Review Thermal Denaturation of Milk Whey Proteins: A Comprehensive Review on Rapid Quantification Methods Being Studied, Developed and Implemented Paulina Freire , Allison Zambrano, Anna Zamora * and Manuel Castillo * Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; paulina.freire@uab.cat (P.F.); allison.zaol@gmail.com (A.Z.) * Correspondence: anna.zamora@uab.cat (A.Z.); manuel.castillo@uab.es (M.C.) Abstract: Heat treatment of milk signifies a certain degree of protein denaturation, which modifies the functional properties of dairy products. Traditional methods for detecting and quantifying the denaturation of whey proteins are slow, complex and require sample preparation and qualified staff. The world’s current trend is to develop rapid, real-time analytical methods that do not destroy the sample and can be applied on/in-line during processing. This review presents the rapid methods that are being studied, developed and/or applied to determine and quantify the thermal denaturation of whey proteins, including spectroscopic, electrochemical and miniaturized methods. The selected methods save a significant amount of time and money compared to the traditional ones. In addition, the review emphasizes the methods being applied directly to milk and/or that have potential for on/in/at-line application. There are interesting options to quantify thermal denaturation of whey proteins such as biosensors, nanosensors and microchips, which have fast responses and could be automated. In addition, electrochemical sensors are simple to use and portable, while spectroscopy alternatives are suitable for on/in/at-line process. Citation: Freire, P.; Zambrano, A.; Zamora, A.; Castillo, M. Thermal Keywords: whey proteins; denaturation; heat treatment; rapid method; on-line; in-line Denaturation of Milk Whey Proteins: A Comprehensive Review on Rapid Quantification Methods Being Studied, Developed and 1. Introduction Implemented. Dairy 2022, 3, 500–512. The milk-protein fraction consists of a large variety of proteins. They are classified https://doi.org/10.3390/dairy3030036 into two big groups: caseins and whey proteins. The group of proteins that precipitate Academic Editor: Burim Ametaj at pH 4.6 are known as caseins, representing approximately 80% of milk proteins. Serum Received: 3 February 2022 or whey proteins are proteins that do not precipitate and remain in the supernatant, Accepted: 15 June 2022 representing about 20% of total protein [1,2]. Four major proteins constitute whey proteins: Published: 18 July 2022 β-lactoglobulin, α-lactalbumin, both being the most abundant [2], bovine serum albumin (BSA) and immunoglobulin. Publisher’s Note: MDPI stays neutral In the dairy industry, raw milk goes through several processes, including standard- with regard to jurisdictional claims in ization and heat treatment. Pasteurization and ultrahigh-temperature (UHT) sterilization, published maps and institutional affil- the most commonly used heat treatments, affect the quality, safety and shelf life of milk iations. and dairy products, inducing physical and chemical changes in protein structure and functionality [3,4]. Thermal processing above 65 ◦ C produces the denaturation of whey proteins such as BSA, while the denaturation of β-lactoglobulin and α-lactalbumin occurs Copyright: © 2022 by the authors. above 70–74 ◦ C [5]. When whey proteins are denatured, their hydrophobic groups are ex- Licensee MDPI, Basel, Switzerland. posed, leading to interactions with themselves, forming aggregates or binding to the casein This article is an open access article micelles through irreversible disulphide bonds (e.g., between β-lactoglobulin and κ-casein distributed under the terms and on the surface of the casein micelle). If the heating process is intensified, irreversible de- conditions of the Creative Commons naturation of α-lactalbumin occurs, enhancing the formation of a complex with denatured Attribution (CC BY) license (https:// β-lactoglobulin [5,6]. Consequently, heat denaturation of proteins induces changes in the creativecommons.org/licenses/by/ functional properties of milk, which may be of significant impact on the dairy industry. 4.0/). In particular, changes in the structure of milk proteins have an significant effect on milk Dairy 2022, 3, 500–512. https://doi.org/10.3390/dairy3030036 https://www.mdpi.com/journal/dairy Dairy 2022, 3 501 structure and stability [6], which might be undesirable or advisable depending on the dairy product being manufactured. For instance, extensive denaturation of whey proteins and subsequent interaction with caseins is pursued in several dairy products due to functional or yield advantages. This is the case on the intensive thermal stabilization of milk required to ensure good heat stability during sterilization of unsweetened condensed milk, as well as to withstand the evaporation processes in sweetened condensed milk. Another example is the intensive whey-protein denaturation prior to coagulation of Mató cheese, a traditional cheese from Catalunya (Spain), to obtain its typical moist and soft texture. Furthermore, the low pasteurization of milk increases the performance in cheese processing and reduces nitrogenous matters of whey, but high pasteurization and UHT milk impairs milk coagula- tion [7,8], yielding high moisture and soft gels that are undesirable for cheese manufacture, while intense denaturation of whey proteins is very desirable in yogurt manufacture, as it aids in improving its texture, preventing wheying-off [7]. Control and quantification of heat-induced protein denaturation are important in the dairy industry. There are different methods and techniques for determining proteins in milk and dairy products. The Kjeldahl, Dumas and Amido Black methods are three chemical procedures to quantify total protein content. The advantages of standard methods such as Kjeldahl or Dumas are that they are precise and have good reproducibility. However, when it comes to quantifying whey-protein content from milk, whey-protein precipitation becomes necessary since these methods quantify the total protein content from the sample. Consequently, this adds more time to the procedure and is not suitable for in-, on-, or at-line determination. The Kjeldahl method can be performed within 30 min to 2 h, while the modern Dumas instrument takes around 5 min [9]. In order to analyze the individual proteins, several classical techniques are used at the laboratory scale: electrophoresis, chro- matography and immunochemistry. These techniques need complex sample preparations before carrying out the analysis. Previously, it