CN106754422A - A kind of Tabin aspergillus and its application in turmeric saponin is prepared - Google Patents
A kind of Tabin aspergillus and its application in turmeric saponin is prepared Download PDFInfo
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Abstract
A kind of application the invention discloses Tabin aspergillus and its in turmeric saponin is prepared, Tabin aspergillus HG 57, its preserving number is CCTCC M2016389.Take appropriate Tabin aspergillus mycelium inoculation to be activated on PDA solid mediums, the collection that added water after spore is produced obtains spore suspension.Spore suspension is linked into amplify in seed culture medium and obtains seed liquor.Seed liquor after spore suspension or amplification is linked into the mixed liquid of yellow ginger Aqueous extracts, the yellow ginger alcohol extracting aqueous solution or yellow ginger water, aerobic fermentation obtains zymotic fluid after 1~7 day；Separation and Extraction obtains saponin product from zymotic fluid.Compared with the saponin content that the direct acid hydrolysis in above-mentioned system is obtained, 1.2~1.4 times, 1.1~1.4 times, 1.4~1.8 times have been respectively increased.The bacterial strain can effectively convert yellow ginger saponin(e for saponin, thoroughly substitute the sour water solution conversion process in traditional handicraft, and be easy to engineering and amplify, with wide industrial applications prospect.
Description
Technical field
The invention belongs to microorganism field and biological technical field, more particularly, to a kind of Tabin aspergillus and its in system
Application in standby turmeric saponin.
Background technology
Diosgenin, also known as saponin, is the important source material for producing steroid hormone class medicine.Global steroid hormone class medicine
In 60% above is with diosgenin as Material synthesis or semi-synthetic production.The method of traditional production diosgenin is
Using strong acid acidolysis, but this method because pollution is big, the water quality safety of high energy consumption and the influence south water to north.The mistake of acidolysis simultaneously
There is saponin(e conversion in journey and not exclusively and easily produce accessory substance so as to influence the quality of saponin product.Current research is generally recognized
For enzymatic isolation method and microbe transformation method have good application prospect, with it is environment-friendly the characteristics of, as current saponin green
The focus of production.
At present, researcher has found that layer goes out fusarium WX12 (A of CN 102703329) and possesses conversion production diosgenin
Function, but it puts into zymotic fluid and is converted again from seed liquor culture thalline, needs to add serial inorganic salts in conversion process
Composition, spent time is oversize, and diosgenin yield is low, and considerable water-soluble of content is have lost during yellow ginger saponin(e is prepared
Property saponin(e.Also some researchers have found that saponin(e can be changed into diosgenin by aspergillus oryzae (B of CN 101012474), but be deposited
Many in intermediate product, the low problem of conversion ratio is difficult to realize commercial application.Separately there is researcher to find, aspergillus awamori (CN
104232724 A) yellow ginger saponin(e production diosgenin can be converted, because starch in yellow ginger dry powder and cellulose are to the beam of saponin(e
Effect is tied up, patent discloses the saponin(e that bacterial strain fails transform portion constraint, causes sapogenin yield relatively low.There is research and utilization acidophilus breast
Acidfast bacilli (B of CN 103497987) conversion yellow ginger saponin(e production diosgenin, wherein employing starch, cellulose and Chinese yam
The method that saponin separation retains Dioscin by nanofiltration again, operating process is complicated, and is wherein not publicly obtained with direct sour water solution
Obtain the comparing of saponin yield, it is impossible to the conversion effect of the bacterial strain is correctly evaluated in the case where different sources yellow ginger quality discrepancy is larger
Rate.During existing patent report microorganism conversion yellow ginger saponin(e production saponin, complex technical process, conversion saponin(e form is single
And transformation efficiency is low, saponin yield is low, it is difficult to realize commercial application.
The A of patent CN 103242418 disclose a kind of cleaning manufacturing technique method of turmeric saponin, make use of saccharification, film
Separate and ultrasonic extraction technology, obtain the film concentrate of saponin(e and the saponin(e extraction concentrate of organic solvent, can be more comprehensive
Saponin(e is obtained in industrialization rank, but sour hydrolyzation catalysis saponin(e hydrolysis is still used during saponin(e is converted into saponin
It is saponin, fails to realize real green production.
The content of the invention
It is the invention provides a kind of Tabin aspergillus and its yellow preparing for the disadvantages described above or Improvement requirement of prior art
Application in ginger saponin, its object is to the yellow ginger saponin(e of the various existence forms of Efficient Conversion, thus can be with existing saponin
Industrialized production is docked, and it is the process of saponin thoroughly to pass through sour hydrolysis saponin(e in replacement traditional handicraft, and is thus solved
Certainly convert the technical bottleneck in yellow ginger saponin(e green production turmeric saponin industrialization process.
