A method for fermenting sunflower bee pollen using composite lactic acid bacteria and its application

Abstract

The present invention discloses a method and application of sunflower bee pollen fermented by composite lactic acid bacteria, and belongs to the technical field of deep processing of bee products. The fermentation method includes inoculating composite lactic acid bacteria into sunflower bee pollen for fermentation; the preparation method of the composite lactic acid bacteria includes: Lactobacillus plantarum and Lactobacillus casei are compounded in a volume ratio of 2: 1-5: 1. The present invention establishes a composite lactic acid bacteria fermentation method suitable for sunflower bee pollen for the first time, provides a new way for the high-value utilization of bee pollen, provides theoretical support for breaking through the bottleneck of bee pollen processing technology, and developing innovative bee pollen products with high absorption rate, high activity and pleasant flavor.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of bee products, in particular to a method for fermenting sunflower bee pollen by utilizing composite lactic acid bacteria and application thereof. Background Art

[0002] Bee pollen is known as a "natural nutrient bank" because it is rich in active ingredients such as protein, phenols and flavonoids. However, its hard spore wall leads to insufficient nutrient absorption rate, and it has a bitter taste and unpleasant odor in its natural state, which seriously limits its application. In the existing technology, lactic acid bacteria fermentation is used to improve the flavor of food. However, different lactic acid bacteria participate in different metabolic reactions, which have different effects on the taste and quality of fermented foods. In addition, the fermentation research on bee pollen is still in its early stages, especially the lack of a targeted fermentation process for sunflower bee pollen. Existing single-species fermentation has problems such as single metabolites and limited improvement in functional activity, and the mechanism of synergistic metabolism of composite strains has not been systematically studied in the bee pollen system.

[0003] At present, the research and development level of bee products in this field is still relatively low, especially in the field of bee pollen. Most of the bee pollen sold on the market is in the form of raw materials, and there is a lack of innovative products that have been deeply processed. Therefore, the added value of bee pollen products is relatively low. At the same time, bee pollen raw materials are often mixed with impurities such as mud and sand, which seriously affects the consumer's eating experience and limits the promotion of bee pollen products in the market. In addition, due to the hard spore wall of pollen, its nutrients are difficult to be effectively absorbed and utilized by the human body. In summary, the existing fermentation process has not fully considered the impact of volatile substance regulation on sensory quality, resulting in low product added value. Therefore, there is an urgent need to develop a composite strain fermentation method that can simultaneously improve the nutritional utilization rate, biological activity and flavor characteristics of bee pollen. Summary of the Invention

[0004] The present invention aims to provide a method for fermenting sunflower bee pollen using a composite lactic acid bacteria and its application to solve the problems existing in the above-mentioned prior art. The fermentation method provided by the present invention can significantly improve the sensory quality and functional properties of bee pollen.

[0005] Technical Solution 1: A method for fermenting sunflower bee pollen using composite lactic acid bacteria, the fermentation method comprising inoculating the composite lactic acid bacteria into the sunflower bee pollen for fermentation; the preparation method of the composite lactic acid bacteria comprising: compounding Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 2:1-5:1.

[0006] Furthermore, the volume ratio of the Lactobacillus plantarum to the Lactobacillus casei is 3:1.

[0007] Furthermore, the inoculation is as follows: the compound lactic acid bacteria is 8%-12% of the mass of the sunflower bee pollen.

[0008] Furthermore, the inoculation is as follows: the compound lactic acid bacteria is 10% of the mass of the sunflower bee pollen.

[0009] Furthermore, the sunflower bee pollen is sprayed with ethanol having a mass concentration of 75% and dried.

[0010] Furthermore, the fermentation further comprises adding sterile water.

[0011] The sterile water accounts for 40% of the mass of the sunflower bee pollen.

[0012] The sterile water accounts for 40% of the mass of the sunflower bee pollen.

[0013] Furthermore, the fermentation is carried out at 37° C. for 72 hours.

[0014] Technical solution 2: Sunflower bee pollen obtained by fermentation using the fermentation method.

[0015] Technical solution three: Application of the sunflower bee pollen in the preparation of functional bee pollen products.

[0016] The present invention discloses the following technical effects:

[0017] In response to the problems of poor palatability, low nutrient utilization, and insufficient biological activity of existing bee pollen, the present invention uses Lactobacillus plantarum and Lactobacillus casei in a ratio of 3:1 as fermentation strains. By optimizing the fermentation parameters (inoculation amount 10%, water addition amount 40%, fermentation at 37°C for 72 hours), the sensory quality and functional properties of bee pollen are significantly improved. After fermentation, the total phenol content reaches 3.51 mg / g and the total flavonoid content reaches 5.58 mg / g, which are 1.09 times and 1.55 times higher than those of unfermented pollen, respectively; the DPPH free radical scavenging rate is increased to 51.29%, the α-glucosidase inhibition rate reaches 53.91%, and the relative content of volatile ester substances is increased by 28.51%. The present invention establishes for the first time a composite lactic acid bacteria fermentation method suitable for sunflower bee pollen, providing a technical solution for the development of functional bee pollen products that combines nutritional enhancement and flavor enhancement. The method invented by this party provides a new way to high-value utilization of bee pollen. It provides theoretical support for breaking through the bottleneck of bee pollen processing technology and developing innovative bee pollen products with high absorption rate, high activity and pleasant flavor, helping the bee industry in this field to transform and upgrade from "scale advantage" to "technological advantage" and promote the development of bee pollen deep processing industry, providing new technical means and innovation directions for the development of the bee pollen industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Effects of fermentation with different strains on the pH value (A), reducing sugar content (B), total phenol content (C), and total flavonoid content (D) of sunflower bee pollen; different lowercase letters indicate significant differences (p < 0.05);

[0020] Figure 2 Effects of different bacterial strain ratios (A), lactic acid bacteria inoculation amounts (B), water addition amounts (C), fermentation time (D), and fermentation temperature (E) on the sensory score, pH value, and viable bacterial count of sunflower bee pollen; different lowercase letters indicate significant differences (p<0.05);

[0021] Figure 3 LOX activity, SOD activity and hyaluronidase inhibition activity of sunflower bee pollen before and after fermentation; different lowercase letters represent significant differences (p<0.05);

