Method for fermenting cistanche deserticola and its application

Abstract

The present invention discloses a method for biofermentation of Cistanche deserticola and its application. The method uses Beauveria bassiana to ferment Cistanche deserticola or crude phenylethanoid glycosides from Cistanche deserticola. By utilizing microbial fermentation technology, the main active ingredients in Cistanche deserticola, such as phenylethanoid glycosides, are bioconverted into small-molecule polyphenols, thereby facilitating the development and application of Cistanche deserticola in health foods and cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a biological fermentation method for Cistanche deserticola and application thereof. Background Art

[0002] Cistanche deserticola is a parasitic plant that lives on the roots of the Haloxylon ammodendron tree in the desert, absorbing nutrients and water from its host. Known as the "Ginseng of the Desert," it possesses exceptional medicinal value and is a precious traditional Chinese medicinal herb. Since the 1980s, extensive research on the components of Cistanche deserticola has been conducted both domestically and internationally, with Japan taking an early lead. Rapid advances in isolation, extraction, and detection technologies have led to the isolation of numerous compounds, including phenylethanoid glycosides, iridoids, lignans, polysaccharides, over a dozen amino acids, and various alkaloids. These compounds are rich in essential trace elements. Total phenylethanoid glycosides are the primary active ingredient in Cistanche deserticola, demonstrating its potential to enhance male sexual function, treat infertility due to uterine coldness, and provide antioxidant, anti-aging, immune, and memory-enhancing properties. While Cistanche deserticola exhibits significant biological activity, its bioavailability is relatively poor. Existing literature indicates that phenylethanoid glycosides are metabolized in the human body into low-molecular-weight polyphenolic compounds such as hydroxytyrosol, caffeic acid, 3-hydroxyphenylpropionic acid, and decaffiened verbascoside, which exert their pharmacological activity. Traditional Chinese medicine fermentation involves the use of microorganisms, under certain environmental conditions (such as humidity and temperature), to modify the original properties of traditional Chinese medicines, enhance their efficacy, or develop new ones, thereby expanding the range of medicinal products and meeting clinical needs. Microbial fermentation of traditional Chinese medicines can significantly improve the utilization rate of their active ingredients. This is because microorganisms produce various extracellular enzymes, such as cellulase and pectinase, during their metabolic process. These enzymes can break down plant cell walls, rupturing the cells and exposing the active ingredients. Furthermore, the various enzymes produced by microbial metabolism can reduce the molecular weight of the active ingredients and remove various macromolecular impurities, enabling better absorption and utilization by the human body.

[0003] There are currently no reports on the fermentation and biotransformation of phenylethanoid glycosides into small-molecule polyphenols, and most studies are on the metabolism of phenylethanoid glycosides by intestinal microorganisms. Summary of the Invention

[0004] The present invention aims to provide a method for bio-fermenting Cistanche deserticola and its application. By utilizing microbial fermentation technology to bio-convert the main active ingredients in Cistanche deserticola, such as phenylethanoid glycosides, into small molecule polyphenols, the method is beneficial to the development and application of Cistanche deserticola in health foods and cosmetics.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0006] The invention discloses a biological fermentation method for Cistanche deserticola, which comprises fermenting Cistanche deserticola or crude phenylethanoid glycosides of Cistanche deserticola by using Beauveria bassiana.

[0007] Furthermore, the Beauveria bassiana is Beauveria bassiana CICC 41021 or Beauveria bassiana CICC 14071.

[0008] Furthermore, the crude phenylethanoid glycosides of Cistanche deserticola are prepared by the following method:

[0009] Take Cistanche deserticola powder, add 70% ethanol, ultrasonically treat for 1 hour, filter, concentrate the filtrate and freeze-dry to obtain crude phenylethanoid glycosides. Preferably, the ratio of Cistanche deserticola powder to 70% ethanol is 200 mL 70% ethanol per 20 g Cistanche deserticola powder.

