CN106834169B - 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 methods that microorganism mixed fermentation prepares yellow ginger saponin(e, belong to field of biotechnology, and emphasis solves the problems, such as that turmeric saponin yield is low in existing production technology.It cultivates bacillus HG224, a kind of Pectinatus or Erwinia, a kind of cellulomonas cartae respectively first, prepares microbial flora after being then inoculated with volume ratio mixing according to 1:0.4~1.2:0~1.2；It is that 1:5~1:15 is configured to uniform yellow ginger slurries according still further to the mass ratio of yellow ginger dry weight and water, it is 10%~40% mentioned microorganism flora that percentage by volume is inoculated in slurries, it then is 30 DEG C~40 DEG C in temperature, revolving speed cultivates 4~6h under conditions of being 100~220r/min.The microbial flora mixed fermentation can make the saponin(e separate out largely fettered, substantially increase turmeric saponin yield, meanwhile, to promote the green production of subsequent turmeric saponin to establish solid foundation, industrial applications have a extensive future.
Description
Technical field
The invention belongs to field of biotechnology, prepare yellow ginger saponin(e more particularly, to a kind of microorganism mixed fermentation
Method.
Background technique
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 in China, is distributed mainly on Hunan, Shaanxi, Sichuan, Guizhou and Yunnan etc.
It saves.The rhizomes of yellow ginger is containing the steroid saponins such as Dioscin constituents, 40% or so starch, 50% cellulose and some
The chemical components 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 (below claim saponin).Saponin is the aglucon of yellow ginger saponin(e, mainly with saponin(e in yellow ginger
Form exists.It is with products such as the steroid hormone intermediate of turmeric saponin synthesis and cortin, sex hormone, protein anabolic hormones
The drug that main hundreds of national economy are special procured is known as " medicinal gold ".It is only secondary that steroid hormone class drug, which has rapidly developed,
In the second major class drug of antibiotic.
The ingredients 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 plant fiber.Thus simple acid hydrolysis process yield is low, and being difficult will be in plant
Chinese yam saponin extracts completely, 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 some saponin(es in conjunction with cellulose, to reduce the yield of saponin.Patent
CN1970785A discloses a kind of method of Chinese yam saponin clean manufacturing and comprehensive utilization, will yellow ginger raw material crush after directly carry out
Solvent extraction can not be obtained with the reference state saponin(e of yellow ginger lignocellulosic structure covalent coupling, and saponin yield, which exists, promotes sky
Between.Patent CN1821380A discloses a kind of effectively compound microbial flora for handling yellow ginger, and the microbial flora used is contained
A variety of complex microorganisms such as lid bacterium, mould, saccharomycete, but it is that fungi growth time is longer there are problem, and bacterium and fungi
There is antagonism during the growth process, most of microbe exists stage by stage in yellow ginger lignocellulosic treatment process
The case where growth, there are uncontrollability as natural fermentation process, and microorganism to sapogenin parent nucleus might have degradation with
Transformation, to reduce saponin yield.
In conclusion traditional acid-hydrolysis method extracts turmeric saponin since the conversion of saponin(e is not specificity, it is simultaneously
Various substances, including lignocellulosic are hydrolyzed, starch, albumen etc., big with acid amount, therefore, low efficiency, energy consumption is high, and needs
High-temperature process；The prior art utilizes microorganism spontaneous fermentation to handle yellow ginger mostly, and the microorganism of spontaneous fermentation is uncontrollable, thus
The microorganism that some saponin parent nucleus that can degrade may be adulterated, so that saponin yield declines to a great extent.
