CN103233056B - Preparation method for great burdock fruit extract through microbial combined fermentation - Google Patents
Preparation method for great burdock fruit extract through microbial combined fermentation Download PDFInfo
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Abstract
The invention specifically discloses a preparation method for a great burdock fruit extract through combined microbial fermentation, which belongs to the technical field of preparation of traditional Chinese medicinal material extracts through microbial fermentation. According to the invention, the great burdock fruit extract is prepared through combined fermentation of Aspergillus awamori var. CICC2203 and Trichoderma reesei CICC40932, optimization study is carried out on fermentation process conditions, so a conversion rate of arctiin in great burdock fruit into arctigenin is substantially improved and reaches 99.9%, the problem of a low conversion rate of arctiin into arctigenin is effectively overcome, and a technical problem of industrial production of the great burdock fruit extract is overcome.
Description
Technical field
The present invention relates to the microorganism method and technology field of preparing Chinese medicinal materials extract of fermenting, be specifically related to a kind of method that Fructus Arctii extract is prepared in microbial association fermentation.
Background technology
Great Burdock Achene (Fructus Arctii), mainly contains the Lignanoids compounds such as Arctiin (Arctiin) and l-arctigenin (Arctigenin, ACT).Arctiin is broken down into l-arctigenin in vivo and produces numerous pharmacological actions.L-arctigenin has antibacterial significantly, antiviral, antitumor, anti-inflammatory and calcium antagonistic activity.And l-arctigenin source is abundant, and its precursor Arctiin content in Chinese medicine burdock and burr burdock fruit is higher, there is very high new drug development and be worth.
The at present domestic existing helicase that utilizes carries out the report of external enzymolysis to the Arctiin extracting, but comes with some shortcomings: complex process, high cost; And early stage research proves that Lignanoids compounds can decompose the in the situation that of acid or alkali already, but can there is isomerization, change original structure, thereby cause the reduction of drug effect.
Therefore, the applicant intends adopting the method for microbial association fermentation to prepare l-arctigenin extract, utilize the enzyme system that microorganisms is abundant, under mild conditions, the ability of decomposition and inversion material is strong, process of preparing Chinese medicine means than general physics or chemistry change the property of medicine by a larger margin, improve curative effect, reduce toxic side effect, expand indication.Also there is the environmental pollution of minimizing simultaneously, the advantage such as reduce production costs, under controlled condition, leniently Arctiin is changed into l-arctigenin effectively in vitro, produce conversion to reach industrialization, effectively improved the content of l-arctigenin in Fructus Arctii extract.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of microbial association fermentation to prepare the method for Fructus Arctii extract.
Object of the present invention is achieved by the following technical programs:
A method for Fructus Arctii extract is prepared in microbial association fermentation, it is characterized in that: described microorganism is Aspergillus awamori mutation and Li's Trichoderma.
Further, described Aspergillus awamori mutation and Li's Trichoderma and be respectively Aspergillus awamori mutation Aspergillus awamori var.CICC2203 and Trichodermareesei Trichoderma reesei CICC40932.
Further, the inoculum size of described Aspergillus awamori mutation and Li's Trichoderma is than being 1:2.
Compared with prior art, the advantage of the inventive method and beneficial effect are:
1, adopt microbe fermentation method prepare Fructus Arctii extract, prepare Fructus Arctii extract with vitro enzyme solution compared with, technique is simple, cost is low, not recurring structure alienation, has intactly kept original chemical structure of Fructus Arctii extract effective constituent, keeps the good drug effect of extract;
2, adopt microbe fermentation method to prepare Fructus Arctii extract, compared with preparing Fructus Arctii extract with physics or chemical process, under mild conditions, utilize the enzyme system that microorganisms is abundant, the advantage that decomposition and inversion physical capacity is strong, change significantly the property of medicine, transformation efficiency and the curative effect of effective constituent are improved, reduce toxic side effect, expand indication, reduced widely the pollution of organic reagent to ecotope;
3, the present invention adopts the method for microbial association fermentation, determine that Aspergillus awamori mutation and Li's Trichoderma are for best fermentation combination, compared with single strain fermentation, improve to a great extent the transformation efficiency that Arctiin in Great Burdock Achene is converted into l-arctigenin, reach 99.9%, effectively solve the problem that Arctiin is converted into l-arctigenin low conversion rate, produced and solved technical barrier for Fructus Arctii extract industrialization.
