A method for extracting rape pollen polysaccharide and an extracted product

Abstract

The present application belongs to the field of traditional Chinese medicine microbial technology, and particularly relates to a method for increasing the content of rapeseed pollen polysaccharide. The method is to increase the content of rapeseed pollen polysaccharide through microbial fermentation, and the microorganism is selected from plant endophytic bacteria and known microbial flora. The microorganism can be a single flora or a mixed flora. The microbial fermentation method can significantly increase the content of plant medicine polysaccharide, create a product probiotic environment, improve the pharmacological activity, and can be widely used in the deep fermentation production of plant medicines.

Description

TECHNICAL FIELD

[0001] The patent belongs to the field of Chinese herbal medicine microbial technology application, and particularly relates to a method for increasing the content of rape pollen polysaccharide. BACKGROUND

[0002] Rape pollen contains rich functional substances (including proteins, vitamins, trace elements, flavonoids), which synergistically act on the body, regulate various functions of the body, balance the body's nutrition, enhance metabolism, and prevent capillary permeability disorders. Pollen can also promote the development of endocrine glands, improve and regulate the secretion function of endocrine glands, and therefore has a therapeutic effect on diseases caused by endocrine function disorders.

[0003] Rape pollen is beneficial to the kidney, solid foundation, and strengthens the waist, and has a high content of flavonols, and has the effects of anti-atherosclerosis, treatment of varicose ulcer, reduction of cholesterol, and anti-radiation. The health care effect, pharmacological effect and efficacy of pollen on human beings. The results show that pollen can prevent and treat cerebrovascular diseases, lower blood lipids, regulate the nervous system, promote sleep, regulate the function of the gastrointestinal system, promote digestion, and treat habitual constipation; it has certain effects on regulating endocrine, improving the immune function of the body, anti-aging, improving sexual function, and treating male infertility. It also has obvious functions of preventing and inhibiting cancer, protecting liver, preventing anemia, diabetes, and anti-fatigue. In addition, the beauty effect of bee pollen is obvious, and its mechanism lies in the overall regulation of the balance of the human endocrine system, from the inside to the outside, fundamentally improving the vitality of skin cells, enhancing the metabolic function of the skin, preventing facial pigmentation, rough skin, and aging, keeping the skin moist, white, shiny, and elastic.

[0004] Polysaccharide is a substance widely existing in organisms, and is a natural high-molecular polymer connected by glycosidic bonds from aldehyde sugar or ketose. It is an important biological macromolecule in organisms and is one of the basic substances for maintaining normal operation of life activities. Plant polysaccharide, also known as plant polysaccharide, is a polysaccharide with a degree of polymerization of more than 10 produced by plant cell metabolism.

[0005] Nowadays, plant polysaccharide research is increasingly attracting attention, and the international scientific community even proposes that the 21st century is the century of polysaccharide. Scientific experimental research shows that many plant polysaccharides have biological and pharmacological activities. It can not only be used as a broad-spectrum immune promoter to regulate the immune function of the body, but also can play a wide range of pharmacological effects in anti-tumor, anti-virus, anti-oxidation, blood sugar reduction, and anti-radiation. Polysaccharide has attracted widespread attention in the medical field due to its wide source, strong pharmacological activity, low cytotoxicity, strong safety, and small toxic and side effects, and has become one of the hotspots of life science research today.

[0006] Glycoprotein is a kind of binding protein, wherein the non-protein part is sugar and is connected with the protein part by covalent bond, and the connected sugar is monosaccharide or relatively short oligosaccharide. Generally, the sugar content of each molecule is less (about 4%). Some glycoproteins only contain one or several glycosyls, and others contain multiple linear or branched oligosaccharide side chains. Glycoproteins are usually secreted into body fluids or membrane proteins, which are located outside the cell and have corresponding functions. Glycoproteins include enzymes, hormones, carriers, agglutinins, antibodies, etc.

