Contrastive Study of the Foaming Properties of N-Acyl Amino Acid Surfactants with Bovine Serum Albumin and Gelatin

: A detailed study on the foamability, foam stability, foam liquid-carrying capacity, and foam morphology of two N-acyl amino acid surfactants with bovine serum albumin (BSA) and gelatin were performed by foam scanning. The results showed that the foamability of the mixed system increased gradually and then tended to be stable with increasing surfactant concentration. The foamability of the high-concentration BSA system was stronger than that of the low-concentration BSA system. The foamability and foam stability of sodium N-lauroyl phenylpropanoic acid (N-C 12 P)/BSA were better than those of sodium N-lauroyl propylamino acid (N-C 12 A)/BSA, and the foamability and foam stability of N-C 12 A/gelatin was better than those of N-C 12 P/gelatin. The liquid-carrying capacity of the foam initially increased and then decreased with increasing time, and the maximum liquid-carrying capacity increased with increasing surfactant concentration. When the concentration of the surfactant was 8 mM, the drainage rate of N-C 12 A/protein was higher than that of N-C 12 P/protein. The morphology of the bubble gradually changed from spherical to polyhedron and the number of bubbles gradually decreased with time increasing. Differences in surfactant structure and protein type had an important effect on the number and area of foam.

Most studies on surfactant/protein systems have focused on the interaction between bovine serum albumin BSA and common surfactants, such as sodium dodecyl sulfate SDS , cetylmethylammonium chloride, and cetylmethylammonium bromide 21 26 . Many techniques, such as UVvisible absorption spectroscopy, circular dichroism CD spectroscopy, fluorescence spectroscopy, surface/interfacial tension measurement 27 , isothermal titration calorimetry, interfacial swelling rheology 28 , and small-angle X-ray scattering can be used to study the interaction between proteins and surfactants. Many valuable insights into the interaction between proteins and surfactants have been obtained through these techniques.
However, studies on the foam interaction between surfactant and protein system are relatively few. Saint-Jalmes et al. studied the foamability, foam stability, and coarsening result made by SDS and milk protein casein solution 29 . Xu et al. studied the foam properties of fatty alcohol polyoxyethylene ether sulfate and weir gum 30 . Zhang et al. studied the foaming properties and dilatational rheology of Tween 20 and bovine serum albumin BSA 31 . However, the foam property of amino acid surfactant and protein system has not been studied. Therefore, the foam properties of N-acyl amino acid surfactants and two kinds of proteins were systematically studied in this article.
In this paper, the foam properties of two kinds of N-alkyl acyl amino acid surfactants with BSA and gelatin were investigated by foam scanning method. The foam properties were monitored by electrical conductivity measurement and image analysis, and the foamability, stability, liquid entrainment, drainage, and foam morphology of the surfactant/protein systems were obtained.

Materials
Sodium N-lauroyl propylamino acid N-C 12 A and sodium N-lauroyl phenylpropanoic acid N-C 12 P were synthesized by methyl ester method, purified by hydrochloric acid acidification, washed with water, then alkalized to pH 8 and dried to obtain the product. the chemical structures were in Scheme 1. Gelatin 99. 5 purity, product code: 20190508 was purchased from Tianjin Fuchen Chemical Reagent Factory, and BSA 97 purity, product code: A8020 was purchased from Beijing Solarbio Science and Technology Co., Ltd. All glassware was thoroughly cleaned with detergent and then extensively rinsed with ultrapure water, and dried in a drying oven at 55 for 24 h. Water used in the experiment was ultrapure water resistivity 18.2 MΩ cm .