Brief description of the drawings
Fig. 1 is l-arctigenin color atlas;
Fig. 2 is different strain and combined fermentation effect comparative result figure thereof;
Fig. 3 is the affect schematic diagram of carbon source on ferment effect;
Fig. 4 is the affect schematic diagram of nitrogenous source on ferment effect;
Fig. 5 is the affect schematic diagram of time on ferment effect;
Fig. 6 is the affect schematic diagram of liquid amount on ferment effect;
Fig. 7 is the affect schematic diagram of pH on ferment effect;
Fig. 8 is the affect schematic diagram of inoculum size on ferment effect;
Fig. 9 is optimization of orthogonal test result schematic diagram.
Embodiment
Following content is for further setting forth technical scheme of the present invention, so that those skilled in the art have more deep understanding to the present invention, but following content should not be understood to the restriction to the claims in the present invention book request protection domain by any way.
This research department is mainly engaged in the research of herbal medicine and Animal diseases aspect, Great Burdock Achene is the traditional Chinese medicine rarity of China, there is very high pharmaceutical use, the research in earlier stage of this research department finds that l-arctigenin has the characteristic of good anti-PCV-II, also there is the good effect such as anti-inflammatory, immunomodulatory simultaneously, these good medicinal characteristics of l-arctigenin, have increased our dense research interest, prepare to be developed as the new veterinary drug of country.But the huge difficult problem that becomes us is extracted in the mass-producing of l-arctigenin; there is no at present this difficult problem of fine solution at home and abroad yet; we adopt chemical extraction method; successfully made l-arctigenin sterling in laboratory; but complex process; cost is high, and a large amount of chemical reagent contaminate environment, cannot realize scale operation.In adopting the fermentation of microorganism single strain, do test of many times, effect is all undesirable; In one time fermentation test, catching sight of Arctiin in battery of tests result, to be converted into the transformation efficiency of l-arctigenin quite high, transformation efficiency almost reaches 100%, finally find it is in the process of plating, in Trichodermareesei bacterial classification flat board, sneak into due to Aspergillus awamori mutation spore, so we intend taking combined ferment, test-results has further been confirmed in test below.
The selection of bacterial classification has very strong purpose really, selected bacterial classification is all that some are conventional, from ancient times to the present, people are producing, in life, all utilizing these fungies to carry out microorganism fermentation, microorganism can produce abundant enzyme system, thereby there is an ability of decomposition and inversion material very powerful under mild conditions, and can produce abundant secondary metabolite, by microbial growth metabolism and the vital movement process of preparing Chinese medicine Chinese medicine that ferments, can change by a larger margin the property of medicine than the process of preparing Chinese medicine means of general physics or chemistry, improve curative effect, reduce toxic side effect, expand indication.Also possess the environmental pollution of minimizing simultaneously, lower the advantages such as production cost.The above-mentioned particular advantages having based on Institute of Micro-biology, this research adopts the mode of enteric microorganism conversion in external microbe fermentation method analogue body effectively to transform Arctiin and generates l-arctigenin, utilize superior strain and the Great Burdock Achene of this six strains beta-glucosidase of screening preservation to carry out common fermentation, utilize the enzyme system effect of microorganism to make the glycosidic link fracture of arctinin and generate l-arctigenin and glucose.Under controlled condition, leniently realize Arctiin is converted into l-arctigenin in vitro, produce and transform to reach to industrialization, effectively improve the content of l-arctigenin in Fructus Arctii extract.
1 test materials
1.1 bacterial classification
Nipa palm aspergillus Aspergillus phoenicis CICC2216; Trichodermareesei Trichoderma reesei CICC40932; Aspergillus awamori mutation Aspergillus awamori var.CICC2203; Aspergillus oryzae Aspergillus oryzae CICC2014 buys in Chinese industrial microbial strains preservation administrative center.Aspergillus niger Aspergillus niger ATCC16404; Viride Trichoderma viride CICC41495 is presented by professor Hao Bo of Life Sci-Tech institute of Hua Zhong Agriculture University.
1.2 instrument and equipment
Bechtop (SW-CJ-1FD), JY2002 type electronic balance, high-pressure sterilizing pot, constant incubator, constant-temperature shaking incubator, Agilent 1100 type high performance liquid chromatographs (Agilent company of the U.S.), KQ-100B type Ultrasonic Cleaners (Kunshan Ultrasonic Instruments Co., Ltd.'s production), the automatic dual pure water distiller of SZ-93 type, RE-5203A type Rotary Evaporators (Shanghai Precision Scientific Apparatus Co., Ltd), AUY120 type analysis balance (Japanese Shimadzu company), CSIO1 type electric drying oven with forced convection (Chongqing testing installation factory), microscope, pH meter, magnetic stirrer, Soxhlet reflux, Erlenmeyer flask (250mL, 50mL), volumetric flask, blood counting chamber, glass sphere, kapillary, development chamber, transfer pipet, liquid-transfering gun, test tube, absorbent cotton, gauze.