[0007] So far, more than 300 polysaccharide compounds have been isolated from natural products. At present, the extraction methods of plant polysaccharides mainly include hot water extraction method, alkali extraction method, enzymatic extraction method, microwave assisted method and ultrasonic method. Among them, the enzyme method is a method combining enzyme and hot water extraction, and the enzyme is mainly a certain amount of pectinase, cellulase and neutral protease. The main methods include complex enzyme method, separate enzyme method and single enzyme method. The enzyme method for extracting plant polysaccharides has many advantages such as mild reaction condition, high efficiency, easy impurity removal, simple process and time saving, and the patent greatly improves the content of polysaccharides on the basis of enzyme extraction by adding microbial fermentation.

[0008] Endophyte is a kind of microorganism that lives in the tissues and organs of healthy plants at some stage or all stages, and can form parasitic, symbiotic, saprophytic and other relationships with plants. Research has found that endophytes can be isolated from various plants, and endophytes have been found in roots, stems, leaves, flowers, fruits and seeds of the same plant. Endophytes are numerous and diverse, including endophytic fungi, endophytic bacteria and endophytic actinomycetes. The endophyte of a plant usually contains several to dozens of endophytic fungi or bacteria, and even up to several hundred. SUMMARY

[0009] Rape pollen polysaccharide is complex in structure and various in types, and the extraction rate of plant polysaccharide is low by using conventional extraction method, and the cost is high. In view of the defects of the prior art, the present application provides a rape pollen polysaccharide extraction method, which aims to improve the content of rape pollen polysaccharide by using the special microbial strain fermentation method of the present application, and increase the drug activity.

[0010] The second object of the present application is to provide a fermentation broth obtained by the extraction method, which contains rich polysaccharide, glycoprotein and the probiotic environment of the microorganism of the present application, and has good medicinal effect.

[0011] A rape pollen polysaccharide extraction method, which is subjected to microbial fermentation treatment to obtain a fermentation broth containing polysaccharide;

[0012] The microorganism comprises at least one of the (Parengyodontium SP.) MD313901 strain (also referred to as MD313901 in the present application) and / or the (Purpureocillium SP.) MD313902 strain (also referred to as MD313902 in the present application), and further comprises at least one of a yeast and a lactic acid bacteria;

[0013] Alternatively, the microorganism comprises at least one yeast and at least one lactic acid bacteria;

[0014] The (Parengyodontium SP.) MD313901 strain has a preservation number of CCTCC M 2018256;

[0015] The (Purpureocillium SP.) MD313902 strain has a preservation number of CCTCC M 2018257.

[0016] The present inventors have found that the fermentation of rapeseed pollen with the microorganism strain according to the present application can significantly improve the polysaccharide extraction rate of rapeseed pollen, increase the polysaccharide content of rapeseed pollen, and create a product probiotic environment and improve the pharmacological activity of rapeseed pollen.

[0017] The (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain according to the present application are both plant endophytic fungi, which are preserved in the China Center for Type Culture Collection (CCTCC) in Wuhan on May 8, 2018, and have preservation numbers of CCTCC M 2018256 and CCTCC M 2018257, respectively.

[0018] The present inventors have further found that the fermentation with a single strain of MD313901 or MD313902 can effectively improve the polysaccharide extraction rate of rapeseed pollen. Furthermore, the combination of MD313901 and MD313902, or the further combination of yeast and lactic acid bacteria, can further synergistically improve the polysaccharide extraction rate of rapeseed pollen. In addition, the present inventors have found that the fermentation of rapeseed pollen with a microbial flora comprising yeast and lactic acid bacteria can also achieve the purpose of synergistically improving the polysaccharide extraction rate of rapeseed pollen.

[0019] As a preferred embodiment, the microorganism comprises a microbial flora of at least two of the (Parengyodontium SP.) MD313901 strain, the (Purpureocillium SP.) MD313902 strain, yeast and lactic acid bacteria. The use of a microbial flora of two or more preferred strains can synergistically improve the polysaccharide extraction rate of rapeseed pollen.

[0020] Further preferably, the microorganism comprises at least one of the (Parengyodontium SP.) MD313901 strain and / or the (Purpureocillium SP.) MD313902 strain, and further comprises a yeast and / or a lactic acid bacteria. The present application has found that the MD313901 strain and / or the MD313902 strain and the yeast and / or the lactic acid bacteria have a synergistic effect in improving the extraction rate of rapeseed pollen polysaccharide. By using the plant endophytic fungi and the yeast and / or the lactic acid bacteria according to the present application, the extraction rate of rapeseed pollen polysaccharide can be further improved.

