CN104046570B - A kind of method improving Paecilomyces cicadae liquid fermentation production output and its lytic activity - Google Patents

Abstract

The invention discloses a kind of method improving Paecilomyces cicadae liquid fermentation production output and its lytic activity, comprise step: the Chinese medicinal materials that is made up of Semen Coicis 10-40 weight part, Radix Astragali 10-40 weight part, matrimony vine 10-40 weight part and bighead atractylodes rhizome 10-40 weight part through extraction with aqueous solution, is obtained Chinese medicine Compound Water extract by (1); (2) Chinese medicine Compound Water extract, atractylodes lactone and tween 80 prepared by step (1) are added in fermentation basic medium, obtain Paecilomyces cicadae fermention medium; (3) by Paecilomyces cicadae fermention medium obtained for Paecilomyces cicadae bacterial classification access step (2), through liquid fermenting, obtain fermented liquid, be then separated and obtain tunning.The present invention can significantly improve biomass and the polysaccharide yield of Paecilomyces cicadae by adding the Chinese medicine Compound Water extract of particular types, atractylodes lactone and tween 80, the activity of polysaccharide also has obvious increase simultaneously.

Description

A method for increasing the yield and activity of Paecilomyces cicadae liquid fermentation product

technical field

The invention belongs to the field of fermentation engineering, and in particular relates to a method for increasing the yield and activity of Paecilomyces cicadae liquid fermentation products.

Background technique

Edible and medicinal fungus Paecilomycescicadae (Mique1) Samson, also known as female cicada, belongs to the same genus of Cordyceps as Cordyceps sinensis and Cordyceps militaris, and is an asexual strain of a precious traditional Chinese medicinal material, Cordyceps sobolifera. The active ingredients and efficacy of Paecilomyces cicadae are similar to those of Cordyceps sinensis, containing nucleosides, myriocin, ergosterol, cordycepic acid, polysaccharides, various essential amino acids, mannitol and alkaloids and other secondary metabolites ( He Liang, Ma Suyun, Cheng Junwen, et al. Research progress on bioactive substances of the medicinal fungus Paecilomyces cicadae [J]. Journal of Food and Biotechnology, 2012,31(1):8-15.). Modern pharmacological studies have shown that Paecilomyces cicadae has many health care functions, such as immune regulation, anti-tumor and improvement of renal function (WengSC, ChouCJ, LinLC, et al. Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordycepscicadae [J]. -85; Chen Anhui, Fan Meizhen, Shao Ying, et al. Research on inhibition of monoamine oxidase and antitumor activity of metabolites of Paecilomyces cicadae[J]. Food Science, 2009,30(11):216-218; ChyauCC, ChenCC, ChenJC, et al.Myceliaglycoproteins from Cordycepssobolifera amelioratecyclosporine-inducedrenal tubuledys function in rats[J].Journalofethnopharmacology,2014.), as well as nourishing and strong, anti-oxidation, anti-virus, etc. .Journal of Wenzhou Medical College, 2005,35(1):13-15; Zhu Yan, Cheng Dongqing. Determination of antioxidant activity of cicadae polysaccharide[J].Chinese Journal of General Medicine,2009,27(7):1552-1554 ; Zhuo Jia, Yang Jiezuan, Jin Liqin, et al. Inhibitory effect of Paecilomyces cicadae polysaccharide on hepatitis B virus replication in vitro[J]. Chinese Journal of Pathophysiology, 2010,26(2):327-332.), therefore , Paecilomyces cicadae can be used as a substitute for Cordyceps sinensis, and has good research and application prospects. In the future, Paecilomyces cicadae preparations may make important contributions to the development of functional foods and medical care, and benefit mankind.

