Coix seed fermented polysaccharide CP1 and its preparation method and use

Abstract

The invention discloses coix seed fermented polysaccharide CP1 and a preparation method and application thereof. Coix seed fermented polysaccharide CP1 is composed of four sugar residues, namely t-Glc(p), 6-Glc(p), 4-Glc(p) and 4,6-Glc(p), with a main chain of α-4-Glc(p)-(1→→4)-α-4-Glc(p)-(1→→4)-β-4,6-Glc(p)-(1→ connected, and a side chain of β-4-Glc(p)-(1→→6 connected. It has a triple helix structure and a spherical structure, and an average molecular weight of 8855 Da. The invention obtains coix seed polysaccharide CP1 by ultracentrifugation and alcohol precipitation of coix seed multi-bacteria fermented broth, and then purifies and freeze-dries the coix seed polysaccharide CP1 by column chromatography separation. Coix seed fermented polysaccharide CP1 has the biological activity of scavenging free radicals and anti-oxidation, and is expected to be developed as an active raw material for application in the fields of food, cosmetics or medicine.

Description

Technical Field

[0001] The present invention relates to the field of biopolymers, and more specifically to coix seed fermented polysaccharide CP1 and a preparation method and application thereof. Background Art

[0002] Coix seed is a kind of medicinal and edible resource with complex chemical composition. It is mainly rich in protein, oil and soluble polysaccharides. In addition, it also contains coix ester, coixol, terpenoid compounds, etc. Coix seed polysaccharides also have a variety of pharmacological effects, including anti-tumor, immunomodulatory, hypoglycemic, antioxidant, etc. The efficacy of coix seed polysaccharides is closely related to its structure. Natural coix seed polysaccharides have problems such as large molecular weight, low solubility, and unexposed active sugar groups, which make them unable to play their active role well. Therefore, coix seed polysaccharides can be appropriately modified by physical, chemical or biological methods to improve their biological activity. Related studies have shown that the soluble polysaccharides of coix seed can be hydrolyzed into a variety of glucans by microbial fermentation, reducing the molecular weight of polysaccharides, changing the chemical structure of polysaccharides, and increasing the diversity of polysaccharides in the fermentation broth.

[0003] Coix seed fermented liquid contains a variety of biologically active ingredients such as organic acids, enzymes, peptidoglycans and nucleotides, which have the advantages of promoting metabolism of the skin, whitening and moisturizing. In addition, coix seed fermented liquid has the advantages of mildness and safety of raw materials, and its macromolecules are fermented to produce small molecular active ingredients, which can greatly improve the absorption and utilization rate of the skin. Therefore, it is very important to develop a coix seed fermented polysaccharide prepared by microbial fermentation technology to overcome the many problems existing in natural coix seed polysaccharides. Summary of the invention

[0004] The first purpose of the present invention is to provide a coix seed fermented polysaccharide CP1 with antioxidant activity, which is a secondary metabolite of coix seed multi-bacteria fermentation. The natural coix seed polysaccharide is modified by microbial fermentation technology to have the advantages of scavenging free radicals and antioxidant effects, which has obvious advantages over natural coix seed polysaccharide.

[0005] The second object of the present invention is to provide a method for preparing coix seed fermented polysaccharide CP1. It includes the process of preparing coix seed fermentation broth and extracting crude polysaccharides from coix seed multi-bacteria fermentation broth and separating and purifying polysaccharide CP1. The method for preparing coix seed multi-bacteria fermentation broth is in accordance with the method disclosed in Example 1 of the specification of the Chinese invention patent with patent application number 2022105295511 and patent name "A coix seed multi-bacteria fermentation broth and its preparation method and application".

[0006] The third object of the present invention is to provide the use of the above-mentioned coix seed fermented polysaccharide CP1 in the preparation of pharmaceutical raw materials with antioxidant functions such as scavenging free radicals.

[0007] In order to achieve the above first object, the present invention adopts the following technical scheme:

[0008] A coix seed fermented polysaccharide CP1, the polysaccharide CP1 mainly consists of four sugar residues, namely, α-4-Glc(p), α-6-Glc(p), β-t-Glc(p), and β-4,6-Glc(p), and its molecular structure is shown in Formula I.

