Cistanche deserticola fermentation liquor with effects of resisting oxidation and regulating immunity as well as preparation method and application of cistanche deserticola fermentation liquor

Abstract

The invention relates to the technical field of microbial fermentation, discloses herba cistanche fermentation liquor with anti-oxidation and immunoregulation effects as well as a preparation method and application thereof, and belongs to the field of microbial fermentation. The preparation method of the cistanche fermentation liquor comprises the following steps: crushing cistanche into powder, and preparing into turbid liquid; adding an enzyme preparation into the turbid liquid, adjusting the pH value, carrying out enzymolysis, and sterilizing to obtain cistanche enzymatic hydrolysate; inoculating a seed solution of a fermentation strain into the cistanche enzymatic hydrolysate, and fermenting to obtain a cistanche fermentation suspension; and sterilizing and centrifuging to obtain the cistanche deserticola fermentation liquor. According to the preparation method disclosed by the invention, the cistanche deserticola fermentation liquor is prepared through a duplex regulation and control technology of enzymolysis and multi-strain fermentation, so that the dissolution of active ingredients such as proteins, polysaccharides and polyphenols in the cistanche deserticola is improved, and the cistanche deserticola has remarkable antioxidant and immune regulation effects; due to the characteristics, the cistanche deserticola fermentation liquor can be widely applied to the fields of dietary supplements, functional foods and health-care products as a functional component.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects, its preparation method, and its application. Background Technology

[0002] Cistanche deserticola is a perennial parasitic herb belonging to the genus Cistanche of the family Orobanchaceae. Rich in bioactive components such as polysaccharides, proteins, and polyphenols, it has garnered significant attention in the functional food and health product sectors. Modern pharmacological research suggests that Cistanche deserticola possesses antioxidant, immunomodulatory, anti-fatigue, and reproductive health protective activities, exhibiting significant medicinal and edible value. With the National Health Commission and the State Administration for Market Regulation officially including it in the list of food and medicine homologous to each other, the application prospects of Cistanche deserticola in dietary supplements, functional foods, and health products within the broader health industry will become even broader.

[0003] Due to limitations in traditional processing technology, equipment, and formulation design concepts, the oral preparations of Cistanche deserticola currently on the market are mainly in the form of decoctions, pills, powders, and alcohol extracts. While these dosage forms conform to traditional medication habits, they have significant shortcomings in terms of retention of active ingredients, applicable scenarios, and efficacy. Decoctions, during the high-temperature boiling process, easily cause degradation, oxidation, and isomerization of active ingredients such as proteins, polysaccharides, phenylethanol glycosides, water-soluble terpenes, and sterols in Cistanche deserticola. Pills are often prepared using honey, rice paste, beeswax, etc., as binders, resulting in a relatively dense matrix that is difficult to effectively disintegrate and release in the gastrointestinal tract after oral administration. Although powders have a large specific surface area, their uneven particle size distribution makes them prone to aggregation due to surface tension during dissolution, affecting bioavailability. The use of alcohol in alcohol extracts severely limits their use by patients with liver disease, those allergic to alcohol, and specific occupational groups (drivers, those working at heights). The aforementioned shortcomings severely limit the expansion of Cistanche deserticola products from the traditional medicinal field to the modern health and functional food field, and also restrict the realization of the industrialization value of Cistanche deserticola, a desert-specific plant resource.

[0004] From the perspective of the compatibility of various Cistanche deserticola dosage forms with their process characteristics and component characteristics, innovation and improvement are necessary. In recent years, enzymatic hydrolysis and microbial fermentation technologies have been widely used to promote the dissolution and transformation of effective components in medicinal and edible homologous substances, and some preliminary attempts have been made in fermenting Cistanche deserticola. Patent CN118059157A discloses a Cistanche deserticola fermentation broth, fermentation method and its application. This invention first enzymatically hydrolyzes Cistanche deserticola using cellulase and pectinase, and then adds Lactobacillus plantarum to the Cistanche deserticola enzymatic hydrolysate. The LZU-J-Q21 method for preparing Cistanche deserticola fermentation broth improved its bitterness and enhanced its antioxidant activity. However, this invention did not optimize the optimal pH of the enzyme preparation used, employed a relatively single fermentation strain, and resulted in a significantly lower total sugar content in the prepared Cistanche deserticola fermentation broth compared to before fermentation. The increase in ABTS free radical scavenging rate was less than 50%. Patent CN116870053A discloses a drug for improving sexual function and its preparation method. This drug, obtained by fermenting Cistanche deserticola with Lactobacillus rhamnosus Y5, can treat erectile dysfunction in men caused by diabetes, with better efficacy than unfermented Cistanche deserticola extract. However, the increase in soluble polysaccharides in the fermentation broth after fermentation with a single strain of Lactobacillus rhamnosus Y5 was only 26.5%. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects, its preparation method, and its application, solving the problems of low dissolution rate and poor bioavailability of active ingredients in existing Cistanche deserticola preparations.

[0006] This invention provides the following technical solution: a method for preparing Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects, comprising the following steps: S1: Cistanche deserticola is pulverized into powder and prepared into a suspension; S2: Add the enzyme preparation to the suspension, adjust the pH, perform enzymatic hydrolysis, and sterilize to obtain Cistanche deserticola hydrolysate; S3: Add the seed liquid of the fermentation strain to the Cistanche deserticola enzymatic hydrolysate, and ferment it using fermentation equipment to obtain fermented Cistanche deserticola suspension; S4: Cistanche fermentation broth is obtained after sterilization and centrifugation.

[0007] Optionally, the particle size of the Cistanche deserticola powder in step S1 is 60-100 mesh, and the suspension is prepared by mixing Cistanche deserticola powder and water at a material-to-liquid ratio of 1:10-1:20, with a total volume of 300 mL.

[0008] Optionally, the enzyme preparation in step S2 is composed of one or more of cellulase and pectinase, with the amount of cellulase added being 30-40 U / g and the amount of pectinase added being 15-20 U / g.