Foam measurement
Foam scanner FoamScan IT Concept, Teclis, France was used to measure foam 32,33 . N 2 gas was blown via the sample solution through the bottom of a porous disk pore size of 14-16 μm and thickness of 3 mm . A 60 mL solution was injected into the circular glass column inside diameter 35 mm with a syringe, and a 20 mL injection volume was given three times. Measurement was carried out at a nitrogen flow rate of 40 mL/min at a constant temperature of 295 K. All the experimental data were taken three times. N 2 addition was stopped when the foam volume reached 130 mL. The duration of bubbling was characterized as the foamability. The foam volume of the sample solution up to 750 s was recorded as foam stability. The liquid-carrying capacity of the liquid film and liquid drainage were recorded from the monitored results of liquid volume change with time. The photos were taken by the camera inside the Foam scanner instrument. The camera position was at the second prisms from the bottom to up. The photos were set every 10 seconds to record the foam morphology. As shown in Fig. 1, the bubbling time of the solution gradually decreased and the foaming capacity increased gradually with the surfactant concentration increased. When the surfactant reached a certain concentration, the change in foaming time tended to be gentle, because a large number of surfactant molecules are adsorbed on the gas-liquid interface with the increase in surfactant concentration. This increase in adsorption is more conducive to the formation of more bubbles and helps increase the foamability. However, when the surfactant concentration continued to increase, the liquid in the foam film resisted Scheme 1 The chemical structures of the surfactants. foamability because of the gravity effect; hence, the foaming time of the system decreased slowly or even changed very little.
When the surfactant concentration was 3 mM, the foaming time of the N-C 12 A/BSA and N-C 12 P/BSA systems in Fig. 1 a were 266 and 264 s, respectively. In Fig. 1 b , the foaming time of N-C 12 A/BSA and N-C 12 P/BSA were 260 and 254 s, respectively. The foaming time of 2 g/L BSA with N-C 12 A and N-C 12 P were shorter than those of the 0.1 g/L BSA system. This law was also applicable to the foaming time of other surfactant concentration systems. The results showed that the foamability of high-concentration BSA was better than that of low-concentration BSA at these two surfactant systems.
A comparison of the different effects of N-C 12 A and N-C 12 P on foamability revealed that the foamability of N-C 12 P/BSA was higher than that of N-C 12 A/BSA at the same BSA concentration except when BSA was 0.1 g/L and surfactant concentration was 2 mM. Foamability depends on the adsorption kinetics of surfactant and the diffusion rate of surfactant molecules on the air-water interface 34 . Therefore, the result indicated that N-C 12 P with BSA had stronger combination at the gas-liquid interface than that of N-C 12 A with BSA. This combination enhanced the hydrophobicity, and molecules more easily gathered on the gasliquid interface; thus, the adsorption capacity of the surfactant and BSA increased, as well as the foamability. 3.1.2 Effect of gelatin on the foamability of N-acyl amino acid/gelatin system Figure 2 a shows that when the mass concentration of gelatin was 0.1 g/L, the foaming time of the two surfactants and gelatin systems initially decreased and then increased with increasing surfactant concentration. The foaming time of the N-C 12 A/gelatin system was the shortest and the foamability was the strongest when the concentration of N-C 12 A was 2 mM. The foaming time of N-C 12 P/gelatin was  the shortest and its foamability was the strongest when the N-C 12 P concentration was 3 mM. The mass concentration of gelatin in Fig. 2 b was 2 g/L. The foamability law of the gelatin system was the same as that of the BSA system; the foamability initially increased and then tended to be stable with increasing surfactant concentration.
The foamability of the two gelatin concentration systems with the same surfactant concentration in Figs. 2 a and 2 b was compared. When the surfactant concentration was 0.8 mM, the foaming time of N-C 12 A with 0.1 g/L gelatin was 247 s, and that of N-C 12 A with 2 g/L gelatin was 269 s. When the surfactant concentration was 4 mM, the foaming time of N-C 12 A with 0.1 g/L gelatin was 248 s, and that of N-C 12 A with 2 g/L gelatin was 260 s. This result showed that higher gelatin concentration prolonged the foaming time and worsened the foamability of the N-C 12 A/gelatin system at low surfactant concentration. However, when the surfactant concentration was more than 6 mM, the foaming time was between 254-257 s, and the change in gelatin concentration had little effect on the foamability. The reason may be that the system carried more liquid and formed resistance with the surfactant concentration increased, which was not conducive to the enhancement of foamability. For the N-C 12 P/gelatin system, when the surfactant concentration was 3 and 4 mM, the foaming time of N-C 12 P with 0.1 g/L gelatin was 251 and 252 s, respectively, and that of N-C 12 P with 2 g/L gelatin was 906 and 533 s, respectively. The result showed that higher gelatin concentration of prolonged the foaming time and worsened the foamability of the N-C 12 P/gelatin system at low surfactant concentration. However, when the surfactant concentration was more than 6 mM, the foaming time was 246-258 s, and the change in gelatin concentration had little effect on the foamability of the system. Therefore, the foamability of the systems with lower gelatin concentration was better at low surfactant concentration, but the influence of gelatin concentration on foamability was no longer obvious with increasing surfactant concentration.
The foamability of the N-C 12 P/gelatin system was remarkably lower than that of N-C 12 A/gelatin system. The reason may be as follows: the combination between N-C 12 A with gelatin had stronger than that of N-C 12 P with gelatin at the gas-liquid interface, and which enhanced the molecules more easily gathered on the gas-liquid interface, so the foamability increased. Figure 3 a shows that when the protein concentration was 0.1 g/L, the foamability of the gelatin system was stronger than that of the BSA system with the same surfactant. When the concentration of N-C 12 A in Fig. 3 b was lower than 2 mM, the foamability of the gelatin system was stronger than that of the BSA system. The foamability of the two protein systems had no substantial difference when the concentration of N-C 12 A was higher than 2 mM. In Fig. 3 b , the foamability of the BSA system was stronger than that of the gelatin system when the concentration of N-C 12 P was lower than 6 mM. The foamability of the two protein systems had little difference when the concentration of N-C 12 P was higher than 6 mM. In general, the foamability of gelatin was stronger than that of BSA, but, the foamability of the BSA system was stronger than that of the gelatin system when N-C 12 P was combined with highconcentration protein. The reasons can be explained from two aspects. One is that high-concentration BSA has stronger combination with N-C 12 P and strengthens the overall hydrophobicity. Therefore, molecules are easier to gather at the interface, and the diffusion rate and adsorption capacity increase. Second, high-concentration gelatin has strong foaming ability. When this gelatin forms a complex with N-C 12 P, it carries a large amount of liquid and forms resistance. This combination is not conducive to the enhancement of foamability.

