A processing method for bee pollen by-products and its application

Abstract

This invention relates to a processing method for bee pollen by-products and their applications. The bee pollen by-products are dried, pulverized, and sieved. They are then soaked in water, homogenized, and sterilized to obtain a bee pollen by-product solution. After inoculation and fermentation, the supernatant of the solution is collected as the fermented bee pollen by-product, thus completing the processing of the bee pollen by-products. The bee pollen by-products of this invention can be used as raw materials in food and health product processing. When fermented with honey, the earthy and bitter taste of the bee pollen by-products is masked, and the fermented wine produces a unique aroma and flavor combining pollen and honey, resulting in a health wine with both floral and wine aromas and rich probiotic functions.

Description

Technical Field

[0001] This invention relates to the field of bee pollen by-product technology, specifically to a processing method for bee pollen by-products and its application. Background Technology

[0002] Bee pollen is a mixture of pollen grains collected by bees from the stamens of nectar-producing plants and processed with secretions from their own glands. It contains nutrients and bioactive components needed by the human body, such as proteins, unsaturated fatty acids, carbohydrates, amino acids, phenolic compounds, various vitamins, trace elements, and pigments (chlorophyll, carotenoids), which have nutritional and physiological value. It is considered "the best food in the world" and has significant effects on improving body fat, lowering blood lipids, regulating lipid metabolism, and anti-inflammation. It has unique research value and broad application prospects in food, medicine, and cosmetics.

[0003] Bee pollen byproducts are the residues after ethanol extraction. Whether and how to reuse them is a challenge and a key issue. Studies have confirmed that different extracts of bee pollen, such as ethanol extracts, water extracts, and fat-soluble extracts, are beneficial to human health. Even after extraction with a single solvent, a large amount of nutrients and active ingredients remain unreleased. Meanwhile, bee pollen cell walls are resistant to acids, alkalis, temperatures, and pressures. Most active ingredients, such as polyphenols and flavonoids, tend to exist in a bound state, making it difficult for the human body to digest and absorb the nutrients in bee pollen, greatly limiting its development and utilization. Currently, the main methods for breaking down bee pollen cell walls include physical, chemical, biological, and combined methods. Because different varieties of bee pollen have significant differences in cell structure, different cell wall breaking methods have different effects on the bioavailability and bioactivity of bee pollen.

[0004] Furthermore, the taste of bee pollen is greatly influenced by the flower species from which it originates, often exhibiting a bitter and earthy flavor, significantly reducing its acceptability. Since bee pollen is derived from plant pollen, certain proteins it contains can act as allergens, triggering allergic reactions and leading to food allergies. These factors pose potential problems for the full development and utilization of bee pollen, and have become a food safety hazard in the development of bee pollen nutritional supplements. Summary of the Invention

[0005] This invention aims to provide a processing method for bee pollen by-products and its application. This invention focuses on bee pollen by-products, and enhances the nutritional components and active functions of bee pollen by-products through probiotic fermentation and cell wall breaking. The fermentation liquid is used as a raw material to co-ferment with honey to produce fermented beverages such as low-alcohol wine, meeting the diversified, functional and health-oriented market demands.

[0006] The technical solution of the present invention is as follows:

[0007] A method for processing bee pollen by-products, the method comprising the following steps:

[0008] S1 Pretreatment: After drying, bee pollen by-products are pulverized and sieved;

[0009] S2 bee pollen by-products were added to water, homogenized, and sterilized to obtain a bee pollen by-product solution.

[0010] S3 Fermentation Cell Wall Breaking: After inoculation and fermentation of bee pollen by-product solution, the supernatant is taken as fermented bee pollen by-product.

[0011] Complete the processing of bee pollen by-products.

[0012] Preferably, the bee pollen byproduct is the solid residue remaining after rapeseed bee pollen has been soaked in ethanol.

[0013] Preferably, the components of the bee pollen by-product include: ash 4.08±0.06 (g / 100g), crude protein 148.2±3.57 (mg / g), crude fat 19.35±0.47 (g / 100g), total sugar 72.28±1.94 (mg / g), total phenols 13.33±0.46 (mg / g), and total flavonoids 12.29±0.35 (mg / g).

[0014] Preferably, the drying conditions in step S1 are: drying at 40°C for 4-6 hours, followed by pulverization and passing through a 50-mesh sieve.

[0015] Preferably, in step S2, the bee pollen by-product and water are soaked and homogenized for 22-26 hours at a ratio of 1:(4-6) (mass-volume ratio g / mL), and sterilized at 65-80℃ for 25-35 minutes.

[0016] More preferably, in step S2, the bee pollen by-product and water are soaked and homogenized in a ratio of 1:5 (mass-volume ratio) for 24 hours, and then pasteurized at 70°C for 30 minutes.

[0017] Preferably, in step S3: the inoculum amount is 8-12%, the mass ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation conditions are 30-38℃ and culturing time is 2.5-4 days;

[0018] Preferably, the inoculum size is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation is carried out at 37°C for 3 days.

