Ultrasound-Treated Dendrobium officinale Polysaccharides as Functional Ingredients for Plant-Based Yogurt: Enhancing Gel Properties of Soy Protein Isolate

Abstract

The application of bioactive polysaccharides from medicine–food homology sources in the food industry still poses a significant challenge. This study investigated the effects of ultrasonically modified polysaccharides from Dendrobium officinale on the physicochemical properties of plant-based yogurt. The Dendrobium officinale polysaccharides were treated with ultrasound at varying power levels (200–600 W) and durations (20–40 min). The modified polysaccharides (0.5%) were then incorporated into soy-protein-isolate-based (5%) yogurt, and the resulting composites were characterized in terms of their structural and functional properties. Results showed that optimal treatment (400 W, 20 min) reduced the particle size of the polysaccharides while enhancing their hydrophilicity and hydroxyl group exposure. The incorporation of these modified polysaccharides into SPI gels promoted probiotic growth, lowered the gel pH, and facilitated the formation of protein gel. Consequently, the resulting gels exhibited a denser microstructure, along with superior gel strength, water-holding capacity, apparent viscosity, storage modulus, deformation resistance, and antioxidant activity (scavenging DPPH and ABTS radicals). These findings suggest that ultrasonic treatment not only modifies polysaccharides from Dendrobium officinale to enhance their bioactivity but also augments their capacity to facilitate protein gel formation. This work provides the evidence that ultrasound-modified polysaccharides from Dendrobium officinale can simultaneously act as prebiotic stimulators and structural reinforcements, offering a novel strategy for designing high-quality plant-based yogurts.

1. Introduction

Medicine–food homology (MFH) refers to a legally defined and regulated category of substances that are officially recognized by authorities such as the Chinese Ministry of Health. In food systems and functional food development, MFH substances are operationally defined by three core attributes: their origin in traditional dietary and medicinal sources; their intended role in long-term, preventive health maintenance through daily intake rather than acute therapeutic intervention; and their incorporation into conventional food matrices to confer physiological benefits beyond basic nutrition [1]. Consequently, the structure and functionality of medicine–food homologous ingredients have drawn considerable research attention. Among these ingredients, medicine–food homologous resources are regarded as important sources of bioactive polysaccharides, since polysaccharides often constitute the primary active components in such materials. Notable examples include polysaccharides derived from Lycium barbarum [2], Dioscorea opposite [3], Polygonatum sibiricum [4], Ziziphus jujuba [5], and Dendrobium officinale [6]. These polysaccharides have demonstrated a range of biological activities, such as hypoglycemic and antihypertensive effects, immunomodulation, antitumor properties, antioxidant capacity, and promotion of probiotic growth. Therefore, integrating these bioactive polysaccharides into food systems could significantly broaden the applications of medicine–food homologous resources.

Dendrobium officinale, a species within the genus Dendrobium of the Orchidaceae family, represents a highly valued medicine-food homologous resource in China [7]. In ancient China, Dendrobium officinale was traditionally described as capable of nourishing the internal organs, alleviating deficiency and fatigue, promoting weight gain in emaciated individuals, enhancing vitality, strengthening the stomach and intestines, contributing to bodily lightness, and extending lifespan [6]. Studies have identified its primary bioactive constituents as polysaccharides [8]. Current research on Dendrobium officinale polysaccharides has demonstrated a range of biological effects, including antitumor, antioxidant, hepatoprotective, choleretic, hypoglycemic, lifespan-extending, immunomodulatory, gastrointestinal protective, and lipid-lowering activities [6]. However, the high molecular weight and aggregation propensity of polysaccharides often limit their bioactivity and bioavailability [9].

To address these challenges, various methods have been employed to reduce the molecular weight and aggregation tendency of polysaccharides, primarily including physical, chemical, and enzymatic approaches [10]. Among them, ultrasound treatment is regarded as a highly promising modification technique due to its distinct advantages. As a physical method, ultrasound can effectively decrease the molecular weight of polysaccharides without altering their chemical structure [11]. Furthermore, this approach offers benefits such as environmental friendliness, high efficiency, ease of operation, and better preservation of polysaccharide bioactivity [12]. Currently, ultrasound treatment has been proved to significantly enhance the antioxidant and hypoglycemic activities of Auricularia auricula polysaccharides [11], as well as the antioxidant activity of Gleditsia sinensis polysaccharides [5]. However, the effect of ultrasound treatment on Dendrobium officinale polysaccharides has not yet been systematically investigated.

