Casein-Based Powders: Characteristics and Rehydration Properties, Lilia Ahrné, Richard Ipsen, and Anni Bygvraa Hougaard

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1 Casein-Based Powders: Characteristics and Rehydration Properties Denise Felix da Silva, Lilia Ahrné, Richard Ipsen, and Anni Bygvraa Hougaard Abstract: Casein-based powders are gaining industrial interest due to their nutritional and functional properties, but they are also known to have poor rehydration abilities. The fundamental physical and chemical mechanisms involved in the rehydration of these powders are essential for determining the critical steps in the manufacturing processes and for developing casein powders with improved rehydration properties. A number of analytical methods have been developed to measure the rehydration ability of powders, but criteria for the selection of methods for casein-based powders have not been provided. This review article provides an overview of the characteristics and methods for the production of casein-based powders, methodologies to measure their rehydration properties, and it summarizes the current state of understanding regarding rehydration. Advancements have been made in the field; however, a fundamental understanding enabling improvement of the rehydration properties of these powders is still lacking. Keywords: dairy powders, inter-linked caseins, rehydration stages Introduction Dairy-based ingredients, such as powders, are used in a great variety of food products due to their functional, nutritional, and sensory properties (Kelly and Fox 2016). Milk protein powders are complex systems that contain caseins, whey proteins, lactose, fat, and minerals in different proportions depending on the applied concentration process (Petit and others 2016). Whey protein powders have received much attention from industry as well as academia during several decades, and their use in a wide range of food products has been extensively explored with the aim of improving nutritional value and product functionality (Ozmihci and Kargi 2008; Hussain and others 2012; Park and others 2014; Crowley and others 2015; Erbay and Koca 2015; Buldo and others 2016). The use of casein-based protein ingredients in food applications is still expanding. In the past few years new production methods have been developed, such as pretreatment and/or design of feed concentration and composition, and their use is gaining interest from the dairy industry due to their nutritive value, functionality, and stability during processing (Bong and Moraru 2014; Carr and Golding 2016). However, casein-based dairy powders, such as milk protein concentrate (MPC), micellar casein isolate (MCI), sodium caseinate (NaCas), and calcium caseinate (CaCas) usually present poor rehydration characteristics. Their low concentrations of lactose and the presence of interlinked casein micelles have been pointed out as CRF Submitted 9/21/2017, Accepted 10/13/2017. Authors are with Dept. of Food Science, Faculty of Science, Univ. of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg C, Denmark. Direct inquiries to author Felix da Silva ( denise@food.ku.dk). responsible for the poor rehydration ability of casein-based powders (Schuck and others 1994; Gaiani and others 2005, 2007, 2009a; Fang and others 2008; Mimouni and others 2010a; Richard and others 2012). However, due to differences in manufacturing processes and composition, as well as the methods used for evaluating their rehydration properties, a fundamental understanding enabling improvement of the rehydration properties of these powders has not been achieved. Previous reviews have focused on the rehydration properties of dairy powders in general (Fang and others 2008; Selomulya and Fang 2013), or more specifically on the effect of salts on rehydration properties of milk protein powders (Hussain and others 2012). Recently, the underlying principles and data outputs of methods to monitor rehydration of dairy powders have been discussed (Crowley and others 2016); nevertheless, no reviews have focused specifically on rehydration challenges of casein powder ingredients and none have discussed techniques to measure their rehydration behavior and how to improve their rehydration properties. The aim of this review is to provide an overview of types and production methods of casein-based powder ingredients and the current state of understanding of their rehydration properties, as well as to discuss and evaluate the suitability of different methods used to assess and improve their rehydration properties. Characteristics and Production of Casein-Based Powders Casein is the main protein in milk (80%) and it exists as 4 different types: α s1, α s2, β, andκ. In milk, most of the casein occurs as spherical particles known as casein micelles. Casein micelles also contain colloidal calcium phosphate, which aids in maintaining the integrity of the casein micelles together with hydrophobic 240 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.17,2018 C 2017 Institute of Food Technologists doi: /

2 Figure 1 Equilibrium modification in different physicochemical and processing conditions. Inspired by Gaucheron (2005). interactions and hydrogen bonds between caseins (Qi 2007; Thomar and Nicolai 2015). Casein micelles are dynamic structures with diameters varying between 30 and 300 nm, and they can be considered as natural colloidal microgels (Sadek and others 2015). However, the exact structure of the casein micelles has not yet been fully elucidated. Several models have been proposed to describe the structure of casein micelles and their interactions with other components (Dalgleish 1998, 2011; Farrell and others 2006; Dalgleish and Corredig 2012; De Kruif and others 2012; De Kruif 2014). Still, some concepts are generally accepted: Most κ-casein is present on the micelle exterior, but the κ- casein is not homogeneously distributed on the surface Hydrophilic segments of κ-casein extend into the serum The interior of the micelle can be considered as a network of α s -andβ-caseins connected to nanoclusters of calcium phosphate Both the content of colloidal calcium phosphate and hydrophobic interactions between casein molecules are necessary for micellar integrity. MPC, MCI, NaCas, and CaCas are all casein-based ingredients that have substantial value due to their protein functionality, and they are most often sold commercially as a spray-dried powder (Dalgleish 1998; Gaiani and others 2006a; Mimouni and others 2009). Generally, these protein-rich powders can be used in the formulation