1.3 medicines and reagent
L-arctigenin reference substance, purity > 99%, lot number 120307, Arctiin reference substance, purity > 99%, lot number 110107, upper Hiroad standing grain medical sci-tech Development Co., Ltd; Great Burdock Achene powder (200 order), lot number 20110402, Wuhan Qiang Kang pharmaceutcal corporation, Ltd; Wheat bran, Semen Maydis powder, bean cake powder, lot number 20120302, Wuhan An You feed corporation,Ltd; Sucrose, methyl alcohol, ethanol, trichloromethane, Glacial acetic acid, propyl carbinol, glucose, agar, sterilized water, potassium primary phosphate, ammonium sulfate, ammonium nitrate, ammonium chloride, urea, magnesium sulfate, calcium chloride, ferrous sulfate, magnesium sulfate, zinc sulfate, cobalt chloride, hydrochloric acid, sodium hydroxide, sulfuric acid, tween 80, peptone, analytical pure, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group; GF254 silica-gel plate, lot number 20120108, Qingdao Marine Chemical Co., Ltd..
2 test methods
2.1 the preparation of substratum
2.1.1 plate culture medium
PDA(Potato Dextrose Agar) its way be first by potato clean peeling, taking 200g is cut into small pieces again, add water and well-donely (boil 20-30min, can be poked by glass stick), by four layers of filtered through gauze, in filtrate, add 20g glucose and 15g agar, supply again moisture to 1000mL, 121 DEG C of sterilizing 15min, make plate culture medium, and cooling rear storage is for subsequent use.
2.1.2 seed culture medium
Mandel nutritive medium: potassium primary phosphate 2g, ammonium sulfate 1.4g, urea 0.3g, magnesium sulfate 0.3g, calcium chloride 0.3g, ferric sulfate 5mg, manganous sulfate 1.56mg, zinc sulfate l.4mg, cobalt chloride 0.2mg, sterilized water is settled to 1000mL.
In Mandel nutritive medium, add tween 80 2mL/L, peptone 1g/L, glucose 10g/L, adjusts pH to 5.0-6.0 with sulfuric acid, and 115 DEG C of sterilizing 20min, obtain seed culture fluid.
2.1.3 fermention medium
Take Great Burdock Achene powder 8g, wheat bran 5g, Semen Maydis powder 5g, peptone 0.6g, packs in 250mL Erlenmeyer flask, 121 DEG C of sterilizing 15min.Then add aseptic Mandel nutritive medium 20mL, and to inject sterilized water be 130mL by liquid amount constant volume, 121 DEG C of sterilizing 15min.
The activation of 2.2 bacterial classifications
Bacterial classification freeze-dried powder storage in vacuum-drying pipe and cryopreservation, returns to room temperature after taking-up, be transferred in Bechtop.To speckle with the cotton of 75% alcohol, wiping outer tube heats outer tube tip on flame.Drip several sterilized waters in heating place, make outer tracheal rupture.Take out heat insulation fiber matter and inner tube, take out inner tube tampon with the tweezers of sterilizing.Draw 0.3-0.5mL sterilized water with aseptic straw, splash in inner tube, after powder dissolution to be dried, draw and splash in the sterilizing test tubes that approximately contains 5mL sterilized water with aseptic straw, slight concussion leaves standstill 30-60min after making it evenly.Get 0.1-0.2mL thallus suspension liquid and evenly coat on the PDA plate culture medium of making, do separation and Culture, culture temperature is 28-30 DEG C.Inspection bacterial classification purity and Activation.
The preparation of 2.3 kinds of daughter bacteria liquid
Get the bacterial classification having activated, adopt aseptic technique to inoculate PDA plate culture medium.In incubator, cultivate 5-6d for 28-30 DEG C.After completing, cultivation can produce ripe spore.
Get cultured bacterial classification flat board, add 10ml sterilized water, rock gently the spore of agar surface is swept away, this spore suspension is placed in sterilizing 50ml Erlenmeyer flask, in bottle, place in advance several aseptic glass spheres, three layers of gauze with sterilizing after shake well filter, and with aseptic water washing filter residue 2-3 time, finally make filtrate volume reach 10.0ml, spore suspension to be measured and get final product.
Sterilized water dilution 100-1000 for spore suspension doubly, is drawn to appropriate diluent to the nucleonics of blood counting chamber, then add cover glass, leave standstill about 5min, first under low power lens, find nucleonics, then convert high power lens to and observe and count.Calculate spore concentration, and to be mixed with concentration be 10
6-10
7the spore suspension of individual/mL is for subsequent use.
