Bee pollen byproduct processing method and application thereof

Abstract

The invention relates to a bee pollen byproduct processing method and application thereof. Bee pollen byproducts are dried, crushed and sieved; s2, adding water into the bee pollen by-product, soaking, homogenizing, and sterilizing to obtain a bee pollen by-product solution; inoculating and fermenting the bee pollen by-product solution, and taking supernate as a fermented bee pollen by-product; the processing of the bee pollen byproducts is completed. The bee pollen byproducts can be applied to processing raw materials of food and health care products, after the bee pollen byproducts and the honey are subjected to compound fermentation, the fishy smell and the bitter taste of the bee pollen byproducts are covered, the fermented wine generates the common faint scent and taste of the pollen and the honey, and the health care wine has the flower fragrance and the wine fragrance and is rich in the probiotic function.

Description

Technical Field

[0001] The invention relates to the technical field of bee pollen by-products, and in particular to a bee pollen by-product processing method and application thereof. Background Art

[0002] Bee pollen is a mixture of pollen grains collected by bees from the stamens of nectar-source plants and their own glandular secretions. It contains nutrients and bioactive ingredients needed by the human body, such as protein, unsaturated fatty acids, carbohydrates, amino acids, phenolic compounds, various vitamins, trace elements, pigments (chlorophyll, carotenoids) and other substances with nutritional and physiological value. It is considered "the best food in the world" and has obvious 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, cosmetics and other aspects.

[0003] Bee pollen byproducts are the residual products after ethanol extraction. Whether and how to utilize them is a difficult and crucial issue. Research has confirmed that different extracts of bee pollen, including 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. Furthermore, the cell walls of bee pollen are resistant to acid, alkali, heat, and pressure. Most of the active ingredients, such as polyphenols and flavonoids, tend to exist in a bound state, making the nutrients in bee pollen difficult to digest and absorb in the human body, greatly limiting its development and utilization. Currently, the main methods for breaking the cell wall of bee pollen include physical, chemical, biological, and combined cell breaking. Because different varieties of bee pollen have significant differences in cell structure, different cell breaking treatments have different effects on the bioavailability and bioactivity of bee pollen.

[0004] Furthermore, the taste of bee pollen is significantly influenced by the source plant and flower, often exhibiting a bitter and earthy flavor, significantly reducing its acceptability. Because bee pollen is derived from plant pollen, certain proteins it contains can act as allergens, potentially triggering allergic reactions and food allergies in humans. These factors pose potential risks to the full development and utilization of bee pollen, and have become a food safety concern for the development of bee pollen-based nutritional supplements. Summary of the Invention

[0005] The present invention aims to provide a bee pollen by-product processing method and application thereof. The present invention takes bee pollen by-product as the object, enhances the nutritional components and active functions in the bee pollen by-product through probiotic fermentation and wall breaking, and uses the fermentation liquid as a raw material to ferment with honey to produce low-alcohol wine and other fermented beverages, meeting the market demand for diversification, functionality and health.

[0006] The technical solutions of the present invention are as follows: A method for processing bee pollen by-products, comprising the following steps: S1 pretreatment: bee pollen by-products are dried, crushed and sieved; S2 bee pollen by-products are added to water, soaked and homogenized, and sterilized to obtain a bee pollen by-product solution; S3 fermentation and cell wall breaking: after inoculation and fermentation of the bee pollen by-product solution, the supernatant is taken as the fermented bee pollen by-product; Complete processing of bee pollen by-products.

[0007] Preferably, the bee pollen by-product is the solid matter remaining after rape bee pollen is soaked in ethanol.

[0008] 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).

[0009] Preferably, the drying condition in step S1 is drying at 40° C. for 4-6 hours, then crushing and passing through a 50-mesh sieve. Preferably, in step S2, the bee pollen byproduct: water = 1: (4-6) (mass volume ratio g / mL) is soaked and homogenized for 22-26 hours, and the sterilization conditions are 65-80°C for 25-35 minutes; Further preferably, in step S2, the bee pollen by-product and water are soaked and homogenized at a ratio of 1:5 (mass to volume ratio) for 24 hours, and the pasteurization condition is 70° C. for 30 minutes.

[0010] Preferably, in step S3, the bacterial inoculation amount is 8-12%, the bacterial mass ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation conditions are 30-38° C. and the culture time is 2.5-4 days; Preferably, the bacterial inoculation amount is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation temperature is 37° C. and the culture time is 3 days.

[0011] The fermented bee pollen by-product obtained by the bee pollen by-product processing method is used for preparing mead.