[0010] Furthermore, the method comprises the following steps:

[0011] Step 1, slant culture: inoculate the original strain of Beauveria bassiana on an agar medium under sterile conditions for cultivation;

[0012] Step 2, primary seed culture: inoculating the cultured bacteria in step 1 into a liquid culture medium for culture to obtain primary seeds;

[0013] Step 3, inoculating the primary seeds into a liquid culture medium for culturing to obtain secondary seeds, and then adding crude phenylethanoid glycosides of Cistanche deserticola for liquid culturing to obtain a fermentation broth;

[0014] Alternatively, the primary seeds are inoculated into a fermentation medium containing Cistanche deserticola powder, and fermented to obtain a fermentation liquid;

[0015] Alternatively, the first-grade seeds are mixed with the sterilized Cistanche deserticola powder, and the mixture is statically cultured to obtain a fermentation product.

[0016] Furthermore, the culture conditions in step 1 are culturing at 30° C. for 3 days; and the culture conditions in step 2 are culturing at 30° C. and 200 r / min on a shaking platform for 2 days.

[0017] Furthermore, in step 3, the first-level seed liquid is inoculated into a Sabouraud glucose liquid medium at a volume ratio of 10%, and the culture is carried out at a stirring speed of 200 r / min at 30°C for 2 days to obtain second-level seeds. 0.5 g of crude phenylethanoid glycosides of Cistanche deserticola is added to every 200 mL of the second-level seed liquid, and the culture is carried out at a stirring speed of 200 r / min at 30°C for 12 days to obtain a fermentation liquid.

[0018] Furthermore, in step 3, the first-level seed liquid is inoculated into a culture medium containing Cistanche deserticola powder at an inoculum volume ratio of 10%, and the culture medium is fermented at 30°C with a stirring speed of 200 r / min for 15 days, and the fermentation liquid is ultrasonically treated. The culture medium containing Cistanche deserticola powder is obtained by adding 5 g of Cistanche deserticola powder to 200 mL of Sabouraud glucose liquid medium and then sterilizing it.

[0019] Furthermore, in step 3, the first-level seeds are mixed with the sterilized Cistanche deserticola powder, and 20 mL of the first-level seed liquid is mixed with 100 g of the sterilized Cistanche deserticola powder, and the mixture is statically cultured. The fermentation product is added with 70% ethanol at a material ratio of 1 g:10 mL and ultrasonically treated.

[0020] The Cistanche deserticola biological fermentation product is prepared by the method.

[0021] Application of the above-mentioned Cistanche deserticola biological fermentation product in the preparation of skin care products.

[0022] The present invention provides a method for preparing a Cistanche deserticola fermentation broth, which has the advantages of simple process, low cost, and easy production. The Cistanche deserticola fermentation broth prepared by the present invention is rich in various pharmaceutical active ingredients such as phenylethanoid glycosides, small molecule polyphenols, and microbial secondary metabolites. The conversion of the pharmaceutical active ingredients is improved under microbial fermentation conditions. The Cistanche deserticola fermentation filtrate obtained by the method is a highly safe and effective cosmetic raw material and health food. The present invention uses Beauveria bassiana to ferment the phenylethanoid glycosides in Cistanche deserticola, converting echinacoside into verbascoside and breaking down some of the verbascoside into compounds such as caffeic acid and decaffeoyl verbascoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC spectrum of the crude phenylethanoid glycosides in the liquid fermentation of Cistanche deserticola by Beauveria bassiana at time 0 in Example 1.

[0024] Figure 2 This is the HPLC spectrum of the crude phenylethanoid glycosides in Cistanche deserticola at the end time of the Beauveria bassiana liquid fermentation in Example 1.

[0025] Figure 3 This is the HPLC spectrum of Cistanche deserticola powder fermented by Beauveria bassiana liquid at time 0 in Example 2.