[0019] The fermented bee pollen by-product obtained by the aforementioned bee pollen by-product processing method is used in the preparation of mead.

[0020] Preferably, the preparation of the mead includes the following process:

[0021] Step 1: Mix fermentation liquid, water, and honey;

[0022] Step 2, Fermentation: After fermentation, collect the supernatant;

[0023] Step 3: Sterilization: Sterilize and can the finished product.

[0024] Preferably, in step one, the mass ratio of fermented bee pollen by-product:water:honey is (13-17):(60-70):(15-32); preferably, the mass ratio of fermented bee pollen by-product:water:honey is 15.6:62.4:30.8.

[0025] Preferably, in step two, the fermentation temperature is 25°C and the fermentation time is 3-7 days;

[0026] In step three: add 30 mg / L potassium sulfite for sterilization.

[0027] Beneficial effects of this invention:

[0028] 1. The byproducts of bee pollen after alcohol extraction can promote the acid and alkali resistance of the strain, increase the growth rate, and improve the antioxidant capacity of the strain, thus exhibiting significant probiotic functions.

[0029] 2. Fermenting bee pollen byproducts with Lactobacillus rhamnosus and yeast produces a combined effect of "probiotics + prebiotics." Lactobacillus rhamnosus exhibits significantly enhanced anti-acid and bile salt effects, antioxidant properties, and the ability to regulate key enzymes in glycolipids. When fermented with honey, the earthy and bitter taste of the bee pollen byproducts is masked, resulting in a pleasant aroma and flavor shared by both pollen and honey. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] Figure 1 Changes in pH of the culture medium at different times;

[0032] Figure 2 The effect of different concentrations of bee pollen byproducts on the growth of Lactobacillus rhamnosus;

[0033] Figure 3 Plate images of Lactobacillus rhamnosus containing different concentrations of bee pollen by-products;

[0034] Figure 4 The effect of pH on the growth curve of Lactobacillus rhamnosus;

[0035] Figure 5 Effects of bee pollen byproducts on the acid tolerance of Lactobacillus rhamnosus

[0036] Figure 6 Effect of bile salt concentration on the growth curve of Lactobacillus rhamnosus;

[0037] Figure 7 The effect of bee pollen byproducts on the bile salt tolerance of Lactobacillus rhamnosus;

[0038] Figure 8 Antioxidant activity of Lactobacillus rhamnosus cultured from bee pollen byproducts: a) ABTS scavenging rate b) DPPH scavenging rate;

[0039] Figure 9 Optical microscope images of bee pollen byproducts before and after fermentation. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. These embodiments are merely some, not all, of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0041] 1. Effects of fermented bee pollen byproducts

[0042] α-glucosidase inhibition rate determination

[0043] The α-glucosidase inhibitory activity of the samples was determined using the PNPG method. Five groups were set up: a sample group, a sample blank group, a blank group, a blank reagent group, and acarbose as a positive control. Repeatability experiments were performed. The absorbance was measured at 405 nm using an ELISA reader, and the α-glucosidase inhibition rate of bee pollen byproducts was calculated according to formula (1). Because the samples are unstable under light conditions, the entire experiment was conducted in the dark.

[0044] (1)

[0045] α-Amylase Inhibition Rate Determination

[0046] The α-amylase inhibitory activity of the samples was determined by the 3,5-dinitrosalicylicacid (DNS) colorimetric method. Five groups were set up: a sample group, a sample blank group, a blank group, a blank reagent group, and acarbose as a positive control. Repeatability experiments were performed. The absorbance was measured at 540 nm using an ELISA reader, and the α-amylase inhibition rate of bee pollen by-products was calculated according to formula (2). The samples were unstable under light conditions, so the entire experiment was conducted in the dark.

[0047] (2)

[0048] Calculation of total inhibition rate

[0049] The total inhibition rate is calculated by taking the inhibition rate of α-glucosidase and the inhibition rate of α-amylase as 50% each, and is calculated according to formula (3).

[0050] (3)

[0051] 2. Analytical methods for nutrients and bioactivity

[0052] Protein content determination: in accordance with national standard GB 5009.5-2016 "Determination of protein in food".

[0053] Determination of total sugar content: in accordance with GB / T 15672-2009 "Determination of total sugar content in edible fungi".

[0054] Determination of total phenolic content: According to GB / T 8313-2018 "Determination of tea polyphenols and catechins in tea".

[0055] Determination of total flavonoid content: in accordance with GB / T 20574-2006 "Determination of total flavonoid content in propolis".

[0056] Determination of DPPH free radical scavenging ability

[0057] According to the national standard GB / T39100-2020 "Determination of Antioxidant Properties of Peptides: DPPH and ABTS Methods"

[0058] The effect of bee pollen byproducts on the growth of Lactobacillus rhamnosus.

[0059] Bee pollen byproducts are the solid substances remaining after rapeseed bee pollen has been soaked in ethanol.

