# Coupled bulk and surface mechanisms lead to whey protein deposits under shear

Margot Grostete, Jeehyun Lee, Françoise Boissel, Maude Jimenez, Romain

Jeantet, Luca Lanotte

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Margot Grostete, Jeehyun Lee, Françoise Boissel, Maude Jimenez, Romain Jeantet, et al.. Coupled bulk and surface mechanisms lead to whey protein deposits under shear. International Journal of Biological Macromolecules, 2026, 340, 10.1016/j.ijbiomac.2026.150196. hal-05462472

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# Coupled bulk and surface mechanisms lead to whey protein deposits under shear

Margot Grostete<sup>a</sup>, Jeehyun Lee<sup>a</sup>, Françoise Boissel<sup>a</sup>, Maude Jimenez<sup>b,c</sup>, Romain Jeantet<sup>a</sup>, Luca Lanotte<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Fouling

Microscopy

Rheology

Denaturation

Interface

Aggregation

## ABSTRACT

Understanding the mechanisms behind fouling deposit formation remains an unsolved challenge for the dairy industry. While the effect of temperature is well-established, few studies have explored the impact of shear rate, a key parameter in dairy processing. Therefore, this study investigates the growth of surficial deposits and bulk aggregates originating from whey protein solutions under shear. Whey protein solutions with different concentrations (5–12 wt%) were sheared at fixed rates (0–175 s⁻¹), using a cone-and-plate rheometer, at 65 °C to minimize thermal denaturation.

Results show that the area covered by the deposits increases by up to fivefold under shear, particularly in concentrated conditions, compared with that observed at rest. This is accompanied by the complexification of deposit morphology, from sparse globular clusters to branches structures interconnecting into gels at 12 wt%. In contrast, solution dynamics are mostly affected by protein concentration rather than shear, resulting in the formation of submicronic aggregates. Two interpretive hypotheses are discussed: i) heat favors whey protein denaturation and the formation of aggregates increasingly colliding with the surface under shear and ii) active surface deposits, formed by these collisions, interconnect with flowing aggregates, particularly at high protein concentrations. The examination of surface and bulk dynamics presented in this study provides deeper insights into fouling mechanisms. They offer potential applications in other areas where protein denaturation/aggregation plays a crucial role.

## 1. Introduction

Fouling is frequently encountered in food processing [1–3] and consists in the accumulation of solid deposits on a surface during the flow of liquid streams. This phenomenon leads to several drawbacks such as higher pressure drops, reduced transfer efficiency and increased risks of biofilm development. Thus, costly and time-consuming cleaning sessions are required to restore the initial efficiency of the equipment.

In the dairy industry, fouling occurs during heat treatments. The deposits observed in the equipment are mainly composed of whey proteins (WP) and minerals, whose relative ratio strongly depends on the operating conditions [4]. Most of the studies in the literature investigated the driving forces governing deposit formation in heat exchangers (i.e., at temperatures higher than 80 °C), typically used for

pasteurization [3,5]. The results revealed how fouling is linked to the thermal denaturation of WPs under such operating conditions. Above ≈ 70 °C, these major components of milk's protein fraction unfold and aggregate before adsorbing onto equipment surfaces [4,6–8]. These findings highlight the predominant influence of temperature [5,9] compared to other parameters, such as physico-chemical characteristics of the products (e.g., protein/mineral concentration, pH) [5,10,11] and equipment surface properties [10,12].

Based on these conclusions, no fouling should occur below WP denaturation temperature. However, the formation of dairy deposits is often observed even at relatively low temperatures. This is the case in falling film evaporators [13–15]. This equipment is used for the operation of vacuum concentration of the treated solutions and operates between 40 and 80 °C [16,17]. The rich literature on heat exchangers,

<footnote><sup>a</sup> INRAE, Institut Agro, STLO, 35042, Rennes, France</footnote>

<footnote><sup>b</sup> Unité Matériaux et Transformations (UMET) - UMR 8207, INRAE, 59651, Villeneuve d'Ascq, France</footnote>

<footnote><sup>c</sup> Institut Universitaire de France (IUF), 1 rue Descartes, 75231 cedex, Paris, France</footnote>

<footnote>* Corresponding author at: INRAE, Institut Agro, STLO, 35042, Rennes, France.</footnote>

<footnote>E-mail addresses: margot.grostete@institut-agro.fr (M. Grostete), jeehyun.lee@institut-agro.fr (J. Lee), francoise.boissel@institut-agro.fr (F. Boissel), maude.jimenez@univ-lille.fr (M. Jimenez), romain.jeantet@institut-agro.fr (R. Jeantet), luca.lanotte@inrae.fr (L. Lanotte).</footnote>
where temperature is the limiting factor, cannot be directly extrapolated to falling film evaporators. Consequently, very limited information is available for deposit growth under vacuum concentration conditions. Certainly, the observation of fouling development in falling film evaporators highlights that temperature is not the only parameter affecting protein denaturation/aggregation. This raises questions about the role of other parameters hitherto neglected, such as shear.

