Impact of Probiotic Fermentation on the Physicochemical Properties of Hemp Seed Protein Gels

2.1. Materials and Chemicals

Hemp seeds were sourced from Shaanxi Chuliang Agricultural Technology Co., Ltd. (Shaanxi, China) and ground using a Breville coffee grinder (Model BCG 300, Breville, Sydney, Australia). The hemp seed powder was defatted with a mixture of n-hexane and ethanol (10:1, v/v), and hemp seed protein isolates (HPI) were extracted following the method described in our previous study [16]. Before the protein solution was freeze-dried, ultrasound (400 W, 12 min) was applied to treat the protein solution to increase its solubility. The hydrophobic fluorescent probe 8-Anilino-1-naphthalenesulfonic acid (ANS) and the fluorescence dye fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and Shanghai Yuanye Biotechnology (Shanghai, China), respectively. Starter cultures, which included Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei, were purchased from Kunshan Baishengyou Biotechnology (Suzhou, China). Other reagents were procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of PSPI-Based Gel

The 8% hemp seed protein solution was prepared by combining HPI with pure water, followed by the dissolution of 5% sugar into the mixture. The solution was stirred thoroughly and kept in the refrigerator overnight. The next day, the mixture was homogenized using a FJ-200 high-speed disperser (Shanghai Specimen Model Factory, Shanghai, China) to ensure uniformity. The solution was then heated in boiling water for 20 min to alter the protein properties, followed by rapid cooling in cold water to 20 °C. A 1% starter culture was added to the solution, which was then mixed well and transferred into 150 mL bottles. The bottles were placed in a fermentation box set to 42 °C for fermentation periods of 0, 4, 8, 12, and 16 h. After fermentation, portions of the samples were used to evaluate gel properties, while the remaining portions were poured into Petri dishes, pre-frozen, and freeze-dried to assess the functional characteristics of the gel powder.

2.3. Effects of Fermentation on Physicochemical Properties of PSPI Gel

2.4. Statistical Analysis

All experiments were conducted in triplicate, and the results were presented as mean ± standard deviation. To identify significant differences (p < 0.05), one-way analysis of variance was used, followed by Duncan’s multiple range test. The statistical analyses were performed using SPSS for Windows version 17.0 (IBM, USA).

3.1. Appearance of Gel and pH Value

As illustrated in Figure 1, probiotic fermentation led to gel formation in HPI, with the extent of gelation depending on the fermentation duration. Gel formation began after 8 h of fermentation and was complete by 12 h. Previous studies showed that the gel of soy proteins was complete by 15 h of fermentation [14] and that the gel of plum seed proteins was complete by 9 h of fermentation [15]. However, after 16 h of fermentation, syneresis (red circle) was observed, suggesting that extended fermentation beyond this point could compromise the gel structure.

Figure 2 shows that the pH value decreased with longer fermentation times, which might be linked to reduced surface charge leading to protein aggregation and gel formation. Plant proteins typically have positive surface charges, and as the pH drops, these positive charges diminish, reducing electrostatic repulsion and promoting aggregation. This phenomenon has been previously documented in studies of acid-induced protein gels, in which protein particles aggregated during acidification due to charge neutralization [23]. Earlier research indicated that HPI had an isoelectric point between pH 4.3 and 6.0 [9]. Therefore, after 4 h of fermentation, the pH approached the isoelectric point of PSPI, suggesting that gel formation occurred through gradual acidification. Previous research on plant-based yogurts, including those from soy protein and plum seed protein, also showed that organic acids produced during probiotic fermentation were key to inducing protein gelation [1,14]. Additionally, the pH value dropped to 4.4 after 12 h of fermentation. This result was similar to that in a previous study, in which hemp-protein-based-yogurt possessed a pH value of 4.5 [24]. Our result also aligned with the pH values of other plant-protein-based yogurts. For instance, pea-protein- and mung-bean-protein-based yogurts typically had a pH of 4.6 [25,26]; soy-protein-based yogurts had a pH value of 4.5 [27]; black soybean yogurt had a pH of 4.7 [28]; and the pH values for soymilk yogurts were in the range of 4.4–4.7 [29] or below 5.0 [30].

3.2. Particle Size

As depicted in Figure 3, probiotic fermentation for 4 h had no significant impact on the particle size of the HPI. However, after 8 h of fermentation, the particle size of the HPI began to increase, reaching its peak at 16 h. This increase in particle size was likely due to the aggregation of HPI induced by fermentation. This finding aligned with the observation that the HPI gelation commenced after 8 h and was fully developed by 12 h of fermentation. Additionally, the particle size of the HPI-based yogurt was notably larger compared to plum-seed-protein-based yogurt [15], soy-protein-based yogurt [9], and pea-protein-based yogurt [31], suggesting that the HPI tended to form larger aggregates during fermentation.