[0022] Figure 4 The inhibitory α-glucosidase activity and tyrosinase activity of sunflower bee pollen before and after fermentation; different lowercase letters represent significant differences (p<0.05);

[0023] Figure 5 To investigate the effects of different types of VOCs on sunflower bee pollen before and after fermentation;

[0024] Figure 6 OPLS-DA model of VOCs of sunflower bee pollen before and after fermentation;

[0025] Figure 7 Cross-validation of the OPLS-DA model for VOCs of sunflower bee pollen before and after fermentation;

[0026] Figure 8 VIP graph of VOCs of sunflower bee pollen before and after fermentation;

[0027] Figure 9 This is the S-plot of VOCs of sunflower bee pollen before and after fermentation. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Screening of Lactic Acid Bacteria Fermentation of Sunflower Bee Pollen

[0032] 1. Experimental Reagents

[0033] Sunflower pollen was purchased from beekeepers and was palynologically tested. Edible alcohol was purchased from Hualin Co., Ltd. The main experimental reagents are detailed in Table 1.

[0034] Table 1 Experimental reagents

[0035]

[0036] 2. Experimental Methods

[0037] 2.1 Pretreatment of sunflower bee pollen

[0038] Spray the sunflower bee pollen with 75% edible alcohol, stirring constantly to evenly distribute the alcohol, and seal it for 2 hours. Then, cover the surface of the bee pollen with sterile gauze and place it in a drying oven at 40°C to remove the alcohol and dry it for subsequent use.

[0039] 2.2 Determination of lactic acid bacteria growth curve

[0040] Use totally 6 kinds of freeze-dried lactic acid bacteria powders of Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactobacillus casei. After thawing, be inoculated in the MRS broth according to inoculum size 1%, and be placed on the activation culture that carries out 12h in a constant temperature incubator. Subsequently, the activated bacterial classification is transferred to the MRS liquid nutrient medium, and is cultivated for 24h under each bacterial classification optimum growth temperature. Measure absorbance at 600nm, calibrate with blank culture medium. In 0-24h, the OD value of bacterial liquid is measured every 2h sampling. Determine that the logarithmic growth phase of Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus casei is in respectively cultivating 2-8h, 4-16h, 2-12h, 2-12h, 6-14h and 2-12h.

[0041] 2.3 Screening of fermentation strains

[0042] Single strain fermentation

[0043] Six bacterial strains were cultured to the logarithmic growth phase, and then sterilized sunflower pollen was fermented in a constant temperature incubator using a water addition of 45%, an inoculum size of 7%, and a fermentation time of 48 hours. The viable bacterial counts in the fermented sunflower pollen were then determined for preliminary strain screening.

[0044] (1) Detection of indicators during fermentation of a single strain

[0045] After activating the strains obtained through preliminary screening, sterilized sunflower bee pollen was fermented in a constant-temperature incubator at a water content of 45% and an inoculum of 7%. Samples were collected at 0, 24, 48, 72, and 96 hours and tested for pH, reducing sugar content, total phenolic content, and total flavonoids.

[0046] (2) Composite bacterial fermentation

[0047] 45% sterile water was added to the sterilized sunflower bee pollen, and the combined strains were mixed at a ratio of 1:1 (volume ratio), with an inoculation rate of 7%, a fermentation temperature of 37°C, and a fermentation time of 48 hours to screen the most suitable strain combination.

[0048] 2.4 Single-factor experiment on fermentation of sunflower bee pollen by complex lactic acid bacteria

[0049] Sterilized sunflower bee pollen was prepared for use. The number of viable bacteria, pH value, and sensory score were used as indicators. A single-factor fermentation experiment was conducted on sunflower bee pollen by controlling the strain ratio (Lactobacillus plantarum: Lactobacillus casei), strain inoculation amount (%), water addition amount (%), fermentation time (h), and fermentation temperature (°C). The experimental design is shown in Table 2.

[0050] Table 2 Single factor experimental design for fermentation process optimization

[0051]

[0052]

[0053] 2.5 Orthogonal experiment of fermentation of sunflower bee pollen by compound lactic acid bacteria

[0054] On the basis of the single-factor experiment, sensory score and viable bacteria count were used as evaluation indicators. The experimental design is shown in Table 3. With viable bacteria count and sensory score as indicators, fermentation temperature (A), inoculation amount of bacteria (B), amount of water added (C), bacteria ratio (D), and fermentation temperature (E) were selected as factors for an orthogonal experiment to determine the optimal fermentation conditions for lactic acid bacteria fermentation of sunflower bee pollen.

[0055] Table 3 Orthogonal design level table for fermentation process optimization

[0056]

[0057] 2.6 Verification Experiment

[0058] Sunflower bee pollen was fermented under the optimal fermentation conditions, and its viable bacteria count was determined and sensory evaluation was performed. The pollen was compared with bee pollen fermented under other fermentation conditions to verify the sensory quality of bee pollen fermented under the optimal fermentation conditions.

[0059] 2.7 Determination method

[0060] (1) Counting of viable lactic acid bacteria

[0061] The count of viable lactic acid bacteria was recorded according to GB4789.35-2016, and the colony count was expressed as LogCFU / g.

[0062] (2) pH determination of fermented bee pollen

[0063] Bee pollen and distilled water were mixed evenly at a ratio of 1:5 (g / v), and the pH was directly measured using a pH meter at room temperature.

[0064] (3) Determination of reducing sugar content

[0065] The reducing sugar content was determined by 3,5-dinitrosalicylic acid (DNS) colorimetric method.

[0066] (4) Determination of total phenolic content and total flavonoid content

[0067] 4g of sunflower bee pollen, both before and after fermentation, was weighed and ultrasonically extracted with 80% methanol at a 1:10 solid-liquid ratio for 1 hour. The extract was then extracted under reflux for 2 hours. The extract was centrifuged at 4000 rpm for 10 minutes, and the supernatant was diluted to 100 mL to obtain the bee pollen methanol extract. The total phenolic content of bee pollen was determined using the Folin-Ciocalteu method and expressed as milligrams of gallic acid equivalents (mg / g). The total flavonoid content of bee pollen was determined according to the method of Ghosh (Ghosh K, Ray M, Adak A, et al. Role of probiotic Lactobacillus fermentum KKL1 in the preparation of a rice-based fermented beverage [J]. Bioresource Technology, 2015, 188:161-168.) and expressed as milligrams of rutin equivalents (mg / g).

