LWT - Food Science and Technology 163 (2022) 113589 Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt Tryptophan front-face fluorescence and functional properties of whey: A preliminary study Yu Ting Teng , Paulina Freire , Anna Zamora *, Manuel Castillo * Centre d’Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), Department of Animal and Food Sciences, Universitat Autònoma de Barcelona, 08193, Bellaterra, (Cerdanyola del Vallès), Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: This study investigated the potential of front-face fluorescence spectroscopy to predict the functional properties Whey of whey. Whey has a commercial interest due to its excellent nutritional value and versatile functional properties. Front-face fluorescence However, its attributes depend dramatically on heat treatment, which may alter its suitability for different food Tryptophan fluorescence applications. Tryptophan front-face fluorescence of whey and its functional properties, i.e., foaming, gel-forming, Heat treatment Functional properties and emulsifying properties, were evaluated after milk heat treatment (at 80 ◦ C with seven holding times) to detect correlations between tryptophan fluorescence and functional parameters and generate predictive models. Whey samples were obtained by isoelectric precipitation of caseins from reconstituted skim milk powder enriched with whey protein isolate. As expected, heat treatments induced an increase in whey protein dena­ turation, as well as a decrease in total whey protein concentration and tryptophan fluorescence intensity. Gel- forming and emulsifying properties of whey significantly correlated with the maximum intensity of trypto­ phan (P < 0.001). Concerning foaming properties, only the foam stability index revealed a weak correlation with tryptophan maximum intensity (P < 0.05). Consequently, models presented strong significances (P < 0.001). Specifically, using only tryptophan fluorescence allowed successful prediction of emulsifying properties but was not enough for foaming and gel forming properties. 1. Introduction possesses several fluorophores naturally or after chemical modification, including tryptophan (Trp), tyrosine and phenylalanine, dityrosine, Front-face fluorescence spectroscopy (FFFS) could be a process NADH/FADH, vitamin A, riboflavin, and some Maillard reaction prod­ analytical technology suitable for estimating rapid and accurately food ucts (Andersen & Mortensen, 2008; Ayala et al., 2020). Among those, properties. It is considered as a potential method that meets the food Trp is one of the most studied markers to monitor changes in proteins qualification requirements since the conventional methods are time- since it has a considerable quantum yield contributing to higher in­ consuming, relatively expensive, labor-intensive, and cannot be used tensity of fluorescence emission (James & Patrik, 2001). Thus, it could for on-line or in-line monitoring (Babu et al., 2018). However, FFFS can give relevant information on the thermal treatment of milk, a funda­ overcome these inconveniences and provides abundant information mental step in manufacturing dairy products to improve microbiological with only one test without destructing or altering the products. More­ quality and extend the shelf life. Several studies have adopted the FFFS over, it can be implemented as a predictive technique for identifying the to investigate the effect of thermal treatment on the milk by analyzing structural and compositional changes (Taterka, 2016) and quantifying the fluorescence spectrum of Trp (Dufour & Riaublanc, 1997; Ayala compounds of interest (Alvarado et al., 2019; Ayala et al., 2017; Liu et al., 2020; Hougaard et al., 2013; Kulmyrzaev et al., 2005; Scham­ et al., 2018) by detecting the deviation of the emission spectrum of berger & Labuza, 2006; Taterka, 2016). Specifically, FFFS at an excita­ fluorophores, since each of them presents a unique excitation and tion wavelength of 290 nm has been corroborated as a potential method emission spectrum, resulting from different treatments or storage con­ for predicting the concentration of undenatured whey protein (UWP) of ditions (Shaikh & O’Donnell, 2017). milk by quantifying the intensity of the peak at 340 nm (Taterka, 2016). This technology is being studied for dairy products since milk However, the degree of heat treatment must be sufficient to induce * Corresponding authors. Departament de ciència animal i dels aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Travessera dels Turons, Edifici V, 08193, Bellaterra (Cerdanyola del Vallès), Barcelona, Spain. E-mail addresses: anna.zamora@uab.cat (A. Zamora), manuel.castillo@uab.es (M. Castillo). https://doi.org/10.1016/j.lwt.2022.113589 Received 27 September 2021; Received in revised form 29 April 2022; Accepted 20 May 2022 Available online 30 May 2022 0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Y.T. Teng et al. LWT 163 (2022) 113589 considerable conformational changes