[0060] The experimental conditions are as follows:

[0061] 1. Activation and proliferation culture of the strain

[0062] (1) Preparation of culture medium

[0063] Prepare and sterilize MRS broth liquid culture medium and sterile physiological saline. Preparation of liquid MRS culture medium: Dissolve the following components in 1 liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g dipotassium hydrogen phosphate, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 1 mL Tween 80, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, and 15 g agar powder. Adjust the pH to 6.2-6.4 and autoclave (101 kPa, 121°C) for 15 min.

[0064] (2) Activation and subculturing of the strain

[0065] Activation steps for the strain: Lactobacillus rhamnosus was inoculated into autoclaved MRS broth and cultured at 37°C for 24 hours until the bacterial concentration reached approximately 10⁻⁶. 7 CFU / mL.

[0066] 2. Effects of bee pollen byproducts on the growth of Lactobacillus rhamnosus

[0067] (1) Effect of bee pollen byproducts on pH value of Lactobacillus rhamnosus

[0068] The bacterial culture was inoculated at a rate of 1.00% (v / v) into MRS liquid medium containing 3.00% (m / m) bee pollen by-products. Ordinary MRS liquid medium was used as a control. The pH value was measured with a pH meter at 0.00, 0.50, 1.00, 1.50, 2.00, and 2.50 h to observe the effect of bee pollen by-products on the pH value of Lactobacillus rhamnosus.

[0069] (2) Effect of bee pollen byproducts on the growth curve of Lactobacillus rhamnosus

[0070] The bacterial culture was inoculated at a ratio of 1.00% (v / v) into MRS liquid medium containing bee pollen by-products at concentrations of 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m). The culture was incubated at 37°C using a CO2 incubator. Samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h, and the OD values ​​were measured using a 600 nm microplate reader. Growth curves of *Lactobacillus rhamnosus* were plotted. The bacterial culture, after 48 h of incubation with different concentrations of bee pollen by-products, was diluted 10⁻⁶ times. 6 The cells were cultured on solid culture medium for 48 hours, and the colony count was calculated.

[0071] (3) Effect of bee pollen byproducts on the acid tolerance of Lactobacillus rhamnosus

[0072] To investigate the acid tolerance of Lactobacillus rhamnosus, bacterial suspension was inoculated into MRS medium at pH 2.00, 3.00, 4.00, 5.00, and 6.00 at an inoculation rate of 1.00% (v / v) and cultured in a CO2 incubator at 37°C. Samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h, and OD values ​​were measured using a 600 nm microplate reader. The acid tolerance curves of Lactobacillus rhamnosus were plotted and analyzed, and subsequent experimental groups were designed.

[0073] The bacteria were inoculated at a rate of 1.00% into MRS liquid medium containing 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m) bee pollen by-products at pH 3.00. The medium was incubated at 37℃ in a CO2 incubator, and samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h. OD values ​​were measured using a 600 nm microplate reader to plot growth curves for *Lactobacillus rhamnosus*, thus comparing the effect of different concentrations of bee pollen by-products on the acid tolerance of *Lactobacillus rhamnosus*.

[0074] (4) Effects of bee pollen byproducts on the bile salt tolerance of Lactobacillus rhamnosus

[0075] To investigate the bile salt tolerance of Lactobacillus rhamnosus, bacterial suspension was inoculated into MRS liquid medium with final bile salt concentrations of 0.20%, 0.25%, 0.30%, 0.35%, and 0.40% (v / v) at an inoculation rate of 1.00%. The medium was incubated at 37°C in a CO2 incubator. Samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h, and OD values ​​were measured using a 600 nm microplate reader. The bile salt tolerance curve of Lactobacillus rhamnosus was plotted and analyzed, and subsequent experimental groups were designed.

[0076] The bacteria were inoculated at a rate of 1.00% into MRS liquid medium containing 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m) bee pollen by-products at a bile salt concentration of 0.30%. The medium was incubated at 37°C in a CO2 incubator. Samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h, and the OD values ​​were measured using a 600 nm microplate reader. Growth curves of *Lactobacillus rhamnosus* were plotted to compare the effect of different concentrations of bee pollen by-products on the bile salt tolerance of *Lactobacillus rhamnosus*.

[0077] 3. Effects of bee pollen byproducts on the antioxidant activity of Lactobacillus rhamnosus

[0078] 3.00% bee pollen by-products were cultured on MRS medium and inoculated with different concentrations of Lactobacillus rhamnosus for 24 h. The supernatant was collected at 3000 rpm for 10 min and then digested in gastric and intestinal fluids, respectively, for 2 h in the stomach and 3 h in the small intestine. Antioxidant activity was then measured. ABTS and DPPH free radical scavenging rates were determined according to the national standard GB / T39100-2020, "Determination of Antioxidant Activity of Peptides: DPPH and ABTS Methods".

[0079] 4. Data Processing

[0080] The experimental results were repeated three times, and the mean ± standard deviation was taken. Data were analyzed using Excel 2020 and IBM SPSS Statistics 27, and plotted using Origin 2022. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant.

