**2. Crystallization of lactose**

Lactose is the principal carbohydrate in the milk of mammalians, which is a reducing disaccharide made up of galactose and glucose joint by a glycosidic bond (β 1–4). Lactose comprises of two stereoisomers α- and β-anomers. In solution, lactose opens and reforms the ring structure interchanging between α and β anomers (mutarotation). The mutarotation equilibrium of lactose at 20°C is attained, when the ratio of β/α isomers is 1.70 (63:37), although this proportion is highly dependent on temperature. In equilibrium, the isomer β form is more abundant and more soluble (500 g L−1) than α-lactose isomer (70 g L−1) [12, 14]. Therefore, the α isomer will crystallize first in a supersaturated solution of lactose, like a whey concentrate. In this section, the three main phases of lactose crystallization are described: supersaturation, nucleation (appearance of crystals), and crystal growth [15].

#### **2.1. Supersaturation**

volume of milk [3]. Therefore, it is estimated that more than 80 million tons of whey are pro-

Most of the small-scale dairy companies dispose of the whey into the municipal sewage, rivers, lakes, or use this by-product as fertilizer and animal feed [4, 5]. The disposal of cheese whey into water bodies and lands should be strongly discouraged because it produces serious environmental problems. The bacterial degradation of whey causes a depletion of oxygen in the water and soil killing aerobic organisms, such as fish, insects, plants, and microorganisms. The high biological and chemical oxygen demand (BOD: 30–50 g L−1; COD: 60–80 g L−1) of the whey arise from its large content of carbohydrates, chiefly lactose (5–6%) [2, 6–8]. In consequence, the removal of lactose reduces more than 80% of the BOD and COD of whey, which

Besides the ecological benefits of lactose removal from whey, this by-product also has a great relevance for the food and pharmaceutical industries [11]. It is estimated that 400,000 tons of crystalline lactose are worldwide produced each year. In comparison with other carbohydrates, lactose has a low caloric value, low glycemic index, good plasticity, compressibility, and low level of sweetness. This sugar is used in the food industries in a wide variety of products such as instant coffee, infant formula, and baked foods. Meanwhile, lactose is used as an excipient for tablets and dry powder inhalers in the pharmaceutical industry [11, 12]. The general steps in the recovery of lactose from the whey involve a step for the partial removal of water followed by a crystallization step. Some of the challenges to overcome in the recovery of lactose from the whey are the long crystallization times, low yields, and low quality of lactose crystals. These problems on lactose crystallization have been approached through the seeding of lactose, the use of antisolvent, and more recently, by the sonocrystallization of lactose [1, 3, 5]. In the last years, the number of research studies of the crystallization of lactose assisted with ultrasound has increased considerably. Hitherto, it has been established that sonocrystallization decreases the size of crystals and improves the crystal size distribution but also might speed up the crystallization process or enhance the purity of lactose crystals. However, the effect that ultrasound has on lactose crystallization is by far not fully understood. This chapter discusses the current knowledge on lactose sonocrystallization (fifth section) but also addresses the basic principles of lactose crystallization (second section) and sonocrystallization (fourth section). Furthermore, the conventional process

minimizes the negative environmental impact of this by-product [9, 10].

of lactose recovery from whey is described in the third section of this chapter.

Lactose is the principal carbohydrate in the milk of mammalians, which is a reducing disaccharide made up of galactose and glucose joint by a glycosidic bond (β 1–4). Lactose comprises of two stereoisomers α- and β-anomers. In solution, lactose opens and reforms the ring structure interchanging between α and β anomers (mutarotation). The mutarotation equilibrium of lactose at 20°C is attained, when the ratio of β/α isomers is 1.70 (63:37), although this proportion is highly dependent on temperature. In equilibrium, the isomer β form is more abundant and more soluble (500 g L−1) than α-lactose isomer (70 g L−1) [12, 14]. Therefore, the

**2. Crystallization of lactose**

duced annually all over the world [4, 5].

