CN109468250A - A method of improving honeysuckle vinasse Content of Chlorogenic Acid - Google Patents
A method of improving honeysuckle vinasse Content of Chlorogenic Acid Download PDFInfo
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Abstract
The invention discloses a kind of methods for improving honeysuckle vinasse Content of Chlorogenic Acid, belong to field of bioconversion.The method of the present invention include the following steps: by deposit number be CCTCC NO:M 2018803 bacillus subtilis hx0210 be inoculated into LB culture medium, 30 DEG C, 150r/min culture 16-18h obtain bacillus subtilis hx0210 bacterium solution；By bacillus subtilis hx0210 bacterium solution, honeysuckle vinasse, LB culture medium by volume 4:8:3 ratio mix, 30 DEG C, 150rpm ferment 6h.The content of the raising fermentation culture medium Content of Chlorogenic Acid of the energy high degree of bacillus subtilis hx0210 used in the present invention；Present invention utilizes the high concentrated organic wastewaters of the distillation workshop section discharge in distilled liquid of honeysuckle production process, reduce pollution and the wasting of resources, provide the new channel of yield of chlorogenic acid.
Description
Technical field
The invention belongs to field of bioconversion, and in particular to a kind of side for improving honeysuckle vinasse Content of Chlorogenic Acid
Method.
Background technique
Honeysuckle (Lonicerae japonica Thunb) is the dry flower for being under the jurisdiction of caprifoliaceae plant, is that one kind is answered
It with the green natural product of extensive " integration of drinking and medicinal herbs ", and is the important Chinese medicine in China, it can be with dietotherapeutic.Honeysuckle tool
There is the effects of clearing heat and detoxicating, wind-dispelling heat-dissipating and antibacterial anti-inflammatory, therefore is referred to as " national treasure a flower ".There is former catechu in honeysuckle
The organic acids compound such as acid, isochlorogenic acid, chlorogenic acid, Content of Chlorogenic Acid are the representative component of organic acid, the presence of chlorogenic acid
So that honeysuckle is provided with the abilities such as anti-inflammatory, antibacterial, removing free radical, anti-oxidant, antiviral.
It currently, can be found everywhere both at home and abroad to the research of honeysuckle, is carried out in terms of the flower of honeysuckle and leaf
Research, honeysuckle distill residue using less.Honeysuckle vinasse is the distillation in commercially available distilled liquid of honeysuckle production process
The high concentrated organic wastewater of workshop section's discharge, in the industrial production, honeysuckle bottoms raffinate is typically directly discarded, wherein containing
Measure it is higher, have a bioactive ingredients such as chlorogenic acid, flavones, saponin(e and tannin with valuable pharmacological effect, honeysuckle distillation
The direct emission of residue not only causes the waste of resource, also creates contaminated wastewater.
Chlorogenic acid is extensive in industrial applications such as medical industry, food, cosmetics, and the demand of chlorogenic acid is larger on the market,
Price is also costly.Residue, which is distilled, using honeysuckle by microorganism conversion produces chlorogenic acid, it can be more economical in creation
The discharge of industrial wastewater is reduced while benefit.
Summary of the invention
The purpose of this invention is to solve the problems associated with the prior art, provides green in a kind of raising honeysuckle vinasse
The bacillus subtilis hx0210 arrived used in the method and this method of Determination of Chlorogenic Acid.
The purpose of the invention is achieved by the following technical solution:
A kind of bacillus subtilis, classification naming are Bacillus subtilis hx0210 bacillus subtilis
Hx0210, deposit number are CCTCC NO:M 2018803, are preserved in China typical culture collection on November 19th, 2018
Center (address: the Chinese Wuhan Wuhan University).
The bacillus subtilis can be used for producing chlorogenic acid；It is especially produced using honeysuckle vinasse as raw material green
The raw material of ortho acid, i.e. production chlorogenic acid preferably includes honeysuckle vinasse.
A method of honeysuckle vinasse Content of Chlorogenic Acid is improved, is included the following steps:
(1) bacillus subtilis hx0210 is inoculated into culture medium and carries out activation culture, obtain bacillus subtilis
Hx0210 bacterium solution.
(2) bacillus subtilis hx0210 bacterium solution, honeysuckle vinasse, culture medium are mixed and carries out fermented and cultured.