Recently, plant-based foods have garnered increasing attention due to their higher environmental sustainability, lower risk of infection and contamination, superior intrinsic ethical attributes, and reduced constraints from cultural and religious dietary habits [13]. Among them, plant-based yogurt has attracted particular interest, with its market size projected to reach USD 2.89 billion by 2026 [14]. However, compared to traditional dairy-based yogurt, plant-based yogurt often faces challenges such as poor textural properties and difficulties in probiotic fermentation. Plant-derived polysaccharides are considered potential prebiotics with the ability to promote probiotic fermentation [15]. Furthermore, studies have confirmed that plant polysaccharides can not only act as thickening agents to reinforce the network structure of yogurt but also improve the texture of the final product, while reducing surface cracking in the curd [15]. Therefore, the addition of plant polysaccharides is a potential strategy to address the current challenges associated with plant-based yogurt. However, the potential of using ultrasonically treated polysaccharides, especially Dendrobium officinale polysaccharides, as an active ingredient, to simultaneously enhance probiotic metabolism and protein gel formation during fermentation, has not been explored.

To address these gaps, this study investigated the effects of ultrasound treatment, specifically varying power levels and durations, on the particle size, exposure of hydroxyl groups and water-holding capacity of Dendrobium officinale polysaccharides. Furthermore, the ultrasonically modified polysaccharides were incorporated into a plant-based fermentation system to evaluate their impact on probiotic growth, as well as the physicochemical properties and antioxidant activity of the resulting plant-based gel. This study aims to elucidate whether and how the physical modification of polysaccharides translates into enhanced probiotic viability and improved gel properties in plant proteins.

2. Results and Discussion

2.1. Particle Size of Dendrobium Officinale Polysaccharides

As shown in Figure 1, a noticeable shift toward lower values in the particle size distribution was observed following ultrasound treatment, compared with the untreated polysaccharides, suggesting a decrease in particle size or aggregation degree. This result was consistent with previous studies, in which ultrasound treatment was reported to effectively reduce the molecular weight of polysaccharides from sweet corncob [16] and Gleditsia sinensis seed [10]. The reduction was mainly attributed to ultrasonic cavitation, which disrupts intermolecular interactions (particularly hydrogen bonds) that promote aggregation during polysaccharide preparation via alcohol precipitation [17].

Moreover, the effect of ultrasound on particle size distribution was found to depend on both power and treatment duration. At a lower power (200 W), prolonged treatment gradually shifted the distribution toward smaller particle size. In contrast, at higher power levels (400 and 600 W), the particle size increased when treatment time was extended from 20 to 40 min. Specifically, when the power was increased from 200 W to 400 W, a clear shift toward smaller particle size region was evident; however, a further increase to 600 W caused the distribution to move back toward larger particle sizes. These outcomes indicate that an optimal ultrasound treatment effectively reduces polysaccharide size, whereas excessive treatment may not lead to further reduction. Two possible reasons may account for this phenomenon. On the one hand, signal attenuation at higher intensities may reduce treatment efficiency [18]. On the other hand, overexposure of active groups on polymer chains could promote new intermolecular interactions and aggregate formation [19].

2.2. Water-Holding Capacity (WHC) of Dendrobium Officinale Polysaccharides

A characteristic feature of polysaccharide structures is their high hydroxyl group content, which theoretically imparts strong hydrophilic properties. However, during preparation, polysaccharides are often isolated by alcohol precipitation, a process that promotes the formation of intermolecular hydrogen bonds between hydroxyl groups. This aggregation diminishes interactions with water molecules and consequently compromises solubility. As shown in Figure 2, the WHC of polysaccharides was significantly enhanced after ultrasound treatment relative to the untreated sample. This observation can be attributed not only to the disruption of intermolecular hydrogen bonds by ultrasound, which exposes additional hydroxyl groups for interaction with water and thereby enhances WHC, but also to the reduction in particle size, as this increases the specific surface area available for water interaction. Consistent with these results, prior research on Auricularia auricula polysaccharides also reported enhanced hydration and solubility following ultrasonic treatment, an effect primarily attributed to cavitation-induced reduction in molecular weight and the increased accessibility of hydroxyl groups [11]. Notably, the improvement in WHC was dependent on both ultrasonic power and treatment duration. The optimal WHC was attained at 400 W for 20 min; further increases in either power or time resulted in a decline in WHC.

