophobic interactions are key in explaining some of the key features of the micelle, such as having κ- casein dominate the outside of the micelle (Horne, 1998, 2002, 2006). Despite the various models that exist and continuously change and evolve, they all seek to be able to explain key behaviors and traits that have been tested and exhibited repeatedly in experimentation. These key features include:

 κ-casein must be located or positioned near surface to be able to stabilize the casein

micelle in aqueous phase.

 Chymosin must be able to rapidly and specifically hydrolyze most of the κ-casein.

 When heated, β-lactoglobluin should interact and form a complex with κ-casein.

 β-casein should be able to migrate out of the micelle as the temperature is lowered and

then reabsorb into the micelle when the temperature is then raised.

Casein micelles are highly hydrated and spongelike, having a hydration ratio of about 3.7 g H20/g protein. Relatively little of this water (about 0.5 g H20/g protein) is actually bonded to

7 the protein; the remainder is contained within and confined by the micelle. This water is also able to move within the confines of the micelle structure (Huppertz et al., 2017; McMahon &

Brown, 1984). The ratio of hydration is a very important property of casein micelles and generally encompasses all water contained in and around the micelle. This ratio of hydration is referred to the voluminosity of the casein micelle (Nöbel et al., 2012, 2016). The voluminosity of the casein micelle varies with pH, ionic strength, temperature and concentration, and it also impacts the apparent viscosity associated with the colloidal system (milk, MCC, etc.). Nöbel et al. (2012) showed that an increase in the voluminosity of the casein micelle was directly correlated to an increase in apparent viscosity. Furthermore, the research done by Nöbel et al.

(2016) found that the voluminosity of the casein micelle decreased with an increase in temperature, thereby reducing the apparent viscosity. This loss in volumniosity of the casein micelle was thought to be paritally due to the hydrophobic nature of many of the casein subspecies and temperature sensitivity of β-casein (Nöbel et al., 2012).

Hydrophobic Properties of β-casein

Hydrophobicity is known as the tendency of nonpolar molecules or particles to aggregate in an aqueous solution and organize themselves in a way that most minimizes contact or interactions with water (Chandler, 2005). It is directly correlated to Gibb’s Free Energy Law:

ΔG=ΔH-TΔS; where ΔG is the change in free energy of the system, ΔH is the change in enthalpy, T is temperature and ΔS is the change in entropy. A reaction will be spontaneous if the

ΔG is a negative value, if ΔG is positive, it doesn’t mean the reaction doesn’t happen, but only that it doesn’t happen spontaneously.

It is well known that non-polar molecules are not miscible in water. This is due to water molecules normally having disordered and highly interchangeable hydrogen bonds with one

8 another at room temperature and higher. When a non-polar molecule is introduced to an aqueous phase the water molecules reorient themselves in respect to the nonpolar surface in order to minimize disruption of the hydrogen bond network of water molecules. This rearrangement leads to a more highly structured water cage or shell around the nonpolar particle’s surface and hence a decrease in entropy (McMahon & Brown, 1984; Silverstein, 1998). Additionally, the water molecules that form the "cage" have restricted mobility which leads to significant losses in the translational and rotational entropy of water molecules making the process unfavorable in terms of the free energy available in the system (Haselmeier et al., 1995; Silverstein, 1998;

Chandler, 2005). Temperature plays an important role in hydrophobicity and solubility. This is driven by the unique properties of water, in that the structure of water becomes more ordered as temperature decreases, additionally, the density of water increases and maxes out at 4°C. The increase in order of the water structure as temperature decreases creates a situation of low entropy. This allows for reactions that would normally not be spontaneous, as they have high enthalpy (ΔH) values (aka they require energy), to become spontaneous due to the increase in entropy (ΔS) of the system, as ΔG will become negative if ΔS is large enough.

The interactions of β-casein with other casein fractions are dominated primarily by hydrophobic interactions, and as a result, some β-casein can dissociate from the casein micelles as water becomes more structured as temperatures are reduced when cooling milk (Sedmerova et al., 1974). Typically, up to 30% of β-casein dissociates from the casein micelles upon cooling, whereas the remaining fraction continues as part of the casein micelles and, when milk is subsequently warmed to adequate temperature (30– 40°C), all dissociated β-casein re-associates with the micelles (Rose, 1968; Downey and Murphy, 1970; Davies and Law, 1983; Ward and

Bastian, 1996; Thienel et al., 2018). The fractions of β-casein that readily dissociate from and

9 re-associate with the micelle during cooling and warming are molecules that are associated with other casein molecules via hydrophobic interactions but are not bound to calcium phosphate nanoclusters, as these interactions are too strong to overcome. Even though a proportion of the

β-casein can dissociate from the casein micelle at lower temperatures, this does not appear to result in disruption of the casein micelle, and it is able to stay intact (Huppertz et al., 2018;

Thienel et al., 2018).

β-caseins are the most hydrophobic of all the casein protein fractions. β-casein has a small 21-residue N-terminal with a net charge of -11.5 comprised of a single anionic cluster.

The remainder of the molecule is largely hydrophobic, this strong polar region only comprises one tenth of the protein molecule but contains over a third of the molecule’s total charge (Sauer and Moraru, 2012; Fox et al., 2015; Horne, 2017; Huppertz et al., 2018). This causes the properties of β-casein to be dominated by the large hydrophobic domain section and causes it to be very sensitive to temperature changes due to the entropic nature of hydrophobicity. These unique properties of β-casein need to be taken into account to determine any relation to the MCC cold-gelation phenomenon, in order to decipher any possible cause.

MCC Production

The concentration of casein began back in the 1960’s by utilizing the binding interactions of casein with calcium. When the binding interactions with calcium are destabilized, the casein micelles are dissociated, and the different protein fractions (casein and whey proteins) can be separated and extracted. The two methods traditionally used for separation of casein have been isoelectric (acid) precipitation and enzymatic hydrolysis of the κ-Caseins (renneting), and combinations of the two (Southward, 1985, 2003a). These processes have frequently been employed in the production of a wide range of casein products, which have applications as food

10 ingredients and in other areas (Southward, 2003b). Caseins produced by isoelectric precipitation

(acid casein) or enzymatic hydrolysis (rennet casein) have been widely available for many decades, and further products can be created with altered functionality through the reaction of acid casein with alkali to yield more soluble products such as sodium and calcium caseinate.

As previously discussed, the casein micelle contains several important and unique properties. The coagulation and heat stability properties of the casein micelle are of particular interest for product development purposes, as the coagulation of the casein micelles enables the production of products like cheese and yogurt. Due to the caseinates having no micellar structure, they were not useful in producing these types of products. Beginning in the 1970’s and culminating in the 1990’s, membrane filtration technology in the dairy industry enabled the creation of novel and superior ingredients (Pouliot, 2008), namely whey concentrates and MCC.

Unlike sodium and calcium caseinates, in MCC the micelle is whole and intact, thus retaining its desired functional properties.

There is no standard of identity of micellar casein concentrates in the US or Europe.

However, the American Dairy Product Institute defines a microfiltered milk product as either microfiltered milk protein or micellar casein (MC) that has an adjusted casein to whey protein ratio, whereas other milk protein ingredients like milk protein isolate (MPI) and milk protein concentrate (MPC) retain the same natural ratio of casein to whey protein found in milk, namely

80:20 (ADPI, 2018a). An MCC will have a higher casein to whey protein ratio than an MPC.

MCC is produced using the aforementioned membrane technology, specifically microfiltration, to separate the casein from the serum proteins and lactose. Microfiltration is a pressure driven separation technique using membranes with pore sizes ranging from 20 µm to 0.1 µm (Maubois,

2002). The composition of MCC is dependent on the method of processing used. MCC is

11 produced almost exclusively with skim milk to minimize fouling of membranes from fat globules. There are multiple types of membranes that can be used in MCC production, the most popular are polymeric spiral wound and ceramic membranes (Zulewska et al., 2009). Recently, stainless steel membranes have been evaluated for their efficacy and possible introduction into production plants (Zhang et al., 2018). These membranes have different structural properties with subsequent advantages and disadvantages, however they all function under the same principle of separating casein micelles and fat globules from serum proteins, lactose, vitamins and minerals, etc. using pore size and pressure (Hurt, Zulewska, Newbold, & Barbano, 2010;

Zulewska et al., 2009; Zhang et al., 2018). The efficacy of microfiltration is that the membrane pore size is such that it exploits the large difference in size between the casein micelles (0.2 µm) and the serum proteins (0.0036 µm). The typical pore size for microfiltration of casein observed in the literature is 0.1 μm with a range of 0.05–0.2 μm (Huppertz et al., 2017; Hurt & Barbano,

2015; Hurt et al., 2010; Zulewska et al., 2009).

Microfiltration in the dairy industry began in the late 1960s using ceramic membranes.

In the 1980s, cross-flow microfiltration became a viable technology due to the creation of uniform and low membrane pressure systems which require a recirculation pumping unit

(Saboya and Maubois, 2000; Maubois, 2002). Around 2000, the need for the extra pumping unit was eliminated, resulting in large energy and cost savings, due to the advent of microfiltration membranes known as Membralox GP® and Isoflux® (Maubois, 2002) which utilize continuous variations in porosity and in membrane thickness, respectively. The many different types of available ceramic membranes necessitate that the user and operator be aware of the specific target application and desired outcome before selecting a membrane, as there are stark differences even between ceramic membranes themselves as shown by Zulewska, Newbold and

Barbano (2009). They conducted serum protein removal tests from skim milk with ceramic

Membralox GP membranes, ceramic uniform transmembrane pressure (UTP) membranes and polymeric spiral wound membranes and found that the GP ceramic membrane allowed the greatest passage of casein into the permeate, but that the UTP ceramic membrane performed the best at not allowing passage of caseins into the permeate. Additionally, the overall removal of serum protein from the milk was slightly higher with the UTP membrane than with the GP membrane with levels of 64.4% and 61.04% respectively.

Currently, a large amount of membrane filtration in the dairy industry is carried out using polymeric spiral wound membranes, which cost less and require less energy, but which also have lower chemical stability and a shorter life when compared with ceramic membranes (Baruah et al., 2006; Zulewska et al., 2009). Polymeric spiral wound membranes are comprised of two flat membrane sheets along with interval spacers wrapped around a perforated tube which collects the permeate (Cheryan, 1998). As the feed material flows over the membrane surface, the permeate spirals its way to the middle of the perforated tube. The interval spacers are included in the assembly to promote turbulent flow, which helps to minimize fouling. The nature of the design and specifically the membrane support allows for operation under high membrane pressures, without causing damage to the membranes themselves (Saboya and Maubois, 2000;

Mistry and Maubois, 2017). While polymeric spiral wound membrane filtration predominates in many sectors, ceramic membranes have traditionally been used to produce MCC for targeted applications. For example, ceramics have traditionally been used in cheese making (Maubois,

2002), the more consistent nature of the pore sizes compared with that of spiral wound material, yield a more selective permeate and retentate (Nelson and Barbano, 2005; Karasu et al., 2010).

Additionally, the amount of polymeric membrane area needed to achieve the same level of

13 concentration for serum protein removal from skim milk is far less for ceramic than for spiral wound membranes (Hurt & Barbano, 2015). Ceramic membranes are also more resistant to extreme chemical and physical conditions and can withstand a pH range from 0.5 to 13.5 and temperatures >100°C (Baruah, Nayak, & Belfort, 2006; Karasu et al., 2010). However, ceramic membranes are more fragile and require a more gradual temperature change (<10°C per min) during processing to avoid cracking the membranes (Zulewska et al., 2009).

Recently, research has been done to analyze the efficacy of using stainless steel membranes for micellar casein and serum protein separation (Zhang et al., 2018) and has shown the membrane to be inert and highly durable. Additionally, it can also withstand elevated temperatures and more rapid temperature changes and pressures, concentrated solvents, and extreme pH ranges than polymeric and ceramic membranes. Stainless steel membranes are also able to sustain higher filtration flux (76.2 L m-2 h-1) than ceramic (54.6 L m-2 h-1) and spiral wound (16.2 L m-2 h-1) membranes (Zhang et al., 2018). The cost and energy usage associated with membrane processing is often the deciding factor for its adoption by the food industry.

Spiral wound systems, being cheaper and requiring less energy than ceramic systems, tend to be more widely used. The costs of an industrial-scale stainless steel membrane system is still uncertain, as this has only recently been studied and further research will be needed to determine efficacy and energy efficiency in order to properly determine return on investments (Zulewska et al., 2009; Hurt & Barbano, 2015; Zhang et al., 2018).

The purity of MCC is most accurately measured by the percentage removal of serum protein in the finished product as compared to the starting milk feed. It is important to note that this is different than referencing the protein content of the finished product, as the protein content can be a combination of both serum and casein proteins. The percent serum protein removal is

14 achieved by the type of membrane being used and by the number of stages with diafiltration being used in a bleed-and-feed method (Nelson & Barbano, 2005; Zulewska et al., 2009; Hurt &

Barbano, 2015). Diafiltration stages are when the retentate of the membrane system is diluted with reverse-osmosis or deionized water and is run through the membrane system a subsequent time to remove additional impurities. Typically, 3 to 4 stages of diafiltration will reach the limit of the system’s capacity to remove any additional impurities (Zhang et al., 2018; Amelia &

Barbano, 2013; Hurt et al., 2010; Lawrence, Kentish, O’Connor, Barber, & Stevens, 2008).

At optimal conditions the levels of serum protein removal from a single pass through a membrane system results in: ≈ 35%, 60% and 65% for spiral wound, stainless steel, and ceramic membranes respectively; and the level of removal after 3 diafiltration stages is about 70%, 90%, and 98% for spiral wound, stainless steel, and ceramic membranes respectively (Zhang et al.,

2018; Zulewska et al., 2009; Beckman et al., 2010). These differences in performance of serum protein removal lead to finished MCC products that can have differing functional properties and sensory characteristics (Nelson & Barbano, 2005; Karasu et al., 2010). The exact differences in chemical, physical and sensory properties between specific levels of serum protein removal have not yet been extensively studied and clarified. More work is necessary in this area to truly determine the need for purification regarding a specific application in order to maximize efficiency and cost to the manufacturer.

