Development of lotus seed-based vegan yogurt: fermentation optimization, quality evaluation and analysis of key bioactive alkaloids

Abstract

Introduction:

Lotus seed is a nutritious and functional food material, yet its utilization in plant-based fermented products remains underdeveloped. This study aimed to develop a novel plant-based yogurt using lotus seeds as the primary raw material, and to systematically optimize the probiotic strain combination and fermentation process to improve product quality and functionality.

Methods:

The optimal lactic acid bacteria strain combination and key fermentation parameters (temperature, time, glucose addition, and inoculum size) were determined through single-factor experiments and response surface methodology.

Results:

The optimal strain combination was Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, and Streptococcus thermophilus at a ratio of 1:2:1. The optimized product, fermented at 38 °C for 7.5 h with 12.2% glucose and a 3.7% inoculum, exhibited high water-holding capacity (68.57%) and favorable sensory acceptance (80.9 points). The yogurt presented a uniform white color, smooth texture, and characteristic lotus seed aroma. Nutritional analysis showed significantly lower fat and calorie content, along with higher dietary fiber, compared to conventional dairy yogurt. Microstructural observations indicated that fermentation promoted the formation of a uniform and compact gel network, enhancing texture and stability. Notably, fermentation effectively preserved key bioactive alkaloids from lotus seeds, including neferine and nuciferine.

Discussion:

This study successfully developed a plant-based yogurt from lotus seeds with desirable physicochemical, sensory, and nutritional properties. The optimized fermentation process also retained important bioactive compounds, offering a theoretical basis and practical guidance for the value-added utilization of lotus seeds and the development of functional plant-based fermented foods with potential health benefits.

1 Introduction

Plant-based vegan yogurt is a fermented food derived from various plant substrates that exhibits a texture and sensory profile comparable to those of conventional dairy yogurt (1). Similar to dairy-based counterparts, plant-based yogurts are capable of maintaining viable lactic acid bacteria throughout extended storage periods (2) and demonstrate improved flavor and mouthfeel, as well as enhanced functional attributes (3). With increasing consumer awareness of healthy dietary patterns, plant-based yogurts have gained considerable attention as suitable alternatives for lactose-intolerant individuals, vegans, and other special population groups (4, 5). Consequently, global market demand for plant-based yogurt products continues to grow steadily (6). However, the development of plant-based yogurts derived from high-starch botanical sources remains challenging, as high starch content during fermentation often leads to weak gel formation, pronounced syneresis (whey separation), and insufficient fermentation activity (7). Among common high-starch ingredients such as corn, cassava, and oats, lotus seed (Nelumbo nucifera Gaertn.) presents a uniquely challenging set of technical hurdles. The amylose content of lotus seed starch is considerably higher than that of typical high-starch plants, making it highly prone to rapid retrogradation after gelatinization (8). This manifests as pronounced syneresis and textural hardening, which severely compromise the gel stability of plant-based yogurt during fermentation and cold storage. Additionally, lotus seed starch has a high gelatinization peak temperature of 72–79 °C and exhibits low swelling power, limiting its ability to fully gelatinize and hindering the formation of a stable gel network (9). Taken together, these factors make the development of lotus seed-based plant yogurt particularly difficult, positioning lotus seed as a quintessential example of a challenging, high-starch ingredient in plant-based yogurt formulation.

Lotus seeds are regarded as a quintessential example of a substance with “food-medicine homology.” Originating from traditional Chinese medicine, this concept refers to edible substances that serve not only as a source of daily nutrition but also as functional components with physiological effects that support health maintenance (10). Although this notion is deeply rooted in traditional medicine, its relevance to modern food science lies in the scientifically validated bioactivities of the constituents found in lotus seeds. Lotus seeds are rich in starch and protein (11) and contain a variety of bioactive compounds, including alkaloids and polyphenols (12). Among these, certain alkaloids—such as nuciferine—have been scientifically validated for their sleep-promoting effects (13), highlighting the potential of lotus seeds as functional ingredients for sleep-related dietary interventions. However, the direct consumption of lotus seeds presents several limitations. Their dense starch granule structure may increase digestive burden, while their astringent characteristics can cause gastrointestinal discomfort (14). Notably, lactic acid bacteria fermentation may provide an effective strategy to overcome these limitations and enhance the nutritional and physiological value of lotus seeds by producing beneficial metabolites (15). Consequently, yogurt fermentation may not only alleviate the adverse effects associated with direct lotus seed consumption, but also facilitate the biotransformation of lotus seed components, thereby improving their bioavailability and functional properties. This approach aligns closely with the core principles of food-medicine homology, leveraging modern food processing techniques to transform a traditionally recognized health-promoting ingredient into a more digestible and bioavailable functional food matrix.

Therefore, the present study aimed to develop a novel plant-based yogurt using lotus seeds as the main raw material, with the goal of achieving a smooth texture, desirable flavor, and good storage stability. Lactic acid bacteria strains were systematically screened, and fermentation conditions were optimized through single-factor experiments and response surface methodology. The resulting product was comprehensively characterized in terms of nutritional composition, rheological properties, flavor evolution during fermentation, microstructural features, and refrigerated storage stability. We hypothesize that optimizing fermentation parameters and probiotic strains will lead to a lotus seed-based yogurt with desirable physicochemical, sensory, and nutritional properties, while also preserving or enhancing bioactive alkaloids. To test this hypothesis, several alkaloids in the finished product were quantitatively analyzed, providing a compositional basis for its potential sleep-aid functionality.

Overall, this study offers a novel plant-based yogurt product that aligns with consumer demand for healthy and functional foods, while also promoting the high-value utilization of lotus seeds. The findings provide both scientific and practical insights into the development of functional plant-based fermented foods and contribute to the diversification and value enhancement of the plant-based food industry.