[0081] 5. Effect of bee pollen byproducts on pH value of Lactobacillus rhamnosus culture medium

[0082] Lactobacillus rhamnosus utilizes carbon sources during growth, metabolizing them to produce lactic acid and other acids, thus causing a decrease in the pH of the culture medium. Figure 1 It was observed that the pH value of *Lactobacillus rhamnosus* gradually decreased during the 0-1.5 h period, and stabilized after 1.5 h. Compared with the group treated with bee pollen by-products (ethanol extract), the pH decrease rate and extent of *Lactobacillus rhamnosus* were significantly higher than those of the bee pollen group and the bee pollen by-product (water extract) group. Furthermore, after 2.0 h, the pH of other groups stabilized, while the pH of the bee pollen by-product (ethanol extract) continued to decrease to below 4.9. This indicates that the ethanol-extracted bee pollen by-product can promote the growth and reproduction of *Lactobacillus rhamnosus*, and its proliferation ability was not inhibited even at pH values ​​below 5.0. Preliminary judgment suggests that the ethanol-extracted bee pollen by-product has strong probiotic function.

[0083] 6. Effects of bee pollen byproducts on the growth curve of Lactobacillus rhamnosus

[0084] OD values ​​of Lactobacillus rhamnosus cultured from bee pollen byproducts at different concentrations are as follows: Figure 2 As shown; the results of spreading cultures at different concentrations on solid culture media are as follows. Figure 3 As shown.

[0085] Depend on Figure 2 It can be seen that the *Lactobacillus rhamnosus* in the control group grew slowly during the 0-6 h period, remaining in a plateau phase. In contrast, after adding bee pollen byproducts, *Lactobacillus rhamnosus* gradually began to grow after 2 h, and entered the exponential growth phase at 4 h. This indicates that the addition of bee pollen byproducts can shorten the lag phase of *Lactobacillus rhamnosus* growth. The bacterial slope of the control group was 0.11 (R0). 2 =0.83), while the bacterial slope of the culture of 5.00% bee pollen by-products was 0.21 (R = 0.83). 2 =0.97), which is 1.9 times that of the control group. This indicates that bee pollen byproducts can promote the proliferation of Lactobacillus rhamnosus, and the higher the concentration of bee pollen byproducts, the more obvious the proliferation effect.

[0086] Depend on Figure 3 It was found that with the increase of bee pollen processing by-product concentration, the colony count of *Lactobacillus rhamnosus* increased significantly (p<0.05), and the colony count in other groups was more than 10-15 times that of the control group, indicating that bee pollen processing by-products have a significant probiotic effect on *Lactobacillus rhamnosus*. Figure 2 The colony growth further validated the conclusions.

[0087] 7. Effects of bee pollen byproducts on the acid tolerance of Lactobacillus rhamnosus

[0088] The changes in OD value of Lactobacillus rhamnosus during culture at different pH levels were plotted in the growth curves, as shown in the figure below. Figure 4 As shown.

[0089] Depend on Figure 4The results showed that the OD value of bacterial growth did not change significantly from 0 to 6 hours. After that, the growth showed a linear upward trend, indicating that although Lactobacillus rhamnosus is acid-resistant, its growth was still inhibited as the acidity increased. After 6 hours, Lactobacillus rhamnosus in the culture medium with pH greater than or equal to 4 entered the exponential growth phase with a slope greater than 0.08, while the slope was smaller at pH 2 and 3, with a slope of only about 0.002.

[0090] The pH of the human stomach is generally around 3, and the growth of *Lactobacillus rhamnosus* is inhibited within this range. Therefore, a culture medium with pH 3 was used as a control group to investigate the effect of different concentrations of bee pollen byproducts on the acid tolerance of *Lactobacillus rhamnosus*.

[0091] Depend on Figure 5 The OD values ​​of the culture systems with added bee pollen byproducts were all higher than those of the control group (the highest OD value was 0.14±0.00), and the bacterial slope of the control group was 0.02 (R²). 2 =0.97), while the slope of bacteria cultured from 5.00% bee pollen byproducts was 0.04 (R = 0.97). 2 =0.99), which is 2.0 times that of the control group. This indicates that bee pollen byproducts can improve the acid resistance of Lactobacillus rhamnosus and significantly increase its growth rate. p<0.05 In a culture medium with a pH of around 3, bee pollen byproducts help Lactobacillus rhamnosus proliferate, thereby improving the survival and colonization of the bacteria in the gut, enabling them to maintain the balance of the gut microbiota and produce beneficial effects on the host.

[0092] 8. Effects of bee pollen byproducts on the bile salt tolerance of Lactobacillus rhamnosus

[0093] The OD values ​​at different times under different bile salt concentrations were measured, and growth curves were plotted as follows: Figure 6 As shown. By Figure 6 The results showed that *Lactobacillus rhamnosus* had a lower OD value in the system containing bile salts, and its growth rate was only one-quarter of that in the control group, indicating that *Lactobacillus rhamnosus* has a weak tolerance to bile salts. Since the concentration of bile salts in the intestine is generally 0.30%, the effect of bee pollen byproduct concentration on the bile salt tolerance of *Lactobacillus rhamnosus* was investigated at a bile salt concentration of 0.30%.

