CN104046570A - Method for improving yield and product activity of liquid fermentation product of Paecilomyces cicadae - Google Patents
Method for improving yield and product activity of liquid fermentation product of Paecilomyces cicadae Download PDFInfo
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Abstract
The invention discloses a method for improving the yield and product activity of a liquid fermentation product of Paecilomyces cicadae, which comprises the following steps: (1) extracting traditional Chinese medicinal materials composed of 10-40 parts by weight of coix seed, 10-40 parts by weight of radix astragali, 10-40 parts by weight of Chinese wolfberry and 10-40 parts by weight of rhizoma atractylodis macrocephalae through a water solution, thus obtaining a traditional Chinese medicine compound water extract liquid; (2) adding the traditional Chinese medicine compound water extract liquid prepared in the step (1), rhizoma atractylodis macrocephalae lactone and Tween-80 into a fermentation basal culture medium, thus obtaining a Paecilomyces cicadae fermentation culture medium; and (3) inoculating a Paecilomyces cicadae strain into the Paecilomyces cicadae fermentation culture medium prepared in the step (2), performing liquid fermentation to obtain a fermentation liquid, and separating to obtain a fermentation product. According to the invention, particular kinds of traditional Chinese medicine compound water extract liquid, rhizoma atractylodis macrocephalae lactone and Tween-80 are added, so that the biomass and polysaccharide yield of the Paecilomyces cicadae can be obviously improved, and meanwhile, the activity of the polysaccharide is also improved obviously.
Description
Technical field
The invention belongs to field of fermentation engineering, be specifically related to a kind of method that improves Paecilomyces cicadae liquid fermentation production output and its lytic activity.
Background technology
Edible medicinal fungus Paecilomyces cicadae (Paecilomyces cicadae (Mique1) Samson) has another name called female cicada fungus, being all Cordyceps with Cordyceps sinensis, Cordyceps militaris (L.) Link., is the anamorphic strain of a kind of famous and precious traditional Chinese medicine material cicada fungus (Cordyceps sobolifera) of China.The activeconstituents of Paecilomyces cicadae is similar to Cordyceps sinensis with effect, contain the secondary metabolite (He Liang such as nucleosides material, myriocin, ergosterol, cordycepic acid, polysaccharide, multiple indispensable amino acid, N.F,USP MANNITOL and alkaloid, Ma Suyun, Cheng Junwen, Deng. the progress [J] of medicinal fungi Paecilomyces cicadae biological active substance. food and biotechnology journal, 2012,31 (1): 8-15.).Modern pharmacology research shows, Paecilomyces cicadae has many medical health care functions, as immunomodulatory, antitumor and improve renal function (Weng S C, Chou C J, Lin L C, et al.Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordyceps cicadae[J] .Journal of ethnopharmacology, 2002,83 (1): 79-85; Chen Anhui, Fan Meizhen, Shao Ying, etc. Paecilomyces cicadae meta-bolites suppresses monoamine oxidase and antitumor activity [J]. Food science, 2009,30 (11): 216-218; Chyau C C, Chen C C, Chen J C, et al.Mycelia glycoproteins from Cordycepssoboliferaameliorate cyclosporine-induced renal tubule dysfunction in rats[J] .Journal of ethnopharmacology, 2014.), also have the strengthening by means of tonics, (Chen Xiufang such as anti-oxidant, antiviral, Jin Liqin, Lv Jianxin, Deng. Paecilomyces cicadae is on the impact of rat nutritional status [J]. Journal of Wenzhou Medical College, 2005,35 (1): 13-15; Zhu Yan, Cheng Dongqing. the mensuration [J] of cicada fungus polysaccharide anti-oxidant activity. the total medical academic periodical of China, 2009,27 (7): 1552-1554; Zhuo Jia, Yang Jie bores, Jin Liqin, etc. paecilomyces cicadae polysaccharide is external to the restraining effect of hepatitis B replication [J]. Chinese Journal of Pathophysiology, 2010,26 (2): 327-332.), therefore, Paecilomyces cicadae can be used as the surrogate of Cordyceps sinensis, has the prospect of good research and application, Paecilomyces cicadae preparation may be made significant contribution on development of functional food and medical health career in the future, brings benefit to the mankind.