Therefore, turmeric saponin yield is improved to greatest extent, and reducing turmeric saponin production cost is then existing production technology face
The outstanding problem faced promotes a large amount of free of yellow ginger saponin(e by innovation microbial process, saponin(e is fettered especially in yellow ginger
Dissociate particularly important, so, it researches and develops new green production process and to carry out industrial application very urgent.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of bacillus, microbial flora
And its method that mixed fermentation prepares yellow ginger saponin(e, its object is to be prepared micro- by selecting suitable microorganism fungus kind
Biological flora enables the microbial flora in yellow ginger fermentation successively to secrete high activity lignocellulolytic enzymes, starch
Enzyme and glycosyl transferase etc., collaboration promote the free of yellow ginger saponin(e, and improve solubility of the saponin(e in water phase, to improve most
Thus the yield of whole saponin solves the low outstanding problem of prior art turmeric saponin yield, be 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, a kind of bacillus is provided, classification naming is
Bacillus sp.HG224, the bacillus are preserved in China typical culture collection center on December 30th, 2016
CCTCC, deposit number are CCTCC M2016791.
Other side according to the invention provides a kind of microbial flora of yellow ginger fermentation, the microbial flora
Including bacillus HG224 and at least one Pectinatus, the Pectinatus can be replaced with Erwinia.
Preferably, the Pectinatus is carrot pectin bacillus, and Latin literary fame scientific name is Pectobacterium
carotovorum。
Preferably, the Erwinia is solution Erzvinia amylovora, and Latin name is Erwinia amylovora.
Preferably, the microbial flora further includes at least one cellulomonas cartae.
Preferably, the cellulomonas cartae is Cellumomonas flavigena, Latin name Cellulomonas
flavigena。
Preferably, bacillus HG224, Pectinatus and cellulomonas cartae described in the microbial flora are in pair
Number growth period, the bacterium solution volume ratio of the bacillus HG224 in logarithmic growth phase, Pectinatus and cellulomonas cartae are
1:0.4~1.2:0~1.2, the Pectinatus can be replaced with Erwinia.
Preferably, the microbial flora is for secreting lignocellulolytic enzymes, amylase and glycosyl transferase.
Preferably, the lignocellulolytic enzymes are cellulase, zytase, mannonase pectase or wood
Lignin-degrading enzymes.
Preferably, the microbial flora yellow ginger ferment when 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.
Other side according to the invention provides the preparation method of microbial flora described in one kind, including as follows
Step: the single colonie of the bacterial strain of the bacillus HG224, Pectinatus and cellulomonas cartae is taken to be inoculated in the LB training respectively
It supports in base, is 30~40 DEG C in temperature, culture bacterial strain obtains respectively to logarithmic growth phase under the conditions of revolving speed is 100~220r/min
The bacillus HG224, Pectinatus and cellulomonas cartae the bacterium solution in logarithmic growth phase, by the bacterium solution according to
Volume ratio 1:0.4~1.2:0~1.2 are mixed and made into the microbial flora, and the Pectinatus can use Erwinia generation
It replaces.
Other side according to the invention provides the application of microbial flora described in one kind, is applied to yellow ginger and sends out
Ferment.
Preferably, the microbial flora is applied to include the following steps: when yellow ginger ferments according to yellow ginger dry weight and water
Mass ratio be 1:5~1:15 be configured to homogeneous slurry, in slurries be inoculated with percentage by volume be 10%~40% it is described
Microbial flora carries out microbial flora mixing fermentation culture, obtains the fermentation liquid containing yellow ginger saponin(e.
Preferably, the fermentation culture conditions are as follows: under the conditions of temperature is 30~40 DEG C, revolving speed is 100~220r/min
Culture 4~for 24 hours.
Preferably, the fermented incubation time is 4~6h.
In general, above-mentioned technical proposal through the invention compared with prior art, can achieve the following beneficial effects.
1, the present invention provides a kind of bacillus HG224, and provide it is a kind of comprising the bacillus for yellow ginger
The microbial flora of fermentation can secrete ligocellulose degradation when using the microbial flora fermentation process yellow ginger raw material
Enzyme system, amylase and glycosyl transferase and enzyme activity vigor is very high, keep lignocellulosic structure loose, and make the saponin(e release of constraint
Out, make to be present in yellow ginger histocyte and sufficiently be dissociated by the saponin(e of the packages such as lignocellulosic, pectin, starch, pass through hair
The control of ferment time releases the package action of starch without influencing glucose content so that Starch Conversion is oligosaccharide or oligosaccharides.
