Enhancement of biological activity and volatile organic compounds of sunflower bee pollen via mixed Lactobacillus fermentation

Abstract

BACKGROUND

Lactobacillus fermentation represents an efficient approach for enhancing the nutritional, bioactive, and sensory properties of bee pollen. However, the biological activities and volatile organic compounds (VOCs) of Lactobacillus‐fermented sunflower bee pollen (FSBP) remain unclear.

RESULTS

In this study, we employed mixed fermentation with Lactiplantibacillus plantarum and Lacticaseibacillus casei, optimizing the process using single factor and orthogonal experiments. We then characterized the physicochemical properties, biological activities, and VOCs of unfermented sunflower bee pollen (USBP) and FSBP. Compared to USBP, FSBP exhibited 9.68% and 54.57% increases in total phenolic and flavonoid contents. Additionally, FSBP exhibited 26.08% and 17.59% increases in α‐glucosidase inhibitory activity (GIA) and tyrosinase inhibitory activity. Notably, antioxidant and anti‐inflammatory activities were significantly improved in FSBP. Headspace‐solid‐phase microextraction‐gas chromatography–mass spectrometry analysis identified 52 VOCs. The orthogonal partial least squares discriminant analysis (OPLS‐DA) revealed 16 key VOC markers distinguishing USBP and FSBP, including (1S)‐(−)‐α‐pinene, camphene, α‐pinene, ethyl butanoate, ethyl caprate, cis‐chrysanthenol, butanoic acid, pentanoic acid, octanoic acid, hexanoic acid, isobutyl methyl ketone, acetophenone, nonanal, tridecane, selina‐5,11‐diene, and p‐cymene.

CONCLUSION

In summary, Lactobacillus fermentation not only improves bioactivity but also modulates flavor, making bee pollen more suitable for food applications. © 2026 Society of Chemical Industry.

INTRODUCTION

Bee pollen is rich in carbohydrates, proteins, amino acids, phenolic compounds, vitamins, and essential nutrients.^{1, 2} It has various biological activities including antioxidant, anti‐inflammatory, and antimicrobial effects, ³ and is acclaimed as ‘the world's best food product’ and ‘only perfectly complete food’.^{4, 5}

Bee pollen is recognized as a nutrient rich natural product and could be used as a food supplement. ⁶ However, unprocessed pollen used in food formulations faces several challenges: (i) food allergy, a serious global health problem because certain pollen proteins can trigger immune responses^{7, 8}; (ii) low bioavailability, because the cell wall structure of sporopollenin reduces bioavailability of pollen nutrients by approximately 50%, which prevents extraction of intracellular bioactive compounds^{9, 10}; (iii) organoleptic issues, with its gritty texture and strong floral notes potentially limiting consumer acceptance. ¹¹ Therefore, adopting advanced wall‐disruption technologies is essential to promote nutrient release and enhance the biological activity of pollen.

Common technologies used to break the wall of bee pollen include physical, chemical, and enzymatic methods. ¹² Physical treatments such as superfine grinding and cobalt‐60 (⁶⁰Co)‐irradiation can effectively break the sporopollenin wall. However, they may damage heat‐sensitive bioactive compounds and increase energy consumption. ¹¹ Chemical approaches using acid or alkali reagents also disrupt the pollen wall structures, but they may cause chemical residues and nutrient loss. ¹³ Enzymatic hydrolysis with cellulases or pectinases provides substrate specificity, but its practical application is still limited because of high cost and strict reaction conditions. Compared with these methods, Lactobacillus fermentation is a milder and more environmentally friendly way to degrade pollen walls. Previous studies showed that Lactobacillus fermentation can break the pollen wall and increase the release of phenolic and flavonoid compounds, which may improve the bioaccessibility of active constituents.^{14, 15} Moreover, it also can reduce allergenic proteins ¹⁶ and promote the formation of beneficial metabolites such as short‐chain fatty acids, esters, and aromatic volatile compounds. ¹⁷

Lactiplantibacillus plantarum can produce cellulases, proteases, and pectinases, which may help degrade plant cell walls and release intracellular nutrients. During fermentation, Lactiplantibacillus plantarum also produces organic acids and other bioactive metabolites and may reduce allergenic proteins through hydrolysis or structural modification. ¹⁸ These metabolic activities contribute to improved fermentation efficiency, microbiological safety, and storage stability. ¹⁹ Lacticaseibacillus casei also plays an important role in regulating microbial metabolism and substrate utilization. Its synergistic interaction with Lactiplantibacillus plantarum may further strengthen pollen wall disruption and enhance the bioavailability of released nutrients. ²⁰ Therefore, mixed fermentation with Lactiplantibacillus plantarum and Lacticaseibacillus casei may improve the nutritional value and biological activity of bee pollen.

Despite recent progress in bee pollen fermentation research, several limitations remain: (i) most studies have focused on single‐strain fermentation, with limited attention to the synergistic effects among different Lactobacillus strains^{16, 17}; (ii) few studies have optimized mixed fermentation conditions for improving the release of bioactive compounds and flavor characteristics; and (iii) the relationship between fermentation‐related changes in volatile organic compounds (VOCs) and biological activities, including antioxidant and anti‐inflammatory activities, still needs further study. ²¹ In this context, mixed fermentation with complementary Lactobacillus strains offers distinct advantages. The strong cell wall‐degrading enzyme system of Lactiplantibacillus plantarum combined with the metabolic regulation capacity of Lacticaseibacillus casei synergistically promotes nutrient release and bioconversion, achieving higher efficiency than single‐strain fermentation. ²¹ Moreover, mixed fermentation diversifies microbial metabolites, thereby enriching bioactive compounds and enhancing the overall flavor profile. ¹⁷

This study investigated the effects of mixed fermentation with Lactiplantibacillus plantarum and Lacticaseibacillus casei on sunflower bee pollen. Changes in β‐carotene, protein hydrolysis, phenolic and flavonoid contents, VOCs, antioxidant activity, and anti‐inflammatory activity were evaluated. These findings may support the future application of fermented bee pollen in food products.