Edible medicinal fungus can be secreted numerous and jumbled enzyme system the abundant nutritive ingredients such as the Mierocrystalline cellulose in Chinese medicine, starch, protein, lipid are used, and promotes fungal growth and product synthetic, thereby obtains abundant meta-bolites.Herb fermenting technical study is found, fungi in metabolic process also likely some active substance such as the terpene in centering medicine, flavonoid, alkaloid, saponin class carry out bio-transformation, form new composition or active higher material, obviously strengthen biological activity and the pharmacological function of tunning.The growth metabolism process of traditional Chinese medicine ingredients and Paecilomyces cicadae exists interrelated, traditional Chinese medicine ingredients likely participates in the pathways metabolism of Paecilomyces cicadae, affect its exocellular polysaccharide accumulation and biosynthetic relevant enzyme, if phosphoglucoisomerase (PGI) is the important enzyme in glycolytic pathway, α-phosphoglucomutase (α-PGM) is the important enzyme synthetic relevant to exocellular polysaccharide (EPS).Gastrodine in bibliographical information gastrodia elata or its composite parts have significant restraining effect to PGI in grifolan metabolic pathway of synthesizing, suppressed glycolytic pathway, thereby be conducive to G-6-P to the (He Zongyi that advances of polysaccharide route of synthesis, Wu Tianxiang, Xu Xiaobao. the impact [J] of gastrodia elata composition and relevant key enzyme synthetic on Extracellular Polysaccharide from Grifola frondosa. Food science, 2013,34 (11): 199-202.).The change of metabolite activity may be to add after Chinese medicine in substratum, stimulate or suppressed the metabolism amount of Paecilomyces cicadae active substance, or Paecilomyces cicadae has carried out bio-transformation to some traditional Chinese medicine ingredients in growth metabolism process, produce new secondary metabolite, thereby changed the activity of meta-bolites.
Research find some oil substances and tensio-active agent in edible medicinal fungus liquid fermenting is produced except as defoamer, can also exert an influence to the growth metabolism of fungi, people just start to attempt to promote as inducible factor with them synthetic (the Hsieh C of edible medicinal fungus given activity composition, Wang H L, Chen C C, et al.Effect of plant oil and surfactant on the production of mycelial biomass and polysaccharides in submerged culture of Grifolafrondosa[J] .Biochemical Engineering Journal, 2008, 38 (2): 198-205, Bi Pengyang, Yang Hailong, Min Weihong. Semen Coicis oil and coixenolide promote the research [J] of deep glossy ganoderma fermenting. foodstuffs industry science and technology, 2011,32 (12): 226-228.).Grease substance promotes fungal growth metabolism and oil or lipid acid change fungal cell's membrane structure and permeability or directly affects in pathways metabolism some important enzymic activity relevant.Research finds that 0.2% Semen Coicis fat contributes to the synthetic of Mycelium Growth of Ganoderma lucidum and intracellular polyse and exocellular polysaccharide, and the activity of some important enzymes in ganoderan biosynthetic pathway is also subject to impact (the Zhou H of Semen Coicis fat, Bi P, Wu X, et al.Improved polysaccharide production in submerged culture of Ganodermalucidumn by the addition of coixenolide[J] .Applied Biochemistry and Biotechnology, 2014,172:1497-1505.).Tensio-active agent tween 80 affects the metabolic mechanism of edible medicinal fungus may (Zhang B B relevant to cross-film Transport Activity the complete structure of mycelial cell film, Cheung P C K.A mechanistic study of the enhancing effect of Tween80on the mycelial growth and exopolysaccharide production by Pleurotus tuber-regium[J] .Bioresource technology, 2011,102 (17): 8323-8326.).The molecular structure of tensio-active agent has amphipathic, and fungal cell's membrane structure is also by the amphipathic Lipid composition of one deck, thereby tensio-active agent