2.3. Fourier Transform Infrared (FTIR) Spectrum of Dendrobium Officinale Polysaccharides

As shown in Figure 3, distinct absorption bands were observed at 1060 cm⁻¹ and 1615 cm⁻¹ in all polysaccharide samples, corresponding to the stretching vibrations of C–OH bonds in sugar molecules, which serve as key functional groups for polysaccharide identification [11]. In addition, a strong and broad absorption band detected around 3400 cm⁻¹ was assigned to O–H stretching vibrations [10]. Following ultrasound treatment, an increase in the intensity of the absorption band at 3400 cm⁻¹ was observed, with the most pronounced enhancement occurring in the sample treated at 400 W for 20 min. These spectral changes suggest that ultrasound treatment promoted the exposure of hydroxyl groups within the polysaccharide molecules. The higher exposure of hydroxyl groups implies that hydrogen bonds between polysaccharide molecules were disrupted by ultrasound, leading to the release of hydroxyl groups. Moreover, the elevated hydroxyl content likely contributed to the improved hydrophilicity of the polysaccharides, which accounts for the enhanced WHC observed after ultrasonic treatment. Previous studies on Auricularia auricula polysaccharides have similarly found that ultrasound treatment can lead to the exposure of hydroxyl groups within the polysaccharide molecules [11].

2.4. Appearance of SPI-Based Gel

Based on the findings from Section 2.1, Section 2.2 and Section 2.3, the polysaccharides treated with ultrasound at 400 W for 20 min exhibited smaller particle sizes, higher WHC, and increased hydroxyl group content. Accordingly, these polysaccharides were incorporated into a soy protein isolate (SPI) gel to examine their influence on the physicochemical properties of gel induced by probiotic fermentation. As shown in Figure 4, a gel was formed from SPI following probiotic fermentation, which aligns with earlier observations that gelation occurs after approximately 10 h of fermentation [20]. Previous studies have indicated that probiotics secrete proteases capable of hydrolyzing soy protein, thereby supporting metabolic activity and the growth of probiotics [21]. For this reason, SPI is widely used as a raw material in plant-based yogurt production [22]. Furthermore, when Dendrobium officinale polysaccharides were added, a gel was also obtained, with no notable macroscopic differences being discernible compared to the control gel.

2.5. Whiteness Index (WI) of SPI-Based Gel

As shown in Figure 5, the WI of all gels was found to exceed 70, which was consistent with the visual observations presented in Figure 4. Following fermentation with probiotics, a gel structure with a smooth surface was formed, thereby enhancing light reflection and resulting in a white appearance [20]. Previous studies on SPI-based yogurt have also reported a whiteness index of approximately 73 [20]. However, a significant reduction in WI was observed upon the incorporation of polysaccharides. This decrease could be attributed to the beige powder nature of the polysaccharides themselves, which likely affected the whiteness of gels. A reduction in the whiteness of yogurt has also been reported upon the addition of polysaccharides, which has been attributed to their inherent color [23]. Furthermore, compared with gels containing untreated polysaccharides, no significant difference in WI was detected when ultrasonically treated polysaccharides were added.

2.6. pH Value of SPI-Based Gel

As shown in Figure 6, the pH values of SPI gels after probiotic fermentation were all approximately 4.5, which is consistent with previous reports on the isoelectric point of SPI [24]. This result indicates that the formation of SPI gels was primarily attributed to the organic acids and short-chain fatty acids produced by probiotics, leading to a decrease in pH [25]. As the pH decreased to near the isoelectric point of the proteins, the net surface charge approached zero, thereby initiating aggregation [26]. These findings are consistent with previous studies on plant-based yogurt production, such as soybean protein yogurt (pH 4.5) [27], pea protein yogurt (pH 4.6) [28], mung bean protein yogurt (pH 4.6) [29], black bean protein yogurt (pH 4.7) [30], and hemp seed protein yogurt (pH 4.5) [31]. Compared with SPI gels (control), the addition of polysaccharides significantly reduced the gel pH. This suggests that polysaccharides may act as prebiotics, enhancing probiotic fermentation and consequently lowering the pH of the gel. The lowest pH was observed when ultrasonically treated polysaccharides were added, implying that ultrasound treatment could potentially improve the prebiotic properties of polysaccharides. Therefore, in subsequent investigations, the microbial count in gel samples was examined.