In this regard, there is no scientific consensus on the impact of shear on protein structural stability [18]. As it concerns WPs, some works argue that shear stress promotes their aggregation and, consequently, fouling deposit formation [19,20]. However, this effect is strongly mitigated at higher concentrations, where increased shear-driven collisions may result in aggregate break-up [9,21–24]. On the other hand, other works do not conclude on the shear effect [25–28]. A possible explanation for such a complex picture is that most of the mentioned studies were carried out at temperatures higher than that of WP denaturation, making it challenging to discriminate the effect of shear. Furthermore, current literature fails to clarify whether bulk (aggregation) and surface (deposit) dynamics are interconnected. Specifically, do proteins first unfold and aggregate in bulk before surface accumulation? Does protein denaturation directly occur at the surfaces, thus initiating fouling? What is the precise role of shear in these dynamics? Very few studies have distinguished between phenomena taking place in the bulk liquid stream and those occurring at the wall-liquid interface. From this point of view, the aggregation of WPs was mainly investigated in the bulk [29,30].

In a previous study [31], we developed an original methodology based on rheometry and microscopy to i) mimic the processing conditions (temperature, shear) of falling film evaporators and ii) study how shear rate influences WP surface deposit below their denaturation temperature. The use of a parallel-plate rheometer enabled the evaluation of WP deposits under a wide range of shear. According to the observations realized by microscopy, shear enhances the formation of WP deposits and their structure complexification with increasing protein concentration. This results in the gradual development of branched protein structures on the surface with hundred micrometers size. However, although highlighting the crucial role of shear on the growth of WP surficial deposit, this study does not dispel the doubts on its direct impact on protein denaturation and on the connected bulk/surface mechanisms.

Thanks to the optimization of the rheofluidic approach adopted in Grostete et al. (2024), [31], we aim at filling this gap with the present study. First, we will quantify the effect of shear on WP aggregation in the bulk and deposit formation at the wall-liquid interface. Then, based on the experimental results, we propose a hypothesis on interconnected bulk and surface mechanisms at the micron-scale leading to deposit formation and its growth.

# 2. Materials and methods

## 2.1. Sample preparation

Suspensions with 5, 10 and 12 wt% (pH = 6.55 ± 0.01) total WP concentrations were prepared by dissolving commercial WP isolate (WPI) powders (Pronativ 95, Lactalis, France) exhibiting 89 wt% protein in deionized water containing 0.02 wt% of sodium azide (NaN₃) as a bacteriostatic agent. The solutions were stirred at 20 °C for 12 h and then stored at 4 °C until use within 7 d.

## 2.2. Rheological tests

### 2.2.1. Shearing of whey protein solutions

Shear tests on the WP solutions were carried out using a rheometer (MCR 301, Anton Paar, Austria) equipped with a cone plate geometry (diameter, D = 50 mm; gap size, e = 0.21 mm) and a Peltier temperature controller (P-PTD 200, Anton Paar, Austria). A transparent glass disc

was used instead of the commercial stainless steel bottom plate to facilitate the observation by microscopy (Fig. 1). The experiments consisted in shearing the WP suspensions for 10 min at 65 °C for fixed values of the shear rate (0, 50, 100, 175 s⁻¹), followed by 5 min of cooling to 20 °C to prevent further protein denaturation. These processing conditions were chosen according to the protocol established in Grostete et al., (2024) [31]. In particular, 65 °C was selected to be slightly below the thermal denaturation threshold, enabling quantification of the shear effect on the formation of WP surficial deposits and/or aggregates in the solutions. This temperature was achieved in the system through a preheating step without shear, and the stability of the set-point temperature was checked by a temperature probe (fluctuations <0.15 °C). Three tests were performed for each concentration and each shear rate. A summary of the experimental conditions during the shear treatments is presented in Table 1. At the end of the treatment, the solutions were collected using a Pasteur pipette and stored in Eppendorf tubes. Then, the surface of the plates was rinsed with deionized water for 30 s on both sides to remove any aggregates sedimented from the solution. The plates were dried in an oven at 40 °C. Both solutions and glass discs were kept at 4 °C and analyzed offline within 24 h after the shear treatment.

