Preparation method and application of broccoli directional germination fermentation product

Abstract

The present application relates to the technical field of fermentation composition, and discloses a preparation method and application of broccoli directional germination fermentation product, which uses broccoli seeds as raw materials, enriches active components such as glucoraphanin and sulforaphane through light-avoiding temperature-control germination process combined with specific culture solution induction, and separates the germination product through physical pressing oil extraction, and then the obtained solid-phase meal is dried and broken, and then inoculated with a composite fermentation inoculum for solid-state fermentation to obtain a high-activity fermentation composition. The present application realizes gradient utilization of all components of broccoli, and improves the comprehensive utilization rate of raw materials. The preparation process is stable and environmentally friendly, and the active components in the obtained fermentation composition are significantly enriched, which can be used as a core substrate for biological agents for intestinal microecological regulation, nerve stress relief or abnormal cell proliferation microenvironment auxiliary nutritional support, and is suitable for industrialized mass production.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation composition technology, and in particular to a method for preparing and applying broccoli directional germination fermentation products. Background Technology

[0002] Broccoli, a plant belonging to the Brassicaceae family and the Brassica genus, is rich in glucosinolates. Among these, sulforaphane can be degraded into isothiocyanates under the action of specific enzymes, with sulforaphane being a representative example. Sulforaphane possesses various biological activities, including regulating cellular oxidative stress and participating in epigenetic modification. Currently, most deep-processing extractions of broccoli use mature plants as raw materials, and the development system for broccoli seeds and their processing byproducts is still underdeveloped.

[0003] Seed germination involves the transformation and synthesis of secondary metabolites within the plant. However, under natural germination conditions, the enrichment efficiency of precursor substances such as glucosinolates within the substrate is low. Existing bio-fermentation technologies mostly employ simple water extraction or direct pulverization fermentation, failing to effectively avoid the oxidative interference of endogenous fatty acids in plants and the non-targeted guidance of enzymatic hydrolysis by extraneous proteins. These limitations result in unsatisfactory yields of effective bioactive components in the final fermentation products, making it difficult to meet the development needs of modern highly active microecological compositions. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a method for preparing broccoli directional germination fermentation products and their application.

[0005] A method for preparing a broccoli fermentation composition includes the following steps: S1. Directional germination induction: Broccoli seeds that have undergone surface disinfection and soaking are placed in a dark environment for germination culture, and induction culture solution is sprayed to replenish them. Germination products are then harvested. S2. Physical pressing and separation: After dehydration of the germinated product, it is subjected to low-temperature physical pressing to separate the free oil phase and the pressed meal. S3. Cell wall breaking treatment: After the pressed meal is dried at low temperature, it is subjected to ultrafine powdering and cell wall breaking treatment to obtain broccoli meal cell wall breaking powder. S4. Directional fermentation: Broccoli meal cell wall-breaking powder or germination freeze-dried powder is sterilized by wet heat, then inoculated with compound fermentation agent and the moisture content is adjusted before fermentation. After fermentation, the liquid phase components are separated and extracted to obtain broccoli fermentation composition.

[0006] Furthermore, in order to better realize the present invention, the culture medium in S1 contains sodium selenite at a concentration of 0.05~0.2 mmol / L, L-methionine at a concentration of 0.1~0.5 g / L, methyl jasmonate at a concentration of 0.01~0.05 mmol / L, and 10%~15% (v / v) kelp mud soaking supernatant.

[0007] Furthermore, in order to better realize the present invention, the germination culture conditions in S1 are a culture temperature of 20~30℃, a relative humidity of 70%~85%, a culture cycle of 2~7 days, and a harvesting standard of hypocotyl length of 0.5~2cm.

[0008] Furthermore, in order to better realize the present invention, the pressing temperature of the low-temperature physical pressing in S2 is not higher than 60°C, and the pressing pressure is 30~50MPa.

[0009] Furthermore, in order to better realize the present invention, the drying conditions in S3 are low-temperature drying at 40~70℃, the total crushing processing temperature is not higher than 70℃, and the particle size of the powder obtained after the cell wall breaking treatment is not less than 200 mesh.