2 Materials and methods

2.1 Materials

Frozen fresh lotus seeds were purchased from Fuzhou Shengdian Food Co., Ltd. (Fuzhou, China), as detailed in Supplementary Table S1. Lactobacillus helveticus LH76, Lactobacillus delbrueckii subsp. bulgaricus LB42, Lactiplantibacillus plantarum Lp90, Lactobacillus acidophilus LA85, Lacticaseibacillus rhamnosus LRa05, and Streptococcus thermophilus ST81 were obtained from Weikang Probiotics (Suzhou) Co., Ltd. (Suzhou, China). MRS agar and MRS broth medium were purchased from Guangdong Huankai Microbial Science & Technology Co., Ltd. (Guangdong, China). Sodium chloride, sodium hydroxide, phenolphthalein, and absolute ethanol were purchased from China National Medicines Corporation Ltd. (Beijing, China). Glucose was supplied by Fuzhou Haiwang Fuyo Pharmaceutical Co., Ltd. (Fuzhou, China). A commercial dairy yogurt (Classy-Kiss) was purchased from Yonghui Supermarket (Fuzhou, China). The product was formulated with raw milk as the primary ingredient, constituting ≥ 80% (w/w).

2.2 Preparation of plant-based lotus seed yogurt

First, the frozen lotus seeds (with embryos removed) were thawed, rinsed and blended with purified water at 1:3 (w/v) to obtain a smooth slurry. Glucose was then added to the slurry, and the mixture was filled into containers, hermetically sealed, and pasteurized at 95 °C for 15 min. After cooling, the sterile slurry was inoculated with the lactic acid bacteria strain and fermented at a controlled temperature. The resulting yogurt was finally cooled to 4 °C and held for 24 h to complete post-ripening, yielding the finished yogurt.

For lactic acid bacteria strain optimization, L. helveticus, L. bulgaricus, L. plantarum, L. acidophilus, L. rhamnosus and S. thermophilus were individually activated in MRS broth through three successive subcultures at 37 °C for 48 h. Each strain was inoculated into lotus seed slurry at 4% (v/v), resulting in an initial viable count of approximately 4 × 106 CFU/g, and fermented at 37 °C for 12 h. Fermented samples were evaluated for water-holding capacity (WHC), pH, acidity, and viable counts according to Zhao et al. (16). Next, three optimal strains (L. bulgaricus, L. plantarum, S. thermophilus) were selected and combined in ratios of 2:1:1, 1:2:1, and 1:1:2. This design was based on the established principle that a dominant strain inevitably emerges in mixed lactic acid bacteria strains (17). In these ratios, the dominant strain was set to comprise 50% of the inoculum, with each auxiliary strain accounting for 25%, in accordance with the approach reported by Barrette et al. (18). A constant total inoculum size was maintained across all formulations to ensure comparability. Fermentation was conducted under the same conditions and evaluated as described above.

2.3 Optimization of preparation process for plant-based lotus seed yogurt

2.3.1 Single-factor experiments

Based on the fermentation process outlined in Section 2.2, single-factor experiments were conducted to evaluate the effects of glucose addition (6%, 9%, 12%, 15%, m/m), inoculum size (2%, 4%, 6%, v/v), fermentation temperature (36 °C, 38 °C, 40 °C, 42 °C), and fermentation time (6 h, 8 h, 10 h, 12 h). Sensory score was used as the evaluation criterion.

2.3.2 Response surface methodology experiment

Based on the results obtained from single-factor experiments, a four-factor, three-level Box-Behnken design was established using Design-Expert 12 software (19). The independent variables were glucose addition (A), inoculum size (B), fermentation temperature (C), and fermentation time (D). WHC (Y₁) and sensory score (Y₂) were selected as response variables. The experimental design is presented in Table 1. The experimental data were fitted to a second-order polynomial model as follows:

2.3.3 Sensory evaluation of plant-based lotus seed yogurt

Sensory evaluation was conducted using the quantitative descriptive analysis (QDA) method, with reference to the description by Talens et al. (20) and with slight modifications. Sensory evaluation was conducted by a panel of 10 assessors under controlled conditions (22 ± 2 °C). All panelists had at least one year of experience in evaluating dairy or plant-based products. Prior to formal evaluation, a calibration session was held to familiarize the panelists with the evaluation criteria and to standardize scoring using reference samples. Samples were presented in a randomized, blind-coded order and evaluated for texture (30 points), mouthfeel (30 points), aroma (20 points), and color (20 points), as shown in Table 2, with higher weights assigned to texture and mouthfeel (30 points each) as these attributes are widely recognized as the most critical factors influencing product stability and consumer perception in plant-based fermented products (3).

2.4 Analysis of nutritional components and quality of plant-based lotus seed yogurt

2.4.1 Determination of nutritional components

The nutritional composition of the plant-based lotus seed yogurt was determined and compared with that of a commercial dairy yogurt (Classy-Kiss). Protein, fat, and moisture contents were analyzed according to AOAC methods (21). Carbohydrates were determined from the difference in the nutrients according to Legislative Decree n. 77 of 16 February 1993, and energy value was calculated using standard conversion factors (9.0 kcal/g for fat, and 4.0 kcal/g for protein and carbohydrate) (22). Starch content was determined following McCleary et al. (23), and dietary fiber was measured using AOAC method 985.29.

2.4.2 Determination of physicochemical and microbiological indicators

E. coli counts were determined according to ISO 7251:2005 (24). Samples were diluted and inoculated into LST broth (37 °C), then positive tubes were transferred to EC broth (44 °C) and confirmed by indole test. The detection limit was 3 MPN/g. Viscosity was measured using an Alpha Re rotational viscometer, and color parameters (L^*, a^*, b^*) were recorded using ADCLP fully automatic colorimeter.