2.7. Microbial Count of SPI-Based Gel

As shown in Figure 7, microbial counts in all gel samples were observed to exceed 1 × 10⁷ CFU/mL, which aligns with the general consensus that the microbial count in yogurt products should be above 10⁷ CFU/mL [32]. Furthermore, studies on plant-based yogurts have reported microbial counts ranging from 0.88×10⁷ to 2.60 × 10⁷ CFU/mL [33]. The microbial count in plum seed protein-based yogurt was 1.02 × 10⁷ CFU/mL [34]. The microbial count of SPI gels fell within this range, indicating their potential suitability as plant-based yogurt alternatives. Moreover, compared with control, a significant increase in the microbial count was observed in samples supplemented with polysaccharides. This suggests that polysaccharide addition can markedly promote the proliferation of probiotics. Previous research on plant-derived polysaccharides has similarly identified their ability to enhance the growth of probiotics, thereby classifying them as novel prebiotics [35]. Such polysaccharides are sourced from beans, grains, fruits, and vegetables (such as wheat, oatmeal, barley, navy bean, white bean, black bean, lentil, kidney bean, and chickpea, tomatoes, onions, garlic, chicory, leafy greens, leeks, shallots, asparagus, spinach, bananas, and berries) [36]. Notably, the highest microbial count was observed in gels incorporated with ultrasonically treated polysaccharides. This indicates that ultrasound treatment may improve the prebiotic properties of polysaccharides. Consistent with this, earlier studies on ultrasonically treated polysaccharides have also reported enhanced bioactivities, such as increased antioxidant and hypoglycemic capacities [10,11]. These improvements have been attributed to the reduction in particle size and aggregation degree, elevated hydrophilicity, and expanded surface area of polysaccharides following ultrasonic treatment. These modifications are likely to increase interaction opportunities between polysaccharides and probiotics, thereby facilitating microbial hydrolysis and ultimately enhancing prebiotic efficacy. Previous studies on ginseng polysaccharides [37] and Naematelia aurantialba polysaccharides [38] have also reported that reducing the particle size or molecular weight of polysaccharides improves their prebiotic activity.

2.8. Particle Size of SPI-Based Gel

As shown in Figure 8, the particle sizes of all samples were primarily distributed within the range of 10–100 μm, which is consistent with previous studies on soybean protein yogurt [20], plum seed protein yogurt [34], and pea protein yogurt [39]. Compared with the control, the incorporation of polysaccharides was found to significantly increase the particle size of gels. This observation aligns with the aforementioned results, indicating that the addition of polysaccharides promotes probiotic fermentation. Previous research on hemp seed protein gels has similarly reported that enhanced fermentation significantly increases the particle size of the resulting gels [40]. Furthermore, gels containing ultrasonically treated polysaccharides exhibited a larger particle size compared to those containing untreated polysaccharides. This result also suggests that ultrasound treatment may improve the ability of polysaccharides to promote fermentation.

2.9. Rheological Behavior of SPI-Based Gel

As shown in Figure 9, all gel samples exhibited shear-thinning behavior, indicating that the applied shear force could disrupt non-covalent interactions within the gel network [41]. Compared with the control, the addition of polysaccharides was found to significantly increase the apparent viscosity of gels. This suggests that polysaccharides functioned as thickening agents. Previous studies on polysaccharides have similarly reported that their incorporation can enhance the viscosity of yogurt [42,43]. This effect is primarily attributed to the ability of polysaccharides to promote protein-protein interactions and polysaccharide-protein interactions, thereby facilitating gel formation and stabilizing the gel structure [23]. Furthermore, gels containing ultrasonically treated polysaccharides exhibited higher apparent viscosity than those prepared with untreated polysaccharides. This implies that ultrasonically treated polysaccharides may interact more readily with proteins. For instance, the release of hydroxyl groups resulting from ultrasonic treatment could promote hydrogen bonding between polysaccharides and proteins, which would enhance yogurt viscosity.

As shown in Figure 10 and Figure 11, all samples were observed to exhibit a higher storage modulus than loss modulus, indicating the characteristic gel-like behavior. Similar findings have been reported in previous studies on coconut milk yogurt [44] and soy milk yogurt [45]. In comparison with the control, an increase in the storage modulus was detected in gels containing polysaccharides. Furthermore, gels prepared with ultrasonically treated polysaccharides exhibited a higher storage modulus than those incorporating untreated polysaccharides. This implies that ultrasonic modification of polysaccharides could further contribute to stabilizing the gel matrix and enhancing its overall structural integrity.

