CN104046570B - A kind of method improving Paecilomyces cicadae liquid fermentation production output and its lytic activity - Google Patents
A kind of method improving Paecilomyces cicadae liquid fermentation production output and its lytic activity Download PDFInfo
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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
Technical field
The invention belongs to field of fermentation engineering, be specifically related to a kind of method improving Paecilomyces cicadae liquid fermentation production output and its lytic activity.
Background technology
Edible medicinal fungus Paecilomyces cicadae (Paecilomycescicadae (Mique1) Samson) has another name called female cicada fungus, being all Cordyceps with Cordyceps sinensis, Cordyceps militaris (L.) Link., is the anamorphic strain of one famous and precious traditional Chinese medicine material cicada fungus (Cordycepssobolifera) of China.The activeconstituents of Paecilomyces cicadae is similar to Cordyceps sinensis with effect, containing secondary metabolite (He Liang such as nucleosides material, myriocin, ergosterol, cordycepic acid, polysaccharide, multiple indispensable amino acid, N.F,USP MANNITOL and alkaloids, 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 (WengSC, ChouCJ, LinLC, etal.ImmunomodulatoryfunctionsofextractsfromtheChineseme dicinalfungusCordycepscicadae [J] .Journalofethnopharmacology, 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; ChyauCC, ChenCC, ChenJC, etal.MyceliaglycoproteinsfromCordycepssoboliferaameliora tecyclosporine-inducedrenaltubuledysfunctioninrats [J] .Journalofethnopharmacology, 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 [J] of rat nutritional status. Journal of Wenzhou Medical College, 2005,35 (1): 13-15; Zhu Yan, Cheng Dongqing. the mensuration [J] of cicada fungus polysaccharide anti-oxidative activity. the total medical academic periodical of China, 2009,27 (7): 1552-1554; Zhuo Jia, Yang Jie bores, Jin Liqin, etc. the external restraining effect to hepatitis B replication of paecilomyces cicadae polysaccharide [J]. Chinese Journal of Pathophysiology, 2010,26 (2): 327-332.), therefore, Paecilomyces cicadae as the surrogate of Cordyceps sinensis, can have the prospect of good investigation and application, Paecilomyces cicadae preparation may make significant contribution in 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 and be used to the nutritive ingredient that the Mierocrystalline cellulose in Chinese medicine, starch, protein, lipid etc. are abundant, promotes fungal growth and Product formation, thus obtains the meta-bolites that enriches.Herb fermenting technical study finds, fungi some active substance such as terpene, flavonoid, alkaloid, soaping agents in metabolic process also likely in centering medicine carries out bio-transformation, form new composition or the higher material of activity, obviously strengthen biological activity and the pharmacological function of tunning.The growth metabolism process of traditional Chinese medicine ingredients and Paecilomyces cicadae also exists interrelated, traditional Chinese medicine ingredients likely participates in the pathways metabolism of Paecilomyces cicadae, affect the accumulation of its exocellular polysaccharide and biosynthetic relevant enzyme, as the important enzyme that phosphoglucoisomerase (PGI) is in glycolytic pathway, α-phosphoglucomutase (α-PGM) synthesizes relevant important enzyme 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, inhibit glycolytic pathway, thus be conducive to the advance (He Zongyi of G-6-P to polysaccharide route of synthesis, Wu Tianxiang, Xu Xiaobao. gastrodia elata composition is on Extracellular Polysaccharide from Grifola frondosa synthesis and the impact [J] of related keyword enzyme. Food science, 2013,34 (11): 199-202.).The change of metabolite activity may be after adding Chinese medicine in substratum, stimulate or inhibit 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, create new secondary metabolite, thus change the activity of meta-bolites.