To achieve the above object, according to one aspect of the present invention, there is provided a kind of Tabin aspergillus, the Tabin aspergillus
Classification And Nomenclature is Aspergillus tubingensis HG-57, and it was preserved in Chinese Typical Representative culture on July 11st, 2016
Collection CCTCC, its deposit number is CCTCC M2016389, and preservation address is：China, Wuhan, Wuhan University.
According to another aspect of the present invention, there is provided a kind of application of described Tabin aspergillus, legal system of fermenting is applied to
Standby turmeric saponin.
Preferably, the application of described Tabin aspergillus, comprises the following steps：
(1) in the spore suspension access yellow ginger saponin(e stoste for obtaining the culture Tabin aspergillus, aerobic fermentation 1~7 day
Afterwards, zymotic fluid is obtained；
(2) the separation and Extraction saponin from the zymotic fluid, as described turmeric saponin.
Preferably, the preparation method of the spore suspension includes：Take described Tabin aspergillus and be inoculated in PDA solid cultures
On base, activated within 2~7 days in 20~37 DEG C of cultures, after spore is grown, added sterilized water, mixed and collect and obtain spore
Suspension.
Preferably, the preparation method also includes：The spore suspension is linked into amplify in seed culture medium and is planted
Sub- liquid, the seed culture medium includes following composition：2~15g/L glucose, 5~15g/L dusty yeasts, 10~25g/L albumen
Peptone.
Preferably, the yellow ginger saponin(e stoste includes that yellow ginger Aqueous extracts, the yellow ginger alcohol extracting aqueous solution and yellow ginger water mix liquid.
Preferably, the preparation method of the yellow ginger Aqueous extracts is：Yellow ginger fresh ginger is crushed into the slurries after homogenate or by yellow ginger
Dry powder adds water after fully mixing and carries out separation of solid and liquid, takes supernatant and obtains yellow ginger Aqueous extracts.
Preferably, the preparation method of the yellow ginger alcohol extracting aqueous solution is：To methyl alcohol or ethanol is added in yellow ginger dry powder, through filling
Dividing after extracting carries out separation of solid and liquid, obtains yellow ginger alcohol extract, and vacuum decompression reclaims methyl alcohol or ethanol, obtains yellow ginger alcohol-extracted extract,
Then it is to obtain the yellow ginger alcohol extracting aqueous solution to inorganic salt solution is added in yellow ginger alcohol-extracted extract.
Preferably, the inorganic salt solution includes following component：0.5~5g/L of potassium nitrate, 0.5~2g/L of magnesium sulfate,
0.2~1.5g/L of potassium dihydrogen phosphate, 0.2~1.5g/L of sodium chloride, 0.2~1.5g/L of disodium hydrogen phosphate.
Preferably, the mixed liquid of the yellow ginger water is after directly the slurries or yellow ginger dry powder after the crushing homogenate of yellow ginger fresh ginger are added water
Re-suspension liquid, the yellow ginger fresh ginger for water content be 50%~80% without dehydration yellow ginger.
In general, by above-mentioned technical proposal of the invention compared with prior art, following beneficial effect can be obtained.
(1) the invention provides one plant of Tabin aspergillus HG-57, can be applied to conversion yellow ginger saponin(e production turmeric saponin；
(2) Tabin aspergillus HG-57 bacterial strains are capable of the yellow ginger saponin(e production turmeric saponin of the various existence forms of Efficient Conversion, tower
Saponin(e in guest aspergillus HG-57 bacterial strains conversion yellow ginger Aqueous extracts, the yellow ginger alcohol extracting aqueous solution or the mixed liquid of yellow ginger water obtains containing for saponin
Amount, compared with the content that the direct acid hydrolysis of above-mentioned system obtains saponin, be respectively increased 1.2~1.4 times, 1.1~1.4 times,
1.4~1.8 times；
(3) saponin(e is converted into during Tabin aspergillus HG-57 bacterial strains can effectively dock existing turmeric saponin industrialized production
The technological process of production of saponin, is thoroughly expected to existing in replacement traditional handicraft by the process that sour hydrolysis saponin(e is saponin
Having saponin industry carries out technology upgrading, realizes green production.