[0026] Figure 4 This is the HPLC spectrum of the termination time of Beauveria bassiana liquid fermentation of Cistanche deserticola powder in Example 2.

[0027] Figure 5 This is the HPLC spectrum of Cistanche deserticola powder fermented by Beauveria bassiana at time 0 in Example 3.

[0028] Figure 6This is the HPLC spectrum of the termination time of Beauveria bassiana solid fermentation of Cistanche deserticola powder in Example 3.

[0029] Figure 7 This is the HPLC spectrum of the crude phenylethanoid glycosides in Cistanche deserticola fermented by Saccharomyces cerevisiae liquid at time 0 in Example 4.

[0030] Figure 8 This is the HPLC spectrum of the termination time of the liquid fermentation of crude phenylethanoid glycosides in Cistanche deserticola by Saccharomyces cerevisiae in Example 4.

[0031] Figure 9 This is the inhibitory effect of the products before and after fermentation in Example 1 on the IL-1α gene expression in HaCaT stimulated by P. acnes.

[0032] Figure 10 This is the inhibitory effect of the products before and after fermentation in Example 1 on the IL-6 gene expression in HaCaT stimulated by P. acnes.

[0033] Figure 11 This is the inhibitory effect of the products before and after fermentation in Example 1 on insulin-induced SZ95 lipid synthesis. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. Experimental methods and reagents for which specific conditions are not specified in the examples are based on conventional conditions in the art.

[0035] The Sabouraud glucose agar medium used in the following examples was purchased from Beijing Aoboxin Biological (Beijing Aoboxin Biological, 02-359), and the Sabouraud glucose liquid medium was purchased from Beijing Aoboxin Biological (Beijing Aoboxin Biological, 02-358).

[0036] In the following examples, the liquid phase conditions adopted are as follows:

[0037] Chromatographic column: DIKMA Diamonsil C18 (4.6×250 mm, 5 μm); mobile phase: 0.1% formic acid aqueous solution (A) and acetonitrile (B); elution program: 0-15 min, 2%-4% B; 15-25 min, 4%-13% B; 25-30 min, 13%-13% B; 30-35 min, 18%-18% B; 35-50 min, 18%-23%; 50-55 min, 23%-4%; 55-60 min, 4%-4% B; flow rate: 1 mL / min; column temperature: 30°C. Injection volume: 20 μL.

[0038] Example 1 Liquid fermentation of crude phenylethanoid glycosides in Cistanche deserticola by Beauveria bassiana

[0039] 20 g of Cistanche deserticola powder (passed through a 20-mesh sieve) was ultrasonically extracted in 200 mL of 70% ethanol for 1 h, and the crude phenylethanoid glycosides were obtained after spin-drying.

[0040] 1. Slant culture: Aseptically inoculate the original strain of Beauveria bassiana CICC 41021 onto Sabouraud dextrose agar and culture at 30°C for 3 days.

[0041] 2. Primary seed culture: The cultured bacteria in step 1 were inoculated into Sabouraud glucose liquid medium under sterile conditions, and cultured at 30°C and 200 rpm for 2 days to obtain primary seeds. The optical density of the Beauveria bassiana suspension at the end of the culture was OD 600 The value reaches about 0.8;

[0042] 3. Secondary seed culture: Inoculate the primary seeds at a volume ratio of 10% of Sabouraud dextrose liquid medium into a 200 mL conical flask at 30°C with a stirring speed of 200 rpm for 2 days to obtain secondary seeds.

[0043] 4. Add 0.5 g of crude phenylethanoid glycosides from Cistanche deserticola to 200 mL of secondary seed solution, incubate at 30°C with a stirring speed of 200 r / min for 12 days.

[0044] The obtained fermentation broth was analyzed and compared by HPLC. Figure 1 and Figure 2 As shown in FIG, after echinacoside is completely converted into verbascoside, verbascoside is again broken down into caffeic acid and decaffeoyl verbascoside.