[0021] Still further preferably, the microorganism comprises the (Parengyodontium SP.) MD313901 strain, at least one yeast and at least one lactic acid bacteria.

[0022] Alternatively, the microorganism is the (Parengyodontium SP.) MD313901 strain, the (Purpureocillium SP.) MD313902 strain, at least one yeast and at least one lactic acid bacteria.

[0023] Alternatively, the microorganism is at least two kinds of yeast and at least two kinds of lactic acid bacteria.

[0024] Alternatively, the microorganism is the (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain. It has been found that the combination of the two strains according to the present application can surprisingly improve the extraction rate of polysaccharide.

[0025] The preferred microorganism has a better synergistic effect, which can further improve the extraction rate of rapeseed pollen polysaccharide.

[0026] As a preferred, the yeast used is one or more of Candida utilis, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Saccharomyces boulardii, and further preferably is Candida utilis and / or Kluyveromyces lactis.

[0027] As a preferred, the lactic acid bacteria used is one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Leuconostoc mesenteroides, Streptococcus lactis, Bacillus stearothermophilus, Bacillus calidolacticus, Bacillus thermolacticus, Bacillus licheniformis, Bacillus mollis, Bacillus polymyxa, and Lactobacillus rhamnosus, and further preferably is at least one of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus rhamnosus.

[0028] More preferably, the microorganism is a strain of (Parengyodontium SP.) MD313901, a strain of (Purpureocillium SP.) MD313902, Candida utilis, Kluyveromyces lactis, Lactobacillus, Bifidobacterium, and a consortium of at least two of Lactobacillus rhamnosus. It has been found that the use of the preferred two or more microorganisms for fermentation of rapeseed pollen can surprisingly further improve the extraction rate of rapeseed pollen polysaccharides.

[0029] Most preferably, the microorganism is a strain of (Parengyodontium SP.) MD313901, Bifidobacterium, and Kluyveromyces lactis; or a strain of (Parengyodontium SP.) MD313901, a strain of (Purpureocillium SP.) MD313902, Kluyveromyces lactis, and Lactobacillus rhamnosus; or Candida utilis, Kluyveromyces lactis, Lactobacillus, and Lactobacillus rhamnosus.

[0030] Preferably, the rapeseed pollen for microbial fermentation is pre-treated by cell wall breaking.

[0031] Further preferably, the cell wall breaking pretreatment is biological enzyme hydrolysis.

[0032] It has been found in the present application that the use of biological enzymes for cell wall breaking of rapeseed pollen followed by fermentation with the microbial strains described in the present application can further effectively promote the content of rapeseed pollen polysaccharides and significantly improve the extraction rate of polysaccharides.

[0033] In the present application, biological enzyme treatment and microbial strain fermentation are combined, biological enzyme treatment is performed first to promote rapid release of effective components, and then microbial strain fermentation is performed to increase the content of plant polysaccharides.

[0034] In the present application, the biological enzyme hydrolysis process is as follows: the rapeseed pollen to be treated is washed, homogenized, and subjected to cell wall breaking by adding microbial enzymes.

[0035] Preferably, the biological enzyme is at least one of pectinase, cellulase, hemicellulase, and papain.

[0036] Preferably, in the enzyme hydrolysis process, the amount of biological enzyme used is 0.01% to 5% by weight of the rapeseed pollen; preferably 2 to 5%.

[0037] Further preferably, the biological enzyme is pectinase and cellulase, wherein the amount of pectinase used is 0.1% to 5% by weight of the rapeseed pollen; and the amount of cellulase used is 0.1% to 5% by weight of the rapeseed pollen.

[0038] The biological enzyme treatment is carried out under suitable temperature and pH conditions.

[0039] The pH of the preferred enzymolysis process is 4-8.

[0040] The temperature of the preferred enzymolysis process is 30-60℃.

[0041] The enzymolysis time is preferably 2-8 hours.

[0042] After the enzymolysis is completed, the enzymolysis system does not need to be separated, and the innovative strain of the present application is directly inoculated and sterilized for fermentation.