As shown in Figure 12, the SPI gels were found to exhibit relatively high strain values, indicating considerable deformation under applied stress. This behavior was attributed primarily to their comparatively weak internal gel structure [46]. Upon the addition of polysaccharides, an obvious reduction in strain was observed, with all values falling below 2%. In contrast, similar studies have reported strain values exceeding 2% for hemp seed protein yogurt, suggesting that the SPI gels incorporated with polysaccharides exhibited greater structural integrity. Furthermore, the lowest deformation was recorded in gels containing ultrasonically treated polysaccharides, compared to those with untreated polysaccharides. This result further indicates that ultrasonication of polysaccharides can reinforce the internal gel network.

2.10. Water-Holding Capacity (WHC) of SPI-Based Gel

As shown in Figure 13, the WHC of SPI gels was similar with plum seed protein-based yogurt [34] and hemp seed protein-based yogurt [40]. The WHC was significantly improved by the addition of polysaccharides. This enhancement could be ascribed to the ability of polysaccharides to form stable hydrogen bonds within the gel matrix under acidic pH conditions, owing to their hydroxyl-rich structure [47]. Such interactions contribute to strengthening the three-dimensional protein gel network and effectively inhibit whey separation. Similar phenomena have been reported in previous studies, in which polysaccharides such as those derived from Tremella fuciformis [48] and Cinnamomum camphora [15] were also observed to markedly enhance the WHC of yogurt. Furthermore, gels supplemented with ultrasonically treated polysaccharides exhibited higher WHC when compared to those containing untreated polysaccharides. This result further indicates that ultrasonically modified polysaccharides contributed to the formation of a more compact and stable gel network structure.

2.11. Gel Strength of SPI-Based Gel

As shown in Figure 14, the gel strength of SPI was similar with plum seed protein-based yogurt [34] and hemp seed protein-based yogurt [40]. The gel strength was significantly enhanced by the addition of polysaccharides. This observation is consistent with previous studies, in which polysaccharides have been reported to improve the textural properties of yogurt [49]. The underlying mechanism is primarily attributed to the ability of polysaccharides to promote cross-linking between protein molecules, thereby facilitating the formation of protein aggregates [50]. Additionally, it has been suggested that probiotics can metabolize plant-derived polysaccharides, leading to increased secretion of exopolysaccharides, which may further contribute to the improvement of gel strength [51]. Furthermore, gels supplemented with ultrasonically treated polysaccharides exhibited higher gel strength than those containing untreated polysaccharides. This result further indicates that ultrasonically modified polysaccharides were more effective in promoting protein cross-linking and supporting probiotic metabolism.

2.12. Microstructure of SPI-Based Gel

As shown in Figure 15, the continuity of the SPI gel structure was significantly improved by the addition of polysaccharides. Particularly in gels formed with ultrasonically treated polysaccharides, the pores were largely eliminated, resulting in a denser and more compact microstructure. This structural observation helps to explain why gels containing ultrasonically treated polysaccharides exhibited superior gel strength, enhanced WHC, higher storage modulus, and increased viscosity, as reported in the preceding sections. Similar findings have been documented in prior studies, where enhanced intermolecular interactions in yogurt were shown to promote the formation of a more continuous and dense gel network, consequently leading to improved gel strength [20].

2.13. Antioxidant Activity of SPI-Based Gel

As shown in Figure 16, the free-radical-scavenging capacity of SPI gel was significantly enhanced by the addition of polysaccharides. This improvement was primarily attributed to the inherent antioxidant properties of Dendrobium officinale polysaccharides. Previous studies have proved that polysaccharides from Dendrobium officinale possess the ability to scavenge DPPH and ABTS radicals [52]. Furthermore, gels supplemented with ultrasonically treated polysaccharides exhibited a higher radical-scavenging capacity than those containing untreated polysaccharides. This result suggests that ultrasound treatment can improve the free-radical-scavenging ability of polysaccharides. Consistent with this finding, prior research has also reported that ultrasonic treatment enhances the antioxidant properties of polysaccharides [10,11]. This enhancement can be explained by three mechanisms. First, ultrasonically treated polysaccharides exhibited significantly improved water solubility and expanded surface areas, which effectively promoted their interaction with free radicals. Second, the ultrasonic treatment disrupted the polysaccharide network structure, resulting in a substantial reduction in particle size. Third, the treatment promoted the exposure of hydroxyl groups along the polysaccharide chains, which directly contributed to their enhanced radical-scavenging capacity.