[0045] Example 2 Beauveria bassiana liquid fermentation of Cistanche deserticola powder

[0046] 1. Prepare a sterilized fermentation medium by mixing 5 g of Cistanche deserticola powder (passed through a 20-mesh sieve) with 200 mL of Sabouraud dextrose liquid medium and sterilizing it at 121°C and 0.1 MPa for 20 min.

[0047] 2. Slant culture: The original strain of Beauveria bassiana CICC 41021 was aseptically inoculated onto Sabouraud dextrose agar and cultured at 30°C for 3 days.

[0048] 3. Primary seed culture: The cultured bacteria in step 2 were inoculated into Sabouraud glucose liquid medium under sterile conditions, and cultured in a shaking incubator at 30°C and 200 rpm for 2 days to obtain primary seeds. The optical density of the Beauveria bassiana suspension at the end of the culture was OD600 The value reaches about 0.8;

[0049] 4. Inoculate the culture medium containing Cistanche deserticola powder at a volume ratio of 10% of the liquid culture medium, and ferment at 30°C with a stirring speed of 200 r / min for 15 days;

[0050] 5. The culture medium was extracted by ultrasonication for 1 hour.

[0051] The fermentation broth after ultrasonication was analyzed and compared by HPLC. Figure 3 and Figure 4 As shown in FIG, after echinacoside is completely converted into verbascoside, verbascoside is again broken down into caffeic acid and decaffeoyl verbascoside.

[0052] Example 3: Solid-state fermentation of Cistanche deserticola powder by Beauveria bassiana

[0053] 1. Take 100g of Cistanche deserticola powder (pass through an 8-mesh sieve but not exceeding a 20-mesh sieve) and sterilize it at 121°C and 0.1 MPa for 20 minutes.

[0054] 2. Slant culture: Aseptically inoculate the original strain of Beauveria bassiana CICC 14071 onto Sabouraud dextrose agar and culture at 30°C for 3 days.

[0055] 3. Primary seed culture: The cultured bacteria in step 2 were inoculated into Sabouraud glucose liquid medium under sterile conditions, and cultured at 30°C and 200 rpm for 2 days to obtain primary seeds. The optical density of the Beauveria bassiana suspension at the end of the culture was OD 600 The values all reached around 0.8;

[0056] 4. Take 20 mL of the first-level seed solution and mix it with the medicinal material powder components in step 1, and then culture it at 30°C for 15 days.

[0057] 5. Ultrasonicate the solid fermentation product in 70% ethanol at a material ratio of 1 g:10 mL for 1 hour. Dry under reduced pressure and store.

[0058] The obtained fermentation broth was analyzed and compared by HPLC. Figure 5 and Figure 6 As shown, echinacoside was completely converted to verbascoside.

[0059] Example 4 Liquid fermentation of crude phenylethanoid glycosides in Cistanche deserticola by Saccharomyces cerevisiae

[0060] 20 g of Cistanche deserticola powder (passed through a 20-mesh sieve) was ultrasonically extracted in 200 mL of 70% ethanol for 1 h, and the crude phenylethanoid glycosides were obtained after spin-drying.

[0061] 1. Primary seed culture: Saccharomyces cerevisiae (CICC 31622) was cultured in a conical flask containing 200 mL of MEB medium (Beijing Luqiao, CM176) at 200 rpm and 30°C to prepare the primary seed. The optical density of the MEB suspension at the end of the culture was OD 600 The values all reach around 1.

[0062] 2. Take 20 mL of the first-grade seed solution and place it in a conical flask containing 200 mL of MEB medium. Incubate it with a shaker at 200 rpm for 2 days. Add 0.5 g of crude phenylethanoid glycosides from Cistanche deserticola and incubate it with a shaker at 30°C and 200 rpm for 12 days.

[0063] The fermentation broth prepared in Example 4 was analyzed and compared by HPLC. The results are shown in Figure 7 and Figure 8 , its composition has not changed significantly.