### 2.2.2. Viscosity measurement

In parallel with the shearing experiments detailed in 2.2.1, the viscosity of WP solutions with different concentrations was monitored to characterize their rheological behavior under shear. Two tests were carried out:

i) measurements of the apparent viscosity throughout the shearing process at 65 °C for each fixed shear rate;

ii) flow sweep tests at 20 °C before (control) and after shearing at 65 °C to calculate the “zero shear” viscosity (η₀) as a function of the shear history. The cooling step to 20 °C after shearing at 65 °C was performed with a controlled duration of 3 min. The viscosity of WP solutions was monitored for shear rates increasing from 0 to 500 s⁻¹. Then, the Carreau model (Eq. 1), widely employed for non-Newtonian fluids with pseudoplastic behavior [32,33], was used to deduce the “zero shear” viscosity:

$$ \eta = \eta_{\infty} + (\eta_{0} - \eta_{\infty})[1 + (\lambda^{*}\dot{\gamma})^{2}]^{\frac{n-1}{2}} \quad (1) $$

With:

* η = measured apparent viscosity (Pa.s)

* η₀ = viscosity at zero shear rate (Pa.s)

* η∞ = viscosity limit at infinite (high) shear rate (Pa.s)

* λ = characteristic time

* n = behavior index

Three tests were carried out for each concentration and each shear rate γ. The model was estimated with the R-Studio 2023 06.0 software, using the minpack.lm package, nlsLM function, and ggplot 2.

## 2.3. Surface analysis

### 2.3.1. Digital microscopy

The surface of the glass plates was scanned using a Keyence digital microscope (VHX 7000 N, Keyence, Japan). The deposit number and shape were characterized as a function of the shear intensity. Ten images were acquired for each glass plate at a magnification of 500× (i.e., 30 images per concentration and shear rate), and successively analyzed by a custom image analysis software (Serie CV-x software). The surface density (Eq. 2), defined as the area occupied by the deposits normalized by the picture area, was chosen to quantify the evolution of WP deposits by number and size.

$$ \text{Surface density} = \frac{\sum \text{Deposit area}}{\text{Picture area}} \quad (2) $$

### 2.3.2. Atomic force microscopy

WP deposits obtained from the shear treatment of 10 wt% samples
<figure><img src="image_3.png" /><figcaption>Fig. 1. Experimental strategy to investigate the impact of shear on whey protein on the surface and in the bulk solution.</figcaption></figure>

<caption>**Table 1**</caption>

Experimental conditions and replicates for whey protein shear treatments (shearing time = 10 min; temperature = 65 °C).

<table><thead><tr><th rowspan="2">Shear rate (s<sup>-1</sup>)</th><th colspan="3">Protein concentration (wt%)</th></tr><tr><th>5</th><th>10</th><th>12</th></tr></thead><tbody><tr><td>0</td><td>3</td><td>3</td><td>3</td></tr><tr><td>50</td><td>3</td><td>3</td><td>3</td></tr><tr><td>100</td><td>3</td><td>3</td><td>3</td></tr><tr><td>175</td><td>3</td><td>3</td><td>3</td></tr></tbody></table>

were characterized by atomic force microscopy (AFM) in resonant intermittent contact-tapping mode with a D3100 microscope (Bruker, USA). AFM investigation allowed us to obtain information on the 3D characteristics of the deposits. After shear treatment and cleaning, the glass plate was fixed on a sample chuck and then explored with a probe (NCHV-A, Bruker) tuned at a frequency of 320 kHz. A laser pointed at the probe was reflected toward sensors, translating the changes in laser wavelength into images that were processed with Gwydion software.

## 2.4. Bulk analysis

### 2.4.1. Digital microscopy

2 µL of sheared solutions were sandwiched between pre-cleaned coverslips after Vortex pre-mixing and scanned using a Keyence digital microscope (VHX 7000 N, Keyence, Japan). Ten images were acquired for each shear rate and each concentration at a magnification of 500×, and successively analyzed by a custom image analysis software (Serie CV-x software). The number of WP aggregates per µL was monitored as an indicator of shear-induced protein denaturation/aggregation.