2.14. Supposed Mechanism

Based on the experimental findings, a mechanistic framework is proposed to explain how ultrasound-treated polysaccharides improve the properties of SPI gels. Ultrasonic disruption of polysaccharide self-aggregation exposes hydroxyl groups (Figure 3) and increases chain flexibility. The resulting unfolded and more hydrophilic conformation appears to render the polysaccharides more susceptible to probiotic metabolism, consistent with the notably higher microbial counts observed in SPU-G (Figure 7). This enhanced metabolic activity accelerates the fermentation-driven pH drop (Figure 6) and may stimulate the production of bacterial exopolysaccharides, both of which contribute to protein interactions and network reinforcement. Concurrently, the greater availability of hydroxyl groups and reduced steric hindrance facilitate hydrogen bonding between polysaccharide chains and the polar amino acid residues of SPI. The cleavage of intramolecular hydrogen bonds (Figure 3) further improves molecular flexibility, allowing the polysaccharides to entangle more effectively with the protein matrix. In addition, the smaller particle size of the modified polysaccharides (Figure 1) offers a larger specific surface area, increasing the frequency of contact and the number of potential binding sites between the two biopolymers. Taken together, ultrasound-induced physicochemical changes lead to the formation of a more interconnected and mechanically stable gel network, reflected in the increased apparent viscosity and storage modulus (Figure 9 and Figure 10), as well as the denser, more continuous microstructure (Figure 15). Such structural integrity also imparts greater resistance to deformation under applied stress, as evidenced by the substantially reduced creep strain (Figure 12).

3. Conclusions

This study investigated the effects of ultrasound treatment on the techno-functional properties of Dendrobium officinale polysaccharides, as well as the effects of treated polysaccharides on the physicochemical properties of SPI gels induced by probiotic fermentation. The results indicated that treatment under optimal ultrasonic conditions (400 W, 20 min) significantly reduced the particle size of polysaccharides from Dendrobium officinale, enhanced their WHC, and increased the exposure of hydroxyl groups. These functional and structural modifications improved the proliferation of probiotics and enhanced the degree of protein aggregation during SPI gelation. Consequently, the resulting gels exhibited a more compact microstructure, which contributed to higher viscosity, storage modulus, deformation resistance, WHC, and gel strength. Furthermore, gels incorporated with ultrasonically treated polysaccharides from Dendrobium officinale showed enhanced antioxidant activity, which was primarily attributed to the reduced particle size and increased hydroxyl exposure induced by ultrasonic treatment. Therefore, the superiority of ultrasound-modified polysaccharides from Dendrobium officinale is attributed to enhanced accessibility to probiotics, leading to higher microbial counts and a lower pH, as well as increased interactions with SPI, resulting in a denser microstructure and improved rheological properties.

Despite the promising findings outlined above, several limitations of the present study should be acknowledged. The mechanistic understanding of how ultrasound modification enhances the prebiotic properties of Dendrobium officinale polysaccharides remains incomplete, particularly because the metabolites generated from probiotic fermentation of the treated polysaccharides were not characterized. Furthermore, while rheological and microstructural analyses offered valuable insights into polysaccharide-induced protein gelation, more direct evidence (such as specific intermolecular interaction forces) is still lacking. In addition, the dynamic changes in polysaccharide structure, polysaccharide–protein interactions, and key physicochemical properties during fermentation were not monitored over time. From an application standpoint, sensory evaluation, storage stability, and in vitro digestion studies of the polysaccharide-fortified plant-based yogurt were not conducted; however, such assessments are essential to support future industrial translation. It should also be noted that all experiments were carried out at laboratory scale; the robustness and stability of the observed effects under pilot or industrial production conditions, as well as their performance in more complex food matrices containing lipids, flavors, or other hydrocolloids, are yet to be validated. Addressing these limitations in future research will not only deepen the mechanistic understanding of ultrasound-modified polysaccharides but also facilitate their rational design and application in plant-based foods.