[0043] The sterilization method can use the existing method. For example, steam sterilization is used, and the time is, for example, 30-60 minutes.

[0044] In the method of the present application, the broken wall system is fermented by the strain or strain group described in the present application, and some components other than polysaccharides in the broken wall system can be further converted into polysaccharides, so as to further increase the content of polysaccharides in the fermentation liquor and improve the extraction rate of polysaccharides.

[0045] In addition to the use of the innovative strain of the present application, further cooperation with the broken wall pretreatment of the rape pollen and the control of the fermentation parameters can further improve the fermentation effect and further improve the extraction rate of polysaccharides.

[0046] As preferred, the inoculation amount of the microorganism in the fermentation process is 0.01-20% of the total raw material (referring to the weight of the rape pollen); further preferably 1-10%; and more preferably 4-8%. Under the preferred inoculation range, the fermentation period can be effectively shortened, and in addition, the strain growth can be prevented from being too fast, the rapid aging and death of the strain can be reduced, and the polysaccharide content of the fermentation product can be improved.

[0047] As preferred, the fermentation temperature is 10-50℃. Controlled in the preferred range, the fermentation effect is better, and not controlled in the preferred range, which is not conducive to the extraction rate of polysaccharides.

[0048] Further preferably, the fermentation temperature is 25-30℃.

[0049] As preferred, the fermentation time is 48h and above; preferably 5-30 days.

[0050] The fermentation process is carried out in a closed container.

[0051] The fermentation mode is preferably static fermentation.

[0052] In the present application, the existing method can be used to improve polysaccharides and the required components from the fermentation liquor.

[0053] As preferred, the rape pollen polysaccharides are extracted from the fermentation liquor;

[0054] Preferably, the extraction process comprises water extraction and alcohol extraction.

[0055] The water extraction is carried out by extracting the fermentation components with water at a temperature of 40-90°C.

[0056] The alcohol extraction is carried out by adding alcohol to the water extract, controlling the alcohol content in the solution system to be 60-90%, and then carrying out alcohol extraction.

[0057] After the water extraction treatment, free proteins in the water extraction product are removed, and then alcohol extraction is carried out.

[0058] A preferred fermentation treatment method of rapeseed pollen is achieved by the following steps:

[0059] (1) Select high-quality rapeseed pollen with impurities removed. Put it into a fermentation tank, add 0.01%-5% biological enzyme, control the enzymolysis temperature to be 30-60°C, add edible acid or edible alkali to adjust the PH value to be 4.0-8.0, and enzymolysis for 2-8 hours. After the enzymolysis is completed, steam sterilization is carried out for 30-60 minutes;

[0060] (2) After the enzymolysis solution is cooled to room temperature, 0.01%-20% of microbial strains by weight of the raw material are added, the stirring speed of the fermentation tank is controlled to be 50-300 rpm, the fermentation temperature is controlled to be 15-50°C, and closed fermentation is carried out for 5-30 days.

[0061] (3) Collect the fermentation liquid, and extract, separate and purify the fermentation liquid to obtain rapeseed pollen glycoprotein and rapeseed pollen polysaccharide.

[0062] Preferably, the fermentation liquid is subjected to water extraction, alcohol precipitation and separation to obtain rapeseed pollen polysaccharide.

[0063] The present application also discloses a fermentation liquid (fermentation product) obtained by the extraction method.

[0064] Preferably, the fermentation liquid contains rapeseed polysaccharide and the microorganism.

[0065] Further preferably, the fermentation liquid further contains glycoprotein.

[0066] The fermentation liquid after fermentation of the present application is subjected to water extraction, alcohol precipitation and separation to obtain rapeseed pollen glycoprotein, which is also an effective active ingredient to be protected by the present patent.

[0067] Advantages

[0068] The application provides a brand new (Parengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain; and it is found innovatively that the brand new strain is helpful for improving the extraction rate of rape pollen polysaccharide and glycoprotein. In addition, it is found by the application that the (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain have a synergistic effect when used in combination, and are helpful for further improving the extraction rate of polysaccharide in rape pollen.

