A strain, microbial preparation and application for oilseed rape pollen fermentation

Abstract

The application belongs to the technical field of microbial fermentation, and discloses a strain, a microbial preparation and application for rapeseed pollen fermentation. Lactococcus lactis The strain is Lactococcus lactis (Lactococcus lactis) HF06, which is preserved in the 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 20252323. The application systematically solves key technical problems in deep development and utilization of rapeseed pollen from allergen elimination, active ingredient enrichment to functional lipid synthesis, and provides a complete microbial fermentation technical solution for production of high-quality and low-sensitivity rapeseed pollen products.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a lactococcus lactis strain HF06 that can be used for fermentation modification of rapeseed pollen, microbial preparations containing this strain, and the application of this strain in reducing rapeseed pollen allergenic proteins and in the preparation of functional products. Background Technology

[0002] Rapeseed pollen is rich in protein, flavonoids, amino acids, vitamins, and various bioactive substances. It possesses multiple pharmacological effects, including antioxidant, immune-enhancing, antibacterial, and prostate-protective properties, and has been included in the list of new resource foods, showing broad development prospects in bee products, health products, and food. However, the deep processing and utilization of rapeseed pollen still faces many technical bottlenecks. First, the dense structure of the pollen cell wall is a key obstacle limiting nutrient release. The outer wall of rapeseed pollen is mainly composed of sporophytin, which is heat-resistant, acid- and alkali-resistant, and enzymatically resistant, exhibiting extremely stable chemical properties; the inner wall is composed of cellulose and pectin. The robust pollen wall makes it difficult for the human body to digest and absorb intracellular nutrients, resulting in low bioavailability when consumed directly. Second, the allergenicity of pollen severely restricts its safety for consumption. Rapeseed pollen has a high protein content, and some proteins (such as Pollen Ole e1 family proteins and proteins containing the SCP domain) have potential allergenicity in susceptible individuals, potentially triggering IgE-mediated hypersensitivity reactions. Current desensitization methods mostly employ enzymatic hydrolysis to treat allergens, which is complex and costly. Furthermore, the lactic acid bacteria strains currently used in pollen fermentation research are not specifically screened for rapeseed pollen substrates. These strains exhibit low utilization efficiency of the pollen substrate, long fermentation cycles, and limited increases in active ingredients and desensitization effects, making them unsuitable for industrial production. Therefore, developing a dedicated fermentation strain with strong adaptability to rapeseed pollen substrates is crucial for simultaneously achieving efficient pollen cell wall disruption, targeted allergen degradation, and comprehensive improvement in nutritional quality. Summary of the Invention

[0003] In view of the current situation where the release of nutrients from rapeseed pollen is difficult and the risk of allergic reactions caused by pollen allergens is high in existing technologies, this invention provides a strain of Lactococcus lactis suitable for solid-state fermentation of rapeseed pollen ( Lactococcus lactis HF06 and its microbial preparations and applications. This invention aims to simultaneously achieve the release of nutritionally active components from rapeseed pollen, the elimination of allergenic proteins, and the conversion and synthesis of rare functional lipids, providing strain resources and technical support for the high-value utilization of rapeseed pollen and the development of low-allergenic functional foods.

[0004] The first aspect of this invention provides a bacterial strain that can be used for rapeseed pollen fermentation, said strain being *Lactococcus lactis* (…). Lactococcus lactisHF06 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 20252323.

[0005] The Lactococcus lactis strain HF06 of this invention was isolated from a sample of homemade fermented sauerkraut. The strain has the following microbiological characteristics: on MRS solid medium, the colonies are milky white, round, smooth and moist, with neat edges.

[0006] A second aspect of the present invention provides a microbial preparation comprising the aforementioned Lactococcus lactis HF06.

[0007] According to a preferred embodiment of the present invention, the microbial preparation comprises lyophilized Lactococcus lactis HF06 bacterial powder and excipients, wherein the excipients are maltodextrin and microcrystalline cellulose, and the viable count of the lyophilized bacterial powder is ≥1.0 × 10⁻⁶. 8 CFU / g. The amounts of maltodextrin and microcrystalline cellulose can be selected conventionally. For example, relative to 1 part by weight of freeze-dried bacterial powder, the amount of maltodextrin can be 1.2-1.5 parts by weight, and the amount of microcrystalline cellulose can be 0.1-0.2 parts by weight.

[0008] A second aspect of the present invention provides the application of the above-mentioned Lactococcus lactis HF06 or the above-mentioned microbial preparation in solid-state fermentation of rapeseed pollen.

[0009] A third aspect of the present invention provides a method for solid-state fermentation of rapeseed pollen, comprising the following steps: (1) Raw material pretreatment: The rapeseed pollen raw material is sieved to remove impurities. The moisture content is adjusted to 35-45% (i.e., the ratio of the weight of water to the weight of rapeseed pollen) based on the weight of the rapeseed pollen raw material after impurity removal, and then left to stand at 4℃ for hydration overnight. (2) Inoculation and fermentation: The lactococcus lactis HF06 described in claim 1 or the microbial preparation described in claim 2 or 3 is inoculated into the pretreated rapeseed pollen raw material at 5%~8% (w / w) for solid-state fermentation. The fermentation conditions include: fermentation temperature 26~30℃, stirring speed 60~100 r / min, fermentation time 68~80 h, and ambient humidity 80%~90%. (3) Post-processing: After fermentation, the fermented material is dried and crushed to obtain fermented rapeseed pollen product.