### 2.4.2. Concentration of native α-lactalbumins and β-lactoglobulins

Samples were diluted at 1% w/v. 800 µL of the solution were mixed with 80 µL of acetic acetate at 0.5 mol.L⁻¹, heated at 30 °C for 5 min and then centrifuged at 13000g for 10 min (20 °C). 100 µL of supernatant were mixed with 900 µL of trifluoroacetate acid (TFA) at 0.106% w/v. It was then filtered through a 0.45 µm filter, to extract native α-lactalbumin (α-lac) and β-lactoglobulin (β-lg). They were separated and identified by HPLC ultimate 3000 (Thermo scientific, USA), equipped with a column compartment oven heated at 40 °C, coupled with DAD (photodiode array detector, 214 nm). The stationary phase was a PLRP-S (Polymeric Reverse Phase-Silicon, Agilent Technologies, USA) column (150 × 2.1 mm², pore size = 300 Å, silicone particle size = 8 µm), equipped with a PLRP-S precolumn made of the same material. The mobile phase was composed of TFA at 0.106% (A) and Acetonitrile + TFA at 0.1% (B), flowing at 0.2 mL.min⁻¹. The gradient started at 84% A, was maintained for 5 min, decreased progressively to 64% in 15 min, to 52% in 1 min and to 20% in 5 min, before returning to 84% A and 16% B, the total run duration being 30 min. The data were processed using

Chromeleon® v7.2 software. The injection volume was 50 µL. Calibration with an external standard method was used for quantification. The calibration range was prepared by diluting a pre-prepared standard solution of α-lac at 2.49 g.L⁻¹ and β-lg at 5.05 g.L⁻¹ with solvent A. The injection volume of the calibration solution varied between 5 and 50 µL. The response was linear with the peak area on the ordinates and the concentration on the abscissa, with forcing the zero-crossing.

### 2.4.3. Turbidity analysis

The turbidity of the samples was calculated from the absorbance at 600 nm (A<sub>600 nm</sub>) measured with a spectrophotometer UVmc² (Safas, Monaco) (n = 3). (Eq. 3). It corresponded to a qualitative evaluation of WP aggregates [34].

$$t = 2.303 \frac{A_{600 \text{ nm}}}{l} \qquad (3)$$

where *l* is the light path length (1 cm) [35].

10 and 12 wt% samples were diluted with deionized water to have an absorbance lower than 1.

### 2.4.4. Particle size distribution by dynamic light scattering

The average particle size was measured by dynamic light scattering (DLS) using the Nanosizer (Nano zeta sizer, Malvern instruments, UK) within 15 min for each sample, at 20 °C (n = 3). The samples were diluted with deionized water to obtain a light attenuation between 5 and 7.

## 2.5. Statistical analysis

Results were divided into two datasets with the same qualitative explanatory variables: concentration (5, 10 and 12 wt%) and shear rate (control, 0, 50, 100, 175 s⁻¹). The first dataset gathered the microscopy data, i.e., two quantitative responses: i) the surface density occupied by the aggregates on the surface ii) the number of aggregates in solution, for a total number of individuals of 360 (an individual being an image for a concentration and a shear). The second dataset gathered the preliminary result on calculation of the “zero shear” viscosity and other analysis of the solution, i.e., five quantitative responses (“zero shear” viscosity, percentage denaturation of α-lac and β-lg, turbidity, average size particle), for a total number of 45 individuals (an individual being one trial for a concentration and a shear rate). The “Linearmodel” function was used to fit a linear model [36]. A one-way analysis of variance (ANOVA) was carried out using R (The R Foundation, 2014) and a Fisher's exact test was performed with a significance level *p* < 0.05. Then, meansComp tests were carried out.
# 3. Results

## 3.1. Rheological characterization

The intensity of the applied shear rate did not affect the viscosity of WP solutions at 65 °C over time (Fig. 2A). On the contrary and as expected, viscosity was influenced by the overall protein concentration. Both 5 wt% and 10 wt% solutions showed no significant viscosity variation during the shear treatment, exhibiting plateau values of 0.6 and 1.4 mPa.s, respectively. At 12 wt%, a gradual increase in the average viscosity from 1.2 to 4.2 mPa.s was registered throughout 10 min of shear. The viscosity increase also resulted in an increased variability, as shown by the higher standard deviations.

whey protein solutions at 5 wt% (blue), 10 wt% (orange) and 12 wt% (green); (B) « Zero shear » viscosity at 20 °C and before treatment (Control □)) and during shear treatment 0 s⁻¹ (iii), 50 s⁻¹ (iii), 100 s⁻¹ (//), 175 s⁻¹ (■) for whey protein solutions at 5 wt% (blue), 10 wt% (orange) and 12 wt% (green) (n = 3).