[0069] The microbial strains are plant endophytic bacteria and known microbial flora. The (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain and yeast bacteria and lactic acid bacteria have a good synergistic effect; the fermentation product can further contain rich polysaccharide and glycoprotein, and at the same time, a product probiotic environment can be created, and the pharmacological activity of a plant medicine is improved. DETAILED DESCRIPTION

[0070] The following examples are specific descriptions of the application, which are intended to further illustrate the application, and cannot be understood as limiting the protection scope of the application, and the improvements and adjustments made by the person skilled in the art based on the application also belong to the protected scope.

[0071] Isolation of (Parengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain

[0072] Preparation Example 1

[0073] The preparation method of the plant endophytic flora is realized through the following steps:

[0074] (1) Plant tissue pretreatment

[0075] The plant tissue (different origin and different variety of polygonatum officinale plant medicinal materials shown in Table 1) is washed with tap water and then washed with sterile water, and after the surface moisture is dried, it is cut into segments with a length of 0.5-2.0 cm, and then the cut material is placed in 70% volume concentration ethanol for 2 min of disinfection, washed with sterile water, soaked in 3% mass concentration sodium hypochlorite for 10 min, and then washed with sterile water, and after the surface moisture is dried, it is ready for use;

[0076] (2) Plant tissue endophytic bacteria leaching

[0077] The sterilized polygonatum officinale tissue is placed in a sterilized mortar, and biological enzyme (cellulase, 5% by weight) is added for grinding. The grinding liquid is transferred into a conical flask, and is oscillated at 200 rpm at 25 DEG C for 2 hours to obtain supernatant and plant residues.

[0078] (3) Isolation and purification of plant endophytic strains

[0079] The supernatant obtained in the leaching process is diluted 10 times with sterile water under sterile conditions, and 200 μL of each is spread on MS medium for culture. After a period of culture to form colonies, different morphological colonies are selected, fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium. The streaking method is used for purification.

[0080] Different origin and different variety of polygonatum officinale medicinal materials are selected, and plant endophytic bacteria are isolated, as shown in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] The results in Table 1 show that the two strains can be obtained by using the separation method of the patent from polygonatum officinale of different origins and different varieties.

[0085] The total raw material weight in the following cases refers to the weight of rapeseed pollen, unless otherwise specified.

[0086] The 16sRNA fragment of the MD313901 strain of the application is analyzed, has a fungal specific ITS2 fragment, and has 99% homology with Parengyodontium SP. It is named MD313901, and the (Parengyodontium SP.) MD313901 strain has been preserved in the China Center for Type Culture Collection on May 8, 2018, with the preservation number CCTCC M2018256.

[0087] The 16sRNA fragment of the MD313902 strain of the application is analyzed, has a fungal specific ITS2 fragment, and has 99% homology with Purpureocillium SP. It is named MD313902, and the (Purpureocillium SP.) MD313902 strain has been preserved in the China Center for Type Culture Collection on May 8, 2018, with the preservation number CCTCC M2018257.

[0088] Example of increasing the content of rapeseed pollen polysaccharide by microbial strain fermentation:

[0089] Example 1

[0090] 1. The breaking of the rapeseed pollen wall:

[0091] Take 10 kg of rapeseed pollen, remove impurities and pour into a fermentation tank, add purified water to cover the medicinal materials, add 1% cellulase (total raw material mass fraction) and 1% hemicellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.7, control the temperature at 46°C, and enzymolysis for 2 hours, then sterilize by steam for 30 min.

[0092] 2. Fermentation:

[0093] After cooling to room temperature, add 4% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain, and ferment at 28°C for 30 days. Filter and collect the medicinal liquid for processing.

[0094] 3. Extraction and content determination of rapeseed pollen glycoprotein

[0095] Add 8 times the amount of purified water to the collected medicinal liquid after fermentation, heat at 100°C for 2 times, 3 hours each time, filter, obtain the extract, concentrate to an appropriate concentration, remove free proteins by Sevag method, then add anhydrous ethanol to make the solution concentration reach 80%, centrifugal precipitate twice, dry the precipitate to obtain rapeseed pollen total polysaccharide.