MATERIALS AND METHODS

Materials and sample preparation

Acetonitrile, ethanol, formic acid and methanol were obtained from Merck (Darmstadt, Germany). Standards for hyaluronidase, β‐carotene, bovine serum albumin, tyrosine, sodium hyaluronate, and other reagents were acquired from Shanghai Yuanye Bio‐Technology Co., Ltd (Shanghai, China). Enzyme assay kits for superoxide dismutase (SOD) and lipoxygenase (LOX) were supplied by Nanjing Jiancheng Biotechnology Co., Ltd (Nanjing, China).

Sunflower (Helianthus annuus L.) bee pollen was collected from apiaries in Inner Mongolia, China, and stored at −20 °C. Pollen species identification was performed using palynological analysis and scanning electron microscopy (SEM, SU8100; Hitachi Inc., Tokyo, Japan). Lactobacillus strains, including Lactobacillus delbrueckii subsp. bulgaricus (LB‐Z16), Lactiplantibacillus plantarum (JYLP‐326), Lactobacillus acidophilus (JYLA‐191), Lacticaseibacillus paracasei (JLPF‐176), Lacticaseibacillus casei (LC‐12) and Lacticaseibacillus rhamnosus (CRL 1505) were purchased from Duoaite Biotechnology Co., Ltd (Beijing, China).

The collected sunflower bee pollen was sterilized under ultraviolet (UV) light for 10 min and then dried in a vacuum oven until the moisture content was below 6%. Fermentation parameters were optimized using viable bacterial count and sensory score as evaluation metrics (Supporting Information Tables S1–S3). Each condition was tested in triplicate to ensure reproducibility. The optimized fermentation conditions were determined as follows: 40% water addition, fermentation temperature of 37 °C, 10% inoculation volume, and a strain ratio of Lactiplantibacillus. plantarum to Lacticaseibacillus casei of 3:1. Unfermented sunflower bee pollen (USBP) and Lactobacillus‐fermented sunflower bee pollen (FSBP) were prepared for subsequent analyses.

SEM of USBP and FSBP

Both USBP and FSBP were analyzed according to the method described by Sun et al., ²¹ and subsequently observed using a field‐emission scanning electron microscope (Zeiss Gemini SEM 360, Jena, Germany).

Basic parameters of USBP and FSBP

Total phenolic content (TPC) and total flavonoid content (TFC)

Total phenolic content (TPC) was determined using the Folin–Ciocalteu assay, and total flavonoid content (TFC) was quantified using the aluminum chloride (AlCl₃) colorimetric method, both in accordance with the processes detailed by Chen et al. ⁵

Reducing sugar content

The reducing sugar content was established using the 3,5‐dinitrosalicylic acid (DNS) colorimetric assay as described by Quoc et al. ²²

β‐Carotene content

The β‐carotene content was determined as described by Ding et al. ²³

Protein content and protein hydrolysis degree

Protein content was evaluated as described by Sun et al. ²¹ The degree of hydrolysis was calculated using the pH‐Stat method, as described by Xiaoli et al. ²⁴

α‐Glucosidase inhibitory activity (GIA)

The α‐glucosidase inhibitory activity (GIA) of bee pollen was assessed using an adapted enzymatic assay from Chen et al. ²⁵ A 100 μL sample was dissolved in α‐glucosidase solution (100 μL, 1 U/mL), and incubated for 10 min at 37 °C in a 0.1 mol/L phosphate buffer (pH 6.9). The mixture was then combined with p‐nitrophenyl‐α‐d‐glucopyranoside (200 μL, 5 mmol/L), and incubated for 20 min at 37 °C. Finally, the reaction was terminated by adding 500 μL of 1 mol/L sodium carbonate (Na₂CO₃) solution. Absorbance was measured at 405 nm and calculated as follows:

$$\mathrm{GIA}\left(\%\right)=\frac{1-A}{A_0}\times 100$$

where A refers to the absorbance of the reaction system containing both enzyme and bee pollen samples, whereas A ₀ represents the control containing enzyme but without the sample.

Antioxidant properties

For this assay, 4 g of sample was extracted with 40 mL of 80% (v/v) methanol. After sonication at 25 °C for 60 min and reflux extraction for 120 min, the mixture was centrifuged at 3000 × g for 10 min at 4 °C. Finally, the supernatant was adjusted to 100 mL and stored at 4 °C.

DPPH radical scavenging activity and FRAP assay

The DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) radical scavenging activity and FRAP (ferric reducing antioxidant power) assay were assessed following the method by Chen et al. ²⁶

Iron(II) ion chelating activity and ABTS ⁺ radical scavenging activity

The iron(II) ion (Fe²⁺) chelating activity and radical cation (2,2′‐azino‐di‐[3‐ethylbenzthiazoline sulphonate]) (ABTS⁺) scavenging activity were conducted following a previously established method. ²⁷

Determination of anti‐inflammatory ability

Hyaluronidase inhibition activity (HIA)

Hyaluronidase inhibition activity (HIA) was determined according to the method of Duan et al. ²⁸ with slight modifications. Tube A contained 0.5 mL of sample and 0.5 mL of hyaluronidase solution (500 U/mL). Tube B contained 0.5 mL of sample and 0.5 mL of acetate buffer (0.1 mol/L, pH 5.6). Tube C contained 0.5 mL of distilled water and 0.5 mL of hyaluronidase solution (500 U/mL), while Tube D contained 0.5 mL of distilled water and 0.5 mL of acetate buffer (0.1 mol/L, pH 5.6). All tubes were incubated at 37 °C for 20 min. Then, 100 μL of 2.5 mol/L calcium chloride (CaCl₂) solution was added and the mixtures were further incubated at 37 °C for 20 min. After that, 500 μL of sodium hyaluronate was added to Tubes A and C, whereas 0.5 mL of acetate buffer was added to Tubes B and D. The mixtures were incubated again at 37 °C for 40 min. Next, 0.5 mL of distilled water, 0.1 mL of 5 mol/L sodium hydroxide (NaOH), and 0.5 mL of acetylacetone were added to each tube. The mixtures were heated in a boiling water bath for 15 min and then cooled to room temperature. Finally, 1 mL of P‐DAB (p‐dimethylaminobenzaldehyde) chromogenic reagent was added, and absorbance was measured at 530 nm. The inhibition rate was calculated as follows:

$$\text{Inhibition rate}\left(\%\right)=\frac{\left(C-D\right)-\left(A-B\right)}{C-D}\times 100$$

