CN106834169A - A kind of method that microorganism mixed fermentation prepares yellow ginger saponin(e - Google Patents
A kind of method that microorganism mixed fermentation prepares yellow ginger saponin(e Download PDFInfo
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Abstract
The invention discloses a kind of method that microorganism mixed fermentation prepares yellow ginger saponin(e, belong to biological technical field, turmeric saponin yield is low during emphasis solves the problems, such as existing production technology.Bacillus HG224, a kind of Pectinatus or Erwinia, a kind of cellulomonas cartae are cultivated respectively first, then according to 1:0.4~1.2:Microorganism species are prepared after 0~1.2 inoculation volume ratio mixing；Mass ratio according still further to yellow ginger dry weight and water is 1:5~1:15 are configured to uniform yellow ginger slurries, and it is 10%~40% mentioned microorganism flora that percentage by volume is inoculated with slurries, are then 30 DEG C~40 DEG C in temperature, and rotating speed is 4~6h of culture under conditions of 100~220r/min.The microorganism species mixed fermentation can make the saponin(e separate out of a large amount of constraints, substantially increase turmeric saponin yield, meanwhile, for the green production for promoting follow-up turmeric saponin has established solid foundation, industrial applications have a extensive future.
Description
Technical field
The invention belongs to biological technical field, yellow ginger saponin(e is prepared more particularly, to a kind of microorganism mixed fermentation
Method.
Background technology
Yellow ginger (Dioscorea zingiberensis C.H.Wright) scientific name dioscorea zingiberensis wright, also known as duration and degree of heating root, for many years
Raw voluble herb plant, is the distinctive wild plant resource of China, is distributed mainly on Hunan, Shaanxi, Sichuan, Guizhou and Yunnan etc.
Save.The root-like stock of yellow ginger containing the steroid saponin constituents such as Dioscin, 40% or so starch, 50% cellulose and some
The chemical compositions such as water-soluble glycoside, alkaloids, flavonoid glycoside, cardiac glycosides, tannin, pigment.The principle active component of yellow ginger
It is diosgenin, also referred to as saponin (claiming saponin below).Saponin is the aglucon of yellow ginger saponin(e, main with saponin(e in yellow ginger
Form is present.The steroid hormone intermediate and the product such as cortin, sex hormone, protein anabolic hormone synthesized with turmeric saponin be
The medicine that main hundreds of national economy are special procured, is described as " medicinal gold ".Steroid hormone class medicine has been developed rapidly as only secondary
In the second major class medicine of antibiotic.
The compositions such as starch, protein, pectin in dioscorea zingiberensis wright plant have certain package action to steroid saponin, and
Steroid saponin is connected further through glycosidic bond with string.Thus simple acid hydrolysis process yield is low, it is difficult to by plant
Chinese yam saponin extracts complete, is only capable of extracting therein 1/4.
Patent CN1515585A discloses a kind of method of environment friendly and pollution-free production Chinese yam saponin, but wherein sieving separating
40% cellulosic sections can take away the saponin(e that some are combined with cellulose, so as to reduce the yield of saponin.Patent
A kind of method that CN1970785A discloses Chinese yam saponin clean manufacturing and comprehensive utilization, is directly carried out after yellow ginger raw material is crushed
Solvent extraction, and the combination state saponin(e of yellow ginger lignocellulosic structure covalent coupling cannot be obtained, and it is empty that saponin yield has lifting
Between.Patent CN1821380A discloses a kind of effectively compound microbial flora for processing yellow ginger, and the microorganism species that it is used are contained
Various complex microorganisms such as lid bacterium, mould, saccharomycete, but there is problem is that fungi growth time is more long, and bacterium and fungi
There is antagonism in growth course, most of microbe exists stage by stage in yellow ginger lignocellulosic processing procedure
, there is uncontrollability as spontaneous fermentation process in the situation of growth, and microorganism sapogenin parent nucleus might have degraded with
Transformation, so as to reduce saponin yield.
In sum, traditional acid-hydrolysis method extracts turmeric saponin because the conversion of saponin(e is not selectivity, is simultaneously
Various materials, including lignocellulosic, starch, albumen etc. are hydrolyzed, it is big with acid amount, therefore, efficiency is low, energy consumption is big, and needs
High-temperature process；Prior art is mostly using microorganism spontaneous fermentation treatment yellow ginger, and the microorganism of spontaneous fermentation is uncontrollable, thus
May be adulterated some microorganisms of saponin parent nucleus that can degrade so that saponin yield declines to a great extent.