Brief description of the drawings
Fig. 1 is enzyme activity curves of the Tabin aspergillus HG-57 during yellow ginger Aqueous extracts are converted；
Fig. 2 is the colonial morphology that Tabin aspergillus HG-57 is cultivated the 3rd day；
Fig. 3 is the colonial morphology that Tabin aspergillus HG-57 is cultivated the 5th day；
Fig. 4 is the systematic evolution tree that Tabin aspergillus HG-57 is built according to ITS sequence using N-j methods.
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 specific embodiment described herein is only used to explain the present invention, not
For limiting the present invention.As long as additionally, technical characteristic involved in invention described below each implementation method that
Conflict is not constituted between this can be just mutually combined.
A kind of Tabin aspergillus HG-57 that the present invention is provided, has delivered to China typical culture collection center preservation.Preservation day
Phase is on July 11st, 2016, and deposit number is CCTCC M 2016389, and its Latin name is Aspergillus
Tubingensis, preservation address：China, Wuhan, Wuhan University.
The invention provides the Tabin aspergillus HG-57 answering in fermentation method conversion yellow ginger saponin(e prepares turmeric saponin
With can effectively convert the soap in the mixed liquid of the saponin(e in yellow ginger Aqueous extracts, the saponin(e in the yellow ginger alcohol extracting aqueous solution and yellow ginger water
Glycosides produces turmeric saponin.
Saponin(e in yellow ginger Aqueous extracts is present in water phase with free monomer or microencapsulated form, is applied to convert yellow ginger water extraction
During saponin(e in liquid, carry out in accordance with the following steps：
(1) yellow ginger Aqueous extracts are prepared：Take yellow ginger fresh ginger appropriate, clean and go after palpus the crushing homogenate that adds water, in slurries centrifuging and taking
Clear liquid, it is also possible to which yellow ginger dry powder adds water with reference to centrifuging and taking supernatant, the supernatant after the treatment of the supplementary means such as ultrasound or microwave
Liquid is yellow ginger Aqueous extracts；The yellow ginger Aqueous extracts are processed into 20min at 121~126 DEG C carries out high-temperature heat sterilization；
(2) Tabin aspergillus spore suspension is prepared：Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane,
Cultivated 2~7 days at 20~37 DEG C, after spore is grown, addition has the sterilized water of bead, shakes 2min, obtains and collect tower
The spore suspension of guest's aspergillus；Spore suspension is linked into seed culture medium, at 25~35 DEG C, 150~220r/min cultures 18
The seed liquor of Tabin aspergillus is obtained after~36h, wherein seed culture medium includes following component：2~15g glucose, 5~15g/L
Dusty yeast, 10~25g/L peptones；
(3) conversion saponin(e production saponin：By the spore suspension of Tabin aspergillus, preferably the seed liquor of Tabin aspergillus is linked into
During step (1) is through the yellow ginger Aqueous extracts of high-temperature heat sterilization, at 25~35 DEG C, 150~220r/min fermented and cultureds 1~7 day
Afterwards, zymotic fluid is obtained；
(4) separation and Extraction saponin：60~90 DEG C of drying of the saponin zymolyte obtained after the zymotic fluid separation of solid and liquid that will be obtained,
Methyl alcohol 10~40min of ultrasonic extraction is added, extraction times are 1~2 time, and wherein methyl alcohol volume is the 1~2 of zymotic fluid initial volume
Times, conventional method concentration, crystal refining obtain saponin product；It is preferred that being extracted using No. 120 gasoline, after extract is dried, obtain
Saponin.
When being applied to convert the saponin(e in the yellow ginger alcohol extracting aqueous solution, carry out in accordance with the following steps：
(1) the yellow ginger alcohol extracting aqueous solution is prepared：The saponin(e in yellow ginger is extracted using methyl alcohol or ethanol, methyl alcohol or ethanol is reclaimed
Afterwards, saponin(e is present in alcohol-extracted extract, plus appropriate inorganic salt solution is made the yellow ginger alcohol extracting aqueous solution.The inorganic salt solution
Including following component, 0.5~5g/L of potassium nitrate, 0.5~2g/L of magnesium sulfate, 0.2~1.5g/L of potassium dihydrogen phosphate, sodium chloride 0.2
~1.5g/L, 0.2~1.5g/L of disodium hydrogen phosphate, is dissolved in water.The yellow ginger alcohol extracting aqueous solution is located at 121~126 DEG C
Reason 20min carries out high-temperature heat sterilization；
(2) Tabin aspergillus spore suspension is prepared：Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane,
Cultivated 2~7 days at 20~37 DEG C, after spore is grown, addition has the sterilized water of bead, shakes 2min, obtains and collect tower
The spore suspension of guest's aspergillus；Spore suspension is linked into seed culture medium, at 25~35 DEG C, 150~220r/min cultures 18
The seed liquor of Tabin aspergillus is obtained after~36h, wherein seed culture medium includes following component：2~15g/L glucose, 5~15g/
L dusty yeasts, 10~25g/L peptones；
(3) conversion saponin(e production saponin：By the spore suspension of Tabin aspergillus, preferably the seed liquor of Tabin aspergillus is linked into
During step (1) is through the yellow ginger alcohol extracting aqueous solution of high-temperature heat sterilization, at 25~35 DEG C, 150~220r/min fermented and cultureds 1~
After 7 days, zymotic fluid is obtained；
(4) separation and Extraction saponin：60~90 DEG C of drying of the saponin zymolyte obtained after the zymotic fluid separation of solid and liquid that will be obtained,
Methyl alcohol 10~40min of ultrasonic extraction is added, extraction times are 1~2 time, and wherein methyl alcohol volume is the 1~2 of zymotic fluid initial volume
Times, conventional method concentration, crystal refining obtain saponin product；It is preferred that being extracted using No. 120 gasoline, after extract is dried, obtain
Saponin.