SOD activity and LOX activity

SOD activity was measured by the xanthine oxidase method ²⁹ and LOX activity was analyzed by colorimetric method. ³⁰

Tyrosinase inhibition activity (TIA)

Tyrosinase inhibition activity (TIA) was measured using the method of Lin et al. ³¹ with slight modifications. Samples were dissolved in methanol (10 mg/mL) and diluted with phosphate buffer (50 mmol/L, pH 6.8). Then, 50 μL of sample solution was mixed with 50 μL of tyrosinase solution (200 U/mL) and incubated at 37 °C for 2 min. Next, 150 μL of L‐DOPA (l‐3,4‐dihydroxyphenylalanine, 0.5 mmol) was added. Absorbance was recorded at 475 nm every 10 s for 5 min.

$$\text{Inhibition rate}\left(\%\right)=\frac{k_0-k_1}{k_0}\times 100$$

where k ₁ represents the slope of the kinetics equation in the presence of the inhibitor, whereas k ₀ represents the slope for the reaction in the absence of the inhibitor.

Determination of VOCs by HS‐SPME‐GC–MS

VOCs were analyzed by headspace‐solid‐phase microextraction‐gas chromatography–mass spectrometry (HS‐SPME‐GC–MS). Briefly, 1 g of sample and 5 mL sodium chloride (NaCl) solution were added into a headspace vial (Agilent, Palo Alto, CA, USA). VOCs separation was carried out using an Agilent HP‐5MS capillary column (30 m × 0.25 mm, 0.25 μm). The injection port was 270 °C. High‐purity helium was used as the carrier gas at a flow rate of 1 mL/min. The MS system was operated in electrospray ionization (EI) mode with an electron energy of 70 eV and a filament emission current of 0.25 mA. The initial temperature was kept at 40 °C for 3 min, increased to 100 °C at 5 °C/min, and then increased to 220 °C at 3 °C/min and held for 10 min. The quadrupole and ionization source temperatures were 150 and 230 °C, respectively. The mass scanning range was 33–350 m/z with a scan time of 250 ms. VOCs were identified using the NIST17 mass spectral library, along with retention index (RI) matching. The calculated retention indices (RI^a) for VOCs were determined using a homologous series of n‐alkanes (C₇–C₃₀) analyzed under identical chromatographic conditions. The obtained RI^a values were then compared with literature retention indices (RI^b) reported under comparable GC–MS conditions.^{32, 33} Compound identification relied on the combined agreement of mass spectral similarity and RI matching. The identified VOCs, along with their calculated and literature RI values, are presented later.

Statistical analyses

Results are expressed as the mean ± standard deviation (SD, n = 3). One‐way analysis of variance (ANOVA) followed by Duncan's multiple range test was carried out using the SPSS software (version 27.0; IBM, Armonk, NY, USA) to analyze differences in physicochemical properties, bioactivities, and relative contents of VOCs between USBP and FSBP. Differences were considered statistically significant at P < 0.05, and significant differences between groups are indicated by different lowercase letters. GraphPad Prism 8.0.2 software (GraphPad Prism Inc., San Diego, CA, USA) was used for data visualization. Orthogonal partial least squares discriminant analysis (OPLS‐DA) was conducted using the SIMCA 14.1 software (Umeå, Västerbotten, Sweden) to identify key VOC markers. For OPLS‐DA, variables with a variable importance in projection (VIP) > 1 and P < 0.05 (determined via ANOVA) were defined as significant discriminant markers distinguishing USBP and FSBP.

RESULTS AND DISCUSSION

Effect of fermentation on bee pollen wall

The morphology of bee pollen grains underwent significant alterations following fermentation, as evidenced by our observations (Fig. 1). Prior to fermentation, the pollen grains exhibited a complete structure integrity, with the USBP wall tightly encapsulating the intracellular contents. The USBP displayed a regular spherical morphology characterized by three distinct germination pores and prominent surface spines as shown in Fig. 1(A1),(A2). However, after the fermentation process, the bee pollen cell walls were observed to have broken down, facilitating the release of the cytoplasmic contents as shown in Fig. 1(B1),(B2). These findings suggest that the fermentation process favors the destruction of the bee pollen cell walls.

Figure 1.

Scanning electron microscopy (SEM) morphology of sunflower bee pollen before and after fermentation. (A1, A2) unfermented sunflower bee pollen (USBP) samples; (B1, B2) Lactobacillus‐fermented sunflower bee pollen (FSBP) samples.

Differences in basic parameters between USBP and FSBP

Table 1 details changes in basic parameters between USBP and FSBP. The reducing sugar content, a key indicator of microbial metabolism, decreased significantly by 13.21% after fermentation (P < 0.05). This decrease may be caused by microbial metabolism during fermentation, where sugars are converted into acids, alcohols, and carbon dioxide. ³⁴

Table 1.

The basic parameters of unfermented sunflower bee pollen (USBP) and Lactobacillus‐fermented sunflower bee pollen (FSBP)

Sample	Total phenolic (g/kg)	Total flavonoid (g/kg)	Reducing sugar (g/kg)	β‐Carotene (g/kg)	Total protein (g/kg)	Protein hydrolysis (%)	GIA (%)
USBP	3.20 ± 0.01a	3.61 ± 0.20a	605.63 ± 9.21b	0.45 ± 0.03a	24.22 ± 0.21b	17.11 ± 0.0039a	42.76 ± 1.00a
FSBP	3.51 ± 0.02b	5.58 ± 0.03b	525.62 ± 6.24a	0.63 ± 0.05b	21.41 ± 1.03a	25.88 ± 0.0026b	53.91 ± 1.32b

Note: Differences were considered statistically significant at P < 0.05, and significant differences between groups are indicated by different lowercase letters. GIA, α‐glucosidase inhibitory activity.