[0068] (5) Sensory evaluation of fermented bee pollen

[0069] Ten students were selected and formed into a sensory evaluation panel after undergoing unified sensory training. They evaluated the color, texture, flavor, and mouthfeel of the fermented bee pollen according to the scoring criteria, ultimately generating a comprehensive score. The sensory scoring criteria are shown in Table 4.

[0070] Table 4 Sensory scoring criteria for fermented bee pollen

[0071]

[0072] 2.8 Data Processing

[0073] Each experiment was repeated three times, and the experimental data were analyzed using IBM SPSS Statistics 26 software. The experimental results were presented as mean ± standard error, and graphs were drawn using GraphPad Prism 8 software.

[0074] 3. Results and Analysis

[0075] 3.1 Screening of lactic acid bacteria and determination of composite fermentation strains

[0076] (1) Growth ability of different bacterial species in sunflower bee pollen

[0077] The experimental results are shown in Table 5. There are significant differences in the growth conditions of the four strains (p < 0.05). Different lactic acid bacteria have different adaptability to the growth environment. Factors such as the reduction of substrate pH value and the consumption of nutrients may affect the growth of lactic acid bacteria. The viable counts of the four lactic acid bacteria, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus paracasei, were 8.06LogCFU / g, 7.25LogCFU / g, 7.41LogCFU / g and 7.30LogCFU / g respectively after fermentation for 48h, all exceeding 7LogCFU / g, while the viable counts of Lactobacillus bulgaricus and Lactobacillus acidophilus were only 6.61LogCFU / g and 6.25LogCFU / g respectively. Therefore, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus paracasei were selected for further study.

[0078] Table 5 Results of viable bacteria counts of sunflower bee pollen fermented by different strains

[0079]

[0080]

[0081] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0082] (2) pH tolerance, hypoglycemic ability, and effects of different bacterial strains on antioxidants in sunflower bee pollen

[0083] Depend on Figure 1 It can be seen from A that as the fermentation time increases, the pH value of each group shows a downward trend. Among them, the pH value of sunflower bee pollen fermented by Lactobacillus plantarum has a faster downward trend, and the final pH value is about 3.3. The pH values ​​of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus casei at 96h were 3.73, 3.36, 3.98 and 3.64 respectively. This shows that the four lactic acid bacteria have different tolerance to pH values. Among them, Lactobacillus plantarum can lower the pH value of the fermented bee pollen system to a lower level than the other three lactic acid bacteria, indicating that it can tolerate a lower pH environment, has a higher pH tolerance, and can withstand acidic conditions in the stomach or intestines. Figure 1 From B, we can see that the reducing sugar content of each group showed a downward trend, and the reducing sugar content of fermented sunflower bee pollen was significantly lower (p < 0.05) than that of unfermented bee pollen. The reducing sugar content of Lactobacillus plantarum dropped sharply in 24-72h, indicating that lactic acid bacteria consumed a large amount of carbon source during this period, and the change in reducing sugar content tended to be stable after 72h. This may be because too much acid accumulated in the first 72h, resulting in the inhibition of bacterial growth, thereby affecting the bacteria's continued consumption of sugars and acid production. In addition, the large accumulation of other metabolic end products such as diacetyl, acetaldehyde and acetyl may also affect the growth of bacteria. Combined with Figure 1 As we know, since Lactobacillus plantarum can tolerate a lower pH environment, the reducing sugar content at the end point is the lowest, which is 47.68g / 100g. Figure 1 As shown by C, the total phenolic content of sunflower bee pollen fermented by Lactobacillus plantarum and Lactobacillus casei increased rapidly between 0 and 24 hours, reaching maximum values ​​of 4.27 mg / g and 3.92 mg / g, respectively. The total phenolic content of Lactobacillus rhamnosus fermented bee pollen reached its maximum value of 3.68 mg / g at 48 hours, and that of Lactobacillus paracasei fermented bee pollen reached its maximum value of 3.64 mg / g at 72 hours. However, the phenolic content of bee pollen fermented by each bacterial species decreased with increasing fermentation time. In their natural form, phenolic compounds are bound to sugars, reducing their availability to organisms. During fermentation, proteolytic enzymes produced by the fermenting bacteria hydrolyze complex phenols into simpler, more bioactive, and easily absorbed phenolic compounds. As fermentation time increases, the growth of lactic acid bacteria is restricted, potentially preventing them from further metabolizing and converting phenolic compounds. Furthermore, other metabolites, such as organic acids and enzymes, may be produced during the metabolic process. These metabolites may react with phenolic substances, causing them to be oxidized and reduced or to form complexes, thereby reducing the total phenol content. Figure 1It can be seen from D that the total flavonoids content of sunflower bee pollen fermented by the four lactic acid bacteria first showed an upward trend. The total flavonoids content of the four lactic acid bacteria reached the maximum value at 72h, which were 6.06mg / g, 5.45mg / g, 5.82mg / g and 5.93mg / g respectively. With the increase of fermentation time, the total flavonoids content gradually decreased.

[0084] (3) Screening of composite strains

[0085] According to the result after single bacteria screening, find that plant lactobacillus fermentation performs better on pH tolerance, hypoglycemic, total phenol content, so choose plant lactobacillus to be composite with Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei respectively.As shown in Table 6 results, contrast single bacterial strain fermentation, the viable count of composite bacterial strain fermentation increases, and all exceeds 8LogCFU / g, and wherein plant lactobacillus and Lactobacillus casei composite after viable count is the highest, is 8.49LogCFU / g.In commercial lactobacillus product, when viable count reaches 8LogCFU / g, can give full play to the beneficial life function of lactobacillus.Therefore the present invention selects plant lactobacillus and Lactobacillus casei to be composite and carry out subsequent experiment.