[0009]

[0010] The coix seed fermented polysaccharide CP1 has the following characteristics:

[0011] (1) The main chain connection mode is α-4-Glc(p)-(1→→4)-α-4-Glc(p)-(1→→4)-β-4,6-Glc(p)-(1→; the side chain connection mode is β-4-Glc(p)-(1→→6;

[0012] (2) Homogeneous glucan composed of glucose, with a molecular weight of 8855 Da;

[0013] (3) Possessing characteristic peaks of polysaccharides, such as hydroxyl, carbonyl, methylene, secondary alcohol, ether bond, etc.;

[0014] (4) The structure is a triple helix structure, which appears as a spherical structure under atomic force microscopy.

[0015] In order to achieve the above second purpose, the present invention adopts the following technical solutions:

[0016] The present invention discloses a method for preparing coix seed fermented polysaccharide CP1, comprising extracting crude polysaccharide from coix seed multi-bacteria fermentation broth and separating and purifying polysaccharide CP1, the steps of which are as follows:

[0017] (1) Preparing coix seed multi-bacteria fermentation broth;

[0018] (2) collecting the supernatant of the coix seed multi-bacteria fermentation broth by centrifugation and concentrating under reduced pressure;

[0019] (3) After the polysaccharide is precipitated with alcohol, the protein is removed and the crude polysaccharide is obtained by freeze-drying;

[0020] (4) The purified fractions were collected and combined by Sephacryl S-100 column chromatography, and then concentrated and freeze-dried to obtain coix seed fermentation polysaccharide CP1.

[0021] Preferably, the method for preparing the coix seed fermented polysaccharide CP1 comprises the following steps:

[0022] (1) Preparing coix seed multi-bacteria fermentation broth;

[0023] (2) The coix seed multi-bacteria fermentation broth was centrifuged at 8000 rpm / min for 20 min and the supernatant was collected. After vacuum concentration, 3 volumes of anhydrous ethanol were added and the solution was allowed to stand at 4°C for precipitation. The alcohol-precipitated polysaccharide solution was centrifuged at 8000 rpm / min for 20 min, the collected precipitate was redissolved in ultrapure water, centrifuged and the supernatant was collected. The alcohol precipitation step was repeated 2 to 3 times to obtain a polysaccharide aqueous solution.

[0024] (3) adding trichloroacetic acid to the polysaccharide aqueous solution, stirring in an ice bath to precipitate the protein, collecting the supernatant by centrifugation, and then freeze-drying to obtain coix seed crude polysaccharide;

[0025] (4) Prepare a 5 mg / ml coix seed crude polysaccharide solution, filter it, and separate and purify it using a Sephacryl S-100 column; elute it with ultrapure water, and draw an elution curve based on the absorbance of the sample at 490 nm. Collect the purified components based on the elution curve, and obtain coix seed fermented polysaccharide CP1 by vacuum concentration and freeze-drying.

[0026] The method for preparing coix seed multi-bacteria fermentation broth is in accordance with the method disclosed in Example 1 of the specification of the Chinese invention patent with patent application number 2022105295511 and patent name "A coix seed multi-bacteria fermentation broth and its preparation method and application". Specifically, it includes the following steps:

[0027] (1) Preparation of bacterial strains:

[0028] a. Lactococcus lactis comes from MG1363 of the NTCC Type Culture Collection and is purchased from NIZO of the Netherlands. After the Lactococcus lactis glycerol bacteria are activated by conventional operation on an M17 plate, a monoclonal colony is selected and inoculated into an M17 medium for overnight culture at 30°C for 20 hours; the bacterial solution obtained by overnight culture is inoculated into a high-density culture medium at a ratio of 1% and cultured for 48 hours to obtain a Lactococcus lactis bacterial solution with a concentration of 8-10 g / L; the formula of the high-density culture medium is: 0.51% crystalline trehalose, 1.531% sucrose, 1.479% bacteriological peptone, 4.438% yeast extract powder, 1.9% sodium β-glycerophosphate, 0.05% sodium ascorbate, and 90.072% deionized water, in terms of mass percentage;

[0029] b. Lactobacillus bulgaricus and Streptococcus thermophilus are commercialized by Shandong Zhongke Jiayi Bioengineering Co., Ltd.; wherein, Lactobacillus bulgaricus - 10 billion CFU / g, Streptococcus thermophilus - 10 billion CFU / g;

[0030] (2) Preparation of fermentation substrate:

[0031] The coix seeds are crushed into powder, passed through a 40-mesh sieve, 3 kg is taken, and mixed with 30 L of water at a solid-liquid ratio of 1:10, and then 2% of the substrate mass of CaCl2 is added, heated and boiled for gelatinization for 1 hour to obtain a gelatinized liquid; the obtained gelatinized liquid is cooled to 75-80°C, α-amylase is added, the enzyme usage is 20 U / g coix seed powder, and it is kept warm at 75-80°C for liquefaction for 2 hours to obtain a liquefied liquid; in the liquefied liquid, the pH is adjusted to 4-4.2, and after cooling to 60°C, saccharifying enzyme is added, the enzyme usage is 300 U / g coix seed powder, and it is kept warm at 75-80°C for saccharification for 2 hours to obtain a saccharified liquid; the obtained saccharified liquid is adjusted to pH 6.0, and sterilized at 117°C for 20 minutes to obtain a coix seed fermentation medium;

[0032] (3) Industrial fermentation:

[0033] The coix seed fermentation medium is cooled to 37° C., and the lactococcus lactis liquid, the Lactobacillus bulgaricus powder and the thermophilic Streptococcus powder obtained in step (1) are added to the coix seed fermentation medium obtained in step (2) at a weight ratio of 1%, respectively, and anaerobically fermented in a 50L fermentation tank for 48 hours to obtain a coix seed multi-bacteria fermentation liquid primary product;

[0034] (4) Final product obtained:

[0035] 1 g / L of lysozyme was added to the initial product of the coix seed multi-bacteria fermentation broth obtained above, and the mixture was stirred at 30°C for 2 hours; the product in the collection tank was homogenized 5 times by a homogenizer at a pressure of 500-800 bar, and then centrifuged at 4°C and 18000 rpm / min for 20 minutes to collect the supernatant; the filtrate was filtered through 0.8 μm and 0.22 μm filter membranes in turn, and the obtained filtrate was the coix seed multi-bacteria fermentation broth of the present invention.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The coix seed fermented polysaccharide CP1 of the present invention is a homogeneous polysaccharide composed of glucose, and structural characterization shows that it is a new type of glucan.

[0038] (2) The coix seed fermented polysaccharide CP1 of the present invention is obtained by fermenting coix seed with multiple bacteria, is a secondary metabolite of coix seed fermentation with multiple bacteria, and makes full use of the fermentation product. Compared with natural coix seed polysaccharide, its biological activity is more superior.

[0039] (3) The coix seed fermented polysaccharide CP1 prepared by the present invention has significant free radical scavenging and antioxidant capabilities, and is expected to be developed into an active raw material for biomedicine, thereby promoting the development and application of coix seed resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1This is a process flow chart for the preparation of coix seed fermented polysaccharide CP1.

[0041] Figure 2 This is the elution curve of coix seed fermented polysaccharide CP1 on Sephadex G-100 gel chromatography column.

[0042] Figure 3 This is the HPGPC detection spectrum of coix seed fermentation polysaccharide CP1.

[0043] Figure 4 This is the GC-MS determination spectrum of coix seed fermentation polysaccharide CP1.

[0044] Figure 5 This is the infrared spectroscopic spectrum of coix seed fermented polysaccharide CP1.

[0045] Figure 6 This is the NMR spectrum of coix seed fermentation polysaccharide CP1. Figure 6 A in is the HSQC spectrum; Figure 6 B in the figure is the COSY spectrum; Figure 6 The C in is the HMBC spectrum.

[0046] Figure 7 2D and 3D AFM images of coix seed fermented polysaccharide CP1.

[0047] Figure 8 These are the results of Congo red experimental analysis of coix seed fermented polysaccharide CP1.

[0048] Fig. 9 The study shows that coix seed fermented polysaccharide CP1 has a scavenging effect on DPPH free radicals, ABTS free radicals and hydroxyl free radicals.

[0049] Fig.10 The scavenging effect of coix seed fermented polysaccharide CP1 on ROS in Caenorhabditis elegans. (n=4, Mean±SD, ** P ≤ 0.01,*** P ≤ 0.001)

[0050] Fig.11 The effect of coix seed fermented polysaccharide CP1 on the antioxidant capacity of Caenorhabditis elegans. (n=4, Mean±SD, ** P ≤ 0.01,*** P ≤ 0.001) DETAILED DESCRIPTION

[0051] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modification and replacement of the details and forms of the technical solution of the present invention by those skilled in the art without departing from the spirit and scope of the present invention belongs to the protection scope of the present invention.