β‐Carotene is an important pigment in bee pollen and has antioxidant, immune‐modulatory, anti‐aging, and chronic disease prevention. ³⁵ Furthermore, Lactobacillus has been showed to synthesize β‐carotene. ³⁵ The β‐carotene content in FSBP significantly increased to 0.63 ± 0.05 g/kg, 1.39 times higher than in USBP (Table 1). This suggests that fermentation enhances the synthesis and stabilization of β‐carotene.

The protein and amino acid content of bee pollen typically ranges from 11% to 35%, ⁴ making it the second most abundant component after carbohydrates. Thus, protein content is widely used to assess the nutritional quality and potential allergenicity of bee pollen. ⁸ Previous studies have demonstrated that fermentation can effectively decrease protein allergen content through hydrolysis while enhancing the physicochemical properties of bee pollen.^{19, 36} Our study found that mixed fermentation enhanced protein content and hydrolysis in sunflower bee pollen, effectively lowering allergen content, consistent with the results of Luan et al. ¹⁹

α‐Glucosidase, a critical enzyme in the small intestine, facilitates carbohydrate hydrolysis, significantly influencing digestion, glucose absorption, and the regulation of postprandial hyperglycemia. Therefore, assessing α‐glucosidase inhibition activity offers a rapid and reliable method to identify potential hypoglycemic substances in vitro.^{37, 38} The GIA of USBP was 42.76% ± 1.00%, whereas that of FSBP was significantly higher at 53.91% ± 1.32%, indicating a stronger inhibitory effect of FSBP compared to USBP. This result agrees with previous studies showing that lactic acid bacteria (LAB)‐fermented camellia pollen enhanced the inhibitory efficacy of α‐glucosidase. ³⁸ Therefore, fermented bee pollen may be an ideal alternative for enhancing the hypoglycemic potential. Phenolic compounds, particularly phenolic acids and flavonoids, are widely reported as major contributors to α‐glucosidase inhibition in plant‐derived matrices, through interactions with catalytic residues or competitive binding at the active site. ³⁹ In this study, mixed Lactobacillus fermentation markedly increased both TPC and TFC (Table 1), which likely contributed to the enhanced GIA observed in FSBP. However, since only TPC and TFC were quantified here, future studies employing ultra‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐QTOF‐MS/MS)‐based targeted or untargeted metabolomics are warranted to identify and quantify individual phenolic and flavonoid inhibitors, and to elucidate their specific structure–activity relationships.

Differences in antioxidant activities between USBP and FSBP

To evaluate the impact of fermentation on antioxidant potential, DPPH, FRAP, ABTS, and Fe²⁺‐chelating activities were compared between USBP and FSBP. As shown in Fig. 2, fermentation significantly enhanced the overall antioxidant capacity of FSBP (P < 0.05). The FRAP value increased by 97.66%, and the DPPH scavenging activity by 58.89%, while ABTS and Fe²⁺‐chelating capacities were 1.30‐ and 1.11‐fold higher, respectively. In addition, both TPC and TFC also increased significantly (Table 1). These results indicate that mixed Lactobacillus fermentation promotes the release and biotransformation of antioxidant compounds in sunflower bee pollen.

Figure 2.

Antioxidant activity of unfermented sunflower bee pollen (USBP) and Lactobacillus‐fermented sunflower bee pollen (FSBP) samples measured by DPPH radical scavenging capacity, FRAP assay, Fe²⁺ chelating activity and ABTS free radical scavenging activity assay.

The enhanced antioxidant activity observed in FSBP may be closely associated with the release and biotransformation of bound phenolic compounds during mixed Lactobacillus fermentation. In plant‐derived matrices, many phenolic acids and flavonoids naturally exist in esterified, glycosylated, or cell wall‐bound forms, which limits their extractability and biological activity. ⁴⁰ During mixed Lactobacillus fermentation, carbohydrate‐active enzymes secreted by Lactiplantibacillus plantarum and Lacticaseibacillus casei are presumed to degrade pollen‐wall polysaccharides, whereas hydrolytic enzymes such as feruloyl esterases (FAEs) and β‐glucosidases may cleave ester and glycosidic linkages, thereby releasing hydroxycinnamic acids and converting flavonoid glycosides into more bioavailable free phenolics and flavonoid aglycones.^{41, 42} Similar microbial biotransformation pathways have been reported in fermented cereals and tea matrices, where LAB fermentation enhanced the liberation of hydroxycinnamic acids and flavonoids with improved antioxidant activity. ⁴³

The conversion of bound phenolics into free forms is thought to improve antioxidant efficiency because free phenolic acids and flavonoid aglycones generally possess stronger electron‐donating capacity and radical‐scavenging ability. The antioxidant activity may be related to hydroxyl groups and conjugated aromatic structures, which can promote hydrogen transfer and stabilize free radicals. Meanwhile, the acidic conditions formed during lactic fermentation may help maintain phenolic stability by reducing oxidative degradation. ⁴⁴ Therefore, the enhanced antioxidant activity of FSBP is likely associated not only with increased TPC and TFC but also with qualitative changes in phenolic composition, molecular form, and bioavailability induced by microbial fermentation.

Nevertheless, the present study quantified only TPC and TFC, and the specific phenolic compounds responsible for the enhanced antioxidant and anti‐inflammatory activities remain unclear. Future studies combining targeted and untargeted UPLC‐QTOF‐MS/MS metabolomics will be valuable to identify differential phenolic and flavonoid compounds and further elucidate their structure–activity relationships.