[0012] Preferably, the preparation of the mead comprises the following process: Step 1: Mix fermentation liquid, water and honey; Step 2: Fermentation: After fermentation, take the supernatant; Step 3: Sterilization: Sterilize and can the finished product.

[0013] Preferably, in step 1, the mass ratio of fermented bee pollen byproduct: water: honey is (13-17): (60-70): (15-32); preferably, the mass ratio of fermented bee pollen byproduct: water: honey is 15.6:62.4:30.8.

[0014] 10. The use according to claim 8, characterized in that in the step 2, the fermentation temperature is 25°C and the fermentation time is 3-7 days; In step 3: add 30 mg / L potassium sulfite for sterilization.

[0015] Beneficial effects of the present invention: 1. The by-products of bee pollen after alcohol extraction can promote the acid and alkali resistance of bacteria, increase the growth rate, and improve the antioxidant capacity of bacteria, and have significant probiotic functions.

[0016] 2. By fermenting bee pollen byproducts with Lactobacillus rhamnosus and yeast, a "probiotic + prebiotic" combination is created. Lactobacillus rhamnosus significantly enhances its anti-acid and anti-bile salt properties, antioxidant capacity, and ability to regulate key glycolipid enzymes. When combined with honey for fermentation, the earthy and bitter flavor of the bee pollen byproduct is masked, resulting in the unique fragrance and flavor of both pollen and honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 Changes in pH value of culture medium at different times; Figure 2 Effects of different concentrations of bee pollen by-products on the growth of Lactobacillus rhamnosus; Figure 3 Plate image of Lactobacillus rhamnosus with different concentrations of bee pollen byproducts; Figure 4 Effect of pH on the growth curve of Lactobacillus rhamnosus; Figure 5 Effects of Bee Pollen By-products on the Acid Resistance of Lactobacillus rhamnosus Figure 6 Effects of bile salt concentration on the growth curve of Lactobacillus rhamnosus; Figure 7 Effects of bee pollen byproducts on bile salt tolerance of Lactobacillus rhamnosus; Figure 8 Antioxidant activity of Lactobacillus rhamnosus cultured with bee pollen byproducts: a) ABTS scavenging rate b) DPPH scavenging rate; Figure 9 Optical microscope images of bee pollen byproducts before and after fermentation. DETAILED DESCRIPTION

[0019] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] 1. Effects of fermented bee pollen by-products α-glucosidase inhibition rate determination The α-glucosidase inhibitory activity of the samples was determined using the PNPG method. Five sample groups, a sample blank group, a blank group, and a blank reagent group were set up, with acarbose used as a positive control. Repeatability experiments were performed. The absorbance was measured at 405 nm using a microplate reader, and the α-glucosidase inhibition rate of the bee pollen byproduct was calculated according to formula (1). Because the samples are unstable under light conditions, the entire experiment was conducted in the dark.

[0021] (1) α-amylase inhibition rate determination The α-amylase inhibitory activity of the samples was determined using a 3,5-dinitrosalicylic acid (DNS) colorimetric assay. Five sample groups, a sample blank group, a blank group, and a blank reagent group were set up for repeatability, with acarbose used as a positive control. The absorbance was measured at 540 nm using a microplate reader, and the α-amylase inhibition rate of the bee pollen byproduct was calculated according to formula (2). Since the byproduct is unstable under light conditions, the entire experiment was conducted in the dark.

[0022] (2) Calculation of total inhibition rate The total inhibition rate was calculated by taking the inhibition rate of α-glucosidase and the inhibition rate of α-amylase as 50%, and calculated according to formula (3): (3) 2. Analytical methods for nutrients and biological activities Determination of protein content: in accordance with the national standard GB 5009.5-2016 "Determination of protein in foods".

[0023] Determination of total sugar content: in accordance with GB / T 15672-2009 “Determination of total sugar content in edible fungi”.

[0024] Determination of total phenol content: in accordance with GB / T 8313-2018 "Determination of tea polyphenols and catechins in tea" Determination of total flavonoids content: in accordance with GB / T 20574-2006 "Determination of total flavonoids content in propolis".

[0025] Determination of DPPH free radical scavenging ability According to the national standard GB / T39100-2020 "Determination of Antioxidant Activity of Peptides by DPPH and ABTS Method" Effects of bee pollen by-products on the growth of Lactobacillus rhamnosus.

[0026] Bee pollen by-product is the solid matter remaining after rape bee pollen is soaked in ethanol.