may be fitted in cytolemma part, thereby accelerated the speed that cell absorbs nourishment from substratum (Chen H B, Huang H C, Chen C I, et al.The use of additives as the stimulator on mycelial biomass and exopolysaccharide productions in submerged culture of Grifolaumbellate[J] .Bioprocess and biosystems engineering, 2010, 33 (3): 401-406.).Research shows, tensio-active agent can effectively promote the synthetic of active metabolite in edible medicinal fungus as tween 80, at the 72h of Antrodia camphorata liquid fermenting, add the tween 80 of 0.1% (w/v), the output of Antrodin C is apparently higher than contrast, by 52.37 ± 1.02mg/L, bring up to 103.76 ± 0.92mg/L, in the coupled fermentation system of tween 80 and soybean oil, the maximum production of Antrodin C is 3.6 times of (Zhang H of contrast, Xia Y, Wang Y L, et al.Coupling use of surfactant and in situ extractant for enhanced production of Antrodin C by submerged fermentation of Antrodiacamphorata[J] .Biochemical Engineering Journal, 2013, 79:194-199.).
A kind of coprinus comatus liquid fermentation process of balsam pear and method of leavened prod and application of adding disclosed in Chinese patent ZL200510068284.9, its processing method is by add appropriate Chinese medicine balsam pear in coprinus comatus liquid fermentation medium, coprinus comatus is carried out bio-transformation to balsam pear composition during the fermentation, has improved the activity of fermented liquid.A kind of cultural method and special culture media thereof of Cordyceps sinensis are disclosed in Chinese patent ZL 200510137457.8, wherein disclose and added the method that soybean oil is carried out Cordyceps sinensis liquid fermentation and culture, this method to fermentative production Cordyceps mycelium there is fast growth, the cycle is short, productive rate is high, technique is simple, low cost and other advantages.By add appropriate traditional Chinese medicine ingredients or oil substances in substratum, significantly promote the growth of edible and medicinal fungi or the research that improves the output of active metabolite, report repeatedly all both at home and abroad, but application on Paecilomyces cicadae liquid fermenting there is not yet report.
Summary of the invention
The object of this invention is to provide a kind of method that improves Paecilomyces cicadae liquid submerged fermentation product output and its lytic activity.
The present invention finds, to the Chinese medicine composite extract, grease and the tensio-active agent that add particular types in fermention medium, carry out the liquid fermentation and culture of Paecilomyces cicadae, contribute to improve the content of Paecilomyces cicadae biological amount and secondary metabolite, promote metabolite activity to improve simultaneously.
A method that improves Paecilomyces cicadae liquid fermentation production output and its lytic activity, comprises step:
(1) by the Chinese medicinal materials being formed by 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, obtain Chinese medicine Compound Water extract;
(2) Chinese medicine Compound Water extract, atractylodes lactone and the tween 80 of being prepared by step (1) adds in fermentation basic medium, obtains Paecilomyces cicadae fermention medium;
(3) in Paecilomyces cicadae fermention medium Paecilomyces cicadae bacterial classification access step (2) being made, through liquid fermenting, obtain fermented liquid, then separation obtains tunning.
The present invention, while carrying out Paecilomyces cicadae liquid fermenting, can not directly Chinese medicinal materials be added in fermentation basic medium, need first Chinese medicinal materials to be prepared as Chinese medicine Compound Water extract, again Chinese medicine Compound Water extract is joined in fermentation basic medium and make the substratum containing Chinese medical extract, contribute to like this to improve the content of Paecilomyces cicadae biological amount and secondary metabolite, promote metabolite activity to improve simultaneously.