When being applied to convert the saponin(e in the mixed liquid of yellow ginger water, carry out in accordance with the following steps：
(1) the mixed liquid of yellow ginger water is prepared：Yellow ginger fresh ginger is crushed the re-suspension liquid after slurries or yellow ginger dry powder after homogenate add water
As yellow ginger water mixes liquid, and the mixed liquid of the yellow ginger water is processed into 20min at 121~126 DEG C carries out high-temperature heat sterilization；
(2) Tabin aspergillus spore suspension is prepared：Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane,
Cultivated 2~7 days at 20~37 DEG C, after spore is grown, addition has the sterilized water of bead, shakes 2min, obtains and collect tower
The spore suspension of guest's aspergillus；Spore suspension is linked into seed culture medium, at 25~35 DEG C, 150~220r/min cultures 18
The seed liquor of Tabin aspergillus is obtained after~36h, wherein seed culture medium includes following component：2~15g/L glucose, 5~15g/
L dusty yeasts, 10~25g/L peptones；
(3) conversion saponin(e production saponin：By the spore suspension of Tabin aspergillus, preferably the seed liquor of Tabin aspergillus is linked into
Step (1) is mixed in liquid through the yellow ginger water of high-temperature heat sterilization, at 25~35 DEG C, 150~220r/min fermented and cultureds 1~7 day
Afterwards, zymotic fluid is obtained；
(4) separation and Extraction saponin：60~90 DEG C of drying of the saponin zymolyte obtained after the zymotic fluid separation of solid and liquid that will be obtained,
Methyl alcohol 10~40min of ultrasonic extraction is added, extraction times are 1~2 time, and wherein methyl alcohol volume is the 1~2 of zymotic fluid initial volume
Times, conventional method concentration, crystal refining obtain saponin product；It is preferred that being extracted using No. 120 gasoline, after extract is dried, obtain
Saponin.
The method for quantitatively determining of turmeric saponin：High performance liquid chromatography, chromatographic condition is：C18 reversed-phase columns (syncronis
250 × 4.6mm of C18), Detection wavelength 204nm, mobile phase is pure methyl alcohol, flow velocity 1ml/min, the μ L of sample size 10.
Fig. 1 is enzyme activity curves of the Tabin aspergillus HG-57 during yellow ginger Aqueous extracts are converted, it can be seen that Tabin aspergillus
Beta-glucosidase, cellulase, the enzymatic activitys of zytase and amylase of the HG-57 during yellow ginger Aqueous extracts are converted,
These four enzymes related to saponin(e hydrolysis have a different degrees of fluctuation in overall process, enzymatic activity be held in one it is higher
Level.Just because of the abundant mutual coordinated of glycosidase enzyme system of bacterial strain, just ensure that Efficient Conversion of the bacterial strain to saponin(e.Simultaneously
Water miscible saponin(e is easily by bacterial strain hydrolysis.
Although beta-glucosidase enzymatic activity is relatively low relative to other three kinds of enzymes, have during bacterial strain conversion saponin(e
Very big fluctuation, illustrates that the enzyme is continually changing in processing procedure with being continually changing for substrate, is one in conversion process
Individual required enzyme, and the enzyme only needs to very low enzyme activity level and can just meet effect of the bacterial strain to substrate.
Fig. 2 and Fig. 3 is colonial morphology when Tabin aspergillus HG-57 is cultivated 3 days and 5 days respectively.Tabin aspergillus HG- of the present invention
57 biology morphology is described as：Rapid in PDA cultured on solid medium, 28 DEG C are cultivated 3 days, 12~15mm of diameter, after 7 days
25~30mm is reached, quality villiform, mycelia nascent is white, palm fibre, black are faded to, without diffusate, reverse side yellowish-brown.