[0009] Optionally, the pH in step S2 is 4.5 to 5.0, adjusted by one or more combinations of citric acid and sodium hydroxide.

[0010] Optionally, the enzymatic hydrolysis temperature in step S2 is 35~55 °C, the enzymatic hydrolysis time is 3~32 h, and the sterilization temperature is 121 °C, the sterilization time is 10~30 min.

[0011] Optionally, the fermentation strain in step S3 is composed of one or more of Bacillus subtilis, lactic acid bacteria and Saccharomyces cerevisiae, and the seed liquid of the fermentation strain is added on the first to eighth day of fermentation.

[0012] Optionally, the inoculation amount of the fermentation strains in step S3 is 2-5%, the OD600 value of Bacillus subtilis is 5-6, the OD600 value of lactic acid bacteria is 1-3, the OD600 value of Saccharomyces cerevisiae is 12-14, the fermentation temperature is 35-40 °C, and the fermentation time is 1-15 days.

[0013] Optionally, the sterilization temperature in step S4 is 121 °C, the sterilization time is 10~30 min, the centrifugation speed is 5000~8000 r / min, and the centrifugation time is 10~20 min.

[0014] A fermented broth of Cistanche deserticola with antioxidant and immunomodulatory effects was prepared according to the above-mentioned preparation method.

[0015] Application of Cistanche deserticola fermented liquid with antioxidant and immunomodulatory effects, wherein the Cistanche deserticola fermented liquid is used in dietary supplements, functional foods and health products.

[0016] Optionally, the fermentation equipment in step S3 includes a fermentation tank, the top of which is hinged to a top cover, a connecting pipe is fixed to the top of the top cover, a first connecting member is slidably connected to the surface of the connecting pipe, a supporting ring is fixed to the top of the first connecting member, and a motor is fixed inside the supporting ring. The first connector has adjustment grooves on both sides, and the connecting pipe has screws fixed on both sides. The screws are slidably connected to the adjustment grooves, and the screws have a washer threadedly connected to their surfaces. The output shaft of the motor is fixed with a connecting shaft, the bottom of the connecting shaft is fixed with a hexagonal prism, the inner wall of the fermenter is fixed with a fixing frame, one end of the fixing frame is fixed with an aeration ring, the inner wall of the aeration ring is fixed with a second connecting member, one end of the second connecting member is fixed with a limit ring, and the inner wall of the limit ring is fixed with a support frame. The limiting ring is internally slidably sealed to a stirring shaft. The top of the stirring shaft has a snap-fit ​​groove, and the bottom of the stirring shaft has a venting groove. An aeration pipe is fixed to the surface of the stirring shaft. A guide tube is fixed to one end of the aeration pipe. A propulsion blade is fixed to the surface of the stirring shaft above the aeration pipe, and an inclined blade turbine is fixed to the surface of the stirring shaft below the aeration pipe.

[0017] Optionally, the fermentation equipment further includes a cooling water discharge pipe: the cooling water discharge pipe is fixed inside the fermentation tank, a sampling pipe is fixed on one side of the fermentation tank, a cooling water inlet pipe is fixed below the sampling pipe on the side of the fermentation tank, an exhaust pipe is fixed on the top of the top cover near the cooling water discharge pipe, a feeding port is fixed on the top of the top cover near the sampling pipe, and an air inlet pipe is fixed on the side of the fermentation tank near the cooling water discharge pipe, and the air inlet pipe is connected to the aeration ring through a fixing frame.

[0018] Optionally, the fermentation equipment further includes auxiliary blades: the auxiliary blades are fixed on the surface of the guide tube, the top diameter of the guide tube is smaller than the bottom diameter of the guide tube, the propulsion blades are located inside the guide tube, the fan blades of the inclined turbine are located below the bottom of the guide tube, and the hexagonal prism is slidably sealed to the snap-fit ​​groove.

[0019] Optionally, the fermentation equipment further includes an electrically controlled valve: the electrically controlled valve is fixed inside the aeration trough, the top space of the aeration trough is isolated into a buffer chamber by the electrically controlled valve, a limit groove is formed on the surface of the buffer chamber, a positioning rod is fixed to the top of the electrically controlled valve, an elastic rope is fixed to the surface of the positioning rod, a third connector is fixed to one end of the elastic rope, a locking ring is slidably connected to the surface of the aeration pipe, and the bottom of the locking ring is connected and fixed by a fourth connector, wherein one locking ring near the third connector is fixed to the third connector.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. This fermented broth of Cistanche deserticola with antioxidant and immunomodulatory effects, its preparation method, and its application significantly improve the content of active ingredients. During the use of the dual-regulation technology of enzymatic hydrolysis and multi-strain fermentation, the efficient and specific hydrolysis by cellulase and pectinase not only promotes the release of active ingredients in Cistanche deserticola but also provides sufficient carbon source for the growth and metabolism of microorganisms during fermentation. The multi-strain fermentation method using Bacillus subtilis, lactic acid bacteria, and Saccharomyces cerevisiae, consisting of two bacteria and one fungus, forms a relatively stable microbial community that mutually promotes each other. This transforms large molecules in Cistanche deserticola that are not absorbed by the body into easily absorbed small molecules and continuously secretes beneficial active ingredients such as polysaccharides, proteins, and polyphenols, thereby effectively improving the content of functional components and bioavailability in the fermented broth of Cistanche deserticola.

[0021] 2. The fermented broth of Cistanche deserticola with antioxidant and immune-regulating effects, its preparation method and application are simple to operate and highly safe. The present invention can obtain the required fermented broth of Cistanche deserticola by simply combining operations such as mixing, enzymatic hydrolysis, fermentation and centrifugation. It has low equipment requirements and is convenient for industrial production. No organic reagents need to be added during the entire preparation process, which meets environmental protection and food safety standards and has broad application prospects and market potential.