In step (1), the preparation of described Chinese medicine Compound Water extract can adopt the water extraction of this area routine, preferably adopt following methods: by described amount, take Chinese medicinal materials, pulverize in (preferred mistake 60 order-100 object powder), add 2 times of-10 times of water soaking 0.5h-1h, then heating decocts, and keeps gentle boiling state 20min-60min, filters and collects extracting solution; Repeat to extract 1 time-4 times, merging filtrate, obtains Chinese medicine Compound Water extract.
In step (2), described atractylodes lactone can adopt commercially available prod, also can adopt existing method preparation; As adopted the supercritical CO of the people such as Yang Lin atractylenolide Ⅰ in the < < bighead atractylodes rhizome
2the atractylodes lactone preparation method who records in extraction process research > > mono-literary composition be prepared (Yang Lin, He Dan. the supercritical CO of atractylenolide Ⅰ in the bighead atractylodes rhizome
2extraction process research. herbal medicine, 2006,37 (9): 1331-1333); Specifically can adopt following preparation method: Rhizoma Atractylodis Macrocephalae is pulverized to (preferred powder is broken to 60 order-100 object powder), the aqueous ethanolic solution that the mass percentage concentration of take is 10%-20% is for carrying agent, extracting pressure 25MPa, resolve pressure 5MPa, 40 ℃-45 ℃ of extraction temperature, under the condition that resolution temperature is 30 ℃-35 ℃, extract 4h-4.5h, make atractylodes lactone.
In the described every 100ml of Paecilomyces cicadae fermention medium, contain Chinese medicine Compound Water extract, the atractylodes lactone of 0.2g-0.6g and the tween 80 of 0.1g-0.3g of counting 1g-2g with raw medicinal herbs amount.
Described fermentation basic medium can adopt the fermentation basic medium that this area liquid fermenting is conventional, and the basic medium that preferably ferments is: glucose 2g/100ml, yeast powder 0.4g/100ml, peptone 0.3g/100ml, KH
2pO
40.1g/100ml and MgSO
40.05g/100ml.
In step (3), described Paecilomyces cicadae bacterial classification can adopt any one Paecilomyces cicadae bacterial classification, can adopt commercially available prod.Paecilomyces cicadae bacterial strain Paecilomyces cicadae (Miq.) Samson CGMCC No.3453 for example, registration preservation that this bacterial strain (is called for short CGMCC) in November, 2009 18 China Committee for Culture Collection of Microorganisms common micro-organisms center.
The ordinary method that the cut-in method of described Paecilomyces cicadae bacterial classification is this area, comprise: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, in liquid seed culture medium after access sterilizing, cultivate, obtain cultured seed liquor, in the Paecilomyces cicadae fermention medium then cultured seed liquor access step (2) being made.Further preferably: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2-8mm
2access in the liquid seed culture medium after sterilizing to 100ml-200ml, 20 ℃-30 ℃ (most preferably 25 ℃), the lower shaking table of 100r/min-150r/min (most preferably 120r/min) is cultivated 3 days-5 days, obtain cultured seed liquor, in the Paecilomyces cicadae fermention medium then cultured seed liquor access step (2) being made.
Described PDA slant medium and liquid seed culture medium all adopt the conventional substratum of this area seed culture, can adopt commercially available prod.Further preferably, described PDA slant medium: potato 200g, glucose 20g and agar 15g-20g, water is settled to 1000 milliliters.Further preferably, described liquid seed culture medium: glucose 20g, yeast powder 5g, KH
2pO
41g and MgSO
40.5g, water is settled to 1000 milliliters.
The bacterial classification inoculum size of described Paecilomyces cicadae bacterial classification is preferably 5%-15%.Liquid fermentation condition is preferred: 20 ℃-30 ℃ of temperature, under rotating speed 60r/min-150r/min condition, shaking table is cultivated 5 days-7 days.
The cultured seed liquor volume of the bacterial classification volume that described bacterial classification inoculum size refers to an access or access and the percentage ratio of the ratio of Paecilomyces cicadae fermention medium volume.
