Strain for rapeseed pollen fermentation modification, microbial preparation and application thereof

Abstract

The application belongs to the technical field of microbial engineering, deep processing of agricultural products and fermentation technology of functional food, and discloses a strain for rapeseed pollen fermentation modification, a microbial preparation and application thereof. Pediococcus The strain is PC08 of Pediococcus sp., which is preserved in China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and has a preservation number of CCTCC M 20252324. The application effectively solves the technical problems of poor substrate adaptability, low wall breaking efficiency, insufficient release of active ingredients and heavy bitter taste of the existing pollen fermentation strains, and has a wide industrial application prospect in the field of functional food raw materials.

Description

Technical Field

[0001] This invention belongs to the fields of microbial engineering, deep processing of agricultural products and fermentation technology of functional foods. Specifically, it relates to a strain of Pediococcus pentosaceus adapted for fermentation modification of rapeseed pollen, a microbial preparation containing this strain, and its application method in the enrichment of active ingredients, enhancement of nutritional value and improvement of flavor in rapeseed pollen. Background Technology

[0002] Rapeseed pollen contains high-quality plant protein, plant polysaccharides, vitamins, minerals, polyphenols and other functional active substances. It has significant physiological effects such as anti-oxidation, anti-inflammation, immune regulation, blood sugar regulation and improvement of intestinal microecology. It has extremely high development value in the fields of health food and biomedicine.

[0003] my country has a large output and abundant resources of rapeseed pollen, but its industrial development and utilization rate is currently extremely low, resulting in a serious waste of resources. The outer wall of rapeseed pollen is composed of dense substances such as cellulose and pectin. The pollen wall is tough and stable, making it difficult to break down using conventional processing methods. This prevents the effective release of nutrients from the pollen, making it difficult for the human body to directly absorb and utilize them. Consequently, the bioavailability is significantly reduced, leading to low content of functional active substances and low product value in the final product.

[0004] Microbial fermentation, with its advantages of being green and safe, operating under mild conditions, and simultaneously achieving cell wall disruption, quality improvement, flavor enhancement, and reduction of anti-nutritional factors, has become the mainstream research direction for high-value pollen processing. However, existing pollen fermentation technologies are mostly concentrated in pine pollen processing, and there is a severe lack of suitable strains and mature technology systems specifically for rapeseed pollen fermentation modification. Directly applying conventional commercial fermentation strains to rapeseed pollen fermentation presents the following significant technical drawbacks: Firstly, the strains lack substrate tolerance. Rapeseed pollen contains high levels of phenolic acids, tannins, and phytic acid, which are substances with antibacterial activity, creating a unique high-stress substrate environment. Conventional brewing yeast and common lactic acid bacteria have difficulty surviving in this system, resulting in unstable fermentation activity and making it difficult to effectively start and sustain the fermentation process.

[0005] Secondly, fermentation quality control is difficult. Due to the poor compatibility between the strain and the pollen substrate, the fermentation process is prone to problems such as rancidity, off-odors, and uncontrollable metabolites, making it difficult to ensure batch-to-batch stability and consistency, which seriously restricts the industrial application and promotion.

[0006] Thirdly, fermented pollen products exhibit a pronounced bitter taste and poor flavor quality. The polyphenols, flavonoids, and alkaloids naturally present in rapeseed pollen possess inherent bitterness. Under improper fermentation conditions, these precursors may undergo unintended transformations or interact with protein degradation products, further generating or intensifying unpleasant flavors. Existing fermentation strains lack the selective metabolic transformation ability for these bitter precursors, making it difficult to effectively reduce the inherent bitterness of the pollen matrix. This results in fermented pollen products with a rough texture and poor flavor harmony, directly impacting consumer acceptance and the market competitiveness of the final product. The difficulty in achieving both flavor and quality has become a significant bottleneck restricting the extension of fermented pollen products from raw material processing to end-consumer applications.

[0007] In summary, addressing the industry pain points in existing rapeseed pollen processing technologies, such as poor survival rate of fermentation strains in pollen matrices, difficulty in reducing bitterness, and inconsistent product quality, this study aims to screen a specific fermentation strain that can adapt well to the unique matrices of rapeseed pollen, possesses efficient cell wall disruption and release capabilities, significantly enriches active ingredients, and simultaneously reduces bitterness and improves flavor. Furthermore, the development of corresponding microbial preparations and fermentation application technologies is of significant practical importance and has broad application prospects for overcoming the technological bottlenecks in rapeseed pollen fermentation processing, achieving high-value utilization of resources, and enhancing product added value and market competitiveness. Summary of the Invention

[0008] This invention aims to overcome the shortcomings of existing rapeseed pollen fermentation and processing technologies, such as poor compatibility of commercially available strains, low viable cell retention rate, limited pollen cell wall disruption, and insufficient release of active ingredients. This invention aims to provide a Pediococcus PC08 strain specifically modified for rapeseed pollen fermentation, a microbial preparation, and its fermentation application method. This achieves efficient cell wall disruption of rapeseed pollen and promotes the release of active substances.

[0009] The first aspect of the present invention provides a strain for rapeseed pollen fermentation, said strain being Pediococcus ( Pediococcus sp.) PC08 is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 20252324.

[0010] This strain was isolated, screened, and domesticated from the traditional naturally fermented pickling brine used in rural areas of the Dabie Mountains. The specific screening process is as follows: (1) Strain isolation: Dilute the traditional natural fermented pickling brine of Dabie Mountain farmhouses (fermentation cycle ≥30 d) with sterile physiological saline, spread it on MRS solid plates, and incubate at 37℃ for 24 h. Select vigorous and regular single colonies, and obtain multiple pure cultures by three consecutive three-zone streak purifications. (2) Gradient acclimatization: The initially screened and purified strains were sequentially transferred to MRS liquid medium containing 5%, 10%, 15%, and 20% (w / v) rapeseed pollen extract, and cultured at 37℃ for 48 h in stages to carry out directional tolerance acclimatization. (3) Screening of superior strains: After gradual domestication, based on the proliferation activity of each strain at different concentration gradients and the colony formation ability and growth status on 20% (w / v) high-concentration pollen solid plates, a domesticated strain with good tolerance to rapeseed pollen substrate and significant growth advantage was selected and named PC08.