[0010] The method of removing impurities in step (1) is usually sieving, preferably sieving through a 40-80 mesh sieve.

[0011] The drying in step (3) is preferably vacuum freeze drying, with the following conditions: pre-freezing at -30℃ to -40℃ for 3 to 6 hours, sublimation drying at a cold trap temperature of -40℃ to -60℃ and a vacuum degree of ≤10 Pa, and then heating to 30℃ for desorption drying until the moisture content drops to below 5%; the pulverization is done by passing through a 60 to 100 mesh sieve.

[0012] A fourth aspect of the present invention provides a fermented rapeseed pollen product prepared by the above method, wherein the fermented rapeseed pollen product has a total flavonoid content ≥60 mg / 100g and a free amino acid content ≥8000 mg / 100g.

[0013] The fifth aspect of the present invention provides the use of the above-mentioned Lactococcus lactis HF06 or the above-mentioned microbial preparation in the degradation of rapeseed pollen allergen proteins; wherein the rapeseed pollen allergen proteins include at least one of Pollen Ole e 1 family proteins and proteins containing SCP domains.

[0014] The sixth aspect of the present invention provides the use of the above-mentioned Lactococcus lactis HF06 or the above-mentioned microbial preparation in the enrichment of hydroxy fatty acids in rapeseed pollen, wherein the hydroxy fatty acids include 10-hydroxypentadecanoic acid and / or 13-hydroxyheptadecanoic acid.

[0015] Compared with the prior art, the present invention has the following advantages: (1) The strain HF06 of this invention has the effect of promoting the release of nutrients such as flavonoids and amino acids. After fermentation by strain HF06 of this invention, the total flavonoid content of rapeseed pollen increased from 10.27 mg / 100g to 69.40 mg / 100g, which is about 5.76 times higher than the blank group and about 1.20 times higher than the commercially available lactococcus group; the free amino acid content increased from 796.32 mg / 100g to 8,603.14 mg / 100g, which is about 9.80 times higher than the blank group.

[0016] (2) The strain HF06 of the present invention has the function of degrading rapeseed pollen allergen proteins, including the core allergen Pollen Ole e1 and allergen proteins containing SCP domains and potential allergens, which are all downregulated, effectively reducing the risk of pollen sensitization.

[0017] (3) The strain HF06 of this invention has a specific fatty acid hydroxylation conversion ability. After fermentation, the contents of rare hydroxy fatty acids 10-hydroxypentadecanoic acid and 13-hydroxyheptadecanoic acid are as high as 249.22 mg / 100g and 187.91 mg / 100g, respectively, which are about 15.2 times and 12.9 times higher than those of commercially available lactococcus lactis group, significantly improving the functional added value of pollen products.

[0018] In summary, this invention systematically solves the key technical challenges in the deep development and utilization of rapeseed pollen, from strain screening, allergen elimination, active ingredient enrichment to functional lipid synthesis, and provides a complete microbial fermentation technology solution for the production of high-quality, low-allergenic rapeseed pollen products.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0020] 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.

[0021] Figure 1 The colony morphology of Lactococcus lactis HF06 is shown.

[0022] Biological Preservation Instructions

[0023] Lactococcus lactis HF06 ( Lactococcus lactis The specimen was deposited on October 24, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC M20252323. Detailed Implementation

[0024] 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.

[0025] Example 1: Isolation and Identification of Lactococcus lactis HF06

[0026] 1. Sample collection: The test samples were taken from homemade sauerkraut that had been naturally fermented for more than 30 days. After aseptic sampling, the samples were sealed and stored at low temperature and quickly transported back to the laboratory for use.

[0027] 2. Enrichment culture: Under aseptic conditions, accurately weigh 10g of sauerkraut sample, add 90 mL of sterile physiological saline, and mix by shaking at a constant temperature for 30 min to fully wash away microorganisms on the sample surface and inside, thus preparing a sample bacterial suspension; take 10 mL of the above bacterial suspension and inoculate it into MRS liquid enrichment medium, and culture at a constant temperature of 28 ℃ for 24 h to complete the enrichment and proliferation of the target strain and obtain the enriched bacterial solution.

[0028] 3. Gradual dilution and plating: The enriched bacterial solution was serially diluted 10-fold using sterile physiological saline to prepare 10-fold plating samples. -110 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 Seven gradient dilutions of bacterial suspension were prepared. 0.1 mL of each gradient dilution was taken and evenly spread on the surface of the directional screening solid medium. The directional screening solid medium was an MRS solid medium containing 5% rapeseed pollen extract and 2% rapeseed pollen, using rapeseed pollen cell wall coarse fibers as an induction substrate for directional screening of strains that can adapt to and utilize the rapeseed pollen matrix. After spreading, the plates were inverted and incubated at 28 °C for 28 h.

[0029] 4. Strain purification: After the culture is completed, larger single colonies with good growth are selected according to the size and growth status. They are continuously passaged for 5 generations to completely remove contaminating bacteria and obtain a single pure strain with stable characteristics, which is named HF06.