2, microorganism conversion saponin(e when due to a large amount of saponin(es water-insoluble state, cause transformation efficiency lower, using this
After the invention microbial flora fermentation yellow ginger, due to the water-solubility saponin largely fettered release and be present in water phase,
Solid foundation has been established for the microorganism progress Efficient Conversion production saponin of subsequent transformation saponin(e.
3, the preparation method of microbial flora provided by the invention and its application in yellow ginger fermentation, promote a large amount of constraints
Saponin(e it is free so that subsequent production of saponin yield is high, technique is environmentally protective, pollution-free, at low cost, and industrial application prospect is wide
It is wealthy.
4, when microbial flora of the invention is applied to yellow ginger fermentation and promotes the release of yellow ginger saponin(e and dissolution, due to wherein not
With the synergistic effect of strain, compared with not adding microorganism or only adding single microorganism, saponin(e yield is greatly improved.
Detailed description of the invention
The enzyme activity dynamic monitoring curve of beta-glucosidase when Fig. 1 is Bacillus sp.HG224 fermentation yellow ginger；
The enzyme activity dynamic monitoring curve of amylase when Fig. 2 is Bacillus sp.HG224 fermentation yellow ginger；
The enzyme activity dynamic monitoring curve of zytase when Fig. 3 is Bacillus sp.HG224 fermentation yellow ginger；
The enzyme activity dynamic monitoring curve of mannase when Fig. 4 is Bacillus sp.HG224 fermentation yellow ginger；
The enzyme activity dynamic monitoring curve of pectase when Fig. 5 is Bacillus sp.HG224 fermentation yellow ginger；
The enzyme activity dynamic monitoring curve of glycosyl transferase when Fig. 6 is Bacillus sp.HG224 fermentation yellow ginger；
Fig. 7 is the phyletic evolution that bacillus sp.HG224 uses the building of N-j method according to 16S rDNA sequence
Tree.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
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 in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
A kind of bacillus provided by the invention, classification naming is Bacillus sp.HG224, in December, 2016
It is preserved within 30th Wuhan, China, China typical culture collection center CCTCC, deposit number is CCTCC M2016791,
Latin name is Bacillus sp..
Fig. 7 is the systematic evolution tree that bacillus HG224 uses the building of N-j method according to 16S rDNA sequence.Bacterial strain gemma
Bacillus HG224 uses the systematic evolution tree of Neighbour-joining method building according to its 16S rDNA sequence, with strain Bacillus
Bacillus M6K gathers on systematic evolution tree for same cluster, thus is classified as Bacillus strain.
Provided by the present invention for the microbial flora of yellow ginger fermentation, including bacillus HG224 and at least one pectin
Bacillus, wherein Pectinatus can be replaced with Erwinia.The microbial flora can also include at least one cellulomonas cartae.
Wherein Pectinatus is preferably carrot pectin bacillus Pectobacterium carotovorum, and Erwinia is excellent
It is selected as solution Erzvinia amylovora Erwinia amylovora, cellulomonas cartae is preferably Cellumomonas flavigena
Cellulomonas flavigena, these three bacterium limit without specific bacterial strain.
Bacillus HG224, Pectinatus and cellulomonas cartae are in logarithmic growth phase, the microbial flora apply with
It is inoculated with when yellow ginger ferments according to bacterium solution volume ratio for 1:0.4~1.2:0~1.2, wherein Pectinatus can use Ou Wenshi
Bacterium replaces.
For secreting lignocellulolytic enzymes, amylase and glycosyl transferase when microbial flora fermentation yellow ginger,
Middle lignocellulolytic enzymes are cellulase, zytase, mannonase pectase or lignin-degrading enzymes.