to modify the fluorescent response 2.2. Sample preparation through a shift on the emission wavelength or a change in the maximum fluorescence intensity. For instance, Ayala et al. (2020) started to 2.2.1. Heat treatment observe changes in fluorescence intensity of Trp once the milk under­ Milk was reconstituted with 12% (w/w) low-heat skim milk powder went 80 ◦ C treatments. and 5% (w/w) WPI by dissolving in 40 ◦ C distilled water and stirring Whey is a fluid by-product produced from the precipitation of pro­ with the aid of a stir bar until entirely mixed and rapidly cooled to 20 ◦ C. teins in milk (Kilara & Vaghela, 2004). Whey proteins are widely used in Then, it was covered with aluminum foil to avoid light-induced oxida­ the food industry due to their excellent nutritional value and versatile tion and left at room temperature for 30 min to ensure full hydration. functional properties (Xu et al., 2019). Therefore, several studies have The reconstituted milk was heated inside steal sealed tubes in a water been intended to identify the structure and functional properties of whey bath at 80 ◦ C for seven holding times: 0, 5, 10, 15, 20, 25 and 30 min. to improve its utilization (Jiang et al., 2018). Heat treatments are The holding times were randomly selected. compulsory processes in dairy manufacturing, and the consequent modifications may have not only desirable but also undesirable impacts 2.2.2. Whey separation on the techno-functional properties of whey (Nunes & Tavares, 2019), Whey separation was performed following a method based on those depending on the temperature and the exposure time to the treatment of Nishanthi et al. (2017) and Bertrand-Harb et al. (2002). After the heat (Nishanthi et al., 2017). In general, whey proteins start to denature at treatment, the pH of the milk was adjusted to 4.5, adding 37% w/w HCl approximately 60 ◦ C. However, the substantial conformational changes as needed with continuous stirring for casein precipitation and centri­ occur at above 80 ◦ C in that they lose their globular structures, exposing fuged at 10,000×g (Sigma Laboratory Centrifuges 4K15, Osterode am the thiol groups and interacting with other whey proteins or casein. Harz, Germany) for 20 min at 20 ◦ C. The supernatant was filtered As indicated above, the denaturation of whey proteins resulting from through a PRAT DUMAS A125210 filter to obtain whey. thermal treatment affects the functional properties (Anema, 2020). To be specific, heating is a prerequisite for foaming properties (Devilbiss et al., 1975) because a partial denaturation leads to molecular rear­ 2.3. Front-face fluorescence spectroscopy of whey rangements and a concomitant high viscosity surface for better stability. On the contrary, emulsifying ability decreases by increasing the thermal Fluorescence measurements of whey samples were collected using in effect due to lower content of native proteins. For gel-forming proper­ duplicate a fluorescence spectrophotometer (Cary Eclipse Fluorescence ties, heating significantly diminishes the gel strength, first due to the Spectrophotometer, Agilent Technologies, Madrid, Spain) equipped denaturation, which provokes less available binding sites for molecular with 15 W lamp “press Xenon lamp” and a “front-face” geometry interaction, and second, to the aggregation that facilitates the formation accessory at 35◦ (Solid Sample Holder Accessory and Cuvette Kit, Agi­ of particulate gel, which is weaker than the fine-stranded gel (Chime lent Technologies). The emission spectrum of tryptophan was acquired et al., 2009). between 300 and 450 nm, with the excitation wavelength set at 290 nm As mentioned before, there are several studies of the fluorescence of (Ayala et al., 2020). The maximum intensity (ITrp) and the emission Trp in milk, however there are very few that applied fluorescence wavelength corresponding to the maximum intensity of Trp (WTrp) were spectroscopy directly in whey. MurilloPulgarín, Alañón, and Alañón collected to detect the shift and intensity change. (2005) presented the fluorescence characteristic of several wheys, where milk was subjected to different heat treatments, showing the full spec­ 2.4. Total protein content of whey trum (with excitation and emission ranges of about 220–320 nm and 260–435 nm, respectively) and remarking that tyrosine and Trp are Total protein content of whey (TWP in % w/w) was analyzed in responsible for the main intrinsic fluorescence of whey in that area. Trp duplicate by the Dumas combustion method (IDF, 2002). Approximately fluorescence on whey was also used by Liu et al. (2017) as a detection 0.5 ml of whey solution obtained as described in section 2.2 above was index to evaluate the adulteration of milk with milk powder. weighted and dried overnight in capsules before analysis with a Leco However, to the best of our knowledge, no studies linking both FFFS analyzer (Leco Corporation, St. Joseph, MI). Total protein content of and functional properties of whey have