Edible and medicinal fungi can secrete complex enzymes to utilize rich nutrients such as cellulose, starch, protein, and lipids in traditional Chinese medicine, promote fungal growth and product synthesis, and obtain rich metabolites. Research on traditional Chinese medicine fermentation technology has found that fungi may also biotransform certain active substances such as terpenoids, flavonoids, alkaloids, and saponins in traditional Chinese medicine during the metabolic process to form new components or substances with higher activity. Significantly enhance the biological activity and pharmacological function of the fermentation product. There is a mutual relationship between the ingredients of traditional Chinese medicine and the growth and metabolism of Paecilomyces cicadae, and the ingredients of Chinese medicines may participate in the metabolic pathway of Paecilomyces cicadae, affecting the accumulation of exopolysaccharides and related enzymes in biosynthesis, such as glucose phosphate isomerase ( PGI) is an important enzyme in the glycolytic pathway, and α-phosphoglucomutase (α-PGM) is an important enzyme related to the synthesis of exopolysaccharide (EPS). It has been reported in the literature that gastrodin or its compound components in the traditional Chinese medicine Gastrodia elata has a significant inhibitory effect on the PGI in the polysaccharide synthesis and metabolism pathway of Grifola frondosa, inhibiting the glycolysis pathway, thereby facilitating the progress of glucose-6-phosphate to the polysaccharide synthesis pathway ( He Zongyi, Wu Tianxiang, Xu Xiaobao. Effects of components of Gastrodia elata on the synthesis of exopolysaccharides from Grifola frondosa and related key enzymes[J]. Food Science, 2013,34(11):199-202.). The change in the activity of metabolites may be that the addition of traditional Chinese medicine to the medium stimulates or inhibits the metabolism of active substances of Paecilomyces cicadae, or that Paecilomyces cicadae biotransforms some Chinese medicine components during growth and metabolism, producing New secondary metabolites are formed, thereby changing the activity of metabolites.

Studies have found that some oily substances and surfactants can not only act as defoamers in the liquid fermentation production of edible and medicinal fungi, but also affect the growth and metabolism of fungi. Synthesis of bacteria-specific active ingredients (HsiehC, WangHL, ChenCC, et al. Effect of plant oil and surfan to the production of mycelial biomass and polysaccharides in submerged culture of Grifola frondosa [J]. Biochemical Engineering Journal, 2008, 38 (2): 198-205; Bi Pengyang, Yang Hailong, Min Weihong. Research on submerged fermentation [J]. Food Industry Science and Technology, 2011,32(12):226-228.). The ability of lipid substances to promote the growth and metabolism of fungi is related to the fact that oil or fatty acids can change the structure and permeability of fungal cell membranes or directly affect some important enzyme activities in metabolic pathways. Studies have found that 0.2% coix seed fat can help the growth of Ganoderma lucidum hyphae and the synthesis of intracellular polysaccharides and exopolysaccharides, and the activities of some important enzymes in the biosynthetic pathway of Ganoderma lucidum polysaccharides are also affected by coix seed fat (ZhouH, BiP , WuX, et al. ImprovedpolysaccharideproductioninsubmergedcultureofGanodermalucidumnbytheadditionofcoixenolide[J].AppliedBiochemistryandBiotechnology,2014,172:1497-1505.). The mechanism of surfactant Tween 80 affecting the metabolism of edible and medicinal fungi may be related to the integrity of mycelium cell membrane structure and transmembrane transport activity (ZhangBB, Cheung PCK. 8323-8326.). The molecular structure of surfactants is amphiphilic, and the fungal cell membrane structure is also composed of a layer of amphiphilic phospholipids, so surfactants may be partially embedded in the cell membrane, thereby accelerating the rate at which cells absorb nutrients from the medium (ChenHB, HuangHC, ChenCI, et al. The use of additives asthestimulator on mycelial biomass and exopolysaccharide productions in submerged culture of Grifolaumbellate [J]. Bioprocess and biosystem engineering, 2010, 33 (3): 401-406.). Studies have shown that surfactants such as Tween 80 can effectively promote the synthesis of active metabolites in edible and medicinal fungi, adding 0.1% (w/v) Tween 80 in the 72h of liquid fermentation of Antrodia camphorata, the production of AntrodinC was significantly higher than that of Contrast, increased to 103.76 ± 0.92mg/L by 52.37 ± 1.02mg/L, in the coupled fermentation system of Tween 80 and soybean oil, the maximum output of AntrodinC is 3.6 times of contrast (ZhangH, XiaY, WangYL, etal. [J]. Biochemical Engineering Journal, 2013, 79:194-199.).