Get the seed culture medium that 50mL prepares and be placed in 250mL Erlenmeyer flask, draw that 2mL prepares 10
6-10
7the spore suspension of individual/mL is inoculated.175rpm, cultivates 1-2d under 28-30 DEG C of condition, obtains kind of a daughter bacteria liquid.
The assay of Arctiin and l-arctigenin in 2.4 Great Burdock Achene powder
2.4.1 the preparation of reference liquid
Accurately take Arctiin standard substance 4.59 ㎎, l-arctigenin standard substance 11.68 ㎎ are dissolved in 25mL methyl alcohol, are mixed with Arctiin and l-arctigenin standard substance volumetric molar concentration is respectively 3.435 × 10
-4mmol/L and 1.246 × 10
-3the mixed standard solution of mmol/L.
2.4.2 chromatogram testing conditions
Fermentation gained sample adopts Agilent high performance liquid chromatograph, under 280nm wavelength, detects; Fig. 1 is l-arctigenin chromatographic behavior in Aspergillus awamori mutation and Li's Trichoderma combined fermentation sample.
2.4.2 the extraction of Arctiin and l-arctigenin
Take 8g Great Burdock Achene powder, carry out refluxing extraction with 80% ethanol of 10 times of quality, extract 3 times, each 1h.Merge the extracting solution of 3 times, with after Rotary Evaporators evaporate to dryness, take out the medicinal extract in flask and dry, use 50mL methanol constant volume, make solution to be measured.Extract three parts of parallel sample by same procedure.By above-mentioned HPLC condition, extract is detected.Adopt external standard method to calculate respectively the amount of substance concentration (C of Arctiin and l-arctigenin in Great Burdock Achene powder
01with C
02), thereby as without the former powder control sample of fermentation medicine, be calculated as follows the transformation efficiency of medicinal powder, solubility rate and rate of loss by fermentation.
T(%)=(C
01-C
e1)/C
01×100；
S(%)=(C
e1+C
e2)/(C
01+C
02)×100；
L(%)=100%-S(%)
In formula: T-transformation efficiency; S-solubility rate; L-rate of loss; C
01-without Arctiin amount of substance concentration (mol/mL) in fermentation medicinal powder; C
e1-arctiin amount of substance concentration (mol/mL) in medicinal powder by fermentation; C
02-without l-arctigenin amount of substance concentration (mol/mL) in fermentation medicinal powder; C
e2-l-arctigenin amount of substance concentration (mol/mL) in medicinal powder by fermentation.
The fermentation of 2.5 Great Burdock Achene powder is concocted
2.5.1 the screening of fermented bacterium
Respectively by the nipa palm aspergillus CICC2216 preparing, Trichodermareesei CICC40932, Aspergillus awamori mutation CICC2203, aspergillus oryzae CICC2014, shown in 41495 kinds of daughter bacteria liquid accordings to the form below 1 of aspergillus niger ATCC 16404 and viride CICC, inoculum size and permutation and combination are seeded in the fermention medium having prepared, and every group is done 3 parallel sample.28-30 DEG C, 175rpm, fermentation time is 168h.
Table 1 inoculation test table
In the backward every bottle of sample of fermentation ends, add the 1mol/L NaOH solution of 10mL to stop fermenting process, get the rear sample of fermentation, 100 DEG C of oven dry, pulverize dry-matter, and methanol constant volume is to 50mL, and filter paper filtering is also collected filtrate, i.e. l-arctigenin crude extract solution.
2.5.2TLC method detects ferment effect
L-arctigenin and Arctiin standard substance are mixed with respectively to 2 ㎎/mL standardized solution for subsequent use.With kapillary by above-mentioned crude extract solution and standard substance solution point sample on GF254 silica-gel plate, point sample baseline is apart from base 2.0cm, point sample diameter is 1-2mm, dot spacing, from being about 1.5cm, being put into development chamber and is launched.
Developping agent is trichloromethane: methyl alcohol: Glacial acetic acid=94.85:5:0.15(volume ratio).In development chamber, add the developping agent of q.s, and on wall, paste two filter paper bars equally high, wide with chamber, one end is immersed in developping agent, smears Vaseline in advance at sealing part, and the lid 30min on sealing chamber top, makes system balancing.The thin layer plate of having put sample is put into the developping agent of development chamber, the degree of depth that immerses developping agent is apart from thin layer plate base 0.5-1.0cm, and sealing chamber cap allows developping agent upwards launch more 90% thin plate length.From launch pond, take out thin plate and mark out with pencil the position, forward position that solvent arrives at once.