Research finds some oil substances and tensio-active agent in edible medicinal fungus liquid fermenting is produced except as except defoamer, can also have an impact to the growth metabolism of fungi, people just start the synthesis (HsiehC attempting promoting edible medicinal fungus given activity composition as inducible factor with them, WangHL, ChenCC, etal.Effectofplantoilandsurfactantontheproductionofmycel ialbiomassandpolysaccharidesinsubmergedcultureofGrifolaf rondosa [J] .BiochemicalEngineeringJournal, 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.).Lipid phase can promote that fungal growth metabolism and oil or lipid acid changes fungal cell's membrane structure and permeability or direct to affect some important enzymic activity in pathways metabolism relevant.The Semen Coicis fat of research discovery 0.2% contributes to the synthesis 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 the impact (ZhouH of Semen Coicis fat, BiP, WuX, etal.Improvedpolysaccharideproductioninsubmergedcultureo fGanodermalucidumnbytheadditionofcoixenolide [J] .AppliedBiochemistryandBiotechnology, 2014,172:1497-1505.).Surface active agent tween 80 affects the metabolic mechanism of edible medicinal fungus may relevant (ZhangBB active in the complete structure of mycelial cell film and transdermal delivery, CheungPCK.AmechanisticstudyoftheenhancingeffectofTween80 onthemycelialgrowthandexopolysaccharideproductionbyPleur otustuber-regium [J] .Bioresourcetechnology, 2011,102 (17): 8323-8326.).The molecular structure of tensio-active agent has amphipathic, and fungal cell's membrane structure is also made up of the phosphatide that one deck is amphipathic, thus tensio-active agent may locally be fitted in cytolemma, thus accelerate the speed (ChenHB that cell absorbs nourishment from substratum, HuangHC, ChenCI, etal.Theuseofadditivesasthestimulatoronmycelialbiomassan dexopolysaccharideproductionsinsubmergedcultureofGrifola umbellate [J] .Bioprocessandbiosystemsengineering, 2010, 33 (3): 401-406.).Research shows, tensio-active agent such as tween 80 effectively can promote the synthesis of active metabolite in edible medicinal fungus, the tween 80 of 0.1% (w/v) is added at the 72h of Antrodia camphorata liquid fermenting, the output of AntrodinC is apparently higher than contrast, 103.76 ± 0.92mg/L is brought up to by 52.37 ± 1.02mg/L, in the coupled fermentation system of tween 80 and soybean oil, the maximum production of AntrodinC is 3.6 times of (ZhangH of contrast, XiaY, WangYL, etal.Couplinguseofsurfactantandinsituextractantforenhanc edproductionofAntrodinCbysubmergedfermentationofAntrodia camphorata [J] .BiochemicalEngineeringJournal, 2013, 79:194-199.).
Disclose in Chinese patent ZL200510068284.9 and a kind ofly add the coprinus comatus liquid fermentation process of balsam pear and the method for leavened prod and application, its processing method is by adding appropriate Chinese medicine balsam pear in coprinus comatus liquid fermentation medium, coprinus comatus carries out bio-transformation to balsam pear composition during the fermentation, improves the activity of fermented liquid.A kind of cultural method and special culture media thereof of Cordyceps sinensis is disclosed in Chinese patent ZL200510137457.8, it is disclosed that and add the method that soybean oil carries out Cordyceps sinensis liquid fermentation and culture, this method has fast growth to fermentative production Cordyceps mycelium, the cycle is short, productive rate is high, technique is simple, low cost and other advantages.By adding appropriate traditional Chinese medicine ingredients or oil substances in substratum, significantly promoting the growth of edible and medicinal fungi or improving the product quantifier elimination of active metabolite, all repeatedly reporting both at home and abroad, but application on Paecilomyces cicadae liquid fermenting there is not been reported.
Summary of the invention
The object of this invention is to provide a kind of method improving Paecilomyces cicadae liquid submerged fermentation Product yields and its lytic activity.
The present invention finds, the Chinese medicine composite extract of particular types, grease and tensio-active agent is added in fermention medium, carry out the liquid fermentation and culture of Paecilomyces cicadae, contribute to the content improving Paecilomyces cicadae biological amount and secondary metabolite, promote that metabolite activity improves simultaneously.
Improve a method for Paecilomyces cicadae liquid fermentation production output and its lytic activity, comprise step:
(1) by 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, obtain Chinese medicine Compound Water extract;
(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, when carrying out Paecilomyces cicadae liquid fermenting, directly Chinese medicinal materials can not 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 the substratum made containing Chinese medical extract, contribute to the content improving Paecilomyces cicadae biological amount and secondary metabolite like this, promote that metabolite activity improves simultaneously.
In step (1), the preparation of described Chinese medicine Compound Water extract can adopt the water extraction of this area routine, preferred employing following methods: take Chinese medicinal materials by described amount, pulverize (preferred mistake 60 order-100 object powder), add 2 times-10 times water soaking 0.5h-1h, then heating decocts, and keeps gentle boiling state 20min-60min, collecting by filtration extracting solution; Repeat extraction 1 time-4 times, merging filtrate, obtain Chinese medicine Compound Water extract.