Chinese patent ZL200510068284.9 discloses a Coprinus comatus liquid fermentation process and fermented products and application method with adding bitter melon. Mushrooms biotransform bitter gourd components and increase the activity of the fermentation broth. Chinese patent ZL200510137457.8 discloses a method for cultivating Cordyceps sinensis and its special medium, which discloses a method of adding soybean oil to the liquid fermentation of Cordyceps sinensis. This method has the advantages of fast growth and short cycle for the production of Cordyceps mycelia , high yield, simple process, low cost and other advantages. The research on significantly promoting the growth of edible and medicinal fungi or increasing the production of active metabolites by adding appropriate amount of traditional Chinese medicine ingredients or oils to the medium has been reported many times at home and abroad, but the research on the liquid fermentation of Paecilomyces cicadae application has not been reported yet.

Contents of the invention

The object of the invention is to provide a method for increasing the yield and activity of Paecilomyces cicadae liquid submerged fermentation products.

The present invention finds that adding a specific type of traditional Chinese medicine compound extract, oil and surfactant to the fermentation medium to carry out the liquid fermentation culture of Paecilomyces cicadae helps to improve the biomass of Paecilomyces cicadae and the production of secondary metabolites. content, and at the same time promote the improvement of the activity of metabolites.

A method for improving the yield and activity of Paecilomyces cicadae liquid fermentation product, comprising the steps of:

(1) extracting Chinese medicinal materials composed of 10-40 parts by weight of coix seed, 10-40 parts by weight of Astragalus membranaceus, 10-40 parts by weight of Chinese wolfberry and 10-40 parts by weight of Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Rhizoma Atractylodes Rhizoma Atractylodes Rhizome;

(2) adding the Chinese medicine composite water extract, Atractylodes lactone and Tween 80 prepared in step (1) into the fermentation basal medium to obtain the Paecilomyces cicadae fermentation medium;

(3) inserting the Paecilomyces cicadae species into the Paecilomyces cicadae fermentation medium prepared in step (2), performing liquid fermentation to obtain a fermentation liquid, and then separating to obtain a fermentation product.

In the present invention, when carrying out liquid fermentation of Paecilomyces cicadae, the Chinese medicinal material cannot be directly added to the fermentation base medium, and the Chinese medicinal material needs to be prepared as a Chinese medicine composite water extract, and then the Chinese medicine composite water extract is added to the fermentation base medium A culture medium containing traditional Chinese medicine extracts is prepared in the medium, which helps to increase the biomass of Paecilomyces cicadae and the content of secondary metabolites, and at the same time promotes the improvement of the activity of metabolites.

In step (1), the preparation of the Chinese medicine compound water extract can adopt the conventional water extraction method in the art, preferably the following method: take the Chinese medicinal material according to the stated amount, pulverize (preferably pass 60 mesh-100 mesh powder ), add 2 times to 10 times of water to soak for 0.5h-1h, then heat and boil, keep a gentle boiling state for 20min-60min, filter and collect the extract; repeat the extraction for 1-4 times, and combine the filtrate to obtain the Chinese medicine composite water extract .

In step (2), described Atractylodes macrolide can adopt commercially available product, also can adopt existing method to prepare; Such as can adopt the supercritical _CO of people such as Yang Lin et al. "The preparation method of Atractylodes lactone recorded in the article is prepared (Yang Lin, He Dan. Supercritical CO ₂ Extraction Technology Research of Atractylodes Atractylodes I in Atractylodes Rhizome. Chinese Herbal Medicine, 2006, 37 (9): 1331-1333); The following preparation method is adopted: pulverize the Atractylodes macrocephala medicinal material (preferably crushed to 60 mesh-100 mesh powder), use ethanol aqueous solution with a mass percentage concentration of 10%-20% as a carrier, extract pressure 25MPa, resolve pressure 5MPa, and extract temperature 40 ℃-45℃, extraction temperature of 30℃-35℃ for 4h-4.5h to obtain atractylodes lactone.

The Paecilomyces cicadae fermentation medium contains 1g-2g of traditional Chinese medicine compound water extract, 0.2g-0.6g of atractylodes lactone and 0.1g-0.3g of Tween 80 in every 100ml.

The fermented basal medium can adopt the commonly used fermented basal medium for liquid fermentation in this field, and the preferred fermented basal medium is: glucose 2g/100ml, yeast powder 0.4g/100ml, peptone 0.3g/100ml, KH ₂ PO ₄ 0.1g/100ml and MgSO ₄ 0.05g/100ml.