been found in literature. whey was used to normalize functional parameters. Therefore, the present study aimed at evaluating the potential of FFFS of Trp to determine the functional properties of whey. Specifically, our objectives were to 1) study the fluorescence response of Trp in whey 2.5. Undenatured whey protein content of milk after milk exposure to a wide range of different heating times, 2) study the effect of heat treatment of milk on whey functionality, and 3) The UWP content of milk was determined following the standard generate models for predicting functional properties based on fluores­ procedure 15.134 documented in Standard Methods for the Examination cence response of Trp in whey. of Dairy Products (Hooi et al., 2004). Milk samples were diluted with distilled water in a ratio of 1:2. Then, 22 ml of the dilution was mixed 2. Materials and methods with 8 g NaCl in the test tubes and placed in a water bath at 37 ◦ C for 30 min. During the first 15 min, for complete saturation of the diluted milk 2.1. Materials with NaCl, the test tubes were shaken every 1.5 min outside the heating bath, and the next 15 min were kept inside the bath constantly. Without Low-heat skim milk powder was chosen to minimize previous whey cooling, the solution was filtered through a Whatman 602H filter. denaturation during the manufacturing process. The standardized Collected 1-ml filtrates were diluted with 10 ml of saturated NaCl so­ skimmed milk powder (low-heat, spray-dried skim milk powder; pH = lution and mixed slowly inverting thrice in a test tube. Then, two drops 6.5, solubility = 99%, WPNI ≥7 mg/g, 800 cfu/g) was supplied by Chr. of HCl solution were added to generate the turbidity and mixed slowly Hansen SL (Barcelona, Spain). Whey protein isolate (WPI) (protein inverting thrice again. The mixed solution was left stand 5–10 min and content >95%, HTST-pasteurized, pH = 6.5, 10,000 cfu/g) was pur­ gently inverted twice before being pipetted to the cuvette. The spectra of chased from Lactalis ingredients (Bourgbarré, France). All other chem­ the samples were collected using a UV–Vis spectrophotometer (Dinko icals used were of analytical grade from either PanReac AppliChem Model UV 4000 spectrophotometer, Barcelona, Spain) with the wave­ (Barcelona, Spain) or Sigma-Aldrich (Merck KGaA, Darmstadt, length set at 420 nm. Then, the percentage of UWP (w/w) was calculated Germany). with a standard curve. The extraction was performed in duplicates and measurements in triplicates. 2 Y.T. Teng et al. LWT 163 (2022) 113589 2.6. Functional properties 2.7. Statistical analysis For the evaluation of the functional properties, whey samples were The complete experiment was replicated four times, except for the stored at 4 ◦ C overnight and warmed to 20 ◦ C before the assessments. EAI, which was repeated twice. Analysis of variance (ANOVA), taking as The emulsifying activity index and the emulsion stability index were factors heat treatment time, replica and their interaction, and Pearson’s measured on the day that whey was obtained. correlations were performed with Statgraphics Centurion XVI software (version 16.1.03, 2010, Statgraphics Technologies, Inc., The Plains, 2.6.1. Foaming properties Virginia, USA). LSD test was carried out for means comparison and Foaming properties, i.e., overrun and foam stability, were evaluated evaluations were based on a significance level of P < 0.05. The predic­ in duplicate with modified methods of Patel and Kilara (1990) and tive models were obtained using the maximum R2 procedure (REG, Jambrak et al. (2008). 40 ml whey sample aliquots were whipped at MAXR) of SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). room temperature with an electric blender (Silvercrest SHMS 300 A1, Kompernass GMBH, Germany) equipped with a wire whip beater at 3. Results and discussions maximum speed for 5 min. Immediately after, 40 ml of foam was weighted. Foam overrun (%) was calculated by the following equation: 3.1. Effects of heat on whey proteins wt. of solution – wt. of foam Overrun (%) = wt. of foam × 100 The percentage of UWP of milk as a function of the heat treatment is presented in Table 1. A significant decrease of UWP (P < 0.05) was where wt. is weight. observed as heating time increased; the more intensive the thermal Foam stability was quantified by transferring a constant volume of treatment was, the greater the denaturation. Indeed, Lamb et al. (2013) the foam to a funnel with glass wool placed in the top of the stem, which observed that UWP followed a first-order response to heating time at retained the foam but allowed drainage. The time taken for the whole 80 ◦ C detected by differential scanning calorimetry. Specifically, in the foam mass to drain was taken as a measurement of foam stability. present study, the concentration of UWP declined dramatically by Both overrun and drainage time were normalized dividing the ob­ ~41% in whey obtained from milk heated for 5 min at 80 ◦ C. tained data by the total protein content of whey. In general, heating the whey proteins above 70 ◦ C even for a short period of time, especially α-Lactalbumin (α-La) and β- Lactoglobulin 2.6.2. Gel-forming properties (β-Lg), two major whey proteins that account for 80% of total whey Gel-forming properties were evaluated in triplicate with modified nitrogen (Farrell et al., 2004; Edwards & Jameson, 2020), is sufficient to method of Luck et al. (2013). 