*3.6. Prospects for the Further Development of Optical Methods and New Devices for Milk Analysis*

The main leading trend in the optical spectroscopy of milk and dairy products is developing affordable, portable, on-site or in-line devices, which can be used outside of a laboratory or even incorporated in milking robots or processing equipment, for the monitoring of milk quality in real-time, either on milking-farms or production facilities. The most promising results so far have been achieved in the NIR technologies, even with conventional spectral equipment, mainly because of the implementation of sophisticated chemometrics algorithms such as artificial neural networks.

The other acute problem in milk analysis is the detection of as many known adulterants as possible. Despite constant improving of the calibration and prediction algorithms it is not always possible even by FTIR spectroscopy because of the inherent limitations of optical spectroscopic techniques [107]. Combination of several different approaches, for example spectrophotometry and acoustic analysis, may be a solution here.

#### **4. Ultrasound Approaches and Techniques for Milk Analysis**

Ultrasound techniques are well-developed instruments used for non-destructive, accurate, and non-invasive measurements [108,109]. In the dairy industry, ultrasound is widely used both for non-destructive monitoring of the quality and parameters of milk [37,110–118], and in the ultrasonic processing of dairy products [110,119,120]. In the first case, low-intensity ultrasound (less than 1 W/cm2) is used at frequencies above 100 kHz, which is a non-destructive instrument for milk characterization. In the second case, high-intensity ultrasound (above 10 W/cm2) is mostly used in the 20–100 kHz frequency range, which is destructive for milk sample: it modifies the properties of milk and is used for the ultrasound treatment at different stages of dairy products processing. In this review, we are interested only in methods of low-power non-destructive ultrasonic characterization of milk.

Ultrasound spectrometry offers the ability to characterize dairy products excluding special preparation or disruption of the liquid sample. In addition, ultrasound methods are of interest for monitoring processes in real time.

#### *4.1. Acoustic Properties of Milk*

Acoustic measurement techniques can provide data about protein [121] and fat [113] content, physiochemical changes of milk with time [122], size distribution of air and fat droplets [123], content of contaminants [112].

When an acoustic wave propagates through a liquid sample, its amplitude decreases, and the phase changes because of the interaction with liquid medium and its dispersed filler. The decline in ultrasound intensity is commonly called attenuation. The phase links to the velocity of propagation of sound through a sample. The variations of sound velocity and attenuation are governed by the physicochemical features of liquid medium and its ingredients. By measuring changes in these acoustic parameters, specific information about the properties of a liquid sample can be accessed. Thus, the thermo-acoustic, physical, and chemical properties of milk can be obtained by measuring ultrasound speed and attenuation. These measurements can be carried out by such methods as transmission, pulse-echo and interferometer techniques (Figure 7). An alternative microbalance approach to acoustic measurements allows access to viscosity and density of milk via measuring of additional mass load of resonant sensor. The analysis and interpretation of acoustic measurements can be done in two steps. The first step is purely phenomenological and involves determination of the ultrasound parameters (speed of sound and attenuation, or viscosity and density) of milk sample. The second step requires the use of model to describe the real milk as a complex multiphase polydisperse system. By solving inverse problems on the basis of appropriate models, it is possible to determine the content of various ingredients in milk and their size information. Such calculations can be done using models of ultrasound attenuation [123] and velocity [124] in dispersed liquids and emulsions.

**Figure 7.** Ultrasound techniques for milk quantitative and qualitative analysis.

The speed of sound can provide some specific information about the composition of a liquid. Speed of sound is a collective value. The simplest mixing rule for sound velocity has been proposed

by [125] and fits well with experimental data in liquid mixtures. Molecule interactions gives to excess Gibb's energy to an ideal mixture, and this affects in an excess compressibility and an excess volume [126]. The speed of sound contains information about the nature of interactions between molecules, because it depends on changes in the shape of molecules and on the type of interaction potentials between the components of liquid mixture [127]. The speed of sound in suspensions and emulsions was studied in detail in [124]. The sound velocity is very sensitive to milk chemistry content and temperature. It is the most suitable parameter for studying milk features on a molecular scale, that is, with characteristic dimensions in angstroms. The speed of sound changes from 1000 to 2000 m/s for a variety of dairy products. For dilute milk, it is about 1500 m/s, close to water sound velocity at room temperature.

