CN102382771A - Strain produced by beta-glucosidase and method for preparing Genipin therefrom - Google Patents
Strain produced by beta-glucosidase and method for preparing Genipin therefrom Download PDFInfo
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Abstract
The invention relates to a method for effectively converting Geniposide into Genipin by stimulating the conversion of microbes in the in vivo intestinal tract through an in vitro microbial fermentation process. The method is characterized in that: a traditional Chinese medicine cape jasmine is fermented by the high yield strain of beta-glucosidase screened and preserved in a laboratory of the inventors, and an optimum condition for obtaining the product Genipin through the microbial conversion is obtained by optimizing through single factor experiments. According to the method of the invention, Geniposide in Genipin in hydrolyzed under mild conditions, so a defect of the effective component loss of traditional methods is avoided; and the scale conversion of the effective component Geniposide of cape jasmine is realized under in vitro controllable conditions with a modern microbial fermentation engineering technology, so the method of the invention has large economic benefits and social benefits, and has a wide market exploitation prospect.
Description
Technical field
The invention belongs to biological technical field, relate to microbiological transformation technology, especially a kind of beta-glucosidase produces bacterium and utilizes this bacterium to transform the method for preparing genipin.
Background technology
Genipin is a kind of iridoids material, and is nontoxic, is soluble in organic solvents such as ethanol, acetone, ETHYLE ACETATE, and the solubleness in water is less.In recent years, since genipin antitumor, aspect such as treatment liver cirrhosis etc. is evident in efficacy, has a lot of novel, important pharmacology as a kind of emerging pharmaceutical intermediate simultaneously and is worth, and therefore has good application prospects at aspects such as injection exploitations.Genipin also is applied in the biomaterial as a kind of novel biological linking agent simultaneously; It can not only form stable crosslinked products; And have that cytotoxicity is little, good biocompatibility, advantage such as anti-degradation capability is strong and Application Areas is extensive, be very promising cross-linked material.
Genipin content in plant is very low, directly extracts to be difficult to realize, is about 0.005% like the content of genipin in cape jasmine, and mainly is to exist with its precursor forms Geniposide form.Because acid and alkali hydrolysis interrupts glycosidic link and generates the method for aglycon and be not suitable for genipin; Its secoiridoid structure can be suffered brokenly ring under acid-base function; Influence its biological activity; So the method for preparing genipin at present mainly is an enzymolysis process, the Geniposide that promptly utilizes the beta-glucoside enzymic hydrolysis to be present in a large number in the plant generates the aglycon genipin.Beta-glucosidase has important use to be worth in medical treatment, food, Wood Adhesives from Biomass as an important component part of cellulase.Cellulase is very extensive in natural distribution; Insect, mollusk, higher plant, bacterium, actinomycetes and fungi can both produce cellulase, even also have in cud of the ruminating animal in the Mammals and the Radix Polygalae Crotalarioidis and can exist by cellulolytic flora.The zymogenic bacteria of cellulase is a lot, mainly contains bacterium, actinomycetes and filamentous fungus.The yield of cellulase of filamentous fungus is higher, and can reach more than the 20g/L, and filamentous fungus product enzyme has plurality of advantages: the cellulase of generation is an extracellular enzyme, is convenient to the separation and the extraction of enzyme; It is high to produce enzyme efficient, and the cellulose enzyme architecture that produces is comparatively reasonable.
Domestic Recent study beta-glucosidase has become focus, has simply extracted the present condition of enzyme production optimization and the purifying of crude enzyme liquid by the research beta-glucosidase in past.The clonal expression of beta-glucosidase gene is accomplished, and the new engineering bacteria that makes up is applied in the production practice.At present domestic having utilizes β one glucuroide that the Geniposide that extracts is carried out enzymolysis.But also exist simultaneously a lot of not enough, too high like: complex process, cost, further develop thereby limited it.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art; A kind of beta-glucosidase generation bacterium is provided and utilizes this bacterium to transform the method for preparing genipin, present method is utilized the mode of enteric microorganism conversion in the external microbe fermentation method simulation human body that Geniposide is effectively transformed and is generated genipin.
