Preparation method of lotus plumule ferment, product and application thereof

Abstract

The application discloses a preparation method and product of lotus plumule fermentation and application thereof, and comprises the following steps: taking dried lotus plumule, crushing the lotus plumule into powder, mixing the powder with water, heating to 40-60 DEG C, and fully stirring and swelling to prepare lotus plumule suspension; adding cellulase, pectinase and papain for hydrolysis to prepare lotus plumule enzymolysis liquid; adding a carbon source, adjusting pH to 6.0-7.0, sterilizing to prepare lotus plumule fermentation medium; preparing fermentation strain seed liquid; inoculating the fermentation strain seed liquid into the lotus plumule fermentation medium, and fermenting to prepare lotus plumule fermentation liquid; and performing ceramic membrane filtration and ultrafiltration membrane filtration to prepare lotus plumule fermentation. The application produces new substances through fermentation and metabolism, improves the bioavailability of active substances in the lotus plumule, and simultaneously, the active substances produced after fermentation have a complex synergistic effect, and can relieve the itching symptoms caused by atopic dermatitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a method for preparing lotus seed heart fermentation product and its application. Background Technology

[0002] Lotus seed heart (Plumula Nelumbinis) is the dried seed kernel and radicle of the mature seed of Nelumbo nucifera Gaertn., a plant in the Nymphaeaceae family. It is also known as lotus heart or lotus seed bark. Lotus seed heart is a plant used for both food and medicine, possessing various biological activities such as anti-inflammatory, anti-tumor, antioxidant, hypoglycemic, hypotensive, and hyperlipidemia prevention. The main active components of lotus seed heart include alkaloids, flavonoids, polysaccharides, and fatty acids. The main components are alkaloids: lotusine, methyl lotusine, and isolivineine.

[0003] Human understanding of the objective world begins with sensation. Sensation is the process by which the brain receives, processes, and recognizes various attributes of external objects through categorized receptors. Sensation includes vision, hearing, smell, taste, and cutaneous sensation (touch, temperature, cold, pain). In the field of sensation, since Joseph Eelnger and Herbert Gasser discovered sensory nerve fibers in 1944, the Nobel Prize in Physiology or Medicine has been awarded in 1961, 1967, and 2004 to scientists who discovered the mechanisms of hearing, vision, and smell, respectively. The mechanism of temperature perception has long been a mystery. In 1997, David Julius of the University of California, San Francisco, studied how capsaicin produces a burning sensation and successfully identified a new ion channel protein, TRPV1, which is sensitive to capsaicin and is also a heat-sensitive receptor activated at temperatures that cause pain. TRPV1 is a non-selective cation channel that transmembrane six times. Its ion micropore region is located between S5 and S6. Capsaicin can directly activate TRPV1 at temperatures above 43°C.

[0004] TRPV1 is also one of the main mediators of inflammation-induced pain. TRPV1 is mainly distributed in class C nerve fibers and can be activated by heat, acid, and vanilla compounds. Activation of class C nerve fibers leads to the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) from sensory nerve secretory granules. The release of substance P drives mast cells to release TNF-α, histamine, leukotriene B4, and prostaglandins, further mediating the pruritus response. Clinically, prolonged topical application of TRPV1 agonists (such as CAPSAICIN) can gradually deplete substance P in C fibers, reducing the pruritus response mediated by mast cell degranulation. Multiple studies have reported that TRPV1 is involved in both skin physiological and pathological functions.

[0005] Atopic dermatitis is a chronic inflammatory disease clinically characterized by prolonged itching. Skin rashes may include erythema, scratch marks, lichenification, and dry skin, often accompanied by elevated serum IgE levels and a family history of allergic rhinitis and asthma. Dry skin and decreased skin barrier function are closely related to the progression of atopic dermatitis. Studies have reported that increased TRPV1 expression may increase the production of pruritus mediators and exacerbate the inflammatory response, while TRPV1 inhibitors can not only alleviate atopic dermatitis symptoms but also promote the recovery of skin barrier function.

[0006] Currently, lotus seed heart is mainly used in drugs for lowering blood sugar and blood pressure. However, its application in cosmetics is extremely limited, and there are no reports of using lotus seed heart in cosmetics to inhibit TRPV1. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing lotus seed heart fermentation.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing lotus seed heart ferment, comprising,

[0011] Take dried lotus seed hearts, grind them into powder using a grinder, and obtain lotus seed heart powder;

[0012] Mix lotus seed heart powder with water evenly, heat to 40-60℃ and stir thoroughly to dissolve and swell, thus obtaining a lotus seed heart suspension.