2.4.3 Determination of rheological properties

Rheological measurements of unfermented lotus seed slurry, plant-based lotus seed yogurt, and commercial dairy yogurt were conducted using an MCR 3031 rheometer (Anton Paar, Austria) following Devnani et al. (25) with minor modifications. A 40-mm parallel plate geometry with a 1-mm gap was used at a constant temperature of 25 °C. Apparent viscosity was evaluated through a shear rate sweep from 0.1 to 100 s⁻¹. Viscoelastic properties were characterized by frequency sweep measurements at a fixed strain of 1% over an angular frequency range of 0.1–10 rad/s, from which the storage modulus (G′) and loss modulus (G″) were determined.

2.4.4 Flavor component analysis

Volatile compounds were analyzed using a PEN3.5 electronic nose (AIRSENSE GmbH, Germany) following a method of Yuan et al. (26) with slight modifications. For each measurement, 5 mL of sample was placed in a 20 mL headspace vial and incubated at 50 °C for 30 min prior to analysis.

Taste profiles were evaluated with an SA402B electronic tongue (INSENT Co., Japan) following a method of Yuan et al. (26) with slight modifications. Sample preparation involved diluting 10 mL of the yogurt 90-fold with distilled water before measurement.

2.4.5 Microstructural analysis

Scanning Electron Microscopy (SEM): Microstructural analysis was performed using a Sigma 300 scanning electron microscope (ZEISS, Germany), following the method described by Li et al. (27) with minor modifications. Samples were freeze-dried and gold-coated prior to observation. Observations were conducted at an accelerating voltage of 30 kV to examine the microstructure of both plant-based lotus seed yogurt and unfermented lotus seed slurry.

FTIR Analysis: The short-range ordered structures of the plant-based lotus seed yogurt and unfermented lotus seed slurry were analyzed by FTIR spectroscopy, following the method described by Li et al. (27) with minor modifications. Briefly, the freeze-dried sample powder was mixed with potassium bromide (KBr) at a ratio of 1:100 (w/w), thoroughly ground, and pressed into a transparent pellet. FTIR spectra were collected at a resolution of 4 cm⁻¹ over the wavenumber range of 400–4000 cm⁻¹.

2.4.6 Storage period experiment

Storage stability was evaluated during 31 days of refrigerated storage (4 °C). Changes in pH, WHC, and viable probiotic counts were monitored at 5-day intervals.

2.5 Analysis of alkaloids by HPLC-MS/MS

2.5.1 Sample preparation

Alkaloids were extracted from yogurt samples using ultrasound-assisted ethanol extraction as described by Shi et al. (28) using ultrasound-assisted extraction method with ethanol as the solvent. Extracts were centrifuged and filtered through a 0.22 μm membrane prior to analysis.

2.5.2 Chromatographic conditions

The analytical separation was performed using an ExionLC UPLC system. The column was equilibrated with a binary gradient consisting of Mobile Phase A (MPA; water with 0.1% (v/v) formic acid) and Mobile Phase B (MPB; acetonitrile) under the initial conditions of 95% MPA and 5% MPB. The flow rate was set at 0.80 mL/min, and the column temperature was maintained at 40 °C throughout the analysis. A linear gradient elution program was applied as follows: 0–4.0 min, 5% MPB; 4.0–6.0 min, 80% MPB; 6.0–6.1 min, 5% MPB; 6.1–8.0 min, 5% MPB. Detection was conducted in positive ion mode using MRM on a QTRAP 4,500 mass spectrometer. The total chromatographic run time was 8 min.

2.5.3 Identification and quantification

Target alkaloids were identified by matching retention times and characteristic MRM transitions with authentic standards. Quantification was performed using the external standard method. Calibration curves were constructed using quadratic regression, and alkaloid concentrations were calculated from the corresponding calibration equations (Tables 3, 4).

2.6 Statistical analysis

All experiments were conducted in triplicate, and results are expressed as mean ± SD. Statistical analysis was performed using SPSS 25 with one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post-hoc test for multiple comparisons. Significance was defined at p < 0.05. Figures were generated using Origin 2024.

3 Results

3.1 Screening of optimal strains and inoculation ratios

The six strains exhibited distinct fermentation performances (Table 5). L. bulgaricus and L. plantarum demonstrated superior WHC, viable counts, and acidity. L. helveticus and L. rhamnosus showed poor overall performance, while S. thermophilus and L. acidophilus exhibited comparable fermentation behavior. Based on these results, L. bulgaricus, L. plantarum, and S. thermophilus were selected as the optimal strains for subsequent mixed-culture fermentation.

Three mixed-culture ratios of L. bulgaricus, L. plantarum, and S. thermophilus (2:1:1, 1:2:1, and 1:1:2) were evaluated (Figure 1). All mixed cultures outperformed single-strain fermentations, suggesting the benefits of microbial synergy. Among them, the 1:2:1 ratio achieved the highest WHC and viable counts; while the other two groups showed inferior performance.

Figure 1

3.2 Optimization of fermentation parameters for lotus seed-based vegan yogurt

The single-factor experimental results showed that the highest sensory scores were achieved at a glucose concentration of 12%, inoculum size of 4%, fermentation temperature of 38 °C, and fermentation time of 8 h (Figure 2). These conditions were selected as the center points for response surface methodology optimization.