Therefore, turmeric saponin yield is improved to greatest extent, and it is then existing production technology face to reduce turmeric saponin production cost
The outstanding problem faced, a large amount of free of yellow ginger saponin(e is promoted by innovating microbial process, and saponin(e is especially fettered in yellow ginger
It is free particularly important, so, research and develop new green production process and to carry out commercial application very urgent.
The content of the invention
For the disadvantages described above or Improvement requirement of prior art, the invention provides a kind of bacillus, microorganism species
And its method that mixed fermentation prepares yellow ginger saponin(e, its object is to pass through to select suitable microorganism fungus kind, prepare micro-
Biological flora so that the microorganism species can successively secrete high activity lignocellulolytic enzymes, starch when yellow ginger ferments
Enzyme and glycosyl transferase etc., collaboration promote the free of yellow ginger saponin(e, and improve solubility of the saponin(e in water phase, so as to improve most
The yield of whole saponin, thus solves the low outstanding problem of prior art turmeric saponin yield, is that the microorganism of next stage is efficient
Conversion saponin(e production saponin has established solid foundation.
To achieve the above object, according to one aspect of the present invention, there is provided a kind of bacillus, Classification And Nomenclature is
Bacillus sp.HG224, the bacillus was preserved in China typical culture collection center on December 30th, 2016
CCTCC, its deposit number is CCTCC M2016791.
According to another aspect of the present invention, there is provided a kind of microorganism species of yellow ginger fermentation, the microorganism species
Including bacillus HG224 and at least one Pectinatus, the Pectinatus can be replaced with Erwinia.
Preferably, the Pectinatus are carrot pectin bacillus, and its Latin literary fame scientific name is Pectobacterium
carotovorum。
Preferably, the Erwinia is solution Erzvinia amylovora, and its Latin name is Erwinia amylovora.
Preferably, the microorganism species also include at least one cellulomonas cartae.
Preferably, the cellulomonas cartae is Cellumomonas flavigena, and its Latin name is Cellulomonas
flavigena。
Preferably, bacillus HG224, Pectinatus and cellulomonas cartae described in the microorganism species are in right
In number growth period, the bacterium solution volume ratio of the bacillus HG224, Pectinatus and cellulomonas cartae in exponential phase is
1:0.4~1.2:0~1.2, the Pectinatus can be replaced with Erwinia.
Preferably, the microorganism species are used to secrete lignocellulolytic enzymes, amylase and glycosyl transferase.
Preferably, the lignocellulolytic enzymes are cellulase, zytase, mannonase pectase or wood
Lignin-degrading enzymes.
Preferably, the microorganism species when yellow ginger ferments for secrete 200~820U/ml amylase, 120~
The beta-glucosidase of 450U/ml, the zytase of 120~410U/ml, the mannase of 130~370U/ml and 110~
The cellulase of the pectase of 390U/ml, the glycosyl transferase of 158~430U/ml and 205~890U/ml.
It is according to another aspect of the present invention, there is provided a kind of preparation method of described microorganism species including as follows
Step：The single bacterium colony for taking the bacterial strain of the bacillus HG224, Pectinatus and cellulomonas cartae is inoculated in the LB trainings respectively
Support in base, be 30~40 DEG C in temperature, rotating speed is obtained respectively to cultivate bacterial strain under the conditions of 100~220r/min to exponential phase
The bacillus HG224, Pectinatus and cellulomonas cartae the bacterium solution in exponential phase, by the bacterium solution according to
Volume ratio 1:0.4~1.2:0~1.2 is mixed and made into the microorganism species, and the Pectinatus can use Erwinia generation
Replace.
According to another aspect of the present invention, there is provided a kind of application of described microorganism species, yellow ginger hair is applied to
Ferment.
Preferably, described microorganism species are applied to comprise the following steps when yellow ginger ferments：According to yellow ginger dry weight and water
Mass ratio be 1:5~1:15 are configured to homogeneous slurry, and it is described in 10%~40% that percentage by volume is inoculated with slurries
Microorganism species, carry out microorganism species mixing fermentation culture, obtain the zymotic fluid containing yellow ginger saponin(e.