Differences in anti‐inflammatory ability between USBP and FSBP

Differences in HIA between USBP and FSBP

Hyaluronic acid, a high molecular weight polysaccharide abundant in soft connective tissues extracellular matrix, is pivotal in regulating cell proliferation and migration and has been associated with various cancer types.^{45, 46} We used hyaluronic acid inhibition in vitro to evaluate its anti‐inflammatory and antiallergic properties. The results demonstrated that the HIA of USBP was 30.72%, whereas that of FSBP was 53.34% (Fig. 3), indicating that the inhibitory activity of USBP was 1.74 times lower than that of FSBP. These findings indicate that fermentation can enhance the anti‐inflammatory potential of bee pollen, improving its health value. Notably, polyphenols and flavonoids have been associated with hyaluronidase inhibition in various natural products, potentially by forming non‐covalent interactions with the enzyme and thereby reducing hyaluronic acid depolymerization. Given that fermentation significantly increased TPC/TFC in FSBP (Table 1), the enhanced HIA may be partially attributed to the improved extractability and/or bioconversion of phenolic/flavonoid constituents during fermentation.

Figure 3.

Anti‐inflammatory ability of unfermented sunflower bee pollen (USBP) and fermented sunflower bee pollen (FSBP) samples tested by hyaluronidase inhibition activity (HIA), superoxide dismutase (SOD) and lipoxygenase (LOX) and tyrosinase inhibition activity (TIA).

Differences in SOD and LOX activity between USBP and FSBP

SOD serves as a key antioxidant enzyme that directly neutralizes superoxide radicals by catalyzing their conversion into hydrogen peroxide and oxygen. This process helps eliminate excess superoxide anions in the human body, ⁴⁷ thereby contributing to anti‐aging, anti‐tumor, and radiation protection functions. Given that SOD activity is prone to degradation in bee pollen, it is important to evaluate how fermentation affects its enzymatic performance. In this study, SOD activity in FSBP increased by 26.95% (Fig. 3), suggesting improved anti‐aging and anti‐inflammatory activities.

LOX plays an important role in inflammation and tumor progression. Inhibiting LOX activity may reduce free radical formation and inflammatory responses.^{48, 49} Figure 3 shows that FSBP has significantly lower LOX activity than USBP, indicating that fermentation may enhance the anti‐inflammatory properties of sunflower bee pollen.

Differences in TIA between USBP and FSBP

Tyrosinase is an important enzyme involved in melanin formation and is related to skin aging, wound healing, insect growth, and browning in fruits and vegetables. Previous studies have shown that bee pollen can inhibit tyrosinase activity and may be used as a natural ingredient to reduce skin pigmentation associated with aging.^{27, 50} However, there are still few studies on the TIA of fermented bee pollen. In this study, clear differences were observed between USBP and FSBP (Fig. 3). FSBP showed significantly higher TIA, which was about 1.18 times higher than that of USBP. These results suggest that fermentation may improve the anti‐tyrosinase activity of bee pollen.

Differences in VOCs between USBP and FSBP

Analysis of the VOCs by HS‐SPME‐GC–MS

Fermentation has an important effect on the flavor profile of sunflower bee pollen. In this study, we employed the VOCs by HS‐SPME‐GC–MS to analyze the composition and changes in VOCs during the fermentation process of sunflower bee pollen (Table 2). A total of 52 VOCs were identified, including 21 terpenes, 13 esters, six alcohols, four acids, three ketones, two aldehydes, one alkane, and two other compounds. Notably, certain original VOCs present in USBP were no longer detected in the FSBP. Conversely, new VOCs were observed after fermentation. Specifically, caproic acid and 4‐isopropyltoluene were absent in FSBP but were replaced by newly synthesized compounds such as α‐selinene, (1S)‐(−)‐α‐pinene, ethyl palmitate, 1‐butanol, 2‐methyl‐(−)‐trans‐pinocarveol, and isobutyl methyl ketone. Importantly, these VOC changes are expected to produce sensory modifications, including the attenuation of unpleasant acidic or fatty notes and the enhancement of fruity and sweet notes, thereby potentially improving the palatability and consumer acceptability of FSBP.

Table 2.

The volatile organic compounds (VOCs) of unfermented sunflower bee pollen (USBP) and Lactobacillus‐fermented sunflower bee pollen (FSBP) using headspace‐solid‐phase microextraction‐gas chromatography–mass spectrometry (HS‐SPME‐GC–MS)