4. Materials and Methods

4.1. Materials and Chemicals

Soy protein isolate (SPI, protein content > 90%) was supplied by the Shandong Yuwang Group (Shandong, China). The molecular weight distribution of SPI subunits ranges from 20 to 210 kDa, the 11S/7S ratio is between 1.6 and 1.8, and the denaturation temperature ranges from 70 to 80 °C. This information was provided by the supplier. Polysaccharides derived from Dendrobium officinale (purity > 90%) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC, purity 90%), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2′-azinobis- (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) were also sourced from the same supplier. The starter culture, containing Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei, was provided by Kunshan Baishengyou Biotechnology (Suzhou, China). All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

4.2. Modification of Dendrobium Officinale Polysaccharides by Ultrasound

The 0.5% (w/v) aqueous solution of Dendrobium officinale polysaccharides was prepared in deionized water. The solution was first homogenized (KINEMATICA PT-MR 2100, Littau-Lucerne, Switzerland) at 15,000 rpm for 1 min to obtain a uniform premix and then stored at 4 °C overnight to ensure complete hydration. Subsequently, the premix was subjected to ultrasonic treatment using a probe sonicator (20 kHz, NingBo Scientz Biotechnology Co., Ltd., Ningbo, China) equipped with a 0.636 cm diameter titanium probe. Sonication was conducted in pulse mode (2 s on, 2 s off) under controlled temperature conditions, with the sample vessel immersed in an ice-water bath to maintain the temperature at 25 °C and prevent thermal degradation. Different ultrasonic intensities (200, 400, and 600 W) and durations (20 and 40 min) were applied. The treatment condition was selected based on previous studies, which reported that ultrasonic treatment at 300 W and 450 W for 40 min significantly improved the bioactivity of polysaccharides derived from Gleditsia sinensis seeds [10]. The untreated sample was prepared in parallel as the control. The sonicated samples were designated as U200-20, U200-40, U400-20, U400-40, U600-20, and U600-40, where the numeric prefix indicates the ultrasonic power (W) and the suffix indicates the treatment time (min).

4.3. Particle Size of Dendrobium Officinale Polysaccharides

Following ultrasonication, the particle size distribution of the polysaccharide samples was analyzed by laser diffraction using a LS13320 particle size analyzer (Beckman Coulter, Indianapolis, IN, USA).

4.4. Water-Holding Capacity (WHC) of Dendrobium Officinale Polysaccharides

WHC was determined by a centrifugation method. Briefly, an empty centrifuge tube was weighed and recorded as $m_0$. Then, 1 g of lyophilized polysaccharides and 5 mL of distilled water were added to the tube, and the total weight was recorded as $m_1$. The tube was centrifuged at 6000× g for 10 min (Beijing Dalong Xingchuang Experimental Instrument Co., Ltd., Beijing, China). After complete removal of the supernatant, the tube containing the sediment was weighed again and recorded as $m_2$. The WHC was calculated according to the following Equation (1):

$$\mathrm{W}\mathrm{H}\mathrm{C}(\%)={\displaystyle \frac{m_2-m_0}{m_1-m_0}}\times 100$$

(1)

4.5. FTIR Spectrum of Dendrobium Officinale Polysaccharides

Lyophilized polysaccharides (2 mg) were thoroughly mixed with potassium bromide at a weight ratio of 1:100. The mixture was then compressed into translucent pellets under a hydraulic pressure of 10 tons. FTIR spectra were recorded on an FTIR-7600 spectrometer (Lambda Scientific, Melbourne, Australia). Spectral data were collected in the range of 500–4000 cm⁻¹ with a resolution of 4 cm⁻¹, accumulating 32 scans per measurement.

4.6. Preparation of SPI-Based Gel

The SPI-based gels were prepared via probiotic fermentation-induced gelation, following a procedure adapted from our previous study with minor modifications [20]. Briefly, a 5% (w/v) protein solution was prepared by dissolving SPI in deionized water. Sucrose (5%) and polysaccharides at 0.5% (w/v) were then added. The mixture was kept at 4 °C overnight to allow full hydration. Subsequently, the solution was homogenized (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at 30 MPa for three cycles and sterilized at 95 °C for 15 min. After cooling to 42 °C, 0.3% (w/v) starter culture was inoculated into the sterilized solution. The inoculated samples were dispensed into sterile containers and fermented at 42 °C for 12 h. Following fermentation, the gels were transferred to a refrigerator and stored at 4 °C for 24 h for post-ripening. The control gel prepared without polysaccharide addition was designated as S-G, while the gels containing Dendrobium officinale polysaccharides and ultrasonicated polysaccharides were labeled SP-G and SPU-G, respectively.

4.7. Appearance of SPI-Based Gel

The appearance of the gel samples was documented using a digital imaging system (Huawei Mate 70, Huawei, Shenzhen, China) for visual characterization.