[0096] The determination of rapeseed pollen polysaccharide content by phenol-sulfuric acid method showed that the extraction rate of rapeseed pollen polysaccharide after fermentation was 14.0%, which was 1.84 times higher than that of the case without the fermentation step 2 (after the breaking step 1, directly proceed to the extraction step 3).

[0097] Example 2

[0098] 1. The breaking of the rapeseed pollen wall:

[0099] Take 10 kg of rapeseed pollen, remove impurities and pour into a fermentation tank, add purified water to cover the medicinal materials, add 1% cellulase (total raw material mass fraction) and 1% hemicellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.7, control the temperature at 46°C, and enzymolysis for 2 hours, then sterilize by steam for 30 min.

[0100] 2. Fermentation:

[0101] After cooling to room temperature, add 4% (total raw material mass fraction) of Purpureocillium SP. MD313902 strain, and ferment at 28°C for 30 days. Filter and collect the medicinal liquid for processing.

[0102] 3. Extraction and content determination of rapeseed pollen glycoprotein

[0103] The collected medicinal liquid after fermentation was added with 8 times of purified water, heated and refluxed at 100°C for 2 times, each for 3 hours, filtered, and the extract was obtained. The extract was concentrated to an appropriate concentration, free protein was removed by Sevag method, anhydrous ethanol was then added to make the solution concentration reach 80%, and the precipitate was obtained by centrifugation twice. The precipitate was dried to obtain the total polysaccharide of rape pollen.

[0104] The content of polysaccharide of rape pollen was determined by phenol-sulfuric acid method. The determination results showed that the extraction rate of polysaccharide of rape pollen after fermentation was 12.3%, which was 1.6 times higher than that of the case without the fermentation step (step 2) of the present case (after the wall breaking treatment of step 1, directly proceed to the extraction of step 3).

[0105] Example 3

[0106] 1. Wall breaking of rape pollen

[0107] 15 kg of rape pollen was taken, impurities were removed, and then put into a fermentation tank. Purified water was added to cover the medicinal materials, 1% pectinase (total raw material mass fraction) and 4% papain (total raw material mass fraction) were added, and the mixture was fully stirred and mixed. The pH value was adjusted to 6.5, the temperature was controlled at 48°C, and the enzymolysis was performed for 4 hours. Then steam sterilization was performed for 40 min.

[0108] 2. Fermentation:

[0109] After cooling to room temperature, 2% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain and 2% (total raw material mass fraction) of Purpureocillium SP. MD313902 strain were added, and the mixture was placed in a 37°C environment for fermentation for 25 days. The medicinal liquid was filtered and collected for treatment.

[0110] 3. Extraction and content determination of rape pollen glycoprotein

[0111] The collected medicinal liquid after fermentation was added with 10 times of purified water, heated and refluxed at 90°C for 3 times, each for 2 hours, filtered, and the extract was obtained. The extract was concentrated to an appropriate concentration, free protein was removed by Sevag method, anhydrous ethanol was then added to make the solution concentration reach 80%, and the precipitate was obtained by centrifugation twice. The precipitate was dried to obtain the total polysaccharide of rape pollen.

[0112] The content of polysaccharide of rape pollen was determined by phenol-sulfuric acid method. The determination results showed that the extraction rate of polysaccharide of rape pollen after fermentation was 16.3%, which was 2.1 times higher than that of the case without the fermentation step (step 2) of the present case (after the wall breaking treatment of step 1, directly proceed to the extraction of step 3).

[0113] Example 4

[0114] 1. Wall breaking of rape pollen:

[0115] Take 10 kg of rapeseed pollen, remove impurities and pour into a fermentation tank, add purified water to cover the medicinal materials, add 1% cellulase (total raw material mass fraction) and 1% hemicellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.7, control the temperature at 46°C, and enzymolysis for 2 hours, then sterilize by steam for 30 min.

[0116] 2. Fermentation:

[0117] After cooling to room temperature, add 2% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain, 3% (total raw material mass fraction) of Bifidobacterium, and 3% (total raw material mass fraction) of Kluyveromyces lactis mixed strains, and place in a 28°C environment for fermentation for 15 days, filter, and collect the medicinal liquid for processing.