The objective of the invention is to realize through following technical scheme:
A kind of beta-glucosidase produces bacterium; It is characterized in that: name is called black mold Y-4; Its classification called after black mold Aspergillus niger, deposit number is CGMCC NO.5141, preservation date: on August 16th, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
A kind of beta-glucosidase produces bacterium and transforms the method for preparing genipin, and step is following:
(1) with the black mold Y-4 spore inoculating of preserving on slant medium, put 25-30 ℃ of thermostat container and cultivated 1-5 days, obtain spore, obtain spore liquid with sterilized water dissolving spore, spore liquid is inserted the shake flask fermentation substratum;
Said shake flask fermentation substratum: the cape jasmine powder is dissolved in the deionized water, and making material-water ratio is g/ml:10%, mould;
Said slant medium is the PDA substratum;
(2) transform the preparation genipin: in temperature is that 25-30 ℃, pH7.0, liquid amount are that 45-55ml, inoculum size are the condition bottom fermentation 80-110h of 8-12%, rotating speed 150-200r/min.
And the concentration of said black mold Y-4 spore liquid is 1.00 ± 0.05 * 108 in every milliliter of spore.
And the condition that said conversion prepares genipin is: temperature is that 28 ℃, pH7.0, liquid amount are that 50ml, inoculum size are 10%, the condition bottom fermentation 96h of rotating speed 180r/min.
Advantage of the present invention and positively effect are:
1, the present invention with the bacterial classification of laboratory screening preservation as starting strain; The fermention medium that adopts the cape jasmine powder to form as sole carbon source carries out the experiment of single factor analysis from several respects such as inoculum size, concentration of substrate, liquid amount, transformation times respectively, thereby confirms the top condition that Geniposide transforms; Improved output effectively; This method is that a large amount of suitability for industrialized production beta-glucosidases at a low price and genipin provide a feasible way, and this working method has reduced production cost, has improved production efficiency; Not only have economic benefit, also have the certain social benefit.
2, the present invention is based on mikrobe and have many-sided enzymatic productivity; Can utilize intravital enzyme system to accomplish the important catalysis and the distinct advantages of metabolic function; Adopt enteric microorganism transforms in the external microbe fermentation method analogue body mode that Geniposide is effectively transformed and generate genipin; The high yield bacterium of β one glucuroide that promptly utilizes preservation that screen in this laboratory is at optimal conditions bottom fermentation Chinese medicine mast; Not only can reduce production costs, beta-glucosidase is widely used in the medicine production process, and effective constituent Geniposide realization scale under external controlled condition of mast is transformed.
3, the present invention utilizes the high yield bacterium fermented tcm cape jasmine of the beta-glucosidase of this laboratory screening preservation; Through experiment of single factor optimization; Confirm that microbial transformation obtains the top condition of product genipin; Hydrolysis Geniposide wherein under mild conditions has avoided traditional method to cause the defective of loss of effective components on the one hand, utilizes modern microbial fermentation engineering technology that effective constituent Geniposide realization scale under external controlled condition of mast is transformed on the other hand; Have bigger economic benefit and social benefit, the prospect of marketing is wide.
Description of drawings:
Fig. 1 is a fermented liquid product TLC analysis of the present invention, wherein: 1, genipin standard substance; 2, cape jasmine powder solution; 3, Y-1 fermented liquid; 4, Y-2 fermented liquid; 5, Y-3 fermented liquid; 6, Y-4 fermented liquid;
Fig. 2 is the high-efficient liquid phase chromatogram of genipin mark article of the present invention;
Fig. 3 obtains the high-efficient liquid phase chromatogram of product genipin for the present invention utilizes the black mold transformation fermentation.
Embodiment
Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
One, soil confirms
Soil sample is taken from cattle farm, Wuqing, Tianjin and is piled up the soil of cow dung for many years; Enzyme reaction substrate is done in mast immersion liquid with 1%; Be equipped with the potato culture medium prescription and form the primary dcreening operation substratum; Utilize jasminoidin after the beta-glucoside enzymic hydrolysis, to generate genipin and glycine reactant generation blueness, judge whether to exist and produce the beta-glucosidase fungal bacterial strain, technology is:
(1) bacterial classification primary dcreening operation: soil is got 1mL and is cultivated to filling in (25mL/250mL) enrichment medium after pre-treatment, and 28 ℃, 180r/min cultivates 36h;
The composition of enrichment medium is counted with g/100mL: (NH
4)
2SO
40.3, MgSO
47H
2O 0.05, NaCl0.2, KH
2PO
40.1, Mierocrystalline cellulose 0.5, ammonia benzyl mycin is an amount of, 115 ℃ of sterilization 30min;
Storage medium: PDA agar slant solid medium;
Primary dcreening operation substratum composition is counted with g/100mL: add cape jasmine powder infusion 1 among the PDA, glycocoll 1 is put into 50 ℃ of incubators behind the cultivation 72h and is incubated 6h;
Through primary dcreening operation (PDA that contains jasminoidin is dull and stereotyped), there are four strain bacterial strains that blue colour developing circle is arranged in the primary dcreening operation flat board, explain that this four strains bacterium can produce beta-glucosidase, with this four strains bacterium called after Y-1, Y-2, Y-3, Y-4;
(2) bacterial classification sieves again: the bacterial classification that primary dcreening operation obtains, utilizes the DNS method to measure enzyme and lives as carbon source through fermentation with the cape jasmine powder
The seed culture medium composition is counted with g/100mL: Zulkovsky starch 2.0, glucose 1.0, KH
2PO
40.1, MgSO
40.1, NaNO
30.2, yeast extract paste 0.5, pH 4;
The liquid fermentation medium composition is counted with g/100mL: wheat bran powder 3.0, (NH
4)
2SO
40.2, KH
2PO
40.2, CaCl
20.04, MgSO
47H
2O 0.04, the pH nature;
Record Y-1, Y-2, Y-3, the enzyme work of Y-4 is counted with IU: 0.3262,0.2463,0.3947,0.9410.