10 g of whey protein solution was heated cause irreversible denaturation (Edwards & Jameson, 2020; Anema, inside the 15 ml centrifuge tubes in the water bath set at 90 ◦ C for 1 h. 2020). Heat induces the exposure of reactive thiol groups of β-Lg, which After gel formation, the sample was centrifuged at 2000 ×g (Sigma were initially buried within the whey protein structure, and allow them Laboratory Centrifuges 4K15) for 10 min at 20 ◦ C. The weight of the to interact with κ-casein or with other denatured β-Lg (Anema, 2020). As pellet was used as a gel-forming index. to the α-La, it is believed that there are no direct interactions between α-La and caseins, except for some particular conditions, such as a high 2.6.3. Emulsifying properties pH environment (Doi et al., 1983). Normally, denatured α-La reacts with The oil-in-water emulsions were prepared by homogenizing 25% of β-Lg first, and the formed complex then interacts with κ-casein (Anema, sunflower oil (8.3 g) and 75% of (25 g) whey with ~0.5 ml silicone for 2020). Subsequently, due to the isoelectric precipitation of casein mi­ preventing the foam formation during emulsification using a homoge­ celles, some denatured whey proteins precipitated with caseins. As a nizer (Heidolph homogenizer DIAX 900, Merck KGaA, Darmstadt, Ger­ result, the concentration of TWP decreased as heating time increased as many) at 15,200 rpm for 5 min. An emulsion aliquot of 20 ml was well (Table 1). In the present study, 80 ◦ C was applied and the con­ transferred gently to a turbiscan tube and measured by Turbiscan centration of UWP and TWP differed significantly (P < 0.05) among the (Turbiscan Lab Expert, Formulaction, Toulouse, France) for 30 min seven holding times, and according to Pearson’s test, a strong positive detecting every 1 min. The slope of Turbiscan stability index (TSI) as a correlation between the concentrations of UWP and TWP was observed function of time from the bottom of the tubes was measured. The whole (r = 0.99, P < 0.0001). analysis was performed in duplicate. TSI values were normalized dividing the obtained data by the total protein content of whey. 3.2. Effect of heat on FFFS response of Trp For the emulsifying activity index (EAI), the method of Pearce and Kinsella (1978) was adopted. EAI represents the amount of oil that can Heat induced a redshift and a decrease of Trp fluorescence intensity be emulsified per unit of protein (Boye et al., 2010). This index was measured in duplicate. The oil-in-water emulsions were prepared by homogenizing 25% of sunflower oil (8 g) and 75% of diluted whey (1:10 Table 1 Effect of heat treatment at 80 ◦ C on the concentration of total protein of whey dilution, 24 ml) with a homogenizer (Heidolph homogenizer DIAX 900, and undenatured whey protein of milk, and front-face fluorescence response of Merck KGaA) at 15,200 rpm for 5 min 100 μl of the emulsions were tryptophan. taken immediately (0 min) from the bottom of the emulsion and added Time (min) TWP (%) UWP (%) WTrp (nm) ITrp (a.u.) into 10 ml of 0.1% (w/v) SDS solution. After mixing the solution by inverting 5 times, the absorbance of the diluted emulsions was measured 0 4.20 ± 0.04a 2.93 ± 0.13a 333.91 ± 0.74b 74.5 ± 5.83a at 500 nm on a UV–Vis spectrophotometer (Dinko Model UV 4000). 5 2.20 ± 0.11b 1.74 ± 0.11b 333.66 ± 0.62b 65.3 ± 5.52b 10 1.83 ± 0.07c 1.42 ± 0.08c 333.15 ± 1.65b 60.8 ± 2.02c The EAI values were calculated with the following equation: 15 1.58 ± 0.04d 1.27 ± 0.09d 335.28 ± 1.26a 59.6 ± 5.07c / 2 × 2.303 × A0 × D 20 1.45 ± 0.03e 1.11 ± 0.04e 335.26 ± 1.06a 55.7 ± 3.14d EAI(m2 g) = , 25 1.33 ± 0.04f 1.02 ± 0.03f 336.01 ± 1.46a 55.2 ± 2.21d c × φ 30 1.25 ± 0.05f 0.97 ± 0.05g 336.53 ± 0.82a 52.3 ± 1.57d where A0 is the initial absorbance (0 min), D is the dilution factor (0.1), c Mean value ± s.d.; n = 54 for UWP, n = 56 for TWP, n = 55 for WTrp and ITrp; is the initial protein content (g), and φ is the volume fraction of oil in the TWP: total protein content of whey; UWP: the concentration of undenaturated emulsion (0.25). whey protein in milk; ITrp: the maximum intensity of tryptophan; WTrp: the emission wavelength corresponding to the maximum intensity of tryptophan. a-g: values per column with different letter were significantly different (P < 0.05). 