Liquid vs Spray Dried MCC

When MCC is produced via membrane filtration, the final product is a liquid protein solution which can later be used downstream in manufacture (e.g. cheese making, yogurt, etc.), or which can be further processed into powder via spray drying for use as a functional dry ingredient (Schuck et al., 2007; Schokker et al., 2011). The permeate from the filtration process

15 also has a lot of value for whey protein production as it contains high quality serum protein

(Amelia and Barbano, 2013).

Liquid MCC can be very useful for vertically integrated dairy companies, such as Greek- style yogurt, sour cream or cheese and whey powder companies, where shipment of liquid MCC isn’t required (Carter, Patel, Barbano, & Drake, 2018). In such a manufacturing setting, milk can be filtered and concentrated into MCC and added directly into the production of cheese, Greek- style yogurt, etc. (Amelia & Barbano, 2013; Bong & Moraru, 2014). Liquid MCC is also desirable from an ingredient addition standpoint because it is already hydrated and so avoids the need for a difficult rehydration step. Liquid MCC can be stored refrigerated for up to 16 weeks allowing for a unique alternative method for the storage of excess dairy milk during peak production seasons (Amelia and Barbano, 2013). At present, liquid MCC is not commonly used in industry outside of vertically integrated systems, and more research is needed to establish its uses and potential applications.

For companies that don’t sell direct to consumer or which are not vertically integrated, turning liquid MCC into a powder may be desirable. This is typically accomplished by spray drying, in combination with other processes. Spray drying ultimately serves to stabilize milk constituents for later use (Schuck, 2002). However, spray drying is an extremely energy intensive process and hence a costly technique. Due to the costly and energy intensive nature of spray drying, liquid MCC (or any other dairy liquid) may undergo a concentration step before spray drying to maximize the efficiency of the process (Schuck et al., 2016, 2015). Two main methods can be used to concentrate the MCC prior to spray drying, namely evaporation and reverse osmosis. Reverse osmosis is prohibitively expensive and subsequently, is not widely used. The most common method of evaporation used for concentration is a multi-stage or multi-

16 effect vacuum evaporation that uses thin film column (falling, rising, or both) evaporation

(Wiegand, 2007; Gourdon and Mura, 2017). Vacuum evaporation uses approximately 10% of the energy per unit of water removal at low solids content vs. spray drying (Schuck et al., 2015;

Grossbier, 2016). The process typically uses vertical columns in order for gravity to assist in the processing by “pulling” the liquid down the tubes under vacuum. Steam is applied to the outside of the tubes to indirectly heat the thin film of product running down the tubes and water is removed via steam evaporation (Gourdon and Mura, 2017).

Powdered MCC is desirable from a cost perspective as it saves on shipping costs, has a long shelf life due to the low water content, and takes up less space for storage; however, the major issue with powdered MCC is that it is extremely difficult to rehydrate (Nasser et al., 2018;

Nasser, Jeantet, et al., 2017; Nasser, Moreau, Jeantet, Hédoux, & Delaplace, 2017; Gaiani,

Schuck, Scher, Desobry, & Banon, 2007; Burgain, Scher, Petit, Francius, & Gaiani, 2016;

Zhang et al., 2018). The insolubility and resulting poor rehydration of MCC powders has long been the limiting factor preventing its wider spread use in the food and beverage industry (Zhang et al., 2018; Burgain et al., 2016). The main theory to explain the poor rehydration properties of

MCC powder is that the casein micelles are slow to release into solution from the powder particle and that this mechanism of casein micelle dissolution should in fact be considered the rate limiting step of MCC solubility rather than wetting of the actual powder (Mimouni et al.,

2009; Crowley et al., 2018).

Storage conditions need to be considered a crucial step in minimizing the loss of solubility of MCC powders over time (Burgain et al., 2016; Nasser et al., 2017; Schokker et al.,

2011). There seems to be many phenomena occurring during storage of MCC powder that directly affect its ability to rehydrate. Burgain et al. (2016) was the first to discover that the

17 surface of the individual MCC powder particles became rougher and hardened over shelf life, forming a skin-like surface layer. This formation of a skin layer at the surface is what is thought to be inhibiting the release of casein micelles into solution. Storage time and temperature were shown to be key components in affecting the severity of the surface hardening. Powders stored constantly at ≤ 20°C showed little change in rehydration rate and maintained a minimum of 80% solubility compared with a control for up to 12 months (Nasser et al., 2017).

Another phenomenon observed over the shelf life of MCC powder is the change in the secondary structure of the proteins with an observable loss of α-helix structure and potential increase in β-sheet formation (Nasser et al., 2018). The loss of α-helix structure is directly correlated to the loss of solubility. These changes are thought to be due to protein-protein interactions and cross linkage causing increased aggregation. The protein aggregation is driven by hydrophobic interactions resulting in an increase in powder particle size which has a direct and detrimental effect on the solubility (Havea, 2006; Schokker et al., 2011).

Despite these factors that contribute to poor MCC powder rehydration, there are steps and treatments that can be applied to improve the solubility of MCC powder. The main treatments currently involve the addition of acids (citrates), phosphates, caseinates and the use of salt ions prior to spray drying (Schuck et al., 2002; Schokker et al., 2011). However, a recent study by

Zhang et al. (2018) showed promising potential in the use of high intensity ultrasound. While rehydration of the casein micelle is increased by these enhanced dissolution methods, the end result for all of them is ultimately disruption or destruction of the casein micelle (Mimouni et al.,

2009; Schokker et al., 2011; Hussain, Gaiani, Aberkane, & Scher, 2011). The deterioration of the micelle structure allows for a more rapid dissolution of the free casein proteins and was shown to cause significantly improved rehydration over untreated powders in the aforementioned

18 studies. A potential problem caused by these pretreatments is that removal of the micelle structure may defeat the entire purpose of using membrane filtration to produce an intact native micellar casein concentrate; as the micelle structure is responsible for most of the functionality of native MCC.

The rehydration issues associated with spray dried MCC indicate that an alternative to powdered MCC needs to be made available before MCC is more widely adopted in the food and beverage industry. A concentrated liquid form of MCC could have the potential to overcome these rehydration issues while still cutting down on shipping costs for a traditional non- concentrated MCC liquid. However, more research is needed to fully calculate and maximize the cost structure for the production and distribution of a liquid MCC product.

Uses and Functionality of MCC

MCC is desirable as an ingredient because of its high nutritional value and its unique and wide ranging functional properties (Amelia & Barbano, 2013; Nasser, Moreau, et al., 2017;

Burgain et al., 2016; Silva, Balakrishnan, Schmitt, Chassenieux, & Nicolai, 2018). MCC has potential to make dairy product manufacturing more efficient, via the pre-removal of serum protein, as in the case of making cheese, Greek-style yogurt, etc. (Amelia & Barbano, 2013; Lu,

McMahon, & Vollmer, 2017; Bong & Moraru, 2014). MCC is heat stable compared to other proteins, especially those globular in nature, like whey protein; MCC also exhibits great water- binding, foaming, emulsifying, whipping and other properties that make it ideal for beverage, bakery, meat and other products (Beliciu, Sauer, & Moraru, 2012; Lu, McMahon, Metzger,

Kommineni, & Vollmer, 2015; Sauer, Doehner, & Moraru, 2012; Zhang et al., 2018).

Research into the use of MCC in cheese manufacturing mainly focuses on Cheddar, mozzarella and cottage cheese (Brandsma and Rizvi, 2001; Nelson and Barbano, 2005; Amelia

19 et al., 2013) . These cheese types represent some of the largest market categories of cheese consumption in the US (USDA, 2017). Due to the extremely high and increasing production volumes of these cheeses, any efficiencies and process improvements are of extreme value to the industry. One such possible process improvement is the use of membrane filtration technology in the cheese manufacture process. The economic feasibility of using membrane filtration prior to cheese making was shown to increase net revenue over traditional cheese making methods and this net revenue difference was shown to increase directly with the price of cream (Papadatos et al., 2003).

The cheese making process requires removal of a large percentage of the serum protein, which produces the byproduct known as sweet or Cheddar fluid whey. Removing the serum protein via membrane filtration before processing helps to reduce the use of rennet and helps make the process more efficient from a yield standpoint (Papadatos et al., 2003; Nelson and

Barbano, 2005; Lu et al., 2015, 2017). In addition to the cheese making process becoming more efficient, the serum protein produced via membrane filtration of skim milk is a superior source for whey protein powder production than that obtained from Cheddar whey. This is because the serum protein obtained from the permeate of skim milk membrane filtration, does not contain or contains very little residual components from the cheese making process (ie. rennet, fat, glycomacropeptides, colorants, acids etc.) leading to a superior tasting product (Zulewska et al.,

2009; Beckman et al., 2010; Evans et al., 2010; Amelia and Barbano, 2013; Amelia et al., 2013).

Research in Cheddar cheese production has shown the potential for liquid MCC use in standardizing protein levels, specifically casein, in milk mixtures before processing which has led to higher yields and superior whey streams as previously mentioned (Nelson and Barbano,

2005). Research into the use of MCC for Cheddar cheese manufacture has shown potential to

20 create a low-fat Cheddar cheese that has superior texture and nutrition compared to full fat

Cheddar cheese. While the flavor was not at parity with full fat cheese, modifications using enzyme modified cheese to enhance aged notes and cover off-flavors could result in consumer acceptance against a full fat Cheddar (Amelia et al., 2013). Brandsma and Rizvi (2001) showed that using liquid MCC in the production of low moisture part skim mozzarella cheese resulted in cheese that had improved texture and functional qualities along with parity of flavor. As previously mentioned, a high-quality whey protein stream is also produced from the skim milk permeate during the filtration step.

Excitement for the use of MCC in the production of cottage cheese has less to do with the final product being improved and more to do with the potential elimination of acid whey, which is a byproduct of cottage cheese, ricotta, and Greek yogurt production and a major waste stream in the dairy processing industry (Bong and Moraru, 2014). By removing the serum protein in the membrane filtration step, the amount of acid whey can be significantly reduced. In turn, permeate produced via filtration becomes a high quality source of whey protein (Nelson and

Barbano, 2005).

Lu et al. (2015, 2017) along with Amelia & Barbano (2013) showed the potential use of a highly concentrated liquid MCC (HC-MCC) varying from 18 to 24% protein in cheese making.

The HC-MCC was produced identically to MCC (using microfiltration of skim milk with either a polymeric or ceramic membrane) except an additional concentration step was added, using either an evaporator in the case of Lu et al. (2017) or an ultrafiltration (UF) system in the case of Amelia and Barbano (2013). HC-MCC can be combined with cream to produce a cheese milk that is very suitable for cheese making using existing cheese making equipment. A phenomenon that occurred in both experiments with HC-MCC was that the highly concentrated liquid would solidify and

21 form a strong, thermos-reversible gel at temperatures as high as 38°C depending on protein concentration. Research into the development of liquid HC-MCC has been limited and more research will be needed to accurately identify its rheological properties and mechanism of gelation.

The use of MCC in yogurt processing has been another application that has seen an increase in research during recent years mainly driven from the meteoric rise in the consumption of Greek-style yogurt (Peng, Serra, Horne, & Lucey, 2009; Bong & Moraru, 2014). The purity of the serum protein permeate from membrane filtration along with the need to remove said serum protein makes Greek-style yogurt an appealing opportunity for the application of MCC

(Bong & Moraru, 2014). Acid whey which is produced as a byproduct of the Greek-yogurt manufacturing process has long been a source of concern from sustainability, cost and efficiency perspectives (Bong and Moraru, 2014). Using membrane filtration to produce a usable, non-acid whey stream from Greek-style yogurt manufacturing would result in outcomes that are multifold, as the farmers, manufacturers, consumers and environment would all potentially benefit.

Spray dried MCC powders have been analyzed for use in yogurt application with the general consensus in most cases being that the fortifying of milk with MCC did not create a better yogurt as measured by gel strength, whey separation, and sometimes fermentation time; conversely, sodium caseinate showed much improved gel strength and whey retention in comparison to MCC and a control (Bong & Moraru, 2014; Peng et al., 2009; Karam and Gaiani,

2012; Karam, Gaiani, Barbar, Hosri and Scher, 2012).

Despite these unpromising results, certain studies have found more specific uses in the type of yogurt production, percent protein MCC, and rehydration time of powders (Bong and

Moraru, 2014). Due to the poor rehydration properties of MCC, it becomes imperative during the yogurt make process to obtain full hydration of the MCC powder. This rehydration time was

22 found to vary between 15 to 24 hours (Karam et al., 2012). With adequate rehydration, the protein content of the powder was shown to play a significant role in the work done by Bong and

Moraru (2014). Their work tested a control Greek-style yogurt against yogurt fortification with

58% and 88% protein MCC powders; with the results indicating that the 88% protein MCC created a weaker yogurt gel and led to an increased rate of whey separation, whereas the 58% protein MCC yogurt maintained parity with the control. These results show that there is potential use for MCC in yogurt manufacture. More research is needed with the use of liquid

MCC, especially in varying protein levels, as all the literature has been focused on yogurt mix fortification with spray dried MCC powder. Additionally, research could be done to see if a liquid HC-MCC could be of potential use when reconstituted to appropriate levels, thus saving on shipping of milk.

MCC has potential use as an ingredient in high protein beverage application products due to its high heat stability, high protein and low carbohydrate content, white color (in liquid), clean flavor and mouthfeel (Beliciu et al., 2012; Sauer & Moraru, 2012; Hurt et al., 2010).

However, a reason that it has not been more readily incorporated is due to the aforementioned difficulty of rehydrating spray dried MCC powder. The high heat stability of MCC compared to that of other proteins has been one of the main reasons it is of interest for protein beverages. In the research done by Beliciu et al. (2012), they compared the stability of spray dried MCC vs liquid MCC ingredients of varying protein content (5 to 10%) at UHT, Retort and Control commercial sterilization processing conditions. Lower protein concentrations as well as being processed at retort conditions vs UHT conditions were generally more favorable from a stability standpoint. The results also indicated that in addition to the protein content and thermal processing conditions, the treatment of the MCC products themselves (liquid vs spray dried) had

23 a significant effect on heat stability to the sterilization processes with spray dried MCC being more unstable than liquid MCC. Some of the other differences observed were in the ζ-potential, pH and apparent viscosity. The change in ζ-potential is crucial as this is believed to be one of the main driving forces in solution stability. The spray dried MCC had a significantly lower ζ- potential which would cause less repulsion between micelles and subsequently lead to more aggregation. These observed differences in the spray dried MCC resulted in products that were significantly more unstable to UHT treatment. In addition to its high solubility, this is another reason why liquid MCC may be of value for protein beverage manufacturers. However, more research is needed in this area to fully understand the mechanisms responsible for the destabilization of liquid and spray dried MCC as a result of UHT treatment.