[0022] 3. The fermentation broth of Cistanche deserticola with antioxidant and immune-regulating effects, its preparation method and application: The fermentation broth inside the fermentation tank is guided by a guide tube, and the fermentation broth is pushed downward inside the guide tube by the propulsion blades. When the fermentation broth overflows from the bottom of the guide tube, it is pushed upward towards the tank wall by the inclined blade turbine, thereby forming radial turbulence around the guide tube inside the fermentation tank, which can further increase the comprehensiveness of gas-liquid contact and thus improve the fermentation efficiency. Attached Figure Description

[0023] Figure 1 The graph shows the change in protein content of the fermentation broth of Cistanche deserticola prepared according to this invention. Figure 2 The graph shows the change in reducing sugar content in the fermentation broth of Cistanche deserticola prepared according to this invention. Figure 3 The graph shows the change in polyphenol content in the fermentation broth of Cistanche deserticola prepared according to this invention. Figure 4 The graph shows the change in ABTS free radical scavenging rate of the fermentation broth of Cistanche deserticola prepared according to this invention. Figure 5 The graph shows the test results of the changes in the content of intracellular antioxidant indicators in the fermentation broth of Cistanche deserticola prepared according to this invention; Figure 6 The figure shows the test results of the changes in the content of intracellular immune regulation-related indicators by the fermentation broth of Cistanche deserticola prepared in this invention; Figure 7 This is a schematic diagram of the fermentation equipment of the present invention; Figure 8 This is a cross-sectional view of the fermenter. Figure 9 This is a schematic diagram of the structure of the stirring shaft and the hexagonal prism; Figure 10 This is a schematic diagram of the flow guide tube and the limiting ring. Figure 11 This is a schematic diagram of the stirring shaft. Figure 12 This is a cross-sectional view of the stirring shaft. Figure 13 for Figure 8 A magnified view of a section at point A in the middle; Figure 14 for Figure 8 A magnified view of a section at point B.

[0024] In the diagram: 1. Fermentation tank; 11. Cooling water discharge pipe; 12. Sampling pipe; 13. Cooling water inlet pipe; 2. Top cover; 21. Exhaust pipe; 22. Feeding port; 23. Connecting pipe; 3. First connecting piece; 31. Support ring; 32. Motor; 33. Adjusting groove; 34. Screw; 35. Gasket; 4. Connecting shaft; 41. Hexagonal prism; 5. Fixing frame; 51. Aeration ring; 52. Second connecting piece; 53. Limiting ring; 54. Support bracket; 55. Air inlet pipe; 6. Stirring shaft; 61. Snap-fit ​​groove; 62. Ventilation groove; 63. Aeration pipe; 64. Guide tube; 65. Propulsion blade; 66. Inclined blade turbine; 7. Auxiliary blade; 8. Electrically controlled valve; 81. Buffer chamber; 811. Limiting groove; 82. Positioning rod; 83. Elastic rope; 84. Third connecting piece; 85. Locking ring; 86. Fourth connecting piece. Detailed Implementation

[0025] 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 some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1:

[0027] Please see Figure 1-6 An embodiment of the preparation method of Cistanche deserticola fermentation liquid according to the present invention includes the following steps: Pretreatment: Fresh Cistanche deserticola is washed several times to remove surface dust and impurities, cut into 2×2 cm squares, dried at 50 °C for 2 days to remove excess moisture, and then pulverized into powder with a particle size of 80 mesh. According to the material-liquid ratio of 1:10, 20 g of Cistanche deserticola powder is mixed with 300 mL of deionized water to prepare a suspension. Enzymatic hydrolysis: Enzyme preparations were added to the Cistanche deserticola suspension at dosages of 35 U / g cellulase and 17.5 U / g pectinase, respectively. The pH was adjusted to 4.8 with citric acid and sodium hydroxide. Enzymatic hydrolysis was carried out at 50 °C for 24 h, and then sterilized at 121 °C for 15 min to obtain Cistanche deserticola enzymatic hydrolysate.

[0028] Fermentation: Add 3% ( ) to the Cistanche deserticola enzymatic hydrolysate. v / v Bacillus subtilis seed culture (OD) 600 =5.65) and lactic acid bacteria seed solution (OD 600 =1.71), fermented at 37 °C, with 3% ( ) added on the 7th day of fermentation. v / v Saccharomyces cerevisiae seed culture (OD) 600=13.03) Continue fermentation until day 14, then sterilize at 121 °C for 15 min, and centrifuge at 6000 r / min for 10 min to obtain the Cistanche deserticola fermentation broth.

[0029] Example 2:

[0030] This invention provides an embodiment of the preparation method of Cistanche deserticola fermentation liquid. The difference between this embodiment and the preparation method described in Example 1 is that the ratio of Cistanche deserticola suspension to liquid in step (1) of this embodiment is 1:15, and the other parameters are the same as in Example 1.

[0031] Comparative Example 1 This comparative example provides a method for preparing Cistanche deserticola extract (water extraction method). The preparation method includes the following steps: Fresh Cistanche deserticola is washed multiple times to remove surface dust and impurities, cut into 2×2 cm cubes, dried at 50 °C for 2 days to remove excess moisture, and then pulverized into powder with a particle size of 80 mesh. 20 g of Cistanche deserticola powder is mixed with 300 mL of deionized water at a material-to-liquid ratio of 1:10 to prepare a suspension. The suspension is stirred for 48 h and centrifuged at 6000 r / min for 10 min to obtain the Cistanche deserticola extract.

[0032] Comparative Example 2 A comparative example of the preparation method of Cistanche deserticola fermentation broth according to the present invention includes the following steps: Pretreatment: Fresh Cistanche deserticola was washed several times to remove surface dust and impurities, cut into 2×2 cm cubes, dried at 50 °C for 2 days to remove excess moisture, and then pulverized into powder with a particle size of 80 mesh. 20 g of Cistanche deserticola powder was mixed with 300 mL of deionized water at a material-to-liquid ratio of 1:10, and sterilized at 121 °C for 15 min to obtain a Cistanche deserticola suspension.