[0027] The experimental conditions are: 1. Activation and proliferation of strains (1) Preparation of culture medium Prepare MRS broth with sterile saline and sterilize. To prepare MRS broth: 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. (2) Activation and subculture of strains Activation step of the strain: inoculate Lactobacillus rhamnosus into MRS broth liquid culture medium that has been sterilized by high temperature and high pressure, and culture it in a constant temperature incubator at 37℃ for 24 hours. The bacterial concentration is about 10 7 CFU / mL.

[0028] 2. Effects of Bee Pollen Byproducts on the Growth of Lactobacillus rhamnosus (1) Effects of bee pollen by-products on the pH value of Lactobacillus rhamnosus The bacterial liquid was inoculated at a 1.00% (v / v) inoculum size into 3.00% (m / m) MRS liquid medium containing bee pollen by-products. Ordinary MRS liquid medium was set as the 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 examine the effect of bee pollen by-products on the pH value of Lactobacillus rhamnosus.

[0029] (2) Effects of bee pollen by-products on the growth curve of Lactobacillus rhamnosus The bacterial solution was inoculated at a ratio of 1.00% (v / v) into MRS liquid medium containing bee pollen by-product concentrations of 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m), and cultured in a 37°C carbon dioxide 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 with a 600 nm microplate reader. The growth curve of Lactobacillus rhamnosus was drawn. The bacteria cultured for 48 h with different concentrations of bee pollen by-products were diluted 10 6 The cells were cultured on solid medium for 48 h and the number of colonies was counted.

[0030] (3) Effects of bee pollen by-products on the acid resistance of Lactobacillus rhamnosus To investigate the acid resistance of Lactobacillus rhamnosus, the bacterial suspension was inoculated into MRS medium with pH values ​​of 2.00, 3.00, 4.00, 5.00, and 6.00 at an inoculum size of 1.00% (v / v). The culture was carried out in a carbon dioxide incubator at 37°C. 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. The acid resistance curve of Lactobacillus rhamnosus was drawn and analyzed, and subsequent experimental groups were designed.

[0031] An inoculum size of 1.00% was inoculated into MRS liquid medium (pH 3.00) containing bee pollen byproducts at concentrations of 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m). Cultures were maintained in a 37°C CO2 incubator. Samples were collected at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h. OD values ​​were measured with a 600 nm microplate reader. Growth curves of Lactobacillus rhamnosus were plotted to compare the effects of different bee pollen byproduct concentrations on the acid tolerance of Lactobacillus rhamnosus.

[0032] (4) Effects of bee pollen by-products on the bile salt tolerance of Lactobacillus rhamnosus To investigate the bile salt tolerance of Lactobacillus rhamnosus, the bacterial suspension was inoculated at an inoculum size of 1.00% (v / v) into MRS liquid culture medium with final bile salt concentrations of 0.20%, 0.25%, 0.30%, 0.35%, and 0.40% (volume ratio). The culture was conducted in a 37°C carbon dioxide 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. The bile salt tolerance curve of Lactobacillus rhamnosus was drawn and analyzed, and subsequent experimental groups were designed.

[0033] The cells were inoculated at a 1.00% inoculum into MRS liquid medium with a bile salt concentration of 0.30% and bee pollen by-product concentrations of 0.00%, 2.00%, 3.00%, 4.00%, 5.00%, and 6.00% (m / m). The cultures were cultured in a carbon dioxide incubator at 37°C. Samples were taken at 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 h, and the OD values ​​were measured with a 600 nm microplate reader. The growth curves of Lactobacillus rhamnosus were plotted to compare the effects of different concentrations of bee pollen by-products on the bile salt tolerance of Lactobacillus rhamnosus.

[0034] 3. Effects of bee pollen by-products on the antioxidant activity of Lactobacillus rhamnosus MRS culture medium containing 3.00% bee pollen byproduct and MRS culture medium inoculated with different concentrations of Lactobacillus rhamnosus were cultured for 24 hours. The supernatant was collected at 3000 rpm for 10 minutes and added to gastric and intestinal digestive fluids. After 2 hours of gastric digestion and 3 hours of small intestinal digestion, antioxidant activity was determined. ABTS and DPPH radical scavenging rates were determined according to the national standard GB / T39100-2020, "Determination of the Antioxidant Activity of Polypeptides by DPPH and ABTS Methods."