is necessary to fractionate the caseins and whey proteins by precipitation, and then these must be filtered to obtain a pure preparation of protein [1]. Moreover, they also need rather expensive reagents and equipment, which means that they are slow protein-detection and quantification methods. Since classical techniques are time-consuming methods, new rapid technologies are being studied for in-, on-, or at-line determination and real-time process control in the dairy industry [10,11]. Thus, it is obvious that at least for small processing plants where more than just one dairy food is manufactured, e.g., cheese and yogurt, knowing if the protein is native or denatured at real time, rather than later in the lab after applying conventional off-line methodologies, will aim with industrial decision making during manufacturing. In other words, rapid methods to directly quantify whey-protein denaturation (i.e., without the need of sample manipulation), and with the potential for in-, on-, or at-line measurements, are compatible with real-time assessment of milk-protein functionality, allowing milk batches to be used for their most suitable purpose, increasing quality of the final product. It would also avoid the need to take samples from the processing line and submit then to either an internal or even external quality-control laboratory. As a result, this review aims at presenting rapid methods being studied, developed and/or applied to determine and quantify the thermal denaturation of whey proteins in milk. Such technologies are considered to present great potential as process analytical technologies to aid with decision-making during heat treatment in the dairy industry. 2. Rapid Methods for Determining the Thermal Denaturation of Whey Proteins in Milk 2.1. Biosensors, Nanosensors and Microchips Biosensors are devices that detect or measure compounds using biological material, i.e., antibodies, enzymes, or whole cells. When the biosensor and the compounds contact, they produce a physical or chemical change, emitting a response transduced into a physical signal, which can be used by a control system [12]. The surface plasmon resonance (SPR) principle has been applied to assess specific solu- ble proteins directly in milk. The antigen–antibody binding produces changes in the refrac- Dairy 2022, 3 502 tive SPR angle which is proportional to the mass of bound material [13]. Dupont et al. [14] used this immunological system (Biacore® 3000 International AB, Uppsala, Sweden) for determining the denaturation index of α-lactalbumin in milk after heat treatment. They produced two specific monoclonal antibodies: Mab 20 and Mab 130. The antigen–antibody binding produces changes in the refractive index on a metal surface that is detected and quantified by an optical system in the form of changes in the reflectance angles, which are expressed as resonance units, represented in a displayed curve on a computer [13]. The study showed that it was possible to determine the heat treatment to which the sample had been subjected by quantifying both native and denatured α-lactalbumin and expressing the results as percentage of denatured α-lactalbumin. Although the denaturation index for sterilization differed drastically from that of pasteurization, 97.2–99.7% vs. 5.2–18.9%, it was harder to differentiate highly pasteurized milk from direct UHT. The same principle (Biacore® Q optical biosensor, GE Healthcare, Uppsala, Sweden) was used to assess the decrease in native BSA after four hours of heat treatment at 70 and 80 ◦ C in diluted skim milk (1:10, v/v) [15]. Sheep polyclonal antibovine BSA was evaluated and selected from four anti-BSA antibodies due to its highest affinity to BSA. Results showed a decrease of 30 and 80% of native BSA, depending on the temperature. The SPR has shown to be very versatile for various purposes, namely clinical [13]. This technology is developing, meaning that investigation is necessary to use the body and antibody that best suits a specific sample. It was recently demonstrated that whey protein’s evaluation is reliable, repeatable, automated, sensible and only requires dilution of the milk in a buffer [16]. These biosensors are faster (~4 min) than the traditional Dumas method, which requires whey separations plus 5 min of the procedure. In addition, biosensors could complement other analytical methods to determine the denaturation of proteins since these biosensors have low detection limits [15,16]. Nanosensors are another sensor type that gather information from nanoparticles [17]. Rezende et al. [18] used an aqueous suspension of vesicles (Polydiacetylene/triblock copoly- mer L64/cholesterol) to detect BSA. This colorimetric sensor changes the color intensity from blue to red according to the concentration of native BSA proteins due to the occur- rence of intermolecular interactions between them (Figure 1). Diluted skim milk, without casein micelles, presented a colorimetric response (CR) average of 50 ± 2.5%, indicating 0.045 ± 0.03 mM of native BSA. The result was compared with the HPLC technique and was well-correlated. Likewise, denatured BSA showed a CR value of 5%. Furthermore, other proteins such as α-lactalbumin and β-lactoglobulin were also evaluated, but they showed unsatisfactory results. As the authors suggest, BSA denaturation may produce the exposure of hydrophobic amino-acid residues that creates a repulsive interaction between the BSA hydrophobic group and the hydrophilic segments of the nanosensor. This lack of blue-to-red transition in the presence of denatured protein allows for the discrimination of native from denatured BSA molecules. L64 nanosensor is a simple, fast (<1 min) and low-cost analytical tool since vesicles are easy to prepare and produce at a large scale [19]. However, milk caseins may cause interferences, and thus, precipitation and separation of micelles before the analysis may be required [20]. A microchip is a miniaturized microfluidic device that utilizes a variety of microchan- nels to assess a sample. A well-known method to separate and analyze milk proteins is the SDS-PAGE electrophoresis, where proteins move through an SDS-polyacrylamide gel according to their electrophoretic mobility as a function of their molecular weight [21]. This technique has been miniaturized by using a microfluidic chip commercialized in different “lab-on-a-chip” versions. It has been proven to separate and quantify whey proteins [22,23] in the presence or absence of reducing agents [24]. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), the ExperionTM Automated Electrophoresis System (Bio Rad, Hercules, CA, USA) or the LabChip® GXII TouchTM (Perkin Elmer, Waltham, MA, USA) are some examples of the commercialization of this technique. They include all necessary reagents/operations: gel matrix solution, protein-dye concentrate solution and marker-protein buffer solution. In addition, they are faster (~30 min) than the tra- Dairy 2022, 3 503 y 2022, 3, x FOR PEER REVIEW ditional method (between 45 to 60 min), automated, require small quantities of sample, consume fewer toxic chemicals and are a potential tool for quality control in the dairy industry [12,24]. Figure Figure 1. Colorimetric 1. Colorimetric response ofresponse of thetoL64 the L64 nanosensor nanosensor the different forms of to BSAthe different proteins. Reprinted/adapted with permission from Ref. [18]. printed/adapted with permission from Ref. [18]. Although biosensors and microchips have some advantages such as fast responses and real-time analysis, they may be of single use or prone to saturation after being used [24], and thusAaremicrochip not suitable for is a miniaturized on-line microfluidic monitoring of whey-protein device that denaturation. uti channels to assess a sample. A well-known method to separate a 2.2. Electrochemical Sensors is the SDS-PAGE Electrochemical sensorselectrophoresis, where ofproteins are based on the measurement move electron transfer duethrough to oxidation/reduction reaction of the analyte through electrodes. gel according to their electrophoretic mobility as a function of the Changes in the electric signal are proportional to the concentration of the target molecule, and the analyte is This technique consumed by the electrode has been [25,26]. miniaturized There are different typesby using a microfluidic of electrochemical transducers chi such as amperometric, conductimetric, impedimetric and potenciometric ferent “lab-on-a-chip” versions. It has been proven to separate and devices, with the amperometric being the most widely employed. These transducers maintain a constant [22,23]to detect potential in the presence variations or absence in the current [12,25–27].of reducing agents [24]. The A Cosio et al. [28] proposed using of an amperometric sensor with a Tungsten electrode (Agilent Technologies, Santa Clara, CA, USA), the ExperionTM A to evaluate the severity of heat treatment to which milk was subjected. This research sisbecause was System (Bio Rad, the sulfhydryl groupsHercules, become free andCA,formUSA) or the thiols after heat LabChip denaturation®ofGXII T proteins. Thiols react with mercury ions and the electrode detects mercury ions that are left. Waltham, MA, USA) are some examples of the commercializatio Therefore, the more whey proteins are denatured, the more thiols can react with mercury include ions, and lessallcurrent necessary reagents/operations: is observed. This study was carried out gel matrix with solution, pro raw, pasteurized and UHT milk after casein precipitation at pH 4.6. The results showed that this method lution and marker-protein buffer solution. In addition, they are differentiates between raw milk or mild treatment and UHT milk. Although this method is traditional fast (90 samples/h), method simple and(between 45ato straightforward, 60 min), previous step toautomated, require isolate whey proteins is sm compulsory. Thus, it is not suitable for on-line control of thermal denaturation. consume fewer The three-omega toxic method chemicals measures and are (k) thermal conductivity a (Figure potential tool 2) [29,30]. for qua It was industry tested with BSA [12,24]. [30] and β-lactoglobulin [29] solutions. These studies demonstrate that alteration in protein conformation, resulting from temperature change, can be correlated Although successfully biosensors with their thermal andwhich conductivity, microchips increased ashave some temperature advantage increased. and The real-time temperature analysis, of denaturation they for BSA andmay be of single β-lactoglobulin in solutionuse or prone (5% w/w, pH 7.00) to sat started at 53 and 61 ◦ C, respectively. The maximum k could be recorded at 68 and 71 ◦ C. [24], This andis thus method are not suitable under development; thus, more for on-line research monitoring is necessary of whey-pr for complex solutions such as milk or whey. This technique is fast (<1 min) and could be used to monitor protein denaturation in real time [29]. 2.2. Electrochemical Sensors Electrochemical sensors are based on the measurement of oxidation/reduction reaction of the analyte through electrodes. C nal are proportional to the concentration of the target molecule protein denaturation in real time [29]. Electrochemical sensors are less popular than optical methods b identify and quantify pathogens, bacterial toxins and pesticides in food Dairy 2022, 3 such as rapid response, simple equipment required, small or moderat 504 tion capabilities and high portability [25–27]. Figure 2. (a) Schematic diagram of 3ω technique for measuring k (thermal conductivity) of liq- Figure 2. (a) Schematic diagram of 3ω technique for measuring k (thermal c uid sample and (b) schematic diagram of thermal conductivity sensor. Reprinted/adapted with sample and permission (b)[29]. from Ref. schematic diagram of thermal conductivity sensor. Reprinted/ sion from Ref. [29]. Electrochemical sensors are less popular than optical methods but they are used to identify and quantify pathogens, bacterial toxins and pesticides in food due to advantages 2.3.asSpectroscopy such rapid response, simple equipment required, small or moderate cost, miniaturization capabilities and high portability [25–27]. Spectroscopy, classified according to the spectrum used, is the st 2.3. Spectroscopy such as absorption, emission or scattering of electromagnetic radiatio Spectroscopy, classified according to the spectrum used, is the study of phenom- teraction ena with matter such as absorption, to obtain emission bothofqualitative or scattering and electromagnetic quantitative radiation caused by inform [26,31]. the Spectroscopy interaction with matter totechniques take less obtain both qualitative andthan 1 mininformation quantitative to be performed on molecules [26,31]. Spectroscopy techniques take less than 1 min to be performed. 2.3.1. 