Described separation adopts the separation method of this area routine, such as selecting one or more in filtrations, alcohol precipitation filtration, the separation method such as centrifugal.
In the present invention, described Chinese medicinal materials meets the requirement of < < Pharmacopoeia of People's Republic of China > >.
Semen Coicis, this product is the dry mature kernal of grass Job's tears Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf.Tap plant during fruit maturation autumn, dries, and lays fruit, then dry, and except decapsidate, tawny kind skin and impurity, collects kind of a benevolence.Proterties: this product is width egg shape or oblong, long 4-8mm, wide 3-6mm.Surface oyster white, smooth, occasionally there is remaining tawny kind skin.The blunt circle in one end, another end is compared with wide and nick has 1 light brown point-like hilum.Back side boss, the outside of belly has 1 compared with wide and dark longitudinal furrow.Matter is solid, section white, mealiness.Gas is micro-, and taste is micro-sweet.
The Radix Astragali, this product is the dry root of leguminous plants Radix Astagali Astragalus membranaceus (Fisch.) Bge.var.mongho-licus (Bge.) Hsiao or Radix Astragali Astragalus membranaceus (Fisch.) Bge..Spring, Qiu Erji excavate, and remove fibrous root and root head, dry.Proterties: this product is cylindrical, what have has a branch, and upper end is thicker, long 30-90cm, diameter 1-3.5cm.Surface light brown yellow or light brown brown, have irregular vertical wrinkle or longitudinal furrow.Matter is hard and tough, frangibility not, and section fibrous is strong, and aobvious mealiness, skin zone's yellow-white, woody part is faint yellow, has radial texture and crack, and Lao Gen center is even a dry and decayed shape, chocolate or be cavity.Gas is micro-, and taste is micro-sweet, and that chews micro-ly has a beany flavor.
Matrimony vine, i.e. wolfberry fruit, this product is the dry mature fruit of plant of Solanaceae lycium barbarum Lycium barbarum L..Summer, autumn gathered when two seasons, fruit took on a red color, and hot-air seasoning, removes carpopodium.Or dry in the air to rhicnosis, dry, remove carpopodium.Proterties: this product is class fusiform or ellipse, long 6-20mm, diameter 3-10mm.Surface redness or garnet, there is the stylar scar of small embossment shape on top, the carpopodium trace of base portion adularescent.Pericarp is pliable and tough, shrinkage; Pulp meat, soft and moist.Seed 20-50 grain, class kidney shape, flat and stick up, long 1.5-1.9mm, wide 1-1.7mm, the light yellow or brown color in surface.Gas is micro-, and taste is sweet.
The bighead atractylodes rhizome, this product is the dry rhizome of feverfew bighead atractylodes rhizome Atractylodes macrocephala Koidz..When withered and yellow, the upper leaf of bottom leaf in winter becomes fragile, excavate, remove silt, dry or dry, then remove fibrous root.Proterties: this product is irregular plump agglomerate, long 3-13cm, diameter 1.5-7cm.Surface lark or taupe brown, have strumae and interrupted vertical wrinkle and rill, and have mark of fibrous root, top to have residual stem foot and bud trace.The hard not frangibility of matter, section is uneven, and yellow-white, to light brown, has the point-like grease chamber of brown color to be dispersed in; Oven dry person's section cutin sample, look dark or have a crack.Gas delicate fragrance, taste is sweet, micro-pungent, the slightly stickiness of chewing.
Beneficial effect of the present invention:
The present invention can significantly improve biomass and the polysaccharide yield of Paecilomyces cicadae by adding Chinese medicine Compound Water extract, atractylodes lactone and the tween 80 of particular types, and the activity of polysaccharide also has obvious increase simultaneously.Wherein compare with cellar culture method, adopt the Paecilomyces cicadae biological amount of the inventive method fermentation culture to improve 37.80%-134.39%, exopolysaccharides has improved 15.86%-37.89%, intracellular polyse has improved 39.74%-134.04%, the active 3.60%-33.5% that improves of exocellular polysaccharide, the active 15.2%-61.67% that improves of intracellular polyse.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details, in embodiment, method therefor is ordinary method if no special instructions.