3 Y.T. Teng et al. LWT 163 (2022) 113589 (Table 1 & Fig. 1). Without heat treatment, the emission spectra of whey 3.3. Effects of heat on functional properties exhibited a Trp maximum emission wavelength at around 333.9 nm, whereas for the sample subjected to thermal treatment at 80 ◦ C for 30 3.3.1. Effects of heat on foaming properties min, it increased to approximately 336.5 nm. Significant differences The effect of heating milk at seven holding times on the foaming were observed only between two groups, those with a heat treatment behavior of whey was investigated. Table 2 illustrates that foams pro­ ≤10 min and those ≥15 min. As to the peak intensity, a significant duced using unheated samples presented the statistically lowest overrun decline was observed as holding heat time increased, dropping from compared with all the whey samples obtained from heated milk, while 74.5 to 52.3 a. u (i.e. a 30% decrease). Accordingly, Ayala et al. (2020) as the heating time increased improvement in the foaming capacity was found that the redshift in milk, followed a quadratic trend with tem­ observed. The obtained results are in accordance with other literature peratures from 70 to 100 ◦ C as a function of holding time (from 0 to 60 data (Bals & Kulozik, 2003; Chime et al., 2009) indicating that a partial min). The redshift could be due to whey protein denaturation caused by denaturation favored the foamability. Devilbiss et al. (1975) stated that the heat since Trp residues become exposed and move from a nonpolar heating is a prerequisite for foaming. Prolonged heating time posed a (folded) to a polar (unfolded) environment; in contrast, proteins that detrimental impact on foaming capacity due to further aggregation and embed into a micelle may result in a blueshift (Caputo & London, 2003). polymerization of whey proteins resulting in less available binding sites Different types of whey proteins possess distinct thermal susceptibility. for protein and air interaction. However, unlike the present study, some Among whey proteins, α-La is generally regarded as one of the most studies demonstrated that unheated whey had better foamability heat-labile whey proteins, whereas β-Lg is one of the most heat-stable (Phillips et al., 1990; Davis & Foegeding, 2004) assuming that native whey proteins (Edwards & Jameson, 2020), but some researchers protein can absorb to the interface more rapidly, which consequently stated that bovine serum albumin and immunoglobulin possess higher contribute to the foamability; and Nicorescu et al. (2009) even showed heat stability (Edwards & Jameson, 2020; Mainer et al., 1997). How­ no difference in overrun between native and heated whey. ever, under the absence of milk fat, all whey proteins denature at 80 ◦ C Foam stability assessed by the time required for the whole foam to (de Wit et al., 1983). Moreover, Trp residues are influenced by their proximity to quenching compounds, resulting in a decrease of fluores­ Table 2 cence intensity (Taterka, 2016). For instance, β-Lg, the most abundant Effect of heat treatment at 80 ◦ C on overrun and drainage time of foam. whey protein, which contains two Trp residues, Trp19 and Trp61, heat prompts the redshift due to the Trp19 exposure from the cavity of β-Lg Time Ov (%) Ov/TWP (% g− 1) tDrain (s) tDrain/TWP (s (min) g− 1) and the decrease of maximum fluorescence intensity is due to the combination of Trp61 and disulfide bond or the proximity of Trp19 and 0 667 ± 94b 15,873 ± 2,180e 461 ± 25c 10,980 ± 657d Arginine124 (Manderson et al., 1999). 5 945 ± 232a 42,684 ± 8,737d 598 ± 126b, 26,650 ± 5,058c c Concerning Pearson’s correlations, the emission wavelength of Trp 10 892 ± 197a 48,839 ± 586 ± 187b, 31,493 ± 8,912c corresponding to the maximum intensity was weakly and negatively 11,244c,d c correlated with UWP (r = − 0.47, 0.01 ≤ P < 0.05) and TWP (r = − 0.43, 15 913 ± 85a 57,590 ± 4,446b, 564 ± 160b, 35,309 ± c c 0.01 ≤ P < 0.05). Redshift at 80 ◦ C occurs only above 15 min and 10,393c a,b b, 20 762 ± 58 52,565 ± 3,750 1,125 ± 77,231 ± presents values of about 2–3 nm provoking the weak correlation with c,d 606a 42,394a both UWP and TWP. At higher temperatures, i.e., 90 and 100 ◦ C, the 25 969 ± 137a 73,270 ± 690 ± 47b 51,782 ± 4,919b emission wavelength moves up to 14 nm (Ayala et al., 2020). In addi­ 12,193a tion, the peak intensity of Trp was strongly and positively correlated 30 819 ± 65,282 ± 967 ± 387a 76,835 ± with the concentration of UWP (r = 0.87, P < 0.0001), as well as that of 232a,b 16,756a,b 32,591a TWP (r = 0.85, P < 0.0001). Mean value ± s.d.; n = 56 for overrun, n = 42 for drainage time. Ov: Overrun of foam; Ov/TWP: Normalized overrun taking into account the total whey protein content; tDrain: Drainage time; tDrain/TWP: Normalized drainage time taking into account the total whey protein content. a-e: values per column with different letter were significantly different (P < 0.05). Fig. 1. Fluorescence spectra of tryptophan, corresponding to whey samples obtained from milk heat-treated at 80 ◦ C for different holding times (0–30 min). 