52 Technological Approaches for Novel Applications in Dairy Processing

Supersaturation of lactose solutions is the first step in the crystallization process, since a nonequilibrium condition is required for the spontaneous birth of nuclei [16]. At any given temperature, a maximum amount of solute can be dissolved in a solvent. When a solution is saturated with a solute, this is considered being in a thermodynamic equilibrium. Any further increase in the concentration above the saturation (solubility) point disturbs the equilibrium and induces a pseudo-equilibrium state or supersaturation. The nucleation and hence crystallization won't occur at the supersaturation point (at least not spontaneously), since the energy available is insufficient to induce the nuclei formation. However, beyond the pseudo-equilibrium state (labile zone), nucleation takes place spontaneously. The region between solubility and supersolubility (supersaturation) is known as the metastable zone (MZ). The width of this region (MZW) is obtained by plotting the solubility and supersolubility of the solute as a function of temperature. From these curves, it is possible to establish the temperature and solute concentration required in a crystallization process [5, 17]. The conventional process of lactose crystallization has a wide MZW, which means that a very high supersaturation is necessary to induce nucleation [18, 19].

#### **2.2. Nucleation**

Nucleation has a major influence on crystallization and consequently on the quality properties of lactose crystals like its structure and size distribution [21]. The formation of a new solid phase from a supersaturated solution is called nucleation, and the nucleation rate is the change in the number of particles in solution with time [22]. There are two kinds of nucleations: the primary and secondary; the former occurs when a crystal is nucleated without an interphase in the solution. Nucleation in the absence of solid surfaces is called homogeneous nucleation, and if there is a foreign interphase in the solution, the process is referred as heterogeneous nucleation. In contrast, the secondary nucleation is induced by pre-existing crystals [13]. Two theories try to explain the nucleation mechanism, the Classical Nucleation Theory (CNT) and the Two-Step Nucleation Theory (TSNT). The basics of the CNT are that from a supersaturated solution, a number of ordered subcritical clusters of solute molecules are formed under certain temperature and concentration conditions. When the number of molecules in the cluster increases (until reaching a critical cluster size *n\**) (∼100 to 1000 atoms), the total free energy (*ΔG*) in the system rises. Above this *n\**, the total free energy decreases continuously and the formation of a crystal nuclei becomes favorable. However, a cluster of size *n\** has equal possibilities to form a crystal nucleus or to disaggregate. Therefore, the height of the free energy barrier for nucleation (*ΔG\**) and the nucleation rate are determined largely by the *n\** [16, 21]. The CNT gives some insights about the *n\** and nucleation rate but does not provide information on the structure of aggregates or pathways leading to the formation of solid crystal from the solution [16]. On the other hand, the major difference between the CNT and the TSNT is that the latter considers the formation of disordered (liquid-like) clusters instead of ordered subcritical clusters. Besides, the TSNT suggests the formation of a crystalline nucleus inside the liquid-like clusters beyond the *n\** [16]. Although the theories of the nucleation process have advanced considerably in recent years, the particular ordering within the solid state via the nucleation process remains ambiguous. Moreover, some of the parameters described by these nucleation theories are difficult to verify experimentally, like the critical cluster size (*n\**). The number of particles can be measured by methods such as light scattering, direct particle counting (microscopy), and turbidity measurements [22]. The problem arises from the fact that *n\** typically falls in a range of 100–1000 atoms, which is hardly accessible to most of the current experimental methods [16].

#### **2.3. Crystal growth**

The growth of lactose crystals is controlled by several factors but the key variable determining the rate of nucleation is the supersaturation [23]. If nucleation is fast, many crystals form simultaneously and they will grow to approximately identical sizes. In contrast, if the nucleation is slow and fewer crystals nucleate at a time, the supersaturation in the solution drops slowly, the nucleation of new crystals continues, and the solution presents a wider crystal size distribution (CSD) [21]. Other variables that affect the crystal growth are the temperature, viscosity, pH, presence of salts, and whey proteins, which modify the levels of supersaturation and consequently the nucleation and crystal growth [24–26]. Speaking of impurities like salts and proteins, these can either accelerate or inhibit the crystal growth. The impurities induce a heterogeneous nucleation and are incorporated frequently into the crystal lattice. In addition, the presence of impurities affects the solubility and supersolubility of the substance being crystalized, modifying the nucleation and crystal growth. It is well established that salts may either increase or decrease the growth rate of lactose crystals. The presence of calcium chloride, calcium lactate, magnesium sulfate, and lithium chloride increases the crystallization velocity, at the difference of potassium phosphate [24]. In the same way, the whey proteins promote nucleation but slow down the growth of lactose crystals. This effect is attributed to its high water-binding capacity that creates areas of lactose supersaturation, which are favorable for nucleation [25].