[0011] The *Pediococcus pentosaceus* PC08 strain of this invention possesses the following biological characteristics: Colonies are spherical, 0.5-2 mm in diameter, with neat, opaque, pale yellow edges, a slightly raised center, a smooth, moist, glossy surface, a soft, viscous texture, and are easily picked up; the bacterial cells are Gram-positive spherical, non-spore-forming, non-flagellated, and non-motile; it is facultatively anaerobic, tolerating weakly acidic environments of pH 3.5-8 and hypertonic environments of 3-8% NaCl; the fermentation type is homolactic fermentation, producing no gas, no hydrogen sulfide, no gelatin liquefaction, and no starch hydrolysis ability, but can utilize glucose, sucrose, and fructose for acid production; the 16S rRNA sequence shows >99% homology with *Pediococcus pentosaceus*, and based on comprehensive morphological, physiological, biochemical, and molecular identification results, it is identified as *Pediococcus pentosaceus*; the *Pediococcus pentosaceus* PC08 strain exhibits high tolerance to endogenous tannins and phytic acid in rapeseed pollen, and can stably colonize and grow in rapeseed pollen fermentation systems, with a fermentation time of 48 days. h can still maintain a high number of viable bacteria.

[0012] A second aspect of the present invention provides a microbial preparation comprising the above-described Pediococcus PC08.

[0013] According to a preferred embodiment of the present invention, the microbial preparation is a solid preparation comprising Pediococcus PC08 cells and a food-grade composite carrier, wherein the viable count of Pediococcus PC08 in the preparation is ≥1.2 × 10⁻⁶. 7 The concentration of CFU / g can be stored at room temperature (4-25℃). The food-grade composite carrier is composed of the following components by mass fraction: 15%-20% skim milk, 2%-5% sucrose, 1%-2% trehalose, 0.5%-2% isomaltooligosaccharide, 3%-5% mannitol, and 0.1%-0.5% vitamin C; the mixing mass ratio of the bacterial cells to the composite carrier is 1:3-5. The microbial preparation can be prepared by mixing the components, followed by low-temperature vacuum drying and pulverization.

[0014] A third aspect of the present invention provides the application of the above-mentioned strains or microbial preparations in the bio-fermentation modification of rapeseed pollen, increasing the content of active nutritional components in rapeseed pollen, reducing bitterness or improving flavor.

[0015] Specifically, the application includes: breaking down the dense pollen walls of rapeseed pollen to increase the content of nutritionally active ingredients; the nutritionally active ingredients include at least one of total pollen phenols, quercetin, kaempferol, luteolin, and disinoyl glucoside.

[0016] A fourth aspect of the present invention provides a method for modifying rapeseed pollen by liquid fermentation based on Pediococcus PC08, comprising the following steps: (1) Raw material pretreatment: Dry rapeseed pollen is sieved to remove impurities and large particles; (2) Preparation of liquid: Mix the sieved rapeseed pollen with sterile pure water and stir thoroughly; (3) Mild sterilization: pasteurization is used, and the mixture is allowed to cool naturally to room temperature after sterilization; (4) Constant temperature fermentation: Inoculate with 1%~3% of the above-mentioned microbial preparation by mass and ferment at a constant temperature of 25~37 ℃ for 24~48 h with stirring; (5) Finished product preparation: After fermentation, the product is dried at low temperature under vacuum, crushed and sieved to obtain fermented modified rapeseed pollen.

[0017] According to the present invention, preferably, in step (1), the mesh size of the sieve is 40-80 mesh; in step (2), the ratio of rapeseed pollen after sieving to sterile pure water is 1:4-6 g / mL; in step (3), the pasteurization conditions include: sterilization temperature of 65-68 ℃ and sterilization time of 10-20 min; in step (5), the temperature of low-temperature vacuum drying is 30-50 ℃ and the mesh size of the sieve is 40-80 mesh.

[0018] The fifth aspect of this invention provides a fermented modified rapeseed pollen product obtained by the above method, wherein the fermented modified rapeseed pollen product has the following characteristics: pollen cell wall breakage rate ≥91%, total flavonoid content ≥520 mg / Kg, total phenol content ≥1020 mg / Kg, quercetin content ≥275 mg / Kg, kaempferol content ≥186 mg / Kg, luteolin content ≥75 mg / Kg, and disinoyl glucoside content ≥875 mg / Kg. All indicators are significantly superior to the blank unfermented group, the brewer's yeast fermented group, and the ordinary lactic acid bacteria fermented group.

[0019] The sixth aspect of this invention provides a method for improving the colonization stability of rapeseed pollen fermentation strains. The method involves applying the aforementioned *Pediococcus PC08* or the aforementioned microbial preparation to a rapeseed pollen fermentation system. Under the stress of endogenous antibacterial substances in pollen, the viable count of the strain can reach 1.52 × 10⁻⁶ after 48 hours of fermentation. 8 The CFU / g is significantly better than that of Saccharomyces cerevisiae and common lactic acid bacteria, solving the defects of traditional fermentation strains in rapeseed pollen systems, such as rapid decline of viable bacteria, poor fermentation stability, and weak metabolic persistence.

[0020] By adopting the above technical solution, the present invention achieves the following beneficial effects: (1) This invention provides a novel fermentation strain with specific adaptability to rapeseed pollen. Specifically, it was selectively isolated from the traditional natural fermentation brine of pickled vegetables in the Dabie Mountains and continuously passaged and domesticated by gradually increasing the concentration of pollen extract (5%→10%→15%→20%, w / v) to obtain Pediococcus PC08, which has excellent tolerance to rapeseed pollen substrate. This strain has extremely strong tolerance to antibacterial stresses such as tannins, phytic acid, and polyphenols in rapeseed pollen, and the viable count in the pollen fermentation system is as high as 1.52×10⁻⁶. 8 The CFU / g was 82.6 times that of the Saccharomyces cerevisiae fermentation group and 12.1 times that of the ordinary lactic acid bacteria fermentation group, respectively, which solved the technical problems of low survival rate and unstable fermentation activity of existing fermentation strains in pollen matrix.