of high-protein nutrition bars, meal replacements, ice cream, bread, processed meat, beverages, and medical nutrition products (Parsons and others 1985; Gallagher and others 2003; Kilic 2003; Agarwal and others 2015). The differences in composition and production methods influence the structure of the caseins and, consequently, their rehydration properties. Examples of how micellar composition and structure can be influenced by processing parameters are shown in Figure 1. Casein ingredients are typically dried by spray-drying, and during the drying process, the proteins in the feed are redistributed in the droplet and water is evaporated, which involves transport of solute and solvent, adsorption, and interactions between solutes and phase transitions. It is well known that the structure of spraydried particles may depend on the composition of the feed as well as the drying conditions. Considering this, it is extremely important to keep in mind that the rehydration properties of powders are strongly affected by powder structure (Barbosa-Cánovas and Juliano 2005). Table 1 presents an overview of casein-based ingredients and their total protein contents, major components, production processes, and applications. The properties of each ingredient are discussed in detail below. MPC/isolate MPCs are produced by heat-treating skim milk and concentrating the protein fractions, both the whey proteins and caseins, using membrane technology (Carr and Golding 2016). Usually, MPC powder contains approximately 82% casein and 18% whey proteins based on the total protein content (Hussain and others C 2017 Institute of Food Technologists Vol.17,2018 ComprehensiveReviewsinFoodScienceandFoodSafety 241

3 Table 1 Overview of casein-based powder ingredients. MPC MPI Total protein content 35% to 90% min 80 % protein Major components Process Structure Application 82% casein and 18% whey proteins Heat treatment of skim milk and concentrating the protein fractions, both whey protein and caseins, using membrane technology MCI >80% 86% casein Membrane filtration of skimmed milk followed by spray-drying NaCas >80% NaCas Produced by acid precipitation of skim milk and resuspension with sodium hydroxide, NaOH CaCas >80% CaCas Produced by acid precipitation of skim milk and resuspension with calcium hydroxide, Ca(OH) 2 MPC, milk protein concentrate; MPI, milk protein isolate; MCI, micellar casein isolate; NaCas, sodium caseinate; CaCas, calcium caseinate. 2012). Caseins are present in micellar structure and whey proteins in their native globular form or with some degree of denaturation due to the thermal treatment during processing. MPCs have been developed with different compositions and used as ingredients in a broad range of dairy products, such as milk for cheese making, ice cream, yogurt, beverages, soups, and salad dressings (Shakeel-Ur- Rehman and others 2003; Guinee and others 2006; Harvey 2006; Patel and others 2006; Caro and others 2011; Jana and Mandal 2011; Agarwal, and others 2015). The ratio of casein to whey proteins in the final dairy ingredient depends on the separation process prior to the drying (Petit and others 2016). MPC powders are commercially available in protein concentrations ranging from 35% to 90% and are denominated accordingly: MPC40 contains 40% protein. However, the drying of such highprotein concentrates can lead to loss of solubility and poor rehydration ability (Carr and Golding 2016). Likewise, MPC can also be called milk protein isolate (MPI) when the amount of protein is higher than 80%. MCI MCI powder is an innovative protein ingredient that belongs to the group of high-protein dairy powders with protein contents higher than 80%. It may also be referred to as native phosphocaseinate, MCI, or micellar casein (Carr and Golding 2016). It is produced by membrane filtration of skim milk followed by spray drying (Schokker and others 2011). During production, whey proteins are removed and caseins are present in the micellar state, containing colloidal calcium phosphate. A microfiltration membrane with pore size of approximately 0.1 μm is typically chosen to allow separation of whey proteins from micellar casein (Carr and Golding 2016). After microfiltration, the micellar casein is spray-dried. Spray-drying is a critical step because the removal of water may enhance the formation of poorly dispersible aggregates through hydrophobic interactions (Havea 2006). Caseins are present in micellar structure and whey proteins are present in their natural globular form or with some degree of denaturation due to the heating during processing. Caseins are present in the micellar state, that is, containing colloidal calcium phosphate Solubilization of precipitated casein into NaCas Calcium is bound to the protein forming aggregates with chargedκ-casein on the surface Milk for cheese making, ice cream, yogurt, beverages, soups and salad dressings andsoon. Yogurt and cheese manufacturing. Nutritional and dietary preparations and in ready meals, baby formulas. Nutritional and dietary preparations, ready meals, infant and geriatric formulas, and so on. NaCas NaCas is produced from skim milk by acid precipitation and resuspension of the precipitate under alkaline conditions (NaOH). Caseinate salts, in general, are known for their ability to form aggregates at low ph. The degree of this aggregation is phdependent (Nakagawa and others 2016). In NaCas, the casein casein interactions are controlled by electrostatic repulsion between the components of the casein molecules. These repulsions are weaker for monovalent cations when compared to divalent cations (such as calcium), and this enables the overcoming of the hydrophobic association energy resulting in the formation of hydrated aggregates (Carr and Golding 2016). Some difficulties are present during NaCas manufacture such as the high viscosity of NaCas solution at moderate concentrations limiting the total solids of the feed for spray-drying to 20%. Likewise, coating of casein micelles with a viscous film delays the dissolution of the caseins after the addition of alkali. To overcome these difficulties, it is important to control the ph and temperature during manufacture (Sarode and others 2016). It is known that during NaCas manufacture calcium phosphate is removed from the casein micelle and the structure is damaged producing individual casein proteins. Therefore, when NaCas powder is reconstituted it may facilitate its solubilization (Holt and others 1986; Smialowska and others 2017). CaCas CaCas is produced by acid precipitation of skim