Number	Compound name	CAS	Molecular formula	RT (min)	Match	Relative content (%)		RIa (calculated)/RIb (literature)
						USBP	FSBP
Terpenoids (21)
1	α‐Thujene	2867‐5‐2	C10H16	3.109	870	0.61 ± 0.07	0.64 ± 0.05	929/929
2	4a,8‐Dimethyl‐2‐(prop‐1‐en‐2‐yl)‐1,2,3,4,4a,5,6,7‐octahydronaphthalene	103827‐22‐1	C15H24	5.41	908	1.35 ± 0.17	1.24 ± 0.42	1491/1492
3	Cedrene	11028‐42‐5	C15H24	8.933	824	2.15 ± 0.50	2.16 ± 0.03	1433/1422
4	β‐Selinene	17066‐67‐0	C15H24	11.786	857	0.30 ± 0.03	0.34 ± 0.03	1493/1486
5	Calarene	17334‐55‐3	C15H24	12.966	936	5.98 ± 1.10	5.17 ± 0.11	1430/1432
6	cis‐β‐Copaene	18252‐44‐3	C15H24	14.297	925	0.93 ± 0.18	0.86 ± 0.04	1437/1432
7	2,4‐Thujadiene	36262‐09‐6	C10H14	15.292	823	0.35 ± 0.04	0.15 ± 0.13	955/956
8	α‐Bulnesene	3691‐11‐0	C15H24	16.308	802	0.02 ± 0.03	0.01 ± 0.02	1508/1505
9	Copaene	3856‐25‐5	C15H24	16.573	847	0.41 ± 0.02	0.40 ± 0.07	1376/1376
10	α‐Cedrene	469‐61‐4	C15H24	20.406	832	0.14 ± 0.02	0.16 ± 0.01	1417/1411
11	α‐Selinene	473‐13‐2	C15H24	20.767	874	—	0.20 ± 0.17	1491/1494
12	Δ‐Cadinene	483‐76‐1	C15H24	20.916	891	0.37 ± 0.01	0.39 ± 0.03	1516/1524
13	β‐Elemene	515‐13‐9	C15H24	21.027	886	0.46 ± 0.06	0.55 ± 0.47	1389/1391
14	β‐Phellandrene	555‐10‐2	C10H16	25.039	933	3.77 ± 0.97	4.45 ± 0.15	1021/1031
15	Terpinolene	586‐62‐9	C10H16	26.525	811	0.15 ± 0.13	0.14 ± 0.12	1090/1088
16	(10S,11S)‐Himachala‐3(12),4‐diene	60909‐28‐6	C15H24	26.877	833	0.39 ± 0.06	0.13 ± 0.22	1399/1399
17	(1S)‐(−)‐ɑ‐Pinene	7785‐26‐4	C10H16	29.295	936	—	3.89 ± 0.28a	936/937
18	Camphene	79‐92‐5	C10H16	29.701	877	0.14 ± 0.04a	0.04 ± 0.00b	950/952
19	α‐Pinene	80‐56‐8	C10H16	30.465	936	7.90 ± 0.46a	2.20 ± 0.52b	933/937
20	γ‐Terpinene	99‐85‐4	C10H16	13.229	882	0.93 ± 0.11	1.08 ± 0.08	1050/1060
21	α‐Terpilene	99‐86‐5	C10H16	14.094	872	0.47 ± 0.07	0.45 ± 0.02	1010/1017
Esters (13)
22	β‐Terpinyl acetate	10198‐23‐9	C12H20O2	3.995	809	0.49 ± 0.02	0.50 ± 0.01	1272/1278
23	Ethyl butanoate	105‐54‐4	C6H12O2	5.488	942	0.77 ± 1.34b	5.64 ± 2.42a	784/802
24	Ethyl caprylate	106‐32‐1	C10H20O2	6.421	922	24.08 ± 3.95	22.77 ± 5.72	1194/1196
25	Ethyl dodecanoate	106‐33‐2	C14H28O2	7.48	896	0.61 ± 0.04b	1.42 ± 0.43a	1593/1595
26	Methyl caproate	106‐70‐7	C7H14O2	7.537	879	0.07 ± 0.12	0.05 ± 0.04	923/925
27	Ethyl isovalerate	108‐64‐5	C7H14O2	8.165	850	0.30 ± 0.09	0.28 ± 0.04	849/854
28	Ethyl caprate	110‐38‐3	C12H24O2	10.262	882	0.36 ± 0.31b	1.49 ± 0.44a	1392/1396
29	Methyl octanoate	111‐11‐5	C9H18O2	9.493	900	1.34 ± 0.18b	4.12 ± 1.84a	1128/1126
30	Ethyl pelargonate	123‐29‐5	C11H22O2	10.781	843	0.23 ± 0.20	0.48 ± 0.07	1280/1296
31	Ethyl palmitate	628‐97‐7	C18H36O2	27.167	811	–	0.03 ± 0.02	1994/1993
32	Ethyl 2‐methylbutyrate	7452‐79‐1	C7H14O2	28.721	850	0.41 ± 0.02	0.42 ± 0.08	848/849
33	Bornyl acetate	76‐49‐3	C12H20O2	28.926	905	0.44 ± 0.03	0.45 ± 0.01	1287/1285
34	Benzoic acid, ethyl ester	93‐89‐0	C9H10O2	33.333	826	0.86 ± 0.03	0.83 ± 0.03	1173/1171
Alcohols (6)
35	Benzyl alcohol	100‐51‐6	C7H8O	3.145	801	1.50 ± 0.25	1.27 ± 0.46	1035/1036
36	1‐Butanol, 2‐methyl‐	137‐32‐6	C5H12O	10.633	830	—	0.06 ± 0.05	736/739
37	trans‐Verbenol	1820‐09‐3	C10H16O	13.612	829	1.30 ± 0.61	0.77 ± 0.11	1151/1144
38	l‐trans‐Pinocarveol	547‐61‐5	C10H16O	24.472	897	—	0.20 ± 0.17	1139/1139
39	cis‐Chrysanthenol	55722‐60‐6	C10H16O	25.265	834	1.42 ± 0.08a	1.09 ± 0.05b	1173/1162
40	Phenylethyl alcohol	60‐12‐8	C8H10O	26.679	817	0.24 ± 0.21	0.22 ± 0.19	1120/1116
Acids (4)
41	Butanoic acid	107‐92‐6	C4H8O2	7.728	820	0.73 ± 0.04a	0.04 ± 0.00b	807/805
42	Pentanoic acid	109‐52‐4	C5H10O2	8.352	875	0.53 ± 0.04a	0.26 ± 0.10b	901/904
43	Octanoic acid	124‐07‐2	C8H16O2	10.262	890	8.87 ± 1.43b	10.28 ± 0.84a	1173/1180
44	Hexanoic acid	142‐62‐1	C6H12O2	11.575	804	0.74 ± 0.12a	—	982/990
Ketones (3)
45	Isobutyl methyl ketone	108‐10‐1	C6H12O	7.726	930	—	1.05 ± 0.04a	722/735
46	2‐Undecanone	112‐12‐9	C11H22O	10.631	885	0.39 ± 0.02	0.46 ± 0.05	1291/1294
47	Acetophenone	98‐86‐2	C8H8O	12.486	805	1.13 ± 0.02a	0.29 ± 0.51b	1068/1065
Aldehydes (2)
48	Nonanal	124‐19‐6	C9H18O	10.505	906	0.66 ± 0.04a	0.31 ± 0.08b	1112/1104
49	Benzaldehyde, 2,5‐dimethyl‐	5779‐94‐2	C9H10O	26.155	876	0.70 ± 0.17	0.33 ± 0.29	1208/1208
Alkanes (1)
50	Tridecane	629‐50‐5	C13H28	28.499	852	0.12 ± 0.02b	0.19 ± 0.00a	1300/1313
Others (2)
51	Selina‐5,11‐diene	52026‐55‐8	C15H24	24.318	841	0.02 ± 0.00b	0.04 ± 0.00a	1446/1447
52	p‐Cymene	99‐87‐6	C10H14	47.167	801	0.16 ± 0.03a	—	1014/1025

Note: RT, retention time; RI^a, retention indices calculated using n‐alkane series (C₇–C₃₀) under experimental conditions; RI^b, retention indices reported in the literature under comparable chromatographic conditions; ‘—’, not detected. The different lowercase letters indicate a significant difference (P < 0.05) among different groups.