Figure 2

A four-factor, three-level Box-Behnken design was applied to optimize glucose addition (A), inoculum size (B), temperature (C), and time (D), with WHC (Y1) and sensory score (Y₂) as responses (Table 6). Both models were highly significant (p < 0.01) with non-significant lack of fit (p > 0.05). High coefficients of determination (R²=0.9679 and 0.9970), the adjusted values (0.9357–0.9940), and low coefficients of variation (CV) values (1.33% and 0.8226%) confirmed a good fit and high reliability of the model. Based on the p-values, the linear terms B and D, the interaction terms BD and CD, and the quadratic terms A², B², C², and D² have a highly significant influence on WHC (p<0.01). The interaction term AD has a significant effect on WHC (p<0.05) (Table 7; Figure 3). For sensory evaluation, the linear terms A, B, C, and D, the interaction terms AB, AC, AD, and BC, and the quadratic terms A², B², C², and D² are highly significant (p<0.01), while the interaction term BD is significant (p<0.05) (Table 8; Figure 4). The remaining terms do not show a significant influence on either WHC or sensory evaluation.

Figure 3

Figure 4

The predicted optimal conditions differed slightly for maximizing WHC and sensory score. Considering practical feasibility, final conditions were set at 12.2% glucose, 3.7% inoculum, 38 °C, and 7.5 h. Validation experiments yielded a WHC of 68.57% and a sensory score of 80.9, closely matching predictions. The resulting yogurt exhibited a uniform structure, smooth mouthfeel, balanced acidity, and characteristic lotus seed aroma (Figure 5).

Figure 5

3.3 Analysis of nutritional profile and physicochemical properties of lotus seed-based vegan yogurt

The lotus seed-based yogurt contained lower protein but significantly higher dietary fiber and carbohydrates compared to dairy yogurt (p < 0.05), with very low fat content (0.25 ± 0.01 g/100 g) and reduced energy value (272.47 ± 7.71 kJ/100 g) (Table 9). The lotus seed-based yogurt exhibited viable probiotic counts comparable to those of commercial dairy yogurt, and no E. coli was detected in either product. It also showed lower acidity (pH 4.01 ± 0.03) but significantly higher WHC (68.57% ± 0.15%) than dairy yogurt, along with higher viscosity (1,447.67 ± 4.29 mPa·s).

Colorimetric analysis revealed that the lotus seed–based yogurt had a warm ivory color, with L^* value of 65.57 ± 0.08, a^* value of −22.85±0.06, and b^* value of 5.85 ± 0.20 (Table 10). Rheological analysis indicated that all tested samples exhibited shear-thinning behavior (Figure 6A). The lotus seed-based yogurt displayed higher apparent viscosity than dairy yogurt, and fermentation significantly increased the viscosity compared to the unfermented lotus seed slurry. Frequency sweep analysis confirmed solid-like gel behavior after fermentation, with storage modulus (G′) consistently exceeding loss modulus (G″) (Figure 6B). In contrast, the unfermented lotus seed slurry showed nearly overlapping G′ and G″ curves.

Figure 6

The flavor evolution during fermentation was monitored using an electronic nose (Figure 7A). During fermentation, increased sensor responses associated with alcohols, aldehydes, and ketones were observed. The combined response intensity of the W1-series sensors (W1W, W1S) was significantly higher than that of the W2S sensor (p < 0.05). Electronic tongue analysis revealed an increase in sourness and a decrease in sweetness after fermentation (Figure 7B). Additionally, noticeable changes in bitterness and umami responses were detected.

Figure 7

Significant microstructural changes were observed when comparing the scanning electron microscopy (SEM) images of unfermented lotus seed slurry and plant-based lotus seed yogurt (Figure 8). The images revealed a transition from a loose, porous structure in the unfermented slurry to a compact, homogeneous gel network in the yogurt after fermentation.

Figure 8

The FTIR spectra (Figure 9) revealed that the primary absorption peaks remained at consistent wavenumbers before and after fermentation. A noticeable broadening and increased intensity of the band at 3300–3500 cm⁻¹ were observed in the plant-based lotus seed yogurt.

Figure 9

The storage stability of the plant-based lotus seed yogurt was evaluated under refrigerated conditions at 4 °C (Figure 10). During storage, the pH gradually decreased to 3.49±0.05. The WHC increased during the first few days of storage, reaching a peak around day 6, before gradually declining. Probiotic counts increased from 133.33 ± 7.23 × 106 CFU/g to 137.33 ± 3.79 × 106 CFU/g during the initial stage of storage, followed by a significant decrease with prolonged storage.

Figure 10

3.4 Quantitative analysis of alkaloids in plant-based lotus seed yogurt

Quantitative analysis confirmed the preservation of key sleep-related alkaloids in the plant-based lotus seed yogurt following fermentation (Figure 11). In the unfermented slurry, the concentrations of nuciferine, liensinine, neferine, and armepavine were measured at 1.983 μg/L, 4.010 μg/L, 48.666 μg/L, and 30.696 μg/L, respectively, while isococlaurine was not detected. After fermentation, these alkaloids were still preserved in the final product. The levels of nuciferine (4.205 μg/L), liensinine (7.883 μg/L), and armepavine (44.138 μg/L) remained present, with neferine also preserved at a measured concentration of 137.122 μg/L in the yogurt. Although the neferine value exceeded the method's upper quantification limit and warrants cautious interpretation, its detection confirms its presence after fermentation. Isococlaurine was not detected at any stage.

Figure 11

4 Discussion

Plant-based yogurt is gaining increasing market favor due to its health and environmental attributes (6). However, developing products from high-starch plants like lotus seeds still faces significant technological bottlenecks. Lotus seeds have an extremely high starch content (typically exceeding 60%), and their unique physicochemical properties lead to inherent difficulties during direct fermentation, including the formation of a fragile gel network, uneven texture, severe whey separation, and insufficient fermentation vigor (29, 30). These challenges have rendered it a “difficult-to-process” raw material for yogurt production. The innovation of this study lies in successfully developing a plant-based yogurt using lotus seeds as the primary matrix by optimizing the fermentation culture (Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus bulgaricus in a 1:2:1 ratio). A comprehensive characterization of its nutritional composition, physicochemical and rheological properties, microstructure, and storage stability was conducted. This work provides a valuable strategy for addressing the technical barriers in applying similar plant resources to fermented foods and offers new insights for developing more diverse and localized plant-based yogurt products.