milk and resuspension with calcium hydroxide (Ca(OH) 2 ). In CaCas, almost all of the calcium is tightly bound to the strong anionic sites of the proteins, as a result of hydrophobic bonds. This causes rearrangement of the caseins by reduction of intermolecular repulsion and formation of aggregates with a predominance of charged κ-casein on the surface. Consequently, CaCas is poorly hydrated and compact (Carr and Golding 2016). CaCas can be used in nutritional and dietary preparations and in ready-to-eat foods such as soups (Moughal and others 2000). However, those applications require good protein dissolution, making the use of CaCas challenging. Principles of Rehydration Considering that most powdered ingredients are dissolved before use, their ability to rehydrate readily in aqueous media is essential (Schuck and others 2002; Gaiani and others 2007; Hussain and others 2011a). Figure 2 describes the 5 stages of rehydration (Crowley and others 2016): Wettability, which is the ability to absorb water Swelling, which is the ability to increase in size with water absorption Sinkability, which is the ability of the swelled particles to sink into the water 242 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.17,2018 C 2017 Institute of Food Technologists

4 Figure 2 Mechanism of 5 stages for powder rehydration. Reproduced from Crowley and others (2016) with permission. Dispersibility, which is the ability to disperse into single particles throughout the water Dissolution, which corresponds to the separation between molecules. More information about Figure 2 can be found in Crowley and others (2016). During rehydration, 2 or more stages may occur simultaneously; wetting and swelling, as well as dispersion and dissolution. Also, for specific powders, some stages may not occur or occur at different times (Barbosa-Cánovas and Juliano 2005; Gaiani and others 2006a; Crowley and others 2016). Likewise, the term instant properties is used for the physical properties of powder linked with these rehydration stages, namely powders that dissolve instantly. Microenvironments such as ph, mineral salts, size and structure of the powders, as well as the rehydration conditions, such as temperature and shear, may affect rehydration behavior (Schuck and others 2007; Hussain and others 2011b). The rehydration properties of dairy powders have been studied using different methods (Fitzpatrick and others 2004; Murrieta- Pazos and others 2011, 2012; Hussain and others 2011a, 2012). Some of the applied standard methods for measuring rehydration of dairy powders are easy to perform and interpret (for example, solubility by centrifugation followed by drying or the insolubility index test), but the results do not represent all the stages of the powder rehydration process and also the reproducibility can be impaired, which makes such methods not applicable for the newly developed casein-based ingredients, such as micellar casein powders, due to their poor rehydration ability (Gaiani and others 2006a; Fang and others 2008). Ji and others (2016a) have discussed the challenge of finding a universal method to measure rehydration of dairy powders. They highlighted wettability and dispersibility as the 2 most important steps during rehydration of milk protein-based powders (Ji and others 2016a). Recent research has focused on dynamic monitoring, such as during the entire process of powder rehydration, which allows identification and quantification of rate-limiting stages for a specific powder. For instance, studies have shown that the ratelimiting stage for casein-based powders is the dispersion time, while for whey-based powders it is the wetting time (Mimouni and others 2009, 2010b). Table 3 describes the strengths and weaknesses of each method. During the concentration and/or drying of casein-based powders, the high activity of calcium ions may facilitate enhanced charge screening between casein micelles, thereby resulting in the formation of poorly dispersible aggregates through hydrophobic interactions (Havea 2006). Also, a partial denaturation of the whey protein present in the samples may lead to a reduction in solubility of the final product (Augustin and others 2012; Fang and others 2012; Zhao and others 2015; Hauser and Amamcharla 2016a). Powder rehydration may be affected by raw material composition, pretreatments, processing methods, and storage conditions (Barbosa-Cánovas and Juliano 2005). In addition, surface and bulk composition, particle structure, and rehydration conditions can influence rehydration ability (Gaiani and others 2009b). Analytical Techniques used to Evaluate Rehydration Properties of Casein Powders The analytical methods to measure hydration can be divided into those which are able to distinguish the various stages (as indicated in Figure 2) and those which are only able to measure the total hydration time and/or a specific stage related to the rehydration. Turbidimetry, light scattering, microscopy analysis, and rheological approaches are methods able to characterize different rehydration stages, while insolubility/solubility index, low-field nuclear magnetic resonance (LF-NMR), focused-beam reflectance, and ultrasound techniques measure total rehydration. A combination of 2 or more methods enables the successful interpretation of rehydration behavior of casein-based powders due to the evaluation of different stages and also the final rehydration. An overview of the methods applied, the casein-based powders studied, as well as the references, is shown in Table 2 and discussed later. Turbidimetry Turbidimetry is a suitable tool to characterize rehydration of powders (Gaiani and others 2007). Turbidity is an optical phenomenon that can be visualized as a cloud or cloudiness in a transparent solution. In other words, turbidimetry measures the loss of intensity of transmitted light due to the scattering effect of C 2017 Institute of Food Technologists Vol.17,2018 ComprehensiveReviewsinFoodScienceandFoodSafety 243