[0030] 5. Colony morphology identification: Using an inoculation loop, the purified HF06 bacterial suspension was streaked in a zigzag pattern on the surface of NB solid medium. The culture dish was then inverted and placed in a microbial incubator, where it was incubated at 28 ℃ until the colonies were stably grown. Observation results showed that the HF06 bacterial colonies were round, white on the front, and had a smooth surface. Figure 1 ).

[0031] 6. Molecular biological identification: Routine morphological observations were performed on the purified HF06 strain; simultaneously, genomic DNA was extracted from the strain, and 16S rRNA gene sequence amplification and sequencing were conducted. The sequencing results were analyzed for homology in the NCBI database, ultimately confirming the strain as *Lactococcus lactis*. Lactococcus lactis The purified, highly active strain was named HF06. It was then stored in an ultra-low temperature freezer at -80 °C for subsequent experiments and applications.

[0032] Example 2: Preparation of Lactococcus lactis HF06 microbial preparation

[0033] This embodiment provides a method for preparing a Lactococcus lactis HF06 microbial preparation suitable for rapeseed pollen fermentation. The obtained microbial preparation can be directly used as a fermentation seed liquid for rapeseed pollen fermentation. The specific preparation steps are as follows: 1. The lactococcus lactis HF06 preserved in Example 1 was inoculated into MRS liquid medium and cultured statically at 28°C for 28 hours to obtain seed culture.

[0034] 2. Transfer the seed culture into a fermenter at an inoculation rate of 3-5% (v / v) for high-density fermentation (add 2% glucose and 1% yeast powder to the culture medium), control the pH at 6.0, and incubate at a constant temperature of 28℃.

[0035] 3. After fermentation, collect the bacterial sludge by centrifugation at 8000 rpm at 4℃.

[0036] 4. Prepare the protective agent: 10% yeast powder, 5% peptone, 2% glycerol, and 1% monosodium glutamate. Sterilize at 121°C for 15 minutes and then cool.

[0037] 5. Mix the mycelium sludge and the preservative at a ratio of 1:1.5 (v / v) until homogeneous, and then freeze-dry under vacuum (pre-freeze at -40℃ for 4 hours, then sublime dry for 24 hours) to obtain a viable count ≥1.0×10⁻⁶. 8 Freeze-dried bacterial powder at CFU / g.

[0038] 6. Take 40 parts of the above-mentioned freeze-dried bacterial powder, 55 parts of maltodextrin, and 5 parts of microcrystalline cellulose and mix them evenly to obtain the microbial preparation.

[0039] Example 3: Application of microbial preparations in solid-state fermentation of rapeseed pollen

[0040] In this embodiment, the microbial preparation prepared in Example 2 is applied to the solid-state fermentation of rapeseed pollen to verify the feasibility and effectiveness of the strain HF06 of the present invention in the actual fermentation process.

[0041] 1. Raw material pretreatment

[0042] The rapeseed pollen raw materials are sourced from reputable bee product companies and are pure rapeseed pollen granules collected that year. Before use, the pollen raw materials are sieved through a 60-mesh sieve to remove impurities and large particles, ensuring uniformity. Take 1.0 kg of rapeseed pollen raw materials and spread them evenly on a sterilized stainless steel tray. Add sterile distilled water at a ratio of 40% of the pollen raw material weight (i.e., 0.4 kg of sterile water per 1.0 kg of pollen), spraying and stirring simultaneously to ensure full contact and even absorption of water with the pollen. The water-adjusted pollen raw materials are moist and loose, forming clumps when squeezed but crumbling easily with a light touch. Place them in a 4℃ refrigerator and let them stand overnight (approximately 12 hours) to allow water to fully penetrate into the pollen grains, providing a suitable water activity environment for subsequent microbial growth.

[0043] To comprehensively evaluate the fermentation effect of the microbial preparation of the present invention, the following three groups of experiments were set up: Experimental group: solid-state fermentation was carried out using the microbial preparation of the present invention (containing strain HF06) prepared in Example 2; Control group: commercially available Lactococcus lactis (… Lactococcus lactis Solid-state fermentation was carried out using the same process. The control group (no microbial agents were added, only an equal volume of sterile water was added and treated under the same conditions) served as a background control. Three parallel samples were included in each group to ensure the reliability of the experimental results.

[0044] 2. Inoculation and Solid-State Fermentation

[0045] The microbial preparation of the present invention prepared in Example 2 was inoculated into the pretreated pollen raw materials of the experimental group at an inoculation rate of 5% (w / w). The mixture was thoroughly stirred using a sterile stirring spatula to ensure uniform distribution of the bacterial solution in the pollen matrix. The control group was inoculated with commercially available Lactococcus lactis bacterial solution at the same inoculation rate; the blank group was inoculated with an equal volume of sterile water. The pollen raw materials of the three groups were then transferred to sterile solid-state fermenters, with the loading volume being 60%–70% of the tank volume to maintain suitable material layer thickness and aeration. The fermentation parameters were set as follows: fermentation temperature: 28℃ (constant temperature control); stirring speed: 80 r / min (intermittent stirring, 5 min per hour to ensure uniform oxygenation and prevent clumping); fermentation time: 72 h; relative humidity: controlled at 80%–90% to prevent surface drying. Samples were taken every 12 h during fermentation to monitor the pH value, viable cell count, and sensory characteristics (color, odor, and mold) of the fermented material to ensure normal fermentation.