[0033] Fermentation: Add 3% ( ) to the Cistanche deserticola enzymatic hydrolysate. v / v Bacillus subtilis seed culture (OD) 600 =5.65) and lactic acid bacteria seed solution (OD 600 =1.71), fermented at 37 °C, with 3% ( ) added on the 7th day of fermentation. v / v Saccharomyces cerevisiae seed culture (OD) 600 =13.03) Continue fermentation until day 14, then sterilize at 121 °C for 15 min, and centrifuge at 6000 r / min for 10 min to obtain the Cistanche deserticola fermentation broth.

[0034] Test Example 1 This test case examines the total protein content of the Cistanche fermentation broth / extract obtained in Examples 1-2 and Comparative Examples 1-2.

[0035] Dilute the sample solution 30 times, add 200 μL of BCA working solution and 20 μL of sample dilution solution to a 96-well plate, shake to mix, incubate at 37 ℃ for 30 min, and detect the absorbance at 562 nm in the ultraviolet light. Calculate the total protein content based on the pre-determined BSA standard curve. The results are as follows Figure 1 As shown, after fermentation, the protein content of all samples increased significantly, with the highest protein content observed in Sample 1. Compared to Comparative Example 1, the protein content of Sample 1 increased by 9.56 times; compared to Comparative Example 2, the protein content of Sample 1 increased by 5.23 times. This demonstrates that the dual-regulation technology of enzymatic hydrolysis and multi-strain fermentation promoted the release of protein components from Cistanche deserticola and increased the protein content of the Cistanche deserticola fermentation broth.

[0036] Test Example 2 This test case examines the reducing sugar content of the Cistanche fermentation broth / extract obtained in Examples 1-2 and Comparative Examples 1-2.

[0037] Dilute the samples 100 times, take 1 mL of the different sample dilutions into a test tube, add 3 mL of 3,5-dinitrosalicylic acid solution to the test tube, heat in a boiling water bath for 9 min, cool immediately to room temperature, take the reaction solution and dilute it 5 times, and detect the absorbance at 540 nm in ultraviolet light. Calculate the reducing sugar content in the samples with different numbers according to the pre-determined glucose standard curve. The results are as follows Figure 2 As shown, after fermentation, the reducing sugar content in each sample increased significantly. The reducing sugar content in the sample of Example 1 was the highest, increasing by 4.72 times compared to the sample of Comparative Example 1. Compared to the sample of Comparative Example 2, the reducing sugar content in the sample of Example 1 increased by 3.12 times. It can be seen that the dual regulation technology of enzymatic hydrolysis and multi-strain fermentation improved the reducing sugar content of Cistanche deserticola fermentation broth.

[0038] Test Example 3 This test case examines the polyphenol content of the Cistanche deserticola fermentation broth / extract obtained in Examples 1-2 and Comparative Examples 1-2.

[0039] All samples were diluted 20 times. 1 mL of each diluted sample was placed in a test tube. 5 mL of Folin-Ciocalteu reagent was added to the test tube and reacted for 5 min. 4 mL of 7.5% Na2CO3 solution was added to the mixture and reacted for 60 min. The absorbance was detected at 765 nm in the ultraviolet light. The polyphenol content in samples with different numbers was calculated based on the pre-determined gallic acid standard curve. The results are as follows Figure 3As shown, after fermentation, the polyphenol content in each sample increased significantly, with the highest polyphenol content observed in Sample 1. Compared to Comparative Example 1, the polyphenol content in Sample 1 increased by 1.73 times; compared to Comparative Example 2, the polyphenol content in Sample 1 increased by 78.02%. This demonstrates that the dual-regulation technology of enzymatic hydrolysis and multi-strain fermentation improved the polyphenol content of the Cistanche deserticola fermentation broth.

[0040] Test Example 4 This test case examines the ABTS free radical scavenging rate of the Cistanche deserticola fermentation broth / extract obtained in Examples 1-2 and Comparative Examples 1-2.

[0041] All samples were diluted 20-fold. 30 μL of sample solution and 120 μL of ABTS working solution were added sequentially to a 96-well plate. The reaction was carried out in the dark for 6 min, and the absorbance was measured at 735 nm. The clearance rate (%) was calculated using the formula: (A0 - A...) i The ABTS radical scavenging rate of the sample solution is calculated as (A0 / A0 × 100%), where A0 is the absorbance value of the blank control group, and A... i The absorbance values ​​of the sample group; The results are as follows Figure 4 As shown, after fermentation, the ABTS free radical scavenging rate of Cistanche deserticola fermentation broth increased significantly. The ABTS free radical scavenging rate of the sample in Example 1 was the highest (70.94%), which was 1.66 times higher than that of the extract (Comparative Example 1 sample) and 31.39% higher than that of the Comparative Example 2 sample (which only underwent multi-strain fermentation). It can be seen that the dual regulation technology of enzymatic hydrolysis and multi-strain fermentation improved the ABTS free radical scavenging ability of Cistanche deserticola fermentation broth.

[0042] Test Example 5 This test case examines the effect of the fermentation broth of Cistanche deserticola obtained in Examples 1-2 and Comparative Examples 1-2 on the changes in the content of intracellular antioxidant-related indicators.

[0043] Testicular interstitial cells were 1×10 5Cells were seeded at a density of cells / well in 12-well plates and incubated for 24 h. The medium was then replaced with 1 mL of medium containing 0.5 mg / mL Cistanche deserticola fermentation broth / extract and incubated for another 24 h. After discarding the medium, 1 mL of medium containing 600 μM H2O2 was added and incubated for another 3 h to induce oxidative damage in the cells. Cells were collected, and 300 μL of PBS was added to each cell aliquot. Cells were sonicated on ice (200 W, 5 s interval, 15 s interval, repeated 5 times), centrifuged at 4000 r / min for 10 min, and the supernatant was collected for later use. Intracellular antioxidant-related indicators were measured according to the instructions of the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) kits provided by Nanjing Jiancheng Bioengineering Research Institute. The results are as follows Figure 5 As shown, after H2O2 treatment, the levels of antioxidant enzymes SOD, CAT, and GSH-Px in testicular interstitial cells decreased significantly, while the level of MDA increased, indicating oxidative damage to the cells. After treatment with the sample from Example 1, the levels of SOD, CAT, and GSH-Px increased significantly, while the level of MDA decreased accordingly. These results indicate that the sample from Example 1 possesses excellent antioxidant capacity and can effectively alleviate oxidative damage to testicular interstitial cells, demonstrating that the dual-regulation technology of enzymatic hydrolysis and multi-strain fermentation improves the antioxidant capacity of Cistanche deserticola fermentation broth.