[0013] Add cellulase, pectinase and papain to the lotus seed heart suspension to hydrolyze it and obtain lotus seed heart enzymatic hydrolysate;

[0014] The lotus seed heart enzymatic hydrolysate was mixed with a carbon source, the pH was adjusted to 6.0-7.0, and then sterilized to obtain the lotus seed heart fermentation medium.

[0015] Prepare a fermentation inoculum solution, wherein the inoculum includes one or more of yeast, lactic acid bacteria, Aspergillus, and Bacillus;

[0016] Inoculate the seed liquid of fermentation bacteria into the lotus seed heart fermentation medium, and ferment to obtain lotus seed heart fermentation liquid;

[0017] The lotus seed heart fermentation liquid was filtered through a ceramic membrane and then through an ultrafiltration membrane to obtain the lotus seed heart fermentation product.

[0018] In a preferred embodiment of the preparation method described in this invention, the lotus seed heart powder is a powder that has passed through a 60-200 mesh sieve.

[0019] In a preferred embodiment of the preparation method described in this invention, the mass ratio of lotus seed heart powder to water is 1:5 to 10.

[0020] In a preferred embodiment of the preparation method described in this invention, the cellulase activity is 10000 U / g, and the cellulase content is 0.01–0.15% based on the weight of the lotus seed heart suspension; the pectinase activity is 100000 U / g, and the pectinase content is 0.05–0.1% based on the weight of the lotus seed heart suspension; the papain activity is 10000 U / g, and the papain content is 0.05–0.1% based on the weight of the lotus seed heart suspension.

[0021] As a preferred embodiment of the preparation method described in this invention, the hydrolysis conditions are: enzymatic hydrolysis at 100 rpm / min and 50–70 °C for 3–6 h.

[0022] In a preferred embodiment of the preparation method described in this invention, the carbon source includes one or more of glucose, xylose, trehalose, maltose, malt extract powder, and glycerol.

[0023] The carbon source is 2-4% of the mass of the lotus seed heart enzymatic hydrolysate.

[0024] As a preferred embodiment of the preparation method described in this invention, the method for preparing Aspergillus oryzae seed solution includes: removing Aspergillus oryzae glycerol cryovials from a -80°C freezer, aseptically streaking them onto a solid PDA plate in a laminar flow hood, placing them in a 30°C incubator, and incubating them upright for 5 days. After spores have grown, the spores are washed with sterile physiological saline in a laminar flow hood, with a spore count of 10... 6 Inoculate 100 cells / ml into a 500ml shake flask containing 100ml of Aspergillus liquid culture medium and incubate at 28℃ and 220rpm for 32h to obtain Aspergillus oryzae seed liquid;

[0025] The method for preparing yeast seed culture includes: taking out yeast glycerol cryovials from a -80℃ freezer, aseptically streaking them onto solid YPD plates in a clean bench, placing them in a 28℃ incubator, inverting them for 2 days, and after colonies have grown, aseptically picking a single colony with an inoculation loop, inoculating it into a 500ml shake flask containing 100ml of YPD liquid culture medium, and incubating it in a shaker at 28℃ and 220rpm for 16 hours to obtain yeast seed culture.

[0026] In a preferred embodiment of the preparation method described in this invention, the inoculation amount of microorganisms during fermentation is 5-10% based on the amount of fermentation culture medium.

[0027] Ceramic membrane filtration, wherein the pore size of the ceramic membrane is 50–200 nm; ultrafiltration membrane filtration, wherein the molecular weight and pore size are 3000–10000 Da.

[0028] Another objective of this invention is to overcome the shortcomings of the prior art and provide a product obtained by a method for preparing lotus seed heart fermentation, the product comprising Aspergillus oryzae lotus seed heart fermentation and yeast lotus seed heart fermentation, with a mass ratio of 2:1.

[0029] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing lotus seed heart fermentation product for use in the preparation of cosmetics or pharmaceuticals with TRPV1 inhibitory effects.