In step (2), described atractylodes lactone can adopt commercially available prod, and existing method also can be adopted to prepare; As the people such as Yang Lin can be adopted at " the supercritical CO of atractylenolide Ⅰ in the bighead atractylodes rhizome
2extraction process is studied " the atractylodes lactone preparation method that records in a literary composition be prepared (Yang Lin, He Dan. the supercritical CO of atractylenolide Ⅰ in the bighead atractylodes rhizome
2extraction process is studied. herbal medicine, 2006,37 (9): 1331-1333); Specifically can adopt following preparation method: Rhizoma Atractylodis Macrocephalae is pulverized (preferred powder is broken to 60 order-100 object powder), be that the aqueous ethanolic solution of 10%-20% is for carrying agent with mass percentage concentration, extracting pressure 25MPa, resolve pressure 5MPa, extraction temperature 40 DEG C-45 DEG C, 4h-4.5h is extracted, obtained atractylodes lactone under the condition that resolution temperature is 30 DEG C-35 DEG C.
Containing the Chinese medicine Compound Water extract, the atractylodes lactone of 0.2g-0.6g and the tween 80 of 0.1g-0.3g that are 1g-2g with raw medicinal herbs gauge in the described every 100ml of Paecilomyces cicadae fermention medium.
Described fermentation basic medium can adopt the fermentation basic medium that this area liquid fermenting is conventional, and preferred fermentation basic medium 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.Such as Paecilomyces cicadae bacterial strain Paecilomycescicadae (Miq.) SamsonCGMCCNo.3453, this bacterial strain registers preservation on November 18th, 2009 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC).
The cut-in method of described Paecilomyces cicadae bacterial classification is the ordinary method of this area, comprise: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, cultivate in liquid seed culture medium after access sterilizing, obtain cultured seed liquor, then by Paecilomyces cicadae fermention medium obtained for cultured seed liquor access step (2).Further preferably: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2-8mm
2to 100ml-200ml in liquid seed culture medium after access sterilizing, 20 DEG C-30 DEG C (most preferably 25 DEG C), under 100r/min-150r/min (most preferably 120r/min), shaking table cultivates 3 days-5 days, obtain cultured seed liquor, then by Paecilomyces cicadae fermention medium obtained for cultured seed liquor access step (2).
The substratum that described PDA slant medium and liquid seed culture medium all adopt this area seed culture conventional, can adopt commercially available prod.Further preferably, described PDA slant medium: potato 200g, glucose 20g and agar 15g-20g, be settled to 1000 milliliters with water.Further preferably, described liquid seed culture medium: glucose 20g, yeast powder 5g, KH
2pO
41g and MgSO
40.5g, is settled to 1000 milliliters with water.
The strain inoculation amount of described Paecilomyces cicadae bacterial classification is preferably 5%-15%.Liquid fermentation condition is preferred: temperature 20 DEG C-30 DEG C, and under rotating speed 60r/min-150r/min condition, shaking table cultivates 5 days-7 days.
The cultured seed liquor volume of the bacterial classification volume that described strain inoculation amount 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, can select filtration, alcohol precipitation filters, one or more in the separation method such as centrifugal.
In the present invention, described Chinese medicinal materials meets the requirement of the Pharmacopoeia of the People's Republic of China.
Semen Coicis, this product is the dry mature kernal of grass Job's tears Coixlacryma-jobiL.var.ma-yuen (Roman.) Stapf.Tap plant during fruit maturation in autumn, dry, lay fruit, then dry, except decapsidate, tawny seed coat and impurity, collect 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 seed coat.The blunt circle in one end, the wider and nick of another end, has 1 light brown point-like hilum.Back side boss, the outside of belly has 1 wider 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 Astragalusmembranaceus (Fisch.) Bge.var.mongho-licus (Bge.) Hsiao or Radix Astragali Astragalusmembranaceus (Fisch.) Bge..Spring, Qiu Erji excavate, and removing fibrous root and root head, dry.Proterties: this product is cylindrical, what have has branch, and upper end is comparatively thick, 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, not frangibility, and section fibrous is strong, and aobvious mealiness, skin zone's yellow-white, and woody part is faint yellow, has radial texture and crack, and Lao Gen center is even dry and decayed shape, chocolate or in cavity.Gas is micro-, and taste is micro-sweet, and that chews micro-ly has beany flavor.
Matrimony vine, i.e. wolfberry fruit, this product is the dry mature fruit of plant of Solanaceae lycium barbarum LyciumbarbarumL..Summer, autumn two gather when season, fruit took on a red color, hot-air seasoning, removing carpopodium.Or dry in the air to rhicnosis, dry, removing 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 AtractylodesmacrocephalaKoidz..Excavate when withered and yellow, the upper leaf of inferior leads in winter becomes fragile, removing silt, dries or dries, 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, there are residual stem foot and bud trace in top.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 comparatively dark or have 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 the Chinese medicine Compound Water extract of particular types, atractylodes lactone and tween 80, the activity of polysaccharide also has obvious increase simultaneously.Wherein compared with conventional culture methods, the Paecilomyces cicadae biological amount of the inventive method fermentation culture is adopted to improve 37.80%-134.39%, exopolysaccharides improves 15.86%-37.89%, intracellular polyse improves 39.74%-134.04%, exocellular polysaccharide activity improves 3.60%-33.5%, and intracellular polyse activity improves 15.2%-61.67%.