The microbial flora can secrete the amylase of 200~820U/ml, the β-of 120~450U/ml when yellow ginger ferments
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 microbial flora includes the following steps: to take the bacillus HG224, Pectinatus and fibre
The single colonie of bacterial strain for tieing up monad is inoculated in respectively in the LB culture medium, is 30~40 DEG C in temperature, and revolving speed is 100~
Under the conditions of 220r/min, obtain the bacillus HG224, Pectinatus and cellulomonas cartae respectively is in logarithmic growth phase
Bacterium solution, the bacterium solution is mixed and made into the microbial flora, the pectin according to volume ratio 1:0.4~1.2:0~1.2
Bacillus can be replaced with Erwinia.
Microbial flora is applied to include the following steps: when yellow ginger ferments according to the mass ratio of yellow ginger dry weight and water to be 1:5
~1:15 is configured to homogeneous slurry, and the above-mentioned microbial flora that percentage by volume is 10%~40% is inoculated in slurries,
Culture 4 under the conditions of temperature is 30~40 DEG C, revolving speed is 100~220r/min~for 24 hours, it is mixed to carry out microbial flora by preferably 4~6h
Fermented and cultured is closed, the fermentation liquid containing yellow ginger saponin(e is obtained.By fermentation liquid after being separated by solid-liquid separation thallus, the saponin(e containing yellow ginger is obtained
Clear liquid, (hydrolysate is washed depickling to sampling analysis saponin content therein by acid hydrolysis, rear to carry out solvent extraction Detection and Extraction
Saponin content in liquid characterizes saponin content), then further obtained by Multistage Membranes filtering containing the dense of a large amount of yellow ginger saponin(es
Contracting liquid, or above-mentioned clear liquid is directly carried out to microorganism conversion production turmeric saponin.
When microbial flora of the invention is applied to yellow ginger fermentation, various enzymes play during yellow ginger ferments dissolution saponin(e
Function analysis it is as follows:
Cellulase: cellulase can effectively degrade the cellulosic structure in yellow ginger material, and cellulose is that glucose is residual
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
Close connection results in the constraint effect to saponin(e, thus cellulase can effectively promote the release of saponin(e.
Amylase: starch degradation is mainly oligomeric glucan or oligosaccharides by amylase, thus can effectively release shallow lake
Powder is to the package of yellow ginger saponin(e, simultaneously because the control microorganism of fermentation time fails after being glucose for starch degradation in time again
It is utilized by oneself, the concentration of glucose of yellow ginger liquid glucose keeps relative constant, so that effective guarantee is next to the comprehensive of liquid glucose
It closes and utilizes.
Zytase, mannase and pectase: due to the complexity of yellow ginger lignocellulosic structure, microorganism is being sent out
It is zytase that microorganism secretes during the fermentation, sweet during the substrate touched during ferment is in continually changing
Dew dextranase, pectase belong to synergistic effect enzyme, if lacking these enzymes, microorganism will be not readily accessible to related substrates, other
Degrading enzyme will not be secreted by Institute of Micro-biology, thus significantly impact the effect that microbial fermentation promotes yellow ginger saponin(e free.
Glycosyl transferase: microorganism during the fermentation, with dissociating for a large amount of yellow ginger saponin(es, promotes microorganism secretion sugared
Based transferase reconnects to monosaccharide on water-insoluble saponin(e, 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 package of lignin,
Microorganism is caused to fail to touch cellulosic structure, thus hardly eccrine fiber element enzyme；The zytase of single bacterial strain secretion,
Mannase and pectase enzyme activity level are very low, influence whole degradation efficiency, the effect that fermentation promotes yellow ginger saponin(e free is very
It is limited.
And two kinds of microorganisms (bacillus HG224 and a kind of Pectinatus or bacillus HG224 and a kind of Ou Wenshi
Bacterium) mixing so that can 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 the above various enzymes 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 mixing of three kinds of microorganisms (bacillus HG224, Erwinia or Pectinatus, cellulomonas cartae)
Since the abundant degree of enzyme system is greatly improved, the synergistic effect between various enzymes is become apparent from, and can obtain higher saponin content.
But the flora can also generate 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 precipitate and mixed in together with solid content again, so that complex technical process, and consume more organic
Solvent, therefore accurately fermentation control just can achieve due performance to the mixed bacterial needs of three kinds of microorganisms, utmostly
Ground promotes the free of saponin(e.