Attenuation of ultrasound propagating through a heterogeneous system is the sum of scattering and absorption. Particle scattering redirects energy of ultrasound wave out of the propagation line. Absorption converts ultrasonic energy into heat because of the interaction effects between the ultrasonic wave and particles and medium. The propagation of acoustic waves through the milk medium is mainly of a thermodynamic character. Big droplets can serve as scattering centers. Submicron particles in milk only absorb ultrasound. Absorption in milk can be divided into so-called internal attenuation and thermal attenuation. The first is measured directly. The second is associated with differences in thermo-mechanical properties between particles and a liquid medium, whereby the particles and liquid expand in ultrasonic field in different ways, creating a temperature gradient at the particle-liquid interface, as a result of which the ultrasonic energy at this interface is converted into heat [123,128]. Thermal part of attenuation in a liquid medium depends on thermal conductivity, thermal expansion, and heat capacity. The total value of the thermal part of attenuation of the multiphase polydisperse liquid depends on these three thermo-mechanical parameters for each component as well as the particle distribution by sizes [123]. Ultrasound attenuation is not good enough to reflect changes in chemical, ionic, and molecular milk composition, but it is sensitive to particles present in milk. Attenuation perceives changes in nanometre-micrometre size level and is suitable for characterizing liquid features associated with inhomogeneity and phase composition of a disperse liquid system [113,123]. Milk can be modelled as a liquid composition of spherical fat droplets of approximately spherical shape in an aqueous solution of proteins and sugars [123,124,129]. Knowing the corresponding physical properties of the constituent milk ingredients, fat particles size distribution in milk can be determined from measurements of attenuation [123]. Attenuation in milk can vary very widely depending on the frequency. In water the attenuation rises with frequency up to 0.2 dB/cm per MHz at 100 MHz. In milk, depending on the fat content, the attenuation can vary from 0.4 to 0.7 dB/cm per MHz for 100 MHz and from 0.1 to 0.4 dB/cm per MHz for 2 MHz. For comparison, for butter and margarine, the attenuation increases from 2 to 7 dB/cm per MHz as the frequency increases from 2 MHz to 100 MHz [113].

Milk density is close to the density of water. Normal milk density ranges from 1027 to 1032 kg/m3, with an average of 1030 kg/m3. The density of milk is higher if it contains more sugar, proteins, and minerals, and lower, with an increase in fat [130]. Density can reflect falsification: decreases with water addition and increases with skimming or diluted with skim milk. For example, a density of 1028 kg/m3 indicates natural milk, 1027 kg/m<sup>3</sup> means suspicious milk, 1027 kg/m3 and below indicates falsified milk with water, more than 1031 kg/m<sup>3</sup> means suspicion of dilution backwards. The addition of water to milk causes a decrease in density of approximately 3 kg/m<sup>3</sup> for every 10% water added. It is characteristic that if the fat is removed from the milk and the same amount of water is added, then the density does not change, and such falsification can only be detected by determining the amount of fat in the milk [22].

Viscosity of milk is understood as the internal friction of liquid layers during their relative movement, which depends on the adhesion forces between the molecules. The viscosity of milk is influenced by emulsified (fats) and colloid-soluble particles (casein), the state of whey proteins, technological modes of milk processing, causing changes in the state of aggregation of its components, etc. [131]. The viscosity of milk and dairy products strongly depends not only on the

composition, but also on the temperature [132]. An increase in temperature leads to an increase in the speed of Brownian motion (random motion [133]) of solution molecules, a weakening of the interaction forces between them, and as a consequence, a decrease in viscosity. However, if the temperature exceeds the coagulation point of whey proteins, the viscosity of these products begins to increase again. This is due to a decrease in the degree of dispersion of whey proteins caused by their thermal denaturation and aggregation [134]. Typical values of milk viscosity are given in Table 2.

