Method for fermenting sunflower bee pollen by using compound lactic acid bacteria and application thereof

Abstract

The invention discloses a method for fermenting sunflower bee pollen by using compound lactic acid bacteria and application of the sunflower bee pollen, and belongs to the technical field of fine and deep processing of bee products. The fermentation method comprises the following steps: inoculating compound lactic acid bacteria into sunflower bee pollen for fermentation; the preparation method of the compound lactic acid bacteria comprises the step of compounding lactobacillus plantarum and lactobacillus casei according to the volume ratio of (2 to 1) to (5 to 1), so as to obtain the compound lactic acid bacteria. The compound lactobacillus fermentation method suitable for the sunflower bee pollen is established for the first time, a new way is provided for high-value utilization of the bee pollen, and theoretical support is provided for breaking through the bottleneck of a bee pollen processing technology and developing an innovative bee pollen product with high absorptivity, high activity and pleasant flavor.

Description

Technical Field

[0001] The invention relates to the technical field of deep processing of bee products, and in particular to a method for fermenting sunflower bee pollen by using 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 bitter taste and bad odor in its natural state, which seriously limits its application. In the prior art, 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 directional fermentation technology for sunflower bee pollen. Existing single-species fermentation has problems such as single metabolites and limited improvement of 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 bee pollen. Most of the bee pollen sold on the market is in the form of raw materials, lacking innovative products with deep processing, so the added value of bee pollen products is 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 does not fully consider 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 purpose of the present invention is to provide a method for fermenting sunflower bee pollen using composite lactic acid bacteria and its application, so as 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 characteristics 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 comprises: 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 composite lactic acid bacteria is 8%-12% of the mass of the sunflower bee pollen.

[0008] Furthermore, the inoculation is as follows: the composite 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 view of the problems of poor palatability, low utilization rate of nutrients, insufficient biological activity and the like of existing bee pollen, the present invention uses Lactobacillus plantarum and Lactobacillus casei as fermentation strains in a ratio of 3:1, and optimizes the fermentation parameters (inoculation amount 10%, water addition amount 40%, fermentation at 37°C for 72 hours), which significantly improves the sensory quality and functional characteristics of bee pollen. 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 is 53.91%, and the relative content of volatile ester substances is increased by 28.51%. The present invention establishes a composite lactic acid bacteria fermentation method suitable for sunflower bee pollen for the first time, and provides a technical solution for the development of functional bee pollen products with both nutritional enhancement and flavor enhancement. The method invented by the present invention provides a new way to utilize bee pollen at a high value. 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. It helps the bee industry in this field to transform and upgrade from "scale advantage" to "technological advantage" and promotes 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0019] Figure 1 The 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 represent significant differences (p<0.05);

[0020] Figure 2 Effects of different bacterial species ratios (A), lactic acid bacteria inoculation amount (B), water addition amount (C), fermentation time (D), and fermentation temperature (E) on the sensory score, pH value, and viable count of sunflower bee pollen; different lowercase letters represent 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] Fig. 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 should be understood 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 bee pollen was purchased from beekeepers and was tested for palynology. Edible alcohol was purchased from Hualin Co., Ltd. The main experimental reagents are shown 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, stir it 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 6 kinds of freeze-dried lactobacillus powders of Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactobacillus casei, after thawing, inoculate in MRS broth according to inoculum size 1%, and be placed in constant temperature incubator and carry out activation culture for 12h. Subsequently, the activated bacterial species is transferred to MRS liquid nutrient medium, and cultivated for 24h under each bacterial species optimum growth temperature. Measure absorbance at 600nm, calibrate with blank culture medium. In 0-24h, sample and measure the OD value of bacterial liquid every 2h. Determine that the logarithmic growth phase of Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactobacillus casei is in cultivation 2-8h, 4-16h, 2-12h, 2-12h, 6-14h and 2-12h respectively.

[0041] 2.3 Screening of fermentation strains

[0042] Single strain fermentation

[0043] The six strains were cultured to the logarithmic growth phase, and the sterilized sunflower bee pollen was placed in a constant temperature incubator for fermentation under the conditions of 45% water addition, 7% inoculation, and 48 hours of fermentation. The number of viable bacteria in the fermented sunflower bee pollen was determined to conduct preliminary screening of the strains.