[0010] Furthermore, to better realize the present invention, the compound fermentation agent in S4 is composed of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Weizmannii coagulans* in a mass ratio of 2-3:1-2:0.5-1, and the total inoculum amount of the compound fermentation agent is 2%-5% of the dry weight of the substrate. The *Lactobacillus plantarum* is *Lactobacillus plantarum* JYZC-LP09, with the accession number CGMCC NO.28057. The other strains are all food-grade public accession standard strains. There are no self-selected specific strains, and no additional strains are required for preservation. They can be obtained publicly through domestic public accession institutions such as CGMCC and CICC or through formal commercial channels, and can be completely reproduced by those skilled in the art.

[0011] Furthermore, in order to better realize the present invention, the fermentation conditions in S4 are a fermentation temperature of 30~39℃, an initial pH of 5.5~6.5, a fermentation time of 48~60h, and a turning operation every 12h during solid-state fermentation.

[0012] Furthermore, in order to better realize the present invention, oat malt and quinoa malt are prepared simultaneously. Oat malt, quinoa malt, mulberry, lemon, pineapple and apple are processed into prebiotic slurry by low-temperature wet cell wall breaking. The slurry is homogenized and mixed with broccoli meal cell wall breaking powder and then co-fermented. Grain sprout prebiotics enhance the probiotic multiplication ability and rely on the natural flavor of fruits and vegetables to neutralize the sour taste, without the need for exogenous sweeteners and acidity regulators.

[0013] The present invention also provides a fermentation composition obtained by the above preparation method.

[0014] The present invention also provides a bioactive preparation comprising the above-mentioned fermentation composition and acceptable edible excipients, wherein the dosage form is a liquid fermentation preparation or a solid compound powder preparation.

[0015] This invention also provides the application of the above-mentioned fermentation composition in the preparation of biological agents for regulating intestinal microecology, relieving nerve stress, and providing nutritional support for the microenvironment.

[0016] The beneficial effects of this invention are as follows: This invention achieves efficient utilization of all components of broccoli seeds. Through a three-step coupling process of epigenetic induction, physical phase separation, and multi-strain co-fermentation, the targeted transformation of active substances is realized, significantly increasing the content of free sulforaphane and short-chain fatty acids in the product. The fermentation composition of this invention has a well-defined composition and stable process, and can be directly used as a core matrix for the preparation of liquid microecological preparations and solid-state targeted sustained-release drug delivery systems. Attached Figure Description

[0017] Figure 1 This is a flowchart of the preparation method of the present invention.

[0018] Figure 2 The total ion chromatogram of SFN and RAA standard solutions in the LC-MS / MS detection method is shown.

[0019] Figure 3 This is the total ion chromatogram of SFN and RAA in the sample of the embodiment of the present invention in the LC-MS / MS detection method.

[0020] Figure 4 This is the MRM diagram of SFN in the LC-MS / MS detection method. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0022] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0023] Example 1: This example provides a basic preparation method for a broccoli fermentation composition, specifically including: S1. Directional Germination Induction: Select plump broccoli seeds and completely immerse them in a 3% hydrogen peroxide solution for 15 minutes for gentle surface disinfection. Then, rinse them three times with pure water and soak them in pure water for 8 hours to activate the dehydrated embryos. Spread the imbibed seeds evenly in porous seedling trays and transfer them to a light-proof incubator equipped with a high-precision environmental monitoring system. Set the incubation temperature to a constant 28 degrees Celsius and the relative humidity to 78%. Prepare an induction culture medium containing 0.1 mmol / L sodium selenite, 0.3 g / L L-methionine, 0.02 mmol / L methyl jasmonate, and 12% (v / v) kelp mud soaking supernatant. Spray this medium onto the seedling trays three times daily at equal intervals. Incubate in the dark for 60 hours, and harvest the entire seedling when the embryos have elongated to approximately 1 cm.

[0024] S2. Physical pressing and separation: The harvested sprouted product is placed in a hot air circulating drying oven for dehydration until the total moisture content drops to 12%. It is then transferred to a twin-screw low-temperature physical cold press. The cooling system is monitored online to ensure that the equipment operating temperature does not exceed 60°C. Under the mechanical extrusion of 40MPa, the oil phase is separated, and the free essential oil phase and the physically degreased pressed solid meal are collected separately.

[0025] S3. Cell wall breaking treatment: The pressed solid phase meal is placed in a 50℃ low temperature drying equipment for deep dehydration to a moisture content of 7%. It is then pulverized at a high shear rate using an airflow ultrafine pulverizer. The powder is passed through a 250-mesh grading sieve to obtain cell wall broken micro powder with increased specific surface area.