The following are embodiments:
Examples 1 and 2 are the blank test in the case where not adding any microorganism, and embodiment 3 is single culture
The comparative test of fermentation yellow ginger, embodiment 4~5 are two kinds of microbial floras (bacillus HG224 and a kind of pectin of the invention
Bacillus or bacillus HG224 and a kind of Erwinia) preparation and 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 the embodiment applied to fermentation yellow ginger.In addition to Examples 1 and 2, each embodiment has monitored each in yellow ginger fermentation process
Microorganism enzyme activity change curve, analysis test the percentage that the saponin content for including 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 for obtaining equivalent yellow ginger material direct acidolysis.
Embodiment 1
In the case where not adding any microorganism, specific steps are as follows:
Yellow ginger fresh ginger 500g is taken, cleans and adds the broken homogenate of pigment after removing palpus, match according to the mass ratio of yellow ginger dry weight and water for 1:5
Homogeneous slurry is made, at 37 DEG C, revolving speed handles the yellow ginger slurries obtained after 4h under the conditions of being 200r/min, includes in 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
Expect the saponin content conversion that direct acidolysis obtains).
Embodiment 2
In the case where not adding any microorganism, specific steps are as follows:
Yellow ginger fresh ginger 500g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:15 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry, at 37 DEG C, revolving speed handles the yellow ginger slurries obtained after 4h under the conditions of being 200r/min, includes in 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 colonie of Bacillus sp.HG224 bacterial strain is taken to be inoculated in LB culture medium, under the conditions of 37 DEG C of 150r/min
Culture, harvest obtains expanding bacterium solution after culture is in logarithmic growth phase to bacterial strain.
When the microbial strains are applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 500g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:10 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora of 20% (v/v) in slurries, cultivates 0.5h under the conditions of 29 DEG C of revolving speeds are 200r/min,
36 DEG C of revolving speeds cultivate 3.5h under the conditions of being 200r/min, obtain yellow ginger fermentation liquid.
FIG. 1 to FIG. 6 is to be applied to each microbial enzyme Mobile Forms when yellow ginger ferments using Bacillus sp.HG224 to monitor song
Line.As can be seen from the figure:
1, the enzyme activity of the cellulase of single bacterium is very low, due to hemicellulose, the package of lignin, microorganism is caused to fail to connect
Cellulosic structure is contacted, thus hardly eccrine fiber element enzyme.
2, the enzyme activity of beta-glucosidase is higher, as shown in Figure 1, being 150~230U.
3, 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 starch can be effectively released to the package of yellow ginger saponin(e, simultaneously because the micro- life of the control of fermentation time
Object fails again in time by starch degradation by being utilized after glucose by oneself, and the concentration of glucose of yellow ginger liquid glucose keeps relatively permanent
It is fixed, so that effective guarantee is next to the comprehensive utilization of liquid glucose.
4, 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 variation, these enzymes that microorganism secretes during the fermentation belong to synergistic effect enzyme, although enzyme activity is in lower water
It is flat, but these enzymes are essential, if lacking these enzymes, microorganism will be not readily accessible to related substrates, other degradations
Enzyme will not be secreted by Institute of Micro-biology, thus significantly impact the effect that microbial fermentation promotes yellow ginger saponin(e free, but simultaneously
It can be seen that the enzyme activity level of these enzymes is lower, whole degradation efficiency is influenced, single bacterial strain has certain degradation capability, but imitates
Fruit is limited.
5, 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 to monosaccharide on water-insoluble saponin(e, effectively improve saponin(e and exist
Solubility in water phase.
The saponin content (being calculated according to saponin parent nucleus content) for including in the yellow ginger fermentation liquid liquid phase that the present embodiment obtains
The 62% of total saponin content is only accounted for, wherein saponin(e is free, improves saponin(e in water phase in yellow ginger fermentation, promotion for single microorganism
The ability of solubility is very limited.