[0094] Depend on Figure 7 The bacterial slope in the control group was 0.03 (R). 2 =0.88), while the bacterial slope of 5.00% bee pollen by-product culture was 0.05 (R = 0.88). 2=0.98), which is 1.7 times that of the control group, indicating that bee pollen by-products improve the bile salt tolerance of Lactobacillus rhamnosus. After 2 hours, the OD value of the medium with 2.00%-5.00% bee pollen by-products increased significantly; and high concentrations of bee pollen by-products made Lactobacillus rhamnosus more tolerant to bile salts, so 3.00% bee pollen by-products were added during simulated gastrointestinal digestion.

[0095] 9. Effects of bee pollen byproducts on the antioxidant activity of Lactobacillus rhamnosus

[0096] The antioxidant activity of *Lactobacillus rhamnosus* cultured in MRS and MRS cultured with 3.00% bee pollen by-products was compared, and the antioxidant activity of the two groups of *Lactobacillus rhamnosus* after gastric and intestinal digestion was also compared. The ABTS and DPPH free radical scavenging rates of *Lactobacillus rhamnosus* cultured in 3.00% bee pollen by-products and MRS culture systems were measured using the above methods. The results are shown in […]. Figure 8 .

[0097] Depend on Figure 8 It can be seen that the ABTS free radical scavenging rate of Lactobacillus rhamnosus cultured from 3.00% bee pollen by-products was 1.1-1.5 times that of the control group, and the difference was significant at cell concentrations of 6.00%-9.00%. p<0.05 The DPPH free radical scavenging rate of *Lactobacillus rhamnosus* cultured from 3.00% bee pollen by-products was 1.1-1.6 times that of the control group, and the difference was significant when the bacterial concentration was 6.00%-8.00%. p<0.05 The difference became less pronounced with higher bacterial concentrations, consistent with the results obtained from ABTS free radical scavenging. This indicates that bacteria cultured from bee pollen byproducts exhibit higher antioxidant activity than those cultured in ordinary culture media. This is because the polyphenols and other active substances in bee pollen byproducts can be transferred to probiotic cells and hydrolyzed and utilized by peptidases in the cells, thus demonstrating good antioxidant activity.

[0098] Based on the aforementioned effect of bee pollen by-products on the growth of *Lactobacillus rhamnosus*, co-fermentation of bee pollen by-products and *Lactobacillus rhamnosus* can break down cell walls. *Lactobacillus rhamnosus* is a normal part of the human gut microbiota and a probiotic proven to have important physiological and health-promoting functions such as regulating intestinal flora, lowering cholesterol, improving immunity, and anti-cancer effects. During the fermentation of bee pollen by-products, *Lactobacillus rhamnosus* can break down large molecular nutrients in the bee pollen by-products into smaller molecules, such as proteins breaking down into free amino acids and small peptides. This promotes the release of antioxidants such as phenolic compounds in the bee pollen by-products, improving their bioavailability. Simultaneously, some metabolites (such as organic acids and esters) produced through fermentation by *Lactobacillus rhamnosus* can impart new flavors to bee pollen, masking its original unpleasant odor and giving it a more pleasant aroma and taste. Therefore, fermented bee pollen by-products have a dual effect of "probiotics + prebiotics." See Example 1 for a specific embodiment.

[0099] Example 1

[0100] A method for processing bee pollen by-products, the method comprising the following steps:

[0101] S1 Pretreatment: After drying, bee pollen by-products are pulverized and sieved;

[0102] S2 bee pollen by-products were added to water, homogenized, and sterilized to obtain a bee pollen by-product solution.

[0103] S3 Fermentation and Cell Wall Breaking: After inoculation and fermentation of bee pollen by-product solution, the supernatant was taken as fermented bee pollen by-product (Table 1 Fermented bee pollen by-product samples).

[0104] Complete the processing of bee pollen by-products.

[0105] The bee pollen by-products are the solid substances remaining after rapeseed bee pollen is soaked in ethanol (Table 1: bee pollen by-product samples).

[0106] The components of the bee pollen by-product include: ash 4.08±0.06 (g / 100g), crude protein 148.2±3.57 (mg / g), crude fat 19.35±0.47 (g / 100g), total sugar 72.28±1.94 (mg / g), total phenols 13.33±0.46 (mg / g), and total flavonoids 12.29±0.35 (mg / g).

[0107] The drying conditions for step S1 are: drying at 40℃ for 5 hours, followed by pulverization and passing through a 50-mesh sieve.

[0108] Step S2: Soak and homogenize bee pollen byproducts and water in a ratio of 1:5 (g / mL) for 24 hours, and sterilize at 70℃ for 30 minutes.