PDA slant medium used: potato 200g, glucose 20g and agar 15g, water is settled to 1000 milliliters, natural pH, sterilizing 20min at 121 ℃.
Atractylodes lactone used, purchased from Rui Fensi bio tech ltd, Chengdu; Paecilomyces cicadae bacterial classification, purchased from Jiade, Jinzhai County Chinese medicinal materials company limited.
Embodiment 1
1. fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 60 orders), by following herbal medicine, formed: calculate by weight 10 parts of Semen Coiciss, 40 parts of the Radixs Astragali, 30 parts of 20 parts of matrimony vines and the bighead atractylodes rhizomes.Add 10 times of water soaking 0.5h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, filter and collect extracting solution.Repeat to extract 2 times, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.2g atractylodes lactone, 0.3g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
(1) mensuration of hypha biomass
In fermented liquid, mycelium obtains through 8 layers of filtered through gauze, and mycelium distilled water flushing 3 times, then put in 60 ℃ of baking ovens and dry to constant weight, obtain thalline dry powder, weigh.
Hypha biomass=thalline dry powder weight ÷ fermentating liquid volume.
(2) mensuration of exocellular polysaccharide content
The supernatant liquor of getting after filtering fermentation liquor mycelium adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, and 3000r/min, after 20min is centrifugal, precipitation is Crude polysaccharides.Precipitation is added to water and be settled to 50mL, by phenolsulfuric acid method, measure polysaccharide in fermentation liquid content, replication is averaged for 3 times, i.e. exocellular polysaccharide content.
Exocellular polysaccharide content=exocellular polysaccharide weight ÷ fermentating liquid volume.
(3) mensuration of intracellular polyse content
Take appropriate thalline dry powder, be placed in beaker, by solid-liquid ratio 1:10 (weight ratio), add distilled water, 90 ℃ of lixiviate 3h, extract twice, filtration under diminished pressure is collected filtrate, concentrating under reduced pressure at 60 ℃, and concentrated solution adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, 3000r/min, after 20min is centrifugal, precipitation is Crude polysaccharides.Precipitation is added to water and be settled to 50mL, by phenolsulfuric acid method, measure polysaccharide in fermentation liquid content, replication is averaged for 3 times, i.e. intracellular polyse content.
Exocellular polysaccharide content=exocellular polysaccharide weight ÷ fermentating liquid volume.
(4) the outer and mensuration of intracellular polyse to DPPH radical scavenging activity of born of the same parents
Taking 7.9mg DPPH, to be dissolved in mass percentage concentration be in 80% aqueous ethanolic solution, and constant volume is in 100mL volumetric flask, obtains the DPPH ethanolic soln of 0.2mM (mmol/L), now with the current.Get the testing sample (the exocellular polysaccharide aqueous solution or the intracellular polyse aqueous solution) of 2mL5mg/mL in test tube, add 2mL0.2mM DPPH ethanolic soln, mix, room temperature lucifuge reaction 30min, at 517nm wavelength place, measure light absorption value, according to following formula, calculate the clearance rate of testing sample to DPPH free radical.
Clearance rate (%)=(1-A
1/ A
0) * 100%
In formula, A
0for the DPPH ethanolic soln of 2mL 0.2mM adds the light absorption value of 2mL distilled water; A
1for the DPPH ethanolic soln of 2mL 0.2mM adds the light absorption value of 2mL testing sample.
Detected result in Table 1, table 2.