Two, the screening of bacterial classification, evaluation, and Optimizing Conditions of Fermentation
1, beta-glucosidase produces the screening of bacterium
(1) soil sampling is divided on a clean paper, respectively gets some soil from four angles and central authorities, behind the mixing, gets the 10g soil sample, adds the 90mL sterilized water, and packing into fills in the triangular flask of granulated glass sphere, jolting a moment, bacterium is disperseed.Get 1mL suspension-s and put into the test tube that fills the 9mL sterilized water, pressure-vaccum three times, and jolting makes it abundant mixing, gets 1mL and cultivates to filling in (25mL/250mL) enrichment medium, 28 ℃, 180r/min cultivates 36h.
Bacterium liquid is connect method with three rides be inoculated in and contain in the antibiotic PDA plate, put into 28 ℃ of incubators and cultivate 4d, obtain single bacterium colony, in petridish, adding microbiotic can bacteria growing inhibiting, thereby reaches the purpose of screening fungi.
Single bacterium colony that (2) will obtain is connected on the primary dcreening operation substratum, puts into 50 ℃ of incubators behind the cultivation 72h and is incubated 6h, takes out.Single bacterium colony of picking out blue colour developing circle is connected to the PDA slant medium.
Primary dcreening operation substratum composition is counted with g/100mL: add cape jasmine powder infusion 1 among the PDA, glycocoll 1 is put into 50 ℃ of incubators behind the cultivation 72h and is incubated 6h;
(3) select for use the slant pore that growth is normal, spore is abundant to add a certain amount of sterilized water, break up spore after, use microscopic counting, and be adjusted to every milliliter of spore (1.00+0.05) * 10
8Individual, insert fermention medium, utilize the DNS method to measure enzyme activity.
(4) draw 1mL sample (rifle head clip) in the EP pipe with liquid-transfering gun, 15000 rev/mins of centrifugal 5min; Get cape jasmine powder 1g, be dissolved in water to the 100mL volumetric flask, get 2mL, add the acetate buffer 1.0mL of pH4.8, add fermentation broth sample solution 1.0mL behind the mixing again, 50 ℃ of water-bath 40min, the sample thief point sample utilizes the analysis of TLC method.
2, the evaluation of bacterial classification
(1) colonial morphology is observed: inclined-plane bacterium point is connected in the plate of PDA substratum, cultivates 2d, the observation colonial morphology in 28 ℃.
(2) thalli morphology is observed: the water logging sheet of fungi is observed: on slide glass, add a sterilized water; With dissecting needle from the sporogenic on a small quantity fungal hyphae of mold colony edge picking (thalline); Place 50% ethanol to soak the spore that comes off with absorption earlier; Be placed on again in the sterilized water on the slide glass, with careful mycelia is scatter of dissecting needle.Covered is put under the low power lens (40 *) and is observed, and changes high power lens in case of necessity and observes.
(3) the 18S rDNA of bacterial strain identifies:
A. 18S rDNA universal primer is adopted in the extraction of genomic dna, the primer of this experiment.
B. adopt the pcr amplification system of 25 μ L:
C. the PCR condition that is used for amplifying target genes:
Through the laggard row agarose gel electrophoresis of amplification with 18S rDNA sequence, obtain its size for about 1800bp, meet the expection amplification of use mould 18S rDNA primer.Finally choose best band and reclaim, the DNA that reclaims is delivered to Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking.Analyze through Nucleotide BLAST, with the 18S rDNA sequence homology of Aspergillus niger strain be 99%, can confirm tentatively that the Y-4 bacterial strain is a black mold.