In step (3), the Paecilomyces cicadae strain can be any Paecilomyces cicadae strain, and commercially available products can be used. For example, Paecilomycescicadae (Miq.) Samson CGMCC No. 3453, which was registered and preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short) on November 18, 2009.

The insertion method of the described Paecilomyces cicadae strain is a conventional method in the art, comprising: the Paecilomyces cicadae strain activated in the PDA slant medium is inserted into the sterilized liquid seed medium for cultivation, Obtain the cultivated seed liquid, then insert the cultivated seed liquid into the Paecilomyces cicadae fermentation medium prepared in step (2). Further preferably: the activated Paecilomyces cicadae strain in the PDA slant culture medium, get 5mm ² -8mm ² and insert in the sterilized liquid seed culture medium to 100ml-200ml, 20 ℃-30 ℃ (most preferably 25 ℃), 100r/min-150r/min (most preferably 120r/min) shaker culture for 3 days-5 days to obtain the cultured seed liquid, then insert the cultured seed liquid into the prepared Paecilomyces cicadae fermentation medium.

The PDA slant culture medium and liquid seed culture medium all adopt the common medium for seed cultivation in the art, and commercially available products can be used. Further preferably, the PDA slant medium: 200g of potatoes, 20g of glucose and 15g-20g of agar, the volume is adjusted to 1000ml with water. Further preferably, the liquid seed culture medium: 20g of glucose, 5g of yeast powder, 1g of KH ₂ PO ₄ and 0.5g of MgSO ₄ , the volume is adjusted to 1000 ml with water.

The inoculum amount of the Paecilomyces cicadae species is preferably 5%-15%. The preferred conditions for liquid fermentation are as follows: at a temperature of 20°C-30°C and a rotation speed of 60r/min-150r/min, culture on a shaker for 5-7 days.

The inoculation amount of the strain refers to the percentage of the ratio of the volume of the strain inserted or the volume of the cultivated seed liquid inserted to the volume of the Paecilomyces cicadae fermentation medium.

The separation adopts conventional separation methods in the art, for example, one or more of separation methods such as filtration, alcohol precipitation filtration, and centrifugation can be selected.

In the present invention, the Chinese medicinal materials meet the requirements of the Pharmacopoeia of the People's Republic of China.

Coix seed, this product is the dry mature seed kernel of Coixlacryma-jobi L.var.ma-yuen (Roman.) Stapf, a grass plant. Harvest the plants when the fruits are ripe in autumn, dry them in the sun, cut the fruits, and then dry them in the sun to remove the shell, yellow-brown seed coat and impurities, and collect the kernels. Properties: This product is broad oval or oblong, 4-8mm long and 3-6mm wide. The surface is milky white, smooth, with occasional yellow-brown seed coat remaining. One end is blunt and round, the other end is wider and slightly concave, with a light brown dotted hilum. The back is round and convex, and there is a wide and deep longitudinal groove on the ventral surface. The quality is solid, the section is white, powdery. Slight gas, slightly sweet taste.

Astragalus, this product is the dry root of the leguminous plant Astragalusmembranaceus (Fisch.) Bge.var.mongho-licus (Bge.) Hsiao or Astragalusmembranaceus (Fisch.) Bge. Excavated in spring and autumn, remove fibrous roots and roots, and dry in the sun. Properties: This product is cylindrical, some have branches, the upper end is thicker, 30-90cm long, 1-3.5cm in diameter. The surface is light brown or light brown, with irregular longitudinal wrinkles or grooves. Hard and tough, not easy to break, strong fibrous cross-section, and powdery, yellowish-white bark, light yellow xylem, radial texture and fissures, old root center occasionally withered, dark brown or hollow. Slight gas, slightly sweet taste, slightly beany smell when chewed.