[0021] (2) This invention achieves efficient cell wall disruption and full release of intracellular active ingredients. Specifically, the *Pediococcus* PC08 strain of this invention can produce highly efficient cell wall degrading enzyme systems (such as cellulase and pectinase) during fermentation, which synergistically disrupt the dense structure of the rapeseed pollen outer wall, achieving a cell wall disruption rate of 91.27%, significantly better than the fermentation group of *Saccharomyces cerevisiae* (83.19%) and the fermentation group of commercially available lactic acid bacteria (86.90%). The effective disruption of the pollen wall promotes the full release of intracellular nutrients and active ingredients, laying a structural foundation for the subsequent enrichment of active ingredients.

[0022] (3) This invention significantly increases the content of polyphenols and characteristic flavonoids in rapeseed pollen. Specifically, after fermentation modification with Pediococcus PC08, the total phenol content in rapeseed pollen reached 1023.5 mg / Kg, which is 7.50 times that of the unfermented blank pollen, 2.59 times that of the Saccharomyces cerevisiae fermentation group, and 2.49 times that of the ordinary lactic acid bacteria fermentation group. The contents of the four characteristic active ingredients, quercetin, kaempferol, luteolin, and disinol glucoside, reached 275.3 mg / Kg, 186.5 mg / Kg, 75.9 mg / Kg, and 879.3 mg / Kg, respectively, which are significantly higher than those of the unfermented blank group. The significant increase in total phenols and characteristic flavonoids provides a solid material basis for enhancing the antioxidant, anti-inflammatory, and other biological activities of fermented pollen.

[0023] (4) During the fermentation process, the Pediococcus PC08 of this invention can effectively metabolize and transform the precursor substances such as polyphenols, flavonoids and alkaloids in pollen that cause bitterness, converting them into flavorful derivatives. At the same time, the lactic acid, short-chain fatty acids and esters produced during fermentation give the product a unique fermented aroma. The PC08 fermented pollen is significantly better than unfermented pollen and the control groups in terms of aroma harmony, smoothness of taste, bitterness intensity and overall acceptability, effectively solving the industry pain points of traditional pollen products such as rough taste, strong bitterness and low consumer acceptance.

[0024] (5) Currently, there are no products or technical solutions on the market for the targeted fermentation modification of rapeseed pollen using Pediococcus. This invention constructs a complete technical chain from strain screening and domestication, microbial preparation, fermentation process optimization to product quality evaluation, providing core strain resources and reliable technical paths for the efficient cell wall breaking modification and functional quality improvement of rapeseed pollen. It has important practical significance and broad application prospects for breaking through the technical bottleneck of rapeseed pollen fermentation and processing, realizing high-value utilization of resources, and enhancing product added value and market competitiveness.

[0025] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0026] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0027] Figure 1 The colony morphology of strain PC08 on MRS plates is shown.

[0028] Biological Preservation Instructions

[0029] Pediococcus PC08 was deposited on October 24, 2025, at the China Center for Type Culture Collection (CCTCC). Address: 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China; Accession number: CCTCC M 20252324. Detailed Implementation

[0030] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0031] Example 1: Isolation, purification, and identification of Pediococcus PC08

[0032] 1. Sampling

[0033] The source of the isolate was the traditional naturally fermented pickled vegetable brine from the Dabie Mountains. The fermentation cycle was ≥30 days. The fermentation system contained no exogenous industrial strains or preservatives. It was sealed and fermented at room temperature in an anaerobic manner. After sampling, the samples were refrigerated at low temperature and the strain isolation operation was completed within 24 hours.

[0034] 2. Initial screening for strain isolation and purification

[0035] 2.1 Sample Pretreatment and Gradient Dilution

[0036] Inside a sterile laminar flow hood, 10 mL of naturally fermented brine for pickled vegetables was quickly added to a 250 mL Erlenmeyer flask containing 90 mL of sterile 0.85% physiological saline. The flask was sealed and placed on a shaker at 180 rpm for 30 min to thoroughly break up the bacterial aggregates, thus preparing a 100% dilution. -1 The sample stock solution was diluted stepwise using a tenfold serial dilution method: 1 mL of 10... -1 The bacterial suspension was transferred to a 9 mL sterile saline tube and thoroughly mixed by inverting to obtain 10... -2 Diluent; prepare 10 in sequence using this method. -3 10 -4 10 -5 10 -6 10 -7 10 -8 A total of 8 graded bacterial suspensions were prepared.

[0037] 2.2 Plate coating and isothermal incubation

[0038] Take sterile MRS solid separation plates and set up 3 parallel plates for each dilution gradient. Use a sterile spreader to take 0.25 mL of the corresponding gradient bacterial solution and spread it evenly on the surface of the plate until the bacterial solution is completely absorbed. After spreading, invert all plates and place them in a constant temperature anaerobic incubator, set the incubation temperature to 37℃, and incubate in the dark for 24 h.

[0039] 2.3 Single colony picking

[0040] After incubation, remove the plates and prioritize dilution gradient plates with dispersed colonies and moderate density. Select vigorous, well-formed single colonies. Remove abnormal colonies such as molds, bacilli, and other miscellaneous bacteria. Use a sterile inoculation loop to pick single colonies that are uniform in shape, color, and size.

[0041] 2.4 Three-zone streak purification

[0042] The selected single colonies were transferred to brand-new MRS solid plates using the three-zone streak method and incubated at 37°C for 24 h. After the single colonies grew on the plates, the streak operation was repeated again by selecting independent single colonies. The complete purification process was repeated 3 times to completely remove symbiotic bacteria and obtain multiple pure cultures of lactic acid bacteria with stable genetic traits and no contamination. All purified strains were numbered and stored at 4°C for short-term use.

[0043] 3. Re-screening of rapeseed pollen compatibility

[0044] 3.1 Preparation of secondary screening culture medium

[0045] The specific steps for preparing MRS liquid and solid screening media containing rapeseed pollen extracts of different concentration gradients (5%, 10%, 15%, 20%, w / v) are as follows: Degreased and impurity-removed rapeseed pollen was mixed with distilled water in a predetermined ratio and extracted in a 45°C water bath for 2 hours. The mixture was then filtered through gauze to remove insoluble pollen residue, and the filtrate was collected as the pollen extract. Appropriate amounts of the extract were measured and added to MRS liquid basal medium, and the volume was adjusted to the target volume to prepare MRS liquid medium with final pollen extract concentrations of 5%, 10%, 15%, and 20% (w / v). The initial pH was adjusted to 6.3 with 1 mol / L HCl or NaOH, and then autoclaved at 121°C for 20 minutes to obtain MRS liquid secondary screening mediums for each concentration gradient. Based on this, 1% (w / v) agar powder was added to each of the above liquid mediums, the initial pH was adjusted to 6.3, and the medium was autoclaved at 121°C for 20 minutes to obtain MRS solid secondary screening mediums for the corresponding concentration gradients.