Fig. 4 is the systematic evolution tree that Tabin aspergillus HG-57 is built according to ITS sequence using N-j methods.Bacterial strain Tabin aspergillus
HG-57 is according to its ITS rDNA sequence, and the systematic evolution tree built using Neighbour-joining methods is bent with bacterial strain tower guest
It is same cluster that mould FJBJ11 gathers on systematic evolution tree.
It is below embodiment：
Embodiment 1
Embodiment 1 is the comparative example of the saponin(e preparation saponin in acid-catalyzed hydrolysis conversion yellow ginger Aqueous extracts.
Yellow ginger dry powder 500g is taken, is added water so that yellow ginger dry weight：Water=1：5, ultrasonically treated 40min, rear 8000r/ after mixing
Min is centrifuged 10min, takes supernatant and obtains yellow ginger Aqueous extracts.Yellow ginger Aqueous extracts are carried out into acid-catalyzed hydrolysis, hydrolysising condition is：Body
It is acid concentration 1mol/L, 100 DEG C of reaction temperature, hydrolysis time 5h；The hydrolysate for obtaining is filtered, and the filter cake for obtaining is using certainly
Water or soda lye wash are to neutrality, then are dried and obtain saponin hydrolysate.The saponin hydrolysate for obtaining is carried using No. 120 gasoline
Take, after extract is dried, obtain saponin.The saponin of gained carries out quantitative analysis and wherein contains saponin using high performance liquid chromatography
1.24g/ is per 100g yellow ginger rhizoma zingiberis.
Embodiment 2
Embodiment 2 is the reality that saponin is prepared using the saponin(e in Tabin aspergillus fermentation method of the invention conversion yellow ginger Aqueous extracts
Apply example.
Yellow ginger dry powder 1000g is taken, is added water so that yellow ginger dry weight：Water=1：5, ultrasonically treated 40min, rear 8000r/ after mixing
Min is centrifuged 10min, and the supernatant that will be obtained obtains yellow ginger Aqueous extracts in humid heat treatment 20min at 121 DEG C.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, is cultivated 3 days at 28 DEG C, spore to be grown
Afterwards, the sterilized water with bead is added, 2min is shaken, the spore suspension of Tabin aspergillus is obtained and collect.
The spore suspension of Tabin aspergillus is linked into yellow ginger Aqueous extracts, at 29 DEG C, 210r/min fermented and cultureds are after 2 days,
Zymotic fluid is obtained, 60~90 DEG C of drying of saponin zymolyte that the zymotic fluid that will be obtained is obtained after 8000r/min centrifugations 10min,
Methyl alcohol ultrasonic extraction 40min is added, extraction times are 2 times, and wherein methyl alcohol volume is 2 times of zymotic fluid initial volume, routine side
Method concentration, crystal refining obtain saponin product.The saponin of gained carries out quantitative analysis and wherein contains using high performance liquid chromatography
Saponin 1.65g/ is per 100g yellow ginger rhizoma zingiberis.
Embodiment 3
Embodiment 3 is the comparative example of the saponin(e preparation saponin in the acid-catalyzed hydrolysis conversion yellow ginger alcohol extracting aqueous solution.
Yellow ginger dry powder 2000g is taken, the saponin(e in yellow ginger is extracted using 75% ethanol, after reclaiming ethanol, saponin(e is present in alcohol
In extracted extract, add water as the yellow ginger alcohol extracting aqueous solution, the yellow ginger alcohol extracting aqueous solution is carried out into acid-catalyzed hydrolysis, hydrolysising condition is：Body
It is acid concentration 1mol/L, 100 DEG C of reaction temperature, hydrolysis time 5h；The hydrolysate for obtaining is filtered, and the filter cake for obtaining is using certainly
Water or soda lye wash are to neutrality, then are dried and obtain saponin hydrolysate.The saponin hydrolysate for obtaining is carried using No. 120 gasoline
Take, after extract is dried, obtain saponin.The saponin of gained carries out quantitative analysis and wherein contains saponin using high performance liquid chromatography
2.14g/ is per 100g yellow ginger rhizoma zingiberis.
Embodiment 4
Embodiment 4 is to prepare saponin using the saponin(e in the Tabin aspergillus fermentation method of the invention conversion yellow ginger alcohol extracting aqueous solution
First embodiment.