2.3.1. Infrared Infrared Spectroscopy Spectroscopy (from 780 to(from 780 50,000 nm) to 50,000 nm) Electromagnetic waves have specific energy according to their wavelength. Molecules Electromagnetic that absorb the energy of infrared waveslights have producespecific vibrations.energy according(IR) Infrared spectroscopy to their cules that expresses absorbofthe the vibration energy covalent bonds ofininfrared lights molecules that produce contains vibrations. quantitative infor- Inf mation about all the constituents that absorb IR radiation [11,32]. Infrared spectrometers (IR) expresses the vibration of covalent bonds in molecules that conta consist of an interferometer and a detector. The interferometer produces infrared light formation over a range of about all the wavelengths, constituents and the that detector registers the absorb IR radiation signals of vibrations. [11,32]. I A Fourier mathematical function transforms these signals to convert ters consist of an interferometer and a detector. The interferometer pro data to a more meaningful absorbance/transmittance response over a frequency/wavelength form. These instruments overfor allow a the range of wavelengths, measuring andsimultaneously of all the frequencies the detector registers [11,32]. Thus, it the signals of v is necessary mathematical to perform statisticalfunction analyses with transforms these signals multivariate statistical to convert software and data to a m apply procedures to correlate changes in infrared waves with a concentration of target molecules [32]. sorbance/transmittance Fourier transform near-infrared response (FT-NIR) over a frequency/wavelength spectroscopy studies the molecules’ ab- form. allow for sorption the in the measuring range of 780–2500of nm, allwhilst the Fourier frequencies transform simultaneously mid-infrared (FT-MIR) [11,32]. spectroscopy studies it in the range of 2500–30,000 nm [26,32]. NIR radiation penetrates to perform statistical analyses with multivariate statistical software an more of the sample; therefore, it produces bands with less absorption than MIR radiation. to correlate Protein absorption changes is due to the in combination infrared waves with of vibrations froma concentration C = O and N-H bonds of oftarget m amide IFourier and amidetransform II with corresponding near-infrared (FT-NIR) spectroscopynm, FT-NIR absorption bands at 2000 and 2222 studies respectively [32]. However, FT-NIR spectra of food represent absorptions of other chemical sorption bonds, in and e.g., C-H theO-H,range of 780–2500 overlapping nm,corresponding in the spectrum, whilst Fourier mainly totransform overtones mid spectroscopy and combinations ofstudies vibrational it mode, in the range which makesofthe 2500–30,000 nm [26,32]. association of frequencies with NIR r specific chemical groups difficult [32,33]. On the contrary, FT-MIR spectra can discriminate more chemical specific of the sample; compositions therefore, and associate it produces frequencies tobands with less slight differences in absorption sample Protein absorption composition, allowing for the is determination due to theofcombination the composition and of vibrations from C = O structure of chemical molecules. In this case, peaks around 6100 and 6500 nm correspond to amide I and amide amide I and amide II with corresponding FT-NIR absorption bands a II bands [32]. respectively [32]. However, FT-NIR spectra of food represent absorpti cal bonds, e.g., C-H and O-H, overlapping in the spectrum, correspond tones and combinations of vibrational mode, which makes the associ with specific chemical groups difficult [32,33]. On the contrary, FT-M uate heat denaturation (80 °C, every 2 min up to 120 min) of skimmed whe centrate with a NIRS 6500 near-infrared spectrophotometer (NIR System Spring, MD, USA) in the range of 1100–2500 nm. Spectra of the different Dairy 2022, 3 calibrated to monitor the whey proteins’ heat denaturation determined 505 b method and expressed as a nitrogen-solubility index at pH 4.6. Obtained i from 95 to 45% after 120 min of treatment. More recently [37], with a FT-N Infrared spectroscopy has been applied in the dairy industry as a tool to quantify most 154S of (Bomem the milk’s technologies, components and establishQuebec, milk quality Canada), used or authenticity the whey-protein [11,33–35]. Due to the ind skim-milk versatility power of this as low technology, at an(70 °Cstage, early 15 s), medium Pouliot et al. [36](85 used °CNIR60tos)evaluate and highheat (>120 denaturation (80 ◦ C, every 2 min up to 120 min) of skimmed whey-protein concentrate with taining nitrogen-index values of ≥6.00, 1.51–5.99 and ≤1.50 for each heat trea a NIRS 6500 near-infrared spectrophotometer (NIR Systems Inc., Silver Spring, MD, USA) tively. in The the range NIR prediction of 1100–2500 nm. Spectra ofofthe the soluble whey-protein different samples index were calibrated was adequat to monitor tion the wheywith known proteins’ heatnitrogen denaturation values determinedthrough by the partial least square Kjeldahl method (PLS)asshowed and expressed a nitrogen-solubility index at pH 4.6. Obtained indexes varied from 95 to 45% after 120 min tions. Briefly, these studies showed that NIR spectroscopy could determin of treatment. More recently [37], with a FT-NIR model MB 154S (Bomem technologies, naturation Quebec, of used Canada), whey theproteins whey-protein through index tocalibrations. classify skim-milk power as low (70 ◦ C ◦ ◦ Recent(85 15 s), medium studies C 60 s)have and highfocused (>120 in C 4using FT-NIRnitrogen-index min), obtaining to detect conformation values of ≥6.00, 1.51–5.99 and ≤1.50 for each heat treatment, respectively. The NIR prediction whey proteins due to heat treatment [3,38]. Whey-protein isolate and solu of the soluble whey-protein index was adequate, and calibration with known nitrogen talbumin values through and β-lactoglobulin, partial least square (PLS) studied showed good with a BrukerTensor correlations. Briefly, these27studies FTIR instru Optik,that showed Ettlingen, Germany) NIR spectroscopy could[38] and atheNexus determine 670 spectrometer heat denaturation (Thermo E of whey proteins through calibrations. son, WI, USA) [3], showed maximum peak intensities at ≈2940, 6040 and 613 Recent studies have focused in using FT-NIR to detect conformational changes in whey type ofdue proteins sample. After heat to heat treatment treatment [3,38]. Whey-protein at isolate temperatures and solutions ofof50α-lactalbumin to 90 °C for 5 m ance and peaks of whey-protein β-lactoglobulin, studied with aisolate and β-lactoglobulin, BrukerTensor 27 FTIR instrumentwhich (Brukerconsists Optik, of 8% Ettlingen, Germany) [38] and a Nexus 670 spectrometer (Thermo Electron, Madison, WI, β-sheet and 47% random coil, decreased, with a concomitant increase in w USA) [3], showed maximum peak intensities at ≈2940, 6040 and 6130 nm, for each type of by theAfter sample. formation of random heat treatment coils [3,38]. at