Allow solvent evaporates on thin plate fall.Speckle displacement on mark silica-gel plate under thin layer chromatograph, and and standard control, find and two kinds of standard substance R
fidentical spot.Choose the sample strain combination used that the spot corresponding with Arctiin spot shoals or disappear and deepen with spot colors corresponding to l-arctigenin spot, as primary election strain combination.
Finally, the content of Arctiin and l-arctigenin in employing HPLC detection primary election strain fermentation sample, and calculate Arctiin transformation efficiency, sample solubility rate and sample loss rate in conjunction with the formula in 2.4.2.Select that Arctiin transformation efficiency is greater than 99%, sample solubility rate be greater than 95% and sample loss rate lower than 5% as selecting eventually fermented bacterium or strain combination.
2.5.3 the optimization of fermentation condition
One-factor experiment: taking Arctiin transformation efficiency, sample solubility rate and rate of loss as index, relatively strain fermentation effect under the culture condition such as different carbon sources, nitrogenous source, liquid amount, pH, incubation time, the fermentation condition of optimization bacterial classification.In the time carrying out every kind of monofactorial test, except Consideration changes by experimental design, other conditions all remain unchanged according to the condition of preparing fermention medium in 2.1.3.
Carbon source: Semen Maydis powder, wheat bran, sucrose, wheat bran+Semen Maydis powder (1:1), wheat bran+sucrose (1:1), sucrose+Semen Maydis powder (1:1), wheat bran+sucrose+Semen Maydis powder (1:1:1), every kind of carbon source numbering is followed successively by C
1-C
7;
Nitrogenous source: peptone, bean cake powder, urea, (NH
4)
2sO
4, NH
4nO
3, NH
4cl, every kind of nitrogenous source numbering is followed successively by N
1-N
6;
Incubation time: 36h, 48h, 72h, 96h, 120h, 144h, 168h;
Liquid amount (250mL Erlenmeyer flask): 30mL, 50mL, 70mL, 90mL, 110mL, 130mL, 150mL;
pH：4，5，6，7，8，9，10；
Inoculum size: 1mL, 2mL, 3mL, 4mL, 5mL
Orthogonal test: on the basis of one-factor experiment, select Great Burdock Achene powder content (A) (content of Great Burdock Achene powder solid matter in whole substratum), planting daughter bacteria liquid inoculates than (B), carbon-nitrogen ratio (C), Mandel nutritive medium add volume (D), solid-to-liquid ratio (E) (described solid-to-liquid ratio refers to the mass ratio that adds solid matter (Great Burdock Achene powder+wheat bran+peptone) and liquid substance (Mandel nutritive medium+distilled water) in substratum) is 5 factors, design 4 different levelss according to table 2 simultaneously, carry out 5 factors, 4 horizontal quadrature tests.Every group of test established parallel 3, measures its l-arctigenin output capacity (every gram of Great Burdock Achene powder is through fermentation later output l-arctigenin quality), averages.Orthogonal test factor, level are in table 2
Table 2 orthogonal test
3 test-results and discussion
3.1 actication of culture
Six kinds of fungies all activate successfully, and bacterial classification purity reaches 100%.Go down to posterity after 3 times its growth cycle and phenotype is basicly stable, can be observed nipa palm aspergillus, aspergillus niger, Aspergillus awamori mutation and aspergillus oryzae 12 h after inoculation just visible mycelia occur, and the growth of Trichodermareesei and viride is comparatively slow, inoculate after 24 hours just mycelial growth as seen.On PDA flat board, Aspergillus awamori mutation bacterium colony surface is black, and velvet shape has radiation rill, and it is yellow to deep yellow that bacterium colony reverse side is; Aspergillus oryzae bacterium colony surface is light brown, thick cotton-shaped, has concentricity, reverse side white; Trichodermareesei is very similar to viride, and without concrete colonial morphology, mycelia yellow-green colour, is the growth of tiling expanded type, and after maturation, lawn surface is green; Aspergillus niger bacterium colony surface is black, and velvet shape has radiation rill, and reverse side is white in color; Nipa palm aspergillus bacterium colony surface is Vandyke brown, and velvet shape has radiation rill, and reverse side is white in color.