Yellow ginger dry powder 1800g is taken, the saponin(e in yellow ginger is extracted using 75% ethanol, after reclaiming ethanol, saponin(e is present in alcohol
In extracted extract, inorganic salt solution (potassium nitrate 0.5g/L, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 0.2g/L, sodium chloride are added
0.2g/L, disodium hydrogen phosphate 0.2g/L), be dissolved in water, natural ph) after 20min processed at 123 DEG C carry out that high temperature is damp and hot to go out
Bacterium..
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, quiescent culture 2 days at 25 DEG C wait to grow
After spore, addition has the sterilized water of bead, shakes 2min, obtains and collect spore suspension.Spore suspension is linked into seed
In culture medium (10g/L glucose, 10g/L dusty yeasts, 15g/L peptones), at 25 DEG C, tower is obtained after 150r/min cultures 18h
The seed liquor of guest's aspergillus.
The seed liquor of Tabin aspergillus is linked into the yellow ginger alcohol extracting aqueous solution, at 25 DEG C, 150r/min fermented and cultureds 1 day
Afterwards, zymotic fluid is obtained, 8000r/min centrifugations 12min obtains sediment.Extracted using No. 120 gasoline, after extract is dried, obtained
To saponin.The saponin of gained carries out quantitative analysis and is wherein done per 100g yellow gingers containing saponin 3.36g/ using high performance liquid chromatography
Ginger.
Embodiment 5
Embodiment 5 is to prepare saponin using the saponin(e in the Tabin aspergillus fermentation method of the invention conversion yellow ginger alcohol extracting aqueous solution
Second embodiment.
Yellow ginger dry powder 1200g is taken, the saponin(e in yellow ginger is extracted using 75% ethanol, after reclaiming ethanol, saponin(e is present in alcohol
In extracted extract, inorganic salt solution (potassium nitrate 5g/L, magnesium sulfate 2g/L, potassium dihydrogen phosphate 1.5g/L, sodium chloride 1.5g/ are added
L, disodium hydrogen phosphate 1.5g/L), it is dissolved in water, 20min is then processed at 123 DEG C carries out high-temperature heat sterilization.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, quiescent culture 7 days at 35 DEG C wait to grow
After spore, addition has the sterilized water of bead, shakes 2min, obtains and collect spore suspension.Spore suspension is linked into seed
In culture medium (2g/L glucose, 5g/L dusty yeasts, 10g/L peptones), at 35 DEG C, tower guest is obtained after 220r/min cultures 36h
The seed liquor of aspergillus.
The seed liquor of Tabin aspergillus is linked into the yellow ginger alcohol extracting aqueous solution, at 35 DEG C, 220r/min fermented and cultureds 7 days
Afterwards, zymotic fluid is obtained, 8000r/min centrifugations 12min obtains sediment.Extracted using No. 120 gasoline, after extract is dried, obtained
To saponin.The saponin of gained carries out quantitative analysis wherein containing saponin 2.354g/ per 100g yellow gingers using high performance liquid chromatography
Rhizoma zingiberis.
Embodiment 6
Embodiment 6 is the comparative example of the saponin(e preparation saponin in the acid-catalyzed hydrolysis mixed liquid of conversion yellow ginger water.
Yellow ginger fresh ginger 5000g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water so that yellow ginger dry weight：Water=1：7, as yellow ginger
Water mixes liquid.The mixed liquid of yellow ginger water is carried out into acid-catalyzed hydrolysis, hydrolysising condition is：System acid concentration 1mol/L, 100 DEG C of reaction temperature,
Hydrolysis time 5h；The hydrolysate for obtaining is filtered, and the filter cake for obtaining uses running water or soda lye wash to neutrality, then is done
It is dry to obtain saponin hydrolysate.The saponin hydrolysate for obtaining is extracted using No. 120 gasoline, after extract is dried, obtains saponin.Gained
Saponin carry out quantitative analysis wherein containing saponin 2.4g/ per 100g yellow ginger rhizoma zingiberis using high performance liquid chromatography.
Embodiment 7
Embodiment 7 is the reality that saponin is prepared using the saponin(e in the Tabin aspergillus fermentation method of the invention mixed liquid of conversion yellow ginger water
Apply example.
Yellow ginger fresh ginger 3000g is taken, is cleaned and is gone after palpus the crushing homogenate that adds water so that yellow ginger dry weight：Water=1：7, at 123 DEG C
Treatment 20min is the mixed liquid of yellow ginger water after carrying out high-temperature heat sterilization.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, quiescent culture 6 days at 30 DEG C wait to grow
After spore, addition has the sterilized water of bead, shakes 2min, obtains and collect spore suspension.Spore suspension is linked into seed
In culture medium (15g/L glucose, 15g/L dusty yeasts, 25g/L peptones), at 29 DEG C, tower is obtained after 200r/min cultures 28h
The seed liquor of guest's aspergillus.