[0035] 4. Data Processing The experiment was repeated three times, and the mean ± standard deviation was taken. Statistics were compiled using Excel 2020, data were analyzed using IBM SPSS Statistics 27, and graphs were drawn using Origin 2022. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0036] 5. Effects of Bee Pollen Byproducts on the pH Value of Lactobacillus rhamnosus Culture Medium During its growth, Lactobacillus rhamnosus utilizes carbon sources and metabolizes to produce lactic acid and other acidic substances, which causes the pH of the culture medium to decrease. Figure 1 The pH value of Lactobacillus rhamnosus gradually decreased between 0 and 1.5 hours, reaching a plateau after 1.5 hours. Compared to the bee pollen by-product (alcohol-extracted), the rate and extent of decrease in Lactobacillus rhamnosus were significantly greater in the bee pollen and water-extracted groups. While the pH in the other groups stabilized after 2.0 hours, the pH in the alcohol-extracted bee pollen by-product continued to decrease, falling below 4.9. This suggests that alcohol-extracted bee pollen by-products can promote the growth and reproduction of Lactobacillus rhamnosus, and even at pH values ​​below 5.0, their proliferation was not inhibited. This suggests that alcohol-extracted bee pollen by-products possesses strong probiotic properties.

[0037] 6. Effects of Bee Pollen Byproducts on the Growth Curve of Lactobacillus rhamnosus OD values ​​of Lactobacillus rhamnosus cultured with different concentrations of bee pollen by-products Figure 2The results after the culture was applied on the solid culture medium at different concentrations are shown in Figure 3 shown.

[0038] Depend on Figure 2 It can be seen that the growth of Lactobacillus rhamnosus in the blank group was slow and in the growth plateau period from 0 to 6 hours. In contrast, after adding bee pollen by-products, Lactobacillus rhamnosus began to grow gradually 2 hours later and entered the exponential growth phase at 4 hours. This shows that the addition of bee pollen by-products can shorten the growth retardation period of Lactobacillus rhamnosus. The bacterial slope of the blank group was 0.11 (R 2 =0.83), while the slope of the bacteria cultured with 5.00% bee pollen byproducts was 0.21 (R 2 =0.97) was 1.9 times that of the blank group. This suggests that bee pollen by-products can promote the proliferation of Lactobacillus rhamnosus, and the higher the concentration of bee pollen by-products, the more obvious the proliferation effect.

[0039] Depend on Figure 3 It can be seen that as the concentration of bee pollen processing by-products increased, the colony count of Lactobacillus rhamnosus showed a significant increase (p<0.05), and the colony counts in other groups were more than 10-15 times that of the blank group, indicating that bee pollen processing by-products have a significant probiotic effect on Lactobacillus rhamnosus. Figure 2 The colony growth results further verified the conclusions.

[0040] 7. Effects of Bee Pollen Byproducts on the Acid Resistance of Lactobacillus rhamnosus The changes in OD value of Lactobacillus rhamnosus during culture at different pH values ​​were analyzed by plotting the growth curve. Figure 4 shown.

[0041] Depend on Figure 4 It was found that there was no obvious change in the OD value of bacterial growth from 0 to 6 hours, and then the growth showed a linear upward trend, indicating that although Lactobacillus rhamnosus itself is acid-resistant, its growth is still inhibited with the increase of acidity; after 6 hours, Lactobacillus rhamnosus in the culture medium with a pH greater than or equal to 4 entered the exponential growth phase, with a slope greater than 0.08, while at pH = 2 and 3, the amplitude was smaller and the slope was only about 0.002.

[0042] The pH of the human stomach is generally around 3, and the growth of Lactobacillus rhamnosus is inhibited within this range. Therefore, using a pH 3 culture medium as a control group, the effect of different bee pollen byproduct concentrations on the acid resistance of Lactobacillus rhamnosus was investigated.

[0043] Depend on Figure 5 The results showed that the OD values ​​of the culture system with bee pollen by-products were higher than those of the control group (the highest OD value was 0.14±0.00), and the slope of the control group was 0.02 (R 2 =0.97), while the slope of the bacteria cultured with 5.00% bee pollen by-products was 0.04 (R2 =0.99), which is 2.0 times that of the control group. This shows that bee pollen by-products can improve the acid resistance of Lactobacillus rhamnosus and significantly increase its growth rate ( p<0.05 ). In an environment where the culture medium pH is around 3, bee pollen byproducts help Lactobacillus rhamnosus proliferate, thereby improving the survival and colonization of the bacterial flora in the intestine, playing its role in maintaining the balance of the intestinal microecological system and producing a probiotic effect on the host body.