Preferably, the fermentation culture conditions are：Temperature be 30~40 DEG C, rotating speed be 100~220r/min under the conditions of
4~24h of culture.
Preferably, the fermented incubation time is 4~6h.
In general, by above-mentioned technical proposal of the invention compared with prior art, following beneficial effect can be obtained.
1st, the invention provides a kind of bacillus HG224, and there is provided it is a kind of comprising the bacillus for yellow ginger
The microorganism species of fermentation, during using the microorganism species fermentation process yellow ginger raw material, it can secrete ligocellulose degradation
Enzyme system, amylase and glycosyl transferase and enzyme activity vigor is very high, make lignocellulosic structure loose, and discharge the saponin(e of constraint
Out, making to be present in fully dissociated by the saponin(e of the parcels such as lignocellulosic, pectin, starch in yellow ginger histocyte, by hair
Ferment time control so that Starch Conversion is oligosaccharide or oligosaccharides, releases the package action of starch without influence glucose content.
2nd, cause transformation efficiency relatively low due to the water-insoluble state of a large amount of saponin(es during microorganism conversion saponin(e, using this
After the described microorganism species fermentation yellow ginger of invention, due to the water-solubility saponins of a large amount of constraints release and be present in water phase,
Solid foundation has been established for the microorganism of subsequent transformation saponin(e carries out Efficient Conversion production saponin.
3rd, the preparation method of the microorganism species that the present invention is provided and its application in yellow ginger fermentation, promote a large amount of constraints
Saponin(e dissociate so that follow-up production of saponin yield is high, and technique environmental protection, pollution-free, low cost, commercial application prospect are wide
It is wealthy.
4th, when microorganism species of the invention are applied to yellow ginger fermentation promotion yellow ginger saponin(e release and dissolve, due to wherein not
With the synergy of strain, with without microorganism or only compared with addition single microorganism, saponin(e yield is greatly improved.
Brief description of the drawings
The enzyme activity dynamic monitoring curve of beta-glucosidase when Fig. 1 is Bacillus sp.HG224 fermentation yellow gingers；
The enzyme activity dynamic monitoring curve of amylase when Fig. 2 is Bacillus sp.HG224 fermentation yellow gingers；
The enzyme activity dynamic monitoring curve of zytase when Fig. 3 is Bacillus sp.HG224 fermentation yellow gingers；
The enzyme activity dynamic monitoring curve of mannase when Fig. 4 is Bacillus sp.HG224 fermentation yellow gingers；
The enzyme activity dynamic monitoring curve of pectase when Fig. 5 is Bacillus sp.HG224 fermentation yellow gingers；
The enzyme activity dynamic monitoring curve of glycosyl transferase when Fig. 6 is Bacillus sp.HG224 fermentation yellow gingers；
Fig. 7 is the phyletic evolution that bacillus sp.HG224 is built according to 16S rDNA sequences using N-j methods
Tree.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as additionally, technical characteristic involved in invention described below each implementation method
Not constituting conflict each other can just be mutually combined.
A kind of bacillus that the present invention is provided, its Classification And Nomenclature is Bacillus sp.HG224, and it is in December, 2016
Wuhan, China, China typical culture collection center CCTCC are preserved within 30th, its deposit number is CCTCC M2016791, its
Latin name is Bacillus sp..
Fig. 7 is the systematic evolution tree that bacillus HG224 is built according to 16S rDNA sequences using N-j methods.Bacterial strain gemma
The systematic evolution tree that bacillus HG224 is built according to its 16S rDNA sequence using Neighbour-joining methods, with strain Bacillus
It is same cluster that bacillus M6K gathers on systematic evolution tree, thus is classified as Bacillus strain.
Provided by the present invention for the microorganism species of yellow ginger fermentation, including bacillus HG224 and at least one pectin
Bacillus, wherein Pectinatus can be replaced with Erwinia.The microorganism species can also include at least one cellulomonas cartae.
Wherein Pectinatus are preferably carrot pectin bacillus Pectobacterium carotovorum, and Erwinia is excellent
Elect solution Erzvinia amylovora Erwinia amylovora as, cellulomonas cartae is preferably Cellumomonas flavigena
Cellulomonas flavigena, these three bacterium limit without specific bacterial strain.