3.3. Rheological Behavior

Figure 4 illustrates that the viscosity of the samples decreased as the shear rate increased, indicating that all the samples exhibited shear-thinning behavior [32]. This behavior suggested that as the shear rate increased, the interactions among the HPI molecules were disrupted [5]. This result was consistent with those of a precious study about soy-protein-based yogurt, in which the viscosity increased with extension of the fermentation time [14]. Additionally, the viscosity of the HPI-based yogurt was comparable to that of soy protein-based yogurt [18], but was lower than those of fava beans, chickpea-based yogurts, and plum-seed-protein-based yogurt [33].

As shown in Figure 5 and Figure 6, the storage modulus (G′) of the HPI-based yogurt was higher than the corresponding loss modulus (G″), suggesting that all the samples showed “gel” like behavior. The previous studies about coconut-milk-based, soy-protein-based, and soy-milk-based yogurts also showed the similar results [14,34,35]. Additionally, the storage modulus of the HPI-based yogurt was lower than those of plum-seed-protein-based [3], pea-protein-based [26], soy-protein-based [14], chickpea-, and lentil-protein-based yogurt [36], indicating that the HPI-based yogurt likely had a weaker internal structure.

The creep recovery test is a method that can be employed to investigate the viscoelastic properties of gel. Typically, a higher strain value indicates a weaker gel structure [37]. As depicted in Figure 7, probiotic fermentation decreased the value of the strain, suggesting an increase in the elasticity and strengthening of HPI-based yogurt. Furthermore, the recovery rates for the HPI-based yogurt from 0 to 16 h of fermentation were 52.04 ± 1.02, 57.32 ± 2.11, 48.15 ± 2.35, 66.69 ± 1.84, 43.47 ± 3.05, respectively. This result indicated that the HPI-based yogurt from 12 h of fermentation exhibited the best resistance to deformation [14].

3.4. Water-Holding Capacity (WHC) and Strength of Gel

As depicted in Figure 8, the water-holding capacity (WHC) of the HPI improved significantly once gel formation began after 4 h of fermentation. This increase in WHC was likely due to the development of a network structure through intramolecular cross-linking or polymerization of the HPI, which could have helped to retain water against centrifugal forces. The highest WHC value was obtained after 12 h of fermentation but declined with further fermentation beyond this time. Previous studies indicated that the WHC of protein-based gels was affected by their microstructure and intramolecular interactions [38,39]. Typically, gels that have more uniform network structures and stronger intramolecular bonds exhibit higher WHC. Our findings aligned with this understanding because the HPI gel fermented for 12 h showed enhanced WHC due to stronger intramolecular interactions. The reduction in WHC after 12 h might have been due to the formation of a more compact, non-porous gel structure, which decreased its water-holding ability. A previous study about soy-protein-based yogurt revealed that syneresis occurred after excessive fermentation due to the formation of non-self-supporting gel [40]. Additionally, the WHC of the HPI-based yogurt was comparable to that of plum-seed-based [15] and soybean-based yogurt [41].

As shown in Figure 9, the gel strength of the HPI improved significantly once gel formation began after 4 h of fermentation. The gel strength augmented as the fermentation time elongated, further validating the observation that the molecular interactions in the gel intensified with prolonged fermentation time. Similar results were also observed in soy-protein- [14] and plum-seed-protein-based yogurt [15].

3.5. Microstructure of Gel

The gel formation during fermentation was examined using fluorescence microscopy. As illustrated in Figure 10, the HPI began to aggregate after 8 h of fermentation, as was evident from the appearance of larger green aggregates and the increase in fluorescence intensity. With an extended fermentation time (from 8 to 16 h), these aggregates became larger and denser, indicating the progression of gel formation. A previous study about plum seed protein-based gel also found that proteins tended to form aggregates with the extension of the fermentation time [15].

The microstructure of the samples fermented for 8 to 16 h was also analyzed using scanning electron microscopy (SEM), with the results shown in Figure 11. After 8 h of fermentation, the HPI gel showed a discontinuous network structure. At 12 h of fermentation, pores began to form on the surface of the HPI gel, indicating gel development. Similar porous structures have been reported in studies of pea-protein-based [17], soy-protein-based [14], and plum-seed-protein-based yogurt [15]. However, the porous structure disappeared and a more compact structure was observed on the surface of the HPI gel after 16 h of fermentation. A previous study about soy-protein-based yogurt showed that the porous structure disappeared with excessive fermentation due to enhanced molecular forces [14]. This result also explained why the gel, from 12 h of fermentation, exhibited a higher water-holding capacity (WHC), and why the WHC decreased as the fermentation time was extended to 16 h. Previous studies also demonstrated that gels with more uniform distributions of pores and homogeneous structures exhibited better ability to hold water [42,43].