[0118] 3. Extraction and content determination of rapeseed pollen glycoprotein

[0119] Add 8 times the amount of purified water to the collected medicinal liquid after fermentation, heat at 100°C for 2 times of reflux, each for 3 hours, filter, obtain the extract, concentrate to an appropriate concentration, remove free proteins by the Sevag method, then add anhydrous ethanol to make the solution concentration reach 80%, centrifugal precipitate twice, dry the precipitate to obtain rapeseed pollen total polysaccharide.

[0120] The content of rapeseed pollen polysaccharide was determined by the phenol-sulfuric acid method, and the determination results showed that the extraction rate of rapeseed pollen polysaccharide after fermentation was 13.6%, which was 1.8 times higher than that of the case without the fermentation step 2 (after the wall breaking treatment in step 1, directly proceed to step 3 extraction).

[0121] Example 5

[0122] 1. Wall breaking of rapeseed pollen

[0123] Take 15 kg of rapeseed pollen, remove impurities and pour into a fermentation tank, add purified water to cover the medicinal materials, add 1% pectinase (total raw material mass fraction) and 4% papain (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.5, control the temperature at 48°C, and enzymolysis for 4 hours, then sterilize by steam for 40 min.

[0124] 2. Fermentation:

[0125] After cooling to room temperature, 2% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain, 2% (total raw material mass fraction) of Purpureocillium SP. MD313902, 2% (total raw material mass fraction) of Kluyveromyces lactis, and 2% (total raw material mass fraction) of Lactobacillus rhamnosus mixed strains are added, and fermentation is carried out at 37°C for 15 days. After filtration, the filtrate is collected for treatment.

[0126] 3. Extraction and content determination of rape pollen glycoprotein

[0127] The filtrate collected after fermentation is added with 10 times the amount of purified water of the medicinal material, heated to reflux at 90°C for 3 times, each for 2 hours, filtered, concentrated to an appropriate concentration, and free proteins are removed by the Sevag method. Then, anhydrous ethanol is added to make the concentration of the solution reach 80%, and the precipitate is obtained by centrifugal precipitation twice. The precipitate is dried to obtain total rape pollen polysaccharide.

[0128] The content of rape pollen polysaccharide is determined by the phenol-sulfuric acid method. The determination results show that the extraction rate of rape pollen polysaccharide after fermentation is 14.5%, which is 1.9 times higher than that of the case without the fermentation step (step 2) of the present case (after the cell wall breaking treatment of step 1, directly proceed to step 3 for extraction).

[0129] Example 6

[0130] 1. Cell wall breaking of rape pollen

[0131] Take 15 kg of rape pollen, remove impurities, and pour into a fermentation tank. Add purified water to cover the medicinal material, add 2% pectinase (total raw material mass fraction), 1% cellulase (total raw material mass fraction), and 1% papain (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.8, control the temperature to 40°C, and enzymatically hydrolyze for 3 hours. Then, steam sterilization is performed for 50 min.

[0132] 2. Fermentation:

[0133] After cooling to room temperature, 1% (total raw material mass fraction) of Candida utilis, 1% (total raw material mass fraction) of Kluyveromyces lactis, 1% (total raw material mass fraction) of Lactobacillus acidophilus, and 1% (total raw material mass fraction) of Lactobacillus rhamnosus mixed strains are added, and fermentation is carried out at 37°C for 20 days. After filtration, the filtrate is collected for treatment.

[0134] 3. Extraction and content determination of rape pollen glycoprotein

[0135] The medicinal liquid collected after fermentation is added with 10 times the amount of purified water of the medicinal material, heated to reflux at 95℃ for 2 times, each time for 2 hours, filtered, concentrated to an appropriate concentration, free proteins are removed by the Sevag method, then anhydrous ethanol is added to make the solution concentration reach 80%, and the precipitate is dried to obtain the total rapeseed pollen polysaccharide.

[0136] The content of rapeseed pollen polysaccharide is determined by the phenol-sulfuric acid method, and the determination result shows that the extraction rate of rapeseed pollen polysaccharide after fermentation is 15.3%, and compared with the case without the fermentation step 2 (after the wall breaking treatment of step 1, directly performing the extraction of step 3), it is found that the polysaccharide content is 2.0 times higher.