4 Y.T. Teng et al. LWT 163 (2022) 113589 drain is shown in Table 2. The worst stability occurred in whey from Table 3 unheated milk, and as the heating time increased, the tendency of Effect of heat treatment at 80 ◦ C on gel formation and emulsifying properties of greater foam stability was observed. Although sample collected from whey. milk heated for 20 min demonstrated the best stability, and no statistical Time Gel (%) TSI (a.u. × TSI/TWP (a.u. EAI (m2 g− 1) difference among heat treatment for 5, 10, and 15 min was observed. (min) 100) g− 1) Richert (1979) also revealed a similar result; with appropriate thermal 0 51.67 ± 0.26 ± 0.05b 0.06 ± 0.01e 1.35 ± 0.01f treatment the foam stability was enhanced, but the excessive heating 19.76a (80 ◦ C, 30 min, pH 7) resulted in an adverse effect. The increase of 5 25.28 ± 0.40 ± 0.05a 0.18 ± 0.03d 2.05 ± 0.00e 7.45b viscosity caused by a previous heat-treatment is one of the major factors 10 21.01 ± 4.62c 0.40 ± 0.03a 0.22 ± 0.01c 2.33 ± 0.15d that improve foam stability (Davis & Foegeding, 2004), since higher 15 16.02 ± 0.38 ± 0.04a 0.24 ± 0.03c 2.73 ± 0.06c viscosity limits the mobility of the aqueous phase which surrounds the 2.22d air bubbles, which consequently enhances the ability of foam against 20 14.69 ± 2.15e 0.42 ± 0.05a 0.29 ± 0.04b 2.83 ± drainage (Wang, 2013). Nevertheless, some studies illustrated 0.03b,c 25 11.21 ± 1.23f 0.40 ± 0.02a 0.30 ± 0.02a,b 3.03 ± conversely that the denaturation and aggregation resulted from the heat 0.17a,b diminished the foam stability (Bals et al., 2003; Chime et al., 2009). 30 9.22 ± 2.95g 0.39 ± 0.03a 0.31 ± 0.01a 3.10 ± 0.37a The inconsistent observations of foaming properties between current Mean value ± s.d.; n = 83 for gel-forming index; n = 56 for TSI; n = 28 for EAI. and previous studies may be due to differences in the temperature of the Gel: gel-forming index; EAI: emulsifying activity index; TSI: Turbiscan stability treatment that resulted in different denaturation levels, the pH value, index; TSI/TWP: normalized TSI taking into account the total whey protein and the whey protein content. Phillips et al. (1990) pointed out that the content. a− g: values per column with different letter were significantly different pH strongly influences the foaming properties. In our case, the pH was (P < 0.05). lower than 4.6, since separation of whey was achieved through precip­ itation of caseins by lowering the pH down to their isoelectric point, particulate gels, which are the aggregation of whey prior to the gel which resulted in reduced foamability since proteins were more stable to formation due to the denaturation arising from the heat treatment against the unfolding for foam generation. Lee et al. (1992) and Chime (Foegeding et al., 1998). The whey protein content plays a critical role in et al. (2009) also demonstrated at different pH, the foaming properties gel strength as well. Lorenzen and Schrader (2006) illustrated that as the differed. As to the whey protein content, in the present study, it whey percentage increased, the gel strength increased significantly, decreased as heating time increased (Table 1), unlike other reports that which was also confirmed in the present study with the concentration of had a constant amount, since the heat treatment was applied to the milk TWP data (Table 1). Consistently, Pearson’s test showed that the and some denatured whey precipitated with casein during the isoelectric gel-forming properties significantly correlated with the concentration of precipitation. In general, higher protein content would have a better TWP (r = 0.87, P < 0.0001). foamability, since more whey proteins can interact with air, as well as a better foam stability which is attributed to the increased thickness of the 3.3.3. Effects of heat on emulsifying properties film surrounding air bubbles (Xiong et al., 2020). The slope of Turbiscan stability index (TSI) from the bottom part of According to Pearson’s test, non-normalized overrun showed no the tube is displayed in Table 3. The higher value indicates less stability, significant correlation (P ≥ 0.05) neither with UWP nor with TWP. because this index represents the phase separation as a function of time. Concerning drainage time, it showed a weak and negative correlation All the whey samples obtained from heated milk had a significantly with UWP (r = − 0.44, 0.01 ≤ P < 0.05), but no correlation (P ≥ 0.05) higher TSI than whey from non-heated milk (P < 0.05), but no statistical with TWP. Denaturation increased simultaneously with decreasing differences among them were observed. This fact may be attributed to whey protein amount, which compensated the effect of decreasing whey the total protein content of all samples from heated milk which might protein amount on foaming properties, since both factors oppositely have been insufficient to cover the oil droplets efficiently. Previous affected the foaming properties. Therefore, for a better understanding of studies investigated the effect of heat on whey directly instead of on the effect of denaturation on foaming properties, the overrun and the milk, which merely altered the denaturation level, but not protein