[0030] Beneficial effects of this invention:

[0031] (1) This invention uses microbial fermentation of lotus seed heart to develop and expand the application range of lotus seed heart in cosmetics; by means of crushing, swelling, enzymatic hydrolysis, fermentation, high pressure homogenization, filtration, concentration and other methods, small molecule substances after lotus seed heart fermentation are collected to obtain lotus seed heart fermentation product rich in highly active small molecules, which solves the stinging and itching symptoms caused by skin diseases.

[0032] (2) The present invention uses fermentation technology to enzymatically ferment lotus seed heart to release active substances in lotus seed heart. Through microbial fermentation and transformation, large molecules that cannot be absorbed by the skin are fermented into small molecules. Through the action of microorganisms, new substances are produced by metabolism, which improves the bioavailability of active substances in lotus seed heart. At the same time, the active substances produced after fermentation have a synergistic effect to relieve itching symptoms caused by atopic dermatitis. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0034] Figure 1 The expression level of TRPV1 in HaCaT cells stimulated by UV in lotus seed hearts fermented by different strains;

[0035] Figure 2 The expression level of TRPV1 in HaCaT cells stimulated by UV was determined by different ultrafiltration membrane treatments of lotus seed heart mold fermentation products;

[0036] Figure 3 The expression level of TRPV1 in HaCaT cells stimulated by UV was determined by different ultrafiltration membrane treatments of lotus seed heart yeast ferment.

[0037] Figure 4 Comparison chart of optimized efficacy ratios for two different fermentation broths;

[0038] Figure 5 The above are liquid chromatograms of lotus seed heart fermentation products from two different bacteria. Detailed Implementation

[0039] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0040] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0042] In this invention: the yeast is Saccharomycopsis fibuligera (CICC31025), the lactic acid bacteria is Lactobacillus pentosus (CICC24202), the Aspergillus oryzae (CICC 40636), and the Bacillus subtilis (CICC 25064), all of which were purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0043] All other raw materials are ordinary commercially available products.

[0044] Example 1

[0045] Preparation of lotus seed heart lactic acid bacteria fermentation product:

[0046] (1) Lotus seed heart powder: Take 700g of dried lotus seed heart and grind it completely into 100 mesh powder using a grinder;

[0047] (2) Preparation of lotus seed heart suspension: Mix 500g of lotus seed heart powder from step 1 with 4000g of purified water, heat to 55℃, and stir for 2h to allow it to fully swell;

[0048] (3) Enzymatic hydrolysis of lotus seed heart suspension: Add 2g cellulase (10000U / g), 3.8g pectinase (100000U / g), and 2.9g papain (10000U / g) to the lotus seed heart suspension obtained in step 2, and stir and hydrolyze for 6h at 60℃ and 100rpm / min.

[0049] (4) Preparation of lotus seed heart fermentation culture medium: Add 90g of glucose to 4500g of lotus seed heart enzymatic hydrolysate obtained in step 3, adjust the pH to 6.8 with 1M NaOH, pour it into a 10L fermenter, and sterilize at 121℃ for 20min.

[0050] (5) Lactobacillus seed culture: Take out the Lactobacillus glycerol cryopreservation tube from the -80℃ freezer, and aseptically streak it onto a solid MRS plate in a clean bench. Place it in a 37℃ incubator and incubate upside down for 1 day. After the colonies grow, aseptically pick a single colony with an inoculation loop and inoculate it into a 500ml shake flask containing 100ml MRS liquid culture medium. Incubate at 37℃ and 220rpm for 16h to obtain Lactobacillus seed culture.

[0051] (6) Inoculation of lotus seed heart fermentation medium with microorganisms: Inoculate 300ml of seed liquid cultured in step (5) into a 10L fermenter that has been sterilized and cooled to 37℃ in step (4), set the temperature to 37℃, pH to 6.8, rotation speed to 180rpm, aeration rate to 0.5vvm, and dissolved oxygen >30% to start fermentation.

[0052] During the fermentation process, pH and dissolved oxygen levels were monitored. When fermentation reached 36 hours, dissolved oxygen increased and pH no longer decreased, indicating the end of fermentation.

[0053] (7) Post-processing of lotus seed heart fermentation product: The lotus seed heart fermentation liquid obtained in step (6) was filtered and clarified by a ceramic membrane with a pore size of 200 nm, and the filtrate was collected; the filtrate was filtered by a 5000 Da ultrafiltration membrane, and the permeate was collected as lotus seed heart fermentation product.