[0109] Preferably, in step S2, the bee pollen by-product and water are soaked and homogenized in a ratio of 1:5 (mass-volume ratio) for 24 hours, and then pasteurized at 70°C for 30 minutes.

[0110] In step S3: the inoculum amount is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation conditions are 37℃ and 3 days.

[0111] Preferably, the inoculum size is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation is carried out at 37°C for 3 days.

[0112] Depend on Figure 9 (The left image shows the product before fermentation, and the right image shows the product after fermentation.) It can be seen that the bee pollen byproducts have an intact structure under a microscope, with the pollen wall tightly enclosing the intracellular material, resulting in a spherical shape. After fermentation, the particles swell, pores appear in the cell walls, and the intracellular material is released, with more nutrients dissolving out. Microscopic observation shows that the specific surface area of ​​the bee pollen byproducts increases after cell wall disruption during fermentation, indicating an increased contact area with the external environment, which may affect its solubility, stability, and other properties.

[0113] Table 1. Changes in nutrient composition and bioactivity of fermentation broth before and after fermentation.

[0114]

[0115] Table 1 shows that the protein content of bee pollen by-products decreased after fermentation, from 148.2 mg / g to 96.8 mg / g, a decrease of 34.6%. This is likely because *Lactobacillus rhamnosus* and yeast degraded the large protein molecules in the bee pollen by-products into smaller peptides and amino acids during fermentation, and also degraded soluble proteins. During fermentation, the total sugar content of the bee pollen by-products decreased by 27.9%, mainly due to *Lactobacillus rhamnosus* consuming fructose and glucose as carbon and energy sources. The bee pollen by-products contained 30.83 mg / g before processing. After industrial extraction of polyphenols with ethanol, a small amount of polyphenols remained. After fermentation, the polyphenol and total flavonoid contents increased, reaching 1.16 times and 1.49 times the levels before fermentation, respectively. The increased content of total phenols and flavonoids may be due to the microbial fermentation process disrupting the cell walls of bee pollen byproducts, thereby releasing nutrients and bioactive substances. Alternatively, fermentation may involve the conversion of glycosides into aglycones and further hydrolysis of the aglycones, which also increases the content of flavonoids. After fermentation, the DPPH free radical scavenging rate increased by 14.91%, and the content of antioxidant components among the bioactive substances was enhanced.

[0116] After fermentation, the α-glucosidase inhibition rate of bee pollen by-products was 93.5%, the α-amylase inhibition rate was 95.7%, and the total inhibition rate was 94.6%. This indicates that fermentation can improve the absorption rate of carbohydrates by increasing the amount of starch converted into glucose, thus playing an important role in regulating sugar metabolism.

[0117] Table 2 Sensory Evaluation Criteria for Bee Pollen

[0118]

[0119] Table 3 Sensory evaluation results of different types of bee pollen

[0120]

[0121] Note: Bee pollen byproducts: These are the solid substances remaining after rapeseed bee pollen has been soaked in ethanol.

[0122] Fermented bee pollen byproduct: Fermented bee pollen byproduct obtained through steps S1-3 of Example 1.

[0123] As shown in Table 3, when bees fly among flowers, they use their unique mouthparts and leg hairs to collect pollen and bring it back to the hive for storage in the cells. The collected bee pollen usually contains impurities such as twigs and bee remains, which need to be cleaned and dried as soon as possible. Because fresh bee pollen has a high moisture content, it is prone to mold and spoilage if not dried promptly. Generally, natural sun-drying or the use of specialized drying equipment is employed to reduce the moisture content of the bee pollen to a certain level for easier storage and processing. During storage, the pollen clusters undergo a series of biochemical reactions, including fermentation, saccharification, and protein hydrolysis. These reactions make the nutrients in the pollen more easily absorbed and utilized by bees. Therefore, the initial color of bee pollen is not significantly different from that of bee pollen byproducts, but it contains more impurities, resulting in a noticeable grainy and astringent texture when chewed, and a relatively rough mouthfeel. After storage and alcohol extraction, impurities are removed, and astringent substances such as tannins, alkaloids, and phenols are dissolved, resulting in a softer and sweeter texture and taste for bee pollen. Simultaneously, ethanol itself has bactericidal and preservative properties; when bee pollen is soaked in ethanol, it inhibits the growth and reproduction of microorganisms, preventing contamination and spoilage by bacteria, mold, and other microorganisms in the natural environment. This also reduces oxidation and microbial metabolism, minimizing changes in the taste and aroma of the bee pollen. Therefore, bee pollen byproducts are superior to original bee pollen in terms of both appearance and taste.

