Apple was bought at local supermarket in Wuhan, China. Wheat gluten was bought from Huafeng Powder Industry Co., Ltd. (Henan, China). Hydrochloric acid, sodium hydroxide, dichloromethane, absolute ethyl alcohol, lithium chloride, potassium acetate, potassium carbonate, sodium bromide, potassium iodide, and sodium chloride were chemical reagents and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Apple powders were accurately weighted (marked as M₀) and placed in a constant weight weighing bottle (25 × 40 mm, the quality was marked as M₁), which was then opened and placed in a sealed desiccator containing different saturated salt solutions at constant temperature (25°C). Specifically, the environment relative humidity of the desiccator containing LiCl, CH₃COOK, MgCl₂, K₂CO₃, NaBr, KI, and NaCl saturated solution was 11.30, 22.51, 32.78, 43.16, 57.57, 68.86, and 75.29%; that is, the corresponding water activity was 0.11, 0.22, 0.33, 0.43, 0.58, 0.69, and 0.75. The weight of the sample-loaded weighing bottle (M₂) was measured at 0, 3, 6, 12, and 24 h, and then, it was measured every 24 h until 240 h (2). All measurements were replicated three times, and the average value was used to draw the moisture adsorption curve. The moisture adsorption content (X) of the sample was calculated as follows:

where M₀ was the weight of the initial sample, M₁ was the weight of the bottle, and M₂ was the weight of the bottle containing the hygroscopic samples.

In the same way as in 2.3, the sample powder was placed in the sealed desiccator with different relative humidity and kept at the constant temperature (25°C). It was measured every 24 h until change in weight loss or gain reached less than 0.0003 g for two successive readings. At this point, the sample reached hygroscopic equilibrium state, the mass of the sample powder (in triplicate) was recorded, and the equilibrium moisture content (EMC) was calculated by Equation (1) (18). Then, GAB, Ferro-Fontan, Smith, Henderson, Oswin, and Peleg models were selected for fitting. The coefficient of determination (R²), residual sum of squares (RSS), and root mean square error (RMSE) were used as goodness-of-fit statistics, and the moisture adsorption isotherm of the sample was drawn by combining the model fitting curve with the highest fitting degree.

When reaching the moisture adsorption equilibrium, the apple powder was placed in a black background and photographed with a digital camera to observe its macroscopic morphology, dispersion, and the degree of adhesion and caking.

A nuclear magnetic resonance analyzer (NMI20-015V-I, Niumag Co., Ltd., Suzhou, China) was used to detect the transverse relaxation time (T₂) of freeze-dried powders of AP/Gli and AP/Glu (0:10, 1:1, and 7:3 in mass ratio) that reached the moisture adsorption balance. Samples were weighted (0.2 ± 0.01 g) and wrapped in PTFE to prevent evaporation of water during the test and put into the nuclear magnetic tube for detection. Carr–Purcell–Meiboom–Gill sequence (CPMG) scanning experiment was conducted for analysis. The parameters of detection settings were as follows: the detection temperature was 32°C; the proton resonance frequency (SF) was 21 MHz; the spectral width was 100 kHz; the repeat interval time (TW) was 3,000 ms; the number of repeated scans (NS) was 16; the relaxation decay time (TE) was 0.16 ms; the number of echoes was 4,000. Multi-Exp Inv Analysis software was used to invert the T₂ decay curve and obtain the T₂ relaxation time spectrum of the sample (19).

The models were fitted by Origin 9. The R², RSS, and RMSE were calculated by Origin 9 and used as goodness-of-fit statistics. In addition, the data points in the figure were all the average values of triplicate measurements. The data were expressed as the mean ± S.D. (standard deviation), and p < 0.05 was considered statistically significant. The statistical analyses were performed by SPSS software (SPSS version 25.0, IBM Institute, USA).

Figure 1 shows the moisture adsorption behaviors of apple pulp (AP) and two kinds of freeze-dried apple powder with protein mass ratios of 0:10, 1:1, and 7:3 at the relative humidity (RH) of 58, 69, and 75%. The EMC of pure gliadin was 5.04% at the RH of 58%, which was significantly (p < 0.05) lower than that of pure glutenin (9.24%) (Figure 1A). When the RH rose to 69%, the two proteins exhibited similar moisture adsorption curves. At the RH of 75%, the moisture adsorption of gliadin was lower than that of glutenin before 103 h. After that, the EMC of gliadin was significantly (p < 0.05) higher than that of glutenin. Those results indicated that the hygroscopicity of the two proteins was quite different under moderate RH, while they are more or less the same at medium and high humidity.

When the apple pulp and protein were mixed at mass ratio of 1:1, the moisture adsorption content of powders with gliadin was lower than those added with glutenin at the RH of 58%. As the storage time increased, their moisture adsorption content became the same when the RH rose to 69%. At the RH of 75%, the moisture adsorption of the sample added with gliadin before 75 h was lower than that of the sample added with glutenin. After 75 h, the moisture adsorption of the sample added with glutenin was lower, and it was 3.1% lower when it reached the moisture adsorption equilibrium, which was 28 h earlier than the intersection point in Figures 1A,B). When the mass ratio of apple pulp to protein was 7:3, the EMC of the sample with two kinds of protein added was almost the same at the RH of 58%. Then, the curve became crossed at the RH of 69%. At the RH of 75%, the intersection point was 30 h earlier than that in Figure 1B, and the EMC difference of the samples with two kinds of protein was increased to 4.7% (Figure 1C).