Fig. 2B shows the “zero shear” viscosity of the solutions at 20 °C, calculated using the Carreau model (Eq. 1), as a function of the shear underwent at 65 °C. Estimating this parameter enabled comparison of sample structural characteristics across treatments: no treatment (control), heat treatment only (0 s⁻¹), and both heat and shear treatments (50, 100, 175 s⁻¹). Contrary to the viscosity measurements (Fig. 2A), the

outcomes highlight an increase in the “zero-shear” viscosity (Fig. 2B) as a function of the shear treatment intensity. However, this effect strongly depended on protein concentration (p = 3.58.10⁻⁵ for the interaction of shear and concentration). At 5 wt%, the viscosity changes slightly with shear (p = 0.04); pairwise comparison shows that the viscosities remain close, and only the 175 s⁻¹ treatment forms a separate group. For 10 wt %, the viscosity is significantly different (p = 2.7.10⁻³) when shear is applied during the treatment, compared to the native, non-sheared solution. Finally, at 12 wt%, the “zero shear” viscosity increased exponentially with the shear rate (p = 1.9.10⁻³). All in all, shear rate impacts the viscosity after the treatment, and this impact is highly visible when the WP concentration increases.

Taken individually, neither Fig. 2A nor B clarifies whether the shear affects the rheological behavior of WP solutions. However, when considering the overall picture, the rapid cooling from 65 to 20 °C possibly reveals WP denaturation and aggregation under shear. When a shear is applied, the protein structures are flow-oriented, thus hindering the viscosity changes [37]. During cooling at rest, the viscosity increases due to sample relaxation and interconnection of the irreversibly denatured WP aggregates and deposits (cold gelation) [38].

Although suggesting possible keys of interpretation, this macroscopic characterization is insufficient to describe the mechanisms occurring on a micron scale. Therefore, systematic analyses were carried out to identify the stages of formation of WP surficial deposits and aggregates in the solution and to link their development to the viscosity increase.

## 3.2. Surface analysis

### 3.2.1. Observation of surficial deposits by digital microscopy

Fig. 3 shows representative images of surficial deposits forming by shearing WP solutions with different concentration. At 5 wt%, the number of deposits increased with shear rate without significant modification of their size. Increasing protein concentration in the system corresponded not only to the rise in deposit number, as summarized in Table 2, but also affected their morphology. At 10 wt%, the surficial deposits exhibited more complex and interconnected structures under increasing shear. These results corroborate our recent study on the formation of WP surficial deposits under shear [31]. At 12 wt%, this phenomenon seemed even more pronounced than at 10 wt%. Larger deposits were observed under the same shear conditions, resulting in gel formation in certain areas of the glass plates at higher shear rates. Nota bene: the presence of deposits was negligible at rest, regardless of protein concentration.

The structural changes described in Fig. 3 were quantified by evaluating the surface density of the surficial deposits under the coupled effect of shear and protein concentration (Fig. 4A). Overall, this analysis confirmed that increasing shear rates promote the development of surficial deposits. On the one hand, the statistical analysis revealed that both shear rate (p < 2.2.10⁻¹⁶) and concentration (p = 1.2.10⁻⁸) individually have a significant effect on the deposit growth. On the other hand, a significant interaction between concentration and shear rate was found (p = 3.1.9.10⁻⁷). At 5 wt%, the effect of shear rate is statistically noticeable in only two groups of shear rates (0 and 50 s⁻¹ vs 100 and 175 s⁻¹) whereas it is distinguished by three and four groups at 10 and 12 wt%, respectively. In a few words, the influence of the shear rate on deposit formation is accentuated by increasing protein concentration. Also, it is worth noting that high standard deviations were obtained for 12 wt% solutions at increasing shear rates. This is likely due to the highly heterogenous deposit distribution on the surfaces, ranging from sparse small deposits (Fig. 4B) to dense interconnected structures (Fig. 4C).

### 3.2.2. Characterization of deposit structure by AFM

The 3D structure of the WP surficial deposits was characterized using AFM. To evaluate structural evolution and avoid the large and complex

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 2. (A) Viscosity at 65 °C during shear treatment at 50 s⁻¹ (○), 100 s⁻¹ (△), 175 s⁻¹ (□) for.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Qualitative characterization of whey protein surficial deposits forming under shear from solutions at 5 wt% (blue), 10 wt% (orange) and 12 wt% (green) (scale bar = 50 µm).</figcaption></figure>

**Table 2**

Number of deposits observed by digital microscopy for each protein concentration and shear rate.