3.2 bacterial screening
Test knownly by TLC, selected six kinds of bacterial classifications and strain combination all have certain conversion capability.Therefore all samples extracting solution need do detection by quantitative and just can determine best fermented bacterium through HPLC.Data statistics shows that a lot of strain combinations such as the mutation of Trichodermareesei+Aspergillus awamori, Trichodermareesei+viride, Trichodermareesei+aspergillus niger, nipa palm aspergillus+Trichodermareesei, Trichodermareesei+aspergillus oryzae, the mutation of nipa palm aspergillus+Aspergillus awamori all have very strong fermentation capacity, be that Arctiin transformation efficiency can reach more than 90%, all be greater than the transformation efficiency of single culture fermentation test group, but it is too low in some fermented product extract, to show solubility rate, or rate of loss crosses high phenomenon, even also can greatly affect finished product output as fermented bacterium.So through relatively comprehensive, in Aspergillus awamori mutation+Trichodermareesei tunning extracting solution, Arctiin transformation efficiency can reach 99.9%, solubility rate and rate of loss are respectively 95.4% and 4.6%, select fermented bacterium the end of therefore selecting this strain combination to produce as combined ferment.Different strain and combined fermentation effect thereof are relatively in table 3 and Fig. 2.
Table 3 different strain and combined fermentation effect comparative data table thereof
The optimization of 3.3 fermentation conditions
In the time carrying out every kind of monofactorial test, except Consideration changes by experimental design, other conditions all remain unchanged according to the condition of preparing fermention medium in 2.1.3.
3.3.1 one-factor experiment
As shown in Fig. 3 and table 4, different carbon sources are for transformation efficiency and do not make significant difference, and all can make fermentation conversion rate maintain between 94%-98%, but for product solubility rate obvious effect, and then make product loss rate uneven.Analyzing its reason may be because some carbon source as the energy that excessive wheat bran not only can provide fermented bacterium to produce beta-glucosidase also may stimulate it to produce other enzymes, makes the Arctiin of stripping and l-arctigenin decompose; Or because suppressed the generation of cellulase system, hindered cell walls decomposition, make the Arctiin cannot stripping, thereby cause the decline of fermentation yield.Comprehensively relatively after, use wheat bran: sucrose: Semen Maydis powder be 1:1:1 during as carbon source fermentation conversion rate can reach 96.6%, solubility rate can reach 98.9%, rate of loss is low to moderate 1.1%, can be considered best fermenting carbon source.
The affect data sheet of table 4 carbon source on fermentation
As shown in Fig. 4 and table 5, different nitrogen sources does not also make significant difference for transformation efficiency, all can make fermentation conversion rate maintain between 96%-98%, but obvious for the impact of product solubility rate yet, and then makes product loss rate have bigger difference.Analyzing its reason may be as (NH because of some nitrogenous source
4)
2sO
4, NH
4nO
3, NH
4cl also may stimulate it to produce other enzymes when the required nitrogen element of fermented bacterium synthetic enzyme is provided, and the Arctiin of stripping and l-arctigenin are decomposed; Or because suppressed the generation of cellulase system, hindered cell walls decomposition, make the Arctiin cannot stripping, thereby cause the decline of fermentation yield.Comprehensively relatively after, use urea during as nitrogenous source fermentation conversion rate can reach 97.5%, solubility rate can reach 93.2%, rate of loss is low to moderate 6.8%, can be considered best fermentation nitrogen source.
The impact of table 5 nitrogenous source on fermentation
As shown in Fig. 5 and table 6, the impact of fermentation time on fermentation, known by the amount concentration-time curve of drug substance, in fermenting process, 0-72h can be considered the cellulase system effect stage, Main Function is to decompose Great Burdock Achene flour cell wall, promotes Arctiin stripping.72h-144h l-arctigenin amount of substance concentration increases gradually, proves that beta-glucosidase acts on the glycosidic link in Arctiin molecule, thereby makes its hydrolysis realize the conversion of Arctiin to l-arctigenin.During this period, Arctiin dissolution rate is less than its hydrolysis speed, causes Arctiin amount of substance concentration to decline gradually.After 144h, Arctiin and l-arctigenin amount of substance concentration are all without noticeable change.Thus, 144h can be considered as to best fermentation time.
The impact of table 6 time on fermentation
The factor such as growing space and the dissolved oxygen amount of fermented liquid of the variation major effect fermented bacterium of liquid amount.From Fig. 6 and table 7, in the time that liquid amount is too small, fermentation strain growing space is little, and in fermented liquid, dissolved oxygen amount is few, makes it cannot normal growth, and production of enzyme is extremely low, causes Great Burdock Achene powder almost not realize conversion, and target product output is extremely low.Along with liquid amount increases, although target product solubility rate increases to some extent, transformation efficiency really presents downtrending, illustrates at liquid amount during at 50mL-90mL that the various enzyme quantum of output of fermented bacterium imbalance cannot reach the conversion requirement of test.In the time that liquid amount reaches 110mL-150mL, not remarkable for the impact of conversion rate of products and solubility rate, rate of loss is also lower.Comprehensively relatively after, in the time that liquid amount is 110mL, transformation efficiency can reach 98%, solubility rate can reach 99.7%, rate of loss is low to moderate 0.3%, can be considered best liquid amount.