Foam stability 3.2.1 Effect of BSA on foam stability
Foam stability refers to the persistence of a stable amount for a period of time after foam production 35 . Foam stability can be expressed in terms of foam volume 32 . Figure 4 shows the foam volume of the surfactant with 0.1 and 2 g/L BSA at 750 s, respectively. Figure 4 shows that the foam volume of N-C 12 P/BSA was between 70 and 85 mL when BSA concentration was 0.1 g/ L, and the range was small. The foam volume of the N-C 12 A/ BSA system varied greatly and increased at 0-100 mL with increasing N-C 12 A concentration when BSA concentration was 0.1 g/L. But when BSA concentration was 2 g/L, the foam volume of the two systems initially increased and then decreased with increasing surfactant concentration. The foam volume range of the N-C 12 P/BSA system was 78-115 mL, and the maximum volume was reached when N-C 12 P concentration was 2 mM. In Fig. 4, the foam volume range of N-C 12 A/BSA system was 60-100 mL, and the maximum volume occurred when N-C 12 A concentration was 3 mM.
A further comparison of the two surfactant foam systems showed that the foam stability of N-C 12 P/BSA was better than that of N-C 12 A/BSA. This result indicated that the structure of the surfactant had great influence on the foam stability of the system, and a strong hydrophobic surfactant is beneficial to maintain foam stability. The results of CD spectra in our experiment showed that, N-C 12 P had a greater influence on BSA and made the BSA molecular chain more unfolded compared with N-C 12 A. The expanded molecular chains were more easily intertwined and linked. Thus, the combination of chains increased and ultimately enhanced foam stability. Figure 5 shows that the foam volume of N-C 12 A/gelatin was maintained at about 110 mL when gelatin concentration was 0.1 g/L, whereas the foam volume of N-C 12 P/ gelatin initially increased and then decreased at 100-108 mL. N-C 12 P/gelatin with 0.1 g/L gelatin obtained the maximum foam volume when N-C 12 P concentration was 2 mM. When gelatin concentration was 2 g/L, the foam volume of the N-C 12 A/gelatin system remained at 112 mL, but that of the N-C 12 P/gelatin system initially increased and then decreased. N-C 12 P/gelatin with 0.2 g/L gelatin obtained the maximum foam volume when N-C 12 P concentration was 4 mM. Different from the BSA system, the N-C 12 A/gelatin system had better foam stability than the N-C 12 P/gelatin system. Figure 6 a show that the foam stability of the gelatin system was better than that of the BSA system with the same surfactant when the protein concentration was 0.1 g/ L. The result indicated that the foam stability of the gelatin system was stronger than that of the BSA system when the protein concentration was low. In Fig. 6 b , for N-C 12 A, the foam stabilities of the gelatin system were greater than those of the BSA system, while for N-C 12 P, it was just the opposite. This result indicated that the structure of surfactant had different effects on the foam stability of the two protein systems when the protein concentration was high. Figures 7 and 8 illustrate the liquid-carrying and drainage capacities of the surfactants with BSA and gelatin, respectively. The liquid-carrying capacity of the foam initially increased and then decreased with increasing time. When  the gas was stopped, the liquid reached the peak and liquid drainage began because of gravity and capillary pressure 36 ; thus, the liquid film became thin and the liquid carrying capacity decreased. The liquid volume gradually became stable after a certain time, and the foam turned to dry foam 37 . In Figs. 7 a and 7 b , the maximum liquid-carrying volumes of the foams in N-C 12 A 0.1 g/L gelatin and N-C 12 P 0.1 g/L gelatin were 30-35 and 25-30 mL, respectively. The maximum liquid-carrying capacity of the N-C 12 A 0.1 g/L BSA system was higher than that of the N-C 12 P 0.1 g/L BSA system. However, in Figs. 7 c and 7 d , the maximum liquid-carrying volumes of the foams were quite different and increased with increasing surfactant concentration. This result indicated that high BSA concentration  had great influence on the maximum liquid-carrying capacity of the foam and increased with the increase in surfactant concentration. The maximum liquid-carrying capacity in Fig. 8 increased with increasing surfactant concentration. In Fig. 8 a -8 c , the maximum liquid-carrying volumes were above 10 mL when the surfactant concentration was low and gelatin concentration was high. On the whole, the liquid-carrying capacity of foam varies greatly with the variety and concentration of the surfactant and protein.