Lycium barbarum, namely Lycium barbarum, this product is the dry and mature fruit of Lycium barbarum L., a plant of the Solanaceae family. Harvest when the fruit is red in summer and autumn, dry with hot air, and remove the fruit stem. Or dry until the skin is wrinkled, then dry and remove the fruit stems. Properties: This product is spindle-shaped or oval, with a length of 6-20mm and a diameter of 3-10mm. The surface is red or dark red, with small raised style scars at the top and white fruit stem scars at the base. The peel is flexible and shrunken; the flesh is fleshy and soft. Seeds 20-50, kidney-like, flat and warped, 1.5-1.9mm long, 1-1.7mm wide, light yellow or brownish yellow on the surface. Slight gas, sweet taste.

Atractylodes macrocephala, this product is the dry rhizome of Atractylodes macrocephala Koidz. In winter, when the lower leaves are yellow and the upper leaves are brittle, they are excavated, removed from the sediment, dried or sun-dried, and then the fibrous roots are removed. Properties: This product is an irregular hypertrophic mass with a length of 3-13cm and a diameter of 1.5-7cm. The surface is gray-yellow or gray-brown, with tumor-like protrusions, intermittent longitudinal wrinkles and grooves, fibrous root scars, and residual stem base and bud scars on the top. The texture is hard and not easy to break, the cross-section is uneven, yellow-white to light brown, with scattered brown-yellow dot-like oil chambers; the cross-section of the dried one is horny-like, darker in color or has cracks. The air is fragrant, sweet, slightly pungent, slightly sticky when chewed.

Beneficial effects of the present invention:

The invention can significantly increase the biomass and polysaccharide yield of Paecilomyces cicadae by adding specific kinds of traditional Chinese medicine compound water extract, atractylodes lactone and Tween 80, and at the same time, the activity of the polysaccharide is also significantly increased. Wherein compared with the conventional culture method, the biomass of Paecilomyces cicadae fermented and cultivated by the method of the present invention has been improved by 37.80%-134.39%, the output of extracellular polysaccharide has been improved by 15.86%-37.89%, and the intracellular polysaccharide has been improved by 39.74%-134.04%. %, the exopolysaccharide activity increased by 3.60%-33.5%, and the intracellular polysaccharide activity increased by 15.2%-61.67%.

detailed description

The present invention will be described in further detail below in conjunction with embodiment, and the method used in the embodiment is conventional method unless otherwise specified.

The PDA slant medium used: 200g of potatoes, 20g of glucose and 15g of agar, the volume was adjusted to 1000ml with water, the natural pH was sterilized at 121°C for 20min.

The Atractylodes lactone used was purchased from Chengdu Ruifensi Biotechnology Co., Ltd.; Paecilomyces cicadae was purchased from Jinzhai Jiade Traditional Chinese Medicine Co., Ltd.

Example 1

1. Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicine (crushed, passed through 60 meshes), which consists of the following Chinese herbal medicines: by weight, 10 parts of Coix seed, 40 parts of Astragalus membranaceus, 20 parts of Lycium barbarum and 30 parts of Atractylodes macrocephala. Add 10 times of water to soak for 0.5h, boil in an electric furnace, adjust the heating temperature properly, keep a gentle boiling state, boil for 60min, filter and collect the extract. The extraction was repeated twice, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a crude drug concentration of 20mg/mL.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.2g atractylodes lactone, 0.3g Tween 80 and 50mL of 20mg/mL traditional Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

(1) Determination of Mycelia Biomass

The mycelium in the fermentation broth was obtained by filtering through 8 layers of gauze, the mycelium was washed with distilled water three times, and then dried in an oven at 60°C until constant weight was obtained to obtain dry powder of the mycelium, which was weighed.

Mycelium biomass = weight of dry powder of bacteria ÷ volume of fermentation broth.

(2) Determination of exopolysaccharide content

Add 4 volumes of absolute ethanol to the supernatant after filtering the mycelia from the fermentation broth, alcohol precipitation overnight, centrifuge at 3000r/min for 20min, and the precipitate is crude polysaccharide. Add water to the precipitate to make it up to 50mL, measure the polysaccharide content in the fermentation broth with the phenol-sulfuric acid method, repeat the measurement 3 times and get the average value, which is the exopolysaccharide content.

Exopolysaccharide content = exopolysaccharide weight ÷ fermentation broth volume.