The impact of table 7 liquid amount on fermentation
During the fermentation, pH has a significant impact for fermented bacterium product enzyme class and enzymic activity tool.As shown in Fig. 7 and table 8, in the time that fermented liquid pH is too small, cellulase system activity is a greater impact, thereby causes solubility rate to reduce; In the time that fermented liquid pH is excessive, activity of beta-glucosidase declines, thereby transformation efficiency is declined greatly.In the time that pH is between 5-7, fermentation conversion rate can maintain 96%-98%, and solubility rate all can reach more than 99%, and rate of loss is extremely low, can be considered best fermentation pH.
The impact of table 8pH on fermentation
In fermentation system, the amount of nutritive substance is certain, in the time that nutritive substance is sufficient, fermented bacterium growth is normal, and can produce the required various enzymes of fermentation test at felicity condition, if crossing stool, inoculum size very likely causes nutrition supply deficiency, make the undesired propagation of fermented bacterium, or the enzyme of the leavened prod output that exerts an influence or other materials.As shown in Fig. 8 and table 9, in the time that inoculum size is greater than 3mL, product solubility rate declines gradually, and rate of loss rises gradually.In the time that inoculum size is too small, growing microorganism is slower, and yield of enzyme is less, also can impact product production.Consider, in the time that inoculum size is 2mL/110mL, transformation efficiency can reach 95.4%, and solubility rate can reach 98.9%, and rate of loss is low to moderate 1.1%, can be considered optimum inoculation amount.
The impact of table 9 inoculum size on fermentation
3.3.2 orthogonal test
By single-factor optimization Test, determine optimum carbon source, nitrogenous source, fermentation time, pH, liquid amount and the inoculum size that fermentation is required.On this basis, it is the further optimization to fermentation condition that orthogonal test is carried out in the combination that factor, level and the table 3 designing according to table 2 designs, orthogonal test combination and l-arctigenin output capacity the results are shown in Table 10, optimization of orthogonal test the results are shown in Table 11, as shown in Figure 9, in the time that carbon source, nitrogenous source, fermentation time, pH, liquid amount and inoculum size are optimum value, the conversion rate of products of all test group all, between 96%-99%, remains on higher level.Different Great Burdock Achene powder content, carbon-nitrogen ratio, inoculum size ratio, solid-to-liquid ratio and Mandel nutritive medium contgs is larger on the impact of product solubility rate.Known by data analysis, 2 test group ferment effect the bests, transformation efficiency can reach 97.8%, and solubility rate can reach 95.7%, and rate of loss is low to moderate 4.3%
The combination of table 10 orthogonal test and l-arctigenin output capacity result
Factors for the size order of l-arctigenin output capacity (being that every gram of Great Burdock Achene powder can extract l-arctigenin quality) impact are as shown in Table 10: Great Burdock Achene powder content > kind daughter bacteria liquid is inoculated than > carbon-nitrogen ratio > solid-to-liquid ratio >Mandel nutritive medium contg (mL).Fermentation top condition is: in fermentation substrate, Great Burdock Achene powder content is 52%, and Aspergillus awamori mutation is with the inoculation of Trichodermareesei kind daughter bacteria liquid than being 1:2, and carbon-nitrogen ratio is 100:6, and Mandel nutritive medium contg is 10mL/110mL, and solid-to-liquid ratio is 1:2.
Table 11 optimization of orthogonal test result
4 brief summaries
4.1 bacterial screening
Filtered out the Aspergillus awamori mutation+Trichodermareesei bacterial classification combination that a group can be l-arctigenin by Arctiin Efficient Conversion, in tunning extracting solution, Arctiin transformation efficiency can reach 99.8%, and solubility rate and rate of loss are respectively 95.7% and 4.3%.
The formulation of 4.2 zymotechniques
Formulate the best zymotechnique of a set of Great Burdock Achene powder, Arctiin transformation efficiency reaches more than 95%; Determine that by single factor experiment best fermenting carbon source is wheat bran: sucrose: Semen Maydis powder is 1:1:1; Best fermentation nitrogen source is urea; Best fermentation time is 144h; Best liquid amount is 110mL; Best fermentation pH is between 5-7; Optimum inoculation amount is 2mL/110mL.Pass through orthogonal test, final definite best fermentation combination condition is: in fermentation substrate, Great Burdock Achene powder content is 52%, and Aspergillus awamori mutation is with the inoculation of Trichodermareesei kind daughter bacteria liquid than being 1:2, and carbon-nitrogen ratio is 100:6, Mandel nutritive medium contg is 10mL/110mL, and solid-to-liquid ratio is 1:2.