temperatures of 50 to 90 ◦ C for 5 min, the absorbance peaks In parallel, of whey-protein a calibration isolate model and β-lactoglobulin, wasconsists which developed by Pabari of 8% α-helix, et al. [39] to 45% β-sheet and dered infant formula changes in casein and whey-protein isolates the 47% random coil, decreased, with a concomitant increase in wave numbers by due to he formation of random coils [3,38]. °C for 10 min In parallel, and 24model a calibration h). In this was study,byaPabari developed MultiEye et al. [39] (Innopharma ® to predict powdered Labs, D ◦ NIR spectrophotometer infant formula changes in casein wasandused in the IR whey-protein range isolates due ofto1515–2200 heat (80 andnm 105 (Figure C 3 for 10 min and 24 h). In this study, a MultiEye ® (Innopharma Labs, Dublin, Ireland) NIR of the spectra variation obtained before and after thermal treatment throug spectrophotometer was used in the IR range of 1515–2200 nm (Figure 3). The analysis component of analysis the spectra variation (PCA) obtained andand before theafter partial thermalleast-square treatment through(PLS)a principal- calibration sho spectroscopy component analysis could (PCA) beand a thegood partialtechnique to determine least-square (PLS) protein calibration showed thatstability NIR af spectroscopy could be a good technique to determine protein ment. However, more data points were needed for the second statistical m stability after heat treatment. However, more data points were needed for the second statistical method. Figure 3. MultiEye®system showing NIR spectrophotometer with four channels attached to four Figure 3. MultiEye® system showing NIR spectrophotometer with four channels reflectance probe fibers. Each probe fiber was attached to a detection and illumination source. reflectance probe Reprinted/adapted withfibers. Each permission probe from fiber was attached to a detection and illumina Ref. [39]. printed/adapted with permission from Ref. [39]. In another study, Alvarez et al. [40] designed an empirical model for p tein aggregation of heat-denatured whey-protein concentrate, according to Dairy 2022, 3 506 Dairy 2022, 3, x FOR PEER REVIEW 7 of 13 In another study, Alvarez et al. [40] designed an empirical model for predicting protein aggregation of heat-denatured whey-protein concentrate, according to three factors: whey-protein concentration (8–12%), time (5–30 min) and temperature (85 to 100 °C) of whey-protein concentration (8–12%), time (5–30 min) and temperature (85 to 100 ◦ C) of heat treatment. The Box–Behnken-response surface method was used to relate them to the heat treatment. The Box–Behnken-response surface method was used to relate them to response of the amide I band with a FTIR Magna IR 560 (Thermo Fisher Scientific Inc., the response of the amide I band with a FTIR Magna IR 560 (Thermo Fisher Scientific Waltham, MA, USA). The resulting model was dominated by changes in processing tem- Inc., Waltham, MA, USA). The resulting model was dominated by changes in processing perature, while temperature, while both heating both time heating and time andprotein proteinconcentration concentrationresulted resultedininno no significant significant model model factors. factors. However, However, the the data data exhibited exhibited aa significant significant linear linear fit. fit. Infrared Infrared spectroscopy spectroscopyisisaccepted acceptedasasananin-line/on-line in-line/on-linedetection detectionmethod in food method be- in food cause it does not require any sample preparation (nondestructive). In addition, it is because it does not require any sample preparation (nondestructive). In addition, it is simple simple to to use, use, rapid, rapid, specific, specific, allows allows several several constituents constituents to to be be measured measured simultaneously simultaneously and and is is reliable for monitoring chemical and physical parameters during a process [11,18,35,41]. reliable for monitoring chemical and physical parameters during a process [11,18,35,41]. Nonetheless, Nonetheless, aa reference-method-calibration reference-method-calibration analysis, analysis, covering covering any any possible possible composition composition fluctuations, is necessary to determine and quantify components fluctuations, is necessary to determine and quantify components [32,36]. [32,36]. 2.3.2. UV-Visible UV-Visible Spectroscopy Ultraviolet Ultraviolet and and visible visible light light absorptions absorptions by by molecules molecules excite excite electrons electrons and produce electronic transitions. The The molecules molecules that that absorb absorb this this radiation have a chromophore. The light that is not absorbed by chromophores is transmitted through the sample sample [26,31]. [26,31]. a. Light-scatter Scatter Scatter light light is is aa part part of of the the light light that that is isnot notabsorbed absorbedor ortransmitted transmittedby bythe themolecules. molecules. Milk has compounds such as casein micelles that scatter light, and therefore Milk has compounds such as casein micelles that scatter light, and therefore light backscatter light backscatter is one of theismost oneused of the most used techniques fortechniques developingfor developing optical sensors optical for foodsensors for food applications. In applications. In these light-scattering these light-scattering sensors, a diode sensors, (LED) aemits dioderadiation, (LED) emits radiation, which which isby is conducted con- an ducted by an optic fiber optic fiber through throughand the sample, thethe sample, and backscatter refracted the refracted (180◦ ) is light backscatter light (180°) received byisa received by a detector detector [42] (Figure 4). [42] (Figure 4). Figure 4. (a) (a) The The light light backscatter backscatter probe; probe; (b) (b) the the optical optical sample sample cell: cell: LL is is the the light light source, source, D D is is the detector; detector; (c) (c) the the complete complete optical optical system. system. Reprinted/adapted Reprinted/adaptedwith withpermission permissionfrom fromRef. Ref.