[0046] 3.2 Stepwise acclimatization and tolerance screening of strains

[0047] All purified strains obtained from the initial screening were inoculated into MRS liquid medium and activated at 37°C for 4 hours to serve as the initial seed culture.

[0048] The strains were subjected to targeted acclimatization and tolerance screening by gradually increasing the concentration of pollen extract: First, the seed culture of each strain was transferred to MRS liquid screening medium containing 5% (w / v) rapeseed pollen extract at an inoculum of 2% (v / v) and cultured at 37℃ for 48 h. Then, the culture was transferred to MRS liquid medium containing 10% (w / v) pollen extract at an inoculum of 2% (v / v) and cultured at 37℃ for another 48 h. Next, the culture was transferred to MRS liquid medium containing 15% (w / v) pollen extract at the same inoculum and cultured at 37℃ for 48 h. Finally, the culture was transferred to MRS liquid medium containing 20% ​​(w / v) pollen extract at the same inoculum and cultured at 37℃ for 48 h, thus completing the stepwise adaptation of the strains to high-concentration pollen matrix.

[0049] After the culture was completed, the fermentation liquid after being cultured with 20% (w / v) pollen extract was dipped into the inoculation loop and evenly spread on the surface of MRS solid screening medium containing 20% ​​(w / v) rapeseed pollen extract, and then placed at 37℃ for 24 h.

[0050] 3.3 Screening and acquisition of superior domesticated strains

[0051] After the above-mentioned step-by-step domestication and solid plate separation, the proliferation activity of each strain in liquid culture medium of various concentration gradients and the colony formation ability and growth status on 20% (w / v) high-concentration pollen solid plates were comprehensively evaluated. Finally, a domesticated strain with good tolerance to rapeseed pollen matrix and showing significant growth advantage was obtained and named PC08. It was used for subsequent morphological observation, physiological and biochemical characteristic analysis and molecular biological identification experiments.

[0052] 4. Morphological identification steps

[0053] 4.1 Colony morphology characteristics

[0054] The purified and preserved strain PC08 was inoculated onto fresh MRS solid medium plates using the streak plating method. The plates were then inverted and incubated at 37°C for 24 hours. After clear single colonies grew on the plates, they were removed and observed and recorded under sterile natural light. Figure 1 As shown: the colonies are regular and round, with a single colony diameter ranging from 0.5 to 2 mm. The strain grows evenly without spreading, and the colony edges are smooth and intact. The colonies are generally opaque, with a uniform light yellow body and no discolored spots. The center of the colony is slightly raised, and the surface is smooth, moist, and glossy without wrinkles. The colonies are soft and viscous in texture, and can be completely lifted by the inoculation loop with a light touch. They are not easily broken or stick to the plate.

[0055] 4.2 Gram staining

[0056] Fresh bacterial suspension was smeared, Gram stained, and observed under an oil immersion microscope. The results showed that the strain was Gram positive, with tetrad spherical cells, no spores, no flagella, and no motility.

[0057] 5. Physiological and biochemical characteristics

[0058] Activated strain PC08 was brought to the logarithmic growth phase, and biochemical tests were carried out. Each group was repeated 3 times, and the following items were tested: (1) Anaerobic growth test: facultative anaerobic, grows more vigorously in anaerobic environment; (2) Acid and alkali tolerance: can grow in medium with pH 3.5-8.0; (3) Salt tolerance test: can proliferate normally under 3-8% NaCl conditions; (4) Fermentation type: homolactic fermentation, no gas production; (5) Characteristic reaction: negative for hydrogen sulfide, gelatin liquefaction, and starch hydrolysis; (6) Carbon source utilization: can utilize glucose, sucrose, and fructose to produce acid, but cannot utilize xylose and lactose.

[0059] 6. Identification of 16S rRNA molecules

[0060] (1) Collection of bacterial cells: PC08 bacterial culture was cultured at 37℃ for 18 h, centrifuged at 10000 r / min for 5 min, and the bacterial cell precipitate was collected; (2) Genomic DNA extraction: DNA was lysed, purified, and eluted using a kit method; integrity was detected by gel electrophoresis; and purity was determined by spectrophotometry. (3) PCR amplification of 16S rRNA, primers: 27F / 1492R; 50μL amplification system; program: 94℃ pre-denaturation for 5 min; 32 cycles (94℃ depolymerization for 30 s → 55℃ extension for 30 s → 72℃ extension for 90 s); 72℃ final extension for 10 min; (4) Sequencing and homology comparison: The recovered product was sequenced, and the sequence was uploaded to NCBI BLAST for comparison; the sequence showed >99.3% homology with the Pediococcus pentosaceus standard strain; (5) Based on the comprehensive morphological, biochemical, and molecular results, it was identified as Pediococcus pentosaceus. Pediococcus pentosaceus .

[0061] 7. Preservation of microbial strains

[0062] The purified, highly active PC08 strain was deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCM 20252324.

[0063] Example 2: Preparation of Pediococcus PC08 microbial preparation

[0064] 1. Strain activation and seed culture preparation

[0065] Preserved *Pediococcus* PC08 was inoculated onto MRS solid medium and incubated at 37 ℃ inverted for 24 h. Single colonies were then picked and inoculated onto MRS liquid medium and incubated at 37 ℃ for 20 h to complete primary activation. The culture was then transferred to fresh MRS liquid medium at a 4-8% inoculation rate and activated a second time under the same conditions for 36-48 h until the viable count was ≥1.0 × 10⁻⁶. 8 The seed culture at CFU / mL was used for subsequent lyophilized powder experiments.