(3) Determination of intracellular polysaccharide content

Weigh an appropriate amount of bacterial dry powder, put it in a beaker, add distilled water according to the ratio of material to liquid 1:10 (weight ratio), extract at 90°C for 3 hours, extract twice, filter under reduced pressure to collect the filtrate, concentrate under reduced pressure at 60°C, and concentrate Add 4 times the volume of absolute ethanol to the solution, ethanol precipitation overnight, 3000r/min, and centrifuge for 20min, the precipitate is crude polysaccharide. Add water to the precipitate to make it up to 50mL, measure the polysaccharide content in the fermentation broth with the phenol-sulfuric acid method, repeat the measurement 3 times and get the average value, that is, the intracellular polysaccharide content.

Exopolysaccharide content = exopolysaccharide weight ÷ fermentation broth volume.

(4) Determination of the ability of extracellular and intracellular polysaccharides to scavenge DPPH free radicals

Weigh 7.9 mg of DPPH and dissolve it in 80% ethanol aqueous solution, and set the volume to a 100 mL volumetric flask to obtain a 0.2 mM (mmol/L) DPPH ethanol solution, which is ready for immediate use. Take 2mL of 5mg/mL sample to be tested (aqueous solution of extracellular polysaccharide or aqueous solution of intracellular polysaccharide) in a test tube, add 2mL of 0.2mM MDPPH ethanol solution, mix well, react at room temperature in the dark for 30min, measure the absorbance value at a wavelength of 517nm, according to the following formula Calculate the scavenging rate of the sample to be tested for DPPH free radicals.

Clearance rate (%)=(1-A ₁ /A ₀ )×100%

In the formula, A ₀ is the absorbance value of 2mL 0.2mM DPPH ethanol solution plus 2mL distilled water; A ₁ is the absorbance value of 2mL 0.2mM DPPH ethanol solution plus 2mL of the sample to be tested.

The test results are shown in Table 1 and Table 2.

Example 2

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicines (crushed, passed through 60 meshes), which consist of the following Chinese herbal medicines: by weight, 20 parts of Coix seed, 30 parts of Astragalus membranaceus, 30 parts of Lycium barbarum and 20 parts of Atractylodes macrocephala. Add 10 times of water to soak for 0.5h, boil in an electric furnace, adjust the heating temperature properly, keep a gentle boiling state, boil for 60min, filter and collect the extract. The extraction was repeated twice, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a crude drug concentration of 20mg/mL.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.4g atractylodes lactone, 0.2g Tween 80 and 50mL of 20mg/mL traditional Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Example 3

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicine (crushed, passed through 60 meshes), which consists of the following Chinese herbal medicines: by weight, 30 parts of coix seed, 10 parts of astragalus, 40 parts of wolfberry and 20 parts of Atractylodes macrocephala. Add 10 times of water to soak for 0.5h, boil in an electric furnace, adjust the heating temperature properly, keep a gentle boiling state, boil for 60min, filter and collect the extract. The extraction was repeated twice, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a crude drug concentration of 20mg/mL.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.6g atractylodes lactone, 0.1g Tween 80 and 50mL 20mg/mL traditional Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Example 4

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicine (crushed, passed through 60 meshes), which consists of the following Chinese herbal medicines: by weight, 40 parts of Coix seed, 30 parts of Astragalus membranaceus, 10 parts of Lycium barbarum and 20 parts of Atractylodes macrocephala. Add 10 times of water to soak for 0.5h, boil in an electric furnace, adjust the heating temperature properly, keep a gentle boiling state, boil for 60min, filter and collect the extract. The extraction was repeated twice, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a crude drug concentration of 20mg/mL.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.3g atractylodes lactone, 0.2g Tween 80 and 50mL of 20mg/mL traditional Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Example 5

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicine (crushed, passed through 60 meshes), which consists of the following Chinese herbal medicines: by weight, 20 parts of Coix seed, 20 parts of Astragalus membranaceus, 30 parts of Lycium barbarum and 30 parts of Atractylodes macrocephala. Add 10 times of water to soak for 0.5h, boil in an electric furnace, adjust the heating temperature properly, keep a gentle boiling state, boil for 60min, filter and collect the extract. The extraction was repeated twice, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a crude drug concentration of 20mg/mL.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.5g atractylodes lactone, 0.2g Tween 80 and 50mL 20mg/mL Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Example 6

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in PDA slant medium, get 8mm ² (ie soybean size) into the liquid seed medium after sterilization, the bottling volume of 500mL Erlenmeyer bottle is 100mL, 20 ℃, 100r/min under the shaker culture for 5 days, to obtain the cultured seed solution.