[0052] Example 1. Preparation of coix seed fermented polysaccharide CP1

[0053] Reference Figure 1 , preparing coix seed fermented polysaccharide CP1, the specific steps are as follows:

[0054] 1. The preparation of coix seed multi-bacteria fermentation broth comprises the following steps:

[0055] (1) Preparation of strains: a. Lactococcus lactis comes from MG1363 of the NTCC Type Culture Collection and is purchased from NIZO of the Netherlands. After activation of Lactococcus lactis glycerol culture on M17 plates, single clones are selected and inoculated into M17 medium for overnight culture at 30°C for 20 h; the bacterial solution obtained by overnight culture is inoculated into high-density culture medium at a ratio of 1% and cultured for 48 h to obtain a Lactococcus lactis bacterial solution with a concentration of 8-10 g / L; the formula of the high-density culture medium is: crystalline trehalose 0 .51%, sucrose 1.531%, bacteriological peptone 1.479%, yeast extract powder 4.438%, sodium β-glycerophosphate 1.9%, sodium ascorbate 0.05%, deionized water 90.072%, according to mass percentage; b. Lactobacillus bulgaricus and Streptococcus thermophilus are commercialized Lactobacillus bulgaricus powder and Streptococcus thermophilus powder by Shandong Zhongke Jiayi Bioengineering Co., Ltd.; among them, Lactobacillus bulgaricus - 10 billion CFU / g, Streptococcus thermophilus - 10 billion CFU / g;

[0056] (2) Preparation of fermentation substrate: crush coix seeds into powder, pass through a 40-mesh sieve, take 3 kg, mix with 30 L of water at a solid-liquid ratio of 1:10, add 2% of the substrate mass of CaCl2, heat and boil for gelatinization for 1 hour, and obtain a gelatinized liquid; cool the obtained gelatinized liquid to 75-80°C, add α-amylase in an amount of 20 U / g coix seed powder, and keep it at 75-80°C for liquefaction for 2 hours to obtain a liquefied liquid; adjust the pH of the liquefied liquid to 4-4.2, cool it to 60°C, add saccharifying enzyme in an amount of 300 U / g coix seed powder, and keep it at 75-80°C for saccharification for 2 hours to obtain a saccharified liquid; adjust the pH of the obtained saccharified liquid to 6.0, sterilize it at 117°C for 20 minutes, and obtain a coix seed fermentation medium;

[0057] (3) Industrial fermentation: The coix seed fermentation medium is cooled to 37° C., and the lactococcus lactis liquid, Lactobacillus bulgaricus powder and thermophilic Streptococcus powder obtained in step (1) are added to the coix seed fermentation medium obtained in step (2) at a weight ratio of 1%, respectively, and anaerobically fermented in a 50L fermentation tank for 48 hours to obtain a coix seed multi-bacteria fermentation liquid primary product;

[0058] (4) Obtaining the final product: adding 1 g / L of lysozyme to the initial product of the coix seed multi-bacteria fermentation broth obtained above, stirring at 30° C. for 2 h; collecting the product in the tank, homogenizing it 5 times with a homogenizer at a pressure of 500-800 bar, centrifuging it at 4° C. and 18,000 rpm / min for 20 min, and collecting the supernatant; filtering it through 0.8 μm and 0.22 μm filter membranes in turn, and the resulting filtrate is the coix seed multi-bacteria fermentation broth of the present invention.

[0059] 2. The coix seed multi-bacteria fermentation liquid was centrifuged at 8000 rpm / min for 20 min and the supernatant was collected. After vacuum concentration, 3 volumes of anhydrous ethanol were added and allowed to stand at 4°C for 12-18 hours. The alcohol-precipitated polysaccharide solution was centrifuged at 8000 rpm / min for 20 min, the collected precipitate was redissolved in 100 ml of ultrapure water, centrifuged and the supernatant was collected. The alcohol precipitation step was repeated 2-3 times to obtain a polysaccharide aqueous solution.