Embodiment
Be described in further details the present invention below in conjunction with embodiment, in embodiment, method therefor is ordinary method if no special instructions.
PDA slant medium used: potato 200g, glucose 20g and agar 15g, be settled to 1000 milliliters with water, natural pH, sterilizing 20min at 121 DEG C.
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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 60 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 10 parts, the Radix Astragali 40 parts, matrimony vine 20 parts and the bighead atractylodes rhizome 30 parts.Add 10 times of water soaking 0.5h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, collecting by filtration extracting solution.Repeat extraction 2 times, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 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 puts and dry to constant weight in 60 DEG C of baking ovens, obtain thalline dry powder, weigh.
Hypha biomass=thalline dry powder weight ÷ fermentating liquid volume.
(2) mensuration of exocellular polysaccharide content
The supernatant liquor got after filtering fermentation liquor mycelium adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, and after 3000r/min, 20min are centrifugal, precipitation is Crude polysaccharides.Precipitation added water and be settled to 50mL, measure polysaccharide in fermentation liquid content with phend-sulphuric acid, 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, add distilled water by solid-liquid ratio 1:10 (weight ratio), 90 DEG C of lixiviate 3h, extract twice, filtration under diminished pressure collects filtrate, concentrating under reduced pressure at 60 DEG C, and concentrated solution adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, after 3000r/min, 20min are centrifugal, precipitation is Crude polysaccharides.Precipitation added water and be settled to 50mL, measure polysaccharide in fermentation liquid content with phend-sulphuric acid, replication is averaged for 3 times, i.e. intracellular polyse content.
Exocellular polysaccharide content=exocellular polysaccharide weight ÷ fermentating liquid volume.
(4) the outer and intracellular polyse of born of the same parents is to the mensuration of DPPH radical scavenging activity
Taking 7.9mgDPPH, to be dissolved in mass percentage concentration be in the aqueous ethanolic solution of 80%, 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 2mL0.2mMDPPH ethanolic soln, mix, room temperature lucifuge reaction 30min, measure light absorption value at 517nm wavelength place, calculate testing sample according to the following formula to the clearance rate of DPPH free radical.
Clearance rate (%)=(1-A
1/ A
0) × 100%
In formula, A
0dPPH ethanolic soln for 2mL0.2mM adds the light absorption value of 2mL distilled water; A
1dPPH ethanolic soln for 2mL0.2mM adds the light absorption value of 2mL testing sample.
Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 60 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 20 parts, the Radix Astragali 30 parts, matrimony vine 30 parts and the bighead atractylodes rhizome 20 parts.Add 10 times of water soaking 0.5h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, collecting by filtration extracting solution.Repeat extraction 2 times, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 60 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 30 parts, the Radix Astragali 10 parts, matrimony vine 40 parts and the bighead atractylodes rhizome 20 parts.Add 10 times of water soaking 0.5h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, collecting by filtration extracting solution.Repeat extraction 2 times, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 60 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 40 parts, the Radix Astragali 30 parts, matrimony vine 10 parts and the bighead atractylodes rhizome 20 parts.Add 10 times of water soaking 0.5h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, collecting by filtration extracting solution.Repeat extraction 2 times, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 60 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 20 parts, the Radix Astragali 20 parts, matrimony vine 30 parts and the bighead atractylodes rhizome 30 parts.Add 10 times of water soaking 0.5h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 60min, collecting by filtration extracting solution.Repeat extraction 2 times, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 8mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 100mL, 20 DEG C, and under 100r/min, 5d cultivated by shaking table, obtains cultured seed liquor.