Milk production has seasonal cycles and having a method for storing a concentrated milk substitute would potentially have value for the industry (Weldon et al., 2003; Amelia and

Barbano, 2013). The variation in protein content of milk along with the shipping costs during peak seasonal cycles are areas in the beverage manufacturing space where opportunities for improvement exist. A liquid MCC with a shelf life of 16 weeks or greater could help meet this need and could also have potential in protein beverage application. Companies that are vertically integrated or who use liquid milk as an ingredient or base in the formulation of their protein beverages would be able to save on shipping and storage of their milk product and be able to standardize the protein content to ensure quality and consistency (Amelia and Barbano, 2013).

The research space for liquid MCC use in beverage application is still relatively limited and more work is needed to find optimal uses and levels of protein concentration and purity of the

MCC for the varying types of commercial beverage processing.

Functional Properties of MCC

While casein ingredients have use as major ingredients or as substitutes for major constituencies of food products due to their nutritional value, they can also be used for their diverse functional properties, namely their ability to gel, foam, and emulsify (Huppertz et al.,

2018). These uses have been dominated by the caseinate ingredients (sodium caseinate, calcium caseinate, etc.) and are used in a wide variety of products e.g. glues, paints, cheese analogs, synthetic whipping creams, meat products and others (Southward, 2003).

However, there is limited literature available regarding the testing of functional properties for liquid MCC specifically. Carter et al. (2018) evaluated various functional aspects of dairy protein products, including spray dried MCC and liquid MCC. Some of the attributes tested in their research were overrun, foam stability, heat stability and sensory characteristics. MCC as an ingredient was shown to have better foaming and higher foaming stability than that produced by

WPI and MPC. Their results also showed significant differences between the liquid and spray dried MCC, with liquid MCC having superior overrun and yield stress but no significant difference was detected for overall foam stability. The sensory qualities, specifically off-flavors, were noticeably more apparent in the spray dried MCC. All of the spray dried powders (WPC,

WPI, MPC, MCC) had significantly higher levels of hexanal, Z-2-heptenal, octanal, 2-nonenal,

E-2-nonenal, pentanal, 2-methyl butanal, and 1-octen-3-one, which previous research has shown to be associated with protein degradation and lipid oxidation (Whitson et al., 2010; Jervis et al.,

2012; Smith et al., 2016). Additionally, only spray dried MCC developed an undesirable corn chip note from the spray dry processing (Carter et al., 2018). Liquid MCC may have potential benefits when used over other dairy protein products, as well as over the more common spray dried forms, due to superior functional and sensory attributes.

One of the main functional uses of casein and casein derivatives is that of emulsification.

Emulsions consist of mixtures of at least two immiscible liquids (such as oil and water), with one of the liquids being dispersed as droplets or micelles in the other. When producing food emulsions, it is important to control both the emulsion formation and the emulsion stability during manufacturing and storage to ensure final quality over the shelf life of the product.

Lazzaro et al. (2017), classified emulsifying agents according to two main properties: their ability to facilitate the blending of the emulsion phases (i.e. emulsifying capacity) and their ability to stabilize the emulsion (i.e. emulsion- stabilizing capacity). The emulsifying capacity of casein is based on its ability to adsorb at the interface of two immiscible liquids (polar and nonpolar) and reduce the surface tension, thereby enabling the blending of the two phases. The stabilization effects of caseins are due to the rigidity provided by the aggregated micelle adsorbed at the interface. Casein products with low aggregation, such as sodium caseinate, have enhanced emulsifying properties but are less effective for the stabilization of emulsions than highly aggregated casein micelles (Courthaudon et al., 1999; McClements, 2016; Lazzaro et al.,

2017).

Protein Concentration and Steric Effects

The rheological properties of a material are the result of the interactions of the molecules that make up said material. An important factor that impacts the molecular interactions are steric effects. Steric effects are nonbonding interactions that influence the conformation and reactivity of ions and molecules. Steric effects result from the interaction between negatively charged electron clouds which produces a repulsive force. This repulsive force makes molecules repel and distance themselves at distances and angles that will minimize contact between the electron clouds of the interacting molecules or ions. The most commonly cited steric effect is steric

26 hindrance, which is a repulsive force experienced when electron clouds between two molecules interact, but it is specific to the situation of chemical reactions. Steric hindrance is a representation of the slowing or hindrance of a chemical reaction due to reducing access of the reaction site for the “attacking” molecule or ion (e.g. a hydroxide ion attacking a methyl bromide vs an ethyl bromide). Steric hindrance is crucial in organic and biochemistry for determining the rate of reactions between molecules, the function of an enzyme, etc. Steric repulsion is the repulsive force experienced by the interacting electron clouds of molecules or ions, but in a situation in which a reaction is not expected to occur (e.g. two methane molecules colliding).

Steric strain is the repulsive force felt between the electron clouds of atoms within a single molecule, but which are not directly bonded to each other. This is responsible for the conformational geometry of molecules, as well as the folding of proteins (Dickinson and

Eriksson, 1991; Damodaran, 2017).

Types of Fluids

In rheology there are many different classifications for fluids and viscoelastic materials.

Fluids can be separated into many different categories based on the properties exhibited by the liquid. These classifications can be characterized by the fluid’s shear stress response to shear rate – Bingham plastic, pseudoplastic fluid (non-Newtonian), dilatant fluid, Newtonian fluid – each of these fluid types has a different shear stress response to the rate of shear applied to the fluid. Newtonian fluids have a linear relationship between shear rate and shear stress, most commonly seen in water and milk; dilatant fluids have an exponentially increased shear stress response to shear rate, referred to as shear thickening; pseudoplastic fluids, also known as power law or Herschel Buckley liquids, exhibit an asymptotic leveling-off in response to shear rate, referred to as shear thinning; Bingham plastics exhibit a linear response to shear rate similar to

27 that of Newtonian fluids except a Bingham plastic has a resting (0 shear rate) shear stress > 0.

Similarly, some non-newtonian fluids, such as Herschel Buckley fluids, also have a resting shear stress > 0, but as mentioned exhibit shear thinning behavior after flow has been achieved.

Milk, and more specifically, MCC are classified as colloidal systems. Colloids are mixtures in which a solute that is too large for a traditional solution (e.g. salt, sugar, etc.), but too small to be visibly seen, is dispersed and suspended throughout a solvent. There are typically two types of colloidal systems, lyophilic (“solvent loving”) and lyophobic (“solvent hating”).

Lyophobic systems will not occur spontaneously and require energy to maintain, making them highly unstable. Lyophilic systems are frequently stabilized via the use of amphiphilic molecules, which generally contain a polar head and hydrophobic tail (Vilet and Walstra, 2017).

In the case of milk, κ-casein acts as the amphiphilic stabilizer for the casein micelle. These amphiphilic molecules will tend to arrange themselves in a traditional micelle structure to reduce the free energy of the system. For the colloidal system of MCC, the solutes are the casein micelles and fat globules and the solvent is water (Zhao and Corredig, 2016).

Colloids can be in the form of a dispersion, gel or foam. The properties of the casein micelle colloid can be seen in all three of these phases – as a dispersion in milk, as a gel in cottage cheese, or as a foam in a milk foam. Depending on concentration and temperature, MCC can be a Newtonian fluid or a non-Newtonian fluid. For example, MCC at lower concentrations exhibits Newtonian behavior, while higher concentrations exhibit power law behavior, and extremely high concentrations have even been shown to exhibit Herschel Buckley behavior; which, as mentioned previously are characterized by their shear thinning properties (Beliciu &

Moraru, 2011; Sauer et al., 2012). In the experiments done by Sauer et al. (2012) they showed

28 that as the rate of shear applied to MCC increased, the apparent viscosity of the MCC decreased.

Understanding these properties are critical for the proper handling in an industrial setting.

Gels

Food gels are usually used for a specific purpose within a food system, often to obtain or maintain a specific consistency or to provide structural stability and physical integrity to the food system. Gels are a two-phase system with molecules of the colloid confining the aqueous phase and also being supported by the aqueous phase. There are many different types of gels that exist in food systems – polymer gels, particle gels, cold-set gels, heat-set gels – the two main divisions of gels in food systems are polymer and particle gels whereas cold-set and heat-set gels are ways to define how the gelation process is induced. Some gels may not necessarily be induced by temperature but by changing the conditions that affect the colloidal interactions of a solution via pH, ionic strength, change in solvent, enzyme activity, etc. This is the case in most milk gels, as the disruption of the casein micelle colloidal system is often the target for gelation. The matrix of a polymer gel consists of long, linear chain molecules, each of which is cross-linked to other molecules at various places along the chain referred to as junctions. These cross-link junction zones consist of many individual bonds which are predominantly non-covalent interactions.

Particle gel networks generally have much larger pore sizes than that of polymer gels and consist of agglomeration of linking of individual particles. In milk or MCC, the particle would be the casein micelle. These gels can form due to aggregation of particles that are made to attract each other due to a change in pH, ionic strength or solvent quality (Vilet and Walstra, 2017). The fracture stress or strain at fracture of a gel is one of the most important physical characteristics of a gel. It is integrally related to the firmness, strength and sensory or eating experience of a gel.

Milk and milk protein fractions have long been used for their ability to create gel structures. Cheese and yogurt are two the most common food gels and both involve the gelation capability of the casein micelle. As previously mentioned, the gelation capacity of the micelle is intricately connected to the outer layer of κ-casein protein strands. The κ-casein strands protruding from the surface of the micelle are negatively charged which allows the micelle to interact with water and also helps to repel other micelles and prevent coalescence. If the κ- casein is removed or altered (via chymosin or pH change), then the casein micelles are no longer repelled and therefore aggregate, forming a particle gel.

It is unclear which type of gel or induction category that MCC cold gelation would be classified as. It is likely that this gelation is a particle gel because most milk and casein gels, such as yogurt, are well-researched particle gels. The properties of MCC ensure that the casein micelle is in its native state and would therefore lend itself to particle gel formation. The induction of the gel does seem to be temperature dependent, but it is also dependent on the casein concentration (Lu et al., 2015, 2017). Additionally, the reversible nature of the gel would imply that it is due to changing conditions of the colloidal stability; in this case, changing the free energy of the MCC system. More research is needed to accurately determine these classifications.

Jammed Systems

The idea described by Lu et al. (2015) that the close packing of the casein micelles is what is causing the solidifying of the HC-MCC is possibly best characterized through the lens of a jammed system. A jammed system is based on the Kepler Conjecture of the packing of hard spheres into a Euclidean geometrical container and that with an ideal arrangement, the spheres can take up a maximum theoretical volume of about ϕ = 0.74%, this was accepted as a

30 mathematical proof in 2017 due to the work of Thomas Hales and his team (Hales et al., 2017).

This is the ideal and theoretical model, using perfectly round and perfectly hard spheres. Under these conditions there is no possibility of movement for the spheres and the system is solid. The complexity of this ideal system increases when a solvent or fluid medium is added to the cylinder to fill the remaining 0.26 volume fraction. The rheology of the system is now dependent on the properties of the solvent itself and with how it interacts with the surrounding spheres. Colloidal hard-sphere particles of narrow-size distribution exhibit crystalline and glassy states beginning at the particle volume fractions ϕ= 0.494 and ϕG = 0.58, respectively (Mezzenga et al., 2005;

Loveday et al., 2007). Consequently, if the spheres are not perfectly hard and are malleable to any degree, the rheology is now also dependent on the internal makeup of the spheres.

Obviously, few real-life products meet the criteria of an ideal system and food is no exception.

In reality, casein micelles are not frictionless monodispersed spheres and Silbert, (2010) indicated that as compressed, soft-sphere packings are decreased in concentration to the point of the jamming transition (i.e., the point where a jammed packing loses mechanical stability), the system may form a random loose packing solid structure that occurs at lower ϕ than the expected jamming ϕ due to friction among the particles. As the friction among the particles increases, the critical ϕ of the particles for solid or gel structure formation occurs will be reduced.

Research into the rheological properties of colloidal systems that have high volume fractions have shown that casein micelles in milk concentrates follow typical ideal hard sphere models up until a specific critical concentration value after which it diverges significantly. This critical fraction volume for casein micelles was at around 54% (Mezzenga et al., 2005). In the research by Loveday et al. (2007) they showed that using viscosity models developed for hard sphere dispersions accurately predicted the trends but not the absolute values of protein

31 dispersions. When increasing the volume fraction of a colloidal system, if the particles have more attraction to each other it will become a gel system, if the particles have less attraction to each other or friction, the volume fraction of the system can continue to increase and lead to a jammed system if enough colloidal particles are added (Mezzenga et al., 2005).

Cold Gelation of MCC

As discussed previously, powdered MCC is notoriously difficult to rehydrate. because

MCC powder presents so many challenges in the rehydration process, and to aid in the cheese making and milk storage processes, Lu et al. (2015) and Amelia et al. (2013) both developed different versions of a liquid HC-MCC. While these products avoid the challenges of powder rehydration, they do present the challenge of a strong, thermoreversible, gelation mechanism at lower temperatures (Amelia and Barbano, 2013; Lu et al., 2016). These products produced by

Lu et al. (2015) and Amelia et al. (2013) had varying protein contents with a range of 18 to 24% and varied with regards to the removal percentage of serum protein (70 to 95%) (Amelia and

Barbano, 2013; Lu et al., 2016).

The difference in composition of the two HC-MCC products is due to the manufacture process. Amelia et al. (2013) produced their HC-MCC using a ceramic membrane filtration system and 3 stage filtration process followed by an additional ultra-filtration step at the end to concentrate the protein content to 18% (w/v). Lu et al. (2015) produced their HC-MCC using a spiral wound polymeric membrane filtration system with a 3-stage filtration process followed by a vacuum concentration step to achieve a protein level of around 23% protein (wt/wt). As previously mentioned, ceramic membranes can achieve higher levels serum protein removal, so the researchers were only able to achieve a serum protein removal of around 70%. It is unclear

32 what impact variations in MCC purity will have on the rheological and functional properties of

MCC, so more research is needed in order to determine what, if any, differences exist.