(2) Preparation of traditional Chinese medicine compound water extract

Weigh 2 g of compound Chinese medicinal materials (crushed, passed through 100 meshes), which consist of the following Chinese herbal medicines: by weight, 30 parts of coix seed, 30 parts of astragalus, 30 parts of wolfberry and 10 parts of Atractylodes macrocephala. Add 2 times the amount of water to soak for 1 hour, boil in an electric stove, adjust the heating temperature properly, keep a gentle boiling state, boil for 20 minutes, filter and collect the extract. The extraction was repeated 4 times, and the filtrates were combined and placed in a 100mL volumetric flask, and water was added to constant volume to obtain a Chinese medicine composite water extract with a concentration of 20 mg/mL in terms of crude drug volume.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.5g atractylodes lactone, 0.2g Tween 80 and 100mL 20mg/mL traditional Chinese medicine compound water extract, add water Fully dissolve and set the volume to 150mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 5%, and cultivated in a shaker at 20° C. and 150 r/min for 7 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Example 7

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Cultivation method: the Paecilomyces cicadae strain after activation in the PDA slant medium, get 6mm ² (ie soybean size) into the liquid seed medium after sterilization, the bottling volume of 500mL Erlenmeyer flask is 200mL, 30 ℃, 150r/min shaker culture for 3 days, to obtain the cultured seed solution.

(3) Preparation of traditional Chinese medicine compound water extract

Weigh 1 g of compound Chinese herbal medicine (crushed, passed through 100 meshes), which consists of the following Chinese herbal medicines: by weight, 20 parts of Coix seed, 30 parts of Astragalus membranaceus, 10 parts of Lycium barbarum and 40 parts of Atractylodes macrocephala. Add 5 times of water to soak for 50 minutes, boil in an electric stove, adjust the heating temperature properly, keep a gentle boiling state, boil for 45 minutes, filter and collect the extract. The extraction was repeated once, and the filtrates were combined and placed in a 50mL volumetric flask, and water was added to make up the volume to obtain a Chinese medicine composite water extract with a concentration of 20mg/mL of crude drug.

(4) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , 0.5g atractylodes lactone, 0.2g Tween 80 and 50mL 20mg/mL Chinese medicine compound water extract, add water Fully dissolve and set the volume to 100mL, the pH is natural, put into a 500mL Erlenmeyer flask, and sterilize at 121°C for 20min.

Cultivation method: the cultured seed liquid is inserted into the liquid fermentation medium with an inoculum amount of 15%, and cultivated in a shaker at 30° C. and 100 r/min for 6 days to obtain a fermentation liquid. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Comparative example

1 Fermentation culture

(1) Shake flask seed culture

1L liquid seed medium: glucose 20g/L, yeast powder 5g/L, KH ₂ PO ₄ 1g/L and MgSO ₄ 0.5g/L, the balance is water, pH is natural, sterilized at 121°C for 20min.

Culture method: the activated Paecilomyces cicadae strain in the PDA slant medium, get 5mm ² (ie soybean size) into the sterilized liquid seed medium, the bottling volume of the 500mL Erlenmeyer flask is 150mL, 25 ℃, 120r/min shaker culture for 3 days, to obtain the cultured seed solution.

(3) Shake flask fermentation culture

Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH ₂ PO ₄ , 0.05gMgSO ₄ , add water to fully dissolve and dilute to 100mL, pH is natural, put into a 500mL Erlenmeyer flask, sterilize at 121°C for 20min .

Cultivation method: the cultivated seed solution is inserted into the liquid fermentation medium with an inoculum amount of 10%, and cultivated in a shaker at 25° C. and 60 r/min for 5 days to obtain a fermentation solution. Three parallels were set up for each experiment.

2 Determination

The measurement of mycelium biomass, the measurement of the content of exopolysaccharide and the measurement of the content of intracellular polysaccharide are the same as in Example 1. The test results are shown in Table 1 and Table 2.