[0044] 8. Effects of Bee Pollen Byproducts on Bile Salt Tolerance of Lactobacillus rhamnosus Measure the OD values ​​at different times under different bile salt concentrations and draw the growth curve. Figure 6 As shown. Figure 6 The results showed that Lactobacillus rhamnosus exhibited a lower OD value in the bile salt-containing system, and its growth rate was one-fourth of that in the blank control group, indicating that Lactobacillus rhamnosus has a weak tolerance to bile salts. The bile salt concentration in the intestine is generally 0.30%, so the effect of bee pollen byproduct concentration on Lactobacillus rhamnosus's bile salt tolerance was investigated at a bile salt concentration of 0.30%.

[0045] Depend on Figure 7 The slope of the control group was 0.03 (R 2 =0.88), while the slope of the bacteria cultured with 5.00% bee pollen by-products was 0.05 (R 2 =0.98), 1.7 times that of the control group, indicating that bee pollen by-products improve Lactobacillus rhamnosus's bile salt tolerance. After 2 hours, the OD values ​​of the culture medium containing 2.00%-5.00% bee pollen by-products increased significantly. High concentrations of bee pollen by-products enhance bile salt tolerance in Lactobacillus rhamnosus, so 3.00% bee pollen by-products was selected for the simulated gastrointestinal digestion.

[0046] 9. Effects of Bee Pollen Byproducts on the Antioxidant Activity of Lactobacillus rhamnosus The antioxidant activity of Lactobacillus rhamnosus cultured in MRS culture and 3.00% bee pollen by-product MRS culture was compared, and the antioxidant activity of the two groups of Lactobacillus rhamnosus after gastric and intestinal digestion was studied. The ABTS and DPPH free radical scavenging rates of Lactobacillus rhamnosus cultured in 3.00% bee pollen by-product and MRS culture systems were measured according to the above method. The results are shown in the figure. Figure 8 .

[0047] Depend on Figure 8 It can be seen that the ABTS free radical scavenging rate of Lactobacillus rhamnosus cultured with 3.00% bee pollen by-products is 1.1-1.5 times that of the control group, and the difference is significant when the bacterial concentration is 6.00%-9.00% ( p<0.05). The DPPH free radical scavenging rate of Lactobacillus rhamnosus cultured with 3.00% bee pollen byproducts was 1.1-1.6 times that of the control group. When the bacterial concentration was 6.00%-8.00%, the difference was significant ( p<0.05 ), while the difference became less pronounced at higher bacterial concentrations, consistent with the results obtained from ABTS free radical scavenging rates. This suggests that bacteria cultured with bee pollen byproducts possess higher antioxidant properties than those cultured in conventional media. This is because active substances such as polyphenols in bee pollen byproducts can be transferred to probiotic cells and hydrolyzed and utilized by cellular peptidases, resulting in excellent antioxidant activity.

[0048] Given the aforementioned beneficial effects of bee pollen byproducts on the growth of Lactobacillus rhamnosus, co-fermentation of bee pollen byproducts with Lactobacillus rhamnosus can not only break down the cell wall, but also contribute to the normal human flora and a probiotic proven to have important physiological health benefits, such as regulating intestinal flora, lowering cholesterol, enhancing immunity, and fighting cancer. During fermentation, Lactobacillus rhamnosus breaks down macromolecular nutrients in the bee pollen byproduct into smaller molecules, such as protein into free amino acids and small peptides. This promotes the release of antioxidants such as phenolic compounds in the bee pollen byproduct, improving their bioavailability. Furthermore, metabolites produced by fermentation with Lactobacillus rhamnosus (such as organic acids and esters) can impart a new flavor to the bee pollen, masking its inherent unpleasant odor and resulting in a more pleasant aroma and taste. Therefore, fermented bee pollen byproducts possess the dual benefits of both a probiotic and a prebiotic. See Example 1 for a specific example.

[0049] Example 1 A method for processing bee pollen by-products, comprising the following steps: S1 pretreatment: bee pollen by-products are dried, crushed and sieved; S2 bee pollen by-products are added to water, soaked and homogenized, and sterilized to obtain a bee pollen by-product solution; S3 fermentation and cell wall breaking: after inoculation and fermentation of the bee pollen by-product solution, the supernatant is taken as the fermented bee pollen by-product (Table 1 fermented bee pollen by-product sample); Complete processing of bee pollen by-products.

[0050] The bee pollen by-product is the solid matter remaining after rape bee pollen is soaked in ethanol (Table 1 Bee pollen by-product sample).

[0051] 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).

[0052] The drying conditions in step S1 are as follows: drying at 40° C. for 5 h, then crushing and passing through a 50-mesh sieve. Step S2: Soak bee pollen byproduct in water at a ratio of 1:5 (mass-to-volume ratio g / mL) for 24 hours and sterilize at 70°C for 30 minutes. Preferably, in step S2, the bee pollen by-product and water are soaked and homogenized in a ratio of 1:5 (mass to volume ratio) for 24 hours, and the pasteurization condition is 70° C. for 30 minutes.