Cold induced gelation presents challenges in manufacturing settings, as storage of HC-

MCC would require refrigeration temperatures, but transportation and pumping would necessitate the HC-MCC to be in a liquid state. It is important to note that 18% protein HC-

MCC is pourable and pumpable at room temperature (22°C) (Amelia and Barbano, 2013); however, the gel of ≥21% protein HC-MCC can only be broken up by heating to around 50°C

(Lu et al., 2017). These observations demonstrate how crucial protein concentration is on the feasibility of HC-MCC in a manufacture setting. The properties and mechanism of this cold gelation phenomena still requires further study and research, but Lu et al. (2015) and Lu et al.

(2017) have done preliminary work in this area to try and explain the properties of this gelation mechanism.

When determining the mechanism(s) determining the cold gelation properties of MCC, it is important to understand the individual functional properties of the differing protein fractions that make up the casein micelle. The differing functionalities of these protein fractions, especially temperature sensitivity, will help to illuminate possible physical and conformational changes to the micelle that may be related to cold gelation. Notably, multiple studies have been conducted to demonstrate and record the unique temperature dependent properties of β-casein

(Livney et al., 2004; Holland et al., 2011; Sauer and Moraru, 2012; Huppertz et al., 2018;

Thienel et al., 2018).

Cold Gelation of HC-MCC

Lu et al. (2015) used transmission electron microscopy to determine the relative distance between casein micelles in an HC-MCC system, measuring between 20 to 50 nm. Lu et al.

(2015) proposed that the cold gelation observed in HC-MCC solutions with protein concentrations >23% (wt/ wt) was caused by steric interference the casein micelles being packed so closely together that their outer tendrils overlapped and inter-penetrate the hydration spheres of each other. The close spatial proximity of the casein micelles and their tendrils may also permit calcium bridging to form between negatively charged domains on the proteins. Lu et al.

(2015) further propose that cold-gelation of HC-MCC occurred when the kinetic energy of the system was sufficiently reduced to inhibit the mobility of casein micelles and their ability to move in relation to other adjacent micelles. At higher temperatures, the resulting increase in kinetic energy of the HC-MCC system was enough to enhance the mobility of surface protuberances and the casein micelles themselves, resulting in less drag while moving past each other causing the gel to melt. When the temperature was again decreased, the reduced kinetic energy lead to restricted movement and gelation of HC-MCC gel (Lu et al., 2015).

HC-MCC is an ingredient designed to be used in the production of other finished products, most commonly cheese. In the production of cheese, the HC-MCC is combined with cream and skim milk to create a recombined concentrated milk (RCM) that has high casein and low serum proteins which makes it ideal for cheese making. Lu et al. (2017) additionally investigated whether a RCM would retain the gelation properties of HC-MCC even with a decreased protein content (around 12%) and the addition of fat. They found that a 12% RCM still experienced cold gelation, but experienced cold gelation at a lower temperature (≤12°C), than that of the high protein HC-MCC. Additionally, the temperature at which the gel formed depended on a variety of factors such as protein level, pH, and calcium and salt additions.

The mechanism for gelation as proposed by Lu et al. (2015) becomes less apparent when using an RCM as the casein micelles are spaced further apart and consequently should

34 experience less steric interference. They hypothesized that the increase in free protein strands might result in entanglements which could restrict movement of micelles and lead to possible calcium bridging. This is thought to be because, as previously explained, at high temperatures, the casein micelles stay more tightly bound due to temperature dependence of hydrophobic interactions. Whereas, at colder temperatures the protein strands are less tightly bound to the micelle. This allows protein strands, most likely β-casein, within the micelle to disassociate and extend further outward allowing for intermolecular interactions with each other (Lu et al., 2017).

The cold gelation temperature (CGT) of the RCM was directly correlated to pH when tested between pH 6.4 to 7.0. For every 0.1 reduction in pH, the CGT decreased by around 7°C, and this was consistent across all protein levels (8 to 12%) (Lu et al., 2017). The reason for a

CGT increase as pH increased was thought to be due to more casein being dissociated from the micelle as pH increased which caused more steric hindrance (Lu et al., 2017). Calcium addition had differing effects depending on the levels of calcium added. If added at ≤0.12mmol/g casein then it had no effect, however if added at ≥0.17mmol/g casein then it caused an increase in CGT.

Citrate addition also showed a significant and linear relation to CGT. As mentioned previously, citrate addition was linked to the destruction of the casein micelle which would result in the release of more free protein strands into solution which could be leading to entanglement of protein strands that can restrict movement. The two studies by Lu et. al (2015 and 2017) do a great job at setting preliminary ground work for the research into MCC cold gelation, however, neither of these studies took the shelf life of the MCC in to account as it relates to the gel strength of the MCC. Additionally, the MCC used in the studies were at a purity of 70 to 75% serum protein reduced, having been manufactured using polymeric spiral wound membranes.

This is much lower purity than can be accomplished using ceramic membranes and MCC with

35 purity levels of >90% serum protein reduced should also be evaluated to limit confounding effects from serum proteins.

The theory proposed by Lu et al. (2015) of casein micelle overcrowding is analogous to what is referred to as a jammed system by rheologists. A “jammed” system contains a particle configuration in which each particle is in “contact” with its nearest neighbors in such a way that structural stability of various types is conferred to the system (Torquato and Stillinger, 2010).

The “packing fraction” (ϕ) is the fraction of space or volume taken up by the particles. The homogeneity of the particle sizes is key in determining how crowded and packed the spheres can become, additionally whether the particles are dry or in a liquid medium, and whether the particles are compressible or not. Non-compressible particles are referred to as “hard” particles.

In a single particle mixture with homogenous size and hard (non-compressible) dry spheres, the packing fraction reaches a natural limit at ϕ=0.64 wherein the material exhibits solid-like behavior, this has been shown to be the repeatable limit for random close packing (Aste et al.,

2005; Kamien and Liu, 2007; Silbert, 2010; Torquato and Stillinger, 2010). For multi particle solutions or for solutions that contain compressible particles, as is the case in most food systems, the value of ϕ that produces similar solid-like characteristics is much higher (Biroli, 2007;

Thomar et al., 2012). Biroli (2007) has even proposed that jamming development is another type of phase transition, akin to glass transition and crystallization.

The complexity of jammed systems increases when a solvent or fluid medium is added to the cylinder to fill the remaining volume fraction. The rheology of the system is now dependent on the properties of the solvent itself and with how it interacts with the surrounding particles.

Colloidal hard-sphere particles of narrow-size distribution exhibit crystalline and glassy states beginning at the particle volume fractions ϕ = 0.494 and ϕ = 0.58, respectively (Mezzenga et al.,

2005; Loveday et al., 2007). Consequently, if the spheres are not perfectly hard and are malleable to any degree, the rheology is now also dependent on the internal makeup of the spheres. Obviously, few real-life products meet the criteria of an ideal system and food is no exception. In the case of HC-MCC the casein micelles are considered the spheres and the solute or medium is water. Research into the rheological properties of colloidal systems that have high volume fractions have shown that casein micelles in milk concentrates follow typical ideal hard sphere models up until a specific critical concentration value after which it diverges significantly

(Mezzenga et al., 2005). This critical fraction volume for casein micelles was at around 54%. In the research by Loveday et al. (2007) they showed that using viscosity models developed for hard sphere dispersions accurately predicted the trends but not the absolute values of protein dispersions. When increasing the volume fraction of a colloidal system if the particles have more attraction to each other it will become a gel system, if the particles have less attraction to each other the volume fraction of the system can continue to increase and lead to a jammed system if enough colloidal particles are added (Mezzenga et al., 2005).

Rheological Measurements

Rheology is the discipline in physics that studies the deformation and flow of matter.

Foods can be classified in different ways, such as solids, gels, homogeneous liquids, suspensions of solids in liquids and emulsions (Gallegos and Franco, 1999; Fischer et al., 2009). These materials will vary in how they respond to an applied force. Some characteristics that are of importance in rheology are the elasticity, viscosity, creep and yield stress. Elasticity is the reaction of solids to a force and is the ability of a material to absorb a force through deformation and then return to its original state after the force is removed. Viscosity is a liquid’s response to force and is the capacity of a material to resist flow. Some materials exhibit both flow and

37 elastic responses to applied forces, depending on the rate at which the force is applied; these materials are considered viscoelastic (Miri, 2010). Rheology measurements typically involve applying a force to a material and measuring the deformation that occurs.

Solids can be broadly characterized as either being crystalline or amorphous. Crystalline solids have a highly ordered structure to their microscopic constituents which form into lattice formations. Amorphous solids have no such wide ranging or pervasive order. Due to this order or lack thereof, they respond very differently to an applied force, specifically in how they yield to stress. Crystalline solids will yield, or break, along one of their lattice lines giving clean smooth fractures, like in a diamond. Amorphous solids tend to shatter randomly once they reach their yield stress limit. All types of solids have some form of elasticity to them, solids that have little elasticity are termed brittle. Creep is how a material responds to a constant load or force, which is below the yield stress, over an extended period, specifically how the material permanently deforms, such as weights bending a barbell.

Liquids do not absorb an applied force in an elastic manner, rather they give way and flow when a force is applied. As previously mentioned, a liquid’s resistance to flow is defined as its viscosity; all liquids have some form of viscosity or thickness, and this property is determined by how the material responds to stress force and the rate or speed at which that force is applied.

A liquid can respond in 3 different ways to an increase in shear rate. It can maintain the same rate of shear stress increase (Newtonian), it can increase the rate of shear stress increase (shear thickening), or it can decrease the rate of shear stress increase (shear thinning) (Fischer et al.,

2009).

Viscoelastic properties are typical of gels and jammed systems. They exhibit solid like behaviors in some situations and liquid like behaviors in other situations. These differences have

38 to do with the shear rate applied to the material and the yield stress of the material. These properties are most commonly measured using a small amplitude, oscillating force via a rheometer attached with parallel plates or cone and plate (Miri, 2010). The oscillating force produces a type sinusoidal wave based on the force being applied and changed. The lag time, or phase angle, between shear stress response verse the strain rate can give information about the viscoelasticity of the material. Solids that exhibit true elastic behavior have a phase angle close to 0°, whereas fluids have a phase angle close to 90°. A material will have equal solid and liquid like behavior at a phase angle of 45°; this value is used to determine the cross over point of a material from a solid to a liquid, where above 45° the material is liquid and below 45° the material is solid (Miri, 2010).

There are two types of moduli that are important to viscoelastic materials; these are the storage modulus and the loss modulus, denoted by G’ and G” respectively. This is another way of measuring the solidness or liquidness of a material by assigning a value to it. The storage modulus or G’, is a measurement of how well the material can store energy in an elastic manner, like a solid. This value gives an indication of how “solid” a material is. Conversely, the loss modulus or G”, is a measurement of how much energy the material loses as heat and gives an indication of how “liquid” a material is. It is important to note that these moduli are intricately linked with the phase shift angle and these numbers are equal it will correspond with a 45° phase angle measurement.

Tests to determine the G’/G” values of a product or the phase shift angle (δ) can be accomplished through very sensitive instruments known as rheometers. These types of rheometer tests are very common for researching the properties of gels, foods, and other viscoelastic materials (Navarro et al., 1997; Baudez and Coussot, 2004; Miri, 2010; Sauer et al.,

2012; Thomar et al., 2012). Rheological properties that are frequency dependent can be used to generate G’ and G” data that provide specific patterns for dilute solutions, concentrated solutions, and gels. In dilute solutions, G” is larger than G’ over the entire frequency range but approach each other at higher frequencies. For a concentrated solution, G” is larger than G’ in the lower frequency range, indicating more liquid-like properties. However, the G” becomes lower than G’ in the higher frequency range, exhibiting more solid-like properties. For gels, G’ is always greater than G” throughout the whole frequency range (Foegeding et al., 2010).

Rheometers can also be used to measure simpler, yet still very important physical attributes, such as viscosity, to determine the flow behavior of a product (Newtonian vs non-

Newtonian). Sauer et al. (2012) evaluated the rheological behavior of 65% and 95% serum protein reduced MCC solutions made using the same process as Zulewska et al. (2009). The

MCC’s were then spray dried and stored before being rehydrated to concentrations of 2.5 to

12.5% casein protein (w/w) according to the procedure set by Beliciu and Moraru (2011). The apparent viscosity was measured using a parallel plate rheometer over the course of different temperatures. The authors found that the apparent viscosity of MCC increased with concentration and decreased with temperature. The MCC exhibited Newtonian behavior below

7.5% protein w/w but at casein concentrations ≥7.5%, the MCC started showing shear-thinning behavior. Serum protein removal (65% vs 95%) showed effects on the viscosity, with higher serum protein reduction leading to higher viscosity in solutions with the same casein concentrations. Beliciu and Moraru (2011) conducted research evaluating the rheological relationship of reconstituted MCC powders that undergo a heat treatment. MCC samples of 2 to

15% protein concentrations (wt/wt) were heated for 5 minutes between 40 to 90°C and then cooled and evaluated at 20°C. Like Sauer et al. (2012), they also found that the MCC solutions

40 displayed shear thinning behavior at concentrations above 7.5% protein w/w and that they exhibited nearly Newtonian behavior below that concentration. The authors also found that the heat treatment of the solutions did not affect the rheological properties of the MCC.

To determine the strength, load capacity, or fracture point of viscoelastic materials, other types of tests and instruments can be used. Uniaxial compression, via a machine such as Instron, is the most popular test for determining the rheological properties of foods, specifically that of fracture, due to the simple sample preparation and execution of the test (Foegeding et al., 2010).

These tests have been used in a wide variety of applications in the food and beverage space, namely for meats, doughs, produce, tofu, and dairy products (Abu-Shakra and Sherman, 1984;

Charalambides et al., 1995; Krokida et al., 1999; Yuan and Chang, 2007; Lee and Chin, 2019).

In the dairy industry, compression tests have been commonly used in studying the firmness properties of cheeses and other dairy protein gels (Shama and Sherman, 1973; Culioli and

Sherman, 1976; Brennan and Bourne, 1994; Charalambides et al., 1995).