3.6. Molecular Interactions

Intrinsic fluorescence is a classic method for investigating changes in THE tertiary structure of protein [44]. As illustrated in Figure 12, fermentation led to a reduction in fluorescence intensity for all THE samples. These changes in intrinsic fluorescence were likely due to the formation of HPI aggregates, which could have obscured the fluorescence and reduced the intensity. The decrease in fluorescence intensity also suggested the formation of aggregates [22], which was also observed in the microstructural analysis and SEM. Previous studies showed that interactions between proteins and polysaccharides could similarly diminish fluorescence due to the shielding effect of polysaccharides [45,46,47]. A previous study also proved that the fluorescence intensity of protein in the aggregated form was lower than that in the unfolded state [48]. Our results were also consistent with the studies about soy-protein-based and plum-seed-protein-based yogurt [14,15].

As shown in Figure 13, fermentation decreased the surface hydrophobicity of the HPI. This result was consistent with the study about soy-protein-based gel, which the author of that study attributed to the decrease in surface hydrophobicity to the formation of protein aggregates [14]. The decrease in the surface hydrophobicity also indicated that hydrophobic interaction might be one of the intermolecular forces that induce HPI to form gel during fermentation, which could be attributed to the unfolding of protein induced by fermentation, followed by the exposure of more hydrophobic groups involved in the formation of the gel [49]. The previous study about pea-protein-based yogurt also found that hydrophobic interaction was the main force inducing the formation of gel [50]. The study about soy-protein-based gel also reported that the hydrophobic interaction was involved in gel formation [51]. However, the study about plum-seed-protein-based gel reported that the fermentation increased the surface hydrophobicity [15]. The inconsistent results might have been due to the occurrence of partial hydrolysis induced by fermentation, which led to the exposure of the hydrophobic groups.

It is well known that disulfide (S-S) bonds are crucial for gel formation, influencing the physicochemical properties and microstructure of gels [52]. Therefore, we measured the free SH content of the HPI during fermentation. As shown in Figure 14, fermentation led to a decrease in the free SH content, likely due to oxidation and the formation of protein–S-S–protein bonds [53]. This decrease indicates that S-S bonds contribute to gel formation. The studies about soy-protein-based [54], myofibrillar-protein-based [55], oat-protein-based [56], and pea protein-based [57] gels proved that disulfide bonds were incorporated into the gel network structure. In addition, our previous study about plum-seed-protein-based yogurt also showed that fermentation decreased the free SH content due to the formation of S-S bonds [15].

To investigate non-covalent molecular interactions, solubility of sample in various buffers was analyzed [58]. Buffer B was used to disrupt electrostatic interactions, while buffer BSU targeted hydrogen bonds and hydrophobic interactions [20]. As shown in Figure 15, the solubility of sample in buffers decreased with increasing fermentation time, indicating that gel formation led to the appearance of insoluble aggregates. For each sample, solubility in buffer B was higher than in DW, indicating that electrostatic interactions were significant in the HPI gel network. In addition, solubility in buffer BSU was much greater than in buffer B, highlighting the importance of hydrogen bonds and hydrophobic interactions in the gel network. Previous studies about plum seed protein-based and pea protein-based yogurt also found that the gel was stabilized by non-covalent interactions, such as hydrogen bonds and hydrophobic interactions [15,25]. This finding aligns with the observation on surface hydrophobicity, indicating that probiotic fermentation reduced the surface hydrophobicity of HPI. Consequently, proteins assembled into gels via hydrophobic interactions. When urea in the buffer BSU disrupted the hydrophobic interactions among protein molecules. It decreased protein aggregation, thereby enhancing protein solubility.

3.7. Subunits Analysis

As depicted in Figure 16, fermentation led to the polymerization of subunits between 55–170 kDa of HPI. This observation contrasts with previous research on lupin oat-based yogurt, which found that fermentation did not affect subunit profile of protein [59]. Similar findings were also reported for rice protein-based [60] and soy protein-based yogurts [14], where protein subunits were not affected by fermentation. In this study, the polymerization of subunits might have been caused by proteolytic enzymes from probiotics, which could induce the formation of covalent crosslinking (i.e., non-disulfide bonds). Previous studies have reported that microbial enzymes (such as transglutaminase) could induce the covalent crosslinking among protein subunits [61,62], which was also used to enhance the gel texture of peanut milk yogurt [63]. We hypothesize that the enzyme-catalyzed cross-linking process might also take place among the amino groups of amino acids. This potential mechanism was illustrated in Figure 17. Unlike these cases of broad enzymatic crosslinking, the polymerization observed in HPI was specific, targeting only subunits of HPI. Future studies should focus on isolating this specific protease and elucidating its mechanism for crosslinking of HPI. Additionally, this crosslinking might contribute to the enhanced molecular interactions and compact structure during fermentation. Thus, HPI gels form not only through gradual acidification but also through special covalent crosslinking.