[0086] Table 6 Results of viable bacteria count of composite bacterial strain fermentation of sunflower bee pollen

[0087]

[0088] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0089] 3.2 Single-factor experimental results

[0090] (1) Strain ratio

[0091] Effects of different bacterial strain ratios on sunflower bee pollen Figure 2 As shown in A. When the ratio of Lactobacillus plantarum is higher, the number of viable bacteria in the fermented bee pollen is also higher. When the ratio of Lactobacillus plantarum to Lactobacillus casei is 2:1, the number of viable bacteria reaches the highest point, which is 7.90LogCFU / g. At this time, the fermented bee pollen has a suitable sweet and sour taste, a bright golden color, a strong floral aroma, and a sensory score of 76.97. As the ratio of Lactobacillus plantarum increases, the number of viable bacteria decreases. There is no significant difference in the number of viable bacteria when the ratio of Lactobacillus plantarum to Lactobacillus casei is 5:1 and 1:1 (p>0.05), but from the perspective of sensory scores, 5:1 is the best choice. In summary, subsequent experiments were carried out with strain ratios of 2:1, 3:1 and 5:1.

[0092] (2) Inoculation amount of bacterial strain

[0093] Effects of different bacterial inoculation rates on sunflower bee pollen Figure 2As shown in B. The inoculation amount of lactic acid bacteria has a great influence on the number of viable bacteria, sensory score and pH value of fermented bee pollen. As the inoculation amount increases, the number of viable bacteria shows a trend of first increasing and then decreasing. When the inoculation amount is 3%, the number of fermented bee pollen in bee pollen is small, the fermentation degree is low, and the acid production is small. At this time, the bitterness and astringency of the bee pollen are obvious, and the sensory score is the lowest, only 63.26. When the inoculation amount reaches 5%, the number of viable bacteria reaches the highest value, which is 7.82LogCFU / g. At this time, the sensory score of bee pollen is 74.54, with a sweet and sour taste, and a floral and slightly sweet aroma. When the inoculation amount is 15%, the pH of the system is too low, resulting in the bee pollen taste being too sour and having a pungent smell. At this time, the number of viable bacteria is only 6.92LogCFU / g, which is not significantly different from the number of viable bacteria when the inoculation amount is 10% (p>0.05). In summary, the subsequent experiments were carried out with inoculation amounts of 5%, 7% and 10% of the strains.

[0094] (3) Amount of water added

[0095] Effects of different water additions on sunflower bee pollen Figure 2 As shown in C. Water is a key factor affecting the growth and metabolism of lactic acid bacteria. When the water addition amount is less than 40%, the bee pollen texture is relatively dry and granular. The insufficient water content in the system leads to incomplete fermentation, and the bee pollen still retains a strong bitter taste, resulting in a low sensory score for the bee pollen. When the water addition amount is 50%, the bee pollen has the highest viable bacterial count (8.40LogCFU / g) and sensory score (78.71). When the water addition amount is greater than 50%, the water content is too high, the bee pollen texture is too thin, the taste is sour and slightly bitter, and the sensory score is reduced. This may be because excessive water addition dilutes the nutrients in the fermentation product, such as carbon and nitrogen sources, which lactic acid bacteria need for growth and reproduction. If the nutrient concentration in the system is too low, the reproductive capacity of lactic acid bacteria will be limited, leading to a decrease in the number of lactic acid bacteria. In summary, subsequent experiments were conducted with water addition amounts of 40%, 45%, and 50%.

[0096] (4) Fermentation time

[0097] The results are as follows Figure 2As shown in Figure 3, the sensory score of fermented bee pollen first increases and then decreases with increasing fermentation time. When the fermentation time is less than 36 hours, the fermentation time of the bee pollen is short, the acid production is low, and the fermentation degree is insufficient. Therefore, the lactic acid bacteria cannot give the fermentation system a unique flavor, which affects the sensory score of the bee pollen. Within the fermentation time range of 36-72 hours, the pH value of the bee pollen is low, the number of viable bacteria is high, and the sensory scores are 70.97, 74.84, and 73.84, respectively, which are significantly different from the other fermentation time groups (p < 0.05). When the fermentation time exceeds 72 hours, due to the prolonged fermentation time, the bee pollen develops a pungent sour taste and unpleasant odor, and the taste is too sour, resulting in a lower sensory score. In addition, due to the prolonged fermentation time, the nutrients in the system are consumed in large quantities, the growth and development of lactic acid bacteria are restricted, and the number of viable bacteria in the system is reduced. In summary, subsequent experiments were carried out with fermentation times of 36 hours, 48 ​​hours, and 72 hours.

[0098] (5) Fermentation temperature

[0099] Effects of different fermentation temperatures on sunflower bee pollen Figure 2 As shown in E. When the fermentation temperature was 37°C, the viable bacterial count (7.00 Log CFU / g) and sensory score (76.04) reached their highest levels. At fermentation temperatures of 35°C and 39°C, the viable bacterial counts were 6.62 Log CFU / g and 6.60 Log CFU / g, respectively, which were significantly different from bee pollen fermented at 33°C and 41°C (p < 0.05). The results indicate that at a fermentation temperature of 37°C, the composite bacterial strain achieves its highest growth and metabolic capacity, with the fastest metabolic rate, enabling it to more efficiently utilize nutrients for growth and lactic acid production. Therefore, 37°C was selected as the fermentation temperature for subsequent experiments.

[0100] 3.3 Orthogonal experimental results

[0101] The experimental results are shown in Table 7. By designing an orthogonal experiment, the fermentation conditions selected for the single-factor experiment were optimized to obtain the optimal fermentation conditions. As can be seen from the R value of the viable count, the order of influence of the five factors on the viable count of bee pollen is fermentation temperature > fermentation time > amount of water added > bacterial strain ratio (Lactobacillus plantarum: Lactobacillus casei) > inoculum size. According to range analysis, the optimal fermentation conditions are A2B2C3D3E2. The order of influence on the sensory score of bee pollen is fermentation time > inoculum size > bacterial strain ratio (Lactobacillus plantarum: Lactobacillus casei) > amount of water added > fermentation temperature. The optimal fermentation conditions are A3B2C3D2E3. The influence of fermentation temperature on the viable count is greater than the influence on the sensory score, so factor A is more suitable for A2. When the amount of water added is 50%, the viable count is higher, but at this time, the amount of water is relatively large, causing the sensory score of bee pollen to decline. Taking the sensory score into consideration, the order of influence of the water added is 45%, i.e., factor D is more suitable for D2. Fermentation time had a greater impact on sensory scores than on viable bacterial counts, so factor E3 is more appropriate. In summary, the optimal fermentation conditions for sunflower bee pollen are A2B2C3D2E3: fermentation temperature 37°C, Lactobacillus plantarum:Lactobacillus casei ratio 3:1, inoculum size 10%, water addition 40%, and fermentation time 72 h.