In a uniaxial compression test, a cylindrical sample of known size is placed between two parallel plates of a Universal Testing Machine, such as an Instron. Usually, the upper plate is moved downward at a constant speed towards the sample, referred to as “cross-head speed”, while the force of the sample against the upper-plate is recorded as a function of time. The resulting force-over-time data can then be converted into stress and/or strain values. When the test and sample preparation are performed correctly (i.e. cylindrical sample with flat and parallel ends and no significant contribution of plate-sample friction, or sample to container friction), the

Young’s Modulus may be calculated. The Young’s Modulus is the slope plot of the stress vs strain values (Nieuwland et al., 2015). If the goal is to only compare various samples under the same testing conditions, then the maximum compression forced can be used to compare the

41 samples. However, if one wants to compare samples across other types of tests, then only results expressed as true stresses and true strains can be used (Foegeding et al., 2010). Not only can compression tests be used to understand the rheological properties of food stuffs, but they can also be used to help correlate the physical properties to the eating and mastication experience.

Sharma and Sherman (1973a) describe a procedure for establishing the conditions that should be used when using an Instron Universal Testing machine so that they simulate those associated with the sensory evaluation, particularly that of “first bite”. However, additional research has shown that instrumental texture analysis is not necessarily accurately correlated with sensory texture perception, and can often be misused or misinterpreted (Szczesniak, 1987; Drake et al.,

1999; Lawless and Heymann, 2010; Technologies, 2020). Despite these correlation and application issues, Drake et al. (1999) found that instrumental rheological testing can still reveal important information on network structure and molecular arrangement. This will be highly valuable in the determination of the cold-gelation mechanism of HC-MCC.

Although uniaxial compression tests are relatively easy and straight forward there are factors to be aware of. Sharma and Sherman (1973b) found that the crosshead speed can be set to a value that is slow enough to allow for stress-relaxation to occur in the sample. This then impacts the perceived firmness. The researchers found this to be particularly true from Edam cheese. Not only is the speed of the cross-head potentially critical, but the amount of friction between the head plate and the sample specimen can also have significant effects (Brennan and

Bourne, 1994). Culioli and Sherman (1976) found that cheese maturity, test temperature, crosshead speed, sample shape, sample height and surface area all influence the resulting behavior of Gouda cheese to some degree when using in an Instron Universal Testing Machine in a force compression test.

While most of the compression research done in dairy has focused on cheese, there have been a few research articles done on casein gels, however there has not been any research done using compression tests with purified (> 90% serum protein reduced) liquid MCC and HC-MCC gel. While compression test research on MCC and HC-MCC gel is very scarce, the research on other casein gels, especially gels made from casein powders, would presumably be similar.

Nieuwland et al. (2015) used a reconstituted micellar protein isolate that was reduced to 3.5% whey protein via addition and washing of 35 mM NaCl and diethylethanolamine, and then filtering out of the sedimented whey proteins. The resulting purified casein solution was diluted to 12% protein by weight and gelation was induced via the addition of transglutaminase. The resulting gel was evaluated using a uniaxial compression test among other analysis. They found that surprisingly, as cross-linking in the gel increased, the Young’s Modulus (and G’) decreased.

The authors hypothesized that this was due to a decrease in the micelle to micelle interactions, leading to an easier sliding of the particles in the gel along each other and thus a decreased modulus.

In addition to gel strength and yield stress testing via uniaxial compression, the flow behaviors of viscoelastic dairy products can be measured by different types of viscometers, among which rotational viscometers are most frequently used (Foegeding et al., 2010). Due to viscoelastic dairy products showing some solid-like properties due to the formation of a weak gel structure, they exhibit a variety of non-Newtonian characteristics as measured by apparent viscosity, or shear rate vs shear stress. There have been numerous research articles that utilize rotational viscometers for the measurement of apparent viscosity (Kindstedt et al., 1989a; b; Fife et al., 1996; Solanki and Rizvi, 2001; Gün and Işiki, 2007; Bara-Herczegh et al., 2013; Fava et al., 2013; Adams and Barbano, 2016; Cheng et al., 2019a; b; Bancalari et al., 2020). Much of the

43 literature deals with skim milk concentrates for the purposes of spray drying (Chang and Hartel,

1997; Velez-Ruiz and Barbosa-Canovas, 1998; Bienvenue et al., 2003; Zisu et al., 2013). The total solids content of these concentrates are extremely high (>40%) (Bienvenue et al., 2003;

Zisu et al., 2013) as manufacturers try to remove as much water possible before spray drying, due to the high energy costs associated with spray drying. These total solids levels are far greater that for that of HC-MCC, but it is important to note that skim milk concentrates still contain whey proteins and lactose, the latter of which is going to account for the majority of the total solids. This makes comparison of skim milk concentrates to MCC and HC-MCC very difficult. Zisu et al. (2013) evaluated treatment of skim milk concentrate (50 to 55% total solids) with ultrasound to reduce age thickening. The ultrasound intensity varied from 130 to 230 W.

The skim milk concentrate was also batch sonicated off-line for 1 min in a stainless-steel cylinder to deliver 40 to 80 W. Apparent viscosity was measured at 50°C over a shear rate profile of 50 to 300 s-1, using a Haake VT550 rotational viscometer, fitted with an MV1 cup and rotor attachment, and the shear rate was increased by 10 s-1 at 10 s intervals. The results showed that ultrasound was able to lower the viscosity of the skim milk concentrates, but the treatment did not prevent age thickening once the ageing process was established. Ultrasound treatment was only able to delay the rate at which concentrated skim milk thickened. Additionally, it was only when ultrasound was applied during the entire evaporation process that the treatment prevented the early onset of age thickening.

Adams & Barbano (2016) did work to evaluate apparent viscosity of MCC obtained through microfiltration using varying types of ceramic membranes, specifically as it related to the channel diameters of the membranes. They measured apparent viscosity via a Brookfield rotational viscometer with a UV adapter that was temperature controlled and held at a constant

44 shear rate of 73 s-1. They found that retentate apparent viscosities at 50°C increased according to increasing retentate protein concentrations and that the MCC solutions with casein concentrations <10% behaved as Newtonian liquids above 40°C, but retentates with casein concentrations above 12.5% (wt/wt) exhibited mild shear thinning at 60°C. However, the apparent viscosity of the retentates were not affected by the membrane channel diameter.

Additionally, the apparent viscosity increased exponentially with decreasing temperature which is also what Sauer et al. (2012) found. These apparent viscosity results are noticeably higher

(about 7%) than the values obtained by Solanki and Rizvi (2001) who produced multiple MCC’s at different pH values (adjusted with glucono-δ-lactone) and which had around 18 to 21% total protein (w/w). These were then diluted to various concentration and apparent viscosity was measured using a Haake RV 100 rotational viscometer. The shear rate used for their research was 1350 s-1 and they found that up to a concentration factor of 4X, the MCC, exhibited

Newtonian behavior like that of milk. Additionally, the viscosity increased with increasing concentration from 1.3 cP of milk to 2.8 cP at 4X MCC (total solids 8.61 to 15.92%, respectively). However, the MCC did exhibit shear thinning behavior at higher concentrations and depending on pH, the onset of shear-thinning varied from 11 to 17% solids, with MCC at pH

6.5 exhibiting such behavior at the lower concentration value. The authors also noted that this shear thinning behavior was different from the findings of Chang and Hartel (1997), who observed Newtonian behavior in freeze-concentrated skim milk up to 25% solids (w/w) and by

Velez-Ruiz and Barbosa-Canovas (1998) who observed non-Newtonian behavior in skim milk concentrate at solids greater than 22.3% (w/w). These differences can be attributed to the differences in composition between skim milk concentrate and MCC as previously mentioned.

Adams and Barbano (2016) stated that the differences in apparent viscosity between their study

45 and that of Solanki and Rizvi (2001) could be due to differences in measurement systems, differences in shear rates used, or a combination of the two.

Cheng et al. (2018a,b) used a very similar method to Adams and Barbano (2016), also using a Brookfield viscometer, and saw increasing apparent viscosity with increasing protein concentration and decreasing temperature. Cheng et al. (2019b) reported an effect of pasteurization and fat, protein, casein to serum protein ratio, and milk temperature on milk beverage color and viscosity and reported that for unpasteurized milk protein beverages within each fat level, variation in casein as a % of total protein dominated the changes in L values, sensory whiteness, and opacity, and decreased a and b* values, sensory color intensity, and yellowness. For HTST pasteurized milk protein beverages, increases in temperature of the beverage (4, 20 and 50°C) decreased viscosity and changes in protein concentration and casein as a % of total protein had a greater effect on viscosity data within each temperature than fat concentration. Cheng et al. (2019a,b) also found that increasing casein as a percentage of total protein and decreasing temperature increased viscosity more than the change in fat level.

Furthermore (Cheng et al., 2019b) showed that increase in casein as a percent of total protein increased viscosity more than increasing total protein through the addition of serum protein.

As previously mentioned, Amelia and Barbano (2013) noted that HC-MCC formed thermo-reversible gels when stored at 4°C. However, none of the MCC in the Adams and

Barbano (2016) study exhibited gel-like behavior when refrigerated for up to 72 h. This is most likely because the highest concentrations used in their study was 12% (wt/wt), which seems to be below the gelation threshold, as well as the time frame of 72 h was not long enough to observe gelation. While Solanki and Rizvi (2001) did use HC-MCC in their research, the temperature at which it was analyzed was at 50°C, this could account for why no gelation was observed, as this

46 temperature seems to be above the melting point for HC-MCC (Amelia and Barbano, 2013; Lu et al., 2015). This would indicate that gelling is not expected to be a problem with skim milk MF retentates (MCC) at total protein concentrations below 12.5% or if the temperature is kept high

(≈50°C).

MCC Protein Fraction Analysis

In addition to analysis of MCC through rheological methods, additional methods can help in understanding the physico-chemical properties of MCC, particularly the protein fraction composition. This is mainly done through the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Farrell et al., 2004). SDS-PAGE is a separation technique that allows for the separation of proteins based on their molecular size after negating their charges through the saturation of the proteins with SDS (Srinivas, 2019). Over the years there have been many variations of this technique, however, the most cited method is that by

Laemmli (1970) to separate and distinguish larger proteins and polypeptides but does not work as well for lower molecular weights (Mr <12000) (Bury, 1981; Fling and Gregerson, 1986).

Fling and Gregerson (1986) created a modification of the Laemmli (1970) procedure by having high concentrations of Tris in the resolving gel (0.75 M) and in the running buffer (0.05 M).

They then had the linear gradient gels (8 to 25% acrylamide) tested with and without varying concentrations of urea and/or glycerol and/or sucrose. At the high molarity of Tris, the use of urea, glycerol, or sucrose proved unnecessary for effective peptide electrophoresis. Gels run without these reagents showed superior resolution throughout the entire molecular weight range when run with Tris at 0.75 and 0.05 M, respectively, removing the need for urea or other additives. The superior resolution of this method enabled the authors to show clear identification of proteins and polypeptides with Mr as low as 1300.

SDS-PAGE analysis has been used in numerous fields of molecular biology and in the research of food proteins, especially dairy proteins (Bury, 1981; Creamer and Richardson, 1984;

Fling and Gregerson, 1986; Politis et al., 1992; Havea et al., 1998; Havea, 2006; Jovanovic et al.,

2007; Roach and Harte, 2008; O’Sullivan et al., 2014). While SDS-PAGE has been of great benefit to the dairy sciences, there have been some complications with the procedure, particularly in relation to the mobility of casein fractions. Creamer and Richardson (1984) found that caseins (especially αS1-casein) have lower mobilities than expected based on their known Mr.

The binding of SDS to both αS1-casein (Mr 23,600) and β-casein (Mr 24,000) reached a maximum at a lower than expected value of 1.3 g SDS/g protein. However, both esterification and dephosphorylation followed by amidation of αS1-casein increased its mobility in SDS-gel electrophoresis, but unfortunately neither modification affected β-casein mobility. The results indicated that the low electrophoretic velocity of αS1-casein in SDS-gel electrophoresis resulted from its uniquely large hydrodynamic radius under the experimental conditions.

This phenomenon of lower casein mobility was also observed in research by Verdi et al.

(1987), who were looking at the effects of proteolysis in high and low somatic cell milks.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in a 10 to 20% gradient was performed on selected milk samples before and after an incubation period. Their procedure for

SDS-PAGE analysis (which was an adaptation of the Laemmli, 1970 method) used stacking gels with 6% acrylamide, 0.16% N,N'-methylenebis-acrylamide, 0.125 M Tris-HC1 pH 6.8, 0.1% T E

M E D, 0.1% SDS, 0.05% ammonium persulphate. The separating gels were linear gradients

10% acrylamide plus 0.27% N,N'-methylenebisacrylamide to 20% acrylamide plus 0.53% N,N'- methylenebisacrylamide, 0.375 M Tris-HC1 pH 8.8, 0.1% SDS, 0.1% TEMED, 0.05% ammonium persulphate. Sample buffer contained 10 mM Tris-HC1 pH 6.8, 1.0% SDS, 20%

48 glycerol, 0.02% bromophenol blue, and 50 mM dithiothreitol. The gels were fixed for 16 h in an aqueous solution of 50% methanol, 8% acetic acid, and stained for 16 h in a 5% methanol, 8% acetic acid, 0.5% Coomassie brilliant blue R250 solution.

Havea (2006) looked at the soluble and insoluble fractions of MPC powders that were reconstituted. The insoluble fraction was characterized using one-dimensional and two- dimensional SDS-PAGE and transmission electron microscopy. Their results showed massive variability in the levels of solubility in MPC powders ranging from 3 to 98%. The TEM results showed that the insoluble material consisted of large particles (≈100 µm) where the casein micelles were conjoined by protein–protein interactions. This material consisted predominantly of α and β-caseins. The 1D PAGE results showed that the amounts of insoluble material in MPC powders increased with storage time at elevated temperatures. Additionally, this material which was formed largely by weak hydrophobic protein–protein interactions were dissociable under

SDS-PAGE conditions.

When using SDS-PAGE analysis, there may be a desire or need to analyze proteins in only a specific portion of sample matrix. To separate out constituents of the various phases or matrices of a sample, centrifugation can be incorporated in combination with SDS-PAGE analysis, to help analyze which proteins are present in which part of a sample or food matrix.