[0053] In step S3, the bacterial inoculation amount is 10%, the number of Lactobacillus rhamnosus and yeast is 1:1, and the fermentation condition is 37° C. and the culture time is 3 days; Preferably, the bacterial inoculation amount is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation temperature is 37° C. and the culture time is 3 days.

[0054] Depend on Figure 9 (Left: before fermentation, right: after fermentation) Microscopically, the bee pollen byproduct exhibits a complete structure, with the pollen wall tightly enveloping the intracellular material, resulting in a spherical shape. After fermentation, the particles swell, and pores appear in the cell walls, releasing intracellular material and allowing more nutrients to dissolve. Microscopic observation reveals an increase in the specific surface area of ​​the bee pollen byproduct after fermentation and cell wall breakage, indicating increased contact area with the outside world, potentially affecting its solubility, stability, and other properties.

[0055] Table 1 Changes in nutritional components and biological activity of fermentation broth before and after fermentation

[0056] Table 1 shows that the protein content of the bee pollen by-product decreased after fermentation, from 148.2 mg / g to 96.8 mg / g, a decrease of 34.6%. This is likely due to the degradation of soluble proteins by Lactobacillus rhamnosus and yeast during fermentation, which degraded the macromolecular proteins in the bee pollen by-product into small peptides and amino acids. The total sugar content of the bee pollen by-product decreased by 27.9% during fermentation, primarily due to the consumption of fructose and glucose as carbon and energy sources by Lactobacillus rhamnosus during fermentation. The pre-processed bee pollen by-product contained 30.83 mg / g of polyphenols. After industrial ethanol extraction, a small amount of polyphenols remained. After fermentation, the polyphenol and total flavonoid contents increased by 1.16 and 1.49 times their pre-fermentation levels, respectively. The increase in total phenolic and flavonoid content may be due to the breakdown of the cell walls of the bee pollen byproduct during microbial fermentation, releasing nutrients and bioactive substances. It may also be due to the conversion of glycosides to aglycones and their further hydrolysis during fermentation, which also increases the content of flavonoids. The DPPH free radical scavenging rate increased by 14.91% after fermentation, and the antioxidant content of bioactive substances was also increased.

[0057] After fermentation, the inhibition rates of α-glucosidase and α-amylase in bee pollen byproducts were 93.5%, 95.7%, and 94.6% respectively. This indicates that fermentation can improve the absorption rate of bee pollen byproducts by reducing the absorption rate of carbohydrates and the amount of starch converted to glucose, which plays an important role in regulating sugar metabolism.

[0058] Table 2 Sensory evaluation standards for bee pollen

[0059] Table 3 Sensory evaluation results of different types of bee pollen

[0060] Note: Bee pollen by-product: the solid matter remaining after rapeseed bee pollen is soaked in ethanol.

[0061] Fermented bee pollen by-product: the fermented bee pollen by-product obtained in step S1-3 of this Example 1.

[0062] As shown in Table 3, bees use their unique mouthparts and leg hairs to collect pollen while flying among flowers. They then bring the collected pollen back to the hive and store it in the cells. Collected pollen is often contaminated with impurities, such as small branches and bee limbs, and needs to be cleaned and dried as soon as possible. Fresh bee pollen has a high moisture content and can easily become moldy and spoil if not dried promptly. Drying the pollen naturally or using specialized drying equipment is generally done to reduce its moisture content to a certain level for subsequent storage and processing. During storage, the pollen mass undergoes a series of biochemical reactions, including fermentation, saccharification, and proteolysis. These reactions make the pollen's nutrients more readily available for bees to absorb and utilize. Therefore, the initial color of bee pollen is similar to that of bee pollen byproducts, but it contains more impurities, resulting in a noticeable graininess and astringency when chewed, resulting in a rough texture. After storage and alcohol extraction, impurities are cleaned and astringent substances such as tannins, alkaloids, and phenols are dissolved, resulting in a softer and sweeter texture and taste. Furthermore, ethanol itself has certain antiseptic and antiseptic properties. When bee pollen is soaked in ethanol, it inhibits the growth and reproduction of microorganisms, preventing contamination and deterioration by bacteria, mold, and other microorganisms in the natural environment. It also reduces oxidation and microbial metabolism that can alter the taste and odor of bee pollen. Therefore, bee pollen byproducts are superior to raw bee pollen in terms of state and taste.