[0044] Test Example 6 This test case examines the effect of the fermentation broth of Cistanche deserticola obtained in Examples 1-2 and Comparative Examples 1-2 on the changes in the content of intracellular immune regulation-related indicators.

[0045] Macrophages were 1×10 5 Cells were seeded at a density of cells / well in 12-well plates and incubated for 24 h. The culture medium was then replaced with 1 mL of medium containing 0.5 mg / mL Cistanche deserticola fermentation broth / extract and incubated for another 24 h. After discarding the culture medium, 1 mL of medium containing 2 μg / mL LPS was added and incubated for another 12 h. Cells were collected, and 300 μL of PBS was added to each cell sample. Cells were sonicated on ice (200 W, 5 s interval, 15 s interval, repeated 5 times), centrifuged at 4000 r / min for 10 min, and the supernatant was collected for later use. Intracellular immune regulation-related indicators were measured according to the instructions of the kits provided by Nanjing Jiancheng Biotechnology Research Institute for inducible nitric oxide synthase (iNOS), nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The results are as follows Figure 6As shown, after LPS induction, the levels of iNOS, NO, IL-6 and TNF-α in cells increased significantly, indicating that the cells underwent an inflammatory response. After treatment with the sample from Example 1, the levels of iNOS, NO, IL-6 and TNF-α in cells decreased significantly, indicating that the dual regulation technology of enzymatic hydrolysis and multi-strain fermentation improved the immunomodulatory capacity of Cistanche deserticola fermentation broth.

[0046] To address the shortcomings of traditional Cistanche deserticola processing techniques, this invention employs a dual-regulation technology combining enzymatic hydrolysis and multi-strain fermentation. In the enzymatic hydrolysis stage, pH and temperature are controlled, utilizing a complex enzyme system of cellulase and pectinase to precisely cleave the β-(1→4) glycosidic bonds of cell wall cellulose and hydrolyze the α-(1→4) galacturonic acid bonds of intercellular pectin, achieving selective degradation of the Cistanche deserticola cell wall and increasing the release rate of active ingredients by nearly 50%. In the multi-strain fermentation stage, a ternary synergistic system composed of Bacillus subtilis, lactic acid bacteria, and Saccharomyces cerevisiae is used. Bacillus subtilis secretes proteases and amylases to degrade large protein and starch molecules into small peptides and oligosaccharides, supplementing its own and other microbial sources of carbon and nitrogen. Lactic acid bacteria continuously produce acid, adjusting the pH of the system... The pH value is stabilized at 4.0-4.5, inhibiting contamination by miscellaneous bacteria and activating endogenous glycosidases in the meat, creating a suitable microenvironment for the transformation of active ingredients. Saccharide yeast utilizes the monosaccharide metabolism produced by enzymatic hydrolysis to convert poorly absorbed large-molecule phenylethanol glycosides into small-molecule aglycones through hydroxylation and demethylation. Simultaneously, the three strains can also secrete extracellular polysaccharides, short-chain fatty acids, and B vitamins, increasing the polysaccharide content of Cistanche fermentation broth by more than 4 times and the total polyphenol content by more than 2 times, significantly enhancing its nutritional value. This synergistic process design forms a complete closed-loop process of "cell wall breaking and release—carbon source supply—microenvironment regulation—active transformation—product value enhancement," providing a new green technology path for improving the efficacy of Cistanche preparations and promoting its deep processing upgrade.