5 Table 2 Overview of methods used for measuring rehydration of casein-based powders. Method Parameters measured Powder Reference Turbidimetry Change in turbidity MCI (Gaiani and others 2005) MPC (Gaiani and others 2007) MCI (Gaiani and others 2009) MCI (Hussain and others 2011a) MCI (Schuck and others 2007) Light scattering Changes in particle size MPC (Mimouni and others 2009) MPC (Jeantet and others 2009) MCI (Hussain and others 2011b) MCI (Gaiani and others 2005) MPC (Gaiani and others 2009) MCI (Richard and others 2013) MCI (Gaiani and others 2006a) NaCas (HadjSadok and others 2008) Microscopy Changes in structure MPC (Mimouni and others 2009) MPC (Yuan and others 2010) MPC (Mimouni and others 2010a) MCI (Richard and others 2013) MCI (Petit and others 2016) MCI (Gaiani and others 2006b) MPI (Gaiani and others 2009) Rheology Changes in viscosity MCI (Gaiani and others 2006b) Insolubility Index Amount of sediment MCI (Davenel and others 2002) MCI (Schuck and others 2002) Solubility test Amount of total solids MPI (Anema and others 2006) MPI (Havea 2006) MPC (Mao and others 2012) MCI (Schokker and others 2011) LF-NMR Relaxation time and relative abundance of water populations with different mobility MCI (Schuck and others 2002) MCI (Davenel and others 2002) MCI (Schuck and others 2007) FBRM Changes in particle size MPC (Fang and others 2010) MPC (Fang and others 2011) MPC (Fang and others 2012) MPC (Hauser and Amamcharla 2016a) MPC (Hauser and Amamcharla 2016b) Ultrasound Acoustic properties NaCas (Povey and others 1999) MCI (Richard and others 2012) MPC (Hauser and Amamcharla 2016a) MPC (Hauser and Amamcharla 2016b) Contact angle Changes in contact angle water/powder bed MPC (Crowley and others 2015) NaCas (Fitzpatrick and others 2016) MPI (Fitzpatrick and others 2016) MPI Ji and others 2016a MCI Ji and others 2016a NaCas Ji and others 2016a MPI MCI NaCas CaCas Washburn Water weight absorbed MPI Ji and others 2016a MCI Ji and others 2016a NaCas Ji and others 2016a MPI MCI NaCas CaCas MPI Ji and others 2015 particles suspended in a solution. Light in the near-infrared region (860 nm) is used and the incident beam is reflected back at 180 by particles in suspension in the solution to an electronic detector located in the equipment. A turbidity meter can be used for measurements. Data are recorded in turbidity units as a function of time, which allows the monitoring of the entire rehydration process. The changes in turbidity occurring during powder rehydration can characterize several stages of the rehydration process (Gaiani and others 2005). Gaiani and others (2005) used turbidimetry together with particle size analysis to study hydration properties of agglomerated and nonagglomerated MCI powders, and they identified wetting, swelling, dispersion, and dissolution stages by analyzing turbidity profiles. Using the same methods, Gaiani and others (2007) demonstrated that the rate-limiting stage for MPCs rehydration was dispersion. The rehydration profile of MCI, characterized by using turbidimetry, has suggested the presence of wetting, swelling, and a slow dispersion stage (Gaiani and others 2009a, 2009b). The obtained results were in agreement with light-scattering and optical microscopy. However, noisy measurements may occur when using turbidimetry. Hussain and others (2011a, 2011b) assigned the noisy turbidity signal of whey protein isolate (WPI) powders during rehydration to the formation of lumps. Also, they reported that rehydration in 0.75% to 3.0% NaCl solution delayed the stabilization of turbidity in MCI, but at higher NaCl concentrations (6% to 12%, w/v) the swelling stage disappeared and a faster stabilization of turbidity was observed. 244 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.17,2018 C 2017 Institute of Food Technologists

6 Turbidimetry is a suitable tool for the characterization of rehydration of powders, and the existence of probes that can be inserted directly into samples or applied during processing, which allows the method to be used in-line. In addition, it is a versatile technique that can be successfully used to understand the rehydration mechanisms of both casein and whey-based powders (Schuck and others 2007; Gaiani and others 2007, 2009a, 2009b; Hussain and others 2011a, 2011b). Wetting time The measurement of wetting time is based on the time required for a given mass of powder to fully submerge below the water surface. The method has been widely reported in pharmaceutical research, but not much for an understanding of the rehydration of casein-based powders. Wetting time measurement is a traditional method in which 10 g of a powder is dropped into 250 ml distilled water at 25 C(GEA Niro 2005). The time required for complete wetting without any agitation is measured. Samples that present a wetting time of less than 60 s are considered easy to wet, while the ones which take more than 120 s are considered nonwettable (GEA Niro 2005). However, this method is of practical use only for powders that are easy to wet, whereas casein-based powders tend to float on the surface of the liquid, and wetting time can be an inappropriate measure (Ji and others 2016a). Dynamic contact angle and Washburn method The dynamic contact angle measurement and the Washburn method (Washburn 1921), which are based on capillary rise wetting behavior, have also been widely used in pharmaceutical industry experiments. More specifically, the dynamic contact angle measurement evaluates a single liquid drop penetrating into a powder bed, and by monitoring the changes in the tangent contact angles over time the wettability can be assessed (Crowley and others 2015; Ji and others 2015, 2016b, 2017). Thus, an optical tensiometer is used to measure contact angle (θ) of a water droplet by the sessile drop technique with dynamic live measurements. Crowley and others (2015) have used the dynamic contact angle technique to understand rehydration properties of MPC powders within a range of protein concentrations (from 35% to 90%). They concluded that high-protein MPC powders (MPC80, MPC85, MPC90) displayed θ values at time zero of 75, which classifies those powders as nonwettable, while powders with 35% and 50% of protein presented θ values around 55. After 5 s,high-protein powders did not show any decrease in the tangent angle in contrast to the low-protein powders (MPC35, MPC50). Nevertheless, it is important to consider that the nonuniform surface topography and composition may affect the results estimating higher θ values (Forny and others 2011; Crowley and others 2015). Rehydration ability of MPI (86% protein) has been widely studied for the last few years (Ji and others 2015, 2016a, 2016b, 2017). The effect of fluid bed agglomeration on the rehydration was studied using the Washburn method, where the powder capillary rise wetting behavior was measured based on the absorbed water weight after 10 min. It was shown that agglomerated powders absorbed significantly more water (Ji and others 2015). The same method, together with contact angle measurements, was used to investigate the rehydration characteristics of a set of dairy powders, including