[0046] 3. Post-processing

[0047] After fermentation, the three groups of fermented pollen materials were taken out separately and evenly spread in freeze-drying trays, with the material thickness controlled at 1-2 cm. Vacuum freeze-drying was used for drying under the following conditions: pre-freezing temperature: -40℃, pre-freezing time: 4 h; sublimation drying stage: cold trap temperature -50℃, vacuum degree ≤10 Pa, temperature rise program from -40℃ to -20℃ slowly, maintained for 12 h; desorption drying stage: temperature rise from -20℃ to 30℃ slowly, maintained for 8 h, until the material moisture content dropped below 5%. After freeze-drying, the dried fermented pollen lumps were taken out and pulverized in a high-speed pulverizer, then passed through an 80-mesh sieve to obtain a uniform powdered fermented rapeseed pollen product. The product was sealed in packaging and stored in a cool, dry place for later use.

[0048] Example 4 Cell wall breakage rate detection

[0049] Cell wall disruption rate is one of the key indicators for evaluating the effect of the strain of this invention on solid-state fermentation of rapeseed pollen. In this embodiment, a hemocytometer combined with staining method was used to directly observe and count the proportion of pollen grains with disrupted cell walls in the pollen sample after fermentation under a microscope, so as to characterize the degree of damage to the cell wall structure of rapeseed pollen by the strain HF06 of this invention.

[0050] 1. Sample preparation

[0051] Pollen samples from the experimental group (fermented by strain HF06 of this invention), control group 1 (fermented by commercially available Lactococcus lactis), and blank group (unfermented protopollen) in Example 3 were taken after fermentation and thoroughly mixed. 1.0 g of fermented pollen sample from each group was accurately weighed and placed in a 10 mL sterile centrifuge tube. 9 mL of sterile physiological saline was added, and the mixture was vortexed for 2 minutes to prepare a homogeneous pollen suspension for later use.

[0052] 2. Staining and slide preparation

[0053] Pollen grains were stained in vivo using trypan blue staining solution. 300 μL of the pollen suspension was added to an equal volume of 0.4% trypan blue staining solution, mixed thoroughly, and allowed to stand at room temperature for 5 min. After staining, 30 μL of the stained pollen suspension was slowly added dropwise to the edge of the counting chamber of a hemocytometer using a micropipette, allowing the suspension to fill the chamber naturally through capillary action. After standing for 1 min to allow the pollen grains to settle and stabilize, the suspension was observed under an optical microscope.

[0054] 3. Microscopic observation and counting

[0055] Observations were performed under a 400x optical microscope. Twelve fields of view were randomly selected from each slide for counting. Three independent slides were prepared for each sample, and the following two types of data were recorded for each field of view: Number of pollen grains with broken cell walls: Pollen grains with obvious cracks, ruptures or disintegration of the outer wall, leakage of contents, and dark blue interior after trypan blue staining (indicating loss of cell membrane integrity); Total pollen grains: The sum of all intact pollen grains and broken pollen grains within the field of view.

[0056] 4. Calculation of cell wall breakage rate

[0057] Calculate the cell wall breakage rate for each group using the following formulas: Cell wall breakage rate (%) = (Number of broken pollen grains / Total number of pollen grains) × 100%.

[0058] 5. Test Results

[0059] The cell wall disruption rates of the experimental group, control group 1, and blank group are shown in Table 1. The cell wall disruption rate of the experimental group was 90.85%, which was significantly higher than that of the control group (83.46%) and the blank group (8.26%). This indicates that the strain HF06 of the present invention can effectively destroy the cell wall structure of rapeseed pollen, significantly improve the cell wall disruption effect of pollen, and provide favorable conditions for the subsequent release of pollen contents and the digestion and absorption of nutrients.

[0060] Table 1. Results of cell wall breakage rate detection in each group of samples.

[0061] Example 5: Determination of Flavonoid Content

[0062] Total flavonoid content is one of the important indicators for evaluating the changes in active ingredients before and after solid-state fermentation of rapeseed pollen. In this example, rutin was used as a reference standard to determine the total flavonoid content in each group of pollen samples.

[0063] 1. Preparation of reagents and solutions

[0064] 70% ethanol solution: Accurately measure 700 mL of anhydrous ethanol, add distilled water to make up to 1000 mL, mix well and set aside; Rutin standard stock solution (0.5 mg / mL): Weigh 25.0 mg of rutin standard (dried at 105℃ to constant weight), dissolve in 70% ethanol and dilute to 50 mL, shake well to obtain a rutin standard solution with a concentration of 0.5 mg / mL. 5% sodium nitrite solution: Weigh 5.0 g of sodium nitrite, dissolve it in distilled water and bring the volume to 100 mL; 10% aluminum nitrate solution: Weigh 10.0 g of aluminum nitrate, dissolve it in distilled water and bring the volume to 100 mL; 1 mol / L sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, dissolve it in distilled water and bring the volume to 100 mL.

[0065] 2. Sample processing

[0066] Pollen samples from the experimental group (fermented by strain HF06 of this invention), control group (fermented by commercially available Lactococcus lactis), and blank group (unfermented) in Example 3 were weighed out at 1.0 g each and placed in 50 mL centrifuge tubes. 20 mL of 70% ethanol solution was added, and the mixture was vortexed and then extracted with ultrasound for 30 min (power 200 W, temperature 40℃). After extraction, the samples were centrifuged at 4000 r / min for 10 min, and the supernatant was transferred to a 25 mL volumetric flask. The residue was extracted once more with 70% ethanol. The supernatants were combined, and the volume was adjusted to 25 mL with 70% ethanol. The mixture was shaken well to obtain the extracts for each pollen sample.