Nutritional analysis confirmed that the final product possesses a favorable profile for health-conscious consumers, being low in fat (0.25 ± 0.01 g/100 g) and calories (272.47 ± 7.71 kJ/100 g) while high in dietary fiber. This composition addresses growing dietary trends focused on weight management and metabolic health, offering a distinct advantage over conventional dairy yogurt for specific consumer groups. Physicochemical evaluation revealed a viscosity of 1,447.67 ± 4.29 mPa·s and a WHC of 68.57% for the lotus seed-based yogurt. These values are superior to those of typical dairy yogurts. The enhanced viscosity and water retention are indicative of a robust gel network, a critical factor for texture and shelf-life. This observation is consistent with the findings of Mäkinen et al. (31). The distinctive warm ivory color (L^* = 65.57 ± 0.08, a^* = −22.85 ± 0.06, b^* = 5.85 ± 0.20) is attributed to the inherent pigments of lotus seeds, differentiating the product visually from commercial dairy counterparts (32).

Rheological assessment confirmed the characteristic shear-thinning behavior of yogurt. Notably, the lotus seed-based yogurt exhibited a higher apparent viscosity than the commercial dairy control across the shear rate range, suggesting a richer, more ‘full-bodied' consistency. Dynamic frequency sweeps revealed that both products maintained a predominant solid-like character (G′ > G″). However, a more detailed comparison showed that the commercial dairy yogurt possessed a significantly higher storage modulus (G′) than the lotus seed-based yogurt, indicating that the casein-based matrix forms a stiffer and more robust gel network compared to the plant-protein/starch complex. While the lotus seed yogurt offers a “softer” gel texture, its structural integrity, as corroborated by SEM evidence showing a transition from a porous, discontinuous slurry to a dense, homogeneous gel after fermentation, is sufficient to resist structural breakdown during handling and storage. This structural integrity likely originates from synergistic interactions between lotus seed starch, proteins, and microbial exopolysaccharides produced during fermentation (33, 34).

The flavor and taste evolution during the fermentation of lotus seed-based yogurt, characterized by E-nose and E-tongue analyses, provides important insights into the formation of sensory quality in starch-rich plant-based fermented foods (35). The increased signals for alcohols, aldehydes, and ketones detected by the E-nose are a direct result of lactic acid bacteria metabolism. These volatile metabolites enrich the flavor complexity of the product, which aligns with the reported patterns of flavor formation in lactic acid bacteria-fermented plant-based beverages (36). The quantitative signal differences between the W1 and W2S sensor arrays confirm the dominant role of the characteristic lotus seed aroma throughout fermentation. The preservation of this core raw material aroma and its synergistic interaction with fermentation-derived volatile compounds are key to flavor optimization in plant-based fermented foods, consistent with findings in other fermented food systems where the raw material's aroma is retained and enhanced (37). The inversely correlated changes in sweetness and sourness revealed by the E-tongue are typical characteristics of plant-based fermentation systems, as has been well-documented in fermented legume products (38). Meanwhile, the alterations in bitterness and umami originate from the proteolytic activity of lactic acid bacteria, generating small-molecule peptides and free amino acids (39) as key taste-active compounds that significantly shape the product's aftertaste and overall flavor profile (40), ultimately enhancing the sensory quality of the plant-based lotus seed yogurt.

The FTIR spectra revealed that the primary absorption peaks remained at consistent wavenumbers before and after fermentation, suggesting that the covalent backbone and chemical integrity of the major components (e.g., proteins and polysaccharides) in lotus seed slurry were largely preserved (30). However, a noticeable broadening and increased intensity of the band at 3300–3500 cm⁻¹ were observed in the plant-based lotus seed yogurt. This band corresponds to the stretching vibrations of -OH and -NH groups, and its intensification indicates the formation of a more extensive hydrogen-bonded network. This structural evolution is attributed to the acidification and protein denaturing during fermentation, which facilitates intermolecular cross-linking and contributes to the formation of the stable gel matrix of the yogurt (31).

The post-acidification observed during storage is commonly reported in fermented dairy and plant-based products and is attributed to the continued metabolic activity of residual lactic acid bacteria even under refrigerated conditions (41). This process contributes to the characteristic tangy flavor of yogurt, and the pH remained within an acceptable range throughout the storage period, indicating that the product retained good quality without excessive acidification. The initial increase in WHC is likely due to starch retrogradation, where amylose and amylopectin chains reassociate through hydrogen bonding, forming a more compact gel network that traps water more effectively (42). As storage continues, the gel network undergoes slow structural relaxation, eventually leading to syneresis and a progressive loss of water retention, a common phenomenon in starch-based gel systems (43).The initial increase in probiotic counts is likely due to continued growth using residual nutrients, while the subsequent decline reflects the combined effects of nutrient depletion, increased acidity, and cold stress during extended refrigerated storage (44).