[0054] Example 2

[0055] Preparation of lotus seed heart mold fermentation product:

[0056] (1) Lotus seed heart powder: Take 1000g of dried lotus seed heart and grind it completely into 100 mesh powder using a grinder;

[0057] (2) Preparation of lotus seed heart suspension: Mix 800g of lotus seed heart powder from step 1 with 6000g of purified water, heat to 58℃, and stir for 2h to allow it to fully swell;

[0058] (3) Enzymatic hydrolysis of lotus seed heart suspension: Add 4g cellulase (10000U / g), 8g pectinase (100000U / g), and 6g papain (10000U / g) to the lotus seed heart suspension obtained in step 2, and stir and hydrolyze for 4h at 60℃ and 100rpm / min.

[0059] (4) Preparation of lotus seed heart fermentation culture medium: Add 230g of malt extract powder to 6800g of lotus seed heart enzymatic hydrolysate obtained in step 3, adjust the pH to 6.0 with 1M NaOH, pour it into a 10L fermentation tank, and sterilize at 121℃ for 20min.

[0060] (5) Aspergillus oryzae seed culture: Remove the Aspergillus oryzae glycerol cryovials from the -80℃ freezer, and aseptically streak them onto a solid PDA plate in a laminar flow hood. Place the plate in a 30℃ incubator and incubate upright for 5 days. After spores have emerged, wash the spores with sterile physiological saline in a laminar flow hood. The spore count should be 10^ 6 Inoculate 100 ml of Aspergillus oryzae seed culture into a 500 ml shake flask containing 100 ml of Aspergillus liquid culture medium (liquid culture medium g / L: rice flour 10, skim milk powder 2, glucose 10, the remainder is purified water, sterilized at 121℃ for 20 min) and incubate at 28℃ and 220 rpm for 32 h.

[0061] (6) Inoculation of lotus seed heart fermentation medium with microorganisms: Inoculate 600ml of Aspergillus oryzae seed liquid cultured in step (5) into a 10L fermenter that has been sterilized and cooled to 30℃ in step (4). Set the temperature to 30℃, pH to 50, rotation speed to 200rpm, aeration rate to 0.8vvm, and dissolved oxygen >40% to start fermentation. Monitor pH and dissolved oxygen during fermentation. When the dissolved oxygen increases and the pH no longer decreases after 72h, fermentation ends.

[0062] (7) Post-processing of lotus seed heart fermentation product: The lotus seed heart fermentation liquid obtained in step (6) was filtered and clarified using a ceramic membrane with a pore size of 50nm, and the filtrate was collected.

[0063] The filtrate was filtered using a 5000Da ultrafiltration membrane, and the permeate was collected as lotus seed heart fermentation product.

[0064] Example 3:

[0065] Preparation of lotus seed heart yeast ferment:

[0066] (1) Lotus seed heart powder: Take 700g of dried lotus seed heart and grind it completely into 120 mesh powder using a grinder;

[0067] (2) Preparation of lotus seed heart suspension: Mix 600g of lotus seed heart powder from step 1 with 6000g of purified water evenly, heat to 60℃, and stir for 2h to allow it to fully swell;

[0068] (3) Enzymatic hydrolysis of lotus seed heart suspension: Add 2.64g cellulase (10000U / g), 5.28g pectinase (100000U / g), and 4.62g papain (10000U / g) to the lotus seed heart suspension obtained in step 2, and stir and hydrolyze for 5h at 60℃ and 100rpm / min.

[0069] (4) Preparation of lotus seed heart fermentation culture medium: Add 198g of malt extract powder to 6600g of lotus seed heart enzymatic hydrolysate obtained in step 3, adjust the pH to 6.5 with 1M NaOH, pour it into a 10L fermentation tank, and sterilize at 121℃ for 20min.

[0070] (5) Yeast seed culture: Take out the yeast glycerol cryopreservation tube from the -80℃ freezer, and aseptically streak it onto a solid YPD plate in a clean bench. Place it in a 28℃ incubator and incubate upside down for 2 days. After the colonies grow, aseptically pick a single colony with an inoculation loop and inoculate it into a 500ml shake flask containing 100ml of YPD liquid culture medium. Incubate at 28℃ and 220rpm for 16h to obtain yeast seed culture.