Embodiment 2
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 60 orders), by following herbal medicine, formed: calculate by weight 20 parts of Semen Coiciss, 30 parts of the Radixs Astragali, 20 parts of 30 parts of matrimony vines and the bighead atractylodes rhizomes.Add 10 times of water soaking 0.5h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, filter and collect extracting solution.Repeat to extract 2 times, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.4g atractylodes lactone, 0.2g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Embodiment 3
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 60 orders), by following herbal medicine, formed: calculate by weight 30 parts of Semen Coiciss, 10 parts of the Radixs Astragali, 20 parts of 40 parts of matrimony vines and the bighead atractylodes rhizomes.Add 10 times of water soaking 0.5h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, filter and collect extracting solution.Repeat to extract 2 times, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.6g atractylodes lactone, 0.1g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Embodiment 4
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 60 orders), by following herbal medicine, formed: calculate by weight 40 parts of Semen Coiciss, 30 parts of the Radixs Astragali, 20 parts of 10 parts of matrimony vines and the bighead atractylodes rhizomes.Add 10 times of water soaking 0.5h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, filter and collect extracting solution.Repeat to extract 2 times, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.3g atractylodes lactone, 0.2g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Embodiment 5
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 60 orders), by following herbal medicine, formed: calculate by weight 20 parts of Semen Coiciss, 20 parts of the Radixs Astragali, 30 parts of 30 parts of matrimony vines and the bighead atractylodes rhizomes.Add 10 times of water soaking 0.5h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, filter and collect extracting solution.Repeat to extract 2 times, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.5g atractylodes lactone, 0.2g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Embodiment 6
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 8mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 100mL, and 20 ℃, under 100r/min, shaking table is cultivated 5d, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 2g compound Chinese medicine material (pulverize, cross 100 orders), by following herbal medicine, formed: calculate by weight 30 parts of Semen Coiciss, 30 parts of the Radixs Astragali, 10 parts of 30 parts of matrimony vines and the bighead atractylodes rhizomes.Add 2 times of water soaking 1h, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 20min, filter and collect extracting solution.Repeat to extract 4 times, filtrate merging is placed in 100mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.5g atractylodes lactone, 0.2g tween 80 and 100mL 20mg/mL, adds water and fully dissolves and be settled to 150mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 5% inoculum size access liquid fermentation medium, at 20 ℃, is cultivated to 7d in 150r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Embodiment 7
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 6mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 200mL, and 30 ℃, under 150r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(3) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicine material (pulverize, cross 100 orders), by following herbal medicine, formed: calculate by weight 20 parts of Semen Coiciss, 30 parts of the Radixs Astragali, 40 parts of 10 parts of matrimony vines and the bighead atractylodes rhizomes.Add 5 times of water soaking 50min, with electric furnace, decoct, adjust suitable Heating temperature, keep gentle boiling state, boil 45min, filter and collect extracting solution.Repeat to extract 1 time, filtrate merging is placed in 50mL volumetric flask, adds water constant volume and is able to the Chinese medicine Compound Water extract that raw medicinal herbs amount meter concentration is 20mg/mL.
(4) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, the Chinese medicine Compound Water extract of 0.5g atractylodes lactone, 0.2g tween 80 and 50mL20mg/mL, adds water and fully dissolves and be settled to 100mL, and pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 15% inoculum size access liquid fermentation medium, at 30 ℃, is cultivated to 6d in 100r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Reference examples
1 fermentation culture
(1) shake-flask seed is cultivated
1L liquid seed culture medium: glucose 20g/L, yeast powder 5g/L, KH
2pO
41g/L and MgSO
40.5g/L, surplus is water, pH nature, sterilizing 20min at 121 ℃.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2after (being soya bean size) access sterilizing, in liquid seed culture medium, the bottling amount of 500mL Erlenmeyer flask is 150mL, and 25 ℃, under 120r/min, shaking table is cultivated 3d, obtains cultured seed liquor.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1g KH
2pO
4, 0.05g MgSO
4, adding water and fully dissolve and be settled to 100mL, pH nature, packs 500mL triangular flask into, 121 ℃ of sterilizing 20min.