Centrifugation involves spinning a sample at high speeds and uses the centrifugal force generated to separate out solutions into different phases based on density, particle size, particle shape and viscosity. Centrifugation can be particularly helpful when it comes to the separation of dairy proteins and more specifically casein micelles (Crowley et al., 2018; Dalgleish, Horne,

& Law, 1989; Davies & Law, 1983; Ferrer, Hill, & Corredig, 2008; Havea, 2006; Lin & Morr,

1971; Y. Lin, Kelly, O’Mahony, & Guinee, 2017; Morr, Lin, Dewan, & Bloomfield, 1973;

Moughal, Munro, & Singh, 2000; Sedmerova et al., 1974; Thienel et al., 2018; Ward & Bastian,

1996). Due to the wide range of micelle sizes, as mentioned in earlier sections, it is possible to separate out the casein micelles into different supernatant, opalescent liquid and pellet phases based on size (Morr, 1973a; b; Morr and Swenson, 1973). In these studies, Morr and colleagues found that integrity of the centrifuge pellet, was not maintained when centrifuged for 147000 x g for 1 h. It was only when done for 2 hours that the pellet maintained its structure after the supernatants had been removed. Additionally, the solvation of the casein micelles were affected by the pH of the skim milks with pH 6.5 having the lowest solvation value (2.85 g H20 / g dry pellet) and skim milks at pH 5.0 and 7.5 having higher values (3.8 g H20 / g dry pellet and 3.5 g

H20 / g dry pellet respectively). They also found that casein micelle pellets from 0°C and 5°C skim milk contained only 66% and 79% as much calcium-phosphate, respectively, as compared to 35°C to 40°C milk pellets. While higher rpms and longer run times will yield better separations and more compact pellets, for general isolation or removal of caseins, centrifugation at 100000 x g for 1 h will sediment 90 to 95% of the casein micelles and this process is even more effective at higher temperatures (30 to 37°C) (Fox et al., 2015).

Lin et al. (1971) were able to use zone ultra-centrifugation and the subsequent fractions in combination with inelastic light scattering techniques to help elucidate the size ranges of casein micelles. The subsequent distribution had about 80% of the casein in micelles with radii between 500 to 1000 Å, and 95% between 400 to 2200 Å, resulting in a most likely radius of about 800 Å. Morr et al. (1973) were able to use centrifugation to identify the sedimentation coefficients of casein micelle fractions. Dalgleish et al. (1989) were able to show that κ-casein must be on the periphery of the casein micelle as the proportion of κ-casein to overall casein increased with decreasing particle size. Davies and Law (1983) looked at casein micelle

50 fractions from skim milk that was ultra-centrifuged at 4°C and 20°C at a speed of 70000 x g for

9, 30, and 120 mins. They found that the casein in the serum phase differed significantly in composition from the casein fractions in the pellet, with the serum proteins being very rich in β- casein and comparatively poor in αS1-caseins, and αS2-caseins. The amount of serum protein present at 4°C was considerably greater than 20°C, with the increase being due almost entirely to

β-casein and β-casein proteolytic fragments. Downey and Murphy (1970) reported comparable results, namely a significant increase in the serum protein concentrations at 5°C, however the increase due specifically to β-casein was lower (46% increase). Davies and Law (1983) hypothesized that the difference in results could be due to the difference centrifugation conditions among the studies, as Downey and Murphy only used 36,000 x g for 2 h. Both of these results also agreed with the findings of Sedmerova et al. (1974) who analyzed the yield and composition of casein micelle fractions isolated under different centrifuge conditions, consisting of 50000 x g to 165000 x g and 5°C and 20°C. The authors found that the casein content of the supernatant obtained at 165000 x g was about 10% of the total casein content. The yield of casein in the pellets was higher at 20°C than at 5°C due to more dissociation of casein into the supernatant phase at 5°C, consisting mainly of β-casein. While these research articles focus on centrifugation and protein fraction determination with skim milk, there has not been much research in the way of temperature dependent fractionation of purified MCC, and so no research has yet been published to show that β-casein behaves identically in skim milk as it does in MCC.

MCC is still a relatively new ingredient for the dairy industry and while it has many promising attributes, it still has yet to see broad integration. The main problem prohibiting larger adoption of MCC is the difficulty of rehydrating spray dried MCC powder. This barrier could be overcome by using liquid MCC. However, to save on high shipping costs, it is necessary to

51 create a highly concentrated liquid MCC (HC-MCC). Few journal articles exist exploring HC-

MCC, but the research performed has shown that HC-MCC exhibits gelation upon cooling, with the gelation point depending on protein concentration, temperature, and time. Additionally, previous research would suggest that either β-casein, or volume fraction (ϕ) may play integral roles in the mechanism of gelation. The objectives of this thesis were to ascertain the rheological behavior of highly pure (>95% serum protein reduced) MCC at varying protein concentrations and temperatures across storage and then compare these results with data obtained through ultracentrifugation and SDS-PAGE analysis to determine if β-casein dissociation may play a role in the cold gelation of HC-MCC.

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CHAPTER 2: VISCOSITY AND GEL FORMATION OF MICELLAR CASEIN CONCENTRATES

M.R. Dunn, D.M. Barbano, and M.A. Drake

Department of Food, Bioprocessing and Nutrition Sciences, Southeast Dairy Foods Research

Center, North Carolina State University, Raleigh, NC 27695

Abstract

This study determined the basis for viscosity increase and cold gelation of liquid MCC at protein concentrations from 6 to 20% during refrigerated storage. Skim milk (ca 350 kg) was pasteurized (72°C for 16 sec) and filtered through a ceramic MF system to make micellar casein concentrate (MCC). The liquid MCC was immediately concentrated via a plate ultrafiltration

(UF) system to 18% protein (w/w). The MCC was then diluted to various concentrations (6 to

18%, w/w). Apparent viscosity readings were collected from liquid MCC samples (6, 8, 10, 12% protein w/w) at 4, 20, and 37°C. Instron compression force of MCC gels (14, 16, 18% protein w/w) was collected over a period of 2 weeks at 4°C. The maximum compressive load was compared at each time point to assess the changes in gel strength over time. Supernatants from

MCC of 6.5 and 10.5% protein were collected after ultracentrifugation (100,605 x g for 2 h at 4,

20 and 37°C) and the nitrogen distribution (total, noncasein, casein, and nonprotein nitrogen) was determined. The entire experiment was replicated 3 times. The highest protein concentrations of MCC formed gels almost immediately on cooling to 4°C, while lower concentrations of MCC were viscous liquids. The protein, casein, and casein as a percent of true protein in the liquid phase around casein micelles in MCC increased with increasing casein concentration of the MCC and with decreasing temperature. Casein as a percent of true protein at 4°C in the liquid phase around casein micelles increased from about 16% for skim milk to about 78% for an MCC containing 10.5% protein. AV of MCC solutions in the range of 6 to

13% casein increased with increasing casein concentration and decreasing temperature. There was a strong temperature by protein concentration interaction for viscosity with AV increasing non-linearly with decreasing temperature at high protein concentration. MCC containing 16 and

18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels

75 increased in strength over 10 days of storage at 4oC, likely due to migration of casein out of the micelles and interaction of the non-micellar casein to form a network that further strengthened the random loose particle jamming gel structure. With an increased understanding of the mechanism of cold thickening of MCC, there may be potential to replace hydrocolloids for thickening and stabilizing beverages with a clean label ingredient and also to provide a high level of protein with superior flavor and color.

Key Words: micellar casein concentrate, viscosity, gelation

Introduction

American consumers continue to seek for more protein in their diets (Euromonitor, 2016;

Nielsen, 2018). Consumers not only want more protein, but they also want to increase the quality of the protein they consume. Dairy proteins are the most desirable types of protein, with levels of 20 to 29 g/serving also being preferred (Harwood and Drake, 2019). This consumer desire has led to the rise in consumption of Greek yogurt, drinkable yogurts, high-protein and meal replacement beverages, protein bars, and other protein fortified foods. Additionally, the dairy industry faces many issues related to the supply and distribution of milk products, including seasonality of production, rising cheese and butter consumption, leading to an excess of fluid skim milk (Barbano, 2017; Weldon et al., 2003).

Many dairy products contain ingredients that are not desired by consumers, but yet are integral to consumer overall liking of the product, particularly hydrocolloids (Varela and

Fiszman, 2013) that provide reduced fat beverages with a more creamy mouthfeel.

Hydrocolloids are critical to the formulation of milk-based beverages, especially those containing cocoa, so as to prevent the settling of cocoa powder. Opportunities for clean label alternatives to hydrocolloids may exist by taking advantage of the unique functional properties of dairy proteins to provide improved viscosity and creamy mouthfeel in easy to consume, high- protein products, such as flavored beverages.

There are a wide variety of milk derived protein ingredients commercially available for use as food ingredients that have been described by the American Dairy Products Institute

(ADPI, 2018b; a). Milk protein ingredients can be produced and used as fresh liquid concentrates or as dried ingredients. Generally, fresh liquid concentrates of milk or whey proteins will have a more neutral flavor in beverage products (Carter et al., 2018). Whey protein

77 ingredients derived from cheese making are a good fit for clear acidic beverages due to their pH stability (i.e., lack of coagulation) under acidic conditions (Mulvihill and Donovan, 1987), while milk protein concentrates (MPC) and milk protein isolate (MPI) are heat stable at neutral pH

(Agarwal et al., 2015; Singh et al., 2019) and are most commonly used in neutral pH protein beverages subjected to high-heat ultrapasteurization (UP) or retort processing.

Caseinates (sodium, calcium and acid) are sometimes used as protein ingredients in beverages, but they have stronger flavor and different functional properties in beverages than casein (CN) in the micellar form. The high heat of UHT processing or retorting produces heat induced sulfur-eggy off-flavors when MPC and MPI ingredients are used. A relatively new milk protein ingredient, micellar casein concentrate (MCC) has had a large percentage of serum proteins (i.e., milk derived whey proteins, MDWP) removed by microfiltration (MF). Jo et al.

(2019) determined that thermal degradation of the MDWP were the origin of sulfur-eggy off- flavors in high temperature short time (HTST) and UP milks. Cheng et al. (2019a) demonstrated that as more MDWP are removed from MCC, the level of sulfur-eggy off flavors can be reduced to very low levels.

MCC is a new dairy protein ingredient produced by MF of skim milk. CN micelles are much larger in size than MDWP and these two groups of proteins can be separated via membrane filtration (Hurt and Barbano, 2010; Hurt et al., 2010). Similar to whey and milk protein products, MCC can be spray dried or used as a fresh concentrated liquid. Depending on the type of process used in the production of MCC (i.e., polymeric versus ceramic membranes), up to 95% of the MDWP can be removed. Generally, a higher MDWP removal can be achieved with ceramic membranes than polymeric membranes (Beckman et al, 2010; Zulewska et al.,

2009; Zulewska and Barbano, 2014).

Amelia and Barbano (2013) produced 95% MDWP reduced MCC by MF and concentrated that MCC to high protein concentration (ca. 18%) using ultrafiltration (UF). Upon cooling the concentrated MCC to temperatures <22oC, the MCC formed a gel very quickly and that gel was thermally reversible. The gel had good microbial stability (<20,000 cfu/mL for 16 weeks) during storage at 4oC. MCC can be used as a protein ingredient as a concentrated liquid or it can be dried to produce an MCC powder. MCC powders are difficult to rehydrate and their rehydration becomes more difficult with age and temperature of storage of the powder documented (Nasser et al., 2017b). A strong correlation was demonstrated between migration of lipids to the surface and evolution of wetting time. Nevertheless, it was clearly established that the wetting step was not the main reason for extended total rehydration time upon ageing. On the contrary, it was shown that delayed release of the CN micelles induced drastic increase of fragmentation and total rehydration time probably due to cross link formation, and should therefore be considered as the key mechanism responsible for extending total rehydration time.

Changes in microstructure of MCC powder occurs during storage (Nasser et al., 2017a).

Starting from skim milk and concentrating to a 18% protein MCC is about 6 to 7 X concentration factor, which means the volume of skim milk has been reduced from 100 kg to about 16.7 to 14.8 kg. A dried MCC would produce about 3 kg of powder from 100 kg of skim milk at 2.6% CN. In the drying process some changes in flavor profile of the reconstituted powder versus fresh liquid MCC will occur (Carter et al., 2018). The liquid concentrate of MCC has very clean flavor and ease of reconstitution advantages over dried MCC, while achieving a substantial reduction of the bulk of shipping of the high value protein in skim milk (from 100 kg for skim to 15 to 17 kg for highly concentrated liquid MCC, HC-MCC). The HC-MCC can be solidified in blocks of various sizes and shipped via refrigerated transportation and then heated to

79 easily convert it back to a liquid by heating to about 50°C upon use without the additional processing cost of drying and reconstitution of powder. Gelled HC-MCC may be an attractive ingredient for high protein beverage manufacture.

Lu et al. (2015) took a different approach to handling HC-MCC by further increasing the protein concentration by vacuum evaporation and then freezing the MCC concentrate. They found that the HC-MCC was completely dispersed by thawing and heating the concentrate to

50°C and speculated that MCC would be a good ingredient for production of high protein liquid food products. Misawa et al. (2016) determined the impact of using fresh liquid MCC on color and viscosity over a range of CN as a percent of true protein (%TP) (5 to 80%) and true protein

(TP) concentrations (3 to 5%) in HTST pasteurized milk systems containing 1 and 2% fat.

Misawa et al. (2016) reported that relative viscosity increased with protein concentration and increasing CN %TP, while increased CN %TP increased whiteness. Cheng et al. (2019b) reported an effect of pasteurization and fat, protein, CN to serum protein ratio, and milk temperature on milk beverage color and viscosity and reported that for unpasteurized milk protein beverages within each fat level, variation in CN %TP dominated the changes in L values, sensory whiteness, and opacity, and decreased a and b* values, sensory color intensity, and yellowness. For HTST pasteurized milk protein beverages, increases in temperature of the beverage (4, 20 and 50°C) decreased viscosity and changes in protein concentration and CN

%TP had a greater effect on viscosity data within each temperature than fat concentration. Vogel

(2019) evaluated 6.5 and 10.5% UP protein beverages made with 95% MDWP reduced liquid

MCC and MPC/MPI. Vogel (2019) reported thickening of the MCC beverages with 4°C storage.