[0066] 2. Preparation of microbial preparations

[0067] The mature PC08 bacterial culture was centrifuged to collect the bacterial cells. A mixture of skim milk (15-20%), sucrose (2-5%), trehalose (1-2%), isomaltooligosaccharide (0.5-2%), mannitol (3-5%), and vitamin C (0.1-0.5%) was used as a carrier. The mixture was thoroughly mixed at a bacterial cell to carrier mass ratio of 1:4, then vacuum dried at low temperature and pulverized to prepare a solid microbial preparation with a viable cell count ≥1.2 × 10⁻⁶. 7 CFU / g, store at room temperature (4-25℃).

[0068] Example 3: Modification of rapeseed pollen by liquid fermentation

[0069] 1. Raw material pretreatment: Dried rapeseed pollen is passed through a 60-mesh sieve to remove impurities; 2. Preparation of liquid solution: Mix rapeseed pollen with sterile pure water at a ratio of 1:5 (g / mL) and stir thoroughly. 3. Sterilization treatment: Pasteurize at 65-68℃ for 15 minutes, then allow to cool naturally to room temperature; 4. Fermentation inoculation: Inoculate with 1-3% (mass fraction) of PC08 microbial preparation, and ferment at a constant temperature of 25-37 ℃ with stirring for 24-48 h; 5. Finished product preparation: After fermentation, the product is dried under vacuum at low temperature (30-50℃), pulverized, and passed through a 60-mesh sieve to obtain rapeseed pollen modified by strain HF06.

[0070] To investigate the superiority of the *Pediococcus* PC08 strain of the present invention, a control experiment was further set up according to the above method, including blank unfermented pollen without any strain, *Saccharomyces cerevisiae* fermented pollen with commercially available yeast strain added, and lactic acid bacteria fermented pollen with commercially available lactic acid bacteria added. The following examples were used to test various indicators of each group of fermented pollen.

[0071] 5. Flavor Improvement: The polyphenols, flavonoids and alkaloids naturally present in rapeseed pollen are the main precursors that cause the bitter taste of pollen products.

[0072] As shown in Table 1, the sensory evaluation results showed that the rapeseed pollen fermented with Pediococcus PC08 had an aroma harmony score of 8.7, exhibiting a unique mellow fermented aroma without any unpleasant rancid or off-odors; a smoothness score of 8.3, with a fine powder texture and no roughness; a bitterness intensity score of 8.6 (the higher the score, the lighter the bitterness), which was significantly lower than the blank unfermented pollen (2.8); and an overall acceptability score of 8.5, which was far superior to the brewer's yeast fermentation group (5.1) and the ordinary lactic acid bacteria fermentation group (5.6).

[0073] In summary, the *Pediococcus* PC08 of this invention, during the fermentation of rapeseed pollen, generates organic acids, primarily lactic acid and acetic acid, through sugar and amino acid metabolism pathways, giving the product a mellow sour taste. Simultaneously, it produces short-chain fatty acids (such as butyric acid and valeric acid) and esters (such as ethyl acetate and ethyl lactate), imparting a unique fermented aroma and fruity fragrance to the product. This results in a harmonious and rounded fermented aroma, effectively masking the original raw pollen taste and unpleasant odors.

[0074] The Pediococcus PC08 fermentation modification technology of this invention achieves efficient pollen wall disruption and significant enrichment of active ingredients, while also effectively reducing the bitterness of rapeseed pollen products and imparting a mellow fermented aroma. This technology effectively solves the industry pain points of traditional pollen products, such as rough taste, strong bitterness, and poor flavor harmony. It lays an important foundation for the flavor and quality of fermented pollen products, extending from raw material processing to end-consumer applications, and significantly improves the market acceptance and industrial competitiveness of the products.

[0075] Table 1. Flavor-improving effects of fermented pollen

[0076] Example 4: Number of viable colonies in fermented pollen

[0077] This embodiment uses rapeseed pollen as a complex substrate. By detecting the number and survival rate of Pediococcus PC08 in the pollen fermentation system, the environmental tolerance of this strain to the pollen substrate is explored. At the same time, the survival effect of Saccharomyces cerevisiae is compared to verify the specific adaptability and application advantages of Pediococcus PC08 to the rapeseed pollen fermentation system.

[0078] 1. Sampling: The experimental samples were selected from three groups of rapeseed pollen samples prepared in Example 3, namely blank unfermented pollen, Saccharomyces cerevisiae-fermented pollen, and Pediococcus PC08-fermented pollen. 1.0 g of each pollen sample was weighed and added to 9 mL of sterile physiological saline, and stirred thoroughly to prepare pollen-bacterial suspension mother liquor.

[0079] 2. Serial dilution: After allowing the solution to stand and stabilize, take 1.0 mL of the stock solution and add 9 mL of sterile physiological saline to perform serial dilution. Prepare sample diluents of appropriate dilution factors sequentially. 3. Dilution and Plating: Colony counting was performed using the serial dilution plate plating method. 0.1 mL of each serial dilution was evenly spread onto the corresponding strain's solid culture medium plates. Each group was tested in triplicate. 4. Strain Culture: After incubation at 37 ℃ for 12 hours, plates with colony counts ranging from 30 to 300 were selected for counting. Each experiment was repeated three times, and the average value was taken. The formula for calculating the number of surviving colonies per gram of pollen sample is:

[0080] In the formula: N is the number of colonies per gram of pollen sample, CFU / g; M is the average number of colonies on the plate; D is the sample dilution factor; V1 is the total volume of sample dilution and final volume, mL; V2 is the volume of sample taken from the plate coating, mL; m is the mass of the pollen sample, g.

[0081] The counting results are shown in Table 2. No viable bacteria were detected in the pollen of the blank group, indicating no contamination by other microorganisms. The substrate background was clean and did not affect the counting results of the experimental groups. In the Saccharomyces cerevisiae group, the viable bacterial count after fermentation was 1.84 × 10⁻⁶. 6 CFU / g; Common lactic acid bacteria group: viable count was 1.26 × 10⁻⁶. 7 CFU / g; Pediococcus PC08 experimental group: viable cell count after fermentation reached 1.52×10⁻⁶. 8 The CFU / g count was significantly higher than that of the three control groups mentioned above.