[0047] On the other hand, the present invention also provides a fermentation device, which is applied to the above-mentioned method for preparing a fermentation liquid of Cistanche deserticola with antioxidant and immunomodulatory effects; Please see Figure 7-14 Step S3: The fermentation equipment includes a fermentation tank 1. A top cover 2 is hinged to the top of the fermentation tank 1. A connecting pipe 23 is fixed to the top of the top cover 2. A first connecting piece 3 is slidably connected to the surface of the connecting pipe 23. A support ring 31 is fixed to the top of the first connecting piece 3. A motor 32 is fixed inside the support ring 31. Adjustment grooves 33 are opened on both sides of the first connecting piece 3. Screws 34 are fixed on both sides of the connecting pipe 23. The screws 34 are slidably connected to the adjustment grooves 33. A gasket 35 is threadedly connected to the surface of the screws 34. The output shaft of motor 32 is fixed with a connecting shaft 4, and a hexagonal prism 41 is fixed at the bottom of the connecting shaft 4. A fixing frame 5 is fixed on the inner wall of fermenter 1. An aeration ring 51 is fixed at one end of the fixing frame 5. A second connecting piece 52 is fixed on the inner wall of the aeration ring 51. A limiting ring 53 is fixed at one end of the second connecting piece 52. A support frame 54 is fixed on the inner wall of the limiting ring 53. A stirring shaft 6 is slidably and sealed inside the limiting ring 53. A snap-fit ​​groove 61 is opened at the top of the stirring shaft 6. A ventilation groove 62 is opened at the bottom of the stirring shaft 6. An aeration pipe 63 is fixed on the surface of the stirring shaft 6. A guide tube 64 is fixed at one end of the aeration pipe 63. A propulsion blade 65 is fixed on the surface of the stirring shaft 6 above the aeration pipe 63. An inclined blade turbine 66 is fixed on the surface of the stirring shaft 6 below the aeration pipe 63. The fermentation equipment also includes a cooling water discharge pipe 11: the cooling water discharge pipe 11 is fixed inside the fermentation tank 1, a sampling pipe 12 is fixed on one side of the fermentation tank 1, a cooling water inlet pipe 13 is fixed below the sampling pipe 12 on the side of the fermentation tank 1, an exhaust pipe 21 is fixed on the top of the top cover 2 near the cooling water discharge pipe 11, a feeding port 22 is fixed on the top of the top cover 2 near the sampling pipe 12, an air inlet pipe 55 is fixed on the side of the fermentation tank 1 near the cooling water discharge pipe 11, and the air inlet pipe 55 is connected to the aeration ring 51 through the fixing frame 5. The fermentation equipment also includes auxiliary blades 7: the auxiliary blades 7 are fixed on the surface of the guide tube 64, the top diameter of the guide tube 64 is smaller than the bottom diameter of the guide tube 64, the propulsion blades 65 are located inside the guide tube 64, the fan blades of the inclined blade turbine 66 are located below the bottom of the guide tube 64, and the hexagonal prism 41 is slidably sealed to the snap-fit ​​groove 61. The fermentation equipment also includes an electrically controlled valve 8: the electrically controlled valve 8 is fixed inside the ventilation trough 62, the top space of the ventilation trough 62 is isolated into a buffer chamber 81 by the electrically controlled valve 8, the surface of the buffer chamber 81 is provided with a limit groove 811, the top of the electrically controlled valve 8 is fixed with a positioning rod 82, the surface of the positioning rod 82 is fixed with an elastic rope 83, one end of the elastic rope 83 is fixed with a third connector 84, the surface of the aeration pipe 63 is slidably connected with a locking ring 85, the bottom of the locking ring 85 is connected and fixed by a fourth connector 86, wherein one locking ring 85 near the third connector 84 is fixed to the third connector 84; During fermentation, the Cistanche deserticola enzymatic hydrolysate and the seed liquid of the fermentation strain are first injected into the fermentation tank 1 through the feeding port 22. Then, the nut on the outside of the screw 34 is rotated to loosen the screw 34, the gasket 35 and the first connecting piece 3. Then, under the action of gravity, the motor 32 drives the support ring 31 and the first connecting piece 3 to descend, so that the screw 34 is adjusted inside the adjustment groove 33. During the descent, the motor 32 drives the connecting shaft 4 and the hexagonal prism 41 to descend synchronously until the hexagonal prism 41 is inserted into the snap-fit ​​groove 61. When the hexagonal prism 41 is fully inserted into the snap-fit ​​groove 61, the motor 32 can no longer descend. At this time, the first connecting piece 3, the adjustment groove 33 and the screw 34 are adjusted to a fixed position. Then, the nut on the outside of the screw 34 is rotated to fix the screw 34 and the first connecting piece 3, thereby fixing the height of the motor 32. Subsequently, the temperature inside the fermenter 1 is adjusted by the controller, and the pH value is adjusted according to the pH meter. Then, the inside of the fermenter 1 is aerated through the controller and the air inlet pipe 55. During the aeration process, the motor 32 is started by the controller. The motor 32 drives the connecting shaft 4 and the hexagonal prism 41 to rotate. The hexagonal prism 41 drives the stirring shaft 6 to rotate inside the limiting ring 53. This is the fermentation operation process of the fermenter 1 in the prior art. Specifically, during the fermentation of Cistanche deserticola hydrolysate, when air is pumped into the air inlet pipe 55, the air enters the aeration ring 51 through the air inlet pipe 55 and the fixed frame 5, and then aeration is carried out on the bottom of the fermentation tank 1 through the aeration ring 51, so that the fermentation liquid can carry out aerobic fermentation. When the motor 32 starts, the motor 32 drives the stirring shaft 6 to rotate synchronously. The stirring shaft 6 then drives the aeration pipe 63, the guide tube 64, the propulsion blade 65, and the inclined blade turbine 66 to rotate synchronously. The propulsion blade 65 generates axial thrust when rotating, which can send the fermentation pressure above the guide tube 64 into the interior of the guide tube 64, forming a main stream from the top to the bottom of the guide tube 64, i.e., downward. When the main stream is pressed to the bottom of the guide tube 64, it diffuses outward from the bottom of the guide tube 64. Subsequently, the radial shear force generated by the rotation of the inclined blade turbine 66 pushes the fermentation liquid diffused from the guide tube 64 toward the tank wall of the fermentation tank 1, causing the fermentation liquid to flow back upward through the annular space formed between the tank wall of the fermentation tank 1 and the guide tube 64, thus forming a cycle. That is, with the guide tube 64 as the boundary, a downward flow stream is formed inside the guide tube 64, and an upward flow stream is formed outside the guide tube 64 and in the middle of the tank wall of the fermentation tank 1, thus forming a strong turbulent circulation of "downward flow from the inner wall of the guide tube 64 + upward flow from the outer wall of the guide tube 64", covering the upper, middle and lower regions inside the tank, eliminating dead corners at the bottom and tank wall; This can further enhance mass transfer efficiency and improve the fermentation conversion efficiency of Cistanche deserticola, allowing for uniform mixing of fermentation liquid, microorganisms, and bubbles. Since the top diameter of the guide tube 64 is smaller than the bottom diameter of the guide tube 64, the resistance to diffusion of the fermentation liquid from the bottom of the guide tube 64 can be reduced. At the same time, the fermentation liquid can be guided to diffuse radially, enhancing the shear interaction with the inclined blade turbine 66. When turbulence is formed, the fermentation liquid can fully contact the oxygen sprayed by the aeration ring 51, further improving the comprehensiveness and efficiency of Cistanche deserticola fermentation. As the aeration pipe 63 rotates, it synchronously drives the guide tube 64 to rotate, causing the guide tube 64 to rotate within the fermentation liquid. This reduces the adhesion of high-viscosity substances in the Cistanche deserticola fermentation liquid to the guide tube 64, further improving the mass transfer efficiency and fermentation efficiency between the Cistanche deserticola fermentation liquid, microbial strains, and bubbles. Furthermore, while radially shearing and pushing the fermentation broth, the inclined blade turbine 66 can break up the bubbles sprayed by the aeration ring 51, preventing the bubbles sprayed by the aeration ring 51 from agglomerating during the upward surge, making the bubbles more dispersed and refined, thereby allowing the bubbles to fully carry out mass transfer fermentation with the fermentation broth, further improving the fermentation efficiency. Based on the aeration of the aeration ring 51, since the interior of the second connecting member 52 is connected to the interior of the limiting ring 53, and the ventilation groove 62 is connected to the interior of the aeration ring 51 through the limiting ring 53, when the air inlet pipe 55 is delivering oxygen, some oxygen sequentially passes through the air inlet pipe 55, the fixing bracket 5, the aeration ring 51, the second connecting member 52, and the limiting ring 53 into the interior of the ventilation groove 62, according to the attached instruction manual. Figure 12 It can be seen that the aeration pipe 63 is connected to the ventilation groove 62, that is, the aeration pipe 63 can be used as a secondary aeration device to perform secondary aeration on the inside of the guide tube 64. Since the aeration pipe 63 is in a rotating state and is set inside the guide tube 64, and the aeration direction of the aeration pipe 63 is opposite to the direction of the main stream inside the guide tube 64, the efficiency of oxygen and fermentation liquid mass transfer inside the guide tube 64 is further increased. Thus, the fermentation liquid below the guide tube 64 and located on the outer wall of the guide tube 64 can be aerated and undergo mass transfer fermentation through the aeration ring 51, and the fermentation liquid in the guide tube 64 can be aerated and undergo mass transfer fermentation through the aeration pipe 63. In this way, with the cooperation of the aeration ring 51 and the aeration pipe 63, the fermentation liquid inside and outside the guide tube 64 can be fully subjected to mass transfer fermentation, thereby improving the aeration and mass transfer range of the present invention. While the guide tube 64 rotates, the guide tube 64 drives the auxiliary blades 7 to rotate synchronously. The tilt direction of the auxiliary blades 7 is consistent with the direction of the inclined blade turbine 66. While the inclined blade turbine 66 pushes the fermentation liquid, it can further increase the backflow speed of the fermentation liquid in the loop flow path between the tank wall of the fermentation tank 1 and the propulsion blades 65. At the same time, the auxiliary blades 7 can break up the bubbles outside the guide tube 64, further reducing the efficiency of bubble aggregation. This allows the bubbles located in the middle of the surface of the guide tube 64 to break up and disperse further, thereby improving the efficiency of aerobic fermentation again. Furthermore, in the actual fermentation process of Cistanche deserticola, since the fermentation strains used in this invention are composed of one or more of Bacillus subtilis, lactic acid bacteria, and Saccharomyces cerevisiae, Bacillus subtilis and Saccharomyces cerevisiae need to undergo aerobic fermentation during the fermentation process, while lactic acid bacteria need to undergo anaerobic fermentation. Therefore, the fermentation of this invention needs to be carried out in stages. In the early stage, aerobic fermentation of Bacillus subtilis and Saccharomyces cerevisiae is carried out, while lactic acid bacteria enter dormancy. In the later stage, anaerobic fermentation is carried out, which activates lactic acid bacteria to produce lactic acid and causes Saccharomyces cerevisiae to switch to anaerobic metabolism to produce active substances. In actual operation, after aerobic fermentation is completed, inert gas can be pumped into the air inlet pipe 55 and the above steps can be repeated so that the inert gas enters the interior of the ventilation trough 62 through the air inlet pipe 55, so that the aeration ring 51 and the aeration pipe 63 work together to spray inert gas to replace the oxygen in the fermentation tank 1, and at the same time, the air is discharged through the exhaust pipe 21. During the filling of inert gas, the electric control valve 8 is activated, allowing the gas to enter the buffer chamber 81 and the limiting groove 811. Under the action of air pressure, the third connector 84 slides outward, which in turn drives the locking ring 85 and the fourth connector 86 to slide along the aeration pipe 63. During the sliding process, the locking ring 85 can clean the air holes of the guide tube 64, thereby reducing the probability that the air holes of the aeration pipe 63 will be blocked by the opposing fermentation liquid, thus improving the efficiency of air replacement in the later stage and improving the efficiency of lactic acid bacteria products. During the above process, the inert gas can be filled intermittently. When the gas pressure inside the electric control valve 8 is insufficient, the third connector 84 can be reset under the action of the elastic rope 83. That is, when the inert gas is filled intermittently, the third connector 84, the locking ring 85 and the fourth connector 86 repeatedly clean the air holes of the aeration pipe 63 under the action of the elastic rope 83 and the gas pressure. During the repeated movement of the locking ring 85, it also guides the bubbles, preventing the bubbles sprayed by the aeration pipe 63 from being too concentrated, thus further expanding the range of mass transfer and fermentation between the bubbles and the fermentation liquid.