[0102] Table 7 Orthogonal experimental results of sunflower bee pollen fermentation

[0103]

[0104] In summary, the optimal fermentation conditions for sunflower bee pollen are: fermentation temperature 37°C, Lactobacillus plantarum:Lactobacillus casei ratio 3:1, inoculum size 10%, water addition 40%, and fermentation time 72 hours. Under these optimal fermentation conditions, the fermented sunflower bee pollen exhibits a bright golden color, a pleasant aroma, and a sweet flavor with the distinctive flavor of lactic acid bacteria fermentation. The texture is smooth and free of noticeable particles, with a viable bacterial count of 8.62 Log CFU / g and a sensory score of 85.38.

[0105] Example 2 Effect of the fermentation method of the present invention on the quality of fermented sunflower bee pollen

[0106] 1. Materials and Reagents

[0107] Sunflower pollen was purchased from a beekeeper and was palynologically tested. The main experimental reagents are shown in Table 8.

[0108] Table 8 Experimental reagents

[0109]

[0110] 2. Experimental Methods

[0111] 2.1 Preparation of fermented bee pollen

[0112] Sterilized sunflower bee pollen was fermented according to the following fermentation conditions: fermentation temperature 37° C., Lactobacillus plantarum: Lactobacillus casei ratio 3:1, inoculation amount 10%, water addition amount 40%, and fermentation time 72 h to obtain a fermented bee pollen sample.

[0113] 2.2 Preparation of bee pollen before and after fermentation

[0114] Preparation of bee pollen before and after fermentation: fermentation temperature 37 ° C, Lactobacillus plantarum: Lactobacillus casei 3:1, bacterial inoculation amount 10%, water addition amount 40%, fermentation time 72h to obtain fermented bee pollen.

[0115] 2.3 Determination of nutrient content

[0116] The determination of reducing sugar content, total phenol content and total flavonoid content is shown in Example 1.

[0117] Determination of β-carotene content: 1 g of bee pollen was mixed evenly with 5 mL of n-hexane:acetone:methanol (2:1:1, v / v / v), and ultrasonically extracted for 30 min. After centrifugation at 6000 r / min for 10 min, the supernatant was taken and made up to 25 mL with n-hexane. The absorbance was measured at 450 nm and quantified using the β-carotene standard curve. The results were expressed as mg / 100 g.

[0118] Determination of lycopene content: 1 g of bee pollen was mixed evenly with 3 mL of n-hexane:methanol (2:1, v / v), allowed to stand, and the upper organic extract was taken. 3 mL of n-hexane was added for secondary extraction, and the extracts were combined. The absorbance was measured at 472 nm and quantified using the lycopene standard curve. The results were expressed as mg / 100 g.

[0119] 2.4 Determination of antioxidant activity

[0120] (1) DPPH free radical scavenging activity: The calculation formula is as follows:

[0121]

[0122] Where: A0, absorbance of 80% methanol solution; A1, absorbance of sample solution.

[0123] (2)Fe 2+ Chelating capacity: The calculation formula is as follows:

[0124]

[0125] Where: A0, absorbance of 80% methanol solution; A1, absorbance of sample solution.

[0126] (3) ABTS free radical scavenging activity: Equal amounts of 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution were mixed and incubated in the dark at room temperature for 12 h to prepare an ABTS stock solution. The stock solution was diluted with methanol to obtain an ABTS working solution with an absorbance of 0.7 ± 0.02 units at 734 nm. Approximately 0.04 mL of sample was reacted with 0.26 mL of ABTS working solution at room temperature in the dark for 5 min, and the absorbance was measured at 734 nm. Ascorbic acid was used as the standard, and the results were expressed as ascorbic acid equivalents in mg / g.

[0127] (4) FRAP: Trolox was used as the standard and the results were expressed in mg / g Trolox equivalent.

[0128] 2.5 Determination of Total Antioxidant Capacity: Mobile phase: methanol (A), 2% acetic acid (B), flow rate: 1.0 mL / min; electrochemical detector (ECD): amperometric detection potential: 0.8 V. Column temperature: 30°C. Gradient elution procedure was based on the method of Cheng Ni (Cheng Ni, Gao Hui, Wang Bini, et al. Study on the Antioxidant Activity of Pine Pollen Extract and Its Phenolic Compounds [J]. Food and Fermentation Industries, 2011, 37:118-122).

[0129] 2.6 Protein content was determined by the Coomassie brilliant blue method with bovine serum albumin as the standard and the results were expressed as mg / g.

[0130] 2.7 Determination of protein hydrolysis degree: Take 5.00 mL of sample and place it in a beaker. Add 60 mL of deionized water and stir until evenly mixed. Measure the pH value and then titrate with 0.1 mol / L NaOH to pH = 8.2. Add 20 mL of neutralized formaldehyde solution to the above solution and titrate with 0.1 mol / L NaOH to pH = 9.2. Record the volume of NaOH solution consumed. The calculation formula is as follows:

[0131]

[0132] Where: When the pH value is 9.2, the volume of NaOH solution consumed is recorded as V1; the blank experiment is recorded as V2; for bee pollen h tot Take 7.44mmol / g.

[0133] 2.8 Determination of anti-inflammatory ability

[0134] (1) Inhibition of hyaluronidase activity: refer to the method of Grabowska (Grabowska K, Podolak I, Galanty A, et al. In vitro anti-denaturation and anti-hyaluronidase activities of extracts and galactolipids from leaves of Impatiens parviflora DC[J]. Nat Prod Res, 2016, 30(10): 1219-1223.).