[0082] Compared to the Pediococcus PC08 of this invention, traditional commercial brewing yeast and lactic acid bacteria exhibit poor survival stability in pollen substrates. The Pediococcus PC08 of this invention demonstrates strong tolerance to antibacterial stresses such as tannins, phytic acid, and polyphenols in the complex matrix of rapeseed pollen. During fermentation, the strain grows well, maintains a high viable cell count, and exhibits excellent colonization stability. It can stably grow and metabolize in high-stress pollen substrates, proving that PC08 possesses specific environmental adaptability for rapeseed pollen fermentation systems. This effectively solves the technical defects of existing fermentation strains, such as low survival rate, unstable fermentation activity, and poor metabolic sustainability, providing a core strain guarantee for efficient, stable, and large-scale fermentation and quality improvement processing of rapeseed pollen.

[0083] Table 2. Colony counts in each pollen sample

[0084] Example 5: Cell wall breakage rate of fermented rapeseed pollen

[0085] In this embodiment, the cell wall disruption rate of four groups of rapeseed pollen samples—blank unfermented pollen, Saccharomyces cerevisiae-fermented pollen, Lactic acid bacteria-fermented pollen, and Pediococcus PC08-fermented pollen of the present invention—was determined using the hemocytometer method. The integrity of pollen grain morphology was used as the evaluation index to systematically investigate the destructive effect of different fermentation treatments on the rapeseed pollen wall structure, providing direct morphological quantitative evidence for the cell wall disruption efficiency of the strain.

[0086] 1. Method for detecting cell wall breakage rate

[0087] 1.1 Count of intact pollen cells before cell wall disruption

[0088] Accurately weigh 0.10 g of blank, unfermented rapeseed pollen sample and place it in a 10 mL centrifuge tube. Add 5 mL of distilled water and vortex thoroughly to evenly disperse the pollen grains, thus preparing a pollen suspension before cell wall disruption. Take 50 μL of the suspension and add it dropwise into the counting chamber of a hemocytometer. Cover with a coverslip and observe and count under an optical microscope. Pollen grains with intact pollen walls, plump morphology, and no leakage of contents are considered intact pollen. Each sample is counted three times, and the average value is recorded as the number of intact pollen grains before cell wall disruption, N0.

[0089] 1.2 Sample pretreatment and intact pollen counting after fermentation

[0090] Three groups of fermented rapeseed pollen samples were collected: one group fermented with *Saccharomyces cerevisiae*, the other with *Lactobacillus simulans*, and the third group fermented with *Pediococcus pyogenes* PC08. Each sample was placed in a 10 mL centrifuge tube, and 5 mL of distilled water was added to each tube. The tubes were vortexed thoroughly to ensure uniform dispersion of the pollen particles, resulting in pollen suspensions after cell wall disruption. 50 μL of each suspension was added to the counting chamber of a hemocytometer and observed and counted under an optical microscope. Pollen particles with intact walls, plump morphology, and no leakage of contents were considered intact pollen. Each sample was counted three times, and the average value was recorded as the number of intact pollen particles (N1) after cell wall disruption in each group.

[0091] 1.3 Calculation of Cell Wall Breakage Rate

[0092] Calculate the cell wall breakage rate of each pollen sample group using the following formula: Cell wall breakage rate (%) = (1 - N1 / N0) × 100% Wherein, N0 is the number of intact pollen in the blank unfermented pollen sample before cell wall disruption, and N1 is the number of intact pollen in the pollen sample after cell wall disruption in each fermentation group.

[0093] 2. Results of cell wall breakage rate determination

[0094] The cell wall breakage rates of the rapeseed pollen samples in each group are shown in Table 3. It can be seen that most pollen grains in the unfermented blank pollen remained intact, with a breakage rate of only 5.21%. This breakage mainly originated from the mechanical forces exerted during pollen collection and processing. After fermentation with *Saccharomyces cerevisiae*, the pollen cell wall breakage rate significantly increased to 83.19%, indicating that the microbial metabolic activity during fermentation effectively destroyed the tough structure of the pollen wall. After fermentation with lactic acid bacteria, the breakage rate reached 86.90%, similar to the effect of the *Saccharomyces cerevisiae* group. However, after fermentation modification with *Pediococcus spp.* PC08 of this invention, the pollen cell wall breakage rate reached as high as 91.27%, the highest among the four groups, significantly better than the ordinary lactic acid bacteria fermentation group, indicating that the strain of this invention more thoroughly destroyed the rapeseed pollen wall.

[0095] The above results demonstrate that *Pediococcus PC08* can produce a highly efficient cell wall degrading enzyme system (such as cellulase and pectinase) during fermentation, effectively disrupting the dense structure of the rapeseed pollen outer wall and promoting the full release of pollen contents. This highly efficient cell wall disruption effect lays the foundation for improving the nutrient content of the strain in this invention during fermentation, and further verifies the significant technical advantages of *Pediococcus PC08* in rapeseed pollen cell wall disruption modification and quality improvement.

[0096] Table 3. Results of Rapeseed Pollen Cell Wall Breakage Rate in Each Sample Group

[0097] Example 6: Determination of total phenolic content in fermented rapeseed pollen

[0098] In this embodiment, the Folin-Ciocalteu colorimetric method was used to establish a standard curve with salicylic acid (SA) as the standard. The total phenol content in four groups of rapeseed pollen samples—blank unfermented pollen, Saccharomyces cerevisiae-fermented pollen, Lactic acid bacteria-fermented pollen, and Pediococcus PC08-fermented pollen of the present invention—was quantitatively detected. The fermentation effect of the strain of the present invention on the release and enrichment of polyphenolic active ingredients in rapeseed pollen was systematically evaluated, and the advantages of the fermentation of this strain on the quality improvement and modification of rapeseed pollen were further verified.

[0099] 1. Establishment of the standard curve for salicylic acid (SA)

[0100] Accurately weigh 50 mg of salicylic acid standard, dissolve it in 50% methanol solution and dilute to 50 mL to prepare a 1 mg / mL SA standard stock solution. Accurately measure 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the above stock solution, and dilute to 10 mL with pure water to prepare SA series standard working solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively.