[0048] It should be noted that all devices of the present invention are controlled by a controller. The aeration pipe 63 is equipped with a control valve to control the delivery of oxygen and inert gas. At the same time, during the delivery of oxygen, the above-mentioned electric control valve 8 can be started and stopped. That is, during the oxygen aeration process of the aeration pipe 63, the air pressure in the buffer chamber 81 and the elastic rope 83 can also drive the third connecting member 84, the locking ring 85 and the fourth connecting member 86 to perform cleaning operations. Meanwhile, the aeration pipe 63 plays a radial stirring role inside the guide tube 64, while the auxiliary blade 7 plays a radial stirring role outside the guide tube 64, so that the stirring of the fermentation liquid in the fermenter 1 can have both axial and radial stirring at the same time, which further improves the efficiency of mass transfer fermentation. During the fermentation process, sampling can be performed through sampling tube 12 to ensure the progress and quality of Cistanche fermentation. At the same time, cooling water can be introduced into the cooling box outside the fermentation tank 1 through cooling water inlet pipe 13 and discharged to the outside through cooling water outlet pipe 11 to regulate the temperature inside the fermentation tank 1. After the fermentation is completed, the Cistanche fermentation liquid can be taken out from the bottom of the fermentation tank 1.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects, characterized in that, Includes the following steps: S1: Cistanche deserticola is pulverized into powder and prepared into a suspension; S2: Add the enzyme preparation to the suspension, adjust the pH, perform enzymatic hydrolysis, and sterilize to obtain Cistanche deserticola hydrolysate; S3: Add the seed liquid of the fermentation strain to the Cistanche deserticola enzymatic hydrolysate, and ferment it using fermentation equipment to obtain fermented Cistanche deserticola suspension; S4: Cistanche fermentation broth is obtained after sterilization and centrifugation.