Terpenes are important contributors to the floral and fruity aroma characteristics of bee pollen. ⁵¹ Although the overall terpene abundance decreased after fermentation, the emergence of newly detected terpenes such as α‐selinene and (1S)‐(−)‐α‐pinene in FSBP suggests active microbial biotransformation during mixed Lactobacillus fermentation. Many terpenes in plant materials are present in glycoside‐bound non‐volatile forms, which limits their direct contribution to aroma. During fermentation, β‐glucosidases and other glycosidases produced by LAB may hydrolyze glycosidic bonds and release free volatile terpenes, including α‐pinene and related compounds. ⁵² This may explain the appearance of new terpene compounds in FSBP despite the lower total terpene content.

Besides glycoside hydrolysis, terpene precursors may also be transformed during fermentation through reactions such as oxidation, reduction, rearrangement, and isomerization. ⁵³ These changes may occur under the acidic conditions formed during fermentation and may be related to microbial enzymes, including monooxygenases and reductases. As a result, some original terpene compounds may be converted into new aroma‐active substances. Similar changes in terpene composition have also been reported during dark tea fermentation, where microbial metabolism altered terpene profiles through glycosidase activity and other metabolic reactions. ⁵⁴ Therefore, mixed Lactobacillus fermentation may not only decrease some native terpenes but also release and transform bound terpene precursors, leading to changes in terpene composition and aroma characteristics in FSBP.

Esters are important aroma compounds that contribute to fruity and sweet flavors. They are usually formed during fermentation through reactions between fatty acids and alcohols. ⁵⁵ In this study, mixed Lactobacillus fermentation increased the levels of ethyl butanoate, methyl octanoate, ethyl caprate, and ethyl dodecanoate, suggesting enhanced ester formation in FSBP. These esters may improve the fruity and sweet aroma characteristics of FSBP. Lipases and esterases produced during fermentation may hydrolyze pollen lipids and release free fatty acids such as butanoic, octanoic, and hexanoic acids. At the same time, carbohydrate fermentation and amino acid metabolism may produce alcohols, including ethanol and branched‐chain alcohols. These fatty acids and alcohols may further react to form volatile esters with fruity aromas through microbial enzymatic reactions.^{56, 57}

The increase in ethyl butanoate in FSBP may be related to ester formation between butanoic acid and ethanol under acidic fermentation conditions. Higher levels of methyl octanoate and ethyl caprate also suggest increased ester formation from medium‐chain fatty acids during fermentation. At the same time, several free fatty acids decreased while ester compounds increased, indicating that fermentation promoted the conversion of fatty acids into esters. These changes may help reduce acidic or rancid odors and improve fruity and sweet aroma characteristics, which could enhance the sensory quality of FSBP. Similar changes in ester‐related aroma compounds have also been reported in other fermented plant foods. ⁵⁵ In addition, the interaction between Lactiplantibacillus plantarum and Lacticaseibacillus casei during fermentation may improve precursor utilization and ester formation, which could contribute to the richer aroma of FSBP. These results suggest that mixed Lactobacillus fermentation can change ester composition and improve the aroma characteristics of sunflower bee pollen.

The relative contents of acids slightly decreased after fermentation, especially several short‐ and medium‐chain fatty acids related to acidic or rancid odors, such as butyric, pentanoic, and hexanoic acids. These changes suggest that active microbial metabolism occurred during mixed Lactobacillus fermentation. Microbial esterases and alcohol acyltransferases may promote reactions between fatty acids and alcohols produced during fermentation. ⁵⁷ As a result, some acid compounds may be converted into volatile esters, leading to changes in the aroma characteristics of sunflower bee pollen.

As shown in Table 2, the relative proportion of acids decreased from 10.87% to 10.56%, accompanied by substantial reductions in butyric, pentanoic, and hexanoic acids, whereas total ester abundance increased by 28.51%. In particular, the disappearance or reduction of hexanoic acid together with the marked accumulation of esters such as ethyl butanoate and ethyl caprate further supports the occurrence of active microbial esterification pathways during fermentation. This metabolic flux shift from free fatty acid accumulation toward ester biosynthesis may reduce unpleasant acidic and fatty off‐notes while simultaneously enhancing fruity and sweet aroma characteristics. Similar aroma optimization mechanisms mediated by mixed LAB fermentation have also been reported in other fermented plant‐derived matrices. ⁵⁸ Therefore, mixed Lactobacillus fermentation appears to improve the sensory quality of sunflower bee pollen through coordinated microbial conversion of fatty acids into aroma‐active esters.