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

[0045] After activating the strains obtained by preliminary screening, the sterilized sunflower bee pollen was placed in a constant temperature incubator for fermentation under the conditions of 45% water addition and 7% inoculation. Samples were taken at 0, 24, 48, 72, and 96 hours respectively to test the pH value, reducing sugar content, total phenol content, and total flavonoid content of the samples.

[0046] (2) Composite bacterial fermentation

[0047] 45% sterile water was added to the sterilized sunflower bee pollen, and the most suitable bacterial strain combination was screened at a ratio of 1:1 (volume ratio) and a 7% inoculation amount, a fermentation temperature of 37° C., and a fermentation time of 48 h.

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

[0049] The sterilized sunflower bee pollen was taken for later use. The number of viable bacteria, pH value and sensory score were used as indicators. A single-factor fermentation experiment was carried out on the sunflower bee pollen by controlling the strain ratio (Lactobacillus plantarum: Lactobacillus casei), strain inoculation amount (%), water addition amount (%), fermentation time (h) and fermentation temperature (℃). 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 scores and viable bacteria counts were used as evaluation indicators. The experimental design is shown in Table 3. Taking viable bacteria counts and sensory scores as indicators, fermentation temperature (A), bacterial inoculation amount (B), water addition amount (C), bacterial strain ratio (D), and fermentation temperature (E) were selected as factors for orthogonal experiments 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 live bacteria count was determined and sensory evaluation was performed. It 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 live lactic acid bacteria

[0061] The counting of live lactic acid bacteria is based on GB4789.35-2016, and the colony count is 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 at room temperature using a pH meter.

[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] Weigh 4g of sunflower bee pollen before and after fermentation, use 80% methanol for ultrasonic extraction at a solid-liquid ratio of 1:10 for 1h, then heat reflux extraction for 2h, take out the extract, centrifuge at 4000r / min for 10min, take the supernatant and dilute to 100mL to obtain bee pollen methanol extract. The total phenol content of bee pollen is determined by the Folin-Ciocalteu method and expressed in milligrams of gallic acid equivalents (mg / g). The total flavonoid content of bee pollen is determined according to Ghosh's method (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.), expressed in milligrams of rutin equivalents (mg / g).

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

[0069] Ten students were selected to form a sensory evaluation team after unified sensory training. The color, texture, flavor, and taste of the fermented bee pollen were evaluated according to the scoring criteria, and a comprehensive score was finally obtained. 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 graphed 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, and 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, and 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 of Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus paracasei are 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 are only 6.61LogCFU / g and 6.25LogCFU / g respectively. Therefore, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus paracasei are selected for further study.

[0078] Table 5 Results of viable bacterial 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 antioxidant substances in sunflower bee pollen

[0083] Depend on Figure 1 It can be seen from A that with the extension of fermentation time, the pH value of each group showed a downward trend. Among them, the pH value of sunflower bee pollen fermented by Lactobacillus plantarum decreased rapidly, and the final pH value was 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 compared with the other three lactic acid bacteria, indicating that it can tolerate a lower pH environment, has a higher pH tolerance, and can withstand the 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 the 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 From the C, it can be seen that the total phenol content of sunflower bee pollen fermented by Lactobacillus plantarum and Lactobacillus casei rose rapidly within 0-24h and reached the maximum value of 4.27mg / g and 3.92mg / g respectively; the total phenol content after fermentation by Lactobacillus rhamnosus reached the maximum value of 3.68mg / g at 48h; the total phenol content after fermentation by Lactobacillus paracasei reached the maximum value of 3.64mg / g at 72h. However, with the extension of fermentation time, the content of phenolic substances in bee pollen fermented by each strain decreased. In natural form, phenolic compounds are combined with sugars, which reduces their availability to organisms. During the fermentation process, the proteolytic enzymes produced by the fermented strains hydrolyze complex phenols into phenolic substances with simple structure, strong biological activity and easy absorption. With the increase of fermentation time, the growth of lactic acid bacteria is restricted, which may cause lactic acid bacteria to be unable to continue to metabolize and convert phenolic substances. At the same time, other metabolites, such as organic acids and enzymes, may be produced during the metabolism of lactic acid bacteria. These metabolites may react with phenolic substances, causing them to be oxidized and reduced or to form complexes, thereby reducing the total phenolic 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, and 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) Composite strain screening

[0085] According to the result after single bacteria screening, it is found that plant lactobacillus fermentation performs better in pH tolerance, hypoglycemic, total phenol content, so plant lactobacillus is selected to be composited with Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei respectively.As shown in Table 6, compared with single strain fermentation, the viable count of composite strain fermentation increases, all exceeds 8LogCFU / g, and the viable count after plant lactobacillus and Lactobacillus casei composite is the highest, which is 8.49LogCFU / g.In commercial lactobacillus products, when the viable count reaches 8LogCFU / g, the beneficial function of lactic acid bacteria can be fully utilized.Therefore the present invention selects plant lactobacillus and Lactobacillus casei to composite and carry out subsequent experiments.