[0067] 3. Preparation of the rutin standard curve

[0068] Take 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 0.5 mg / mL rutin standard solution and place them into seven 25 mL stoppered colorimetric tubes, numbered 0-6 respectively. Each tube is brought to a final volume of 25.0 mL with 70% ethanol solution. Perform the colorimetric reaction as follows: Add 0.3 mL of 5% sodium nitrite solution to each tube, shake well, and let stand for 5 min; then add 0.3 mL of 10% aluminum nitrate solution, shake well, and let stand for 5 min; finally, add 2.0 mL of 1 mol / L sodium hydroxide solution, shake well, and bring the volume to 10 mL with 70% ethanol solution. Shake thoroughly and let stand at room temperature for 10 min. Zero the tube (blank control) and measure the absorbance at 510 nm. Plot a standard curve with rutin concentration (mg / mL) on the x-axis and absorbance on the y-axis, and perform linear regression to obtain the regression equation.

[0069] 4. Determination of total flavonoid content in samples

[0070] Take 1.0 mL of pollen extract from each group and place it in a 25 mL stoppered colorimetric tube. Follow the same colorimetric steps as described above and measure the absorbance at a wavelength of 510 nm. Each sample was measured in triplicate, and the average value was taken.

[0071] The total flavonoid concentration (mg / mL) in each sample's test solution was calculated based on the regression equation of the standard curve, and then converted to the total flavonoid content in the sample using the following formula: Total flavonoid content (mg / g) = M1 / ​​M2 × 100 M1—Flavone content in the sample, in mg; M2 – Total mass of pollen sample, in g; 5. Results The flavonoid content in each pollen sample is shown in Table 2. The total flavonoid content of the unfermented pollen in the control group was only 10.27 mg / 100g. After fermentation with commercially available Lactococcus lactis, the total flavonoid content increased to 31.53 mg / 100g, approximately 2.07 times higher than the control group. After fermentation with the strain HF06 of this invention, the total flavonoid content significantly increased to 69.40 mg / 100g, approximately 5.76 times higher than the control group and approximately 1.20 times higher than the commercially available Lactococcus lactis control group. This indicates that solid-state fermentation can effectively promote the release and dissolution of flavonoids in rapeseed pollen. More importantly, the effect of strain HF06 of this invention on increasing the total flavonoid content of pollen is significantly better than that of commercially available Lactococcus lactis, indicating that the strain of this invention has unique advantages in disrupting the pollen cell wall structure and promoting the conversion of bound flavonoids to free flavonoids. It can be used as a highly efficient and dedicated strain for solid-state fermentation of rapeseed pollen and has broad application prospects.

[0072] Table 2 Flavonoid content in each group of samples

[0073] Example 6: Detection of Free Amino Acid Content

[0074] The content of free amino acids is one of the important indicators for evaluating the degree of protein degradation and nutritional quality of rapeseed pollen before and after solid-state fermentation. In this example, the total amount of free amino acids in each group of pollen samples was determined by the ninhydrin colorimetric method with leucine as a reference.

[0075] 1. Sample processing

[0076] Accurately weigh 0.5 g of each of the freeze-dried pollen samples from the experimental group (fermented by the strain HF06 of this invention), control group (fermented by commercially available Lactococcus lactis), and blank group (unfermented) in Example 3, place them in a 50 mL centrifuge tube, add 20 mL of 70% ethanol solution, vortex to mix, and then extract with ultrasonic assistance for 30 min (power 200 W, temperature 40℃). After extraction, centrifuge at 4,000 r / min for 10 min, transfer the supernatant to a 25 mL volumetric flask, and repeat the extraction with 70% ethanol once on the residue. Combine the supernatants, make up to 25 mL with 70% ethanol, and shake well to obtain the test extract for each pollen sample.

[0077] 2. Preparation of leucine standard curve

[0078] Take 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 1.0 mg / mL leucine standard solution and place them into seven 25 mL stoppered colorimetric tubes, numbered 0 to 6 respectively. Make up to 1.0 mL of distilled water in each tube. Add 1.0 mL of ninhydrin colorimetric reagent to each tube, mix well, and heat in a boiling water bath for 15 min. Remove and rapidly cool to room temperature, then make up to 10 mL with distilled water and shake thoroughly. Zero the tube using tube 0 (blank control) and measure the absorbance at 570 nm. Plot a standard curve with leucine concentration (mg / mL) on the x-axis and absorbance on the y-axis.

[0079] 3. Determination of free amino acid content in samples

[0080] Accurately measure 1.0 mL of each pollen extract and place it in a 25 mL stoppered colorimetric tube. Add ninhydrin as described above for color development, and measure the absorbance at 570 nm. Each sample was measured in triplicate, and the average value was taken.

[0081] Calculate the mass concentration (mg / mL) of free amino acids in each sample's test solution based on the standard curve, and convert it to the free amino acid content in the sample using the following formula: Free amino acid content (mg / 100g) = (C×25×N) / M×100 In the formula: C—The mass concentration of free amino acids in the test solution obtained from the standard curve, in mg / mL; N – Dilution factor; M – The weight of the pollen sample, in grams.