The seed liquor of Tabin aspergillus is linked into the mixed liquid of yellow ginger water, at 31 DEG C, 200r/min fermented and cultureds are obtained after 7 days
To zymotic fluid, 8000r/min centrifugation 12min acquisition sediments.Sediment is extracted using No. 120 gasoline, after extract is dried, is obtained
To saponin.The saponin of gained carries out quantitative analysis and is wherein done per 100g yellow gingers containing saponin 4.32g/ using high performance liquid chromatography
Ginger.
Embodiment 8
Embodiment 8 is using the saponin(e system in Tabin aspergillus fermentation method of the invention conversion industrially scalable yellow ginger Aqueous extracts
The embodiment of standby saponin.
Take yellow ginger fresh ginger some (dry weight 2000kg), the crushing homogenate that adds water causes yellow ginger dry weight：Water=1：5, passed through after mixing
Plate-frame filtering squeezing is crossed, the clear liquid that will be obtained obtains yellow ginger Aqueous extracts in humid heat treatment 20min at 121 DEG C.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, is cultivated 3 days at 28 DEG C, spore to be grown
Afterwards, the sterilized water with bead is added, 2min is shaken, the spore suspension of Tabin aspergillus is obtained and collect.
The spore suspension of Tabin aspergillus is inoculated in seed culture medium (5g/L glucose, 10g/L dusty yeasts, 12g/L albumen
Peptone) in, 30 DEG C, 200r/min culture 24h after obtain primary seed solution, after by two grades amplify obtain secondary seed solutions, will put
Seed liquor after big is linked into yellow ginger Aqueous extracts, at 28 DEG C, 210r/min, and fermented and cultured is obtained after 2 days under throughput 0.5vvm
To zymotic fluid, the saponin zymolyte that the zymotic fluid that will be obtained is obtained after being squeezed through plate-frame filtering dry at 60~90 DEG C, addition
Methyl alcohol ultrasonic extraction 40min, extraction times are 2 times, and wherein methyl alcohol volume is 2 times of zymotic fluid initial volume, and conventional method is dense
Contracting, crystal refining obtain saponin product.The saponin of gained carries out quantitative analysis and wherein contains saponin using high performance liquid chromatography
1.62g/ is per 100g yellow ginger rhizoma zingiberis.
Embodiment 9
Embodiment 9 is using the soap in the Tabin aspergillus fermentation method of the invention conversion industrially scalable yellow ginger alcohol extracting aqueous solution
Glycosides prepares the embodiment of saponin.
Yellow ginger dry powder 1680kg is taken, the saponin(e in yellow ginger is extracted using 75% ethanol, after reclaiming ethanol, saponin(e is present in alcohol
In extracted extract, inorganic salt solution (potassium nitrate 4g/L, magnesium sulfate 1.2g/L, potassium dihydrogen phosphate 1.3g/L, sodium chloride are added
1.5g/L, disodium hydrogen phosphate 1.3g/L), it is dissolved in water, 20min is then processed at 123 DEG C carries out high-temperature heat sterilization.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, quiescent culture 7 days at 35 DEG C wait to grow
After spore, addition has the sterilized water of bead, shakes 2min, obtains and collect spore suspension.Spore suspension is linked into seed
In culture medium (5g/L glucose, 10g/L dusty yeasts, 10g/L peptones), at 35 DEG C, tower guest is obtained after 220r/min cultures 36h
The primary seed solution of aspergillus, after by two grades amplify obtain secondary seed solutions.Seed liquor after amplification is linked into yellow ginger alcohol extracting
In the aqueous solution, at 35 DEG C, 220r/min, fermented and cultured obtains zymotic fluid, plate-frame filtering squeezing after 7 days under throughput 0.35vvm
Obtain saponin zymolyte.Extracted using No. 120 gasoline, after extract is dried, obtain saponin.The saponin of gained uses efficient liquid phase
Chromatography carries out quantitative analysis wherein containing saponin 2.2g/ per 100g yellow ginger rhizoma zingiberis.
Embodiment 10
Embodiment 10 is using the saponin(e in the Tabin aspergillus fermentation method of the invention mixed liquid of conversion industrially scalable yellow ginger water
Prepare the embodiment of saponin.