2. The preparation method according to claim 1, characterized in that, The particle size of the Cistanche deserticola powder in step S1 is 60-100 mesh, and the suspension is prepared by mixing Cistanche deserticola powder and water at a ratio of 1:10 to 1:20, with a total volume of 300 mL.

3. The preparation method according to claim 1, characterized in that, The enzyme preparation described in step S2 is composed of one or more of cellulase and pectinase, with the amount of cellulase added being 30-40 U / g and the amount of pectinase added being 15-20 U / g.

4. The preparation method according to claim 1, characterized in that, The pH in step S2 is 4.5 to 5.0, which is adjusted by one or more combinations of citric acid and sodium hydroxide.

5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature in step S2 is 35~55 °C, the enzymatic hydrolysis time is 3~32 h, and the sterilization temperature is 121 °C, the sterilization time is 10~30 min.

6. The preparation method according to claim 1, characterized in that, The fermentation strain mentioned in step S3 is composed of one or more of Bacillus subtilis, lactic acid bacteria and brewer's yeast, and the seed liquid of the fermentation strain is added on the first to eighth day of fermentation.

7. The preparation method according to claim 1, characterized in that, The inoculation amount of the fermentation strains in step S3 is 2-5% (v / v), the OD600 value of Bacillus subtilis is 5-6, the OD600 value of lactic acid bacteria is 1-3, and the OD600 value of Saccharomyces cerevisiae is 12-14. The fermentation temperature is 35-40 °C, and the fermentation time is 1-15 days.

8. The preparation method according to claim 1, characterized in that, The sterilization temperature in step S4 is 121 °C, the sterilization time is 10~30 min, the centrifugation speed is 5000~8000 r / min, and the centrifugation time is 10~20 min.

9. A fermented liquid of Cistanche deserticola with antioxidant and immunomodulatory effects, characterized in that, Prepared by the method according to any one of claims 1 to 8.

10. The application of a Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects, comprising using the Cistanche deserticola fermentation broth with antioxidant and immunomodulatory effects as described in claim 9, characterized in that, The fermented Cistanche deserticola liquid is used in dietary supplements, functional foods, and health products.

11. The preparation method according to claim 1, characterized in that: The fermentation equipment in step S3 includes a fermentation tank (1), a top cover (2) is hinged to the top of the fermentation tank (1), a connecting pipe (23) is fixed to the top of the top cover (2), a first connecting piece (3) is slidably connected to the surface of the connecting pipe (23), a support ring (31) is fixed to the top of the first connecting piece (3), and a motor (32) is fixed inside the support ring (31). The first connector (3) has adjustment grooves (33) on both sides, and the connecting pipe (23) has screws (34) fixed on both sides. The screws (34) are slidably connected to the adjustment grooves (33), and the surface of the screws (34) is threaded with a washer (35). The output shaft of the motor (32) is fixed with a connecting shaft (4), and a hexagonal prism (41) is fixed at the bottom of the connecting shaft (4). A fixing frame (5) is fixed on the inner wall of the fermentation tank (1). An aeration ring (51) is fixed at one end of the fixing frame (5). A second connecting piece (52) is fixed on the inner wall of the aeration ring (51). A limit ring (53) is fixed at one end of the second connecting piece (52). A support frame (54) is fixed on the inner wall of the limit ring (53). The limiting ring (53) is internally slidably sealed to a stirring shaft (6). The top of the stirring shaft (6) is provided with a snap-fit ​​groove (61), and the bottom of the stirring shaft (6) is provided with a ventilation groove (62). An aeration pipe (63) is fixed on the surface of the stirring shaft (6). A guide tube (64) is fixed at one end of the aeration pipe (63). A propulsion blade (65) is fixed on the surface of the stirring shaft (6) above the aeration pipe (63), and a slanted blade turbine (66) is fixed on the surface of the stirring shaft (6) below the aeration pipe (63).

12. The preparation method according to claim 11, characterized in that: The fermentation equipment also includes a cooling water discharge pipe (11): the cooling water discharge pipe (11) is fixed inside the fermentation tank (1), a sampling pipe (12) is fixed on one side of the fermentation tank (1), a cooling water inlet pipe (13) is fixed below the sampling pipe (12) on the side of the fermentation tank (1), an exhaust pipe (21) is fixed on the top of the top cover (2) near the cooling water discharge pipe (11), a feeding port (22) is fixed on the top of the top cover (2) near the sampling pipe (12), an air inlet pipe (55) is fixed on the side of the (1) near the cooling water discharge pipe (11), and the air inlet pipe (55) is connected to the aeration ring (51) through a fixing frame (5).

13. The preparation method according to claim 12, characterized in that: The fermentation equipment also includes auxiliary blades (7): the auxiliary blades (7) are fixed on the surface of the guide tube (64), the top diameter of the guide tube (64) is smaller than the bottom diameter of the guide tube (64), the propulsion blades (65) are located inside the guide tube (64), the fan blades of the inclined blade turbine (66) are located below the bottom of the guide tube (64), and the hexagonal prism (41) is slidably sealed to the snap-fit ​​groove (61).

14. The preparation method according to claim 13, characterized in that: The fermentation equipment also includes an electric control valve (8): the electric control valve (8) is fixed inside the ventilation trough (62), the top space of the ventilation trough (62) is isolated into a buffer chamber (81) by the electric control valve (8), a limit groove (811) is opened on the surface of the buffer chamber (81), a positioning rod (82) is fixed on the top of the electric control valve (8), an elastic rope (83) is fixed on the surface of the positioning rod (82), a third connector (84) is fixed at one end of the elastic rope (83), a locking ring (85) is slidably connected to the surface of the aeration pipe (63), and the bottom of the locking ring (85) is connected and fixed by a fourth connector (86), wherein one locking ring (85) near the third connector (84) is fixed to the third connector (84).

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