The role of protein adsorption on bubble formation and stabilisation

This research summary written by Boxin Deng is based on the peer-reviewed article ‘Onsite coalescence behavior of whey protein-stabilised bubbles generated at parallel microscale pores: Role of pore geometry and liquid phase properties’, authored by Boxin Deng, Dirk Wijnen, Karin Schroën, and Jolet de Ruiter.

The research explores foam formulation for bubble formation and stabilization and has been recently published in Food Hydrocolloids (2023). 138, 108435.

Abstract

Foams are dispersed systems with large surface areas leading to thermodynamical instability. In the formulation for food foams, where proteins are often the choice, an excess is used to suppress the coalescence of bubbles formed at (sub)millisecond time scales. However, the effect of protein adsorption on bubble coalescence has rarely been investigated under these short time scales. Here, the coalescence stability of whey protein isolate-stabilised bubbles was studied in a microfluidic device. Most importantly, the extent of bubble coalescence was investigated as a function of the bubble formation time for different liquid phase properties, including protein concentration and continuous phase viscosity. The results show that the bubble formation time varies in the range of 0.01-2.8 ms, and the extent of bubble coalescence can be suppressed by increasing the protein concentration (till 7.5% wt.) and the continuous phase viscosity (till 4 mPa·s). Besides, we proposed a coalescence model, and it nicely captures the relationship between the extent of bubble coalescence and the bubble formation time, yielding a realistic fitting for the minimum period of time required to instantly stabilise a fresh bubble, which varies in the range of 0.5-0.1 ms and decreases with protein concentration. This microfluidic study provides close insights concerning protein adsorption and its roles in bubble formation and stabilisation under conditions relevant to industrial-scale food foam production.

Introduction | Bubble formation and stabilisation for foam production

During foam production, bubbles are formed and compacted, leading to a large increase in surface area and, thus, surface-free energy. As a result, bubble coalescence occurs, through which the surface area and, thus, the surface free energy reduces. In short, the final properties of a foam represent a balance between interface creation (i.e., bubble formation) and timely interface stabilisation (through emulsifier adsorption).

In practice, the bubble formation time is very short (e.g., < 0.3 ms ¹). To compensate for these fast dynamics and thus suppress bubble coalescence at time scales relevant to bubble formation – i.e., instant coalescence, typically, an excess of emulsifier is used, especially for slow-adsorbing proteins. To date, due to the limitation in analytical techniques, the influence of protein adsorption rate on bubble coalescence has rarely been investigated under relevant process conditions: high protein concentration and (sub)millisecond time scales.

Microfluidic platforms have been widely used to make and characterise droplets and bubbles and to some extent, also characterise some dynamic behaviours. So far, in a number of studies, droplet coalescence has been studied in the so-called coalescence chamber, in which monodisperse droplets are formed at a T-junction at first and then collide and coalesce in a wide chamber ^2–4.

The T-junction and the wide chamber are connected by a channel that varies in length. In the coalescence chamber, the extent of coalescence can be tuned by the type and concentration of emulsifiers and also by the adsorption time that is delivered by the length of the connecting channel. However, this microfluidic platform is not suitable for investigating the instant coalescence of bubbles as the approachable adsorption time (11-173 ms ⁵) does not match the fast bubble formation time (e.g., < 0.3 ms ¹).

As an alternative, we recently introduced a microfluidic device – the ‘partitioned-EDGE’, in which monodisperse bubbles are formed at a number of pores that are placed in close, well-defined proximity and coalesce upon their snap-off at the pores and subsequent collision with existing bubbles that are sitting in front of the pores ⁶. In this device, bubbles are formed at time scales down to tens of microseconds, and bubble formation and coalescence occur simultaneously. In the current study, the partitioned-EDGE device was used, and bubble coalescence was investigated for the effects of device geometry (i.e., pore geometry) and liquid phase properties related to protein adsorption for which a semi-empirical model was introduced to capture its dynamics.

Aims

• To investigate bubble coalescence at time scales relevant to large-scale production.
• To monitor the coalescence stability of whey protein-stabilised bubbles.
• To research the effects of liquid phase properties such as protein concentration and liquid phase viscosity on the coalescence stability of whey-protein stabilised bubbles.
• To link device design to its effects on the physical interaction and thus the coalescence of whey protein-stabilised bubbles.

Experiment setup | Designing an Edge-based Droplet GEneration device

The bubble formation time was varied systematically through the applied pressure, and the corresponding extent of bubble coalescence was quantified by analysis of bubble sizes through high-speed recordings. Moreover, in the presence of various concentrations of protein, protein adsorption and its influences on bubble coalescence were captured in a semi-empirical model, which relies on the mass balance of proteins accumulated at the surface of the coalesced bubble, assuming that protein adsorption at the surface of the coalesced bubble follows the Langmuir-type adsorption.