[0086] Table 6 Results of viable bacterial counts of composite bacterial 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) Proportion of bacterial strains

[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 live bacteria in fermented bee pollen is also higher. When the ratio of Lactobacillus plantarum to Lactobacillus casei is 2:1, the number of live bacteria reaches the highest point, which is 7.90LogCFU / g. At this time, the taste of fermented bee pollen is appropriately sweet and sour, the color is bright golden yellow, the floral fragrance is strong, and the sensory score is 76.97. As the ratio of Lactobacillus plantarum increases, the number of live bacteria decreases. There is no significant difference in the number of live 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, the bacterial strain ratios of 2:1, 3:1 and 5:1 were taken for subsequent experiments.

[0092] (2) Inoculation volume

[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 live bacteria, sensory score and pH value of fermented bee pollen. With the increase of inoculation amount, the number of live bacteria shows a trend of first rising and then falling. When the inoculation amount is 3%, the number of fermented bee pollen of bee pollen is small, the fermentation degree is low, and the acid production is small. At this time, the bitterness and astringency of bee pollen are obvious, and the sensory score is the lowest, only 63.26. When the inoculation amount reaches 5%, the number of live 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 live bacteria is only 6.92LogCFU / g, which is not significantly different from the number of live bacteria when the inoculation amount is 10% (p>0.05). In summary, the inoculation amounts of 5%, 7% and 10% of the strains were taken for subsequent experiments.

[0094] (3) Amount of water added

[0095] Effects of different water addition amounts on sunflower bee pollen Figure 2 As shown in C. Water is an important factor affecting the growth and metabolism of lactic acid bacteria. When the amount of water added is less than 40%, the bee pollen tissue state is relatively dry and granular. The insufficient water content of the system leads to insufficient fermentation, and the bee pollen still retains a heavy bitter taste, which makes the sensory score of the bee pollen low. When the amount of water added is 50%, the live bacteria count (8.40LogCFU / g) and sensory score (78.71) of bee pollen are both the highest. When the amount of water added is greater than 50%, the water content is too high, the bee pollen tissue state is too thin, the taste is sour and slightly bitter, and the sensory score is reduced. This may be because adding too much water will dilute the nutrients in the fermentation, such as carbon sources and nitrogen sources, which lactic acid bacteria need to grow and reproduce. If the concentration of nutrients in the system is too low, the reproductive capacity of lactic acid bacteria will be limited, which will lead to a decrease in the number of lactic acid bacteria. In summary, the water addition amounts of 40%, 45% and 50% were taken for subsequent experiments.

[0096] (4) Fermentation time

[0097] The results are as follows Figure 2As shown in D, the sensory score of fermented bee pollen shows a trend of first rising and then falling with the increase of fermentation time. When the fermentation time is less than 36h, the fermentation time of bee pollen is short, the acid production is small, and the fermentation degree is insufficient. Therefore, lactic acid bacteria cannot make the fermentation system form a special flavor, thereby affecting the sensory score of bee pollen. Within the fermentation time range of 36-72h, the pH value of 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 other fermentation time groups (p < 0.05). When the fermentation time exceeds 72h, due to the long fermentation time, bee pollen has a pungent sour taste and unpleasant odor, and the taste is too sour, and the sensory score is reduced. In addition, due to the long 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, the fermentation time of 36h, 48h and 72h was taken for subsequent experiments.

[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 number of viable bacteria (7.00LogCFU / g) and the sensory score (76.04) reached the highest. When the fermentation temperature was 35°C and 39°C, the number of viable bacteria was 6.62LogCFU / g and 6.60LogCFU / g, respectively, which was significantly different from the fermented bee pollen at 33°C and 41°C (p < 0.05). From the results, it can be seen that when the fermentation temperature is 37°C, the growth and metabolic capacity of the composite bacteria reaches the highest, the metabolic rate is the fastest, and it can more effectively utilize nutrients for growth and production of lactic acid. Therefore, the fermentation temperature of 37°C was selected for subsequent experiments.