[0082] 4. Test Results

[0083] The free amino acid content in each group of pollen samples is shown in Table 3. The free amino acid content of the unfermented pollen in the control group was 796.32 mg / 100g; after fermentation with commercially available Lactococcus lactis, the free amino acid content significantly increased to 4,168.22 mg / 100g, which was about 4.23 times higher than that of the control group; while after fermentation with the strain HF06 of this invention, the free amino acid content reached as high as 8,603.14 mg / 100g, which was about 9.80 times higher than that of the control group and about 1.06 times higher than that of the commercially available Lactococcus lactis control group.

[0084] The above results indicate that the protease system secreted by lactic acid bacteria during solid-state fermentation can gradually degrade large protein molecules in pollen into small peptides and free amino acids, thereby significantly increasing the content of free amino acids in pollen. The proteolytic ability of strain HF06 in this invention is significantly superior to that of commercially available lactococci, resulting in a more complete release of free amino acids from pollen after fermentation. This significant increase in free amino acid content not only enhances the nutritional value of pollen products but also improves their flavor characteristics (such as the release of umami and sweet amino acids), providing a foundation for the application of fermented rapeseed pollen in the field of functional foods.

[0085] Table 3. Free amino acid content in each group of samples

[0086] Example 7 Proteomics Analysis

[0087] To reveal the effects of solid-state fermentation of strain HF06 of this invention on the protein composition of rapeseed pollen at the molecular level, especially its regulatory role on allergy-related proteins, this embodiment employs data-independent acquisition (DIA) proteomics technology to systematically compare and analyze the protein expression profiles of rapeseed pollen before and after fermentation.

[0088] 1. Protein extraction

[0089] Take appropriate amounts of samples from the blank group (unfermented protopollen) and the experimental group (fermented pollen from strain HF06 of this invention), place them in a pre-cooled mortar, add liquid nitrogen, and grind thoroughly into a fine powder. Weigh approximately 100 mg of each sample powder, add pre-cooled acetone solution containing 10% trichloroacetic acid (containing 0.07% β-mercaptoethanol), and precipitate the protein at -20℃ for 2 h. After precipitation, centrifuge at 4℃, 12,000 r / min for 15 min, discard the supernatant, and collect the protein precipitate. Wash the precipitate twice with pre-cooled acetone, centrifuging at 4℃, 12,000 r / min for 10 min each time, discard the supernatant, and air dry at room temperature.

[0090] SDT lysis buffer was added to the protein precipitate, and the precipitate was vortexed to dissolve it completely. Then, sonication-assisted lysis was performed using an ultrasonic homogenizer (200 W power, 5 s sonication, 10 s interval, 3 cycles) to fully disrupt residual cell structures and release proteins. After sonication, the sample was boiled in a water bath for 15 min to fully denature the proteins. After denaturation, the sample was centrifuged at 11,000 r / min for 40 min at room temperature, and the supernatant was collected as the total protein extract.

[0091] 2. Proteolytic activity and peptide preparation

[0092] Take an appropriate amount of protein extract from each sample (containing approximately 100 μg of protein), add dithiothreitol (DTT) to a final concentration of 40 mM, and perform a reduction reaction in a 37°C water bath for 1.5 h to break the disulfide bonds within the protein molecules. After reduction, add iodoacetamide (IAA) to a final concentration of 20 mM, and perform an alkylation reaction at room temperature in the dark for 30 min to block the thiol groups and prevent the reformation of disulfide bonds.

[0093] The protein solution was subjected to buffer replacement using ultrafiltration centrifuge tubes (molecular weight cutoff 10 kDa): 100 μL of urea buffer (8 M urea, 100 mM Tris-HCl, pH 8.0) was added, and the solution was centrifuged at 14,000 g for 15 min, repeated three times; then 100 μL of 25 mM NH4HCO3 buffer was added, and the solution was centrifuged at 14,000 g for 15 min, repeated twice.

[0094] Trypsin was added at a ratio of 1:50 (trypsin to protein by mass), and the mixture was incubated at 37°C for 15–18 h (overnight). After enzymatic hydrolysis, the hydrolysate was acidified with 0.1% formic acid solution to terminate the reaction. The hydrolyzed peptides were desalted using a C18 solid-phase extraction column: the C18 column was activated sequentially with methanol and 0.1% formic acid solution, followed by washing with 0.1% formic acid solution to remove salts, and finally eluted with 80% acetonitrile / 0.1% formic acid solution. The eluent was collected, concentrated to dryness in a vacuum concentrator, and the peptides were redissolved in 0.1% formic acid solution. The peptide concentration was determined using a micro-spectrophotometer and stored at -80°C for later use.

[0095] 3. LC-MS / MS analysis

[0096] Chromatographic separation was performed using a Vanquish Neo ultra-high performance liquid chromatography system (Thermo Scientific) with a nanoliter flow rate. Peptide samples were first loaded onto a trapping column (100 μm × 2 cm, ReproSil-Pur C18-AQ, 5 μm), washed and desalted with the mobile phase, and then transferred to an analytical column (75 μm × 25 cm, ReproSil-Pur C18-AQ, 1.9 μm) for gradient separation. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The chromatographic gradient elution program was as follows: 0–5 min, 4% B; 5–55 min, 4%–22% B; 55–70 min, 22%–35% B; 70–75 min, 35%–95% B; 75–90 min, 95% B. The flow rate was maintained at 300 nL / min.