<table><thead><tr><th rowspan="2">Shear rate (s<sup>-1</sup>)</th><th colspan="3">Protein concentration (wt%)</th></tr><tr><th>5</th><th>10</th><th>12</th></tr></thead><tbody><tr><td>0</td><td>2769</td><td>3332</td><td>3166</td></tr><tr><td>50</td><td>5626</td><td>7934</td><td>10,141</td></tr><tr><td>100</td><td>13,096</td><td>11,929</td><td>13,107</td></tr><tr><td>175</td><td>17,991</td><td>16,639</td><td>20,671</td></tr></tbody></table>

gel-like structures observed at 12 wt%, analyses were carried out on 10 wt% samples across all applied shear rates (Fig. 5). Consistent with Figs. 3 and 4, the results highlighted the evolution of deposit size and structure with shear, shifting from globular micrometric objects (Fig. 5A and B) to highly branched structures (Fig. 5C and D). Moreover, the gradual morphological complexification also corresponded to thickening in specific regions of the deposits. The height of these regions doubled under shear (1–1.4 µm) compared to the deposits observed at rest (≈ 0.5 µm). Based on height measurements, the characteristic branched structures at 100 s⁻¹ and 175 s⁻¹ consisted of thicker nodes interconnected through thin arborescent branches (0.5–0.7 µm).

In our recent study [31], the presence of thicker nodes and arborescent branches at high shear rates was considered as an outcome of a multi-step mechanism in deposit formation. First, micrometric primary deposits would form on the surface mainly due to chemical adsorption. Then, the deposits would propagate due to physical short-range interactions, thus explaining the formation of thinner branches [39]. At higher WP concentrations (12 wt%), this phenomenon would be enhanced by the shorter average distance between branched structure, resulting in a transition from deposits to gel. In this light, the viscosity of WP solutions under shear (Fig. 2) could be strongly linked to the development of the surficial deposits. At 5 and 10 wt%, the micrometric deposits and branched structures were not dense enough to affect the rheological behavior of the samples. On the contrary, the gel gradually forming at 12 wt% would be at the origin of the constant increase in viscosity with time.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** (A) Quantitative characterization by surface density, of surficial whey protein deposits from solutions at 5 wt% (blue), 10 wt% (orange) and 12 wt% (green), sheared at 0 s⁻¹ (!!!), 50 s⁻¹ (!!!), 100 s⁻¹ (//), 175 s⁻¹ (■) (n = 3); (B) Observation of high surface density for 12 wt% sheared at 175 s⁻¹ and (C) Observation of low surface density for 12 wt% sheared at 175 s⁻¹.</figcaption></figure>

### 3.3. Bulk analysis

#### 3.3.1. Turbidity analysis of WP solutions

The solutions undergoing thermal and shear treatment displayed a milky colour, whereas the control WP solutions were yellowish and transparent. The higher the protein concentration, the more turbid the treated solutions. This is likely due to the formation of aggregates. To
<figure><img src="image_9.png" /><figcaption>Fig. 5. Qualitative observation of the shear rate impact on the morphology of 10 wt% whey protein surficial deposits.</figcaption></figure>

quantify this phenomenon, the turbidity of the solutions was evaluated by spectrophotometry (Fig. 6A). Despite the high dilution required for this kind of analysis (≈ 1 wt%), the outcomes were in agreement with qualitative observations. The turbidity of the control solutions was almost equal to 0, thus underlining their transparency. Concerning the treated WP solutions, their turbidity was higher compared to the control

and significantly increasing for each concentration (p = 4.18.10⁻⁵), irrespective of the applied shear (p = 0.53). To shed light on the micron-scale dynamics linked to these chromatic changes, the presence of micrometric and nanometric WP aggregates was evaluated by digital microscopy and DLS, respectively. The related protein denaturation in the solutions was quantified by HPLC.