[0026] S4. Directional Fermentation: The cell-wall-broken micro-powder was added to pure water to adjust the moisture content to 50%, and subjected to moist heat treatment at 121℃ for 20 minutes to inactivate endogenous proteins. After cooling to room temperature, a compound fermentation agent consisting of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Weizmannii coagulans* in a mass ratio of 2:1:0.5 was inoculated under aseptic conditions, with a total inoculum amount of 3% of the substrate dry weight. The substrate was placed in a 32℃ biochemical incubator, the initial pH was adjusted to 6.0, and fermentation was started for 60 hours. During fermentation, the substrate was turned over every 12 hours to dissipate metabolic heat. After fermentation, pure water was added at a solid-liquid ratio of 1:5, and the mixture was dynamically soaked at room temperature for 2 hours. The mixture was then centrifuged at 8000 rpm for 10 minutes and clarified using a 0.45-micron filter membrane. The liquid phase was collected to obtain the original fermentation solution. The product was found to contain 590.5 μg / mL sulforaphane, 812.6 μg / mL sulforaphane, 84.0 mg / mL total phenols, and 57.3 mg / mL γ-aminobutyric acid.

[0027] Example 2: The process steps in this example are basically the same as in Example 1, except that the induction signal parameters are optimized and adjusted: In S1, the concentration of sodium selenite in the induction culture medium is increased to 0.15 mmol / L, and the concentration of methyl jasmonate is increased to 0.04 mmol / L; based on the metabolic acceleration brought about by the concentration adjustment, the germination culture cycle is shortened to 24 hours accordingly. Other operating parameters remain completely consistent with those in Example 1. Measurements showed that the concentration of glucosinolates in the fermentation composition obtained in this example increased to 782.6 μg / mL, the concentration of sulforaphane increased to 1021.6 μg / mL, the concentration of total phenols was 83.5 mg / mL, and the concentration of γ-aminobutyric acid (GABA) was 54.3 mg / mL. This verifies that parameter optimization has a significant enrichment effect on glucosinolates, while having little effect on total phenols and GABA.

[0028] Example 3: Preparation of a liquid fermentation formulation: 100 parts by weight of the fermentation composition stock solution obtained in Example 2 were accurately measured as the core matrix. To improve the physicochemical stability of the liquid formulation, the specific preparation steps are as follows: 1. Preparation of prebiotic fruit and vegetable pulp: Prepare 30 parts by weight of oat malt, 12 parts by weight of quinoa malt, 20 parts by weight of mulberry, 3 parts by weight of lemon, 10 parts by weight of pineapple, and 15 parts by weight of apple. All raw materials are subjected to low-temperature wet cell wall breaking at 40℃ and filtered to remove coarse fiber to obtain a natural blended pulp. This pulp is rich in citric acid, malic acid, fructooligosaccharides and β-glucan.

[0029] 2. Homogenization and mixing: The above-mentioned prebiotic fruit and vegetable slurry is used to replace pure water and is homogenized and mixed with the broccoli meal cell wall-breaking powder in Example 2 at high speed to adjust the moisture content of the fermentation system to 50%. This step relies on the natural organic acids in the slurry to pre-buffer the pH of the system in order to neutralize the lactic acid produced in the subsequent fermentation.

[0030] 3. Sterilization, inoculation, fermentation and extraction: The relevant operating parameters are the same as in Example 1.

[0031] Detection Indicators and Evaluation: The fermentation broth obtained in this example contained 659.1 μg / mL glucoraphane, 1228.7 μg / mL sulforaphane, 338.4 mg / mL total phenols, and 98.6 mg / mL γ-aminobutyric acid (GABA). Compared to Example 2, the glucoraphane content remained relatively stable, but the total phenol and GABA contents significantly increased. The final pH of the fermentation system was 5.5. Sensory evaluation showed a mild sweet and sour taste, without a strong fermented sour or thiol flavor, requiring no artificial acidity regulators or sweeteners.