[0064] Example 5 Determination of the anti-inflammatory efficacy of the product of Example 1 before and after fermentation

[0065] RT-RCR was used to detect the inhibitory effect of the Beauveria bassiana fermentation broth of the present invention on the pro-inflammatory factors IL-1α and IL-6 in the inflammatory response.

[0066] 1. Preparation of a heat-killed P. acnes suspension: Resuscitate freeze-dried P. acnes ATCC 6919 cells and dissolve them in 0.1 mL of sterile water. Once completely dissolved, streak four colonies onto 1.5% RCM agar. Place the plate in an anaerobic bag and incubate at 37°C for 7 days. Pick a single colony from the streaked RCM plate and inoculate it into a culture tube containing 5 mL of RCM liquid medium. Place the culture tube in an anaerobic environment and incubate at 37°C for 7 days. Collect the cultured cells, inactivate them at 100°C for 45 minutes, centrifuge at 12,000 g for 10 minutes, and discard the supernatant. Resuspend the cells in sterile PBS and centrifuge at 12,000 g for 10 minutes. Discard the supernatant. Resuspend the cells in sterile PBS. Repeat the centrifugation and resuspension three times to wash the cells. Collect the inactivated cells and confirm complete inactivation by spot-plating them on RCM agar plates.

[0067] 2. After HaCaT cells were cultured to the logarithmic phase, they were digested and inoculated. When the cells reached 70-80% fusion rate, the supernatant was discarded and the cells were gently washed twice with PBS. The cells were plated on a six-well plate at a CFU of 1.5x10 6 2 mL of HacaT cells were cultured for 24 h.

[0068] 3. Add 2 mL of the prepared DMEM suspension containing Propionibacterium acnes (CFU of 1.5x10 8The cells were cultured for a period of time and RNA was extracted.

[0069] 4. Perform fluorescence quantitative PCR to detect the gene expression of inflammatory factors IL-1a and IL-6.

[0070] PHG is the crude product of phenylethanoid glycosides before fermentation by Beauveria bassiana in Example 1, and BJJF is the crude product of phenylethanoid glycosides after fermentation by Beauveria bassiana in Example 1 (the same below). Figure 9 and Figure 10 In contrast, it was found that BJJF could inhibit the gene expression of IL-1a and IL-6 in stratum corneum cells induced by Propionibacterium acnes, showing better anti-inflammatory efficacy.

[0071] Example 6 The in vitro oil control efficacy of the Beauveria bassiana fermented Cistanche deserticola obtained in Example 1 was measured

[0072] 1. In a six-well plate, add 2 mL of culture medium to each well and seed approximately 200,000 sebaceous gland cells per well. Set up three parallel wells for each drug concentration. Use medium containing 2% DMSO and no drug as a blank control, and label it accordingly.

[0073] 2. After culturing at 37°C and 5% CO2 for 24 hours, the cells were washed twice with PBS and culture medium containing 1%, 0.5%, and 0.25% of the fermentation product of Beauveria bassiana was added, along with 20 μg / mL of insulin as a modeling agent.

[0074] 3. After 48 hours of continued culture, wash twice with PBS, add 10 μL of Nile Red fluorescent storage dye solution to a final concentration of 100 ng / mL, and incubate at room temperature for 15 minutes.

[0075] 4. Add 0.5 mL of 0.25% trypsin containing 0.02% EDTA and digest for 5 minutes;

[0076] 5. Add 0.5 mL of culture medium containing 10% calf serum to terminate digestion and aspirate the culture medium containing cells into a 1.5 mL eppendorf tube;

[0077] Centrifuge at 800-1000 rpm for 5 minutes, carefully discard the supernatant, add 0.5 mL of DPBS, and gently pipette to wash the cells.

[0078] 7. After passing through a 70-mesh cell sieve, the cells were placed on a flow cytometer and the average fluorescence intensity of each cell in 10,000 cells in each specimen was measured using an excitation wavelength of 485 nm and an emission wavelength of 565 nm (i.e., the fluorescence wavelength was PE).