carried out with this MWCO, the fat and protein fraction are retained in retentate; meanwhile, permeate keeps the lactose, vitamins, and minerals. The major drawback of this technology is the high cost of UF equipment and membranes because most of the small and mediumscale dairy processors cannot afford it [4]. Finally, if the deproteinized whey is not evaporated immediately, this must be pasteurized to avoid the fermentation of lactose by microorganisms. The clarified, defatted, and deproteinized whey is sent to the evaporators for concentration. Evaporation is performed under reduced pressure in falling film (single and multiple effect) evaporators. These evaporation units allow the concentration of total solids in the whey by nearly ten folds (concentration factor Q = 9.5). The content of dry matter in the whey is measured during the whole evaporation process through the refractive index *n.* When the whey reaches 40 to 65% of dry matter, the evaporation process is stopped. The temperature of the final concentrate is ∼60°C, and the lactose content ranges from 39 to 56%. At this point, lactose is supersaturated in the whey concentrate but won't crystallize as the temperature is high [1, 4, 28, 29]. The whey concentrate (still being hot) is then transferred into a large stirred tank where it is cooled fast enough to induce crystallization of lactose. Once in the crystallizer, the whey is first cooled rapidly from 60 to 30°C and then slowly from 30 to 20–25°C (1–3°C h−1) [17, 26, 29]. The nucleation and crystallization of lactose will occur spontaneously just beyond the metastable zone (MZ), that is a region between the supersaturation point where nucleation occurs and the saturation equilibrium of lactose [14, 30]. This MZ is attained mostly during the second cooling stage when the temperature drops below 30°C, and the lactose supersaturation rises considerably [26]. The progress of crystallization can be followed measuring the changes of lactose concentration in the liquor either by refractive index, density, or conductivity [31]. The complete process of crystallization is prolonged and may take up to 48 h. Crude lactose crystals are separated from the liquor by centrifugation, filtration, or both and then washed with a nonsolvent compound (such as ethanol) to remove impurities and water. The resulting crystals are air dried and further characterized by its size distribution and purity. The yield of crystallization depends upon many variables, but typically 65% of lactose is recovered from this process [12, 17, 29]. Crude lactose is further recrystallized, if some quality parameters are not achieved such as the crystal size distribution (CSD), form,

Sonocrystallization of Lactose from Whey http://dx.doi.org/10.5772/intechopen.74759 55

**Figure 1.** Schematic procedure for the recovery of lactose from whey.

#### **3. Conventional recovering of lactose from whey**

The process of recovery of lactose from cheese whey is described in **Figure 1**. Before whey processing, curd fines and fat are separated from the whey by centrifugation [27]. This clarified and defatted (0.07%) whey must be deproteinized in advance to the concentration step. The presence of whey proteins decreases the solubility of lactose [24], promotes nucleation, accelerates the lactose crystallization [25], and reduces the purity of lactose crystals [5]. Besides, proteins increase significantly the viscosity of the concentrated whey, hindering the recovery of lactose crystals [5]. The heat-acid precipitation of whey proteins is the easiest and cheapest method for whey deproteinization, although this method leaves between 0.1 and 0.2% of the residual protein in the whey [28]. Proteins can also be removed by ultrafiltration (UF) using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 10 kDa. When UF is

#### Sonocrystallization of Lactose from Whey http://dx.doi.org/10.5772/intechopen.74759 55

**Figure 1.** Schematic procedure for the recovery of lactose from whey.

that the latter considers the formation of disordered (liquid-like) clusters instead of ordered subcritical clusters. Besides, the TSNT suggests the formation of a crystalline nucleus inside the liquid-like clusters beyond the *n\** [16]. Although the theories of the nucleation process have advanced considerably in recent years, the particular ordering within the solid state via the nucleation process remains ambiguous. Moreover, some of the parameters described by these nucleation theories are difficult to verify experimentally, like the critical cluster size (*n\**). The number of particles can be measured by methods such as light scattering, direct particle counting (microscopy), and turbidity measurements [22]. The problem arises from the fact that *n\** typically falls in a range of 100–1000 atoms, which is hardly accessible to most of the