[0032] Example 4: Preparation of a solid compound powder formulation: The fermentation composition stock solution obtained in Example 2 was placed in a vacuum concentration reactor and concentrated under mild conditions until the solid content reached 30%. Subsequently, it was prepared into an active freeze-dried powder using vacuum freeze-drying technology. 40 parts by weight of germinated brown rice powder, 30 parts by weight of germinated oat powder, and 20 parts by weight of germinated quinoa powder were added together with 10 parts by weight of the active freeze-dried powder and dry-mixed uniformly in a three-dimensional powder mixer. This compound powder has good flowability and can be directly used as a matrix for hard capsule filling or as a dispersant main ingredient.

[0033] Comparative Example 1 provides a conventional natural hydroponic fermentation product as a control. It employs the exact same physical pressing, cell wall disruption, and solid-state fermentation process as Example 1, differing only in the pre-treatment S1: this comparative example omits the light-protected, temperature-controlled, and induction culture medium interventions, placing broccoli seeds in a conventional natural light environment and using ordinary water for conventional germination treatment for 5 days. Analysis showed that the product obtained in this comparative example contained 476.5 μg / mL sulforaphane, 701.7 μg / mL sulforaphane, 69.6 mg / mL total phenols, and 61.8 mg / mL γ-aminobutyric acid. These figures are significantly lower than those in Examples 1, 2, and 3 of this invention, fully verifying the necessity and significant advantages of the directional germination induction process of this invention for enriching key active ingredients.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a broccoli fermentation composition, characterized in that, Includes the following steps: S1. Directional germination induction: Broccoli seeds that have undergone surface disinfection and soaking are placed in a dark environment for germination culture, and the induction culture solution is replenished by atomized spraying. Germination products are then harvested. S2. Physical pressing and separation: After dehydration of the germinated product, it is subjected to low-temperature physical pressing to separate the free oil phase and the pressed meal; the pressing temperature of low-temperature physical pressing is not higher than 60℃, and the pressing pressure is 30~50MPa; S3. Cell wall breaking treatment: After the pressed meal is dried at low temperature, it is subjected to ultrafine powdering and cell wall breaking treatment to obtain broccoli meal cell wall breaking powder. S4. Directional fermentation: Broccoli meal cell wall-breaking powder or germination freeze-dried powder is sterilized by wet heat, then inoculated with compound fermentation agent and the moisture content is adjusted before fermentation. After fermentation, the liquid phase components are separated and extracted to obtain broccoli fermentation composition.

2. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: The induction culture medium in S1 contains sodium selenite at a concentration of 0.05-0.2 mmol / L, L-methionine at a concentration of 0.1-0.5 g / L, methyl jasmonate at a concentration of 0.01-0.05 mmol / L, and 10%-15% (v / v) of kelp mud soaking supernatant.

3. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: The conditions for germination culture in S1 are a culture temperature of 20-30℃, a relative humidity of 70%-85%, a culture cycle of 2-7 days, and a harvesting standard when the hypocotyl length reaches 0.5-2cm.

4. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: The drying conditions in S3 are low-temperature drying at 40~70℃, the total crushing temperature is not higher than 70℃, and the particle size of the powder obtained after cell wall breaking is not less than 200 mesh.

5. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: The S4 compound fermentation agent is composed of Lactobacillus plantarum, Lactobacillus rhamnosus and Weizmannii coagulans mixed in a mass ratio of 2-3:1-2:0.5-1, and the total inoculum amount of the compound fermentation agent is 2%-5% of the dry weight of the substrate.

6. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: The fermentation conditions in S4 are: fermentation temperature of 30~39℃, initial pH adjusted to 5.5~6.5, fermentation time of 48~60h, and turning operation every 12h during the fermentation process.

7. The method for preparing the broccoli fermentation composition according to claim 1, characterized in that: Oat malt and quinoa malt are prepared simultaneously. Oat malt, quinoa malt, mulberry, lemon, pineapple and apple are processed into prebiotic slurry by low-temperature wet cell wall breaking. The slurry is then homogenized and mixed with broccoli meal cell wall breaking powder and fermented together. Grain sprout prebiotics enhance the probiotic multiplication capacity. The acidity is neutralized by the natural flavor of fruits and vegetables, without the need for exogenous sweeteners and acidity regulators.

8. A fermentation composition, characterized in that, The broccoli fermentation composition is obtained by the preparation method of any one of claims 1 to 7.

9. A bioactive preparation, characterized in that: It comprises the fermentation composition of claim 8 and acceptable edible adjuvants, and is in the form of a liquid fermentation preparation or a solid compound powder preparation.

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