The partitioned Edge-based Droplet GEneration (partitioned-EDGE) device was designed by the research group of Prof. Schroën at Wageningen University and fabricated by Micronit Microtechnologies B.V. (Enschede, the Netherlands). The design and the performance of the partitioned-EDGE device are described as follows:

• The partitioned-EDGE device (Figure 1A) consists of two deep channels and one shallow plateau (whose height is only 1 µm, and about 1/200^th that of the deep channel).
• The two deep channels are used to flow the two phases, with the dispersed phase flowing through the straight channel (pressurised at P_d) and the continuous phase through the meandering channel (pressurised at P_c).
• The shallow plateau connects these two deep channels, and on the continuous phase side of the shallow plateau, it is partitioned into identical pores, where the two phases meet, and the monodisperse bubbles are formed (as illustrated in Figure 1B).
• The bubble formation is driven by a pressure difference across the shallow plateau and pores, which is P_d* = P_d – P_c /2.
• The interaction between two adjacent growing bubbles can be adjusted by varying both the pore width and the partition width (Figure 1C).

The flow of the two phases was tuned with a pressure-driven flow controller (Elveflow OB1, Mk3) via Smart Interface software (Elveflow, France). To analyse bubble formation and coalescence at time scales down to tens of microseconds, videos were recorded with a recording system consisting of an inverted microscope (Axiovert 200 MAT, Carl Zeiss B.V., the Netherlands) and a high-speed camera (FASTCAM SA-Z, Photron Limited, Japan).

The coalescence stability of monodisperse whey protein-stabilised bubbles for foam formulation was analysed for varied protein concentration (2.5-10% wt. whey protein isolate), continuous phase viscosity that is tuned with glycerol (1.4-10 mPa·s based on 5% wt. whey protein isolate), and device geometry, including plateau length (L that is varied based on a fixed length ratio between the pore and the plateau, i.e.,  l/L; 20/100, 40/200 and 80/400), pore width (w; 5, 10, 20, 40, and 80 µm, based on l/L = 40/200 and s = 20 µm), and partition width (s; 5 and 20 µm, based on l/L = 40/200 for w = 5 and 40 µm). Our standard condition for liquid phase properties was set to 5% wt. whey protein isolates with a viscosity of 2 mPa·s.

Materials

• Elveflow OB1 MK3+ pressure controller
• Bronkhorst coriolis flow sensor
• Smart Interface software (Elveflow, France)
• Inverted microscope (Axiovert 200 MAT, Carl Zeiss B.V., the Netherlands)
• High-speed camera (FASTCAM SA-Z, Photron Limited, Japan)
• Glycerol (1.4-10 mPa·s based on 5% wt. whey protein isolate)
• The partitioned-EDGE device (Figure 1A)

Key findings | Foam formulation influences bubble formation

An important feature: The partitioned-EDGE device has extreme shallowness in the plateau and pores. This introduces high Laplace pressures that dominate bubble formation at the pores, allowing rich observations concerning bubble formation and coalescence.

Bubble formation and coalescence. In the partitioned-EDGE device, bubble formation takes place in two regimes, namely low- and high-pressure regimes, and the transition between the regimes occurs at the Laplace pressure of the meniscus devoid of any proteins (e.g., 1400 mbar).

In the low-pressure regime, bubbles are formed only when the Laplace pressure of the meniscus (constricted inside the pores) is reduced via protein adsorption (at the meniscus, through lowering the surface tension of the meniscus) below the applied pressure (P_d*). Bubbles (with size d₀) are formed slowly and stabilised before bubble-bubble collisions take place (Figure 2A, C).

In the high-pressure regime, protein adsorption at the meniscus is not a necessity to initiate bubble formation, and bubbles (with size d₀) are formed at an increasing frequency as a function of applied pressure. As a result, bubble formation co-exists with bubble coalescence, which results in a larger bubble size, d_coal (Figure 2B, C). The coalescence stability of whey protein-stabilised bubbles was studied for applied pressures in the range of P_tran – P_cross (i.e., in the high-pressure regime), at which initially identical, monodisperse bubbles are formed.

Bubble formation characterisation. Typically, one bubble formation can be divided into two processes: the pore filling and bubble necking process, which correspond to time scales of t_fill and t_n, respectively.

The bubble formation time, , is the sum of these two-time scales. In the high-pressure regime, t_fill decreases as function of applied pressure and converges to t_n,, thus  = t_fill + t_n; the size of initial bubbles – d₀, is dominated by t_n. Accordingly, d₀ slightly decreases and then levels off as function of applied pressure; at a given applied pressure, it increases only with pore width and continuous phase viscosity above 4 mPa·s.