[7]. [7]. Lamb Lambetetal.al. [7][7] studied the relationship studied between the relationship changeschanges between in the sizeinofthe casein sizemicelles and mi- of casein the denaturation of milk whey proteins. This technique relies on variations of backscatter celles and the denaturation of milk whey proteins. This technique relies on variations light intensity according to wavelength of backscatter and particle light intensity diameter. according to After heat denaturation, wavelength and particle β-lactoglobulin aggregates diameter. After heat and binds to casein micelles, whose diameter increases. Although a priori, the wavelength intervals denaturation, β-lactoglobulin aggregates and binds to casein micelles, whose diameter used varied from 200–1100 nm; the highest response was observed at wavelengths ranging 500–900 increases. Although a priori, the wavelength intervals used varied from 200–1100 nm; the nm. This optical method proposed by [7] showed a proper correlation with data collected with cur- highest response was observed at wavelengths ranging 500–900 nm. This optical method rently used techniques (SDS-PAGE and differential scanning calorimetry). Nevertheless, the optical proposed method by [7] tended to showed a proper correlation slightly overestimate the amountwith data collected of protein with currently denaturation. used Furthermore, tech- protein niques (SDS-PAGE aggregates produced and differential during scanning denaturation calorimetry). may also Nevertheless, affect this technique [5]. the optical method tween light backscatter measurements and the size of casein micelles at pH after 10 min of heat treatment at 80 and 90 °C. On one hand, they found a lation between treatment temperatures, the intensity of light backscatter an Dairy 2022, 3 at pH lower than 7.1. On the other hand, at pH 7.1, denatured whey507protein aggregates, and the temperature treatment does not change micelle size. The same authors recently studied the relation between light backsca tended ment to andslightly the overestimate size of thethe amount casein of proteinbut micelles, denaturation. Furthermore, as a function protein of whey-protein aggregates produced during denaturation may also affect this technique [5]. casein micelles and the formation of whey-protein soluble aggregates [42]. T The binding of β-lactoglobulin strongly depends on pH, and the highest diameter firmed of a positive casein micelles correlation was found at pH 6.3between [5]. Taterka bound whey and Castillo proteins, [42] studied themicelle relation size an backscattered light. At pH lower than 7.1, a proportion of β-lactoglobulin between light backscatter measurements and the size of casein micelles at pH 6.3, 6.7 and 7.1 after 10 min of heat treatment at 80 and 90 ◦ C. On one hand, they found a positive gates and the rest binds at the casein micelle depending on pH. Consequen correlation between treatment temperatures, the intensity of light backscatter and micelle micelle size at pH size lowerand theOn than 7.1. intensity of light the other hand, at pHbackscatter 7.1, denaturedalso wheydepend on the proteins tend to num aggregates form aggregates, present in the heat-treated and the temperature treatment does milknot [42]. changeOptical sensors using a lig micelle size. The same authors recently studied the relation between light backscatter measurement are currently in development. Different factors that affect whey-protein and the size of the casein micelles, but as a function of whey-protein binding to the must micelles casein be considered (e.g., temperature, and the formation of whey-proteinpH andaggregates soluble the amount of whey-protei [42]. This study However, this optical method could be used in-line in the dairy industry confirmed a positive correlation between bound whey proteins, micelle size and intensity of to backscattered light. At pH lower than 7.1, a proportion of β-lactoglobulin forms aggregates treated milk to make cheese or yoghurt depending on the level of denatur and the rest binds at the casein micelle depending on pH. Consequently, the casein micelle teins, size andsince thereofislight the intensity no backscatter need to alter the milk also depend on thesample number ofand the aggregates protein measurement real time present in the[7]. heat-treated milk [42]. Optical sensors using a light backscatter are currently in development. Different factors that affect whey-protein denaturation must be considered b. temperature, (e.g., Fluorescence spectroscopy pH and the amount of whey-protein aggregates). However, this optical method could be used in-line Fluorescence occurs in the dairy industry when specifictomolecules evaluate heat-treated absorbmilk to makefrom l photons cheese or yoghurt depending on the level of denatured whey proteins, since there is no release need thetheexcess to alter energy milk sample and by the emitting measurement light at a longer is provided wavelength in real time [7]. [10]. Mil fluorescent b. components Fluorescence spectroscopy such as vitamin A, aromatic amino acids, NADH vanced Maillard compounds [43]. Whey proteins such as β-lactoglobulin a Fluorescence occurs when specific molecules absorb photons from light, and they min have release at least the excess energyone amino-acid by emitting light at a tryptophan residue, longer wavelength responsible [10]. Milk for whe has intrinsic flu- orescent trinsic components fluorescence such [44]. as vitamin A, aromatic fluorescence Tryptophan amino acids, NADH and some depends onadvanced the environm Maillard compounds [43]. Whey proteins such as β-lactoglobulin and α-lactalbumin have on milk characteristics [10]. at least one amino-acid tryptophan residue, responsible for whey proteins’ intrinsic flu- Right-angle orescence fluorescence [44]. Tryptophan spectroscopy fluorescence depends on the environment, and thus on milk characteristics [10]. Right-angle fluorescence spectroscopy, using 90° configuration (Figu Right-angle fluorescence spectroscopy dilution of milk samples because if the absorbance of the sample is higher Right-angle fluorescence spectroscopy, using 90◦ configuration (Figure 5), requires sorption dilution of emitted of milk radiation samples because if theoccurs andofdecreases absorbance the sample fluorescence is higher than 0.1,intensity re- ( absorption fect) [10].of emitted radiation occurs and decreases fluorescence intensity (inner-filter effect) [10]. Figure 5. Principles of right-angle fluorescence and front-face fluorescence. Figure 5. Principles of right-angle fluorescence and front-face fluorescence. Birlouez-Aragon et al. [45] evaluated milk heat treatment using the right-angle con- Birlouez-Aragon figuration on whey obtainedet byal. [45] evaluated precipitation milk heat and separation treatment of milk using caseins at pH 4.6. the ri The FAST method (Fluorescence of Advanced Maillard products and Soluble Tryptophan) figuration on whey obtained by precipitation and separation of milk case was applied, where the FAST index was used as an indicator of the damage during a heat The FAST process. method This method (Fluorescence measured