[0101] Take 0.1 mL of each concentration gradient standard working solution into a graduated test tube, add 0.25 mL of Folin-Ciocalteu reagent, vortex to mix, and let stand for 3 min. Then add 0.5 mL of saturated sodium carbonate solution, add pure water to make up to 5 mL, and shake well. Incubate the above reaction solution in the dark at room temperature for 60 min. After the reaction, centrifuge at 3000 r / min for 10 min, and measure the absorbance of the supernatant at 725 nm. Perform linear regression fitting with salicylic acid mass concentration (mg / mL) as the abscissa (x) and the corresponding absorbance value as the ordinate (y) to establish the standard curve equation for the determination of total phenols.

[0102] 2. Extraction of total phenols from pollen samples and preparation of test solutions

[0103] Accurately weigh 0.10 g of each of the following four groups of rapeseed pollen samples: blank unfermented pollen, Saccharomyces cerevisiae-fermented pollen, Lactobacillus-fermented pollen, and Pediococcus PC08-fermented pollen. Place each sample in a sterile centrifuge tube, add 3 mL of 70% methanol solution to each tube, and vortex thoroughly for 20 min. After extraction, centrifuge at 3000 r / min for 10 min and collect the supernatant. Repeat the above extraction and centrifugation steps three times, combine all the supernatants obtained from the three extractions, transfer them to a 10 mL volumetric flask, dilute to the mark with pure water, and mix thoroughly to obtain the total phenols test solution for each group of pollen.

[0104] 3. Sample colorimetric reaction and absorbance measurement

[0105] Accurately pipette 0.1 mL of each pollen test solution into a graduated test tube, add 0.25 mL of Folin-Ciocalteu reagent, vortex to mix, and let stand for 3 min. Then add 0.5 mL of saturated sodium carbonate solution, dilute to 5 mL with pure water, and shake thoroughly. Incubate the reaction system in the dark at room temperature for 60 min. After the reaction, centrifuge at 3000 r / min for 10 min, and measure the absorbance of the supernatant at 725 nm. Three parallel samples were prepared for each group, and the average value of the results was used to calculate the total phenol content of each group of samples using the salicylic acid standard curve equation.

[0106] 4. Results of total phenol content determination

[0107] The results of the total phenol content determination of each group of rapeseed pollen samples are shown in Table 4. In natural, unfermented rapeseed pollen, polyphenols are mostly present in bound form within the pollen wall and intracellular matrix, with a low dissolution rate; the total phenol content is only 136.4 mg / kg. After fermentation with *Saccharomyces cerevisiae*, the total phenol content of the pollen increased to 394.7 mg / kg, but the improvement was limited. After fermentation with lactic acid bacteria, the total phenol content was 410.9 mg / kg, which, although somewhat higher than the control group, was still not significant. However, the rapeseed pollen fermented with *Pediococcus pyogenes* PC08 of this invention had a total phenol content as high as 1023.5 mg / kg, which is 7.50 times that of the unfermented control pollen, 2.59 times that of the *Saccharomyces cerevisiae* fermentation group, and 2.49 times that of the lactic acid bacteria fermentation group, showing a highly significant improvement.

[0108] The above results demonstrate that *Pediococcus PC08* can effectively disrupt the dense structure of rapeseed pollen walls during fermentation, breaking the bonds between polyphenols and macromolecular components such as proteins and polysaccharides, significantly promoting the dissociation and dissolution of bound polyphenols, thereby significantly improving the extractability of total phenolic active ingredients. This fermentation modification technology can effectively remove the encapsulation and binding effect of rapeseed pollen matrix on polyphenolic active ingredients, significantly increasing the dissolution and effective utilization rate of total phenolic substances, and synergistically enhancing the antioxidant, anti-inflammatory, and other biological activities of rapeseed pollen with flavonoid components. These results fully confirm that *Pediococcus PC08* of this invention has a highly efficient ability to improve and modify rapeseed pollen, and has excellent application value in the industrial development of functional pollen products.

[0109] Table 4. Colony counts in each pollen sample

[0110] Example 7: Determination of the contents of quercetin, kaempferol, luteolin and disinoyl glucoside in fermented rapeseed pollen

[0111] This embodiment employs ultra-high performance liquid chromatography (UPLC) to qualitatively and quantitatively detect the contents of four characteristic active components—quercetin, kaempferol, luteolin, and 1,2-disinapoylgentiobiose—in rapeseed pollen from the blank unfermented group, the Saccharomyces cerevisiae fermentation group, the common lactic acid bacteria fermentation group, and the Pediococcus PC08 fermentation group of this invention. This accurately evaluates the release and enrichment effect of PC08 strain fermentation on rapeseed pollen-specific flavonoid monomers and glycoside functional components, verifying the quality improvement and modification technology advantages of the strain fermentation of this invention at the microscopic active substance level.

[0112] 1. Sample pretreatment and preparation of test solution

[0113] Accurately weigh 0.10 g of each of the four groups of rapeseed pollen samples: blank unfermented pollen, Saccharomyces cerevisiae-fermented pollen, common lactic acid bacteria-fermented pollen, and Pediococcus PC08-fermented pollen. Place them in sterile centrifuge tubes, add 3 mL of 70% methanol solution to each, and vortex thoroughly for 20 min. After extraction, centrifuge at 3000 r / min for 10 min and carefully collect the supernatant. Repeat the above extraction and centrifugation operation three times, combine all the supernatants from the three extractions, transfer them to 10 mL volumetric flasks, and dilute to the mark with ultrapure water. Mix thoroughly to obtain the UPLC test extracts for each group of samples, and let stand for later use.

[0114] 2. UPLC chromatographic detection conditions

[0115] Ultra-high performance liquid chromatography (UHPLC) was used for separation and analysis. A Waters ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 µm, Waters, Milford, USA) was used. The detection wavelength was set to 330 nm, and the mobile phase flow rate was kept constant at 0.21 mL / min. The mobile phase system consisted of 45% mobile phase A and 55% mobile phase B, where mobile phase A was methanol and mobile phase B was an acetic acid-water solution (2:98, v / v).