Embodiment 4
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
The single colonie of Bacillus sp.HG224 and Pectobacterium carotovorum bacterial strain is taken to be inoculated in respectively
It in LB culture medium, at 30 DEG C, is cultivated under the conditions of 100r/min, is cultivated respectively to each bacterial strain to be in after logarithmic growth phase to harvest and obtain
Expand bacterium solution, be mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio, ratio is as follows: Bacillus sp.50% and
Pectobacterium carotovorum 50%.
When microbial flora mixed liquor is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 1000g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:5 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the mentioned microorganism flora mixed liquor of 10% (v/v) in slurries, is 100r/min condition in 30 DEG C of revolving speeds
Lower culture 4h carries out fermented and cultured, obtains yellow ginger fermentation liquid, the saponin content for including 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 utilize different substrates simultaneously, thus quick release wrapped up and
Covalently bound yellow ginger saponin(e.
Embodiment 5
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
The single colonie of Bacillus sp.HG224 and Erwinia amylovora bacterial strain is taken to be inoculated in LB culture medium respectively
In, it at 30 DEG C, is cultivated under the conditions of 100r/min, cultivates harvest after being in logarithmic growth phase to each bacterial strain respectively and obtain expanding bacterium
Liquid is mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio, and volume ratio is as follows: Bacillus sp.70% and
Erwinia amylovora 30%.
When microbial flora mixed liquor is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 1000g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:5 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the mentioned microorganism flora mixed liquor of 10% (v/v) in slurries, is 100r/min condition in 30 DEG C of revolving speeds
Lower culture 4h carries out fermented and cultured, obtains yellow ginger fermentation liquid, the saponin content for including 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 utilize different substrates simultaneously, thus quick release wrapped up and
Covalently bound yellow ginger saponin(e.
Embodiment 6
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strain
Bacterium colony is inoculated in respectively in LB culture medium, at 37 DEG C, is cultivated under the conditions of 200r/min, is cultivated respectively to each bacterial strain and is in logarithm life
Harvest obtains expanding bacterium solution after long-term, is mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio, volume ratio is as follows:
Bacillus sp.35%, Erwinia amylovora 40% and Cellulomonas flavigena 25%.
When microbial flora is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 800g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:12 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora mixed liquor of 30% (v/v) in slurries, is trained under the conditions of 37 DEG C of revolving speeds are 210r/min
It supports 5h and carries out fermented and cultured, obtain yellow ginger fermentation liquid, the saponin content for including in liquid phase (calculates) according to saponin parent nucleus content
Account for the 83% of total saponin content；
Embodiment 7
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strain
Bacterium colony is inoculated in respectively in LB culture medium, at 30 DEG C, is cultivated under the conditions of 100r/min, is cultivated respectively to each bacterial strain and is in logarithm life
Harvest obtains expanding bacterium solution after long-term, is mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio, volume ratio is as follows:
Bacillus sp.40%, Erwinia amylovora 25% and Cellulomonas flavigena 35%.
When microbial flora is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 1500g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:5 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora mixed liquor of 10% (v/v) in slurries, is trained under the conditions of 30 DEG C of revolving speeds are 100r/min
It supports 4h and carries out fermented and cultured, obtain yellow ginger fermentation liquid, the saponin content for including in liquid phase (calculates) according to saponin parent nucleus content
Account for the 73% of total saponin content；
Embodiment 8
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strain
Bacterium colony is inoculated in respectively in LB culture medium, at 40 DEG C, is cultivated under the conditions of 220r/min, is cultivated respectively to each bacterial strain and is in logarithm life
Harvest obtains expanding bacterium solution after long-term, is mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio, volume ratio is as follows:
Bacillus sp.30%, Erwinia amylovora 35% and Cellulomonas flavigena 35%.
When microbial flora is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
Yellow ginger fresh ginger 1800g is taken, cleans and adds the broken homogenate of pigment after removing palpus, is 1:15 according to the mass ratio of yellow ginger dry weight and water
It is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora mixed liquor of 40% (v/v) in slurries, is trained under the conditions of 40 DEG C of revolving speeds are 220r/min
It supports 6h and carries out fermented and cultured, obtain yellow ginger fermentation liquid, the saponin content for including in liquid phase (calculates) according to saponin parent nucleus content
Account for the 85% of total saponin content.