[0135] (2) LOX activity and SOD activity: The LOX activity and SOD activity of bee pollen were determined according to the method of the kit, and the enzyme activity of bee pollen before fermentation was recorded as a control.

[0136] 2.9 Determination of inhibition of α-glucosidase activity: 1.0 U / mL α-glucosidase solution was prepared in 0.1 mol / L phosphate buffer at pH 6.9, 0.1 mL of bee pollen extract was mixed with 0.1 mL of α-glucosidase solution, and incubated at 37°C for 10 min. Then, 0.2 mL of 5 mmol / L p-nitrophenyl-D-glucoside was added and incubated at 37°C for 20 min. Subsequently, 0.5 mL of 1 mol / L Na2CO3 solution was added to interrupt the reaction, and the absorbance was recorded at 405 nm. The inhibition rate of α-glucosidase activity of sunflower bee pollen before and after fermentation was expressed as the inhibition rate, and the calculation formula is as follows:

[0137]

[0138] Where: A0, absorbance of enzyme solution; A1, absorbance of mixed solution of sample and enzyme.

[0139] 2.10 Determination of Tyrosinase Inhibition: A 100 U / mL tyrosinase solution was prepared in 0.1 mol / L sodium phosphate buffer (pH 6.8). 0.3 mL of bee pollen extract was mixed with the tyrosinase solution and incubated at 37°C for 10 min. Then, 0.3 mL of 15% L-DOPA was added, mixed thoroughly, and incubated at 37°C for 20 min. The absorbance was recorded at 475 nm. The inhibition rate of tyrosinase activity by sunflower bee pollen before and after fermentation was expressed as the inhibition rate, calculated as follows:

[0140]

[0141] Where: A0, absorbance of enzyme solution; A1, absorbance of mixed solution of sample and enzyme.

[0142] 2.11 Determination of antibacterial ability: The antibacterial ability of sunflower bee pollen against Escherichia coli and Staphylococcus aureus before and after fermentation was determined according to the method of Duan Qianqian (Duan Qianqian, Cheng Ni, Zhao Cheng, et al. Study on fermentation technology and biological activity of rapeseed bee pollen [J]. Food and Fermentation Industries, 2023, 49: 135-142.).

[0143] 2.12 Determination of Volatile Organic Compounds (VOCs): VOCs in sunflower bee pollen before and after fermentation were determined using solid-phase microextraction-gas chromatography / mass spectrometry (SPME-GC / MS). Sample preparation: Sunflower bee pollen before and after fermentation was mixed with a saturated NaCl solution at a ratio of 1:5 (g / v) and stored in a headspace vial. A new SPME tip was conditioned at 270°C for 1 hour in the gas chromatograph inlet. The tip was then placed in the headspace of the sample for 30 minutes, removed, and inserted into the gas chromatograph inlet for desorption at 250°C for 5 minutes. Chromatographic conditions: HP-5ms column (30 m × 250 μm × 0.25 μm), inlet temperature 250°C, splitless flow, and a flow rate of 1 mL min / L. Temperature program: column temperature was maintained at 50°C for 3 min, then increased to 100°C at a rate of 5°C / min, maintained for 3 min, then increased to 220°C at a rate of 3°C / min, maintained for 10 min; mass spectrometry conditions: EI ion source, ion source temperature was 230°C, quadrupole temperature was 150°C, and the mass number scanning range was 33-350 m / z.

[0144] 3. Data Processing

[0145] All samples were tested in triplicate. Data were qualitatively analyzed using the NIST chemical structure library for compounds with a match of 800 or more. Data processing, plotting, and statistical analysis were performed using IBM SPSS Statistics 26, GraphPad Prism 8, and SIMCA 14.1.

[0146] 4. Results and Analysis

[0147] 4.1 The effects of fermentation on the nutrient content of sunflower bee pollen are shown in Table 9.

[0148] Table 9 Effect of fermentation on the nutrient content of sunflower bee pollen

[0149]

[0150] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0151] It can be seen that the reducing sugar content of bee pollen decreased significantly after fermentation (p < 0.05), and decreased by 13.21% compared with before fermentation. The total phenol content and total flavonoid content of sunflower bee pollen after fermentation increased significantly to 3.51 mg / g and 5.58 mg / g, which were 1.09 and 1.55 times that before fermentation. Phenolic and flavonoid substances increased during the fermentation process. In summary, fermentation according to the method of the present invention can dissolve phenolic substances in bee pollen, release bound phenolic substances, and biotransform complex phenolic compounds, and create an environment suitable for phenolic compounds, thereby increasing the content of phenolic substances, and ultimately affecting the bioactivity and bioavailability of the resulting fermented food. In addition, the palatability of bee pollen increased after fermentation, and the bitterness was reduced. β-carotene is one of the most common carotenoids and an important antioxidant in plants and animals. It plays an important role in organ development, vision, vitamin A synthesis, etc. Lycopene, a carotenoid, possesses strong antioxidant properties, scavenging free radicals and promoting cell growth and division, delaying aging, and enhancing immunity. Table 9 shows that the β-carotene content of fermented sunflower bee pollen is 1.39 times higher than the original, while the lycopene content is 0.31 times higher.

[0152] 4.2 Effect of fermentation on the antioxidant capacity of sunflower bee pollen: The antioxidant activity of sunflower bee pollen before and after fermentation was determined by DPPH free radical scavenging activity, Fe 2+ As shown in Table 10, the DPPH free radical scavenging activity of bee pollen increased from 32.28% to 51.29% after fermentation, which is 1.59 times that before fermentation. 2+ The chelating ability increased from 34.61% to 38.39%, which is 1.11 times that before fermentation; the ABTS free radical scavenging activity increased from 4.01 mg / g to 5.21 mg / g, which is 1.30 times that before fermentation; the FRAP value increased from 2.56 mg / g to 5.06 mg / g, which is 1.98 times that before fermentation. This proves that fermentation can improve the antioxidant capacity of bee pollen. Phenolic compounds have antioxidant properties and can efficiently eliminate harmful free radicals and reactive oxygen species, including singlet oxygen, superoxide radicals and hydroxyl radicals. Studies have shown that antioxidant activity is significantly positively correlated with the total phenol content in the sample. In addition, from the results of the FRAP value, it can be seen that lactic acid bacteria fermentation enhances the electron donating ability of sunflower bee pollen and promotes Fe 2+ The reduction of ions hinders the oxidation process of free radicals and other compounds.