[0097] The separated peptides were subjected to DIA data acquisition using an Astral high-resolution mass spectrometer (Thermo Scientific). The detection mode was ESI positive ion mode, with a precursor ion scan range (m / z) of 380–980. The primary mass spectrometry resolution was 240,000 (at 200 m / z), the normalized automatic gain control (AGC) target was 500%, and the maximum ion implantation time (IT) was 5 ms. The secondary mass spectrometry used DIA data acquisition mode, with 299 scan windows, an isolation window width of 2 m / z, a high-energy collision dissociation (HCD) collision energy of 25 eV, a normalized AGC target of 500%, and a maximum IT of 3 ms.

[0098] To ensure data quality, three biological replicates were performed for each sample group. Raw mass spectrometry data were processed using Spectronaut software for DIA data processing and qualitative and quantitative protein analysis. Protein identification criteria included the presence of at least one specific peptide and a false detection rate (FDR) of <1%.

[0099] 4. Screening of differentially expressed proteins

[0100] As shown in Table 4, the expression level of Pollen Ole e 1 (Accession: A0A3P6BR47), a core pollen allergen, significantly decreased after fermentation. This protein is a key molecule triggering human pollen allergic reactions, and its significant downregulation directly reduced the probability of rapeseed pollen binding to human sensitization receptors, which is the core reason for the decrease in pollen sensitization after fermentation. Furthermore, the expression of three allergen proteins containing the SCP domain (Accession: A0A397YGZ2, A0A398AM12, and A0A3P6CM06) was downregulated after fermentation. The SCP domain is a typical characteristic structure of pollen allergens, and the family-like downregulation of this type of protein indicates that the fermentation process has a targeted regulatory effect on proteins with sensitizing characteristic structures in pollen. Meanwhile, the expression of four potential allergen proteins (Accession: A0A397Y3I1, A0A397YCW0, A0A397YQQ8 and the aforementioned A0A3P6BR47) was also downregulated after fermentation. As potential sensitizing factors, their widespread downregulation further reduced the types of pollen sensitizing proteins.

[0101] In summary, lactic acid bacteria fermentation has a targeted downregulation effect on rapeseed pollen allergen proteins, achieving significant downexpression of core allergenic proteins, as well as comprehensive downregulation of allergenic characteristic structural proteins and potential allergenic proteins, without any upregulation compensation of core allergenic proteins. Ultimately, this effectively reduces the core allergenic risk of rapeseed pollen and provides a theoretical basis at the protein level for the development of hypoallergenic pollen products.

[0102] Table 4. Differentially expressed allergenic proteins after rapeseed pollen fermentation

[0103] Example 8 Lipidomics Analysis

[0104] To systematically investigate the effects of solid-state fermentation of strain HF06 of this invention on the lipid composition of rapeseed pollen, this embodiment uses ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-TripleTOF MS / MS) to perform non-targeted lipidomics analysis on the lipid metabolism profile of rapeseed pollen before and after fermentation, in order to reveal the regulatory characteristics of the fermentation process on pollen lipid components.

[0105] 1. Sample grouping and pretreatment

[0106] Three lyophilized powder samples were taken from both the blank group (unfermented protopollen) and the experimental group (fermented pollen from strain HF06 of this invention), each with three biological replicates. Each sample was accurately weighed at 10.0 mg and placed in a 15 mL glass centrifuge tube. 2.0 mL of methanol was added, and the tube was vortexed to fully disperse the sample. Then, 4.0 mL of chloroform and 1.6 mL of distilled water were added, the tube was tightly capped, and the tube was vigorously vortexed for 10 min to fully extract the lipids into the organic phase. After extraction, the tube was centrifuged at 4,000 r / min for 5 min to separate the organic and aqueous phases. The lower organic phase (chloroform layer) was carefully transferred to a new glass centrifuge tube using a pipette, and the solvent was dried using a DC-24 nitrogen dryer with a gentle nitrogen stream. The residual lipid extract was reconstituted with 1.0 mL of chloroform-methanol mixture (2:1, V / V), and 10.0 µL of lipid internal standard (containing various lipid internal standards for subsequent quantitative correction) was added. After filtration through a 0.22 μm organic phase filter membrane, the solution was transferred to a vial for UPLC-MS / MS analysis.

[0107] 2. Chromatographic conditions

[0108] Lipid separation was performed using a Phenomenex Kinetex C18 column (100 × 2.1 mm, particle size 2.6 μm, Phenomenex, USA). The column oven temperature was set to 60 °C, and the mobile phase flow rate was 0.4 mL / min. Mobile phase A was methanol-water-acetonitrile (1:1:1, V / V / V, containing 5 mM ammonium acetate), and mobile phase B was isopropanol-acetonitrile (5:1, V / V, containing 5 mM ammonium acetate). The gradient elution program was as follows: 0–0.5 min, 20% B; 0.5–1.5 min, 20%–40% B; 1.5–3.0 min, 40%–60% B; 3.0–13.0 min, 60%–98% B; 13.0–17.0 min, 98%–20% B; 17.0–20.0 min, 20% B (equilibration). The injection volumes for positive ion mode and negative ion mode are 2.0 µL and 6.0 µL, respectively.