[0071] (6) Inoculation of lotus seed heart fermentation medium with microorganisms: Inoculate 500ml of seed liquid cultured in step (5) into a 10L fermenter that has been sterilized and cooled to 28℃ in step (4), set the temperature to 28℃, pH to 5.8, rotation speed to 200rpm, aeration rate to 0.5vvm, and dissolved oxygen >30% to start fermentation.

[0072] pH and dissolved oxygen levels were monitored during the fermentation process. When the dissolved oxygen level increased after 28 hours of fermentation and the pH level stopped decreasing, the fermentation was considered complete.

[0073] (7) Post-processing of lotus seed heart fermentation product: The lotus seed heart fermentation liquid obtained in step (6) was filtered and clarified using a ceramic membrane with a pore size of 200nm, and the filtrate was collected.

[0074] The filtrate was filtered using a 3000Da ultrafiltration membrane, and the permeate was collected as lotus seed heart fermentation product.

[0075] Example 4

[0076] Preparation of Bacillus subtilis ferment:

[0077] (1) Lotus seed heart powder: Take 1000g of dried lotus seed heart and grind it completely into 120 mesh powder using a grinder;

[0078] (2) Preparation of lotus seed heart suspension: Mix 800g of lotus seed heart powder from step 1 with 6000g of purified water, heat to 58℃, and stir for 2h to allow it to fully swell;

[0079] (3) Enzymatic hydrolysis of lotus seed heart suspension: Add 6g cellulase (10000U / g), 6g pectinase (100000U / g), and 5g papain (10000U / g) to the lotus seed heart suspension obtained in step 2, and stir and hydrolyze for 5h at 60℃ and 100rpm / min.

[0080] (4) Preparation of lotus seed heart fermentation culture medium: Add 140g of glucose to 6800g of lotus seed heart enzymatic hydrolysate obtained in step 3, adjust the pH to 7.0 with 1M NaOH, pour it into a 10L fermenter, and sterilize at 121℃ for 20min.

[0081] (5) Bacillus subtilis seed culture: Take out the Bacillus subtilis glycerol cryopreservation tube from the -80℃ freezer, and aseptically streak it onto a solid LB plate in a clean bench. Place it in a 37℃ incubator and incubate upside down for 1 day. After the colonies grow, aseptically pick a single colony with an inoculation loop and inoculate it into a 500ml shake flask containing 100ml of LB liquid medium. Incubate at 37℃ and 220rpm for 15h to obtain Bacillus subtilis seed culture.

[0082] (6) Inoculation of lotus seed heart fermentation medium with microorganisms: Inoculate 400ml of seed liquid cultured in step (5) into a 10L fermenter that has been sterilized and cooled to 37℃ in step (4), set the temperature to 37℃, pH to 6.0, rotation speed to 210rpm, aeration rate to 0.5vvm, and dissolved oxygen to >35% to start fermentation.

[0083] During the fermentation process, pH and dissolved oxygen levels were monitored. When fermentation reached 32 hours, dissolved oxygen increased and pH no longer decreased, indicating the end of fermentation.

[0084] (7) Post-processing of lotus seed heart fermentation product: The lotus seed heart fermentation liquid obtained in step (6) was filtered and clarified by a ceramic membrane with a pore size of 200 nm, and the filtrate was collected; the filtrate was filtered by a 5000 Da ultrafiltration membrane, and the permeate was collected as lotus seed heart fermentation product.

[0085] Comparative Example 1

[0086] Based on the preparation process in Example 2, Aspergillus oryzae lotus seed heart fermentation broth was prepared and filtered using a 3000Da ultrafiltration membrane to obtain the fermentation broth product.

[0087] Comparative Example 2

[0088] Based on the preparation process in Example 2, Aspergillus oryzae lotus seed heart fermentation broth was prepared and filtered using a 10000Da ultrafiltration membrane to obtain the fermentation broth product.

[0089] Comparative Example 3

[0090] Based on the preparation process of Example 3, lotus seed heart yeast ferment was prepared, and the fermentation broth product was obtained by filtration using a 5000Da ultrafiltration membrane.

[0091] Comparative Example 4

[0092] Based on the preparation process of Example 3, lotus seed heart yeast ferment was prepared and filtered through a 10000Da ultrafiltration membrane to obtain the fermentation broth product.