4.8. Whiteness Index of SPI-Based Gel

The color parameters (L, a*, and b*) of the gel samples were measured using a benchtop colorimeter (HunterLab, Reston, VA, USA). The whiteness index (WI) was subsequently calculated according to the following Equation (2):

$$\mathrm{WI}=100-\sqrt{(100-L{)}^2+(a^{\ast }{)}^2+(b^{\ast }{)}^2}$$

(2)

4.9. pH Value of SPI-Based Gel

The gel samples were first homogenized by stirring at 40× g for 5 min, after which the pH was determined with a calibrated pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China).

4.10. Microbial Count of SPI-Based Gel

The total viable count in the gel samples was determined according to a previously described method [33]. Briefly, each sample was serially diluted with sterile saline, and appropriate dilutions were spread onto plates. All plates were then incubated at 37 °C for 48 h (BD23 incubator, Binder, Tuttlingen, Germany) prior to colony enumeration.

4.11. Particle Size of SPI-Based Gel

The gel samples were reconstituted in deionized water to a final concentration of 1% (w/v). Particle size distribution was then determined by laser diffraction using a LS13320 analyzer (Beckman, Indianapolis, IN, USA).

4.12. Rheological Behavior of SPI-Based Gel

The rheological properties of the gel samples were measured using a Discovery HR-1 rheometer (TA Instruments, Leatherhead, UK) equipped with parallel plate geometry (60 mm diameter, 100 μm gap). Steady shear tests were performed by linearly increasing the shear rate from 0.1 to 1000 1/s at 25 °C, and the corresponding apparent viscosity was recorded [20]. Dynamic oscillatory measurements were carried out within an angular frequency range of 0.1–100 rad/s at a fixed strain amplitude of 0.1%, during which the storage modulus and loss modulus were monitored at 25 °C. Additionally, creep and recovery tests were conducted under a constant shear stress of 5 Pa at 25 °C, following the procedure described in a previous study [40]. A constant stress was applied for 300 s, after which the recovery response was recorded over a further 300 s period.

4.13. Water-Holding Capacity (WHC) of SPI-Based Gel

For determination of WHC, the gel samples were centrifuged at 10,000× g for 10 min (Beijing Dalong Xingchuang Experimental Instrument Co., Ltd., Beijing, China). After removal of the supernatant, WHC was calculated as the percentage of water retained in the pellet relative to the initial sample weight, according to the following Equation (3):

$$\mathrm{WHC}(\%)={\displaystyle \frac{W_2}{W_1}}\times 100$$

(3)

where W₁ is the weight of the sample before centrifugation and W₂ is the weight of the pellet after supernatant removal.

4.14. Gel Strength of SPI-Based Gel

Gel strength was evaluated using a texture analyzer (StableMicro Systems, TA-XT2i, Godalming, UK). A 100 mL gel sample was placed in a glass cylinder (50 mm diameter) and compressed to 50% of its original height at a constant speed of 1 mm/s using a cylindrical probe (36 mm diameter). The maximum force (g) recorded during penetration was reported as the gel strength.

4.15. Microstructure of SPI-Based Gel

For identification of the protein component within the gel matrix, 1 g of fresh gel was thoroughly mixed with 10 μL of fluorescein isothiocyanate (FITC) solution (0.1 mg/mL in dimethyl sulfoxide). The labeled samples were subsequently observed under a fluorescence microscope (DM2500, Leica, Wetzlar, Germany).

4.16. Antioxidant Activity of SPI-Based Gel

The antioxidant activity of the gel samples was determined based on a reported method with minor modifications [53]. In brief, 1 mg of gel was homogenized in 2 mL of either absolute ethanol or an aqueous solution of ammonium persulfate (2.45 mM). Then, 2 mL of each homogenate was combined with 2 mL of 0.2 mM DPPH solution or 7 mM ABTS solution, respectively. Following incubation in the dark at 37 °C for 30 min, the mixtures were centrifuged at 5000× g at 4 °C for 20 min (Allegra 64R, Beckman, Indianapolis, IN, USA). The absorbance of the resulting supernatant was then measured at 517 nm (for DPPH assay) or 734 nm (for ABTS assay) using a Spark 10M microplate spectrophotometer (Tecan, Männedorf, Switzerland).

4.17. Statistical Analysis

All measurements were expressed as a mean ± standard deviation (SD) of three independent replicates. Statistical analysis was performed with SPSS Statistics software (Version 25.0, IBM Corp., Armonk, NY, USA). Significant differences among sample groups were assessed using one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test for multiple comparisons. Differences were considered statistically significant at p < 0.05.