The causes of cold gelation and age thinking of MCC are not fully understood. CN micelles in MCC range in size from about 40 to 300 nm in diameter and are highly hydrated structures. This property is referred to as voluminosity, with an estimated range from 2 to 4 mL of hydrated volume per gram of dry CN (Walstra, 1999), meaning that about 65 to 75% of the hydrated micelle is milk serum and as result their phase volume (ϕ) is much higher than the concentration of dry CN in solution. A theory posed by Lu et al. (2015) was that the HC-MCC system becomes jammed, as water or permeate, is removed from skim milk and the casein CN micelles are forced into extremely close proximity with each other. Solidification of aqueous liquids containing particles can occur at high particle concentration by a phenomenon called particle jamming as discussed by Biroli, (2007). From a theoretical perspective, modeling of particle jamming often assumes spherical, monodisperse, frictionless spheres in water with calculated ϕ for solidification ranging from 0.6 to 0.88. In reality, CN micelles are not frictionless monodispersed spheres and Silbert (2010) indicated that as compressed, soft-sphere packings are decreased in concentration to the point of the jamming transition (i.e., the point where a jammed packing loses mechanical stability), the system may form a random loose packing solid structure that occurs at lower ϕ than the expected jamming ϕ due to friction among the particles. As the friction among the particles increases, the critical ϕ of the particles for solid or gel structure formation occurs will be reduced.

The thickening from very close proximity of particles is exacerbated as energy is removed from the system (via cooling) and Brownian motion of the CN micelles is reduced, resulting in gelation by particle jamming at high ϕ. Additionally, another theory for cold gelation which occurs below ϕ concentrations necessary to cause jamming is that caseins that have disassociated from the micelle create an entangled gel network (i.e., more friction among

81 the particles). This was supported by Lu et al. (2017) who saw CN strand entanglement in lower

MCC concentrated solutions (≈12% w/w) via electron microscopy. By controlling age thickening of MCC, there may be potential to replace hydrocolloids for thickening and stabilizing beverages with a clean label ingredient and also to provide a high level of protein with superior flavor and color; however, we need a better understanding of the rheological properties of MCC. Our objective was to determine the basis for viscosity increase and gelation of liquid

MCC at protein concentrations from 6 to 20% during refrigerated storage.

Materials and Methods

Experimental Design

Skim milk (ca 350 kg) was pasteurized (72°C for 16 sec) and filtered through a ceramic

MF system as described by Cheng et al. (2018) with minor modifications. The MCC was immediately concentrated via a plate UF system to 18% protein (w/w). The entire process was completed in 1 day, approximately 14 hours. The MCC was then diluted into various concentrations (6 to 18%, w/w) and preserved with the addition of thimerosal. The highest protein concentration of MCC formed gels almost immediately on cooling to 4°C, while lower concentrations of MCC were viscous liquids. The preserved MCC was stored in plastic containers at 4°C for analysis. Testing consisted of apparent viscosity (AV) determination using a rotational viscometer (Brookfield), gel strength using a compression test (Instron), and protein analysis of supernatants from ultracentrifugation. Brookfield data was collected from MCC samples (6, 8, 10, 12% protein w/w) at 4, 20, and 37°C. Instron compression force data was collected over a period of 2 weeks storage at 4°C with time points every 48 to 72 h. The maximum compressive load was compared at each time point to assess the changes in gel strength over time. For protein analysis following ultracentrifugation, the supernatants from

MCC (6.3 and 10.5% protein w/w) were collected after ultracentrifugation (100,605 x g for 2 h at 4, 20 and 37°C) and the nitrogen distribution (total, noncasein, CN, and nonprotein nitrogen) were compared across the different temperatures. The entire experiment was replicated three times.

Processing of Micellar Casein Concentrate (MCC)

Liquid MCC with about 95% milk serum removal was produced at about 7 to 8% protein concentration using a ceramic MF system as described by Cheng et al. (2018), with minor modifications. Raw skim milk (350kg) was prefiltered before pasteurization at 4°C using a

Nexis T-filter (NXT 10-30U-M7S, Pall Corp., Port Washington, NY.), pasteurized (720 kg/h) with a plate heat exchanger (model T4 RGS- 16/2, SPX Flow Technology, Greensboro, NC) at

72°C for 16 s, and then heated to 50°C for processing. One thousand grams of the pasteurized milk was collected and stored at 4°C for future ultracentrifugation and SDS-PAGE analysis (to be reported separately). The remaining milk was weighed and kept at 50°C during processing via a jacketed 409.15 L stainless steel, feed tank. pre-filtered, HTST pasteurized skim milk (30 kg) was used to flush out the de-ionized (DI) water from the membrane system before collection began. A 3 stage, 3x MF process described by Zulewska and Barbano (2014) was used to produce a 95% MDWP reduced MCC with a true protein concentration between 7.3 to 7.5% using a pilot scale MF system (Tetra Alcross MFS-7, TetraPak Filtration Systems) equipped with

0.1-μm nominal pore diameter graded permeability ceramic Membralox (model EP1940GL0.1u,

AGP1020, alumina, Pall Corp.) membranes. The final 95% MDWP reduced MCC was about

6.85% (±0.15%) TP (w/w) as determined by mid-infrared (MIR) spectrophotometer (Lactoscope

FTA, Delta Instruments, Drachten, Netherlands). Samples of the stage 3 MCC retentate were collected and stored at 4°C for later compositional analysis to confirm true protein and the level

83 of serum protein removal. MCC was ultrafiltered (UF) to increase the protein concentration from 6.85% to 18%. The same cleaning procedure as described by Zulewska et al. (2009) was used to restore the system to the initial water flux before processing. Aerobic plate counts (APC) and coliform counts (AOAC International, 2012; method 989.10) (Petrifilm Aerobic Count Plate and Petrifilm Coliform Count Plate, 3M Food Safety, Saint Paul, MN, USA) were taken throughout the MF and UF process runs to ensure the microbial quality of the final product prior to preservation with thimerosal.

UF processing was done using a Pellicon® 2, 10K plate ultrafiltration apparatus

(Millipore Sigma, Burlington, Massachusetts). The plates were Biomax® polyethersulfone with a 10,000 Dalton cut-off. The assembly was limited to 5 plates in a stack for optimal pressure performance. Each plate had a surface area of 0.5 m2 and the volume of the entire system was approximately 2 liters. The plates were assembled using a stainless-steel sanitary design plate holder (Steel Pellicon® 2 Cassette filter holders, Millipore Sigma, Burlington, Massachusetts).

The MF stage 3 MCC retentate was weighed and poured into a jacketed stainless-steel feed tank

(Meyer-Blank Company, St Louis, MO) recirculating with 50°C water. The feed tank was connected to the membrane stack by a feed pump (Baldor Industrial Motor, single phase, 1.5

H.P., 3450 RPM, 60hz, Baldor Electric Co., Ft. Smith, AR). The protein content and known weight of the feed were used to create a permeate weight removal goal to achieve the

concentration target of 18% TP (w/w). The calculation used was P = 푊 − , where P = permeate to be removed, WF = weight of feed MCC, and CF = concentration factor. The target

CF (e.g., 2.48) was determined by dividing the desired final protein (18%) by the starting protein

(e.g., 7.28%).

The inlet pressure to the plate stack was 138 kPa and the retentate outlet pressure was

27.6 kPa. The retentate was recirculated back into the feed tank and the permeate was collected separately for weighing. Protein analysis was conducted on the permeate every 10 min with a mid-infrared milk analyzer (Lactiscope FTA, Delta Instruments, Drachten, The Netherlands) to check for protein leakage through the UF membrane. When the weight of permeate removed reached the calculated target, a sample of the MCC was diluted with DI water in a 1:2 (MCC to water) for final confirmation via MIR. The dilution was to ensure that no gelation of the MCC would occur inside the flow system of the MIR milk analyzer. The resulting protein content was multiplied by 3 to estimate the actual protein concentration of the MCC (ca. 18% w/w). The

MCC was then drained out of the UF feed tank for analyses. The UF system was then cleaned and sanitized. Briefly, the system was rinsed with deionized (DI) water at 50°C for 10 min followed by application of an alkaline cleaner (Ultrasil 110 and 01 (Ecolab, St. Paul, Minnesota) at 50°C for 10 min. The system was then flushed with DI water at 50°C for 10 min followed by an acid rinse at 50°C (Ultrasil 76, Ecolab). The system was then rinsed once again with 50°C DI water for 10 min, sanitized (Ecolab XY-12 Liquid Sanitizer (Ecolab) at 21°C and then flushed with DI water (21°C) for 10 min.

Preparation of MCC Dilutions for Analysis

Portions of UF concentrated MCC were weighed into 13.64 L round plastic storage containers (Rubbermaid FG572624CLR, Amazon, Seattle, WA) and diluted to 6, 6.3, 8, 10,

10.5, 12, 14, 16, 18% TP (w/w) with DI water. Each diluted MCC was preserved with the addition of thimerosal (Thermo Fisher Scientific, Ward Hill, Massachusetts) at a rate of 1 mL of

10% w/v aqueous thimerosal per 1000 grams of diluted MCC). Polypropylene flip top 170.5 mL vials (capitol vial polypropylene flip top 08CL, Thermo Scientific) were filled with each MCC

85 dilution, flash frozen in liquid nitrogen, and stored at -80°C for composition analysis at a later time.

MCC dilutions for Brookfield analysis were stored in lidded 170.5 mL vials (capitol vial polypropylene flip top 08CL, Thermo Scientific). Dilutions of 6, 8, 10, 12 % TP solutions were stored in the vials in triplicate, with each vial being marked for measurement at either 4, 20, or

37°C. MCC dilutions at 14, 16, and 18% true protein for Instron analysis were measured out

(150 mL) using a volumetric pipettor (Drummond Scientific Company, Broomall, PA into 25 (5 samples for 5 different time points for a total of 75 containers) separate 227.3 mL plastic deli dish containers (3.81 cm height x 11.43 cm top diameter), (Dura Home, ASIN - B01FKGJ5NG,

Amazon, Seattle, WA). The containers were then sealed with airtight lids such that the lid was not in contact with the MCC solution to prevent fracture of the gel when the lid was removed for analysis. The containers were placed onto metal trays and then placed on a flat, leveled surface

(to ensure that MCC gelation creates a flat surface for compression testing) inside a 4°C walk-in cooler. Approximately 1000 g of MCC diluted to 6.3 and 10.5% protein and pasteurized skim milk were weighed into HDPE half-gallon containers. The containers were refrigerated at 4°C until ultracentrifugation.

Ultracentrifugation

For the centrifugation of the pasteurized skim milk, the centrifuge rotor (T29-8x50 Super

Speed Rotor, Thermo Fischer Scientific, Waltham, MA) and skim milk were both tempered at

4°C for at least 12 h prior to centrifugation. The centrifuge (Sorvall Lynx 6000 Ultracentrifuge

System, Thermo Fischer Scientific) was set to 4°C and allowed to temper for one hour prior to use. Approximately 40 g of skim milk was weighed into each centrifuge tube (Nalgene Oak

Ridge PPCO 3139-0050 Tube, Thermo Fischer Scientific). Centrifuge tubes were paired based

86 on weight (0.02 g difference max) to balance the centrifuge rotor (4 pairs total). Tube pairs were placed in the rotor opposite one another and were centrifuged at 100,605 x g for 2 h at 4°C. The speed of the rotor for the runs was determined using the following equation:

푛 = 푛 where, nper= the max possible speed for the load, nmax= the max possible speed for the rotor and machine, Lmax= the max load for the rotor, and Lact= the actual load placed in the rotor. The centrifuge tubes were removed from the rotor and a side by side picture of all the tubes was taken. The top 15 mL of supernatant was collected from each tube and combined into a single 170.5 mL vial (Nalgene Oak Ridge PPCO 3139-0050 Tube, Thermo

Fischer Scientific, Waltham, MA). This process was repeated two more times so that there were three vials each containing 40 mL of supernatant from the skim milk. The remaining supernatant was discarded, and a single pellet of precipitate was removed, and a photo of the pellet was recorded collected (Figure 1). This process was replicated for ultracentrifuge runs at 20°C and

37°C. The same ultracentrifugation runs were repeated to collect ultracentrifugation supernatants for the 6.3% and 10.5% TP MCC for further analysis. The centrifuge rotor (T29-

8x50 Super Speed Rotor, Thermo Fischer Scientific) and MCC were both tempered at 4°C for at least 12 h prior to centrifugation. The centrifuge (Sorvall Lynx 6000 Ultracentrifuge System), itself, was set to 4°C and allowed to temper for one hour prior to use. Approximately 40 g of 6.3

% MCC was weighed into each centrifuge tube (Nalgene Oak Ridge PPCO 3139-0050 Tube,

Thermo Fischer Scientific, Waltham, MA) (total of 4 tubes) and 40 g of 10.5% MCC was weighed into another set of 4 tubes. Centrifuge tubes were paired based on weight (0.02 g difference max) and type of MCC to balance the centrifuge rotor (4 pairs total). Tube pairs were placed in the rotor opposite one another and were centrifuged at 29,000 rpm for two hours at

4°C. The centrifuge tubes were removed from the rotor and a side by side picture of all the tubes

87 was taken. The top 15 mL of supernatant was collected from each tube of 6.3% MCC and combined into a single 170.5 mL vial (Thermo Scientific). The same process was repeated for the tubes of 10.5% MCC. The remaining supernatant was discarded, and a single pellet of precipitate was removed from a 6.3% MCC tube and a 10.5% MCC tube and a picture was collected of both pellets. This process was then replicated for runs at 20°C and 37°C.

Instron Analysis

Compression testing was carried out using a universal testing machine (5565 series, serial# Q3220, Instron, Norwood, Massachusetts) with a 5 kN force capacity and a 5 kN static load cell (serial# 50096, Instron). Measurements were taken every 48 to 72 h over the course of

10 days, for a total of 5 time points for each experimental replication. Sample containers (11.4 cm diameter) were removed individually from refrigeration and tested immediately at 4°C.

Analysis was carried out using BlueHill 2 software (version 2.33.893, 2005, Instron). Testing conditions for single compression per container were 1.25 mm penetration depth and a crosshead speed of 0.1mm/sec and the resulting maximum compressive loads (N) were recorded.