In this study, HPLC-MS/MS analysis was also employed to characterize the alkaloid components in lotus seed-based yogurt. A key finding of this work is the significant fermentation-mediated enrichment of bioactive isoquinoline alkaloids native to lotus seeds, such as nuciferine and liensinine. We propose that this enrichment results from the biotransformative activity of the lactic acid bacteria consortium (45). Specifically, bacterial enzymes such as glycosidases may hydrolyze conjugated or glycosylated forms of these alkaloids, releasing free, bioaccessible monomers (46). Similar biotransformation mechanisms have been reported in fermented plant matrices, where lactic acid bacteria mediate the conversion of bound phenolic compounds and alkaloids into their free forms (46, 47). This positions fermentation not just as a processing step for texture and preservation, but as a targeted bioprocessing strategy to amplify the intrinsic functional properties of the plant matrix. It should be noted, however, that the detected concentration of neferine exceeded the upper limit of quantification of the analytical method used; therefore, the reported value should be interpreted as semi-quantitative. Nevertheless, the detection above this threshold qualitatively confirms its persistence after fermentation. Given the documented pharmacological activities of these alkaloids (e.g., sedative, anxiolytic) (13, 48), their enrichment following fermentation suggests the potential for functional retention. This compositional shift provides a basis for further investigation into the bioactivity of fermented lotus seed products. Nuciferine, for instance, has been shown to exert sedative and anxiolytic effects in animal models at oral doses of 50 and 100 mg/kg in a dose-dependent manner (13). It should be noted that the nuciferine concentrations detected in this study are substantially lower than the pharmacological doses used in animal studies, and the product is intended for regular dietary consumption rather than acute pharmacological intervention.

Despite these promising findings, this study has several limitations. The quantification of neferine was semi-quantitative due to concentrations exceeding the analytical range. The investigation was confined to in vitro analyses, lacking in vivo validation of bioavailability or functional activity. Additionally, microbiological safety assessment was limited to E. coli, without evaluation of other pathogens or probiotic viability during storage. Future work should address these gaps through improved quantification methods, in vivo studies, and expanded safety evaluations, alongside process scale-up to support industrial application.

5 Conclusion

In this study, we successfully established an optimized process for producing high-quality plant-based yogurt using lotus seeds as the raw material. The final product demonstrated distinct nutritional characteristics, including low fat, low calorie, and high dietary fiber content, aligning with modern demands for healthy diets. The uniform and dense gel network formed during fermentation was key to achieving the product's excellent WHC and favorable rheological properties. Notably, fermentation significantly increased the concentrations of multiple alkaloids associated with neuroregulation, providing a compositional basis for the potential development of functional plant-based foods with sleep-promoting properties.

Overall, this study offers a novel plant-based yogurt product that aligns with consumer demand for healthy and functional foods, while also promoting the high-value utilization of lotus seeds. In the context of the rapidly expanding plant-based food sector, this work introduces a novel application of lotus seeds—a traditional “food-medicine homology” ingredient—as a viable substrate for plant-based yogurt production. The innovative integration of fermentation optimization, quality evaluation, and bioactive alkaloid analysis provides a scientifically grounded framework for transforming underutilized agricultural resources into functional food products. This approach not only expands the diversity of plant-based yogurt offerings but also demonstrates a strategy for enhancing the functional value of plant-based matrices through targeted fermentation. By bridging traditional food heritage with modern food technology, this study offers a scalable and value-added solution that supports the sustainable development of the plant-based food industry.

Future research could focus on process scale-up, precise flavor modulation, and further validation of functional activities such as sleep promotion through in vitro or in vivo models.

References

• 1.

  WangQLiQZhangHWuJQinWCaiJ. The physicochemical, nutritional, and antioxidative properties of plant-based quinoa yogurt. Food Sci Nutr. (2025) 13:e70910. doi: 10.1002/fsn3.70910

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 2.

  MontemurroMVerniMRizzelloCGPontonioE. Design of a plant-based yogurt-like product fortified with hemp flour: formulation and characterization. Foods. (2023) 12:485. doi: 10.3390/foods12030485

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 3.

  RenWLiangHLiBLiJ. Towards ideal plant-based yogurts: evaluating component and processing effects on mouthfeel and stability. J Future Foods. (2025) 5:455–69. doi: 10.1016/j.jfutfo.2024.08.003

  • CrossRef
  • Google Scholar

• 4.

  CraigWJBrothersCJ. Nutritional content and health profile of non-dairy plant-based yogurt alternatives. Nutrients. (2021) 13:4069. doi: 10.3390/nu13114069

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 5.

  UedaJMFernández-RuizVMoralesPFerreiraAHelenoSACarochoMR. An update on fermented plant-based dairy alternatives: advances towards consumer acceptability and modern diets. Food Res Int. (2025) 221:117504. doi: 10.1016/j.foodres.2025.117504

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 6.

  DharPSanghviGSinghRRoopashreeRBhowmikASharmaMet al. Emerging trends in plant-based yogurt: novel raw materials, bio-functionality, stability, and sustainability perspectives – a review. Trends Food Sci Technol. (2025) 166:105402. doi: 10.1016/j.tifs.2025.105402

  • CrossRef
  • Google Scholar

• 7.

  ZangJLinTShiYLinYXuCMaKet al. Recent advances in exopolysaccharide–protein interactions in fermented dairy- and plant-based yogurts: mechanisms, influencing factors, health benefits, analytical techniques, and future directions. Compr Rev Food Sci Food Saf. (2025) 24:e70219. doi: 10.1111/1541-4337.70219

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 8.

  ZhengMSuHYouQZengSZhengBZhangYet al. An insight into the retrogradation behaviors and molecular structures of lotus seed starch-hydrocolloid blends. Food Chem. (2019) 295:548–55. doi: 10.1016/j.foodchem.2019.05.166

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 9.

  ChenCLiGZhuF. A novel starch from lotus (Nelumbo nucifera) seeds: composition, structure, properties and modifications. Food Hydrocoll. (2021) 120:106899. doi: 10.1016/j.foodhyd.2021.106899

  • CrossRef
  • Google Scholar

• 10.

  WangLZhuXLiuHSunB. Medicine and food homology substances: a review of bioactive ingredients, pharmacological effects and applications. Food Chem. (2025) 463:141111. doi: 10.1016/j.foodchem.2024.141111

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 11.