SC, MCI, and MPI (Ji and others 2016a). The results showed that the Washburn method was applicable for powders with a porous structure such as agglomerated MPI, but it was not able to measure wettability of NaCas powders. Contact angles over time showed a different tendency for the powders; for example, agglomerated MPI did not present a faster decrease in θ value over time. The authors also indicated that this method is simple to perform as it presents straightforward observation and high selectivity for dairy powders (Crowley and others 2015; Ji and others 2016a). However, limitations of the optical tensiometer method, such as the difficulty in measuring the dynamic contact angles with high reproducibility and accuracy at high speed rate were discussed. Both methods were used to understand rehydration of agglomerated MPI, MCI, NaCas, and CaCas (). Agglomeration improved the droplet penetration process during contact angle measurement and during the capillary rise wetting procedure for all powders. This improvement was more significant for MPI when using the contact angle method, while it was more significant for MPI and MCI when using the Washburn method. Conductivity Conductivity is a technique to evaluate the mineral release or ionic strength of a protein-containing solution. It has been used in rehydration studies to demonstrate that low ionic strength has a strong negative effect on the rehydration properties of casein-based powders, and that the synergistic effect of increasing ionic strength and temperature can significantly accelerate rehydration (Crowley and others 2015). Likewise, according to Ji and others (2016a), the conductivity of suspensions is a useful indirect indicator for quantification of dispersibility by the release of minerals during rehydration. It is assumed that calcium, phosphorus, magnesium, together with caseins, all belong to the slow-dissolving fractions in casein-based powders (Mimouni and others 2010b) coming from the casein micelle structures and exhibiting similar kinetics of solubilization (Mimouni and others 2010b). It is, therefore, possible to compare the kinetics of solubilization of casein-based powders based on the changes in conductivity during rehydration. Ji and others (2016a) have shown that agglomerated MPI presented a faster increase in conductivity compared to nonagglomerated MPI. Similarly, skim milk powder and WPI, which are considered easy-to-wet powders, reached their steady conductivity values in a very short time. Ligh-scattering Light-scattering (LS), also known as laser diffractometry or small-angle LS, has been used to determine particle size and particle size distribution in both dry and wet systems. This technique estimates particle sizes based on the specific angle at which the particles present scatter light; in other words, small particles scatter at large angles, while large particles scatter at small angles (Mimouni and others 2009). The prediction of a light-scattering pattern is based on the refractive index (RI) of the particles. Changes in particle size distributions during the dissolution of commercial CaCas were investigated using LS. After rehydration in water for 180 min, the solutions were introduced to an equipment with an LS system and analyzed for changes in the volume distribution and obscuration for 100 min. Two peaks were observed, the first with a size range between 0.1 and 2.0 μmandthe second ranging between 2 and 80 μm. During analysis the first peak increased, while the second one decreased, coinciding with a decrease in the obscuration, which indicated the dissolution of particles (Moughal and others 2000). The same method was used to study hydration of MCI powders. In the beginning of rehydration, an increase in particle sizes was observed, but after some time the particle sizes decreased significantly, followed by a continuous reading of small particles C 2017 Institute of Food Technologists Vol.17,2018 ComprehensiveReviewsinFoodScienceandFoodSafety 245

7 showing the dissolution of MCI powder (Gaiani and others 2005). Mimouni and others (2009) used LS for measuring hydration of agglomerated and nonagglomerated MPC powder at different temperatures. They showed that the volume concentration of agglomerated powders decreased faster than that of nonagglomerated powders. In addition, they noted the disruption of agglomerated particles into primary powder particles and, simultaneously, the release of material from the powder particles into the surrounding aqueous phase, and they concluded that these were the 2 rehydration stages occurring during MPC rehydration. The release of material from the particles was considered to be the rate-limiting step of dissolution of MPC (Mimouni and others 2009). The use of LS has been used mainly to validate data from other methods such as turbidimetry and rheometry. However, the method does not provide any information about wetting of powders, which is a disadvantage if the powder studied has wettability as the rate-limiting stage (McSweeney 2004; Gaiani and others 2007; Schuck and others 2007; Hussain and others 2011a). Also, for measuring the hydration of powders the solution must be diluted, which makes its utilization in production lines more difficult. Furthermore, the dilution increases the risk of introducing artifacts. LS has other limitations as well. The choice of RI varies between studies and affects data analysis. Proteins can be evaluated with many RI values ranging from 1.36 to 1.57 (Regnault and others 2004) to 1.57 (Griffin and Griffin 1985). Mimouni and others (2009) have noted that, with the low values of the RI (such as 1.36 or 1.38), the particle size distribution of a MPC85 powder suspension during rehydration became bimodal as a submicron peak appeared, and the lower the value of the RI, the higher the submicron peak area. Microscopy analysis Optic microscopy during powder dissolution can be used to monitor MCI rehydration (Gaiani and others 2009a, 2009b). The microscopy images clearly showed the swelling stage, where the particles increased in size from 250 to 400 μm with water absorption followed by dispersion and disintegration of the particles. Mimouni and others (2009) used a light microscope with a video camera to study structural changes in MPC powders after contact with a drop of water. This method allowed visualization of air vacuoles in the particles entrapped during spray-drying. Also using light microscopy, Fang and others (2010) studied commercial MPC powders and concluded that agglomeration behavior persisted after