Cultural method: cultured seed liquor, with in 10% inoculum size access liquid fermentation medium, at 25 ℃, is cultivated to 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of the mensuration of hypha biomass, exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result in Table 1, table 2.
Table 1 Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity detected result
Table 2
Numerical value in table 2 is the per-cent that in each embodiment, in Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity relative comparison example, corresponding Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity improve.
The data presentation of table 1 and table 2, the present invention can significantly improve biomass and the polysaccharide yield of Paecilomyces cicadae by adding Chinese medicine Compound Water extract, atractylodes lactone and the tween 80 of particular types, and the activity of polysaccharide also has obvious increase simultaneously.Compare with cellar culture method, adopt the Paecilomyces cicadae biological amount of the inventive method fermentation culture to improve 37.80%-134.39%, exopolysaccharides has improved 15.86%-37.89%, intracellular polyse has improved 39.74%-134.04%, the active 3.60%-33.5% that improves of exocellular polysaccharide, the active 15.2%-61.67% that improves of intracellular polyse.
In the scope limiting in preparation method of the present invention, the variation of each parameter does not affect the raising of Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity, so in preparation method of the present invention, the combination of arbitrary parameter all can realize the raising of Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity.Do not repeat them here.
Claims (8)
1. a method that improves Paecilomyces cicadae liquid fermentation production output and its lytic activity, is characterized in that, comprises step:
(1) by the Chinese medicinal materials being formed by 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, obtain Chinese medicine Compound Water extract;
(2) Chinese medicine Compound Water extract, atractylodes lactone and the tween 80 of being prepared by step (1) adds in fermentation basic medium, obtains Paecilomyces cicadae fermention medium;
(3) in Paecilomyces cicadae fermention medium Paecilomyces cicadae bacterial classification access step (2) being made, through liquid fermenting, obtain fermented liquid, then separation obtains tunning.
2. method according to claim 1, it is characterized in that, the preparation method of described Chinese medicine Compound Water extract comprises: by described amount, take Chinese medicinal materials, pulverize, add 2 times of-10 times of water soaking 0.5h-1h, then heating decocts, and keeps gentle boiling state 20min-60min, filters and collects extracting solution; Repeat to extract 1 time-4 times, merging filtrate, obtains Chinese medicine Compound Water extract.
3. method according to claim 1, is characterized in that, contains Chinese medicine Compound Water extract, the atractylodes lactone of 0.2g-0.6g and the tween 80 of 0.1g-0.3g of counting 1g-2g with raw medicinal herbs amount in the described every 100ml of Paecilomyces cicadae fermention medium.
4. method according to claim 1, is characterized in that, described fermentation basic medium: glucose 2g/100ml, yeast powder 0.4g/100ml, peptone 0.3g/100ml, KH
2pO
40.1g/100ml and MgSO
40.05g/100ml.
5. method according to claim 1, it is characterized in that, the cut-in method of described Paecilomyces cicadae bacterial classification comprises: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, in liquid seed culture medium after access sterilizing, cultivate, obtain cultured seed liquor, in the Paecilomyces cicadae fermention medium then cultured seed liquor access step (2) being made.
6. method according to claim 5, is characterized in that, the cut-in method of described Paecilomyces cicadae bacterial classification comprises: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2-8mm
2access in the liquid seed culture medium after sterilizing to 100ml-200ml, 20 ℃-30 ℃, under 100r/min-150r/min, shaking table is cultivated 3 days-5 days, obtains cultured seed liquor, in the Paecilomyces cicadae fermention medium then cultured seed liquor access step (2) being made.
7. method according to claim 1, is characterized in that, the bacterial classification inoculum size of described Paecilomyces cicadae bacterial classification is 5%-15%.
8. method according to claim 1, is characterized in that, described liquid fermentation condition: 20 ℃-30 ℃ of temperature, under rotating speed 60r/min-150r/min condition, shaking table is cultivated 5 days-7 days.
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