  LiuXDongWYiYWangLHouWAiYet al. Comparison of nutritional quality and functional active substances in different parts of eight lotus seed cultivars. Foods. (2024) 13:2335. doi: 10.3390/foods13152335

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 12.

  RajputMAKhanRA. Phytochemical screening, acute toxicity, anxiolytic and antidepressant activities of the Nelumbo nucifera fruit. Metab Brain Dis. (2017) 32:743–49. doi: 10.1007/s11011-017-9963-x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 13.

  ZhangHShiXWuYYangJQiuMLinQet al. Lotus leaf nuciferine improves sleep and reduces the low neuronal activity and brain tissue abnormalities associated with insomnia. Food Biosci. (2024) 62:105345. doi: 10.1016/j.fbio.2024.105345

  • CrossRef
  • Google Scholar

• 14.

  ChandakADhullSBChawlaPGoksenGRosePKAl ObaidSet al. Lotus (Nelumbo nucifera G) seed starch: understanding the impact of physical modification sequence (ultrasonication and HMT) on properties and in vitro digestibility. Int J Biol Macromol. (2024) 278:135032. doi: 10.1016/j.ijbiomac.2024.135032

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 15.

  WangZLinYLiuLZhengBZhangYZengH. Effect of lotus seed resistant starch on lactic acid conversion to butyric acid fermented by rat fecal microbiota. J Agric Food Chem. (2022) 70:1525–35. doi: 10.1021/acs.jafc.1c06000

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 16.

  ZhaoLLiaoJWangTZhaoH. Enhancement of nutritional value and sensory characteristics of quinoa fermented milk via fermentation with specific lactic acid bacteria. Foods. (2025) 14:1406. doi: 10.3390/foods14081406

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 17.

  ReddyMSVedamuthuERWashamCJReinboldGW. Associative growth studies in three-strain mixtures of lactic streptococci. Appl Microbiol. (1972) 24:953–57. doi: 10.1128/am.24.6.953-957.1972

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 18.

  BarretteJChampagneCPRoyDRodrigueN. The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media. J Ind Microbiol Biotechnol. (2000) 25:288–97. doi: 10.1038/sj.jim.7000105

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 19.

  Babacan CevikSKahramanKEkiciL. Production of oven-baked wheat chips enriched with red lentil: an optimization study by response surface methodology. J Food Sci Technol. (2022) 59:2243–54. doi: 10.1007/s13197-021-05237-8

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 20.

  TalensCVilliotSRiojaPAlvarez-SabatelSUrzainkiIAPaciulliM. Statistical modelling of yogurt alternatives with chickpea, oat, and hydrolyzed insect proteins: improving texture, protein quality, and sensory performance. Food Bioproc Tech. (2025) 19:22. doi: 10.1007/s11947-025-04112-0

  • CrossRef
  • Google Scholar

• 21.

  Joy UjirogheneOLiuLZhangSLuJZhangCLvJet al. Antioxidant capacity of germinated quinoa-based yoghurt and concomitant effect of sprouting on its functional properties. LWT Food Sci Technol. (2019) 116:108592. doi: 10.1016/j.lwt.2019.108592

  • CrossRef
  • Google Scholar

• 22.

  HuangKLiuYZhangYCaoHLuo DK YiCet al. Formulation of plant-based yoghurt from soybean and quinoa and evaluation of physicochemical, rheological, sensory and functional properties. Food Biosci. (2022) 49:101831. doi: 10.1016/j.fbio.2022.101831

  • CrossRef
  • Google Scholar

• 23.

  McClearyBVCharmierLMJMcKieVA. Measurement of starch: critical evaluation of current methodology. Starke. (2019) 71:1800146. doi: 10.1002/star.201800146

  • CrossRef
  • Google Scholar

• 24.

  ISO. ISO 7251:2005 – Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Detection and Enumeration of Presumptive Escherichia coli — Most Probable Number Technique (2005). Available online at: https://www.iso.org/standard/34568.html (Accessed April 18, 2026).

  • Google Scholar

• 25.

  DevnaniBOngLKentishSEScalesPJGrasSL. Physicochemical and rheological properties of commercial almond-based yoghurt alternatives to dairy and soy yoghurts. Future Foods. (2022) 6:100185. doi: 10.1016/j.fufo.2022.100185

  • CrossRef
  • Google Scholar

• 26.

  YuanNChiXYeQLiuHZhengN. Analysis of volatile organic compounds in milk during heat treatment based on e-nose, e-tongue and HS-SPME-GC-MS. Foods. (2023) 12:1071. doi: 10.3390/foods12051071

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 27.

  LiXChenWGaoJGaoWZhangYZengHet al. Structural changes of butyrylated lotus seed starch and its impact on the gut microbiota of rat in vitro fermentation. Food Hydrocoll. (2023) 139:108501. doi: 10.1016/j.foodhyd.2023.108501

  • CrossRef
  • Google Scholar

• 28.

  Shi MZ YuYLZhuSCGuYXYueZXYanTCet al. Boron nitride nanosheet-assisted matrix solid-phase dispersion microextraction of alkaloids from lotus plumule by high-performance liquid chromatography coupled with ultraviolet detection and ion mobility quadrupole time-of-flight mass spectrometry. Electrophoresis. (2022) 43:581–89. doi: 10.1002/elps.202100286

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 29.

  SuJQiuXPeiYZhangZLiuGLuanJet al. Physical stability of lotus seed and lily bulb beverage: the effects of homogenisation on particle size distribution, microstructure, rheological behaviour, and sensory properties. Foods. (2024) 13:769. doi: 10.3390/foods13050769

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 30.

  XuWWuXXiaCGuoZZhaiZChengYet al. Optimization of enzymatic hydrolysis and fermentation processing for set-type oat yogurt with favorable acidity and coagulated texture. Foods. (2024) 13:4180. doi: 10.3390/foods13244180

  • CrossRef
  • Google Scholar

• 31.