Bacillus HG224, Pectinatus and cellulomonas cartae are in exponential phase, the microorganism species application with
Yellow ginger ferment when according to bacterium solution volume ratio be 1:0.4~1.2:0~1.2 is inoculated with, and wherein Pectinatus can use Ou Wenshi
Bacterium replaces.
For secreting lignocellulolytic enzymes, amylase and glycosyl transferase during microorganism species fermentation yellow ginger, its
Middle lignocellulolytic enzymes are cellulase, zytase, mannonase pectase or lignin-degrading enzymes.
The microorganism species can be secreted when yellow ginger ferments the amylase of 200~820U/ml, 120~450U/ml β-
The fruit of glucuroide, the zytase of 120~410U/ml, the mannase of 130~370U/ml and 110~390U/ml
The cellulase of glue enzyme, the glycosyl transferase of 158~430U/ml and 205~890U/ml.
The preparation method of the microorganism species, comprises the following steps：Take the bacillus HG224, Pectinatus and fibre
The single bacterium colony of bacterial strain for tieing up monad is inoculated in the LB culture mediums respectively, is 30~40 DEG C in temperature, and rotating speed is 100~
Under the conditions of 220r/min, obtain respectively the bacillus HG224, Pectinatus and cellulomonas cartae in exponential phase
Bacterium solution, by the bacterium solution according to volume ratio 1:0.4~1.2:0~1.2 is mixed and made into the microorganism species, the pectin
Bacillus can be replaced with Erwinia.
Microorganism species are applied to comprise the following steps when yellow ginger ferments：Mass ratio according to yellow ginger dry weight and water is 1:5
~1:15 are configured to homogeneous slurry, and the above-mentioned microorganism species that percentage by volume is 10%~40% are inoculated with slurries,
Temperature is 30~40 DEG C, rotating speed is culture 4~24h, preferably 4~6h under the conditions of 100~220r/min, carries out microorganism species and mixes
Fermented and cultured is closed, the zymotic fluid containing yellow ginger saponin(e is obtained.By zymotic fluid through separation of solid and liquid thalline after, obtain containing yellow ginger saponin(e
Clear liquid, sampling analysis saponin content therein (acid hydrolysis, depickling is washed by hydrolysate, after carry out solvent extraction Detection and Extraction
Saponin content in liquid characterizes saponin content), then further obtain dense containing a large amount of yellow ginger saponin(es by multistage membrane filtration
Contracting liquid, or above-mentioned clear liquid is directly carried out microorganism conversion production turmeric saponin.
When microorganism species of the invention are applied to yellow ginger fermentation, various enzymes are played during yellow ginger fermentation dissolution saponin(e
Function analysis it is as follows：
Cellulase：Cellulase can effectively degrade the cellulosic structure in yellow ginger material, and cellulose is residual glucose
The polysaccharose substance that base is formed with β-Isosorbide-5-Nitrae-glycosidic bond high polymeric, further through hydrogen bond, Van der Waals force etc. between different sugar chains
Closely connection, result in the constraint effect to saponin(e, thus cellulase can be effectively promoted the release of saponin(e.
Amylase：Starch degradation is mainly oligomeric glucan or oligosaccharides by amylase, thus can effectively release shallow lake
Powder to the parcel of yellow ginger saponin(e, simultaneously because fermentation time control microorganism to fail to be glucose by starch degradation in time again after
Utilized by oneself, the concentration of glucose of yellow ginger liquid glucose keeps relative constancy, so that effective guarantee is next to the comprehensive of liquid glucose
Close and utilize.
Zytase, mannase and pectase：Due to the complexity of yellow ginger lignocellulosic structure, microorganism is in hair
The substrate touched during ferment is in during being continually changing, zytase that microorganism secretes during the fermentation, sweet
Dew dextranase, pectase belong to synergy enzyme, if lacking these enzymes, microorganism will be not readily accessible to related substrates, other
Digestive enzyme will not be secreted by Institute of Micro-biology, thus significantly impact the effect that microbial fermentation promotes yellow ginger saponin(e to dissociate.
Glycosyl transferase：Microorganism during the fermentation, with dissociating for a large amount of yellow ginger saponin(es, promotes microorganism secretion sugar
Based transferase reconnects on water-insoluble saponin(e monose, effectively improves solubility of the saponin(e in water phase.