Liquid-carrying and drainage capacities of foam
Moreover, the coordinates of the systems at the maximum liquid-carrying capacity and about 500 s when the surfactant concentration was 8 mM from which the liquid drainage rate can be calculated are shown in Figs. 7 and 8, respectively. The drainage rate of the N-C 12 A/protein system was faster than that of the N-C 12 P/protein system. Figure 9 shows that the foam morphology of N-C 12 A 0.1 g/L BSA changed with time when the surfactant concentration was 8 mM. Table 1 shows the result of the bubble number and foam area of the three systems at different time. Figure 9 shows that the thickness of the foam film grad-ually decreased, and the color of the foam gradually became shallower; thus, the foam became bigger and the shape of foam gradually changed from a sphere to a polyhedron 38 . As shown in Table 1, the number of bubble in three systems gradually decreased, and the maximum bubble area and total bubble area increased with increasing time. The solution did not stop blowing gas at 200 s. The amount of liquid entrained by the foam was large and the number of bubble was small because of the action of gas power. The number of bubble in the N-C 12 A 0.1 g/L BSA, N-C 12 P 0.1 g/L BSA, and N-C 12 P 0.1 g/L gelatin systems was 585, 698, and 743, respectively, and the total area of their foam was 9.58, 7.66, and 8.06 mm 2 respectively. After 300 s, the gas stopped blowing. The foam film became thinner because of the effect of gravity and the capillary pressure drained in the foam. When gravity and capillary pressure reached equilibrium, the adjacent bubbles coalesced dominated by Oswald ripening and bubble merging processes 39,40 . However, the foam area and maximum foam area increased substantially.

Form morphology
We further compared the number of bubble and foam area of the three systems. The number of bubble of the N-C 12 P/BSA system was higher than that of the N-C 12 A/BSA system at the same time, but its maximum bubble area and total foam area were smaller than those of the N-C 12 A/BSA system. This result indicated that the structure of the surfactant has great influence on the number of bubble and the area of foam in the same protein system. The reason may be that the benzene ring structure of N-C 12 P crosses at the interface to form more dense arrangement so that the water capacity of foam increases, which is conducive to the stability of small and numerous bubbles. The number of foam of the N-C 12 P/gelatin system was higher than that of the N-C 12 P/BSA system, but the resulting maximum bubble area was just the opposite. The total foam area was not very different after 300 s. This finding indicated that the effect of the same surfactant on the number of foam and the formation of large bubble was greatly influenced by the different proteins.

Conclusion
The foamability of surfactant/protein system with 0.1 g/L gelatin initially increased and then decreased, whereas those of the other systems increased gradually and then stabilized with the increase in surfactant concentration. The foamability of the high-concentration BSA system was stronger than that of the low-concentration BSA system, but the low-concentration gelatin system was stronger than the high-concentration gelatin system when the surfactant concentration was low. The foamability and foam stability of N-C 12 P were better than those of N-C 12 A for the BSA system but the opposite for the gelatin system. The foamability and foam stability of the surfactant/gelatin system were better than those of the surfactant/BSA system when protein concentration was low. The liquid-carrying capacity of the surfactant/protein system initially increased and then decreased with increasing time. The maximum liquidcarrying capacity of the system increased with increasing surfactant concentration. The drainage rate of the N-C 12 A/ protein system was higher than that of the N-C 12 P/protein system when the surfactant concentration was 8 mM. The number of bubbles decreased with increasing time; never-  The standard deviation σ values are σ (number of bubble) = 1.24-2.03, σ (maximum bubble area) = 0.0054-0.0063 mm 2 , σ (foam area) = 0.057-0.064 mm 2 .
theless, the maximum area of the bubble and the total foam area increased, and the morphologies of the bubbles gradually changed from spherical to polygonal. This study showed that differences in surfactant structure and protein type have great influence on the number and area of foam.