  MäkinenOEWanhalinnaVZanniniEArendtEK. Foods for special dietary needs: non-dairy plant-based milk substitutes and fermented dairy-type products. Crit Rev Food Sci Nutr. (2016) 56:339–49. doi: 10.1080/10408398.2012.761950

  • CrossRef
  • Google Scholar

• 32.

  MarlapatiLBashaRFSNavarreAKinchlaAJNoldenAA. Comparison of physical and compositional attributes between commercial plant-based and dairy yogurts. Foods. (2024) 13:984. doi: 10.3390/foods13070984

  • CrossRef
  • Google Scholar

• 33.

  ShaoDZhangJShaoTLiYHeHWangYet al. Modification of structure, pasting, and in vitro digestion properties of glutinous rice starch by different lactic acid bacteria fermentation. Foods. (2025) 14:367. doi: 10.3390/foods14030367

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 34.

  NaibahoJButulaNJonuziEKorzeniowskaMGLaaksonenOFösteMet al. Potential of brewers' spent grain in yogurt fermentation and evaluation of its impact in rheological behaviour, consistency, microstructural properties and acidity profile during the refrigerated storage. Food Hydrocoll. (2022) 125:107412. doi: 10.1016/j.foodhyd.2021.107412

  • CrossRef
  • Google Scholar

• 35.

  VanarajR. P BI, Mayakrishnan G, Kim IS, Seong-CheolKim. A systematic review of the applications of electronic nose and electronic tongue in food quality assessment and safety. Chemosensors. (2025) 13:161. doi: 10.3390/chemosensors13050161

  • CrossRef
  • Google Scholar

• 36.

  MaJDuJZhangWZhaoHMaYHongHet al. Analysis of flavor metabolism characteristics of quinoa (Chenopodium quinoa Willd) sprout plant beverage by GC-IMS combined with intelligent sensory technology. Food Res Int. (2025) 221:117373. doi: 10.1016/j.foodres.2025.117373

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 37.

  SunXYuYSalehASMYangXMaJGaoZet al. Characterization of aroma profiles of Chinese four most famous traditional red-cooked chickens using GC-MS, GC-IMS, and E-nose. Food Res Int. (2023) 173:113335. doi: 10.1016/j.foodres.2023.113335

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 38.

  EngelsWSiuJvan SchalkwijkSWesselinkWJacobsSBachmannH. Metabolic conversions by lactic acid bacteria during plant protein fermentations. Foods. (2022) 11:1005. doi: 10.3390/foods11071005

  • CrossRef
  • Google Scholar

• 39.

  YangXHongJWangLCaiCMoHWangJet al. Effect of lactic acid bacteria fermentation on plant-based products. Fermentation. (2024) 10:48. doi: 10.3390/fermentation10010048

  • CrossRef
  • Google Scholar

• 40.

  SchindlerSWittigMZelenaKKringsUBezJEisnerPet al. Lactic fermentation to improve the aroma of protein extracts of sweet lupin (Lupinus angustifolius). Food Chem. (2011) 128:330–37. doi: 10.1016/j.foodchem.2011.03.024

  • CrossRef
  • Google Scholar

• 41.

  AryanaKJOlsonDW. A 100-year review: yogurt and other cultured dairy products. J Dairy Sci. (2017) 100:9987–10013. doi: 10.3168/jds.2017-12981

  • CrossRef
  • Google Scholar

• 42.

  Dircio-MoralesMAVelazquezGSifuentes-NievesIFlores-SilvaPCFonseca-FloridoHAMendez-MontealvoG. Effect of retrograded starch with different amylose content on the rheological properties of stored yogurt. J Food Sci Technol. (2023) 60:2012–22. doi: 10.1007/s13197-023-05735-x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 43.

  AlmusallamIAAhmedIAMBabikerEEAl-JuhaimiFYSalehAQasemAAet al. Effect of date palm (Phoenix dactylifera L) spikelets extract on the physicochemical and microbial properties of set-type yogurt during cold storage. LWT Food Sci Technol. (2021) 148:111762. doi: 10.1016/j.lwt.2021.111762

  • CrossRef
  • Google Scholar

• 44.

  LiuXMaoBTangXZhangQZhaoJChenWet al. Bacterial viability retention in probiotic foods: a review. Crit Rev Food Sci Nutr. (2025) 65:7964–86. doi: 10.1080/10408398.2025.2488228

  • CrossRef
  • Google Scholar

• 45.

  LarsenNHenriksenMGCrocollCLiQLametschRPoojaryMMet al. Enhancing the sensory and nutritional properties of faba bean protein through fermentation with lactic acid bacteria and Bacillus spp. Int J Food Microbiol. (2025) 446:111549. doi: 10.1016/j.ijfoodmicro.2025.111549

  • CrossRef
  • Google Scholar

• 46.

  GayaPPeiroténÁLandeteJM. Expression of a β-glucosidase in bacteria with biotechnological interest confers them the ability to deglycosylate lignans and flavonoids in vegetal foods. Appl Microbiol Biotechnol. (2020) 104:4903–13. doi: 10.1007/s00253-020-10588-x

  • CrossRef
  • Google Scholar

• 47.

  ZhangLLiALiuHMoQZhongZ. Effects of lactic acid bacteria fermentation on the release and biotransformation of bound phenolics in ma bamboo shoots (Dendrocalamus latiflorus Munro). Foods. (2025) 14:2573. doi: 10.3390/foods14152573

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 48.

  PyneMEGoldNDMartinVJJ. Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera). Metab Eng. (2023) 77:162–73. doi: 10.1016/j.ymben.2023.03.010

  • CrossRef
  • Google Scholar