[0100] 3.3 Orthogonal experimental results

[0101] The experimental results are shown in Table 7. The fermentation conditions selected by the single-factor experiment were optimized by designing an orthogonal experiment to obtain the optimal fermentation conditions. From the R value of the viable bacteria count, it can be seen that the order of the five factors affecting the viable bacteria count of bee pollen is fermentation temperature > fermentation time > water addition > strain ratio (Lactobacillus plantarum: Lactobacillus casei) > inoculation amount. According to the range analysis, the optimal fermentation condition is A 2 B 2 C 3 D 3 E 2 The order of influence on the sensory score of bee pollen is fermentation time > inoculation amount > bacterial species ratio (Lactobacillus plantarum: Lactobacillus casei) > water addition amount > fermentation temperature. The best fermentation condition is A 3 B 2 C 3 D 2 E 3The fermentation temperature has a greater impact on the number of viable bacteria than on the sensory score, so factor A is A. 2 When the water addition amount is 50%, the number of live bacteria is higher, but the amount of water is too much at this time, which leads to a decrease in the sensory score of bee pollen. Considering the sensory score comprehensively, the water addition amount is selected as 45%, that is, factor D is D 2 The fermentation time has a greater impact on the sensory score than on the number of viable bacteria, so factor E is E 3 In summary, the best fermentation conditions for sunflower bee pollen are A 2 B 2 C 3 D 2 E 3 That is, the fermentation temperature is 37°C, the ratio of Lactobacillus plantarum to Lactobacillus casei is 3:1, the inoculation amount is 10%, the amount of water added is 40%, and the fermentation time is 72h.

[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 3:1, inoculation amount 10%, water addition amount 40%, and fermentation time 72h. Under the optimal fermentation conditions, fermented sunflower bee pollen is bright golden yellow, has a pleasant aroma, tastes sweet and has a special flavor after lactic acid bacteria fermentation, has a smooth texture and no obvious particles, has a viable count of 8.62LogCFU / 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 beekeepers and was tested for palynology. 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] The sterilized sunflower bee pollen was fermented according to the fermentation conditions of fermentation temperature 37° C., Lactobacillus plantarum: Lactobacillus casei 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 result was expressed as mg / 100 g.

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

[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: Mix equal amounts of 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution and incubate at room temperature in the dark for 12 h to prepare ABTS stock solution. Dilute the stock solution with methanol to obtain ABTS working solution with an absorbance of 0.7 ± 0.02 units at 734 nm. Take about 0.04 mL of sample and react with 0.26 mL of ABTS working solution at room temperature in the dark for 5 min, and detect the absorbance at 734 nm. Ascorbic acid is used as the standard, and the results are expressed as ascorbic acid equivalents in mg / g.

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

[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 refers to Cheng Ni's method (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 5.00mL of sample in a beaker, add 60mL of deionized water, stir evenly and measure its pH value, then titrate with 0.1mol / LNaOH to pH=8.2. Add 20mL of neutralized formaldehyde solution to the above solution, then titrate with 0.1mol / L NaOH to pH=9.2, record the volume of NaOH solution consumed, and 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 V 1 ; Blank experiment is V 2 ; 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 measured 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: Prepare 1.0U / mL α-glucosidase solution in 0.1mol / L phosphate buffer with a pH of 6.9, mix 0.1mL of bee pollen extract with 0.1mL of α-glucosidase solution, and incubate at 37°C for 10min. Then, add 0.2mL of 5mmol / L p-nitrophenyl-D-glucoside and incubate at 37°C for 20min. Subsequently, add 0.5mL of 1mol / LNa2CO3 solution to interrupt the reaction, and record the absorbance at 405nm. The inhibition rate of α-glucosidase activity of sunflower bee pollen before and after fermentation is 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 inhibition of tyrosinase activity: 100U / mL tyrosinase solution was prepared in 0.1mol / L sodium phosphate buffer at pH 6.8, 0.3mL bee pollen extract was mixed with the tyrosinase solution, incubated at 37°C for 10min, then 0.3mL of 15% L-DOPA was added, mixed evenly, incubated at 37°C for 20min, and the absorbance was recorded at 475nm. The inhibition rate of tyrosinase activity of sunflower bee pollen before and after fermentation was expressed as the inhibition rate, and the calculation formula is 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 Duan Qianqian's method (Duan Qianqian, Cheng Ni, Zhao Cheng, et al. Research 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): The VOCs of sunflower pollen before and after fermentation were determined by solid phase microextraction-gas chromatography / mass spectrometry (SPME-GC / MS). Sample preparation: Sunflower pollen before and after fermentation was mixed with saturated NaCl solution at a ratio of 1:5 (g / v) and placed in a headspace bottle for later use. After a new solid phase microextraction head was aged at 270°C for 1 h in the injection port of the gas chromatograph, the extraction head was used to extract the sample in the headspace of the sample for 30 min, then pulled out and inserted into the injection port of the gas chromatograph for analysis at 250°C for 5 min; Chromatographic conditions: chromatographic column HP-5ms (30m×250μm×0.25μm), injection port temperature 250°C, no split, flow rate 1mL 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 230°C, quadrupole temperature 150°C, mass number scanning range 33-350 m / z.