The growth of lactose crystals is controlled by several factors but the key variable determining the rate of nucleation is the supersaturation [23]. If nucleation is fast, many crystals form simultaneously and they will grow to approximately identical sizes. In contrast, if the nucleation is slow and fewer crystals nucleate at a time, the supersaturation in the solution drops slowly, the nucleation of new crystals continues, and the solution presents a wider crystal size distribution (CSD) [21]. Other variables that affect the crystal growth are the temperature, viscosity, pH, presence of salts, and whey proteins, which modify the levels of supersaturation and consequently the nucleation and crystal growth [24–26]. Speaking of impurities like salts and proteins, these can either accelerate or inhibit the crystal growth. The impurities induce a heterogeneous nucleation and are incorporated frequently into the crystal lattice. In addition, the presence of impurities affects the solubility and supersolubility of the substance being crystalized, modifying the nucleation and crystal growth. It is well established that salts may either increase or decrease the growth rate of lactose crystals. The presence of calcium chloride, calcium lactate, magnesium sulfate, and lithium chloride increases the crystallization velocity, at the difference of potassium phosphate [24]. In the same way, the whey proteins promote nucleation but slow down the growth of lactose crystals. This effect is attributed to its high water-binding capacity

that creates areas of lactose supersaturation, which are favorable for nucleation [25].

The process of recovery of lactose from cheese whey is described in **Figure 1**. Before whey processing, curd fines and fat are separated from the whey by centrifugation [27]. This clarified and defatted (0.07%) whey must be deproteinized in advance to the concentration step. The presence of whey proteins decreases the solubility of lactose [24], promotes nucleation, accelerates the lactose crystallization [25], and reduces the purity of lactose crystals [5]. Besides, proteins increase significantly the viscosity of the concentrated whey, hindering the recovery of lactose crystals [5]. The heat-acid precipitation of whey proteins is the easiest and cheapest method for whey deproteinization, although this method leaves between 0.1 and 0.2% of the residual protein in the whey [28]. Proteins can also be removed by ultrafiltration (UF) using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 10 kDa. When UF is

**3. Conventional recovering of lactose from whey**

current experimental methods [16].

54 Technological Approaches for Novel Applications in Dairy Processing

**2.3. Crystal growth**

carried out with this MWCO, the fat and protein fraction are retained in retentate; meanwhile, permeate keeps the lactose, vitamins, and minerals. The major drawback of this technology is the high cost of UF equipment and membranes because most of the small and mediumscale dairy processors cannot afford it [4]. Finally, if the deproteinized whey is not evaporated immediately, this must be pasteurized to avoid the fermentation of lactose by microorganisms.

The clarified, defatted, and deproteinized whey is sent to the evaporators for concentration. Evaporation is performed under reduced pressure in falling film (single and multiple effect) evaporators. These evaporation units allow the concentration of total solids in the whey by nearly ten folds (concentration factor Q = 9.5). The content of dry matter in the whey is measured during the whole evaporation process through the refractive index *n.* When the whey reaches 40 to 65% of dry matter, the evaporation process is stopped. The temperature of the final concentrate is ∼60°C, and the lactose content ranges from 39 to 56%. At this point, lactose is supersaturated in the whey concentrate but won't crystallize as the temperature is high [1, 4, 28, 29]. The whey concentrate (still being hot) is then transferred into a large stirred tank where it is cooled fast enough to induce crystallization of lactose. Once in the crystallizer, the whey is first cooled rapidly from 60 to 30°C and then slowly from 30 to 20–25°C (1–3°C h−1) [17, 26, 29]. The nucleation and crystallization of lactose will occur spontaneously just beyond the metastable zone (MZ), that is a region between the supersaturation point where nucleation occurs and the saturation equilibrium of lactose [14, 30]. This MZ is attained mostly during the second cooling stage when the temperature drops below 30°C, and the lactose supersaturation rises considerably [26]. The progress of crystallization can be followed measuring the changes of lactose concentration in the liquor either by refractive index, density, or conductivity [31]. The complete process of crystallization is prolonged and may take up to 48 h. Crude lactose crystals are separated from the liquor by centrifugation, filtration, or both and then washed with a nonsolvent compound (such as ethanol) to remove impurities and water. The resulting crystals are air dried and further characterized by its size distribution and purity. The yield of crystallization depends upon many variables, but typically 65% of lactose is recovered from this process [12, 17, 29]. Crude lactose is further recrystallized, if some quality parameters are not achieved such as the crystal size distribution (CSD), form, and purity. For this lactose refining, the crystals are re-dissolved, treated with charcoal to remove impurities (salts and proteins), and recrystallized as previously described [4, 31].