[0153] Table 10 Antioxidant activity of sunflower bee pollen before and after fermentation

[0154]

[0155] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0156] 4.3 Effect of Fermentation on the Total Antioxidant Capacity of Sunflower Bee Pollen: The present invention used HPLC-ECD to determine phenolic compounds. Table 11 shows that the total peak area of ​​sunflower bee pollen increased by 29.02% after fermentation. The larger total peak area in the HPLC-ECD fingerprint indicates a high electron transfer rate during the reaction and a stronger antioxidant capacity. This is consistent with previous measurements of the total phenolic content and antioxidant capacity of sunflower bee pollen, indicating that fermentation can increase the antioxidant content of sunflower bee pollen.

[0157] Table 11 HPLC-ECD total peak area of ​​sunflower bee pollen before and after fermentation

[0158]

[0159] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0160] 4.4 Effects of fermentation on protein content and hydrolysis degree of sunflower bee pollen

[0161] As shown in Table 12, the protein content of sunflower bee pollen decreased significantly (p < 0.05) and the degree of protein hydrolysis increased significantly (p < 0.05) after fermentation, which means that the digestibility and utilization of bee pollen protein were improved after fermentation.

[0162] Table 12 Protein content and hydrolysis degree of sunflower bee pollen before and after fermentation

[0163]

[0164] Note: Different lowercase letters indicate significant differences among the groups (P<0.05).

[0165] 4.5 Effect of fermentation on the anti-inflammatory activity of sunflower bee pollen

[0166] Depend on Figure 3 The results showed that after fermentation, the LOX activity of bee pollen decreased from 100% to 74.40%, a decrease of 25.60%. However, the SOD activity increased from 100% to 126.95%, a rise of 26.95%. This suggests that fermentation can enhance the effect of sunflower bee pollen in reducing or eliminating inflammatory responses in the body. The results also showed that the inhibitory effect of hyaluronidase activity of fermented sunflower bee pollen increased from 30.72% to 53.34%, a 73.63% increase.

[0167] 4.6 Effects of fermentation on the inhibition of α-glucosidase and tyrosinase activities of sunflower bee pollen

[0168] Inhibition of α-glucosidase can reduce intestinal glucose absorption and postprandial hyperglycemia, which is very important for controlling type II diabetes. Therefore, inhibition of the activity of this enzyme is an indirect indicator for evaluating the anti-diabetic properties of fermentation products. Figure 4 It can be seen that the inhibitory effect of bee pollen on α-glucosidase activity increased from 42.76% to 53.91% after fermentation, an increase of 26.08%. The enzymatic reaction catalyzed by tyrosinase is the key rate-limiting step in the synthesis of melanin and an important mechanism for the accumulation of melanin. Figure 4 It can be seen that after fermentation, the inhibitory effect of bee pollen on tyrosinase activity increased from 75.84% to 89.18%, an increase of 17.59%.

[0169] 4.7 Effect of fermentation on the antibacterial activity of sunflower bee pollen

[0170] Table 13 shows that both unfermented and fermented bee pollen exhibited antibacterial effects against Escherichia coli and Staphylococcus aureus. Fermentation significantly expanded the inhibition zones for both harmful bacteria (p < 0.05), indicating that fermentation can enhance bee pollen's antibacterial effects. Therefore, fermentation can increase the content of phenolic compounds and flavonoids in sunflower bee pollen. These compounds have been shown to degrade bacterial cytoplasmic membranes, leading to potassium ion loss and the initiation of cell autolysis, thereby achieving an antibacterial effect.

[0171] Table 13 Antibacterial activity of sunflower bee pollen before and after fermentation

[0172]

[0173] Note: Different lowercase letters represent significant differences (p<0.05), and “-” means not detected.

[0174] 4.8 Effect of fermentation on VOCs of sunflower bee pollen

[0175] The present invention performs SPME-GC / MS analysis on the VOCs of sunflower bee pollen before and after fermentation, selects substances with a Match degree of 800 or above, and uses the NIST database for analysis. The statistical results are as follows: Figure 5 and as shown in Table 14.

[0176] Table 14 Effect of fermentation on VOCs of sunflower bee pollen

[0177]

[0178]

[0179] Note: Different lowercase letters represent significant differences (p<0.05); “-” represents not detected.