[0079] pass Figure 11 By comparison, it was found that the fermented product in Example 1 can stimulate the secretion of oil in sebaceous gland cells induced by insulin, showing better oil control effect.

Claims

1. A method for biological fermentation of Cistanche deserticola, characterized in that: Beauveria bassiana ( Beauveria bassiana ) fermenting Cistanche deserticola or crude phenylethanoid glycosides of Cistanche deserticola; The Beauveria bassiana ( Beauveria bassiana ) is Beauveria bassiana ( Beauveria bassiana ) CICC 41021 or Beauveria bassiana ( Beauveria bassiana )CICC 14071.

2. The method according to claim 1, wherein: The preparation process of the crude phenylethanoid glycosides of Cistanche deserticola comprises the following steps: taking Cistanche deserticola powder, adding 70% ethanol, adding 200 mL of 70% ethanol to every 20 g of Cistanche deserticola powder, ultrasonically treating for 1 h, filtering, concentrating the filtrate, and freeze-drying to obtain the crude phenylethanoid glycosides.

3. The method according to claim 1, wherein: The method comprises the following steps: Step 1, slant culture: Beauveria bassiana ( Beauveria bassiana ) The original strain was inoculated on Sabouraud dextrose agar medium under sterile conditions for cultivation; Step 2, primary seed culture: inoculating the cultured bacteria in step 1 into Sabouraud glucose liquid medium for culture to obtain primary seeds; Step 3, inoculating the primary seeds into Sabouraud glucose liquid medium for cultivation to obtain secondary seeds, which are then added to the crude phenylethanoid glycosides of Cistanche deserticola for liquid culture to obtain a fermentation broth; Alternatively, the primary seeds are inoculated into a culture medium containing Cistanche deserticola powder, fermented, and the fermentation liquid is subjected to ultrasonic treatment; Alternatively, the first-grade seeds are mixed with sterilized Cistanche deserticola powder, and the mixture is statically cultured. The fermentation product is then added with 70% ethanol and ultrasonically treated.

4. The method according to claim 3, wherein: The culture conditions in step 1 are 30°C for 3 days; the culture conditions in step 2 are 30°C and 200 r / min shaking for 2 days.

5. The method according to claim 3, wherein: In step 3, specifically, the first-level seed liquid is inoculated into Sabouraud glucose liquid medium at a volume ratio of 10%, and the culture is carried out at a stirring speed of 200 r / min at 30°C for 2 days to obtain second-level seeds. 0.5 g of crude phenylethanoid glycosides of Cistanche deserticola is added to every 200 mL of second-level seed liquid, and the culture is carried out at a stirring speed of 200 r / min at 30°C for 12 days to obtain a fermentation liquid.

6. The method according to claim 3, wherein: In step 3, the first-level seed liquid is inoculated into a culture medium containing Cistanche deserticola powder at an inoculum volume ratio of 10%, and the culture medium is stirred at 200 r / min at 30°C for 15 days. The fermentation liquid is ultrasonically treated, and the culture medium containing Cistanche deserticola powder is obtained by adding 5 g of Cistanche deserticola powder to 200 mL of Sabouraud glucose liquid medium and sterilizing it.

7. The method according to claim 3, wherein: In step 3, specifically, after the first-level seeds are mixed with the sterilized Cistanche deserticola powder, every 20 mL of the first-level seed liquid is mixed with 100 g of the sterilized Cistanche deserticola powder, and the mixture is statically cultured. The fermentation product is added with 70% ethanol at a material ratio of 1 g:10 mL and ultrasonically treated.

8. The Cistanche deserticola biofermentation product prepared by the method according to any one of claims 1 to 7.

9. Use of the Cistanche deserticola biofermentation product according to claim 8 in the preparation of skin care products.

Images