The process of lactose crystallization is very slow (up to 72 h), the quality of lactose is usually poor, and the yields of crystallization are very low. One of the oldest methods used to improve the process of crystallization is the seeding of lactose. This approach consists in the addition of small lactose crystals into whey concentrate (seeding of nuclei) just before the second cooling step. The addition of lactose crystals may induce a secondary nucleation that accelerates the crystallization process and reduces the CSD [5]. However, this method has low reproducibility because its success depends on the addition of crystals in the appropriate timing [13]. More recently, alternative methods such as the use of antisolvent or sonocrystallization have been explored to assist the crystallization of lactose. The addition of nonsolvent compounds into whey concentrate (antisolvent crystallization) decreases the solubility of lactose, narrows the metastable zone, and reduces the induction times of nucleation. In general, the antisolvent crystallization improves the yield of crystallization and reduces the size of lactose crystals [20, 32]. The main drawbacks of antisolvent crystallization are the large amounts of solvent used, and the expensive separation and purification steps required to remove the antisolvent from the product [5, 9]. The crystallization of lactose assisted with low-frequency power ultrasound (sonocrystallization) is discussed later in the chapter.

diffusion" which is defined as slow growth of the acoustic bubble as a function of time due to unequal mass transfer across the air/water interface [35]. There are two types of cavitation bubbles that exist depending upon the ultrasonic intensity, i.e., transient cavitation bubbles and stable cavitation bubbles. When the ultrasonic intensity is very high, transient cavitation bubbles last for a few acoustic cycles. On the other hand, stable cavitation bubbles can oscillate for many acoustic cycles. The size of stable cavitation bubbles grows over time due to coalescence and also by rectified diffusion until the size is reached, where the coupling of bubble's resonance frequency and driving frequency of the ultrasound occurs. In a multibubble cavitation field, bubbles with a range of size are generated and grown toward the critical size. Miinaert's equation (Eq. 1) provides a relationship between linear

*<sup>f</sup>* (1)

Sonocrystallization of Lactose from Whey http://dx.doi.org/10.5772/intechopen.74759 57

*Pv* } (2)

resonance radius (critical size) of the bubble with frequency [36].

where Rres is the linear resonance radius (m) and f is the ultrasonic frequency (Hz).

Transient cavitation bubbles dominate at the lower frequency where they can grow rapidly above a threshold size during the rarefaction cycle. The nature of cavitation bubble is controlled by numerous parameters, such as acoustic pressure, frequency, type of reactor, and

The collapse of cavitation bubble leads to the generation of a very high temperature of >5000 K and pressure (>1000 atm) within the bubble (**Figure 2**) [34, 36, 39]. The collapse of the bubble takes place in a very short period of time, and thermodynamically, the work done leads to a near adiabatic heating of the bubble contents, which lead to extreme conditions [36–38]. The maximum temperature and pressure generated within the cavitation bubble can be theoretically calculated using Eqs. (2) and (3), respectively, to a near adiabatic heating of the bubble

*Pm*(*<sup>γ</sup>* <sup>−</sup> <sup>1</sup>) \_\_\_\_\_\_\_

*Rres* <sup>=</sup> \_\_<sup>3</sup>

**Figure 2.** Schematic representation of acoustic cavitation.

**4.3. Chemical and physical effects of ultrasound**

contents which lead to extreme conditions [36–38].

T*max* = *T*0{

bubble size.