two indicatorsof Advanced of heat treatment:Maillard fluorescenceproducts of advancedand So phan) was applied, where the FAST index was used as an indicator Maillard products (excitation/emission wavelengths of 330/420 nm) and soluble of the d trypto- phan (290/340 nm) to calculate the FAST index, which is obtained by dividing advanced a heat process. This method measured two indicators of heat treatment: f Maillard fluorescence compounds by tryptophan fluorescence and multiplying the result advanced Maillard products (excitation/emission wavelengths of 330/420 nm tryptophan (290/340 nm) to calculate the FAST index, which is obtained b vanced Maillard fluorescence compounds by tryptophan fluorescence an the result by 100. The results of this study showed a good correlation betw Dairy 2022, 3 508 Dairy 2022, 3, x FOR PEER REVIEW 9 of 13 by 100. The results of this study showed a good correlation between the FAST index and HPLC [45].protein-quantification traditional Later on, Birlouez-Aragon methods et such al. [46] validated as the Lowry the FASTand method method HPLCwith [45]. stand- Later ardized on, HPLC methods Birlouez-Aragon et al.and [46]studied validatedits capacity the FASTtomethod discriminate milk heat treatments. with standardized HPLC meth- The ods studyand studiedthat showed its capacity a milderto discriminate heat treatmentmilk heat treatments. provoked The studywith lower denaturation showed that a a concom- milder heat treatment itant higher tryptophan provoked lower denaturation fluorescence intensity and with a concomitant higher a corresponding low FASTtryptophan index. fluorescence intensity and a corresponding low FAST index. Tryptophan Tryptophan fluorescence allowed for the distinguishing of thermalized milk (68 °C, 24 s) fluorescence allowed for the distinguishing of thermalized milk ◦ C, 24 s) from pasteurized (68 ◦ C, (68Furthermore, from pasteurized (68 °C, 2 min and 72 °C, 2 min) milk. the FAST index could 2discriminate ◦ min and 72 between C, 2 min) milk.indirect Furthermore, the treatments. FAST indexHence, could discriminate between direct, and bottle it was proven that the direct, indirect FAST index and bottle allows for thetreatments. estimationHence, it was proven of the intensity of heatthat the FASTinindex treatment milk allows for by quanti- the estimation of the intensity of heat treatment in milk fying protein denaturation through changes in fluorescence intensity. by quantifying protein denaturation through changes Murillo et al.in[44] fluorescence evaluatedintensity. total fluorescence spectra (bandpasses of 4 nm for both Murillo et al. [44] evaluated excitation and emission with 3.2 increments total fluorescence and 0.4spectra nm step (bandpasses size) of wheyof 4obtained nm for both from excitation and emission with 3.2 increments and 0.4 nm step milk samples subjected to different treatments (raw, pasteurized and UHT). In the size) of whey obtained from UV milk samples subjected to different treatments (raw, pasteurized region, two fluorescent bands were obtained. The liquid chromatographic analysis with and UHT). In the UV region, two fluorescent bands were obtained. The liquid chromatographic analysis with fluorescence detection showed that the two fluorescent amino acids, tyrosine and trypto- fluorescence detection showed that the two fluorescent amino acids, tyrosine and trypto- phan, which were present at a soluble fraction of milk, were responsible for the intrinsic phan, which were present at a soluble fraction of milk, were responsible for the intrinsic fluorescence of whey. Heat treatment did not modify the position of the two bands but fluorescence of whey. Heat treatment did not modify the position of the two bands but did did modify the fluorescence intensity with the smaller corresponding to UHT milk. modify the fluorescence intensity with the smaller corresponding to UHT milk. Not long ago, the research group of Birlouez-Aragon [8] implemented a fluorometric Not long ago, the research group of Birlouez-Aragon [8] implemented a fluorometric method in a compact and portable right-angle fluorometer named Amaltheys®® (Spectralys method in a compact and portable right-angle fluorometer named Amaltheys (Spectralys Innovation, Romainville, France) (Figure 6). This device is at-line and provides three key Innovation, Romainville, France) (Figure 6). This device is at-line and provides three key indicators on indicators on milk, milk, powders powders and and dairy dairy derivatives derivatives withwith high highaccuracy: accuracy: soluble solubleproteins, proteins, whey-protein nitrogen index and FAST index. This tool is fast but whey-protein nitrogen index and FAST index. This tool is fast but needs calibration modelsneeds calibration mod- to determine the amount of denatured whey proteins as a way to qualify the effect of heatof els to determine the amount of denatured whey proteins as a way to qualify the effect heat treatment treatment [8]. [8]. Figure 6. Amaltheys® ® device. Even though devices based based onon the the right-angle right-angle fluorescence fluorescencespectroscopy spectroscopyarearebeing being developed to offer control of milk protein for the dairy industry at-line, they developed to offer control of milk protein for the dairy industry at-line, they cannot cannot be used be on-line since they used on-line sincerequire either either they require the dilution of milkofsamples the dilution or precipitation milk samples of caseins or precipitation to of ca- analyze whey. whey. seins to analyze Front-face Front-face fluorescence fluorescence spectroscopy spectroscopy Front-face ◦ ◦ Front-face fluorescence fluorescencespectroscopy spectroscopyuses usesananillumination illuminationangle anglebetween between3030°andand60 60° (Figure 5) [47]. (Figure 5) [47]. This Thisposition positionminimizes minimizeslight’s light’s reflection, reflection, scatter scatter andand polarization polarization [10], [10], de- decreasing the real intensity of fluorescence. Light does not go through creasing the real intensity of fluorescence. Light does not go through the sample; it isthe sample; it re- is recovered just from the sample’s surface. As a result, front-face fluorescence covered just from the sample’s surface. As a result, front-face fluorescence can be used can be used directly on turbid, solid, powder and concentrated samples [10,47]. directly on turbid, solid, powder and concentrated samples [10,47]. Dufour and Riaublanc [10] studied the emission fluorescence of different molecules Dufour and Riaublanc [10] studied the emission fluorescence of different