Preferably, the condition of activation culture described in step (1) is 30 DEG C, 150r/min cultivates 16-18h.
Preferably, step (2) bacillus subtilis hx0210 bacterium solution, the volume ratio of honeysuckle vinasse are 1:2；Into one
Step is preferred, bacillus subtilis hx0210 bacterium solution, honeysuckle vinasse, culture medium volume ratio be 4:8:3.
Preferably, the condition of fermented and cultured described in step (2) be 30 DEG C, 150rpm ferment 6h.
Preferably, step (1), culture medium described in (2) are LB culture medium.
Preferably, the method for the raising honeysuckle vinasse Content of Chlorogenic Acid, includes the following steps:
(1) bacillus subtilis hx0210 is inoculated into LB culture medium, 30 DEG C, 150r/min culture 16-18h obtain it is withered
Careless bacillus hx0210 bacterium solution.
(2) by the ratio of bacillus subtilis hx0210 bacterium solution, honeysuckle vinasse, LB culture medium 4:8:3 by volume
Example mixing, 30 DEG C, 150rpm fermentation 6h.
The present invention has the following advantages compared with the prior art and the utility model has the advantages that bacillus subtilis hx0210 of the invention
The content of the raising fermentation culture medium Content of Chlorogenic Acid of energy high degree；Present invention utilizes the distillations in distilled liquid of honeysuckle production process
The high concentrated organic wastewater of workshop section's discharge, reduces pollution and the wasting of resources, provides the new channel of yield of chlorogenic acid.
Detailed description of the invention
Fig. 1 is chlorogenic acid canonical plotting.
Fig. 2 is result figure of the fermentation temperature to chlorogenic acid yield effect.
Fig. 3 is result figure of the fermentation time to chlorogenic acid yield effect.
Fig. 4 is result figure of the inoculum concentration to chlorogenic acid yield effect.
Fig. 5 is result figure of the revolving speed to chlorogenic acid yield effect.
Specific embodiment
Following embodiment should not be construed as limiting the invention for further illustrating the present invention.If not referring in particular to
Conventional means bright, that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1
Natural storage 20 days or more honeysuckle vinasses are filtered, clarification stoste are obtained, in superclean bench, with shifting
The stoste that liquid rifle draws 1mL moves into the test tube equipped with 9mL sterile water, sufficiently shakes up, and is made 10-1Honeysuckle raffinate dilution,
10 are produced in the same way-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Honeysuckle vinasse dilution.It prepares
PDA culture medium, coating concentration respectively in PDA plate is 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Dilution
After liquid, in 37 DEG C of culture 72h, bacterium colony is grown to it, picking individual colonies are crossed in new PDA culture medium, until being purified
Single colonie.
As procedure described above by repeatedly screening, the bacillus subtilis of a plant height yield of chlorogenic acid is obtained, withered grass is named as
Bacillus hx0210.
Bacillus subtilis hx0210 is rounded, and dirty white, edge is imperfect, and centre is slightly swelled, and rough surface is impermeable
It is bright, it is not easy to provoke, forms wrinkle mould, oil is in the shape of a rod under the microscope.
Bacillus subtilis hx0210 be preserved on November 19th, 2018 China typical culture collection center (address:
The Chinese Wuhan Wuhan University), classification naming is Bacillus subtilis hx0210 bacillus subtilis hx0210, preservation
Number is CCTCC NO:M 2018803.
The measurement of 2 chlorogenic acid yield of embodiment
(1) drafting of chlorogenic acid standard curve
Precise 6.20mg chlorogenic acid standard items shake up spare with RO water constant volume into 100mL volumetric flask.Use liquid relief
Rifle draws the chlorogenic acid standard solution of 0mL, 1mL, 2mL, 3mL, 4mL, 5mL in 10mL volumetric flask, chlorogenic acid mark is not added
One group of quasi- product solution is used as blank, using chlorogenic acid standard solution concentration (C) as abscissa, with the chlorogenic acid of various concentration
The absorbance (A) of standard solution draws standard curve as ordinate, and finds out corresponding regression equation.
The absorbance of the chlorogenic acid standard items of various concentration is measured at wavelength 320nm, the results are shown in Table 1.
The absorbance of 1 various concentration chlorogenic acid standard items of table
Standard curve is as shown in Figure 1, chlorogenic acid calibration curve equation is A=58.721C-0.1199, R2=0.9994.