dissolution. Scanning electron microscopy (SEM) was used as a tool to investigate the effect of storage on the microstructure of rehydrated MPC powder particles after long (80 min) and short (10 min) hydration times (Mimouni and others 2010a). For this methodology, several steps of sample preparation have to be carried out, which may affect the sample structure. Using SEM it was shown that a combination of different interactions, such as bridging between casein micelles, resulted in a porous, gel-like structure that restrained the dispersion of individual micelles into the surrounding liquid phase without preventing water penetration and solubilization of nonmicellar components (Mimouni and others 2010a). During storage of the powder, interactions increased between and within micelles, which led to compaction of micelles and the formation of a monolayer skin of casein micelles packed close together. This combination was proposed to be the reason for the slow dissolution of stored MPC powders (Mimouni and others 2010a). The same method was used to investigate the effect of changes during storage on the microstructure of MPC powders during rehydration. The images showed that poorly rehydrated MPC exhibited a surface skin consisting mainly of interlinked casein micelles, which were able to imbibe water but not to release casein micelles (Mimouni and others 2010b). Richard and others (2013) studied the effect of temperature, agitators, and stirring speed on particle size reduction during the rehydration of MCI powders. Using images from a granulomorphometer during reconstitution, they showed that the mixing system design had a strong influence on the course of the rehydration process (Richard and others 2013). Relations between particle surface hardening and rehydration impairment during micellar casein powder storage were investigated using SEM images (Petit and others 2016). The ability of stored powder to adsorb water was affected compared to fresh powders. This impaired ability to absorb high amounts of water can be related to the structural modifications such as the formation of an inter-linked network of casein micelles at the particle surfaces. Also from SEM images, it was observed that the powder particle surface is not uniformly affected by the rehydration medium, as some parts were solubilized while other parts remained unaffected. However, small exposed particle surface modifications during storage are not visible on SEM, but they can be observed on topographical images by atomic force microscopy (AFM) (Petit and others 2016). Rheological approaches Rheology can be used to monitor powder rehydration by identifying rehydration stages based on viscosity changes (Crowley and others 2016). For rennet casein, peaks in the viscosity index have been related to particle size, inner particle interactions, and protein-water interactions (Ennis and others 1998). Wetting and swelling stages were identified as an initial increase in viscosity and a minor subsequent increase in viscosity was assigned to interparticle interactions, followed by network formation characterized by a further increase in viscosity. The maximum viscosity was related to absorption and a continuous low viscosity was taken as indicative of complete rehydration. This method was also used as a tool to evaluate rehydration of rennet casein manufactured from milk in a pilot-scale manufacture of mozzarella cheese analogs (Ennis and Mulvihill 1999, 2001). Gaiani and others (2006b) developed a method based on rheology to study the dissolution behavior of MCI powders as a function of concentration and temperature. Solutions (2%, 5%, 10%, and 12% at 15, 24, and 55 C) with MCI powders were subjected to a shear rate of 100 s 1 and viscosity profiles were used to interpret the rehydration process. At low concentration (2%) only the wetting was noticeable. No swelling peak was observed. LS measurements and microscopy images were used to validate the method. However, LS showed that the swelling phenomena appeared even at 2% MPC, which indicated a limitation for the rheometer method. At 5% MPC, the viscosity profile showed wetting and swelling followed by a slow dissolution of the particles. The events, indicated by viscosity measurements, were also related to particle size variations. Dispersion of powder led to a rapid increase in viscosity due to particle-wetting. The wetting stage was followed by swelling of the particles corresponding to a new peak in viscosity profile. As a consequence of the swelling, a disintegration of the wet particles occurred and their progressive 246 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.17,2018 C 2017 Institute of Food Technologists

8 dissolution explained viscosity and particle size decrease. After 13.3 h no further changes in particle size (0.36 μm) or viscosity were observed due to completion of dissolution. Concentration affected the viscosity profile. Two peaks on the viscosity profile for swelling and 2 peaks for disintegration were observed at a concentration of 10% and 12%, the first one was related to the swelling of the initial particles, and the second one to the swelling of the remaining particles. Temperature influenced the duration of each stage: higher temperatures led to shorter total rehydration time. Gaiani and others (2006b) concluded that temperature, concentration, and time were the major factors influencing MPC powder dissolution. Insolubility index test and solubility by total solids of supernatant Hydration properties of powders are directly related to the solubility in the aqueous phase (Crowley and others 2016), and standard methods, based on the total solids present in the supernatant and the amount of sediment measured after stirring and centrifugation, have been used widely to describe the rehydration behavior of dairy powders. The insolubility index test (IIT) consists of measuring the remaining insoluble material in a graduated standard glass cylinder (IDF standard 1988). The most widely used method consists of preparing a 5% (w/v) solution of powder in water, stirring for 30 min at a defined temperature, centrifuging at 700 g for 10 min, placing an aliquot of the supernatant on paper and drying it overnight at 105 C. The solubility is calculated based on the total solids. Hydration of high-protein powders has been studied using the methods described by Anema and others (2006), Havea (2006), Schokker and others (2011), and Mao and others (2012). The amount of insoluble material is easily determined. However, the reproducibility may be impaired and the