Aldehydes, primarily lipid‐derived volatile compounds, generally exhibit low odor thresholds. At low concentrations, they contribute pleasant aroma notes, whereas excessive accumulation may generate undesirable fatty, waxy, rancid, or irritating off‐flavors. ⁵⁹ Therefore, reducing aldehyde levels is beneficial for improving overall aroma quality. In this study, both aldehydes detected in sunflower bee pollen, namely nonanal and 2,5‐dimethylbenzaldehyde, decreased significantly after fermentation, suggesting active microbial redox metabolism during mixed Lactobacillus fermentation. Nonanal is commonly formed through lipid oxidation pathways and is frequently associated with fatty or waxy off‐notes at elevated concentrations. Mechanistically, LAB possess alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH), which can interconvert aldehydes, alcohols, and acids depending on the intracellular NADH/NAD⁺ balance. Under fermentative conditions, reducing equivalents (NADH) favor the reduction of aldehydes into their corresponding alcohols via ADH, whereas ALDH‐mediated oxidation can convert aldehydes into acids that subsequently participate in ester biosynthesis.^{60, 61, 62} These enzymatic transformations not only reduce aldehyde‐derived off‐flavors but also generate alcohol and acid precursors for ester formation. Consequently, mixed Lactobacillus fermentation appears to redirect metabolic flux away from aldehydes and toward alcohols and esters, thereby contributing to aroma rebalancing and improved sensory quality of FSBP.

Multivariate analysis of identified VOCs between USBP and FSBP

To further elucidate the changes in VOCs following fermentation and to link these changes with the bioactivity of sunflower bee pollen, we employed multivariate statistical analysis to process the VOC‐related data. We used the OPLS‐DA model to compare the VOCs profiles of USBP and FSBP. We conducted a random permutation of the sample sequence, which was randomly arranged 200 times when we established the OPLS‐DA model to assess the reliability of this model. The prediction parameters for the OPLS‐DA model were R ² X, R ² Y, and Q ². The closer the three parameters were to 1, the more stable and reliable the model. The OPLS‐DA model exhibited high explanatory and predictive capability, with R ² Y = 0.998 and Q ² = 0.944 (Fig. 4(A)). More importantly, USBP and FSBP were distinctly separated, suggesting that fermentation substantially alters the VOCs of bee pollen. Additionally, we conducted a permutation test to prevent overfitting (Fig. 4(B)). The Q ² regression line intersected the y‐axis below zero, indicating that the model was not overfitted and possessed good predictive reliability.

Figure 4.

Multivariate analysis of volatile organic compound (VOC) profiles between unfermented sunflower bee pollen (USBP) and Lactobacillus‐fermented sunflower bee pollen (FSBP) based on orthogonal partial least squares discriminant analysis (OPLS‐DA). (A) OPLS‐DA score plot. (B) Validation plot of 200 permutation tests for the OPLS‐DA model. (C) Variable importance in projection (VIP) plot, blue substance represented VIP > 1 and P < 0.05. (D) OPLS‐DA S‐plot. Variables with higher absolute p[1] and p(corr)[1] values, which were located at the lower left corner or the upper right corner, explained the separation between USBP and FSBP. The substances represented by the numbers in the figure are shown in Table 2.

To further assess the contribution of each VOC to the classification, we calculated the VIP predicted by the OPLS‐DA model. VOCs with a VIP > 1 were considered significant for classification. Based on the VIP values and P < 0.05, we identified 16 VOCs ((1S)‐(−)‐α‐pinene, camphene, α‐pinene, ethyl butanoate, ethyl caprate, cis‐chrysanthenol, butanoic acid, pentanoic acid, octanoic acid, hexanoic acid, isobutyl methyl ketone, acetophenone, nonanal, tridecane, selina‐5,11‐diene, p‐cymene) were identified as key markers for distinguishing between USBP and FSBP (Fig. 4(C)).

We employed an S‐plot to visualize the influence of the sensitivity indices on USBP and FSBP (Fig. 4(D)). Discriminatory markers are shown in the upper right and lower left corners of the S‐plot, with higher absolute p[1] and p(corr) values used to select potential markers. In this model, the sensitive markers included butanoic acid, α‐pinene, hexanoic acid, p‐cymene, cis‐chrysanthenol, nonanal, isobutyl methyl ketone, (1S)‐(−)‐α‐pinene, and tridecane, all of which had VIP values > 1.4, suggesting that fermentation substantially influenced their abundance. Notably, the accumulation of terpenes (e.g., α‐pinene) and esters (e.g., ethyl butanoate) correlated with the increased TPC and TFC in FSBP, whereas the reduction of fatty acids (e.g., butanoic acid) aligns with enhanced antioxidant and anti‐inflammatory activities. ¹⁷ These observations are consistent with the metabolic logic that fermentation‐induced cell wall disruption and microbial metabolism simultaneously modulate bioactive components and VOC profiles.

CONCLUSION

This study examined the effects of mixed fermentation with Lactiplantibacillus plantarum and Lacticaseibacillus casei on the nutritional composition, biological activities, and VOCs of sunflower bee pollen. Fermentation disrupted the pollen wall, promoted the release of intracellular nutrients, and improved the bioactivity and flavor characteristics of bee pollen. Compared with USBP, FSBP showed higher β‐carotene content and a higher degree of protein hydrolysis, which may help reduce the allergenicity of bee pollen proteins. TPC and TFC also increased after fermentation. In addition, SOD activity, HIA, GIA, and TIA were significantly higher in FSBP, suggesting improved antioxidant and anti‐inflammatory activities after fermentation. A total of 52 VOCs were identified by HS‐SPME‐GC–MS, and 16 VOCs were identified as important markers distinguishing USBP and FSBP. Fermentation changed both the nutritional composition and volatile compounds of bee pollen. In particular, mixed Lactobacillus fermentation reduced acidic and fatty off‐flavor compounds and increased fruity and sweet esters, which improved the aroma characteristics and palatability of bee pollen. Overall, FSBP showed improved bioactivity and flavor quality, indicating its potential application in functional foods. Future studies should further analyze the phenolic and flavonoid composition of FSBP using targeted and untargeted UPLC‐QTOF‐MS/MS metabolomics. Differential compounds should also be correlated with α‐glucosidase, tyrosinase, and hyaluronidase to identify the main compounds related to enzyme inhibitory activity.

CONFLICT OF INTEREST

The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

Supporting information

Table S1. Orthogonal test levels of fermentation process.

Table S2. Sensory evaluation criteria for fermented bee pollen.

Table S3. Results of orthogonal experiments on fermentation of sunflower bee pollen.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.