Embodiment 9
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strain
Bacterium colony is inoculated in respectively in LB culture medium, at 30 DEG C, is cultivated under the conditions of 100r/min, is cultivated respectively to each bacterial strain and is in logarithm life
Harvest after long-term obtains expanding bacterium solution, is mixed and made into microbial flora mixed liquor according to bacterium solution volume ratio using amplification twice,
Volume ratio is as follows: Bacillus sp.40%, Erwinia amylovora 25% and Cellulomonas flavigena
35%.
When microbial flora is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
It takes yellow ginger fresh ginger several (dry weight 1750kg), cleans and go after palpus plus the broken homogenate of pigment, according to yellow ginger dry weight and water
Mass ratio is that 1:5 is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora mixed liquor of 10% (v/v) in slurries, is trained under the conditions of 30 DEG C of revolving speeds are 100r/min
It supports 4h and carries out fermented and cultured, obtain yellow ginger fermentation liquid, the saponin content for including in liquid phase (calculates) according to saponin parent nucleus content
Account for the 76% of total saponin content；
Embodiment 10
Method preparation is prepared as follows in a kind of microbial flora for yellow ginger fermentation:
Take the list of Bacillus sp.HG224, Erwinia amylovora and Cellulomonas flavigena bacterial strain
Bacterium colony is inoculated in respectively in LB culture medium, at 40 DEG C, is cultivated under the conditions of 220r/min, is cultivated respectively to each bacterial strain and is in logarithm life
After long-term, obtain expanding bacterium solution using amplification twice, be mixed and made into microbial flora mixed liquor, volume according to bacterium solution volume ratio
Ratio is as follows: Bacillus sp.30%, Erwinia amylovora 35% and Cellulomonas flavigena
35%.
When microbial flora is applied to yellow ginger fermentation, specific steps are as follows:
(1) yellow ginger pre-processes:
It takes yellow ginger fresh ginger several (dry weight 2250kg), cleans and go after palpus plus the broken homogenate of pigment, according to yellow ginger dry weight and water
Mass ratio is that 1:15 is configured to homogeneous slurry；
(2) yellow ginger ferments
It is inoculated with the microbial flora mixed liquor of 40% (v/v) in slurries, is trained under the conditions of 40 DEG C of revolving speeds are 220r/min
It supports 6h and carries out fermented and cultured, obtain yellow ginger fermentation liquid, the saponin content for including in liquid phase (calculates) according to saponin parent nucleus content
Account for the 86% of total saponin content.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (12)
1. a kind of bacillus (Bacillus sp.) HG224, which is characterized in that the bacillus was on December 30th, 2016
It is preserved in China typical culture collection center CCTCC, deposit number is CCTCC M2016791.
2. a kind of microbial flora of yellow ginger fermentation, which is characterized in that the microbial flora includes as described in claim 1
Bacillus (Bacillus sp.) HG224 and at least one Pectinatus (Pectobacterium sp.).
3. microbial flora as claimed in claim 2, which is characterized in that the Pectinatus is soft rotten Pectinatus
(Pectobacterium carotovorum)。
4. a kind of microbial flora of yellow ginger fermentation, which is characterized in that the microbial flora includes as described in claim 1
Bacillus (Bacillus sp.) HG224 and at least one Erwinia (Erwinia sp.).
5. microbial flora as claimed in claim 4, which is characterized in that the Erwinia is solution Erzvinia amylovora
(Erwinia amylovora)。
6. such as the described in any item microbial floras of claim 2 to 5, which is characterized in that the microbial flora further include to
A kind of few cellulomonas cartae (Cellulomonas sp.).