[0063] Raw bee pollen, derived from rapeseed flowers, has a light, refreshing aroma. However, the aroma of raw bee pollen can be affected by impurities or storage conditions, with hints of grass or mild mustiness. Ethanol-soaked bee pollen's active ingredients may undergo structural changes, making them more readily absorbed and utilized by humans and animals. Ethanol extraction also slightly improves the state and flavor of the bee pollen's byproducts. Fermentation with yeast and Lactobacillus rhamnosus significantly alters the aroma of raw bee pollen, making it richer and more complex than raw bee pollen, masking any unpleasant odors. This is because microorganisms during the fermentation process break down astringent substances, such as tannins, thereby reducing the astringency. Furthermore, fermentation-derived microbial metabolites impart a sour aroma similar to fermented foods. Organic acids and enzymes, for example, enhance the sour and umami flavors of bee pollen, making it softer and more delicate, with a better balance of sourness and sweetness, and a richer flavor. Therefore, fermented bee pollen by-products scored the highest in the sensory evaluation.

[0064] The fermented bee pollen by-product obtained in Example 1 was used to conduct a mouse experiment. The specific experimental conditions were: 1. Animal and Experimental Preparation 1. Mouse Grouping and Housing Forty 7-week-old male C57BL / 6J mice were housed in a controlled room with a temperature of approximately 25°C and a relative humidity of approximately 50%. The mice were kept on a 12-hour light-dark cycle with bedding changed twice weekly. During this period, the mice had free access to food. After one week of acclimation, they were randomly assigned to four groups of 10 mice each (split into three cages in a 3-3-4 pattern). Each cage was ear-tagged. Each mouse was weighed daily, food intake was measured every two days, and water intake was measured weekly. The experimental period lasted eight weeks. Specific treatments are shown in the table below.

[0065] Table 4 Animal group feeding method

[0066] Note: Mice and feed were purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0067] 3% fermented bee pollen by-product mixed feed refers to 3g bee pollen by-product mixed with 97g 60% high-fat control feed, which is HFD+PBP feed; The 3% fermented bee pollen by-product mixed feed refers to 3g of fermented bee pollen by-product mixed with 97g of 60% high-fat control feed, which is HFD+FPBP feed.

[0068] 2. Animal Sacrifice and Collection After the glucose tolerance test, collect fresh feces from the mice on ice, snap-freeze in liquid nitrogen, and store at -80°C. The night before sacrifice, remove the mice's feed, replace the bedding, and deprive them of food but not water. After 12 hours, weigh the mice.

[0069] Blood was collected by enucleation of the eyeballs and collected in a 2 mL sterile centrifuge tube. The mice were then allowed to rest overnight at 4°C. The mice were then stretched out and their body length (distance from the nose tip to the anus) was measured. The Lees index was calculated according to formula (2-3). The mice were fixed on a dissecting table, the abdominal cavity was opened, and the epididymal fat, pancreas, and liver were removed. The entire intestine was removed from the stomach to the anus. The peri-intestinal fat was carefully scraped with forceps and weighed. The intestine was flushed with a 1 mL syringe. The kidneys were removed, and the peri-renal fat was removed and weighed. The thoracic and abdominal cavities were removed, and subcutaneous fat was removed and weighed. A small, relatively intact portion of the epididymal fat, pancreas, liver, and colon was selected and fixed with 4% paraformaldehyde. The tissues were washed with PBS, blotted dry with absorbent paper, snap-frozen in liquid nitrogen, aliquoted into 2 mL sterile EP tubes, and stored at -80°C. The blood that had been allowed to stand overnight was centrifuged at 4000 rpm and 4°C for 10 min. The supernatant obtained was the mouse fasting serum, which was divided and stored in a -80°C refrigerator.

[0070]

[0071] Wherein, the unit of weight is gram (g) and the unit of body length is centimeter (cm).

[0072] 3. Detection of blood lipid related indicators (TG, T-CHO, HDL-C, LDL-C) Biological kits and microplate reader were used to detect and calculate the levels of triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the fasting serum of mice.

[0073] 4. Experimental Results Table 5 Effects of fermented bee pollen by-products on growth parameters of mice

[0074] Note: * indicates P < 0.05 compared with NC, # indicates P < 0.05 compared with HFD, P < 0.05 indicates significant difference As shown in Table 5, the obese group showed increased weight, decreased body length, and increased body fat percentage and Lees score compared to the normal group, demonstrating the validity of the experimental model for obesity. Compared to the obese group, mice fed fermented bee pollen by-products showed significantly lower body weight and body fat, longer and heavier colons, larger thymuses, and decreased serum triglyceride, total cholesterol, and high-density lipoprotein (HDL) levels (P < 0.05), demonstrating that bee pollen by-products are effective in alleviating high-fat diet-induced hyperlipidemia. In particular, fermented bee pollen by-products were superior to unfermented bee pollen by-products in multiple parameters, including weight, body fat, and blood lipids. They significantly inhibited high-fat diet-induced weight gain, improved inflammation, and promoted the growth of immune organs. In summary, fermented bee pollen by-products have beneficial effects on a variety of physiological functions, including improving immunity and regulating blood lipids.