Bubble coalescence. Bubble coalescence was captured by both the coalesced bubble size, d_coal, and the number of coalesced bubbles, N+1. It is defined as N+1 = V_coal/V₀, and monitored as function of applied pressure. Typically, d_coal increases with the applied pressure. Compared to d_coal, which hints at the final properties of a foam product, N+1 reflects the extent of bubble coalescence, i.e., the stabilising effects of protein adsorption at the bubble surface. N+1 was expressed as function of the bubble formation time, , which sets the time scale for protein adsorption and thus the interplay between bubble formation (i.e., interface creation) and protein adsorption (i.e., interface stabilisation).

• Device geometry (Figure 3A-C). Bubble coalescence was studied for varied plateau length, as well as pore width and partition width. At a given bubble formation time (i.e., a given pore frequency of bubble formation), the unit frequency of bubble formation is independent of plateau length, and increases with reduced pore and partition width. Compared to varied plateau length, N+1 increases with reduced pore and partition width, due to the higher level of bubble crowding in front of the pores in the continuous phase channel and the physical interactions between bubbles growing at adjacent pores (e.g., in the scenario when d₀ > w + s; the most left image in Figure 1C).
• Liquid phase properties (Figure 3D-E). In the design of chosen, where d₀ < w + s and coalescence occurs mainly between bubbles that are formed subsequently from the same pore, bubble coalescence was investigated for the effects of protein concentration and continuous phase viscosity. N+1 reduces with increased protein concentration up to 7.5% wt. and continuous phase viscosity up to 4 mPa·s, due to enhanced interface stabilisation and slowed process of drainage, respectively.

A semi-empirical model to describe protein adsorption and bubble coalescence stability. During one coalescence event, a bubble grows at the pore (e.g., the daughter bubble) and merges into the coalesced bubble (e.g., the mother bubble) that is sitting in front of it upon snap-off (as illustrated in Figure 4). Protein adsorption occurs at the surface of these two types of bubbles; until the moment when the daughter bubble snaps off at the pore and obtains a surface concentration of , the surface concentration of the mother bubble gradually increases.

Upon collision and merging of these two bubbles, the surface concentration of the mother bubble is diluted, although it is still higher in comparison to its value after the last cycle of coalescence.

The mother bubble ‘stops’ coalescing further when a total number of k = N + 1 daughter bubbles have coalesced into forming this mother bubble, and its surface concentration reaches the value for stabilisation, . Assuming that protein adsorption at the surface of the mother bubble follows the Langmuir-type adsorption, a semi-empirical model was proposed based on a mass balance of proteins accumulated at the surface of the mother bubble.

• The mass balance is a function of the bulk protein concentration and the bubble formation time.
• The model was fitted simultaneously to the four datasets of (N, ) obtained for protein concentrations in the range of 2.5-7.5% wt. As one of the three fitting parameters, t₁ represents the minimum period required to instantly stabilise a daughter bubble. The t₁ decreases as protein concentration increases and varies in the range of 0.1-0.5 ms. The model shows good agreement with our experimental results (Figure 5); in an independent test in the presence of 10% wt. whey protein, the daughter bubbles are formed and stabilised at time scales down to 0.1 ms.

Conclusions | Time and protein concentration are critical for proper bubble stabilisation

In this study, the instant coalescence of whey protein-stabilised bubbles was studied in a microfluidic device – the partitioned-EDGE. There, the extent of bubble coalescence was linked to the bubble formation time that varies between 0.01 and 2.8 ms.

The bubble coalescence was studied for the effects of device geometry and liquid phase properties. In terms of device geometry, we found two modes of bubble coalescence; namely, bubble coalescence occurs either vertically between subsequent bubbles formed from the same pore or horizontally between bubbles growing at neighbouring pores. As an illustration, the latter scenario appears more when the distance between the centre of two pores is smaller than the bubble size upon its snap-off.

The former mode of bubble coalescence was studied for varied liquid phase properties; the bubble coalescence can be suppressed, to a certain extent, by increasing the protein concentration (up to 7.5% wt.) and the continuous phase viscosity (up to 4 mPa·s). Besides, our semi-empirical model shows good agreement with the experimental results, making it a relevant predicting tool for bubble coalescence. To instantly stabilise a freshly-formed bubble at the pore, the minimum period of time required varies in the range of 0.1-0.5 ms, and it decreases when protein concentration increases from 2.5% wt. to 7.5% wt. This is in line with our experimental observation that 0.1 ms ensures the immediate stabilisation of monodisperse (initial) bubbles in the presence of 10% wt. whey protein.

This study proves that our microfluidic system – the partitioned-EDGE, can be used as a useful platform for high-throughput screening of emulsifiers in terms of its capacity for interface stabilisation under relevant process conditions. The obtained insights are expected to provide guidelines for formulation optimisation in practical applications of bubble formation.