[0060] 3. Add 5% (m / V) trichloroacetic acid to the polysaccharide aqueous solution, stir under ice bath for 3 h, and collect the supernatant by centrifugation; place the polysaccharide aqueous solution after protein removal in a dialysis bag and dialyze at 4°C for 48 h, and then freeze-dry to obtain coix seed fermentation crude polysaccharide;

[0061] 4. Prepare 5 mg / mL coix seed fermented crude polysaccharide solution, filter with 0.22 μm water filter membrane, and separate and purify with Sephacryl S-100 column. Elute with ultrapure water, and draw an elution curve according to the absorbance of the sample at 490 nm. Collect the purified components according to the elution curve, and obtain coix seed fermented polysaccharide CP1 by reduced pressure concentration and freeze drying.

[0062] The elution curve of coix seed fermented polysaccharide CP1 on Sephacryl S-100 column is as follows Figure 2 As shown in Figure 2, the elution curve is a single main peak; the high performance gel permeation chromatogram (HPGPC) of the purified product is shown in Figure 2 Figure 3 The result shows a single symmetrical peak, indicating that coix seed fermented polysaccharide CP1 is a uniform polysaccharide. The molecular weight of coix seed fermented polysaccharide CP1 measured by HPGPC is 8855 Da.

[0063] Example 2. Chemical structure identification of coix seed fermented polysaccharide CP1

[0064] The chemical structure of the coix seed fermented polysaccharide CP1 prepared in Example 1 was identified by the following tests, as follows:

[0065] (1) Analysis of monosaccharide composition of coix seed fermented polysaccharide CP1

[0066] Guangzhou Analysis and Testing Center was commissioned to use GC-MS (Agilent, 7820A-5977B) to determine the monosaccharide composition of coix seed fermented polysaccharide CP1. The GC-MS analysis results are as follows Figure 4 As shown, the monosaccharide composition of coix seed fermented polysaccharide CP1 was determined to be glucose by comparison with the standard monosaccharide spectrum.

[0067] (2) Fourier transform infrared spectroscopy (FT-IR) analysis of coix seed fermented polysaccharide CP1

[0068] 1.0 mg of coix seed fermented polysaccharide CP1 was mixed with 150 mg of dry KBr powder and ground into powder. The mixture was pressed into tablets using an infrared tablet press and spectrally scanned using an infrared spectrophotometer (Bruker, VERTEX70) with a scanning wavelength range of 400–4000 cm -1 .

[0069] The infrared spectrum of CP1 is as follows Figure 5 As shown: CP1 contains the characteristic infrared absorption peak of glucose: 3415.65 cm -1 is the stretching vibration of hydroxyl (OH), 2927.76 cm -1 is the stretching vibration of the carbon-hydrogen bond (CH), 1654.07 cm -1 is the stretching vibration of carbonyl (C=O), 1420.19 cm -1 is the shear vibration of methylene (-CH), 1370.10 cm -1 is the variable angle vibration of methylene (CH), 1155.36 cm -1 The shear vibration peak of the secondary alcohol group (OH) is 1024.13 cm -1 It is the stretching vibration peak of the ether bond (COC) on the polysaccharide ring, 849.18 cm -1 It is the symmetric and asymmetric stretching vibration absorption peak of the pyranose ring, 576.52- 1 - CH bending vibration.

[0070] (3) Nuclear magnetic resonance (NMR) analysis of coix seed fermented polysaccharide CP1

[0071] 100.0 mg of coix seed fermented polysaccharide CP1 was dissolved in 1 mL of heavy water (D2O), filtered through a 0.22 μm filter membrane, and transferred to a nuclear magnetic resonance tube. The results were detected using a Bruker ascend-600 nuclear magnetic resonance instrument. 1 H NMR,13 C NMR, HSQC, COSY and HMBC spectra, the results are as follows Figure 6 And as shown in Table 1.