Techniques for measuring the above acoustic properties of milk are briefly discussed below.

#### *4.2. Transmission Technique*

Figure 8a shows in a simplified way the idea of the transmission measurement method. To the left and to the right of the cuvette with the liquid sample, two piezoelectric transducers are located at the same level, opposite to each other. The first acts as a transmitter, and the second as a receiver of an ultrasound waves. The transmitter sends an ultrasonic wave with a certain frequency and amplitude into the liquid-filled cuvette, and the receiver gets a signal weakened in intensity and changed in phase as a result of the wave propagation through the liquid sample and the interaction between ultrasound wave and liquid medium and its dispersed filler. A piezoelectric receiver converts this ultrasonic signal into an electrical one, which is then compared with the input signal. The result of such a comparison makes it possible to determine the values of speed of sound and attenuation in a liquid sample, according to the equations [108,135]:

$$V = \text{x/t}; A\_{\text{out}} = A\_{\text{in}} \exp(-a\mathbf{x}), \tag{2}$$

where *V* is the speed of sound; α is attenuation; *x* is the traveling distance; *t* is the traveling time of the wave; *A*in and *A*out are the amplitudes of ultrasound wave at the beginning and at the end of the traveling distance. For the transmission approach (Figure 8a) *x* = *L*.

**Figure 8.** Ultrasound measuring approaches: simplified illustrations of transmission (**a**), pulse-echo (**b**), interferometer (**c**) and quartz crystal microbalance (**d**) techniques.

Sound velocity and attenuation measurements can be made over a wide frequency range, typically for milk in the 1 to 100 MHz range.

The most informative frequency range for detecting fat in milk is from 40 to 50 MHz [113]. Relatively big particles, such as air droplets in milk, can be detected by measuring the attenuation

spectrum at low frequencies up to 10 MHz. Small particles down to 5 nm can be detected by studying and interpretation of the attenuation spectra in the higher frequency part of the spectrum up to 100 MHz [123]. For a sufficiently accurate control of the fat particle size distribution in milk, an attenuation measurement accuracy of level about 0.01 dB/cm per MHz is required [113].

The accuracy of sound velocity measurements is limited by the accuracy of temperature control. A 1 ◦C change in temperature leads to a change in the velocity of sound in water by 2.4 m/s, while maintaining a uniform temperature of the liquid sample with an accuracy less than 0.01 ◦C is impossible.

The transmission method has been used by various researchers to determine the water content, fat content [113], particle size distribution [123], and various chemical compounds [113], melamine [114], microorganisms [136] in milk.

## *4.3. Pulse–Echo Technique*

In a pulse-echo method [110,137], the only one piezoelectric transducer is used (see Figure 8b). It is both a transmitter and a receiver of the ultrasonic signals. On the opposite side of the cuvette with a liquid sample, there is a reflector. The wall of the cuvette can act as a reflector, while for good reflection it is important to reach a high acoustic contrast and low acoustic losses at the liquid-wall interface. The pulsed ultrasound wave is directed from the transducer to the opposite wall of the cuvette, reflected from it, and returns to the transducer with weakened intensity. The wave partially reflected from the transducer travels the same path again and returns to the transducer as a second echo. Knowing the travel time of the wave and the distance, it is possible to calculate the speed of sound and attenuation based on Equation (2), only in this case, unlike the transmission method, the travel distance *x* is equal to 2 *L* for the first echo and 4 *L* for the second one. Also, when using Equation (2) to calculate attenuation, it is necessary to take into account multiple reflections and the respective reflection coefficients at the interfaces.