[0124] Raw bee pollen, specifically rapeseed bee pollen, has a faint, refreshing rapeseed aroma. However, the aroma of raw bee pollen can be affected by impurities or storage conditions, sometimes carrying a faint grassy or slightly musty smell. After being soaked in ethanol, the structure of the active ingredients in bee pollen may change to some extent under the influence of ethanol, making these components easier to absorb and utilize in humans or animals. Additionally, the byproducts of bee pollen extraction with ethanol show slight improvements in appearance and taste. Fermentation with yeast and Lactobacillus rhamnosus significantly alters the aroma of bee pollen, making it more concentrated and complex than raw bee pollen, masking any unpleasant odors present in the original bee pollen. This is because during fermentation, microorganisms decompose substances that cause astringency, such as tannins, thus reducing astringency; simultaneously, some microbial metabolites produced during fermentation give bee pollen a sour and fragrant aroma similar to fermented foods. Components such as organic acids and enzymes increase the sourness and umami flavor of bee pollen, making its texture smoother, more delicate, and its sour and sweet flavors more balanced and richer. Therefore, fermented bee pollen byproducts scored the highest in sensory evaluation.

[0125] The fermented bee pollen byproduct obtained in Example 1 was used in mouse experiments. The specific experimental conditions were as follows:

[0126] I. Preparation of Animals and Experiments

[0127] 1. Mouse grouping and feeding

[0128] Forty 7-week-old male C57BL / 6J mice were housed in a controlled environment with a temperature of approximately 25°C and a relative humidity of approximately 50%. They were provided with 12 hours of light and 12 hours of darkness daily, and their bedding was changed twice a week. The mice had free access to food. After one week of acclimatization, they were randomly divided into four groups of 10 mice each (divided into three cages using a 3-3-4 pattern). Each cage was marked with an ear tag. The weight of each mouse was measured daily, food intake was measured every two days, and water intake was measured weekly. The experiment lasted for eight weeks. Specific treatments are shown in the table below.

[0129] Table 4 Animal grouping and feeding methods

[0130]

[0131] Note: Mice and feed were purchased from Xiaoshuyoutai (Beijing) Biotechnology Co., Ltd.

[0132] The 3% fermented bee pollen by-product mixed feed refers to 3g of bee pollen by-product mixed with 97g of 60% high-fat control feed, which is HFD+PBP feed.

[0133] The 3% fermented bee pollen by-product mixed feed refers to the mixture of 3g of fermented bee pollen by-product and 97g of 60% high-fat control feed, which is HFD+FPBP feed.

[0134] 2. Animal euthanasia and material collection

[0135] After the glucose tolerance test, fresh feces from the mice were collected on ice, flash-frozen in liquid nitrogen, and stored at -80°C. The night before the mice were euthanized, their food was removed, their bedding was replaced, and they were kept on a fast but allowed free access to water. Twelve hours later, the mice were weighed.

[0136] Blood was collected by enucleation into 2 mL sterile centrifuge tubes and incubated overnight at 4°C. Mice were laid out naturally, and their body length (distance from nose to anus) was measured. The Lees index was calculated using formula (2-3). Mice were fixed on a dissection table, the abdominal cavity was opened, and epididymal fat, pancreas, and liver were harvested. The entire intestine was removed from the stomach to the anus, and the periintestinal fat was carefully scraped off with forceps and weighed. The intestines were flushed with a 1 mL syringe. The kidneys were removed, and the perirenal fat was harvested and weighed. The thoracic and abdominal cavities were removed, and subcutaneous fat was harvested and weighed. A relatively intact small portion of the epididymal fat, pancreas, liver, and colon from the same location was selected and fixed with 4% paraformaldehyde fixative. The collected tissues were washed with PBS buffer, blotted dry with absorbent paper, flash-frozen in liquid nitrogen, aliquoted into 2 mL sterile Eppendorf tubes, and stored at -80°C. Blood that has been left to stand overnight is centrifuged at 4000 rpm and 4°C for 10 min. The resulting supernatant is the fasting serum of mice, which is aliquoted and stored at -80°C.

[0137]

[0138] In the formula, the unit of weight is grams (g) and the unit of body length is centimeters (cm).

[0139] 3. Testing of lipid-related indicators (TG, T-CHO, HDL-C, LDL-C)

[0140] The levels of triglycerides (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in fasting mouse serum were calculated using biological reagent kits and an enzyme-linked immunosorbent assay (ELISA) reader.

[0141] 4. Experimental Results

[0142] Table 5. Effects of fermented bee pollen byproducts on mouse growth parameters

[0143]

[0144] Note: # indicates P < 0.05 compared to NC, # indicates P < 0.05 compared to HFD. P < 0.05 indicates a significant difference.

[0145] Table 5 shows that compared with the normal group, the obese group had increased weight, shortened body length, increased body fat percentage, and increased Lees value, indicating the significance of the obesity animal model. Compared with mice fed fermented bee pollen by-products, the obese group showed significantly lower weight and body fat, longer and heavier colons, enlarged thymus, and decreased serum triglyceride, total cholesterol, and high-density lipoprotein levels (P < 0.05), indicating that bee pollen by-products have an effect on alleviating hyperlipidemia induced by a high-fat diet. In particular, fermented bee pollen by-products were superior to unfermented bee pollen by-products in multiple indicators such as mouse weight, body fat, and blood lipids, significantly inhibiting weight gain caused by a high-fat diet, improving inflammation levels, and promoting the growth of immune organs. In conclusion, fermented bee pollen by-products are beneficial for improving obesity caused by a high-fat diet, enhancing immunity, and regulating blood lipids, among other physiological functions.