In conclusion, for pristine protein samples, the moisture adsorption of gliadin was lower than that of glutenin in an environment of less than the RH of 69%, while the moisture adsorption of gliadin was higher than that of glutenin in an environment of more than the RH of 69%. When the mass ratio of AP to two kinds of protein was 7:3, the inhibition effect of gliadin on hygroscopicity was better when the relative humidity of the storage environment was lower than 58%. If the relative humidity of the storage environment was higher than 58%, the inhibition effect of glutenin on hygroscopicity of the system was better. In addition, when the moisture adsorption of freeze-dried apple powder with the same amount of two kinds of protein was less than 15%, the moisture adsorption of the sample added with gliadin was lower.

Macroscopic observations of gliadin and glutenin at their moisture adsorption equilibrium under seven different relative humidity environments are shown in Figure 3A. Agglomerate occurred for glutenin at the RH of 43%. When the RH attained 69%, the proteins shrank into a chunk owing to their excessive moisture adsorption. However, glutenin was loose under various relative humidity conditions, and there was still only a slight agglomeration even at the RH of 75%. In consistent with the results in Section 3.1, the moisture content of glutenin was higher than that of gliadin at the RH of 58% and 69%. At the RH of 75%, glutenin was more effective in maintaining the state of powder particles. At the mass ratio of AP/Gli and AP/Glu of 7:3, the gliadin-containing powders began to agglomerate at RH higher than 33% (Figure 3B). At increased RH (58%), the sample powder became contracted and chunked, and the color also deepened gradually. In terms of glutenin-added powders, no caking was observed even at the RH of 58%. At the mass ratio of AP/Gli and AP/Glu of 1:1 (Figure 3C), the caking of freeze-dried apple powder added with gliadin could be crushed by slight external force at the RH of 33% and 43%. The sample added with gliadin formed a hard agglomerate when the RH reached 58%. The freeze-dried apple powder added with glutenin did not agglomerate until the RH reached 69%. All these results indicated that glutenin had a significant inhibitory effect on the caking of freeze-dried apple powder during storage at high relative humidity compared with gliadin. This might be due to the superior power fluidity of gluten after absorbing moisture.

The T₂ relaxation time distribution curves of the samples made from apple pulp mixed with gliadin and glutenin are shown in Figure 4, which could be used to compare the differences of the effects of the two proteins on the water state in the system. The relaxation time of bound water (T₂₁), stagnated water (T₂₂), and free water (T₂₃) was 0.01 ms–1 ms, 1 ms–20 ms, and 10 ms–1000 ms, respectively.

As shown in Figure 4A, the state of water molecules in glutenin was relatively stable as RH increased. The change mainly showed the gradual increase of bound water content, and the peak time gradually moved to the right from 0.11 ms to 0.66 ms. However, gliadin began to appear double humps at the RH of 58%, which indicated the dynamic migration of bound water to stagnant water. Figure 4C shows the T₂ relaxation time distribution curves of freeze-dried apple powder with the mass ratio of apple pulp to protein of 7:3. Gliadin-added powder began to show water migration at the RH of 58%. However, the water content of freeze-dried apple powder added with glutenin was transformed into stagnant water at the RH of 75%. At the mass ratio of apple pulp to protein of 1:1, higher proportion of gliadin could effectively reduce the amount of water migration in the freeze-dried apple powder sample to higher degree of freedom, while higher proportion of glutenin had little effect on the water migration (Figure 4B).

To better display the dynamic migration law of water and the conversion of water types in the hygroscopic process of different samples, the peak areas of corresponding signals of bound water, stagnated water, and free water (A₂₁, A₂₂, and A₂₃) were compared. As shown in Figure 5A, when RH increased from 11% to 58%, the proportion of stagnated water and free water in gliadin gradually decreased, while the proportion of bound water kept increasing, up to 90.51%. It demonstrated that the absorbed water constantly migrated into the state of bound water. When RH was greater than 69%, the proportion of hysteretic water increased, indicating that the water began to migrate to the stagnated water. However, the proportion of bound water in glutenin was always the highest, which might be the reason for the agglomeration of glutenin under different RH conditions. Figure 5B shows that the migration of bound water to stagnated water occurred in the freeze-dried apple flour with AP/Gli and AP/Glu of 7:3. The difference was that the migration of gliadin-added freeze-dried apple powder occurred earlier and the system contained more free water, which resulted in a greater degree of agglomeration of the powder. As shown in Figure 5C, compared with the first two kinds of freeze-dried apple powder, the proportion of bound water in the freeze-dried apple powder with the mass ratio of AP to Gli 1:1 increased, while the proportion of free water decreased under higher RH. However, the distribution of water types in freeze-dried apple powder with the mass ratio of AP to Glu 1:1 had no obvious change. The three types of water converted to each other with the increase of ambient RH. The reason for this may be that at the initial stage of hygroscopic behavior, capillary action within and between powder particles had the ability to absorb water, and water adhered to the surface of particles or filled the gap. As more water was absorbed, water gradually penetrated and bonded with proteins and sugars by hydrogen bonds. When the hydrophilic groups of glucose residues in freeze-dried apple powder absorbed water and expanded, more hydrophilic binding sites might be exposed, and the water molecules would migrate to a more free state, resulting in adhesion between particles.

To sum up, the water in the gliadin-added sample migrated to the direction of high degree of freedom earlier. Before the water migration in the system, the proportion of bound water in the gliadin-added sample was significantly higher than that of the glutenin-added sample, which could maintain the powder quality better.