Entire flow:
1, the preparation of bacterial classification: take the peeling potato that 200g is cut into small pieces, add water boil 20-30min, by four layers of filtered through gauze, in filtrate, add 20g glucose and 15g agar, then supply moisture to 1000mL, 121 DEG C of sterilizing 15min, make plate culture medium, inoculate respectively Aspergillus awamori mutation and Trichodermareesei bacterial classification on PDA plate culture medium, cultivate 120-144h, after having cultivated, can produce ripe spore for 28-30 DEG C.
2, seed culture medium preparation: accurately take potassium primary phosphate 2g, ammonium sulfate 1.4g, urea 0.3g, magnesium sulfate 0.3g, calcium chloride 0.3g, ferric sulfate 5mg, manganous sulfate 1.56mg, zinc sulfate l.4mg, cobalt chloride 0.2mg is in 1000mL volumetric flask, sterilized water constant volume, obtains Mandel nutritive medium; In Mandel nutritive medium, add respectively tween 80 2mL/L, peptone 1g/L, glucose 10g/L, adjust pH to 5.0-6.0 with sulfuric acid, 115 DEG C of sterilizing 20min, obtain seed culture fluid.
3, plant the preparation of daughter bacteria liquid: get cultured Aspergillus awamori mutation and Trichodermareesei bacterial classification flat board, add 10mL sterilized water, rock gently the spore of agar surface is swept away, this spore suspension is placed in sterilizing 50mL Erlenmeyer flask, in bottle, place in advance several aseptic glass spheres, three layers of gauze with sterilizing after shake well filter, and with aseptic water washing filter residue 2-3 time, finally make filtrate volume reach 10.0mL, spore suspension to be measured and get final product.
Sterilized water dilution 100-1000 for spore suspension doubly, is drawn to appropriate diluent to the nucleonics of blood counting chamber, then add cover glass, leave standstill about 5min, first under low power lens, find nucleonics, then convert high power lens to and observe and count.Calculate spore concentration, and to be mixed with concentration be 10
6-10
7the Aspergillus awamori mutation of individual/mL and the spore suspension of Li's Trichoderma, respectively get respectively in the seed culture medium that 2mL is inoculated into 50mL, and be placed in 250mL Erlenmeyer flask, and 175rpm, cultivates 24-48h under 28-30 DEG C of condition, obtains kind of a daughter bacteria liquid.
4, the preparation of fermention medium: accurately take respectively wheat bran 8.3g, sucrose 8.3g, Semen Maydis powder 8.3g, urea 1.5g, Great Burdock Achene powder 28.6g in 250mL Erlenmeyer flask, 121 DEG C of sterilizing 15min, after cooling, then add aseptic Mandel nutritive medium 10mL, add again sterilized water 100mL, regulate pH between 5.0-7.0, finally inoculate respectively 0.7mL Aspergillus awamori mutation kind daughter bacteria liquid and 1.3mL Trichodermareesei kind daughter bacteria liquid, 175rpm, 28-30 DEG C of condition bottom fermentation cultivated 144h, obtains final fermented liquid.
5, l-arctigenin crude extract preparation: add the 1mol/LNaOH solution of 10mL to stop fermenting process in the backward every bottle of fermented liquid of fermentation ends, get the rear sample of fermentation, 100 DEG C of oven dry, pulverize dry-matter, methanol constant volume is to 50mL, filter paper filtering is also collected filtrate, i.e. l-arctigenin crude extract solution adopts HPLC to carry out content detection.
6, test-results: obtain Arctiin transformation efficiency in Great Burdock Achene tunning extracting solution by this set of best zymotechnique of screening and can reach 99.8%, solubility rate and rate of loss are respectively 95.7% and 4.3%.
Claims (2)
1. a method for Fructus Arctii extract is prepared in microbial association fermentation, it is characterized in that: described microorganism is Aspergillus awamori mutation and Li's Trichoderma;
Described Aspergillus awamori mutation and Li's Trichoderma are respectively Aspergillus awamori mutation
aspergillus awamori var. CICC2203and Trichodermareesei
trichoderma reesei CICC40932.
2. the method for claim 1, is characterized in that: the inoculum size of described Aspergillus awamori mutation and Li's Trichoderma is than being 1:2.
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