The enzyme activity of the cellulase of single bacterial strain bacillus HG224 is very low, due to hemicellulose, the parcel of lignin,
Microorganism is caused to fail to touch cellulosic structure, so that hardly eccrine fiber element enzyme；The zytase of single bacterial strain secretion,
Mannase and pectase enzyme activity level are very low, and the overall degradation efficiency of influence, fermentation promotes the free effect of yellow ginger saponin(e very
It is limited.
And two kinds of microorganisms (bacillus HG224 and a kind of Pectinatus, or a kind of bacillus HG224 and Ou Wenshi
Bacterium) mixing cause to share out the work and help one another between the flora microorganism, both avoided not same species of microorganism to the competing of narrow substrate
The effect of striving, also improves the degradation efficiency of different substrates, while secreting any of the above enzyme of enzymatic activity high, the association between various enzymes
Same-action becomes apparent from, the bound yellow ginger saponin(e of quick release.
A kind of a kind of three kinds of mixing of microorganism (bacillus HG224, Erwinia or Pectinatus, cellulomonas cartae)
Because the abundant degree of enzyme system is greatly improved, the synergy between various enzymes becomes apparent from, and can obtain saponin content higher.
But the flora can also be produced while promoting saponin(e free and continue the side effect that hydrolyzing saponin causes saponin(e water solubility to decline,
So that free saponin(e is precipitated and mixed in together with solid content again so that complex technical process, and consume more organic
Solvent, therefore the mixed bacterial of three kinds of microorganisms needs the accurately fermentation control can just to reach due performance, at utmost
Ground promotes the free of saponin(e.
It is below embodiment：
Embodiment 1 and 2 be without any microorganism in the case of blank experiment, embodiment 3 be single culture
The contrast test of fermentation yellow ginger, embodiment 4~5 is two kinds of microorganism species (bacillus HG224 and a kind of pectin of the invention
Bacillus, or bacillus HG224 and a kind of Erwinia) preparation and be applied to yellow ginger fermentation embodiment, embodiment 6~
8 use a kind of a kind of preparation of three kinds of microorganisms (bacillus HG224, Pectinatus or Erwinia, cellulomonas cartae) with
And it is applied to the embodiment of fermentation yellow ginger.In addition to embodiment 1 and 2, each embodiment has been monitored each in yellow ginger fermentation process
Microorganism enzyme activity change curve, analysis tests the percentage that the saponin content included in yellow ginger fermentation clear liquid accounts for total saponin content
Than wherein total saponin content is to convert to obtain by the saponin content that obtains equivalent yellow ginger material direct acidolysis.
Embodiment 1
In the case of without any microorganism, concrete operation step is as follows：
Yellow ginger fresh ginger 500g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:5 match somebody with somebody
Homogeneous slurry is made, at 37 DEG C, rotating speed is included to process the yellow ginger slurries obtained after 4h under the conditions of 200r/min in its liquid phase
Saponin content (being calculated according to saponin parent nucleus content) account for total saponin content 29% (total saponin content is by by equivalent yellow ginger material
The saponin content conversion that material direct acidolysis are obtained).
Embodiment 2
In the case of without any microorganism, concrete operation step is as follows：
Yellow ginger fresh ginger 500g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:15
Homogeneous slurry is configured to, at 37 DEG C, rotating speed is included to process the yellow ginger slurries obtained after 4h under the conditions of 200r/min in its liquid phase
Saponin content (being calculated according to saponin parent nucleus content) account for the 42% of total saponin content.
Embodiment 3
A kind of microbial strains of yellow ginger fermentation, are prepared as follows method preparation：
The single bacterium colony for taking Bacillus sp.HG224 bacterial strains is inoculated in LB culture mediums, under the conditions of 37 DEG C of 150r/min
Culture, results obtain expanding bacterium solution after culture to bacterial strain is in exponential phase.