<figure><img src="image_10.png" /><figcaption>Fig. 6. (A) Turbidity of whey protein solutions (B) Average particle size of whey protein solution (C) Concentration of native α-lactalbumin and (D) β-lactoglobulin (D) for solutions at 5 wt% (blue), 10 wt% (orange), 12 wt% (green), before treatment (Control (□)) and after shear treatment at 0 s⁻¹ (■■■), 50 s⁻¹ (■■), 100 s⁻¹ (//), 175 s⁻¹ (■) (n = 3).</figcaption></figure>
### 3.3.2. Detection of WP aggregates by digital microscopy

Overall, all solutions exhibited a low density of aggregates (≈200 aggregates·µL⁻¹), independent of protein concentration (p = 0.07) but rather influenced by the combined influence of heat and shear (p = 1.049·10⁻⁶). The latter seemed to foster the aggregation at 65 °C compared to the control, although no clear tendency related to the intensity of the shear could be observed (Fig. S1).

### 3.3.3. Characterization of the average size of WP aggregates by DLS

Scanning the solutions by digital microscopy after the shear treatment enabled the detection of micrometric WP aggregates only. To investigate the presence of WP submicronic aggregates, the average particle size of WP aggregates forming under shear was measured by DLS for each concentration. The results summarized in Fig. 6B underline the predominant presence of submicronic structures (≈ 100–250 nm) in the solutions. A slight but statistically significant increase in the average aggregate size was observed with higher protein concentrations (p = 3.98·10⁻⁷). In contrast, the shear did not show any effect (p = 0.56).

### 3.3.4. Concentration of native α-lactalbumin and β-lactoglobulin by HPLC

The concentrations of native β-lg (Fig. 6C) and α-lac (Fig. 6D) were determined in control samples and treated solutions as a function of WP concentration and shear rate. The goal was to explore the link between the formation of WP aggregates and protein denaturation in the solutions. Consistent with the previous analyses, no shear effect was detected on WP denaturation in the bulk (p β-lg = 0.30, p α-lac = 0.11). When comparing the native protein content in control samples and in solutions sheared at 65 °C, it is evident that the higher the concentration, the more the reduction in proteins with native structure. In other words, the denaturation process was significantly fostered by higher concentrations (p β-lg = 7.10·10⁻¹³, p α-lac < 2.10⁻¹⁶), due to the higher probability of collisions between molecules. This finds a possible explanation in the current literature, which indicates that denaturation and aggregation are, respectively, a first-order and a second-order reaction with WP concentration [40–42]. During sample preparation, denatured and aggregated proteins were extracted to measure the remaining native proteins by HPLC. There was then no differentiation between denatured and aggregated proteins. The reaction therefore exhibits an order intermediate between 1 and 2, taking into account both states.

To sum up, the investigation of WP sheared solutions revealed that the denaturation process was likely triggered by heat and further accentuated by protein concentration. This results in the formation of many aggregates with a few hundred-nanometer size and a minor fraction of micronic aggregates, conferring a milky colour to the samples. Unlike the observations of WP surficial deposits, shear had a significant influence exclusively on the development of micronic aggregates, which however represent a minor fraction of the population in solution.

## 4. Discussion

Contrary to what has been proposed in most previous studies focusing on the role of heat-induced denaturation [43], our results highlight that the formation of WP deposits originates from the combined effects of temperature and shear. Distinct yet interconnected dynamics occur in the bulk and at the surface, whose kinetics vary depending on protein concentration possibly due to the different molecule-aggregate interactions [20].

On the one hand, shear increased the number of deposits and the complexity of their structure, in agreement with our previous study [31]. This was highlighted by the formation of ramified branches at first, followed by a thick gel above the critical concentration of 12 wt% (Fig. 3). On the other hand, shear solicitation had no effect on WP stability in the bulk, where a minor fraction of micrometric aggregates was observed. Instead, a majority of submicronic WP aggregates, whose average size depended on protein concentration, resulted from heating

the solution up to 65 °C. Could these different deposit and aggregation dynamics be linked?

The experimental approach adopted in this work does not enable real-time observation of the events occurring in the bulk and at the surface under shear. However, some keys to understanding can be deduced from the interpretation of these offline results:

1. Previous works investigated WP aggregation under shear at high temperatures (≥ 80 °C) [29,44,45]. Under these experimental conditions, shear modulates the average size of the heat-induced aggregates due to enhanced collisions in the bulk, resulting in a population of micrometric structures [24]. In the present study, the relatively low temperature hindered thermal denaturation [43] and shear did not significantly contribute to foster aggregate growth, as illustrated in Fig. 6. Studies on β-lg amyloid formation showed that, at sub-denaturation temperatures, shear limits aggregation and promotes the formation of active protofibrils of few hundred nanometers. By analogy, we can hypothesize a similar chemical activity in the submicronic aggregates found in our experiments.