(2) Chinese medicine Compound Water extract preparation
Take 2g compound Chinese medicinal material (pulverize, cross 100 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 30 parts, the Radix Astragali 30 parts, matrimony vine 30 parts and the bighead atractylodes rhizome 10 parts.Add 2 times of water soaking 1h, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 20min, collecting by filtration extracting solution.Repeat extraction 4 times, filtrate merging is placed in 100mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
4, the Chinese medicine Compound Water extract of 0.5g atractylodes lactone, 0.2g tween 80 and 100mL20mg/mL, adds water and fully dissolves and be settled to 150mL, and pH nature, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 5%, at 20 DEG C, cultivate 7d in 150r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 6mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 200mL, 30 DEG C, and under 150r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(3) Chinese medicine Compound Water extract preparation
Take 1g compound Chinese medicinal material (pulverize, cross 100 orders), be made up of following herbal medicine: calculate by weight, Semen Coicis 20 parts, the Radix Astragali 30 parts, matrimony vine 10 parts and the bighead atractylodes rhizome 40 parts.Add 5 times of water soaking 50min, decoct with electric furnace, adjust suitable Heating temperature, keep gentle boiling state, boil 45min, collecting by filtration extracting solution.Repeat extraction 1 time, filtrate merging is placed in 50mL volumetric flask, and the constant volume that adds water is able to the Chinese medicine Compound Water extract that raw medicinal herbs gauge concentration is 20mg/mL.
(4) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
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, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 15%, at 30 DEG C, cultivate 6d in 100r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 DEG C.
Cultural method: by the Paecilomyces cicadae bacterial classification after activation in PDA slant medium, get 5mm
2(i.e. soya bean size) accesses after sterilizing in liquid seed culture medium, and the bottling amount of 500mL Erlenmeyer flask is 150mL, 25 DEG C, and under 120r/min, 3d cultivated by shaking table, obtains cultured seed liquor.
(3) shake flask fermentation is cultivated
Liquid fermentation medium: 2g glucose, 0.4g yeast powder, 0.3g peptone, 0.1gKH
2pO
4, 0.05gMgSO
4, add water and fully dissolve and be settled to 100mL, pH nature, loads 500mL triangular flask, 121 DEG C of sterilizing 20min.
Cultural method: by cultured seed liquor with in the inoculum size access liquid fermentation medium of 10%, at 25 DEG C, cultivate 5d in 60r/min shaking table, obtain fermented liquid.Each test establish 3 parallel.
2 measure
The mensuration of the mensuration of hypha biomass, the mensuration of exocellular polysaccharide content and intracellular polyse content is with embodiment 1.Detected result is 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 Paecilomyces cicadae liquid fermenting meta-bolites output corresponding in Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity relative comparison example in each embodiment 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 the Chinese medicine Compound Water extract of particular types, atractylodes lactone and tween 80, the activity of polysaccharide also has obvious increase simultaneously.Compared with conventional culture methods, the Paecilomyces cicadae biological amount of the inventive method fermentation culture is adopted to improve 37.80%-134.39%, exopolysaccharides improves 15.86%-37.89%, intracellular polyse improves 39.74%-134.04%, exocellular polysaccharide activity improves 3.60%-33.5%, and intracellular polyse activity improves 15.2%-61.67%.
In the scope that preparation method of the present invention limits, the change of each parameter does not affect the raising of Paecilomyces cicadae liquid fermenting meta-bolites output and its lytic activity, and therefore 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. improve a method for Paecilomyces cicadae liquid fermentation production output and its lytic activity, it is characterized in that, comprise step:
(1) by 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, obtain Chinese medicine Compound Water extract;
(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.
2. method according to claim 1, it is characterized in that, the preparation method of described Chinese medicine Compound Water extract comprises: take Chinese medicinal materials by described amount, pulverize, add 2 times-10 times water soaking 0.5h-1h, then heating decocts, and keeps gentle boiling state 20min-60min, collecting by filtration extracting solution; Repeat extraction 1 time-4 times, merging filtrate, obtain Chinese medicine Compound Water extract.
3. method according to claim 1, is characterized in that, containing the Chinese medicine Compound Water extract, the atractylodes lactone of 0.2g-0.6g and the tween 80 of 0.1g-0.3g that are 1g-2g with raw medicinal herbs gauge 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, cultivate in liquid seed culture medium after access sterilizing, obtain cultured seed liquor, then by Paecilomyces cicadae fermention medium obtained for cultured seed liquor access step (2).
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
2to 100ml-200ml in liquid seed culture medium after access sterilizing, 20 DEG C-30 DEG C, under 100r/min-150r/min, shaking table cultivates 3 days-5 days, obtains cultured seed liquor, then by Paecilomyces cicadae fermention medium obtained for cultured seed liquor access step (2).
7. method according to claim 1, is characterized in that, the strain inoculation amount of described Paecilomyces cicadae bacterial classification is 5%-15%.
8. method according to claim 1, is characterized in that, described liquid fermentation condition: temperature 20 DEG C-30 DEG C, and under rotating speed 60r/min-150r/min condition, shaking table cultivates 5 days-7 days.
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