When the microbial strains are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 500g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:10
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species of 20% (v/v) are inoculated with slurries, 0.5h are cultivated under the conditions of 29 DEG C of rotating speeds are 200r/min,
36 DEG C of rotating speeds obtain yellow ginger zymotic fluid to cultivate 3.5h under the conditions of 200r/min.
Fig. 1~Fig. 6 is to be applied to each microbial enzyme Mobile Forms monitoring song when yellow ginger ferments using Bacillus sp.HG224
Line.As can be seen from the figure：
1st, the enzyme activity of the cellulase of single bacterium is very low, due to hemicellulose, the parcel of lignin, causes microorganism to fail to connect
Cellulosic structure is contacted, so that hardly eccrine fiber element enzyme.
2nd, the enzyme activity of beta-glucosidase is higher, as shown in figure 1, being 150~230U.
3rd, the enzyme activity of amylase is higher, as shown in Fig. 2 400~550U, starch degradation is mainly oligomeric Portugal by amylase
Glycan or oligosaccharides, thus parcel of the starch to yellow ginger saponin(e can be effectively released, simultaneously because the micro- life of the control of fermentation time
Thing fails starch degradation in time by being utilized by oneself after glucose again, and the concentration of glucose of yellow ginger liquid glucose keeps relatively permanent
It is fixed, so that the comprehensive utilization following to liquid glucose of effective guarantee.
4th, the enzyme activity (Fig. 3) of zytase, 10~30U；The enzyme activity (Fig. 4) of mannase, 60~80U；Pectase
Enzyme activity (Fig. 5), 12~32U.
Due to the complexity of yellow ginger lignocellulosic structure, the substrate that microorganism touches during the fermentation is in continuous
During change, these enzymes that microorganism secretes during the fermentation belong to synergy enzyme, although enzyme activity is in relatively low water
It is flat, but these enzymes are essential, and if lacking these enzymes, microorganism will be not readily accessible to related substrates, other degradeds
Enzyme will not be secreted by Institute of Micro-biology, thus significantly impact the effect that microbial fermentation promotes yellow ginger saponin(e to dissociate, but simultaneously
It can be seen that the enzyme activity level of these enzymes is relatively low, the overall degradation efficiency of influence, single bacterial strain possesses certain degradation capability, but effect
It is really limited.
5th, the enzyme activity of glycosyl transferase is 50~380U (Fig. 6).Microorganism during the fermentation, with a large amount of yellow ginger saponin(es
It is free, promote microorganism secretion glycosyl transferase to reconnect on water-insoluble saponin(e monose, effectively improve saponin(e and exist
Solubility in water phase.
The saponin content (being calculated according to saponin parent nucleus content) included in the yellow ginger zymotic fluid liquid phase that the present embodiment is obtained
Only account for the 62% of total saponin content, single microorganism ferments in yellow ginger, promote that wherein saponin(e is free, improve saponin(e in water phase
The ability of solubility is very limited.
Embodiment 4
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
The single bacterium colony for taking Bacillus sp.HG224 and Pectobacterium carotovorum bacterial strains is inoculated in respectively
In LB culture mediums, at 30 DEG C, cultivated under the conditions of 100r/min, cultivate respectively and harvest and obtain after being in exponential phase to each bacterial strain
Expand bacterium solution, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio, ratio is as follows：Bacillus sp.50% and
Pectobacterium carotovorum 50%.
When microorganism species mixed liquor is applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 1000g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:5
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The mentioned microorganism flora mixed liquor of 10% (v/v) is inoculated with slurries, is 100r/min conditions in 30 DEG C of rotating speeds
Lower culture 4h carries out fermented and cultured, obtains yellow ginger zymotic fluid, and the saponin content included in liquid phase (is counted according to saponin parent nucleus content
Calculate) account for the 70% of total saponin content.
Different microorganisms in mixed microorganism flora can simultaneously utilize different substrates, thus quick release be wrapped and
Covalently bound yellow ginger saponin(e.
Embodiment 5
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
The single bacterium colony for taking Bacillus sp.HG224 and Erwinia amylovora bacterial strains is inoculated in LB culture mediums respectively
In, at 30 DEG C, cultivated under the conditions of 100r/min, results after being in exponential phase to each bacterial strain are cultivated respectively to be obtained expanding bacterium
Liquid, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio, and volume ratio is as follows：Bacillus sp.70% and
Erwinia amylovora 30%.
When microorganism species mixed liquor is applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 1000g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:5
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The mentioned microorganism flora mixed liquor of 10% (v/v) is inoculated with slurries, is 100r/min conditions in 30 DEG C of rotating speeds
Lower culture 4h carries out fermented and cultured, obtains yellow ginger zymotic fluid, and the saponin content included in liquid phase (is counted according to saponin parent nucleus content
Calculate) account for the 74% of total saponin content.