results may not reflect all processes involved in powder rehydration. Anema and others (2006) used the solubility test to evaluate the effects of storage temperature on the solubility of MPC85. They found that the solubility of MPC85 decreased exponentially with an increase in storage temperature and time. Likewise, MPCs produced with different processing condition were evaluated using this method (Havea 2006). It was shown that solubility is related to the formation of insoluble material such as disulfide-linked aggregates consisting mainly of β-lactoglobulin. Furthermore, this method has limitations because powders with low density or high fat content may not sediment after centrifugation, even if the particles are not totally soluble. It can provide reproducible results for instant powders, but the method is not readily suitable for casein-based powders. Low-field nuclear magnetic resonance spectroscopy Low-field nuclear magnetic resonance is a nondestructive method that is also called NMR time domain, wide line, pulsed spectroscopy or relaxometry. This method has been widely used to study powder rehydration and has proved to be extremely sensitive to protein water interactions (Brosio and others 1983; Davenel and others 2002; Schuck and others 2002; Gaiani and others 2007; Lin and others 2016). The method uses a pulsed magnetic field to determine the transverse relaxation time (T 2 ) of water via the transverse relaxation of 1 H protons. T 2 and relative abundance (M 2 ) of water fractions provide information about the exchange of water protons with protein protons and/or the exchange of water between water fractions. All mobile protons present in the sample contribute to the NMR signal. In other words, the mobility of water molecules is affected by the interaction of water with macromolecules. During caseinbased powder rehydration, water molecules may interact dynamically with proteins, thereby reducing their freedom of movement. Water populations that are bound to protein have their freedom restricted, which implies smaller relaxation time values (T 2 )(fast relaxation), while populations of water that are not bound (free water) present higher relaxation time values (slow relaxation). M 2 indicates which percentage of the total water is present in bound or free water form (Davenel and others 2002). Deconvolution of decay curves using bi-exponential equations is used to analyze the contributions to the overall signal of protons in lipids, protons in free water, or water interacting with carbohydrates and proteins (Davenel and others 2002; Hoffmann and Reger 2014; Torres and others 2016). Using NMR relaxometry, Brosio and others (1983) studied water-binding in milk powder. Schuck and others (2002) investigated the effects of added mineral salts on water transfer of MCI powder during rehydration. T 2 of rehydrated powder with added NaCl slightly decreased compared to the control samples, while the addition of citrate or phosphate solution considerably decreased T 2 values, proposing a stronger interaction between powder and water, and thus the improvement of rehydration. The authors also concluded that the NMR method was mostly sensitive to protein water interactions associated with the advanced stages of rehydration, and not able to recognize primary stages of rehydration (Brosio and others 1983; Davenel and others 2002). A larger reduction in relaxation time of MCI powders has been observed with the addition of citrates compared to the addition of phosphates, and freeze-dried powders showed delayed rehydration compared to spray-dried MCI powder (Schuck and others 2002). These authors also proposed that NMR relaxometry was able to provide novel and useful information about the reconstitution process of MCI under industrial application conditions. The interpretation of T 2 data from NMR measurements was correlated with the results from the insolubility index test (IIT). However, the IIT did not indicate if the powder could be totally reconstituted in water after a long reconstitution period or if the powder contained insoluble substances. For this reason, the authors considered NMR more appropriate to identify later rehydration steps. In agreement with that, Gaiani and others (2009a, 2009b) noted that the NMR method was not able to identify the wetting stage of total powder rehydration; moreover, its potential to be used as on-line method is still remote. Focused-beam reflectance Focused-beam reflectance measurement (FBRM) is based on laser light back-scattering and has been used to measure particle size distribution and the kinetics of particles in suspension. In this method, a laser (λ = 780 nm) is directed through an optics module that focuses the beam at a narrow point near a sapphire window at the base of the probe. The rotation of the optics directs the laser away from the middle axis of the probe, making the beam scan the particles in a circular manner. This rotational movement increases the area covered by the laser providing more accurate data. During the measurement, laser light is back-scattered from particles and then coupled by a beam splitter to an optical fiber and conducted to the detector. The duration of the pulse depends on the time taken to scan an entire particle. The pulse is then multiplied by the scan speed to yield the chord length. The chord length distribution is calculated as a number or volume/weighted particle size distribution. This method computes aggregates and agglomerates as a single big particle, but can be used for nonspherical C 2017 Institute of Food Technologists Vol.17,2018 ComprehensiveReviewsinFoodScienceandFoodSafety 247