[0146] Example 2

[0147] Honey wine was prepared using the fermented bee pollen byproducts obtained in Example 1.

[0148] The preparation of the mead includes the following process:

[0149] Step 1: Mix fermentation liquid, water, and honey;

[0150] Step 2, Fermentation: After fermentation, collect the supernatant;

[0151] Step 3: Sterilization: Sterilize and can the finished product.

[0152] Fermented bee pollen byproducts: Water:Honey in a mass ratio of 15.6:62.4:30.8 were mixed and fermented. The fermentation temperature was 25℃, and the fermentation time was 7 days. The supernatant was then collected, and 30 mg / L potassium sulfite was added for sterilization before bottling into the finished product.

[0153] The fermentation process for ordinary mead is as follows: the ratio of water to honey is 78:30.8 (by weight), the amount of dry yeast inoculated is 0.10%, and the mixture is fermented (25℃ for 7 days). The supernatant is collected, and 30mg / L potassium sulfite is added for sterilization. The finished product is then bottled.

[0154] Body indicators:

[0155] Table 6

[0156]

[0157] As shown in Table 6, fermented bee pollen byproduct honey wine has significantly higher levels of flavonoids, total amino acids, DPPH free radical scavenging rate, and sensory evaluation than ordinary honey wine. It contains higher levels of bioactive substances, antioxidant activity, and lower sugar content. It also has a rich and delicate aroma, a mellow and lingering taste, and a unique flavor that is refreshing, sweet, and long-lasting.

[0158] The technical solutions of the present invention have been explained through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above specific embodiments to be implemented. Any improvements made by those skilled in the art based on the present invention, or equivalent substitutions for the materials selected in the present invention, fall within the scope of patent protection.

Claims

1. A method for processing bee pollen by-products, characterized in that: The processing method includes the following steps: S1 Pretreatment: After drying and removing impurities, the bee pollen by-products are pulverized and sieved. The bee pollen by-products are the solid substances remaining after rapeseed bee pollen is soaked in ethanol. S2 bee pollen by-products were added to water, soaked and homogenized, and sterilized to obtain a bee pollen by-product solution. The mass-to-volume ratio of bee pollen by-products to water was 1:(4-6)g / mL. Soaking and homogenizing were carried out for 22-26 hours, and the sterilization conditions were 65-80℃ for 25-35 minutes. S3 Fermentation and Cell Wall Breaking: After inoculating the bee pollen by-product solution with fermentation, the supernatant is taken as the fermented bee pollen by-product. The inoculation amount is 8-12%, and the mass ratio of Lactobacillus rhamnosus and yeast is 1:

1. The fermentation conditions are 30-38℃ and the culture time is 2.5-4 days. Complete the processing of bee pollen by-products.

2. The processing method for bee pollen by-products according to claim 1, characterized in that: The components of the bee pollen by-product include: ash 4.08±0.06 (g / 100g), crude protein 148.2±3.57 (mg / g), crude fat 19.35±0.47 (g / 100g), total sugar 72.28±1.94 (mg / g), total phenols 13.33±0.46 (mg / g), and total flavonoids 12.29±0.35 (mg / g).

3. The processing method for bee pollen by-products according to claim 1, characterized in that: The drying conditions for step S1 are: drying at 40℃ for 4-6 hours, followed by pulverization and passing through a 50-mesh sieve.

4. The method for processing bee pollen by-products according to claim 1, characterized in that: In step S2, the mass-to-volume ratio of bee pollen by-product to water is 1:5 g / mL. The product is soaked and homogenized for 24 hours, and then pasteurized at 70°C for 30 minutes.

5. The method for processing bee pollen by-products according to claim 1, characterized in that: In step S3: the inoculum amount is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation is carried out at 37 ℃ for 3 days.

6. The application of the bee pollen by-product obtained by the bee pollen by-product processing method according to any one of claims 1-5, wherein the bee pollen by-product is used in the preparation of mead.

7. The application according to claim 6, characterized in that, The preparation of the mead includes the following process: Step 1: Mix fermentation liquid, water, and honey; Step 2: Fermentation; after fermentation, collect the supernatant. Step 3: Sterilize and bottle the finished product.

8. The application according to claim 7, characterized in that, In step one, the mass ratio of fermented bee pollen by-product: water: honey is (13-17):(60-70):(15-32).

9. The application according to claim 8, characterized in that, The mass ratio of fermented bee pollen byproducts to water to honey is 15.6:62.4:30.

8.

10. The application according to claim 7, characterized in that, In step two, the fermentation temperature is 25℃ and the fermentation time is 3-7 days. In step three: add 30 mg / L potassium sulfite for sterilization.

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