Different microorganisms in mixed microorganism flora can simultaneously utilize different substrates, thus quick release be wrapped and
Covalently bound yellow ginger saponin(e.
Embodiment 6
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strains
Bacterium colony is inoculated in LB culture mediums respectively, at 37 DEG C, is cultivated under the conditions of 200r/min, cultivates respectively to each bacterial strain and is given birth in logarithm
Harvested after long-term and obtain expanding bacterium solution, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio, volume ratio is as follows：
Bacillus sp.35%, Erwinia amylovora 40% and Cellulomonas flavigena 25%.
When microorganism species are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 800g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:12
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species mixed liquor of 30% (v/v) is inoculated with slurries, is trained under the conditions of 37 DEG C of rotating speeds are 210r/min
Foster 5h carries out fermented and cultured, obtains yellow ginger zymotic fluid, the saponin content (being calculated according to saponin parent nucleus content) included in liquid phase
Account for the 83% of total saponin content；
Embodiment 7
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strains
Bacterium colony is inoculated in LB culture mediums respectively, at 30 DEG C, is cultivated under the conditions of 100r/min, cultivates respectively to each bacterial strain and is given birth in logarithm
Harvested after long-term and obtain expanding bacterium solution, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio, volume ratio is as follows：
Bacillus sp.40%, Erwinia amylovora 25% and Cellulomonas flavigena 35%.
When microorganism species are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 1500g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:5
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species mixed liquor of 10% (v/v) is inoculated with slurries, is trained under the conditions of 30 DEG C of rotating speeds are 100r/min
Foster 4h carries out fermented and cultured, obtains yellow ginger zymotic fluid, the saponin content (being calculated according to saponin parent nucleus content) included in liquid phase
Account for the 73% of total saponin content；
Embodiment 8
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strains
Bacterium colony is inoculated in LB culture mediums respectively, at 40 DEG C, is cultivated under the conditions of 220r/min, cultivates respectively to each bacterial strain and is given birth in logarithm
Harvested after long-term and obtain expanding bacterium solution, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio, volume ratio is as follows：
Bacillus sp.30%, Erwinia amylovora 35% and Cellulomonas flavigena 35%.
When microorganism species are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Yellow ginger fresh ginger 1800g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water, be 1 according to the mass ratio of yellow ginger dry weight and water:15
It is configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species mixed liquor of 40% (v/v) is inoculated with slurries, is trained under the conditions of 40 DEG C of rotating speeds are 220r/min
Foster 6h carries out fermented and cultured, obtains yellow ginger zymotic fluid, the saponin content (being calculated according to saponin parent nucleus content) included in liquid phase
Account for the 85% of total saponin content.
Embodiment 9
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strains
Bacterium colony is inoculated in LB culture mediums respectively, at 30 DEG C, is cultivated under the conditions of 100r/min, cultivates respectively to each bacterial strain and is given birth in logarithm
Harvested after long-term, then obtain expanding bacterium solution by amplifying twice, microorganism species mixed liquor is mixed and made into according to bacterium solution volume ratio,
Volume ratio is as follows：Bacillus sp.40%, Erwinia amylovora 25% and Cellulomonas flavigena
35%.
When microorganism species are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Take yellow ginger fresh ginger some (dry weight 1750kg), clean and go after palpus the crushing homogenate that adds water, according to yellow ginger dry weight and water
Mass ratio is 1:5 are configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species mixed liquor of 10% (v/v) is inoculated with slurries, is trained under the conditions of 30 DEG C of rotating speeds are 100r/min
Foster 4h carries out fermented and cultured, obtains yellow ginger zymotic fluid, the saponin content (being calculated according to saponin parent nucleus content) included in liquid phase
Account for the 76% of total saponin content；
Embodiment 10
A kind of microorganism species for yellow ginger fermentation, are prepared as follows method preparation：
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strains
Bacterium colony is inoculated in LB culture mediums respectively, at 40 DEG C, is cultivated under the conditions of 220r/min, cultivates respectively to each bacterial strain and is given birth in logarithm
After long-term, then obtain expanding bacterium solution by amplifying twice, microorganism species mixed liquor, volume are mixed and made into according to bacterium solution volume ratio
Ratio is as follows：Bacillus sp.30%, Erwinia amylovora 35% and Cellulomonas flavigena
35%.