[0093] The inhibitory effect of the prepared lotus seed heart fermentation broth on TRPV-1 was determined using a human TRPV-1 ELISA kit (Aviva Systems Biology: OKEH02208).

[0094] Cell culture: Human immortalized epidermal cells (HaCaT; BNCC339817) were seeded at 1.5 × 10⁵ cells / mL into 6-well plates. 1.0 mL of DMEM complete medium containing 10% FBS and 0.1% antibiotics was added to each well. The cells were cultured at 37°C in a 5% CO₂ incubator until the cell confluence reached 90%.

[0095] Sample preparation: Remove the culture medium, add DMEM to each well to prepare the fermentation sample, and incubate in an incubator for 24 h. The control group and blank group cells were cultured in DMEM.

[0096] UV stimulation: Remove the culture medium, add PBS buffer, and administer 50 mJ / cm² under UV-B (315 nm) conditions. 2 Energy irradiation. Remove PBS buffer and continue culturing in DMEM medium for 12 hours. The control group consisted of samples without UV stimulation (blank) and samples without UV stimulation (control).

[0097] ELISA assay: Remove the culture medium, add 150 μL of RIPA cell lysis buffer (containing 1% PMSF) to each well, collect the cells, centrifuge at 3000 rpm for 20 min, take the supernatant, and use an ELISA kit to detect TRPV-1 at 450 nm.

[0098] It was also confirmed that, among different fermentation broths of lotus seed hearts, fermentation with Aspergillus oryzae showed the best inhibitory effect on TRPV-1. Figure 1 The second most abundant component is yeast fermentation broth.

[0099] To further enhance its efficacy, the inhibitory effect of filtration of mold and yeast with membranes at 3000 Da, 5000 Da, and 10000 Da was investigated. The results showed that the inhibitory effect of TRPV-1 was best in the mold fermentation broth at 5000 Da and in the yeast fermentation broth at 3000 Da. Figure 2-3 The results indicate that the major TRPV-1 inhibitor produced by yeast fermentation broth has a larger molecular weight than the major inhibitor produced by mold fermentation broth.

[0100] In this study, lotus seed heart fermentation broth containing mold-5000Da and yeast-3000Da was selected. The synergistic effect of these two broths was evaluated after mixing them at volume ratios of 3:0, 2:1, 1.5:1.5, 1:2, and 0:3, with a 1% cell treatment volume. Figure 4 The results showed that the best effect was achieved when the ratio of mold fermentation broth to yeast fermentation broth was 2:1, followed by 1.5:1.5.

[0101] Example 5

[0102] The changes in the main active ingredients after fermentation were determined by high performance liquid chromatography.

[0103] High performance liquid chromatography: Thermo Fisher Scientific UltiMate 3000 series (DAD detector); Column: Thermo Fisher Scientific Acclaim™ 120C18 column (5μm, 4.6mm x 250mm); Mobile phase: H2O (containing 0.02% ammonia) as phase A, methanol (containing 0.02% ammonia) as phase B, phase A at 35%–20%, for 30 min; Injection volume: 20μL.

[0104] Analyze the changes in its metabolites during fermentation ( Figure 5 );

[0105] The results showed that fermentation by molds and yeasts can metabolize methyl lotusine, lotusine, and isolivineine in lotus seeds, and generate their alkaloid derivatives. These derivatives exhibit the characteristic absorption of 282 nm, similar to methyl lotusine, lotusine, and isolivineine. However, the conversion rate by yeast was lower than that by molds.

[0106] Further mass spectrometry analysis revealed [M+H] + =314 and [M+H] + The peak at 300 also indicates that its main compounds, methylnepenone and isolipenone, can be metabolized into dibenzylisoquinoline derivatives 1, 2, and 3, respectively.

[0107] Furthermore, derivatives 1 and 2 are isomers, so their peaks are higher than those of derivative 3. Also, derivatives 1 and 2 have the same structural polarity, making them difficult to separate in high-performance liquid chromatography (HPLC).