Table 1 The yield and activity detection results of liquid fermentation metabolites of Paecilomyces cicadae

Table 2

The numerical values in Table 2 are the percentages that the yield and product activity of Paecilomyces cicadae liquid fermentation metabolites in each embodiment are increased relative to the corresponding control examples.

The data of table 1 and table 2 shows, the present invention can significantly improve the biomass and the polysaccharide output of Paecilomyces cicadae by adding specific kinds of traditional Chinese medicine composite water extract, atractylodes lactone and Tween 80, and the activity of polysaccharide also has obvious simultaneously increase. Compared with the conventional culture method, the biomass of Paecilomyces cicadae fermented and cultivated by the method of the present invention is increased by 37.80%-134.39%, the output of exopolysaccharide is increased by 15.86%-37.89%, and the amount of intracellular polysaccharide is increased by 39.74%-134.04%. , the exopolysaccharide activity increased by 3.60%-33.5%, and the intracellular polysaccharide activity increased by 15.2%-61.67%.

The change of each parameter within the scope limited by the preparation method of the present invention does not affect the yield of Paecilomyces cicadae liquid fermentation metabolites and the improvement of product activity, so any combination of parameters in the preparation method of the present invention can realize the liquid fermentation of Paecilomyces cicadae Improvement of metabolite production and product activity. I won't repeat them here.

Claims (8)

1. a method for improving Paecilomyces cicadae liquid fermentation product output and product activity, is characterized in that, comprises steps: (1) extracting Chinese medicinal materials composed of 10-40 parts by weight of coix seed, 10-40 parts by weight of Astragalus membranaceus, 10-40 parts by weight of Chinese wolfberry and 10-40 parts by weight of Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizoma Rhizoma Atractylodes Rhizoma Atractylodes Rhizome; (2) adding the Chinese medicine composite water extract, Atractylodes lactone and Tween 80 prepared in step (1) into the fermentation basal medium to obtain the Paecilomyces cicadae fermentation medium; (3) inserting the Paecilomyces cicadae species into the Paecilomyces cicadae fermentation medium prepared in step (2), performing liquid fermentation to obtain a fermentation liquid, and then separating to obtain a fermentation product. 2. The method according to claim 1, characterized in that, the preparation method of the Chinese medicine compound water extract comprises: taking the Chinese medicinal material by said amount, pulverizing, adding 2-10 times of water soaking for 0.5h- 1h, then heat and decoct, keep a gentle boiling state for 20min-60min, filter and collect the extract; repeat the extraction 1-4 times, combine the filtrate to obtain the Chinese medicine composite water extract. 3. the method according to claim 1, is characterized in that, every 100ml of described Paecilomyces cicadae fermentation medium contains the Chinese medicine composite water extract of 1g-2g, 0.2g-0.6g Atractylodes lactone and 0.1g-0.3g Tween 80. 4. method according to claim 1, is characterized in that, described fermentation basal medium: glucose 2g/100ml, yeast powder 0.4g/100ml, peptone 0.3g/100ml, KH ₂ PO ₄ 0.1g/100ml and _MgSO4 0.05g/100ml. 5. method according to claim 1, is characterized in that, the access method of described Paecilomyces cicadae bacterial classification comprises: the Paecilomyces cicadae bacterial classification after being activated in PDA slant culture medium, after inserting sterilization Cultivate in the liquid seed culture medium, obtain the seed liquid of cultivating, then insert the seed liquid of cultivating in the Paecilomyces cicadae fermentation medium that step (2) makes. 6. method according to claim 5, is characterized in that, the inserting method of described Paecilomyces cicadae strain comprises: the Paecilomyces cicadae strain after activation in PDA slant medium, takes 5mm ^{2-8mm 2.} Insert the sterilized liquid seed medium into 100ml-200ml, 20°C-30°C, 100r/min-150r/min and culture on a shaker for 3 days to 5 days to obtain the cultured seed liquid, and then culture Good seed liquid is inserted in the Paecilomyces cicadae fermentation medium that step (2) makes. 7. The method according to claim 1, characterized in that the inoculum amount of the Paecilomyces cicadae species is 5%-15%. 8 . The method according to claim 1 , characterized in that, the liquid fermentation conditions: culture on a shaker for 5 days to 7 days at a temperature of 20° C. to 30° C. and a rotation speed of 60 r/min to 150 r/min.