[0180] Specifically, a total of 52 volatile compounds were identified in the two samples before and after fermentation, including 21 terpenes, 13 esters, 7 acids, 6 alcohols, 4 acids, 3 ketones, and 2 aldehydes. The fermentation method of the present invention can reduce the relative content of terpenes in sunflower bee pollen. The decrease in the concentration of these compounds may be due to their conversion to secondary products through processes such as oxidation, hydroxylation, acylation, or isomerization. After fermentation, the number of terpenes increased from 19 to 21. This change in composition is likely to increase complexity, enhance floral and fruity aromas, and improve the aroma of bee pollen. α-Pinene has a pine resinous odor and is the most abundant in bee pollen, followed by calendula and cedrene, which have a woody and slightly citrus aroma. After fermentation, two new terpenes, α-caryophyllene and (1S)-(-)-α-pinene, are produced, both of which have a refreshing pine aroma. The relative content of esters in fermented bee pollen increased significantly after fermentation, from 29.95% to 38.49%, a 28.51% increase. The number of ester types increased from 12 before fermentation to 13. Ethyl caprylate had the highest relative content among bee pollen lipids, with a fruity and brandy aroma. This relative content did not decrease significantly after fermentation (p>0.05). The relative contents of ethyl butyrate, ethyl laurate, and ethyl caprate all increased significantly (p<0.05). Ethyl butyrate has a sweet aroma reminiscent of cherry, strawberry, pineapple, or banana; ethyl laurate has a mild fruity and floral aroma with a slightly oily undertone; and ethyl caprate has a fruity and winey aroma, with notes reminiscent of pear and brandy. Furthermore, ethyl palmitate, a newly produced ester after fermentation, has a sweet, fruity, or waxy odor. After fermentation, the relative content of alcohols decreased from 4.68% to 3.44%, but the difference was not significant (p>0.05). Benzyl alcohol has a mild aromatic odor and has the highest relative content. 2-Methylbutanol and (-)-trans-carveol are newly produced alcohols after fermentation, providing the fermented bee pollen with a richer aroma. The present invention found that fermentation has the potential to inhibit the unpleasant flavor of bee pollen. After fermentation, the organic acid content decreased from 10.87% to 10.56%, and n-butyric acid, n-valeric acid, and hexanoic acid, which cause the rancidity and odor of bee pollen, were significantly reduced (p<0.05). n-Butyric acid has a rancid sour smell, but it is also an indispensable substance in flavoring raw materials. When used as a flavoring agent, even a very small amount of butyric acid can produce a strong and intense odor. n-Valeric acid is present in flue-cured tobacco leaves, oriental tobacco leaves, and smoke, and has an unpleasant, pungent odor. Because its odor is not widely accepted, it is not used as a flavoring. Hexanoic acid has an unpleasant coconut oil odor and a spicy taste. Caprylic acid has been described as having a rancid, sour, milky, or wooly odor and is commonly used in food additives, spices, and medicine. The relative content of caprylic acid increased significantly (p < 0.05) after fermentation, from 8.87% to 10.28%.Table 14 shows that acids participate in two chemical reactions during the fermentation process: (1) Acids react with esters to form esters, such as the increase in the relative content of bee pollen methyl octanoate after fermentation; (2) Esters degrade to form acids, such as the decrease in the relative content of bee pollen ethyl octanoate and the increase in the relative content of octanoic acid after fermentation. Ketones have a strong smell even at very low levels. After fermentation, methyl nonyl ketone is newly produced, and the relative content of acetophenone increases significantly (p < 0.05). Methyl nonyl ketone has a citrus and oily smell. Acetophenone has a hawthorn-like smell. The present invention also found that the relative content of aldehydes decreased significantly after fermentation (p < 0.05). This is because aldehydes are formed from amino acids through transamination or Strecker degradation. Due to the presence of carbonyl groups, aldehydes have relatively active chemical properties and can be easily reduced to alcohols or oxidized to acids.

[0181] 4.9 Model Analysis and Evaluation of VOCs in Sunflower Bee Pollen Before and After Fermentation

[0182] Orthogonal partial least squares discriminant analysis (OPLS-DA) model was used to distinguish between unfermented and fermented sunflower bee pollen. Figure 6 It can be seen that the unfermented and fermented bee pollen samples are within the 95% confidence interval, and the OPLS-DA score graph shows obvious separation between the groups, indicating that there is a big difference in the overall aroma of bee pollen before and after fermentation. In order to avoid overfitting that may lead to unusable results, the reliability of the model was verified by permutation test and cross-validation analysis. Figure 7 As shown, R 2 Curve and Q 2 The intercept of the curve on the vertical axis is less than 1, Q 2 The intercept of the curve on the ordinate is less than 0, indicating that the OPLS-DA model has good model fitness and predictability.

[0183] 4.10 Difference Analysis of VOCs in Sunflower Bee Pollen Before and After Fermentation

[0184] Variable projection importance (VIP) can be used to evaluate the influence and explanatory power of a single variable in a sample on the classification and discrimination ability of each group of samples. Variables with VIP>1 can be regarded as a signature substance for distinguishing between samples. Figure 8As shown in the figure, 16 substances with VIP > 1 and p < 0.05 can be considered as signature compounds that distinguish between pre- and post-fermentation sunflower bee pollen. These substances include 3 terpenes, 2 esters, 1 alcohol, 4 acids, 2 ketones, 1 aldehyde, 1 alkane, and 2 other compounds: (1S)-(-)-α-pinene, camphene, α-pinene, ethyl butyrate, ethyl decanoate, cis-chrysanthene alcohol, n-butyric acid, n-pentanoic acid, octanoic acid, hexanoic acid, methylnonyl ketone, acetophenone, n-nonanal, n-tridecane, 1,4a-dimethyl-7-propenyl-1,2,3,4,4a,5,6,7-octahydronaphthalene, and 4-isopropyltoluene. S-plots were used to identify differences in chemical composition between the two samples and to help identify metabolites with statistical and potential biochemical significance. The points at both ends of the S-plots represent the variables that contribute the most to the model, while the variables with smaller contributions are clustered near the origin. Figure 9 The blue dots in the figure represent substances with VIP>1.4. It can be seen that the nine components with the most significant differences in sunflower bee pollen before and after fermentation are n-butyric acid, α-pinene, hexanoic acid, 4-isopropyltoluene, cis-chrysanthene alcohol, n-nonanal, methyl nonyl ketone, (1S)-(-)-α-pinene, and n-tridecane.

[0185] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for fermenting sunflower bee pollen using a composite lactic acid bacteria, characterized in that: The fermentation method comprises inoculating composite lactic acid bacteria into sunflower bee pollen for fermentation; the preparation method of the composite lactic acid bacteria comprises: compounding Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 3:1; The sunflower bee pollen is sprayed with ethanol with a mass concentration of 75% and dried.

2. The fermentation method according to claim 1, characterized in that The inoculation is as follows: the composite lactic acid bacteria accounts for 8%-12% of the mass of the sunflower bee pollen.

3. The fermentation method according to claim 1, characterized in that The inoculation is as follows: the composite lactic acid bacteria accounts for 10% of the mass of the sunflower bee pollen.

4. The fermentation method according to claim 1, characterized in that The fermentation further comprises adding sterile water.

5. The fermentation method according to claim 4, characterized in that The sterile water accounts for 40% of the mass of the sunflower bee pollen.

6. The fermentation method according to claim 1, characterized in that The fermentation was carried out at 37°C for 72 hours.

7. Sunflower bee pollen obtained by fermentation according to any one of claims 1 to 6.

8. Use of the sunflower bee pollen according to claim 7 in preparing functional bee pollen products.

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