7. microbial flora as claimed in claim 6, which is characterized in that the cellulomonas cartae is Cellumomonas flavigena
(Cellulomonas flavigena)。
8. microbial flora as claimed in claim 6, which is characterized in that the microbial flora includes the bacillus
When (Bacillus sp.) HG224, Pectinatus and cellulomonas cartae, bacillus described in the microbial flora
(Bacillus sp.) HG224, Pectinatus and cellulomonas cartae is in logarithmic growth phase, described in logarithmic growth phase
The bacterium solution volume ratio of bacillus (Bacillus sp.) HG224, Pectinatus and cellulomonas cartae be 1:0.4~1.2:0~
1.2；
When the microbial flora includes the bacillus (Bacillus sp.) HG224, Erwinia and cellulomonas cartae,
Bacillus described in the microbial flora (Bacillus sp.) HG224, Erwinia and cellulomonas cartae is in pair
Number growth period, described bacillus (Bacillus sp.) HG224, Erwinia and cellulomonas cartae in logarithmic growth phase
Bacterium solution volume ratio be 1:0.4~1.2:0~1.2.
9. the preparation method of the microbial flora as described in claim 2~8 any one, which is characterized in that the microorganism
When flora includes the bacillus (Bacillus sp.) HG224, Pectinatus and cellulomonas cartae, which includes
Following steps: the single colonie of the bacterial strain of the bacillus (Bacillus sp.) HG224, Pectinatus and cellulomonas cartae is taken
It is inoculated in the LB culture medium respectively, is 30~40 DEG C in temperature, revolving speed cultivates bacterial strain extremely under the conditions of being 100~220r/min
Logarithmic growth phase obtains being in for the bacillus (Bacillus sp.) HG224, Pectinatus and cellulomonas cartae respectively
The bacterium solution is mixed and made into the microbial bacteria according to volume ratio 1:0.4~1.2:0~1.2 by the bacterium solution of logarithmic growth phase
Group；
When the microbial flora includes the bacillus (Bacillus sp.) HG224, Erwinia and cellulomonas cartae,
The preparation method includes the following steps: to take the bacillus (Bacillus sp.) HG224, Erwinia and cellulomonas cartae
The single colonie of bacterial strain be inoculated in the LB culture medium respectively, be 30~40 DEG C in temperature, revolving speed is 100~220r/min item
Bacterial strain is cultivated under part to logarithmic growth phase, obtains the bacillus (Bacillus sp.) HG224, Erwinia and fibre respectively
The bacterium solution is mixed according to volume ratio 1:0.4~1.2:0~1.2 and is made by the bacterium solution in logarithmic growth phase for tieing up monad
At the microbial flora.
10. the application of the microbial flora as described in claim 2~8 any one, which is characterized in that be applied to yellow ginger and send out
Ferment.
11. the application of microbial flora as claimed in claim 10, which is characterized in that the microbial flora is applied to yellow ginger
Include the following steps: to be that 1:5~1:15 is configured to homogeneous slurry according to the mass ratio of yellow ginger dry weight and water when fermentation, in slurries
It is inoculated with the microbial flora as described in claim 2~8 any one that percentage by volume is 10%~40%, carries out microorganism
Flora mixing fermentation culture obtains the fermentation liquid containing free yellow ginger saponin(e.
12. the application of microbial flora as claimed in claim 11, which is characterized in that the fermentation culture conditions are as follows: in temperature
Degree for 30~40 DEG C, revolving speed be 100~220r/min under the conditions of culture 4~for 24 hours.
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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 |
| CN104109644A (en) * | 2014-05-29 | 2014-10-22 | 华中科技大学 | Bacillus sp. and its use in natural product extraction |
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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 |
| CN104109644A (en) * | 2014-05-29 | 2014-10-22 | 华中科技大学 | Bacillus sp. and its use in natural product extraction |
Non-Patent Citations (2)
| Title |
|---|
| Novel method utilizing microbial treatment for cleaner production of diosgenin from Dioscorea zingiberensis CH Wright (DZW);Wei, M等;《BIORESOURCE TECHNOLOGY》;20130801;第146卷;第549-555页 * |
| 生物技术提取黄姜皂素的研究进展;李文君等;《生物质化学工程》;20131130;第47卷(第6期);第46-52页 * |
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