molecules such as tryptophan in proteins to characterize the changes in fluorescence of raw and heat- such as tryptophan in proteins to characterize the changes in fluorescence of raw and heat- treated milk. The authors used a 56◦ configuration and determined that the fluorescence treated milk. The authors used a 56° configuration and determined that the fluorescence properties were modified when applying a heat treatment (70 ◦ C, 20 min). Furthermore, properties were modified when applying a heat treatment (70 °C, 20 min). Furthermore, these authors suggested the use of a matrix with fluorescence results of tryptophan (excita- these authors suggested the use of a matrix with fluorescence results of tryptophan (exci- tion: 290 nm, emission: 305–400 nm) and others fluorescent compounds, such as retinol tation: 290 nm, emission: 305–400 nm) and others fluorescent compounds, such as retinol (excitation: 321 nm, emission: 260–350 nm), could improve discrimination between raw (excitation: milk 321 nm, emission: and heat-treated milk. 260–350 nm), could improve discrimination between raw milk and heat-treated milk. Dairy 2022, 3 509 Fluorescence changes of whey protein after denaturation by several heat treatments were studied by Kulmyrzaev et al. [48]. In this study, together with the front face spec- troscopy (56◦ configuration) and the statistical technique, principal-component regression was used to create regression models for both qualitatively and quantitatively evaluating the thermal denaturation of whey proteins. This study revealed that NADH and FADH spectra (excitation: 360 nm, emission: 460 and 518 nm, respectively) were good predictors of the content of the native β-lactoglobulin. Furthermore, front-face fluorescence correlated with immunological methods to determine residual native whey proteins subjected to different temperatures of low-pasteurization treatment. Higher treatment temperatures (72–120 ◦ C) were studied with front-face fluores- cence excitation–emission matrices (60◦ configuration, excitation: 250–350 nm, emission: 260–500 nm) by Hougaard et al. [49]. In this case, a parallel factor analysis (PARAFAC) was used to estimate the different concentrations of the fluorophores of samples subjected to different heat treatments. Another model evaluated for heat treatment was the partial least-squares model used by Diez et al. [47]. This study used front-face spectroscopy (32◦ configuration) to obtain emission spectra of tryptophan (excitation: 290 nm, emission: 312–398 nm) and advanced Maillard products (excitation: 350 nm, emission: 402–498 nm), and to predict FAST index and soluble whey protein from heat-treated milk (72–115 ◦ C) at six different times (from 2 min to 9.3 min). A good regression was obtained between the concentration of soluble whey protein measured with conventional methodologies and those predicted by partial least squares. The FAST index had a deviation at high tempera- tures due to the predominance of Maillard fluorescence compounds over tryptophan. The authors highlight the need for more samples with high heat charge to create another model and correctly predict the FAST index. Ayala et al. [50] also studied the tryptophan fluorescence emission by using front- face fluorescence (56◦ configuration, excitation: 290 nm, emission: 300–450 nm) with heat-treated skim-milk powder. Significant changes were observed at 90 and 100 ◦ C. A decrease in tryptophan fluorescence intensity and a red shift was found when the heat- treatment intensity increased. In this study, principal-component analysis was performed and suggested using well-selected specific wavelengths or narrowband array. Front-face fluorescence spectroscopy and multivariate statistical analysis have a great potential to become a method for distinguishing between milk samples subjected to dif- ferent heat treatments based on the changes in fluorescence of intrinsic fluorophores of milk [10,43,47–49]. More studies and models are needed to validate the method to become a fast at-line or on-line method [10]. The advantages of this method are that no prelimi- nary sampling is needed (nondestructive) since it can operate on opaque samples such as nondiluted milk, and it is fast, relatively inexpensive and sensitive [10,43,47]. 3. Conclusions Some methods are well-established in the dairy industry such as optical methods based on infrared spectroscopy, which are fast, do not require sample manipulation and detect in/on-line once properly calibrated. Furthermore, microchips, nanosensors and biosensors are well implemented in the industry. Nondestructive rapid methods such as those used to detect antibodies enable one to obtain the denaturing index of α-lactalbumin directly from a milk sample. They are fast, nondestructive and easy to use, but cannot perform continuous detection because they saturate. Optical methods using fluorescent spectroscopy are fast and easy to apply methods that allow the detection and quantification of thermally denatured whey proteins. Espe- cially the front-face fluorescence spectroscopy configuration can be developed as an at-, in-, on-line technique since it allows to analyze a milk sample directly without being previously manipulated. In addition, the backscatter of visible light is being studied to develop a sensor capable of detecting and quantifying the denaturation of whey proteins in/on-line during processing. The electrochemical method had been developed to differentiate ther- Dairy 2022, 3 510 mal treatments on milk based on the denaturation of the whey proteins. It is a fast, simple and direct method, but the sample needs to be prepared to obtain a whey-protein solution. The current trend is the development of rapid methods that can work in-, on-, or at-line to be able to correct processes to which the milk is subjected, such as heat treatment, in time. A method able to rapidly detect and quantify the denaturation of whey proteins as a manner to predict the real-time functional quality of the heat-treated milk would aim not only with process optimization but also with decision making, assisting dairy plants in the efficient use of milk depending on the product being manufactured, ensuring, in turn, both higher dairy foods’ yield and quality. Funding: This research was funded by [Secretaría de Educación Superior, Ciencia, Tecnología e Innovación of the Ecuadorian Government] grant number [106-2017 (SENESCYT-SDFC-DSEFC-2017- 3855-O)]. The mentioned funds corresponded to P. Freire´s PhD fellowship. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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