The preservation information of the aspergillus niger strain that filters out is following: name is called black mold Y-4; Its classification called after black mold Aspergillus niger; Deposit number is CGMCC NO.5141; Preservation date: on August 16th, 2011, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
3, the subconditional optimization of black mold Y-4 fermentation mast
(1) medium component: the cape jasmine powder that will contain 3% Geniposide mixes with deionized water according to different ratios and is configured, and material-water ratio is counted with g/mL: 5%, 10%, 15%, 20%, 25%, and boil, filter, the pH nature;
(2) confirming of fermentation time: in temperature is that 28 ℃, pH7.0, material-water ratio are 10%, liquid amount is 20%; Inoculum size 1mL; Ferment 48,72,84,96,108 under the condition of rotating speed 180r/min respectively, 120h, press the content that the HPLC method is measured genipin in the fermented liquid;
(3) confirming of inoculum size: the condition bottom fermentation 120h that be in temperature that 28 ℃, pH7.0, material-water ratio are 10%, liquid amount is 20%, inoculum size is respectively 1mL, 2mL, 4mL under the condition of rotating speed 180r/min, press the content of genipin in the HPLC method mensuration fermented liquid;
(4) confirming of liquid amount: the condition bottom fermentation 120h that be in temperature that 28 ℃, pH7.0, material-water ratio are 10%, liquid amount is respectively 30mL, 50mL, 70mL, 90mL under the condition of inoculum size 1mL, rotating speed 180r/min, press the content of genipin in the HPLC method mensuration fermented liquid;
(5) confirming of fermentation material-water ratio: be in temperature that 28 ℃, pH7.0, liquid amount are 20%, under the condition of inoculum size 1mL, rotating speed 180r/min; Material-water ratio is respectively 5%, 10%, 15%, 20%, 25% condition bottom fermentation 120h, presses the content that the HPLC method is measured genipin in the fermented liquid;
Detecting the optimal conditions of fermentation of confirming fermentation of Aspergillus niger conversion Geniposide through experiment of single factor optimization, HPLC is: fermentation time 96h, inoculum size is 10%, liquid amount 50mL in the 250mL triangular flask, material-water ratio 10%.
4, the determination and analysis of product evaluation and transformation efficiency
Each factor top condition with experiment of single factor is confirmed is fermented; Get fermentation broth sample 1mL then in the EP pipe, add equal volume of ethyl acetate, the centrifugal 5min of 12000r/min; The HPLC method detects Geniposide transformation efficiency in the fermented liquid: get supernatant in another EP pipe; After treating that ETHYLE ACETATE volatilizees fully, in methyl alcohol: water=dissolving in 45: 55, detect with the HPLC method then.
Chromatographic condition: ODS-2HPERSIL C18 chromatographic column (4.6mm * 250mm, 5.0 μ m), methyl alcohol, the ultrasonic 15min degassing of deionized water, mobile phase methanol: water=45: 55; The detection wavelength is 238nm; Flow velocity 1mL/min; Column temperature is 25 ℃; Sample size 20 μ L.
Precision takes by weighing the EP pipe that genipin 1.00mg places 1.5mL; Add methyl alcohol: water=dissolving in 45: 55; The reference substance solution of accurately drawing 20,40,60,80,100 μ L and is used methyl alcohol in the EP pipe of 1.5mL: water=45: 55 solution dilution to 100 μ L, and draw 20 μ L then and measure.With peak area (Y) content (X) is done regression Calculation.The result shows that in 0.2～1mg/mL scope, genipin has good linear relationship, and regression equation is: Y=7868.9X-389.19, R
2=0.963.
The product TLC method of genipin is analyzed: with cape jasmine powder solution, genipin standardized solution as reference substance solution; Point sample 5 μ L are in GF254 thin layer plate (50 * 100mm) respectively; With vinyl acetic monomer (acetate): sherwood oil (1: 1) is a developping agent, and uv lamp 254nm detects down.
Three, utilize black mold Y-4 transformation fermentation to prepare the method for genipin, step is following:
(1) configuration of substratum: slant medium (PDA) (1000mL): yam 200g stripping and slicing, added the 1000mL water boil 30 minutes, the elimination potato ball complements to 1000mL with filtrating, adds glucose 20g, agar 20g, dissolve the back packing, 115 ℃ of sterilizations, 15min;
The shake flask fermentation substratum: 50g cape jasmine powder is dissolved in the 500mL deionized water, and making material-water ratio is (g/ml) 10%, boils, filters, and the pH nature divides to install in the triangular flask of 10 250mL 121 ℃ of sterilizations, 20min;
(2) seed preparation: with the black mold Y-4 spore inoculating of preserving on slant medium; Putting 28 ℃ of thermostat containers cultivated 3 days; Obtain female inclined-plane, select for use the slant pore that growth is normal, spore is abundant to add a certain amount of sterilized water, break up spore after; Count one by one with microscope, and be adjusted to every milliliter of spore (1.00 ± 0.05) * 10
8Individual, insert the shake flask fermentation substratum;
(3) microbial transformation prepares genipin: be in temperature that 28 ℃, pH7.0, material-water ratio are 10%, liquid amount is that 50ml, inoculum size are 10%, the condition bottom fermentation 96h of rotating speed 180r/min; Press the content that the HPLC method is measured genipin in the fermented liquid, and calculate its transformation efficiency.Assay determination transformation efficiency, transformation efficiency are 14%.