Pulse–echo technique has been used to determine the water content, carbohydrates, and total fat content, fat globular, and casein micelles sizes distribution in milk [110,124,137].

#### *4.4. Ultrasonic Interferometer Technique*

At first glance, the ultrasonic interferometer technique is very close to the previous approach, but in reality, it has differences both in the method of collecting raw data, in the approach to their analysis, and in sensitivity to changes in the speed of sound and attenuation. An ultrasonic interferometer uses a single transducer that serves both transmitter and receiver roles. On the opposite side there is a movable reflector (Figure 8c) that allows profiling the distance at which standing acoustic waves are generated in the liquid sample because of the interference of the incident and reflected waves. When the distance between transducer and reflector changes, the intensity oscillations are measured, corresponding to the maxima and minima of the ultrasonic wave along its propagation path with a step of the half wavelength. Description of devices, principles of use, and improvements of the ultrasonic interferometer technique can be found in [138–140]. Furthermore, some devices of the ultrasonic interferometer include optical diffraction, which allows expanding its analytical capabilities [141]. Examples of using an ultrasonic interferometer for milk analysis are given in [111,142].

#### *4.5. Microbalance Technique*

Acoustic resonant sensor principles are broadly used as biochemical sensors. They are typically understood to convert a surface mass change into a frequency change of a resonant device. They are therefore often called microbalance with the quartz crystal microbalance (QCM) being its most prominent sensor. However, a resonance sensor is far more than a mass balance. Shear bulk acoustic resonators are commonly used for biosensing because of low in-liquid radiation losses. This allows to keep relatively high quality factor of the resonant sensor even while interfacing viscous liquids. QCM is a recognized technology that is used to detect interactions at the surface [143]. Excited at a

shear bulk vibration mode, its resonant frequency shifts in accordance with interface loading. For milk characterization, QCM is commonly used to detect proteins [121,144,145].

The QCM measurements are based on a frequency shift (Δ*f*) detection from the fundamental resonant frequency (*f* 0) of quartz resonator when it is loaded by a small additional mass (Δ*m*) per area of electrode (*A*), according to the equation:

$$
\Delta f = -\mathbf{K} \cdot f\_0^2 \, (\Delta m/A),
\tag{3}
$$

where *K* is a constant and depends on the properties of quartz crystal and its cut.

Small mass and high Q-factor of the resonator give access to a mass detection limit Δ*m*/*m*res of 10−<sup>6</sup> to 10−<sup>9</sup> with Δ*m* and *m*res being the added mass and resonator mass per unit area, respectively.

When a quartz crystal sensor is in direct contact with a liquid, the shear wave penetrates into it to a small depth and the frequency shift is determined by the acoustic properties of the near-surface liquid with the thickness of δ = (η/πρ*f*) <sup>1</sup>/<sup>2</sup> (Figure 8d), where η and ρ are the liquid viscosity and density. For reliable separate measurements of liquid sample viscosity and density, in [143] a method for comparing the results of measuring a liquid using smooth and corrugated electrodes of QCM was proposed. The respective frequency shifts for a smooth- (Δ*f* 1) and corrugated-surface (Δ*f* 2) QCM liquid sensors are given as [143]:

$$
\Delta f\_1 = -c\_1 \sqrt{\rho \eta}; \quad \Delta f\_2 = -c\_1 \sqrt{\rho \eta} - c\_2 h \rho,\tag{4}
$$

where *c*<sup>1</sup> and *c*<sup>2</sup> are constants, *h* is the depth of the grooves on the corrugated surface of QCM electrode.

Some modern QCM biosensors use a sensitive or recognition layer to enhance sensitivity and selectivity for specific ingredients in liquid sample. Such QCM biosensors with specially prepared sensitized surfaces have been used to detect specific proteins in milk [144,145].

Table 7 summarizes information about ultrasonic measurements of milk by various research groups.