[0072] The four groups of H / C signals in the anomeric region identified by HSQC spectrum were 5.31 / 99.77, 5.16 / 91.87, 4.90 / 98.54 and 4.58 / 95.79 ppm, which were respectively attributed to the residual sugars α-4-Glc(p), α-6-Glc(p), β-t-Glc(p) and β-4,6-Glc(p); 1 H- 1 The H COSY spectrum determined the positions of the above four major residual sugars as shown in Table 1; the HMBC spectrum was used to infer the connection mode of the glycosidic bonds between the residual sugars. For example, in the α-4-Glc(p) residual sugar with the highest content, the anomeric hydrogen signals 5.31 / 71.46 and 5.31 / 76.76 were long-range correlations between the anomeric hydrogen of the residual sugar and C2, C5 in the same ring, C4 of another α-4-Glc(p) residue, and C4 of β-4,6-Glc(p), confirming the 1→4 connected glycosidic bonds. The same method was used to determine that the connection mode of the CP1 main chain was α-4-Glc(p)-(1→→4)-α-4-Glc(p)-(1→→4)-β-4,6-Glc(p)-(1→→4), and the connection mode of the side chain was β-4-Glc(p)-(1→→6.

[0073]

[0074] (4) Atomic force microscopy (AFM) analysis of coix seed fermented polysaccharide CP1

[0075] A 10 μg / mL CP1 aqueous solution was dropped onto a mica sheet, dried in an oven, and scanned with an atomic force microscope (Park, Dimension Fastscan) to collect microscopic images. The AFM three-dimensional structure of CP1 is shown in Figure 7 As shown, CP1 is distributed evenly without any height differences, indicating that the surface morphology of CP1 is not obvious and there are some spherical particles or protrusions.

[0076] (5) Congo red analysis of coix seed fermented polysaccharide CP1

[0077] 5.0 mg CP1, 2.0 mL ultrapure water and 2.0 mL 100 μmol / L Congo red reagent were mixed evenly, and 1 mol / L NaOH was gradually added dropwise to make the final concentration of NaOH solution 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mol / L, respectively. The maximum absorption wavelength was scanned in the range of 200-800 nm (UV-visible spectrophotometer Evolutio 350, Thermo Fisher). The results are shown in Figure 2. Figure 8 As shown, by comparing the changing trend of the maximum absorption wavelength of the CP1 solution when the NaOH concentration changes from low to high, it can be judged that CP1 has a triple helix structure.

[0078] Example 3. Antioxidant activity test of coix seed fermented polysaccharide CP1

[0079] (1) Scavenging effect of coix seed fermented polysaccharide CP1 on DPPH free radicals

[0080] 1 mL of anhydrous ethanol and 4 mL of DPPH solution were mixed, incubated at room temperature in the dark for 30 min, and the absorbance of the mixture at 517 nm was measured (UV-visible spectrophotometer Evolutio 350, Thermo Fisher Scientific) and recorded as A0; 1 mL of CP1 solution of different concentrations and 4 mL of DPPH solution were mixed respectively, incubated at room temperature in the dark for 30 min, and the absorbance of the mixture at 517 nm was measured and recorded as A; clearance rate %=1-(A0-A) / A0.

[0081] The results are as follows Fig. 9 As shown, under the experimental concentration conditions, the scavenging rate of CP1 on DPPH free radicals was dose-dependent.

[0082] (2) Scavenging effect of coix seed fermented polysaccharide CP1 on ABTS free radicals

[0083] The ABTS free radical stock solution was diluted with anhydrous ethanol until its absorbance at 734 nm reached 0.70 (denoted as A0). This solution was the ABTS free radical working solution. 1 mL of CP1 solution of different concentrations and 4 mL of ABTS free radical working solution were mixed respectively, and the mixture was allowed to stand at room temperature in the dark for 30 min. The absorbance at 734 nm was measured (UV-visible spectrophotometer Evolutio350, Thermo Fisher Scientific) and denoted as A. The clearance rate (%) = 1-(A0-A) / A0.

[0084] The results are as follows Fig. 9 As shown in the figure, within the experimental concentration range of CP1, the scavenging rate of ABTS free radicals by CP1 was dose-dependent, and when the concentration was 5.0 mg / mL, the scavenging rate could reach 71.30%.

[0085] (3) Scavenging effect of coix seed fermented polysaccharide CP1 on hydroxyl free radicals

[0086] 1 mL of ultrapure water was mixed with 1 mL of 9 mmol / L FeSO4 solution, 9 mmol / L salicylic acid-ethanol solution, and 30% H2O2 solution, and reacted in a 37°C water bath for 30 min. After cooling to room temperature, the absorbance at 510 nm was measured (UV-visible spectrophotometer Evolutio 350, Thermo Fisher) and recorded as A0. 1 mL of CP1 solution of different concentrations was mixed with 1 mL of 9 mmol / LFeSO4 solution, 9 mmol / L salicylic acid-ethanol solution, and 30% H2O2 solution, and reacted in a 37°C water bath for 30 min. After cooling to room temperature, the absorbance at 510 nm was measured and recorded as A; clearance rate % = 1-(A0-A) / A0.