Preliminary tests confirmed that no fracture of the gel occurred to produce minimal frictional interference forces from the sides of the container. The load cell was calibrated before any head attachments were connected. A flat, circular, metal head attachment (6 cm diameter) was used and connected to the Instron crosshead. Force readings were calibrated with the head attachment by using a foam standard and ensuring the output reading was the same (± 2 N) before each measurement timepoint. Samples were placed below the crosshead and centered. The crosshead was lowered until the metal head attachment was visibly close to the surface of the sample. The load cell force was balanced at zero, and the metal head was then lowered onto the sample to achieve a value of -0.2N (+/- 0.05). Crosshead height and load cell force were then tared to 0 at

88 that position and the test was carried out to completion and the max compressive loads were recorded. Measurements were taken in triplicate for each MCC protein concentration (14, 16,

18%) at each time point. The average max compression values of each test were recorded and plotted using Microsoft Excel software (Microsoft Corp., Redmond, Washington) to show gel strength (max compression value) as a function of time and protein concentration.

Brookfield Analysis

AV testing was conducted using a rotational viscometer (LV-DV2T, Brookfield

Engineering Laboratories Inc., Middleboro, Massachusetts) equipped with a jacketed cup-and- bob fixture (Enhanced UL Adapter, Brookfield Engineering Laboratories Inc.) according to the procedure specified by Adams and Barbano (2016) with the following alterations.

Measurements were conducted on MCC concentrations of 6, 8, 10, and 12% TP at constant temperatures of 4, 20 and 37°C ±1°C. The samples were first tempered at 50°C for 10 min to erase any thermal history experienced during refrigerated storage before being placed in a water bath set to the temperature for measurement to allow for 5 mins of equilibration. The shear rates were varied based on protein concentration and temperature measurement according to Table 1.

The differences in shear rate were due to the wide-ranging viscosities for MCC solutions from 6 to 12% protein concentration. It was necessary to alter the shear rates in accordance with the concentration and temperature of the MCC solutions to stay within linear range of the instrument. Each sample was tested in triplicate for each of the 3 experimental replicates. The averaged viscosities of each MCC sample were log-transformed and plotted using Microsoft

Excel software (Microsoft Corp.) at each temperature to derive linear regression equations to predict viscosity as a function of protein concentration.

Chemical Analysis Methods

MCC were analyzed in triplicate using the following analytical methods: Total solids was analyzed by direct forced-air method (AOACI 2016, method number 990.20), fat by ether extraction (AOACI 2016, method number 989.05), lactose by an enzymatic method (AOACI,

2016; method number 2006.06), total nitrogen (TN) (AOACI 2016, method number 990.20), nonprotein nitrogen (NPN) (AOACI, 2016; method number 990.21), noncasein nitrogen (NCN)

AOACI, 2016; method number 998.05). TP was calculated as TN minus NPN multiplied by

6.38, CN was calculated as TN minus NCN multiplied by 6.38, and SP content was calculated by subtracting NPN from NCN and multiplying by 6.38.

For monitoring, skim milk, MCC and MF permeate, and UF permeate composition (i.e., fat, protein, and lactose concentration g/100 g) during the MF and UF processing runs, samples were analyzed using a mid-infrared (MIR) spectrophotometer (Lactoscope FTA, Delta

Instruments, Drachten, Netherlands). The MIR was calibrated with milk, MF permeate and MCC standards produced at Cornell University. The reference chemistry for the milk calibration samples was all lab mean reference chemistry for the modified milk calibration samples as described by Wojciechowski et al. (2016). The wavelengths, scale factors (i.e., primary slope) and intercorrection factors for each virtual filter model (fat A, fat B, protein, and lactose) were as described by Kaylegian et al. (2009)

Statistical Analysis

The GLM procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) was used to determine the effect of temperature (4, 20, and 37°C; a category variable), protein concentration

(about 6, 8, 10 and 12%), and experimental replicate (n= 3) on MCC AV measured with a

Brookfield viscometer. Actual measured protein concentrations (total nitrogen x 6.38) were

90 used. All interactions of these parameters were included in the model. The protein concentration was transformed by mean-centering (Misawa et al., 2016) to avoid co-linearity effects on statistical analysis (Glantz and Slinker, 2001). If the F-value for the full model was significant

(P < 0.05), then significance of each parameter (P < 0.05) and their interactions was determined. For Instron data, the firmness of 4°C MCC samples with 14, 16, and 18% protein

(category variable) was measured at days 1, 3, 6, 8 and 10 days (continuous variable that was mean centered) of storage at 4°C. For CN and protein concentration in the ultracentrifugation supernatants of skim milk and MCC's, the impact of temperature (4, 20, and 37°C; category variable) and protein concentration (starting skim milk or MCC; 3.5, 6.5 and 10.5%; continuous variable that was mean center) were determined.

Results and Discussion

Protein Content of Micellar Casein Concentrates

The results of the chemical analysis of the MCC from each production run are shown in

Table 2. In general, the protein concentration in all MCC dilutions were slightly higher than the target values but provided the range of concentrations desired for each product functionality testing method. The protein concentration among the three experimental replications were consistent as indicated by the low standard deviations.

Protein Composition of the Aqueous Phase Around the Casein Micelles

The amount of protein, CN, and milk serum proteins in the aqueous phase outside the CN micelles was determined via Kjeldahl analysis of ultracentrifugation supernatants from skim milk (used for a control) and 6.93% and 11.51% protein MCC. The protein contents of the skim milk and 6.93% and 11.51% MCC supernatants were measured as true protein, CN and CN as a percentage of total protein in solution (Table 3). The effects of temperature and protein

91 concentration on protein and CN movement out of the micelles into the serum phase of skim milk and MCC are clearly seen in Figures 2 and 3. Protein and CN concentration in the supernatant both increased (P <0.05) with decreasing temperature. There was an effect of both

MCC protein concentration and temperature (P <0.05) and a very strong interaction effect of these two variables (P <0.05) on true protein (Figure 2) and CN (Figure 3) content of the supernatants. CN as a percentage of true protein (Table 3) of the ultracentrifugation supernatants increased (P <0.05) with both increasing MCC protein concentration and decreasing temperature.

The proportion of CN to true protein in the supernatants increased (P <0.05) from about

16.7 to 77.52% (Table 3) when protein concentration was increased from skim milk to an MCC containing 11.51% protein at 4°C. This change was also seen at the other temperatures with increases of 3.19% to 34.45% and 1.15% to 13.55% at 20°C and 37°C respectively. When looking at the effect of temperature within each of the protein concentrations (Table 3), the CN as a percentage of true protein increased (P <0.05) from 1.15% at 37°C to 16.67% at 4°C for skim milk and 8.14% to 37.86% and 13.55% to 77.52% for the 6.93% and 11.51% MCC solutions, respectively. Both the increase in protein and CN as a percent of true protein in the serum phase around CN micelles with decreasing temperature would be expected to cause an increase in viscosity with both decreased temperature and increasing protein concentration.

The protein composition results are consistent with previous work done by Davies and

Law (1983) who also used centrifugation (70,000 x g for 2 to 4 h) to measure protein in different fractions from milk. They found that the protein concentration in solution around the CN micelles changed dramatically at 4°C when compared with 20°C with the serum CN concentration increasing from 9.8% to 23.2% of the total CN in the milk. In our study for skim

92 milk, results are presented as the CN as a percentage of protein in the supernatant at 4, 20, and

37°C and decreased from 16.7 to 1.15% (as temperature increased from 4 to 37°C) percent of the protein in supernatant produced from skim milk, showing a trend of increasing CN in the serum phase around the CN micelles as temperature decreased. Additionally, Davies and Law (1983) reported that almost the entire increase of serum protein came from β-casein (87% of increase) and γ-casein (7.4% of increase), which is now known to be a proteolysis byproduct of β-casein.

Downey and Murphy (1970) reported comparable results, namely a significant increase in the serum protein concentrations at 5°C, however the increase due specifically to β-casein was lower

(46% increase). Davies and Law (1983) hypothesized that the difference in results could be due to the difference centrifugation conditions among the studies, as Downey and Murphy only used

36,000 x g for 2 h.

Impact of Protein Concentration and Temperature on Apparent Viscosity

Both temperature and protein concentration of MCC and their interaction had an impact

(P <0.05) on AV (Figure 4) and log of AV (Figure 5) with a very strong interaction effect (P

<0.05) for MCC ranging from 6.5 to 13.2% true protein content at temperatures of 4, 20 and

37°C. MCC with high protein concentration had more than a 10-fold increase in AV when the temperature was decreased from 37 to 4°C, than for skim milk (Figures 4 and 5). The AV for the

10.66% MCC increased from 3.9 to 25.1 Pa*s when temperature decreased from 37 to 4°C whereas the 13.21% MCC showed a much larger increase, (7.3 to 133.2 Pa*s) in AV over the same temperature range. The ϕ occupied by the CN micelles increased as protein content of the

MCC increased and can be seen in Figure 1 by the difference in volume of the CN micelle pellet in the centrifuge tubes for the MCC containing 6.9 and 11.5% protein (Figure 1). The observed increase in AV at protein concentrations from 6.5 to 13.2% MCC (Figure 4), when temperature

93 was decreased from 37 to 4°C, could be due to both the higher ϕ of the CN micelles in solution with increasing protein concentration, or it could be that as the protein concentration in the serum phase (Figure 2) and the CN as a percent of true protein (Table 3) increased in the serum phase, it resulted in an increase in AV, or a combination of both (interaction effect) at 4°C.

When looking at the data points in Figure 5, showing the log AV, we were able to have a line of best fit and equation for the prediction of AV that matched the data points with extremely high precision. All the equations and resulting lines of best fit for log AV in Figure 5 had R2 values of ≥ 0.993. This data is very consistent with that reported by Adams and Barbano (2016) and likewise, allows for the prediction of log AV for variable MCC protein concentrations at different temperatures with high accuracy. Additionally, the AV findings agree with the research by Misawa et al. (2016) and Cheng et al. (2019a,b) who saw increasing AV with increasing protein concentration and decreasing temperature. Cheng et al. (2019a,b) also found that increasing CN as a percentage of TP and decreasing temperature increased viscosity more than the change in fat level. Furthermore Cheng et al. (2019b) showed that increase in CN as a percent of TP increased viscosity more than increasing TP (using MDWP). This would indicate that CN, particularly its temperature dependent reversible disassociation in and out of CN micelles has a large influence on the rheological properties of milk and MCC. These relationships between protein concentration and viscosity need to be cross referenced to sensory mouth feel and stability of dairy based beverages using MCC as a novel protein ingredient in future research targeted at using protein to build mouthfeel in dairy based beverages instead of hydrocolloids and enable the dairy industry to achieve clean label beverages that consumers desire.

Impact of Protein Concentration and Temperature on MCC Gel Strength

The MCC solutions contained roughly 14.5, 16.7, and 18.7% CN and had calculated CN particle ϕ (assuming the CN micelle is 2/3 by volume serum phase) of ϕ = 0.437, 0.501, and

0.56, respectively, as seen in Table 4. When the liquid MCC concentrates were filled into containers and cooled, it was visually apparent that the treatment containing the highest protein concentration (20.3%) immediately had visible gelation when cooled to 4°C. We wanted to determine if gel strength was constant across time of storage at 4°C over the range of MCC protein concentrations (from about 15 to 20%), or if MCC gels increased in strength with time of storage. Compression force as an index of gel strength over 10 d of storage is shown in Figure 6.

Both MCC with 17.9 and 20.3% protein were gels at 4°C on day 1 of storage, while the 15.6%

MCC was not. Both protein content and time of storage influenced gel strength with higher gel strength at higher protein (P <0.05) and gel strength increasing (P <0.05) with time of storage at

4°C with an interaction effect of protein by time of storage (P <0.05). The increase in gel strength with time of storage was larger for MCC with higher protein content. For the 20.3% and 17.9% protein MCC samples, the gel strength increased by 4.3 and 5.1 N respectively over the 10-day shelf life, while the 15.6% sample only showed a gel strength increase of 1.9 N. We hypothesize that the observed gel structure for the 16.7 and 18.7% CN MCC solutions at day 1 was due to the high ϕ of the CN micelles in solution and could be attributed to a gel formation resembling a jammed particle gel when the ϕ was in the range of ϕ = 0.5 to 0.6 (i.e., 50 to 60% of the solution volume occupied by particles). Additionally, over time of storage, all 3 MCC concentration increased (P <0.05) in gel firmness and we hypothesize that this increase in gel strength with time was due to the structuring of the proteins in solution around the CN micelles

(Table 3 and Figure 2) where a large portion of the protein in solution around the micelles was

95 disassociated nonmicellar CN, presumably β-casein (Downey and Murphy, 1970; Davies and

Law, 1983). High CN concentration in the liquid phase around the micelles created friction between micelles and lead to a random loose packed gel system (Silbert, 2010).

The gelation data from the Instron (Figure 6) is consistent with the theory of a particle jamming gel (18.7% CN) and solid like properties related to random loose packing of particles

(16.7% CN), particularly for the rapid development of solid properties at higher protein concentration. Previous research on cold gelation of MCC done by Lu et al. (2017) provided electron micrographs of ≈20% protein (wt/wt) MCC and showed a structure consistent with that of a jammed system with hard spheres. This would explain the rapid onset of gelation of higher protein MCC’s.

Conclusions

The protein, CN, and CN as a percent of TP in the liquid phase around casein micelles in

MCC increase with increasing casein concentration of the MCC and with decreasing temperature. CN as a percent of TP at 4°C in the liquid phase around CN milk cells increased from about 16% for skim milk to about 78% for an MCC containing 11.5% protein. AV of micellar concentrate solutions in the range of 6 to 13% CN increased with increasing CN concentration and decreasing temperature. There was strong temperature by protein interaction with AV increasing non-linearly with decreasing temperature at high protein concentration.

MCC containing about 16 and 18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels increased in strength over 10 days of storage at 4°C and this was like due migration of CN out of the micelles and interaction of the non-micellar CN to form a network that further strengthened the random loose jamming get structure. Learning how to

96 control viscosity changes in beverages using MCC could be a useful approach to use dairy protein as a clean label viscosity modifier in high protein beverages.

Acknowledgments

Funding was provided in part by Dairy West (Meridian, ID), the National Dairy Council

(Rosement, IL), and the Northeast Dairy Foods Research Center (Cornell University, Ithaca,

NY). The technical assistance of laboratory staff members Chassidy Coon, Michelle Bilotta, and

Sara Hatch from the Department of Food Science at Cornell University (Ithaca, NY) with analytical testing was greatly appreciated.