[0109] 3. Mass spectrometry conditions

[0110] Electrospray ionization (ESI) was used for data acquisition in both positive and negative ion modes. The mass spectrometry parameters were set as follows: ion spray voltage: +5,500 V for positive ion mode and -4,500 V for negative ion mode; ion transfer tube temperature (i.e., desolventization temperature): 600 °C; declustering potential (DP): +80 V for positive ion mode and -80 V for negative ion mode; collision energy (CE): +10 V for positive ion mode and -30 V for negative ion mode; primary mass spectrometry scan range (m / z): 50–1,200. In Information-Dependent Acquisition (IDA) mode, the 10 strongest precursor ions in each scan cycle were analyzed by secondary mass spectrometry. The secondary collision energies were 35 eV (positive ion) and -35 eV (negative ion), and the dynamic exclusion time was set to 15 s.

[0111] 4. Data processing and lipid identification

[0112] The raw mass spectrometry data were analyzed using LipidSearch software (Thermo Scientific) for peak identification, extraction, lipid type identification, and relative quantification.

[0113] Table 5 shows that 10-hydroxypentadecanoic acid and 13-hydroxyheptadecanoic acid were not detected in the unfermented pollen of the blank group. After fermentation with common lactococcus, the contents of 10-hydroxypentadecanoic acid and 13-hydroxyheptadecanoic acid were 16.41 mg / 100g and 14.53 mg / 100g, respectively. However, after fermentation with the strain HF06 of this invention, the contents of the two were as high as 249.22 mg / 100g and 187.91 mg / 100g, respectively. The HF06 group of this invention showed an increase of approximately 15.2 times and 12.9 times compared with the common lactococcus group, respectively.

[0114] In summary, the solid-state fermentation of strain HF06 of this invention can significantly alter the lipid metabolism profile of rapeseed pollen, particularly achieving ultra-efficient enrichment of two rare hydroxy fatty acids, 10-hydroxypentadecanoic acid and 13-hydroxyheptadecanoic acid. Lipidomics data further confirm, at the lipid molecular level, the promoting effect of fermentation on the release of pollen cell contents, providing a scientific basis from a lipidomics perspective for the application of strain HF06 in improving the nutritional quality of rapeseed pollen and developing functional lipid products.

[0115] Table 5. Detection results of lipid content in each group of samples

[0116] 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 that can be used for rapeseed pollen fermentation, characterized in that, The strain is Lactococcus lactis ( Lactococcus lactis HF06 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 20252323.

2. A microbial preparation, characterized in that, The microbial preparation comprises Lactococcus lactis HF06 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation comprises lyophilized Lactococcus lactis HF06 bacterial powder and excipients, wherein the excipients are maltodextrin and microcrystalline cellulose, and the viable count of the lyophilized bacterial powder is ≥1.0×10⁻⁶. 8 CFU / g.

4. The application of Lactococcus lactis HF06 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in solid-state fermentation of rapeseed pollen.

5. A method for solid-state fermentation of rapeseed pollen, characterized in that, Includes the following steps: (1) Raw material pretreatment: The rapeseed pollen raw material is sieved to remove impurities. The moisture content is adjusted to 35-45% based on the weight of the rapeseed pollen raw material after impurity removal, and then left to stand at 4℃ overnight for hydration. (2) Inoculation and fermentation: The lactococcus lactis HF06 described in claim 1 or the microbial preparation described in claim 2 or 3 is inoculated into the pretreated rapeseed pollen raw material at 5%~8% (w / w) for solid-state fermentation. The fermentation conditions include: fermentation temperature 26~30℃, stirring speed 60~100 r / min, fermentation time 68~80 h, and ambient humidity 80%~90%. (3) Post-processing: After fermentation, the fermented material is dried and crushed to obtain fermented rapeseed pollen product.

6. The solid-state fermentation method for rapeseed pollen according to claim 5, characterized in that, In step (1), the impurity removal process involves passing the material through a 40-80 mesh sieve.

7. The solid-state fermentation method for rapeseed pollen according to claim 5, characterized in that, The drying in step (3) is vacuum freeze drying, with conditions including: pre-freezing at -30℃ to -40℃ for 3~6 h, sublimation drying at a cold trap temperature of -40℃ to -60℃ and a vacuum degree of ≤10Pa, and then heating to 30℃ for desorption drying until the moisture content drops to below 5%; the pulverization is passed through a 60~100 mesh sieve.

8. A fermented rapeseed pollen product prepared by the method according to any one of claims 5-7, wherein the fermented rapeseed pollen product contains a total flavonoid content ≥60 mg / 100g and a free amino acid content ≥8000 mg / 100g.

9. The use of Lactococcus lactis HF06 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the degradation of rapeseed pollen allergen proteins; wherein the rapeseed pollen allergen proteins include at least one of Pollen Ole e 1 family proteins and proteins containing SCP domains.

10. The application of Lactococcus lactis HF06 according to claim 1 or the microbial preparation according to claim 2 or 3 in the enrichment of hydroxy fatty acids in rapeseed pollen, characterized in that, The hydroxy fatty acids include 10-hydroxypentadecanoic acid and / or 13-hydroxyheptadecanoic acid.

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