[0087] The results are as follows Fig. 9 As shown in the figure, under the experimental concentration conditions, the scavenging rate of CP1 on hydroxyl free radicals was dose-dependent. When the concentration was 5.0 mg / mL, its scavenging rate reached 68.40%.

[0088] Example 4. Effect of coix seed fermented polysaccharide CP1 on the antioxidant activity of Caenorhabditis elegans

[0089] (1) CP1 clears ROS in Caenorhabditis elegans

[0090] The growth density of Caenorhabditis elegans was adjusted to 5 / 10 μL, and the worm solution and Escherichia coli NA22 were added to a 24-well culture plate at a ratio of 9:1, 1000 μL / well, and cultured at 20°C, 120 rpm / min for about 40 h to the L4 stage; 80 μL NA22, 25 μL 5-FUdR solution and CP1 solution were added to each well, and cultured for about 40 h to the L4 stage; 200 μL 0.5 mmol / L azobisisobutyramidine hydrochloride (AAPH) solution was added to each well of the model group and the drug-treated group, and 200 μL S. Medium culture medium was used for further cultivation for 1 hour; the nematodes were collected after natural sedimentation; the volume was fixed to 500 μL after washing twice with M9 buffer, and the supernatant was obtained after lysis; the supernatant was mixed with DCFH / DA probe at a ratio of 1:1 in a black 96-well plate, and kinetic detection was performed at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, 10 min / time, for a total of 120 times; the Max V value of the sample was divided by the Max V value of the blank control group to obtain the relative ROS level.

[0091] The results are as follows Fig.10 As shown in Table 2, compared with the model group, when C. elegans was treated with CP1, the ROS signal intensity in its body was significantly reduced, indicating that CP1 has the ability to inhibit cell apoptosis caused by AAPS oxidative damage.

[0092] (2) Effect of CP1 on the anti-oxidative stress capacity of Caenorhabditis elegans

[0093] The growth density of Caenorhabditis elegans in the early egg-laying stage was adjusted to 5 / 10 μL, and the worm solution and Escherichia coli NA22 were added to a 24-well culture plate at a ratio of 9:1, 1000 μL / well, and cultured at 20°C, 120 rpm / min for about 40 h to the L4 stage; 80 μL NA22, 25 μL 5-FUdR solution and CP1 solution were added to each well, and the culture was continued for about 40 h to the L4 stage; 200 μL 0.5 mmol / L AAPH solution was added to each well of the model group and the drug-treated group, and 200 μL S. Medium medium was added to the blank control group. After continuing to culture for 4 h, the number of surviving nematodes was recorded.

[0094] The results are as follows Fig.11 As shown in Table 3, compared with the model group, the survival rate of C. elegans in the CP1-treated group was significantly improved, indicating that CP1 can significantly improve the anti-oxidative stress ability of C. elegans.

[0095] .

Claims

1. A coix seed fermented polysaccharide CP1, characterized in that The polysaccharide CP1 is mainly composed of four sugar residues, namely, α-4-Glc(p), α-6-Glc(p), β-t-Glc(p), and β-4,6-Glc(p), and its molecular structure is shown in Formula I, and its molecular weight is 8855 Da; 。 2. The coix seed fermented polysaccharide CP1 according to claim 1, characterized in that It has the following characteristics: (1) Homogeneous glucan composed of glucose; (2) Having characteristic peaks of polysaccharides; (3) The structure is a triple helix structure, which appears as a spherical structure under atomic force microscopy.