drainage time had been normalized by the total protein content of whey, amount (El-Shibiny et al., 2007; Ghanimah & Ibrahim, 2018; Patel & and the outcomes became more statistically different among the seven Kilara, 1990). In the present study, although the oil to whey ratio used holding times (Table 2). The normalized overrun illustrated a strong and was the same as other studies, the protein amount reduced significantly negative correlation with UWP (r = − 0.85, P < 0.0001) and TWP (r = (Table 1) due to the thermal treatment since the whey samples applied − 0.85, P < 0.0001). Similarly, strong and negative correlations between were obtained after the isoelectric precipitation of caseins (pH 4.6) from normalized drainage time and UWP (r = − 0.65, 0.001 ≤ P < 0.01) as the heated milk. Therefore, comparing with other research, the oil to well as drainage time and TWP (r = − 0.61, 0.001 ≤ P < 0.01) were whey protein ratio was not kept constant. However, in general, partial demonstrated. denaturation is conducive to the emulsifying stability, but in the meanwhile, the decrease of the concentration of TWP posed an adverse 3.3.2. Effects of heat on gel-forming properties impact, which had been confirmed by Jiang et al. (2018) demonstrating Milk heating significantly deteriorated the gel formation (Table 3). that WPI had significantly better emulsifying stability than whey protein The longer the heating time, the lesser the gel strength, due to greater concentrate (WPC). denaturation of whey, especially β-Lg, the most relevant whey protein According to Pearson’s test, the slope of TSI negatively correlated for gelation (Mulvihill & Donovan, 1987). Therefore, a positive and with both UWP (r = − 0.72, P < 0.0001) and TWP (r = − 0.64, 0.0001 ≤ statistically significant correlation between gel-forming parameter and P < 0.001). In order to take only the denaturation into account, TSI had the concentration of UWP (r = 0.87, P < 0.0001) was observed. Besides, been normalized with the protein content, and a tendency for TSI to a pronounced drop of gel formation took place between samples ob­ increase was observed, in other words, a decrease in emulsifying sta­ tained from unheated milk and milk heated for 5 min, while further heat bility with increasing holding time was found. After normalization, treatment led to smaller reduction of the gel-forming parameter, which higher correlation coefficients were obtained (r = − 0.95 with P < is in agreement with Hongsprabhas and Barbut (1996) and Chime et al. 0.0001 for TWP and r = − 0.94 with P < 0.0001 for UWP). However, this (2009). Extending heating time resulted in further denaturation that in observation differed from Jiang et al. (2018), who found better emul­ turn increased the protein insolubility (Kilara & Mangino, 1991), which sifying stability after heating due to a greater zeta potential, that facilitated the formation of particulate gel (Veith & Reynolds, 2004). generated a larger electrostatic repulsion to retard the coalescence and The weak gel strength is strongly influenced by the presence of 5 Y.T. Teng et al. LWT 163 (2022) 113589 creaming, while Raikos (2010) stated that further denaturation caused significant with P ≤ 0.001, and those models with the higher R2 were large aggregation of whey proteins, resulting in the inefficiency in model II with both WTrp and ITrp as predictors, which were considered as covering the fat droplets, which explained the decreasing of emulsion better models. However, only the models for TSI and EAI had a R2 higher stability in the current study. than 0.8 and 0.9, respectively, which indicates that applying merely one The EAI significantly increased by prolonging the heating time fluorescent marker was not enough to generate a robust predictive (Table 3), and whey obtained from unheated milk possessed the lowest model. For instance, Ayala (2018) required four fluorescent compounds emulsifying capacity. This observation agreed with Dissanayake and in order to predict successfully the concentration of lactulose and Vasiljevic (2009) and Farrag et al. (2016), who also displayed an furosine in milk, and Babu and Amamcharla (2018) obtained a good R2 improvement of EAI once the samples had been heated, since the for predicting the solubility of whey protein concentrate with FFFS of denaturation made the hidden hydrophobic groups in the globular Trp and Maillard products. Not to mention, in the present study, pre­ proteins to expose (Harper, 1992). However, some researchers stated dicting the functional properties is more challenging since their per­ differently (Chime et al., 2009; Jiang et al., 2018), that the denaturation formances are based on many factors, not only on the concentration of caused by heat impairs the emulsifying ability. In general, heat would UWP and TWP. lead to a decrease in emulsifying capacity due to irreversible protein denaturation, but a partial protein unfolding may enhance the interfa­ 4. Conclusions cial properties and accordingly improves the emulsifying ability, which explains the results of the current study (Phillips et al., 1990). Since whey functional properties were distinctively affected by Furthermore, the protein content influenced the emulsifying ability as different thermal treatments and front-face fluorescence spectroscopy well since whey proteins acted as emulsifiers, that adsorbed at the sur­ detected differences in tryptophan response, significant correlations face of the newly formed oil droplets (Singh & Ye, 2020). Thus, WPI between tryptophan fluorescence and functional properties of whey presented significantly higher EAI than WPC after the heat treatment at were observed. 