[0108] 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. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a Ploughshare kernel ferment, characterized by: The application relates to a method for preparing lotus plumule fermentation product. The lotus plumule powder is obtained by drying lotus plumule, crushing and powdering the lotus plumule; The lotus plumule powder is mixed with water, heated to 40-60 DEG C, and stirred to swell, thereby obtaining lotus plumule suspension; The lotus plumule suspension is hydrolyzed by adding cellulase, pectinase and papain, thereby obtaining lotus plumule enzymatic hydrolysate; The lotus plumule enzymatic hydrolysate is added with carbon source, and the pH is adjusted to 6.0-7.0, thereby obtaining lotus plumule fermentation medium, wherein the carbon source is malt extract powder, and the carbon source accounts for 2-4% of the mass of the lotus plumule enzymatic hydrolysate. Separately prepared yeast seed culture and Aspergillus seed culture, wherein the yeast was *Diplostomum cuspidatum* (Saccharomyces cerevisiae). Saccharomycopsis fibuligera CICC31025, the Aspergillus species is Aspergillus oryzae ( Aspergillus oryzae CICC40636, all purchased from the China Industrial Microbial Culture Collection Center; The yeast seed liquid is inoculated into the lotus plumule fermentation medium, and fermentation is carried out, thereby obtaining yeast lotus plumule fermentation liquid; The yeast lotus plumule fermentation liquid is filtered through a ceramic membrane and an ultrafiltration membrane, thereby obtaining yeast lotus plumule fermentation product, wherein the pore size of the ceramic membrane is 200 nm, and the molecular weight pore size of the ultrafiltration membrane is 3000 Da. The yeast seed liquid is inoculated into the lotus plumule fermentation medium, and fermentation is carried out, thereby obtaining yeast lotus plumule fermentation liquid; The yeast lotus plumule fermentation liquid is filtered through a ceramic membrane and an ultrafiltration membrane, thereby obtaining yeast lotus plumule fermentation product, wherein the pore size of the ceramic membrane is 200 nm, and the molecular weight pore size of the ultrafiltration membrane is 3000 Da. The yeast lotus plumule fermentation product and the yeast lotus plumule fermentation product are mixed, thereby obtaining the lotus plumule fermentation product, wherein the mass ratio of the yeast lotus plumule fermentation product and the yeast lotus plumule fermentation product is 2:

1.

2. The production method according to claim 1, characterized by: The lotus plumule powder is sieved through a 60-200 mesh sieve.

3. The production method according to claim 1 or 2, characterized by: The mass ratio of the lotus plumule powder and water is 1:5-10.

4. The production method according to claim 1 or 2, characterized by: The enzyme activity of the cellulase is 10000 U / g, the cellulase accounts for 0.01-0.15% of the weight of the lotus plumule suspension; the enzyme activity of the pectinase is 100000 U / g, the pectinase accounts for 0.05-0.1% of the weight of the lotus plumule suspension; and the enzyme activity of the papain is 10000 U / g, the papain accounts for 0.05-0.1% of the weight of the lotus plumule suspension.

5. The production method according to claim 4, characterized by: The hydrolysis condition is that the enzyme hydrolysis is carried out at 100 rpm / min and 50-70 DEG C for 3-6 h.

6. The production method according to claim 1 or 2, characterized by: The method for configuring the Aspergillus oryzae seed liquid comprises the following steps: taking the Aspergillus oryzae glycerol freezing tube from a-80 DEG C refrigerator, streaking to a solid PDA plate in a super-clean table in a sterile operation, placing in a 30 DEG C incubator, and vertically culturing for 5 days; after spores grow, the spores are washed with sterile normal saline in the super-clean table; 10 6 ml of the Aspergillus oryzae liquid medium is inoculated into a 500 ml flask containing 100 ml of the Aspergillus liquid medium according to the spore quantity of 10 6 individuals / ml, and is placed in a 28 DEG C, 220 rpm shaking table for culturing for 32 h to obtain the Aspergillus oryzae seed liquid. The method for preparing the yeast seed liquid comprises the following steps: taking the yeast glycerol freezing tube from a-80 DEG C refrigerator, performing sterile operation to draw lines on a solid YPD plate in an ultra-clean bench, placing the YPD plate in a 28 DEG C incubator, and performing inverted culture for 2 days; after the colonies grow, the single colony is picked up by a sterile inoculation loop, and inoculated into a 500 ml flask containing 100 ml YPD liquid medium, and placed in a 28 DEG C, 220 rpm shaking bed to culture for 16 h, thereby obtaining the yeast seed liquid.

7. The production method according to claim 1 or 2, wherein: The inoculation amount of the microorganism during fermentation is 5-10% based on the amount of the fermentation medium.

Images