2. In the reference work (31), numerous WP deposits measuring a few hundred nanometers were observed on the glass surfaces after the shear treatment. It could be hypothesized that these deposits form as a result of the adsorption of the aggregates of comparable size observed in the bulk (Fig. 6B) [21,23,46,47]. In this case, applying a shear would lead to increased collisions and, consequently, to faster and enhanced deposit initiation at the surface depending on protein concentration (Fig. 7). In dilute conditions (5 wt%), the active sites were sparsely distributed on the surface even at increasing shear rates, leading merely to the formation of primary deposits of micrometric size. At 10 wt%, the increasing number of collisions between active aggregates from the bulk resulted in the interconnection of the mentioned primary deposits below a critical distance. This explains the development of complex branched structures. Above a critical concentration (≈ 12 wt%), all these ramified structures merged into a gel-like matrix.

3. The veracity of the hypotheses linked to dynamics from bulk to surface does not exclude the possibility that protein denaturation and accumulation can take place directly at the surface due to the shear effect. Although it is unlikely that shear alone could provide the activation energy required for WP full unfolding [48], it has been proven that their denaturation can occur in case of prolonged shear treatments [18].

## 5. Conclusions

This paper contributes to unravel the role of shear on the development of WP deposits by discriminating bulk and surface dynamics under sub-denaturation temperature. On the surface, the effect of the shear rate on protein deposit formation was quantitatively significant and concentration-dependent. While under dilute conditions (5 wt%) increasing shear results merely in a higher number of micrometric surface deposits, at higher concentrations (10–12 wt%) shear fosters deposit interconnection and morphological complexification, up to gel-like structure formation. In the solution, where only submicronic aggregates were observed, shear did not have any significant effect on the aggregation of WPs, which was rather affected by increasing concentration.

Based on these outcomes, we hypothesized a two-steps dynamic of development of whey protein deposits. First, WPs would undergo heat-induced denaturation and aggregation in the solution. The collision of such submicronic aggregates on the surface is enhanced under shear, resulting in the formation of initiation points for deposit growth. Second, such active preliminary deposits would serve as “hooking” points for other active aggregates flowing in the solution. The aggregate-deposit and deposit-deposit interconnection is promoted by increasing protein concentration.

The rheofluidic approach adopted in the present study has proven
<figure><img src="image_11.png" /><figcaption>Fig. 7. Scheme of solution and surface deposition mechanism as a function of concentration and shear rate.</figcaption></figure>

effective due to the small volumes required for testing and the precise control of experimental conditions. Nevertheless, real-time observations would be necessary to validate the proposed mechanisms and identify the dynamics of fouling initiation and propagation. From this point of view, a microfluidic approach would enable i) real-time observation of aggregates colliding from the bulk, using fluorescent probes for protein denaturation (e.g., Thioflavin T) [18] and ii) quantitative analysis of deposit growth kinetics as a function of flowing aggregates by microscopy. The development of microdevices with metallic surfaces would mark a significant step toward more accurately replicating the operational conditions of falling film evaporators.

Certainly, our findings need further investigation in more heterogeneous systems, starting with milk itself. Nevertheless, this new perspective on the effect of shear on WP deposit formation paves the way for a possible generalization of fouling dynamics involving globular proteins in other domains. For example, the rheofluidic strategy could be useful to explore the mechanisms of blood vessel clogging or fouling during purification processes in the pharmaceutical industry.

### CRediT authorship contribution statement

**Margot Grostete:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Jeehyun Lee:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Conceptualization.
**Françoise Boissel:** Investigation. **Maude Jimenez:** Writing – review & editing, Supervision, Conceptualization.
**Romain Jeantet:** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.
**Luca Lanotte:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

### Funding

This project was supported by grants from INRAE and Allocations de Recherche Doctoral (ARED) of Bretagne region.

### Declaration of competing interest

The authors declare no conflicts of interest.

### Acknowledgements

The authors thank Ghazi Ben Messaoud and Florence Rousseau for their help in rheology, Pascaline Hamon for her technical support with HPLC and Manon Perrignon for her advice in statistical analysis. The

authors also thank Valentin De Ryuter for the analysis of turbidity and the quantification of native whey proteins. The authors wish to thank Louis Thomas for atomic force microscopy observations. This work was supported by RENATECH, the IEMN-PCMD-PCP.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2026.150196.

### Data availability

Data will be made available on request.

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