[0116] 3. Establishment of standard curve and content calculation

[0117] A series of mixed standard gradient solutions containing quercetin, kaempferol, luteolin, and disinoyl glucoside were prepared and sequentially injected under UPLC chromatographic conditions identical to those used for sample detection. The retention time and peak area of ​​each characteristic component were recorded. Linear regression fitting was performed with the mass concentration (mg / L) of each standard as the x-axis and the corresponding peak area as the y-axis to establish standard curve equations for the four active ingredients, ensuring good linearity (R² ≥ 0.999) within the corresponding concentration gradient range. Using the external standard method, the content of the four characteristic active ingredients in each pollen sample was quantitatively calculated by substituting the peak areas of each characteristic component in the sample into the corresponding standard curve equations.

[0118] 4. Content determination results

[0119] The results of the content determination of the four characteristic active ingredients in each group of rapeseed pollen samples are shown in Table 5. As can be seen from the results in Table 5, in unfermented rapeseed pollen, quercetin (12.1 mg / kg), kaempferol (36.4 mg / kg), luteolin (12.8 mg / kg), and disinol glucoside (0.5 mg / kg) are mostly bound in the pollen wall and intracellular matrix in a densely bound form, and the content of free active ingredients is extremely low.

[0120] After fermentation with *Saccharomyces cerevisiae*, the contents of the four components were 9.24 mg / kg, 16.0 mg / kg, 13.3 mg / kg, and 0.02 mg / kg, respectively, all showing a decrease. After fermentation with lactic acid bacteria, the contents of the four components were 25.6 mg / kg, 58.1 mg / kg, 25.4 mg / kg, and 6.81 mg / kg, respectively, showing a certain increase compared to the control group, but the overall effect was still not significant.

[0121] After fermentation modification with Pediococcus PC08 according to this invention, the dense wall structure of rapeseed pollen was effectively destroyed, and macromolecular bound flavonoids and glycosides were fully dissociated and transformed. The contents of the four characteristic active ingredients reached 275.3 mg / kg, 186.5 mg / kg, 75.9 mg / kg and 879.3 mg / kg, respectively, which were 22.8 times, 5.1 times, 5.9 times and 1759 times higher than the blank unfermented group, respectively; 29.8 times, 11.7 times, 5.7 times and 43965 times higher than the Saccharomyces cerevisiae fermentation group, respectively; and 10.8 times, 3.2 times, 3.0 times and 129 times higher than the ordinary lactic acid bacteria fermentation group, respectively, showing extremely significant improvement.

[0122] The above results demonstrate that the Pediococcus PC08 fermentation system of this invention can precisely break through the encapsulation restrictions of characteristic flavonoids and glycosides in the rapeseed pollen matrix. Through enzymatic hydrolysis during fermentation, it effectively promotes the release of bound and enrichment of free forms of core functional components such as quercetin, kaempferol, luteolin, and disinol glucoside. These results further confirm the efficient quality improvement and modification capabilities of the strain of this invention on rapeseed pollen at the monomeric active ingredient level, verifying the superiority of this technology compared to traditional fermentation processes.

[0123] Table 5. Detection results of cell wall breakage rate and active substance content in each group of samples.

[0124] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A bacterial strain for modifying rapeseed pollen through fermentation, characterized in that, The strain is Pediococcus ( Pediococcus sp.) PC08 is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 20252324.

2. A microbial preparation, characterized in that, The microbial preparation comprises Pediococcus PC08 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation comprises Pediococcus PC08 cells and a food-grade composite carrier, wherein the viable count of Pediococcus PC08 in the preparation is ≥1.2 × 10⁻⁶. 7 CFU / g.

4. The microbial preparation according to claim 3, characterized in that, The food-grade composite carrier is composed of the following components in the indicated mass fractions: 15%~20% skim milk, 2%~5% sucrose, 1%~2% trehalose, 0.5%~2% isomaltooligosaccharide, 3%~5% mannitol, and 0.1%~0.5% vitamin C; the mass ratio of the bacterial cells to the composite carrier is 1:3~5.

5. The application of the strain described in claim 1 or the microbial preparation described in any one of claims 2-4 in the bio-fermentation modification of rapeseed pollen, the enhancement of the content of active nutritional components in rapeseed pollen, the reduction of bitterness or the improvement of flavor.

6. The application according to claim 5, characterized in that, The application includes: breaking down the dense pollen walls of rapeseed pollen to increase the content of nutritionally active ingredients; the nutritionally active ingredients include at least one of total pollen phenols, quercetin, kaempferol, luteolin, and disinol glucoside.

7. A method for liquid fermentation modification of rapeseed pollen based on Pediococcus PC08, characterized in that, Includes the following steps: (1) Raw material pretreatment: Dry rapeseed pollen is sieved to remove impurities and large particles; (2) Preparation of liquid: Mix the sieved rapeseed pollen with sterile pure water and stir thoroughly; (3) Mild sterilization: pasteurization is used, and the mixture is allowed to cool naturally to room temperature after sterilization; (4) Constant temperature fermentation: Inoculate with 1% to 3% by mass of the microbial preparation described in any one of claims 2-4, and ferment at a constant temperature of 25 to 37 °C for 24 to 48 h with stirring. (5) Finished product preparation: After fermentation, the product is dried at low temperature under vacuum, crushed and sieved to obtain fermented modified rapeseed pollen.

8. The method according to claim 7, characterized in that, In step (1), the sieve mesh size is 40~80 mesh; In step (2), the ratio of sieved rapeseed pollen to sterile pure water is 1:4~6 g / mL; In step (3), the pasteurization conditions include: sterilization temperature of 65~68 ℃ and sterilization time of 10~20 min; In step (5), the temperature of low-temperature vacuum drying is 30~50 ℃, and the mesh size of the sieve is 40~80 mesh.

9. The fermented modified rapeseed pollen product obtained by the method of claim 7 or 8, characterized in that, The fermented modified rapeseed pollen product has the following characteristics: pollen cell wall breakage rate ≥91%, total flavonoid content ≥520 mg / Kg, total phenol content ≥1020 mg / Kg, quercetin content ≥275 mg / Kg, kaempferol content ≥186 mg / Kg, luteolin content ≥75 mg / Kg, and disinoyl glucoside content ≥875 mg / Kg.

10. A method for improving the colonization stability of rapeseed pollen fermentation strains, characterized in that, The Pediococcus PC08 of claim 1 or the microbial preparation of any one of claims 2-4 is applied to the rapeseed pollen fermentation system.

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