When microorganism species are applied into yellow ginger fermentation, concrete operation step is as follows：
(1) yellow ginger pretreatment：
Take yellow ginger fresh ginger some (dry weight 2250kg), clean and go after palpus the crushing homogenate that adds water, according to yellow ginger dry weight and water
Mass ratio is 1:15 are configured to homogeneous slurry；
(2) yellow ginger fermentation
The microorganism species mixed liquor of 40% (v/v) is inoculated with slurries, is trained under the conditions of 40 DEG C of rotating speeds are 220r/min
Foster 6h carries out fermented and cultured, obtains yellow ginger zymotic fluid, the saponin content (being calculated according to saponin parent nucleus content) included in liquid phase
Account for the 86% of total saponin content.
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention, it is not used to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc., all should include
Within protection scope of the present invention.
Claims (9)
1. a kind of bacillus, it is characterised in that the Classification And Nomenclature of the bacillus is Bacillus sp.HG224, described
Bacillus was preserved in China typical culture collection center CCTCC on December 30th, 2016, and its deposit number is CCTCC
M2016791。
2. the microorganism species that a kind of yellow ginger ferments, it is characterised in that the microorganism species are including bacillus HG224 and extremely
A kind of few Pectinatus, the Pectinatus can be replaced with Erwinia, and the Pectinatus are preferably
Pectobacterium carotovorum, the Erwinia is preferably Erwinia amylovora.
3. microorganism species as claimed in claim 2, it is characterised in that the microorganism species also include at least one fiber
Monad, the cellulomonas cartae is preferably Cellulomonas flavigena.
4. microorganism species as claimed in claim 2 or claim 3, it is characterised in that bacillus described in the microorganism species
HG224, Pectinatus and cellulomonas cartae are in exponential phase, the bacillus HG224 in exponential phase,
The bacterium solution volume ratio of Pectinatus and cellulomonas cartae is 1:0.4~1.2:0~1.2, the Pectinatus can use Ou Wenshi
Bacterium replaces.
5. microorganism species as described in claim 2~4 any one, it is characterised in that the microorganism species are used for point
Secrete lignocellulolytic enzymes, amylase and glycosyl transferase, it is preferable that the lignocellulolytic enzymes be cellulase,
Zytase, mannonase pectase or lignin-degrading enzymes.
6. the preparation method of the microorganism species as described in claim 2~5 any one, it is characterised in that including following step
Suddenly：The single bacterium colony for taking the bacterial strain of the bacillus HG224, Pectinatus and cellulomonas cartae is inoculated in the LB cultures respectively
It it is 30~40 DEG C in temperature in base, rotating speed is obtained respectively to cultivate bacterial strain under the conditions of 100~220r/min to exponential phase
The bacterium solution in exponential phase of the bacillus HG224, Pectinatus and cellulomonas cartae, by the bacterium solution according to body
Product ratio 1:0.4~1.2:0~1.2 is mixed and made into the microorganism species, and the Pectinatus can be replaced with Erwinia.
7. the application of the microorganism species as described in claim 2~5 any one, it is characterised in that be applied to yellow ginger fermentation.
8. the application of microorganism species as claimed in claim 7, it is characterised in that the microorganism species are applied to yellow ginger hair
Comprise the following steps during ferment：Mass ratio according to yellow ginger dry weight and water is 1:5~1:15 are configured to homogeneous slurry, are connect in slurries
The microorganism species as described in claim 2~5 any one that percentage by volume is 10%~40% are planted, microbial bacteria is carried out
Group's mixing fermentation culture, obtains the zymotic fluid containing free yellow ginger saponin(e.
9. the application of microorganism species as claimed in claim 8, it is characterised in that the fermentation culture conditions are：In temperature
For 30~40 DEG C, rotating speed are culture 4~24h, preferably 4~6h under the conditions of 100~220r/min.
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| CN103146795A (en) * | 2012-12-27 | 2013-06-12 | 西安绿泉生物技术有限公司 | Method for producing diosgenin through microbial fermentation of peltate yam rhizome-yellow ginger |
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