[0144] 3. Data processing

[0145] All samples were tested in triplicate. The compounds with matches above 800 were qualitatively analyzed using the NIST chemical structure library. IBM SPSS Statistics 26, GraphPad Prism 8, and SIMCA 14.1 were used for data processing, plotting, and statistical analysis.

[0146] 4. Results and Analysis

[0147] 4.1 The effect of fermentation on the nutrient content of sunflower bee pollen is 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), which was 13.21% lower than that 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 produce an environment suitable for phenolic compounds, increase the content of phenolic substances, and ultimately affect the biological activity 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. As one of the carotenoids, lycopene has strong antioxidant capacity, can remove free radicals, and has the functions of promoting cell growth and division, delaying aging, and improving immunity. As shown in Table 9, the β-carotene content of fermented sunflower bee pollen is 1.39 times the original, while the lycopene content is 0.31 times the original.

[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 analyzed 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; Fe 2+ The chelating capacity 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. And studies have shown that the 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 donation capacity of sunflower bee pollen and promotes Fe 2+ The reduction of ions is inhibited, thereby hindering 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 uses HPLC-ECD to determine phenolic compounds. As shown in Table 11, the total peak area of ​​sunflower bee pollen increased by 29.02% after fermentation. The larger total peak area in the HPLC-ECD fingerprint spectrum means that it has more electron transfer and stronger antioxidant capacity during the reaction process, which is consistent with the previous results of the determination of the total phenol content and antioxidant capacity of sunflower bee pollen, that is, fermentation can increase the content of antioxidant substances in 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 It can be seen that the LOX activity of bee pollen after fermentation decreased from 100% to 74.40%, a decrease of 25.60%; the SOD activity after fermentation increased from 100% to 126.95%, an increase of 26.95%. This shows that fermentation can enhance the effect of sunflower bee pollen on reducing or eliminating inflammatory responses in the body. The results showed that the inhibitory hyaluronidase activity of fermented sunflower bee pollen increased from 30.72% to 53.34%, an increase of 73.63%.

[0167] 4.6 Effect 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 fermented 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 a 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] As shown in Table 13, both unfermented and fermented bee pollen have antibacterial effects on Escherichia coli and Staphylococcus aureus, and the range of the inhibition zone of the two harmful bacteria is significantly expanded after fermentation (p < 0.05), which means that fermentation can improve the antibacterial effect of bee pollen. Therefore, fermentation can increase the content of phenolic substances and flavonoids in sunflower bee pollen, which have been proven to degrade bacterial cell plasma membranes, cause potassium ion loss and initiate cell autolysis, thereby achieving antibacterial effects.