3. A method for preparing coix seed fermented polysaccharide CP1 as claimed in claim 1, characterized in that The method comprises extracting crude polysaccharide from coix seed multi-bacteria fermentation broth and separating and purifying polysaccharide CP1, and the steps are as follows: (1) Preparing coix seed multi-bacteria fermentation broth; (2) The coix seed multi-bacteria fermentation broth was centrifuged at 8000 rpm / min for 20 min and the supernatant was collected. After vacuum concentration, 3 volumes of anhydrous ethanol were added and the mixture was allowed to stand at 4°C for precipitation. The alcohol-precipitated polysaccharide solution was centrifuged at 8000 rpm / min for 20 min, the collected precipitate was redissolved in ultrapure water, centrifuged and the supernatant was collected. Repeat the alcohol precipitation step 2 to 3 times to obtain a polysaccharide aqueous solution; (3) adding trichloroacetic acid to the polysaccharide aqueous solution, stirring in an ice bath to precipitate the protein, collecting the supernatant by centrifugation, and then freeze-drying to obtain coix seed crude polysaccharide; (4) A 5 mg / mL coix seed crude polysaccharide solution was prepared, filtered and purified by Sephacryl S-100 column; eluted with ultrapure water, and an elution curve was drawn according to the absorbance of the sample at 490 nm. According to the elution curve, the purified components were collected, and the coix seed fermented polysaccharide CP1 was obtained by vacuum concentration and freeze-drying; The method for preparing coix seed multi-bacteria fermentation broth described in step (1) comprises the following steps: (1) Preparation of bacterial strains: a. Lactococcus lactis comes from MG1363 of the NTCC Type Culture Collection and is purchased from NIZO of the Netherlands. After the Lactococcus lactis glycerol bacteria are activated by conventional operation on an M17 plate, a monoclonal colony is selected and inoculated into an M17 medium for overnight culture at 30°C for 20 hours; the bacterial solution obtained by overnight culture is inoculated into a high-density culture medium at a ratio of 1% and cultured for 48 hours to obtain a Lactococcus lactis bacterial solution with a concentration of 8-10 g / L; the formula of the high-density culture medium is: 0.51% crystalline trehalose, 1.531% sucrose, 1.479% bacteriological peptone, 4.438% yeast extract powder, 1.9% sodium β-glycerophosphate, 0.05% sodium ascorbate, and 90.072% deionized water, in terms of mass percentage; b. Lactobacillus bulgaricus and Streptococcus thermophilus are commercialized by Shandong Zhongke Jiayi Bioengineering Co., Ltd.; wherein, Lactobacillus bulgaricus - 10 billion CFU / g, Streptococcus thermophilus - 10 billion CFU / g; (2) Preparation of fermentation substrate: The coix seeds are crushed into powder, passed through a 40-mesh sieve, 3 kg is taken, and mixed with 30 L of water at a solid-liquid ratio of 1:10, and then 2% of the substrate mass of CaCl2 is added, heated and boiled for gelatinization for 1 hour to obtain a gelatinized liquid; the obtained gelatinized liquid is cooled to 75-80°C, α-amylase is added, the enzyme usage is 20 U / g coix seed powder, and it is kept warm at 75-80°C for liquefaction for 2 hours to obtain a liquefied liquid; in the liquefied liquid, the pH is adjusted to 4-4.2, and after cooling to 60°C, saccharifying enzyme is added, the enzyme usage is 300 U / g coix seed powder, and it is kept warm at 75-80°C for saccharification for 2 hours to obtain a saccharified liquid; the obtained saccharified liquid is adjusted to pH 6.0, and sterilized at 117°C for 20 minutes to obtain a coix seed fermentation medium; (3) Industrial fermentation: The coix seed fermentation medium is cooled to 37° C., and the lactococcus lactis liquid, the Lactobacillus bulgaricus powder and the thermophilic Streptococcus powder obtained in step (1) are added to the coix seed fermentation medium obtained in step (2) at a weight ratio of 1%, respectively, and anaerobically fermented in a 50L fermentation tank for 48 hours to obtain a coix seed multi-bacteria fermentation liquid primary product; (4) Final product obtained: 1 g / L of lysozyme was added to the initial product of the coix seed multi-bacteria fermentation broth obtained above, and the mixture was stirred at 30°C for 2 hours; the product in the collection tank was homogenized 5 times by a homogenizer at a pressure of 500-800 bar, and then centrifuged at 4°C and 18000 rpm / min for 20 minutes to collect the supernatant; the filtrate was filtered through 0.8 μm and 0.22 μm filter membranes in turn, and the obtained filtrate was the coix seed multi-bacteria fermentation broth.

4. Use of the coix seed fermented polysaccharide CP1 according to claim 1 or 2 in the preparation of antioxidant pharmaceutical raw materials.

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