Claims (4)
1. a beta-glucosidase produces bacterium; It is characterized in that: name is called black mold Y-4; Its classification called after black mold Aspergillus niger, deposit number is CGMCC NO.5141, preservation date: on August 16th, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. one kind is utilized beta-glucosidase according to claim 1 to produce bacterium to transform the method for preparing genipin, and it is characterized in that: step is following:
(1) with the black mold Y-4 spore inoculating of preserving on slant medium, put 25-30 ℃ of thermostat container and cultivated 1-5 days, obtain spore, obtain spore liquid with sterilized water dissolving spore, spore liquid is inserted the shake flask fermentation substratum;
Said shake flask fermentation substratum: the cape jasmine powder is dissolved in the deionized water, and making material-water ratio is g/ml:10%, mould;
Said slant medium is the PDA substratum;
(2) transform the preparation genipin: in temperature is that 25-30 ℃, pH7.0, liquid amount are that 45-55ml, inoculum size are the condition bottom fermentation 80-110h of 8-12%, rotating speed 150-200r/min.
3. beta-glucosidase according to claim 2 produces bacterium and transforms the method for preparing genipin, and be characterised in that: the concentration of said black mold Y-4 spore liquid is 1.00 ± 0.05 * 108 in every milliliter of spore.
4. beta-glucosidase according to claim 2 produces bacterium and transforms the method for preparing genipin, and be characterised in that: the condition that said conversion prepares genipin is: temperature is that 28 ℃, pH7.0, liquid amount are that 50ml, inoculum size are 10%, the condition bottom fermentation 96h of rotating speed 180r/min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865803A (en) * | 2014-01-26 | 2014-06-18 | 玉林师范学院 | Beta-glucosidase high-yielding strain and application thereof in conversion preparation of genipin and resveratrol |
| CN105505829A (en) * | 2016-01-20 | 2016-04-20 | 黑龙江省中医药科学院 | Fermented traditional Chinese medicine dominant bacterial community selective culture medium and preparation method thereof, and screening method of fermented traditional Chinese medicine dominant bacteria |
| CN105838622A (en) * | 2016-04-14 | 2016-08-10 | 浙江树人大学 | Aspergillus niger HC306 and application of aspergillus niger HC306 to prepare naringenin through naringin conversion |
| CN110771876A (en) * | 2019-11-05 | 2020-02-11 | 质每(中国)有限公司 | Method for fermenting ginseng by cordyceps militaris |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899484A (en) * | 2009-12-28 | 2010-12-01 | 抚州市临川之信生物科技有限公司 | Preparation method of genipin |
-
2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899484A (en) * | 2009-12-28 | 2010-12-01 | 抚州市临川之信生物科技有限公司 | Preparation method of genipin |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865803A (en) * | 2014-01-26 | 2014-06-18 | 玉林师范学院 | Beta-glucosidase high-yielding strain and application thereof in conversion preparation of genipin and resveratrol |
| CN103865803B (en) * | 2014-01-26 | 2016-01-27 | 玉林师范学院 | Beta-glucosidase Producing Strain and the application prepared in genipin and trans-resveratrol in conversion thereof |
| CN105505829A (en) * | 2016-01-20 | 2016-04-20 | 黑龙江省中医药科学院 | Fermented traditional Chinese medicine dominant bacterial community selective culture medium and preparation method thereof, and screening method of fermented traditional Chinese medicine dominant bacteria |
| CN105838622A (en) * | 2016-04-14 | 2016-08-10 | 浙江树人大学 | Aspergillus niger HC306 and application of aspergillus niger HC306 to prepare naringenin through naringin conversion |
| CN105838622B (en) * | 2016-04-14 | 2019-04-12 | 浙江树人大学 | Aspergillus niger HC306 and conversion aurantiin prepare the application in naringenin |
| CN110771876A (en) * | 2019-11-05 | 2020-02-11 | 质每(中国)有限公司 | Method for fermenting ginseng by cordyceps militaris |
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