[0137] Example 7

[0138] Compared with Example 1, the only difference is that the wall breaking treatment of step 1 is not performed, and the dandelion medicinal material is directly washed clean by removing impurities with tap water, poured into a fermentation tank, inoculated with the strain after sterilization, and then subjected to the fermentation of step 2, followed by the polysaccharide extraction of step 3. The content of polysaccharide in the fermentation liquid is tested by the method of Example 1, which is 10.4%.

[0139] Example 8

[0140] Compared with Example 1, the only difference is that the enzyme breaking wall method of Example 1 is replaced by the ultrafine grinding breaking wall method in step 1. Other operations and parameters are the same as those of Example 1. The content of polysaccharide in the fermentation liquid is tested by the method of Example 1, which is 12.1%.

[0141] It is found by comparing Example 1, Example 7 and Example 8 that the polysaccharide content of the fermentation product after wall breaking treatment is significantly improved compared with the fermentation experiment without wall breaking treatment. In addition, it is also found by the present application that the breaking wall technology adopted is biological enzyme breaking wall, and the ultrafine grinding breaking wall method is selected for comparison during the experiment implementation. Compared with the ultrafine grinding breaking wall technology, the biological enzyme breaking wall is more gentle and the breaking wall effect is more sufficient, and has no effect on the active ingredients.

Claims

1. A method for extracting Brassica pollen polysaccharides, characterized in that, The rape pollen is pretreated by wall breaking and then subjected to microbial fermentation to obtain a fermentation liquor containing polysaccharides; The microorganism comprises Parengyodontium SP at least one of the MD313901 strain and / or Purpureocillium SP. the MD313902 strain, and optionally at least one of a yeast, a lactic acid bacteria Parengyodontium SP . The preservation number of the MD313901 strain is CCTCC M 2018256; Purpureocillium SP. The preservation number of the MD313902 strain is CCTCC M 2018257; The yeast is one or more of Candida utilis and Kluyveromyces lactis. The lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus, Lactococcus, Lactobacillus bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, Streptococcus lactis, Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus polymyxa, and Lactobacillus rhamnosus. The wall breaking pretreatment is biological enzyme hydrolysis. The biological enzyme is one or more of pectinase, cellulase, hemicellulase, and papain.

2. The method of claim 1, wherein the Brassica pollen polysaccharides are extracted from Brassica pollen. The microorganism comprises Parengyodontium SP . MD313901 strain, at least one yeast and at least one lactic acid bacteria or is Parengyodontium SP . the MD313901 strain and Purpureocillium SP. the MD313902 strain, at least one yeast and at least one lactic acid bacteria or is Parengyodontium SP . MD313901 strain and Purpureocillium SP. MD313902 strain.

3. The method of claim 1, wherein the Brassica pollen polysaccharide is extracted by the steps of: During the enzymolysis process, the amount of biological enzyme is 0.01%-5% of the weight of rape pollen.

4. The method of claim 1, wherein the Brassica pollen polysaccharide is extracted by the steps of: The pH of the enzymolysis process is 4-8.

5. The method of claim 1, wherein the Brassica pollen polysaccharide is extracted by the steps of: During the fermentation process, the inoculation amount of microorganism is 0.01-20% of the mass of rape pollen.

6. The method of claim 5, wherein the Brassica pollen polysaccharides are extracted from Brassica pollen by the steps of: The fermentation temperature is 10-50℃, and the fermentation time is 48h or more.

7. The method of claim 1, wherein the Brassica pollen polysaccharide is extracted by the steps of: Rape pollen polysaccharides are extracted from the fermentation liquor.

8. The method of claim 7, wherein the Brassica pollen polysaccharides are extracted from Brassica pollen by the method comprising the steps of: The extraction process includes water extraction and alcohol extraction performed in sequence.

9. The fermentation broth obtained by the extraction method according to any one of claims 1-8; characterized in that, The fermentation liquor contains rape pollen polysaccharides and the microorganism.

10. The fermentation broth obtained by the extraction method according to claim 9, characterized in that, The fermentation liquor also contains glycoprotein.

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