80 ◦ C (Jiang et al., 2018). As to the predictive models, which did not present a sufficient determination coefficient for predicting the functional properties 3.4. Prediction of whey protein concentration and functional properties robustly, it is suggested to exploit more fluorescence markers in order to obtain reliable predictive models since front-face fluorescence spec­ A great number of correlations were observed between Trp fluores­ troscopy is a useful, non-invasive, rapid, and relatively cheap technique. cence and functional parameters (see Appendix), which indicated that Furthermore, owing to the feasibility of measuring turbid samples with the two Trp fluorescence parameters may have the potential to be used front-face fluorescence spectroscopy, it may be interesting to investigate as predictors. the front-face fluorescence response of the whey powder directly, which On one hand, the emission wavelength corresponding to maximum would be more convenient for commercial utilization. fluorescence intensity of Trp correlated with normalized drainage time Finally, due to the advantages of front-face fluorescence spectros­ (r = 0.54, 0.01 ≤ P < 0.05), gel formation (r = − 0.44, 0.01 ≤ P < 0.05), copy and the increasing popularity of whey application by the industries EAI (r = 0.61, 0.01 ≤ P < 0.05), and normalized TSI (r = 0.53, 0.001 ≤ P due to its functional properties, further research studying the prediction < 0.01). On the other, the maximum intensity of Trp correlated with of functional properties of whey applying front-face fluorescence tech­ normalized overrun (r = − 0.78, P < 0.0001), drainage (r = − 0.58, nology with more fluorescence markers is valuable. 0.0001 ≤ P < 0.001, and r = − 0.74, 0.0001 ≤ P < 0.001, for non- and normalized data respectively), gel-forming properties (r = 0.79, P < Funding sources 0.0001), and TSI (r = − 0.65, 0.0001 ≤ P < 0.001, and r = − 0.89, P < 0.0001, for non- and normalized data respectively). This research was partially supported by Secretaría de Educación Since Trp fluorescence gave two parameters, two predictive models Superior, Ciencia, Tecnología e Innovación (SENESCYT, Ecuador). were obtained for each variable (Table 4). All models were strongly Table 4 Models for the prediction of undenatured whey protein concentration of milk, total whey protein content and functional properties with front-face fluorescence response of tryptophan. Model β0 β1 β2 R2 SEP CV I*** UWP = β0 + β1ITrp − 2.82*** 0.07*** 0.75 0.33 21.92 II*** UWP = β0 + β1ITrp + β2WTrp 7.13 0.07*** − 0.05 0.76 0.33 22.18 I*** TWP = β0 + β1ITrp − 4.40*** 0.11*** 0.73 0.52 26.12 II*** TWP = β0 + β1ITrp + β2WTrp 1.66 0.10*** − 0.02 0.73 0.53 26.60 I*** Ov/TWP = β0 + β1ITrp 166,121*** − 1,904*** 0.60 12,572 24.71 II*** Ov/TWP = β0 + β1ITrp + β2WTrp − 489,586 − 1,721*** 1,925 0.62 12,522 24.62 I*** tDrain/TWP = β0 + β1ITrp 228,952*** − 3,047*** 0.55 20,534 46.33 II*** tDrain/TWP = β0 + β1ITrp + β2WTrp − 1,446,857 − 2,565*** 4,918 0.59 20,042 45.22 I*** Gel = β0 + β1ITrp − 72.96*** 1.56*** 0.63 9.61 45.01 II*** Gel = β0 + β1ITrp + β2WTrp 168.98 1.49*** − 0.71 0.63 9.75 45.65 I*** TSI/TWP = β0 + β1ITrp 0.81*** − 0.01*** 0.79 0.04 17.29 II*** TSI/TWP = β0 + β1ITrp + β2WTrp − 1.64 − 0.010*** 0.01 0.80 0.04 17.07 I*** EAI = β0 + β1ITrp 7.83*** − 0.09*** 0.90 0.20 7.17 II*** EAI = β0 + β1ITrp + β2WTrp − 2.95 − 0.09*** 0.03 0.90 0.21 7.39 n = 28 for UWP, TWP, Ov/TWP, Gel and TSI/TWP; n = 21 for tDrain/TWP; n = 14 for EAI. β0-2: regression coefficients; R2: determination coefficient; SEP: standard error of prediction (units correspond to those of predicted parameters); CV: coefficient of variation (%); ITrp: maximum fluorescence intensity of tryptophan (a.u.); WTrp: emission wavelength corresponding to maximum fluorescence intensity of tryptophan (nm); UWP: undenatured whey protein of milk (%); TWP: total protein content of whey (%); Ov/TWP: normalized overrun taking into account the total whey protein content (% g− 1); tDrain/TWP: normalized drainage time taking into account the total whey protein content (s g− 1); Gel: gel-forming index (%); TSI/TWP: normalized turbiscan stability index taking into account the total whey protein content (a.u. g− 1); EAI: emulsifying activity index (m2 g− 1). 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