[0075] Example 2 The fermented bee pollen by-product obtained in Example 1 was used to prepare mead.

[0076] The preparation of the mead comprises the following processes: Step 1: Mix fermentation liquid, water and honey; Step 2: Fermentation: After fermentation, take the supernatant; Step 3: Sterilization: Sterilize and can the finished product.

[0077] Fermentation was performed by mixing bee pollen byproducts, water, and honey in a mass ratio of 15.6:62.4:30.8 (mass ratio). The fermentation temperature was 25°C, and after 7 days of fermentation, the supernatant was removed and sterilized with 30 mg / L potassium sulfite. The finished product was canned.

[0078] Ordinary mead fermentation process: water: honey ratio is 78:30.8 (mass ratio), dry yeast inoculation amount is 0.10%, mixed fermentation (fermentation at 25℃ for 7 days), take the supernatant, add 30mg / L potassium sulfite for sterilization, and can the finished product.

[0079] Wine body index: Table 6

[0080] As shown in Table 6, the flavonoids, total amino acid content, DPPH free radical scavenging rate and sensory evaluation of fermented bee pollen by-product honey wine are far greater than those of ordinary honey wine. It contains higher bioactive substances, antioxidant activity and lower sugar content, and has a rich and delicate aroma, a mellow and long taste, and has a unique flavor that is refreshing, sweet and long-lasting.

[0081] The above embodiments are used to explain the technical solutions of the present invention. However, 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 of materials used in the present invention, etc., fall within the scope of protection of the patent.

Claims

1. A method for processing bee pollen by-products, characterized by: The processing method comprises the following steps: S1 Pretreatment: Dry and remove impurities from bee pollen by-products, crush them, and sieve them; S2 bee pollen by-products are added to water, soaked and homogenized, and sterilized to obtain a bee pollen by-product solution; S3 fermentation and cell wall breaking: after inoculation and fermentation of the bee pollen by-product solution, the supernatant is taken as the fermented bee pollen by-product; Complete processing of bee pollen by-products.

2. The bee pollen by-product processing method according to claim 1, characterized in that: The bee pollen by-product is the solid matter remaining after the rape bee pollen is soaked in ethanol.

3. The bee pollen by-product processing method 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).

4. The bee pollen by-product processing method according to claim 1, characterized in that: The drying condition of step S1 is to dry at 40° C. for 4-6 hours, then crush and pass through a 50-mesh sieve.

5. The bee pollen by-product processing method according to claim 1, characterized in that: In step S2, the bee pollen byproduct is soaked in water at a ratio of 1: (4-6) (mass-to-volume ratio g / mL) for 22-26 hours and homogenized, and the sterilization conditions are 65-80°C for 25-35 minutes; Preferably, in step S2, the bee pollen by-product and water are soaked and homogenized in a ratio of 1:5 (mass to volume ratio) for 24 hours, and the pasteurization condition is 70° C. for 30 minutes.

6. The bee pollen by-product processing method according to claim 1, characterized in that: In step S3, the bacterial inoculation amount is 8-12%, the bacterial mass ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation conditions are 30-38° C. and the culture time is 2.5-4 days; Preferably, the bacterial inoculation amount is 10%, the ratio of Lactobacillus rhamnosus to yeast is 1:1, and the fermentation temperature is 37° C. and the culture time is 3 days.

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

8. The use according to claim 7, characterized in that The preparation of the mead comprises the following processes: Step 1: Mix fermentation liquid, water and honey; Step 2: Fermentation: After fermentation, take the supernatant; Step 3: Sterilization: Sterilize and can the finished product.

9. The use according to claim 8, characterized in that In the step 1, the mass ratio of fermented bee pollen byproduct: water: honey is (13-17): (60-70): (15-32); preferably, the mass ratio of fermented bee pollen byproduct: water: honey is 15.6:62.4:30.

8.

10. The use according to claim 8, characterized in that In the step 2, the fermentation temperature is 25°C and the fermentation time is 3-7 days; In step 3: add 30 mg / L potassium sulfite for sterilization.

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