9 particles (Crowley and others 2016; Fang and others 2010, 2011). FBRM was used to characterize MPC powder solubility at varying concentration and thermal and storage aspects (Fang and others 2010). The authors showed that a faster rate of the chord length reduction implied better solubility, as more particles broke down and were dissolved in solution. For this study, FBRM provided the ability to monitor the in situ changes in chord length with time at different powder concentrations, reflecting the solubility of MPC powders. Using the same method, Fang and others (2011) observed that the solubility of agglomerated MPC powders decreased after 2 mo of storage at 35 C. Also, chord length decreased faster for rehydration at 50 C compared to 20 C. FBRM showed that the number of large particles (150 to 300 μm) decreased rapidly up to 300 s, while the number of small particles (1 to 10 μm) increased significantly during 1800 s of rehydration. Fang and others (2012) investigated the effect of spray-drying conditions on the solubility of MPC using FBRM. A smaller chord length suggested a better solubility, and the chord lengths of MPC particles dried at lower temperatures (inlet temperatures of 77 and 107 C) decreased more quickly compared to MPC powders dried at higher temperatures (inlet temperatures of 155 and 178 C). The authors proposed that higher temperatures may induce structural changes in the proteins on the particles, consequently increasing the formation of insoluble material. Hauser and Amamcharla (2016b) determined the effect of protein content and dissolution temperature on the dissolution characteristics of MPC and MPI using FBRM. They showed that after adding the powders into the water, the number of large particles (50 to 150 μm) decreased as they disintegrated into small and medium-sized particles. Subsequently, the number of small (<10 μm) and medium-sized particles (10 to 50 μm) increased. The large-particle counts for powder with 85% or 87% of protein decreased rapidly, whereas the large-particle counts for powders with 88% and 90% protein showed a gradual reduction. FBRM showed that an increase in protein content negatively affected the solubility (Hauser and Amamcharla 2016b). Low-intensity ultrasound Methods based on low-intensity ultrasound can measure the sound transmission that is related to the number and types of bonds present in the food sample, and the acoustic properties depend on the physical and chemical composition of the material (Zheng and Sun 2006). In other words, ultrasonic analysis associates ultrasonic properties with the properties of a specific food material. This association can be done by fitting the measured ultrasonic data of a well-characterized sample in an empirical equation that best fits the results (Pico 2012). The techniques based on low-intensity ultrasound are precise and nondestructive considering that the low-intensity ultrasound waves do not cause physical or chemical alterations in the properties of the material (Pico 2012). Ultrasonic spectroscopy has been used to identify the degree of rehydration of NaCas powders (Povey and others 1999). Acoustic modeling of NaCas was used to analyze the particle size distribution of a 4% NaCas solution. The authors concluded that the method was very sensitive to the degree of hydration of casein and, therefore, is a potential qualitative measure of the dissolution of casein (Povey and others 1999). Typical ultrasound test equipment consists basically of 3 units: a pulser-receiver, which generates electrical pulses; a transducer, which converts the electric pulses into short ultrasonic pulses; and display devices. The 2 most-used modal types of pulser-receiver are pulse-echo (PE) and through-transmission (TT). Mcclements and Gunasekaran (1997) concluded that the pulse-echo mode is automated and easier to design and operate. In addition, the pulseecho method has the lowest cost for ultrasonic testing (Mcclements and Gunasekaran 1997). Meyer and others (2006) used an ultrasound spectrometer in pulse-echo mode to correlate the data from ultrasound spectroscopy with a visual reconstitution test in milk powder (Meyer and others 2006). Richard and others (2012) confirmed that ultrasound spectroscopy in a through-transmission mode can monitor rehydration of MPC and MCI powders. The development of the ultrasound attenuation parameter was used to determine relaxation time. Analysis of the ultrasound signal and images obtained by a granulomorphometer in the course of dispersion showed that the time required by the ultrasound attenuation parameter to relax was strongly associated with the time required for water penetration into the powder particles. MPC and MCI were classified according to their relaxation times (ultrasound test) and rehydration times (rehydration test, by LS) (Richard and others 2012). Several studies have shown the efficiency of ultrasound spectroscopy to evaluate and characterize powder rehydration. However, according to Hauser and Amamcharla (2016a), an ultrasound spectroscope is expensive, which makes the use of this technique by small companies unfeasible. A portable ultrasonic flaw detector (UFD) is an economical alternative to ultrasound spectroscopy and has been used as a tool to study dissolution behavior of MPC80 powder (Hauser and Amamcharla 2016a). A method using UFD was developed to characterize the dissolution behavior of MPC of powders stored at 25 and 40 C for up to 4 wk. Relative ultrasound velocity and ultrasound attenuation were used as parameters to evaluate dissolution properties. During rehydration, the ultrasound signal gradually reappeared and the time for the signal to reappear increased as the storage time of powder increased, thus indicating a loss of solubility. The ultrasound attenuation for a fresh powder increased during the first 510 s and then remained relatively stable. As the storage time increased, the ultrasound attenuation increased at a slower rate. Samples stored for 1 and 4 wk at 25 C showed stabilization at 750 and 950 s, respectively. At 40 C, the increase in the ultrasound attenuation was delayed as the powder storage time increased. The area under the attenuation curve was also used as an indicator of solubility. As the storage time and temperature increased, the area under the curve decreased, indicating loss of solubility. It was concluded that the method proposed was effective to identify the changes in dissolution characteristics for powders stored at 25 and 40 C. UFD was also used to determine the effect of protein content (MPC85, MPC87, MPC88, and MPC90) and temperature (40 and 48 C) on the dissolution characteristics of MPC (Hauser and Amamcharla 2016b). In this study, the relative velocity standard deviation between 900 and 1800 s was extracted from the relative ultrasound velocity in order to quantify the relative ultrasound velocity trend. As the protein content of MPC increased from 85% to 90%, an increase in relative ultrasound velocity standard deviation and ultrasound fluctuation time was observed. The authors explained this behavior based on the lactose content of the powders. This means that increasing the protein content from 85% to 90% led to a reduction in the lactose amount, which proved to affect the dissolution of the MPC powders negatively. In addition, the authors proposed that increasing the dissolution temperature 248 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.17,2018 C 2017 Institute of Food Technologists