Take yellow ginger fresh ginger some (dry weight 2120kg), add water crushing homogenate so that yellow ginger dry weight：Water=1：7, at 123 DEG C
Lower treatment 20min is the mixed liquid of yellow ginger water after carrying out high-temperature heat sterilization.
Tabin aspergillus HG-57 is transferred on PDA solid mediums from inclined-plane, quiescent culture 6 days at 30 DEG C wait to grow
After spore, addition has the sterilized water of bead, shakes 2min, obtains and collect spore suspension.Spore suspension is linked into seed
In culture medium (15g/L glucose, 15g/L dusty yeasts, 25g/L peptones), at 29 DEG C, tower is obtained after 200r/min cultures 28h
The seed liquor of guest's aspergillus, after by two grades amplify obtain secondary seed solutions.Seed liquor after amplification is linked into the mixed liquid of yellow ginger water
In, at 31 DEG C, 200r/min, fermented and cultured obtains zymotic fluid after 7 days under throughput 0.6vvm, and plate-frame filtering squeezing obtains soap
Plain zymolyte.Extracted using No. 120 gasoline, after extract is dried, obtain saponin.The saponin of gained uses high performance liquid chromatography
Quantitative analysis is carried out wherein containing saponin 4.15g/ per 100g yellow ginger rhizoma zingiberis.
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 (10)
1. a kind of Tabin aspergillus, it is characterised in that the Classification And Nomenclature of the Tabin aspergillus is Aspergillus tubingensis
HG-57, it was preserved in China typical culture collection center CCTCC on July 11st, 2016, and its deposit number is CCTCC M
2016389。
2. a kind of application of Tabin aspergillus as claimed in claim 1, it is characterised in that be applied to fermentation method and prepare brown windsor soap
Element.
3. the application of Tabin aspergillus as claimed in claim 2, it is characterised in that comprise the following steps：
(1) will cultivate in the spore suspension access yellow ginger saponin(e stoste that the Tabin aspergillus are obtained, aerobic fermentation is obtained after 1~7 day
To zymotic fluid；
(2) the separation and Extraction saponin from the zymotic fluid, as described turmeric saponin.
4. the application of Tabin aspergillus as claimed in claim 3, it is characterised in that the preparation method of the spore suspension includes：
Take Tabin aspergillus as claimed in claim 1 to be inoculated on PDA solid mediums, lived within 2~7 days in 20~37 DEG C of cultures
Change, after spore is grown, add sterilized water, mix and collect and obtain spore suspension.
5. the application of Tabin aspergillus as claimed in claim 4, it is characterised in that the preparation method also includes：By the spore
Amplification obtains seed liquor during sub- suspension is linked into seed culture medium, and the seed culture medium includes following composition：2~15g/L Portugals
Grape sugar, 5~15g/L dusty yeasts, 10~25g/L peptones.
6. the application of Tabin aspergillus as claimed in claim 3, it is characterised in that the yellow ginger saponin(e stoste includes yellow ginger water extraction
Liquid, the yellow ginger alcohol extracting aqueous solution and yellow ginger water mix liquid.
7. the application of Tabin aspergillus as claimed in claim 6, it is characterised in that the preparation method of the yellow ginger Aqueous extracts is：
Added water by the slurries after the crushing homogenate of yellow ginger fresh ginger or by yellow ginger dry powder and carry out separation of solid and liquid after mixing, taken supernatant and obtain Huang
Ginger Aqueous extracts.
8. the application of Tabin aspergillus as claimed in claim 6, it is characterised in that the preparation method of the yellow ginger alcohol extracting aqueous solution
For：To adding methyl alcohol or ethanol in yellow ginger dry powder, it is extracted after carry out separation of solid and liquid, obtain yellow ginger alcohol extract, vacuum decompression is returned
Methyl alcohol or ethanol are received, yellow ginger alcohol-extracted extract is obtained, is then to obtain yellow ginger to inorganic salt solution is added in yellow ginger alcohol-extracted extract
The alcohol extracting aqueous solution.
9. the application of Tabin aspergillus as claimed in claim 8, it is characterised in that the inorganic salt solution includes such as the following group
Point：0.5~5g/L of potassium nitrate, 0.5~2g/L of magnesium sulfate, 0.2~1.5g/L of potassium dihydrogen phosphate, 0.2~1.5g/L of sodium chloride, phosphorus
Sour 0.2~1.5g/L of disodium hydrogen.
10. the application of Tabin aspergillus as claimed in claim 6, it is characterised in that the mixed liquid of the yellow ginger water is by yellow ginger fresh ginger
The re-suspension liquid that the slurries or yellow ginger dry powder obtained after crushing homogenate are obtained after adding water.
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