[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 more than 800, and uses the NIST database for analysis. The statistical results are as follows: Figure 5 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, 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, 2 aldehydes, etc. 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 into secondary products through oxidation, hydroxylation, acylation or isomerization. After fermentation, the number of terpenes increased from 19 to 21. This change in composition is very likely to increase complexity, enhance floral and fruity aromas, and improve the aroma of bee pollen. α-pinene has a rosin smell and has the highest content in bee pollen, followed by white calendula and cedrene, which have woody and slightly citrus aromas. After fermentation, two new terpenes, α-caryophyllene and (1S)-(-)-α-pinene, are produced, both of which have a fresh pine aroma. The relative content of esters in bee pollen after fermentation increased significantly, from 29.95% to 38.49%, an increase of 28.51%, and the types of esters increased from 12 before fermentation to 13. The relative content of ethyl caprylate was the highest among bee pollen lipids, with the aroma of fruit and brandy, and the relative content did not decrease significantly after fermentation (p>0.05). The relative content of ethyl butyrate, ethyl laurate, and ethyl caprate all increased significantly (p<0.05), among which ethyl butyrate had the aroma of cherry, strawberry, pineapple or banana, with a certain sweetness; ethyl laurate had a mild fruity and floral aroma, with a slight oily smell; ethyl caprate had a fruity and wine aroma, with a pear and brandy-like aroma. In addition, ethyl palmitate is a new ester produced after fermentation, with a sweet, fruity or waxy smell. 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 smell and its relative content is the highest. 2-Methylbutanol and (-)-trans-carvacrol are newly added alcohols after fermentation, providing a richer smell for fermented bee pollen. The present invention found that fermentation has the potential to inhibit the bad flavor of bee pollen. After fermentation, the content of organic acids decreased from 10.87% to 10.56%, and the n-butyric acid, n-valeric acid and caproic acid that cause the rancidity and odor of bee pollen were significantly reduced (p<0.05). n-Butyric acid has a rancid sour taste, but it is also an indispensable substance for flavoring raw materials. When used as a flavoring agent, a very small amount of butyric acid can emit a very strong and strong smell. n-Valeric acid exists in flue-cured tobacco leaves, oriental tobacco leaves, and smoke, and has an unpleasant irritating smell. Because the smell is not generally accepted, it is not used as a fragrance; caproic acid has an unpleasant coconut oil smell and a spicy taste. The smell of caprylic acid is described as rancid, sour, milky or wool-like, and is often 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 relative content of bee pollen octanoic acid methyl ester increases after fermentation; (2) esters degrade to form acids, such as the relative content of bee pollen octanoic acid ethyl ester decreases and the relative content of octanoic acid increases after fermentation. Ketones have a strong taste 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 the smell of citrus and oil. Acetophenone has a smell similar to hawthorn. The present invention also found that the relative content of aldehydes decreased significantly after fermentation (p < 0.05). This is because aldehydes are formed by 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 of 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 inter-group separation, indicating that there is a large difference in the overall aroma of bee pollen before and after fermentation. In order to avoid overfitting resulting in 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 ordinate 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, there are 16 signature substances that can be regarded as distinguishing sunflower bee pollen before and after fermentation with VIP>1 and p<0.05, including 3 terpenes, 2 esters, 1 alcohol, 4 acids, 2 ketones, 1 aldehyde, 1 alkane, and 2 other types, namely (1S)-(-)-α-pinene, camphene, α-pinene, ethyl butyrate, ethyl decanoate, cis-chrysanthene alcohol, n-butyric acid, n-pentanoic acid, octanoic acid, hexanoic acid, methyl nonyl 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 the differences in chemical composition between the two samples and help identify metabolites with statistical and potential biochemical significance. The points at both ends of the S-plots represent the variables that contribute most to the model, while variables with smaller contributions are clustered near the origin. Fig. 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-chrysenol, n-nonanal, methyl nonyl ketone, (1S)-(-)-α-pinene, and n-tridecane.

[0185] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for fermenting sunflower bee pollen using composite lactic acid bacteria, characterized in that: The fermentation method comprises inoculating composite lactic acid bacteria into sunflower bee pollen for fermentation; and the preparation method of the composite lactic acid bacteria comprises compounding Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 2:1-5:

1.

2. The fermentation method according to claim 1, characterized in that The volume ratio of the plant lactobacillus to the casei lactobacillus is 3:

1.

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

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

5. The fermentation method according to claim 1, characterized in that The sunflower bee pollen is sprayed with ethanol with a mass concentration of 75% and dried.

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

7. The fermentation method according to claim 6, characterized in that The sterile water accounts for 40% of the mass of the sunflower bee pollen.

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

9. Sunflower bee pollen obtained by fermentation according to any one of claims 1 to 8.

10. Use of the sunflower bee pollen according to claim 9 in preparing functional bee pollen products.

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