Chlorogenic acid standard concentration is good with absorbance value linear relationship within the scope of 0.0062~0.0310mg/mL.
(2) measurement of sample Content of Chlorogenic Acid yield
Sample to be tested 1mL is drawn in 100mL volumetric flask with liquid-transfering gun, is added RO water to be diluted to scale, is obtained sample to be tested
Liquid.Make blank with RO water, the absorbance of measurement sample to be tested at the maximum absorption wavelength 320nm of chlorogenic acid standard items, then by
Standard curve calculates sample to be tested Content of Chlorogenic Acid, tests in triplicate.Chlorogenic acid yield is calculated by formula (1):
E=(CSample liquidV-CMother liquor·V)·1000(1)
In formula: E: the yield of chlorogenic acid, μ g/mL；CSample liquid: three parallel laboratory test fermentation broth sample Content of Chlorogenic Acid mass concentrations,
mg/mL；CMother liquor: the mixed liquor Content of Chlorogenic Acid mass concentration without bacterium solution fermentation corresponding to three parallel samples, mg/mL；V: liquid
The volume of body fermentation liquid, mL.
3 experiment of single factor of embodiment
Extracting honeysuckle vinasse (Hubei sky above Hubei and Hunan relax pharmaceutcal corporation, Ltd) high temperature under the conditions of 121 DEG C in high-pressure sterilizing pot
Sterilize 20min, is used for subsequent experimental.
Bacillus subtilis is inoculated into LB culture medium in the shaking table culture that revolving speed is 150r/min, temperature is 30 DEG C
The bacillus subtilis bacterium solution that 18h is activated.
Liquid fermentation liquid container is set as 500mL conical flask according to dissolved oxygen amount, and total fermentation system is set as 150mL, includes
Bacillus subtilis bacterium solution, honeysuckle vinasse and LB culture medium, wherein LB culture medium 30mL.By certain inoculation than (bacterium solution:
Honeysuckle vinasse, V:V) fermentation system is put into shaking table culture, fermentation liquid is taken out after culture, centrifuging and taking supernatant measures
Absorbance obtains chlorogenic acid concentration according to chlorogenic acid standard curve regression equation, is obtained by chlorogenic acid yield formula green
The yield of ortho acid.It investigates Bacillus subtilis strain respectively by single factor experiment, fermentation temperature (DEG C), fermentation time (h), connect
Each horizontal influence to chlorogenic acid yield under the kind amount factors such as (V:V) and revolving speed (r/min).
(1) influence of the different Bacillus subtilis strains to chlorogenic acid yield
Bacillus subtilis (commercially available, CCTCC AB 90008) bacterium solution, bacillus subtilis hx0210 bacterium solution are taken respectively, are pressed
Bacterium solution: honeysuckle vinasse is the inoculum concentration of 1:2 (V:V), and 30mL LB culture medium, 40mL bacterium solution, 80mL honeysuckle are distilled
Raffinate is added in 500mL conical flask, is put into the shaking table culture 6h that revolving speed is 150r/min, temperature is 30 DEG C.It is taken out after 6h
Ferment mixed liquor, and centrifuging and taking supernatant simultaneously surveys absorbance, by the way that chlorogenic acid yield is calculated, does 3 groups of experiments in parallel, investigates same
Under the conditions of influence of two kinds of Bacillus subtilis strains to chlorogenic acid yield.The results are shown in Table 2, through bacillus subtilis
Chlorogenic acid yield is much higher than bacillus subtilis (commercially available) after hx0210 fermentation.
Absorbance and chlorogenic acid yield after 2 two kinds of Bacillus subtilis strain fermentations of table
(2) influence of the fermentation temperature to chlorogenic acid yield
By bacillus subtilis hx0210 bacterium solution: honeysuckle vinasse is the inoculum concentration of 1:2 (V:V), and 30mL LB is trained
It supports base, 40mL bacterium solution, 80mL honeysuckle vinasse to be added in 500mL conical flask, being put into revolving speed is 150r/min, temperature point
Not Wei 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, cultivate 6h in 40 DEG C of shaking table.Fermentation mixed liquor is taken out after 6h, centrifuging and taking supernatant is simultaneously
Absorbance is surveyed, by the way that chlorogenic acid yield is calculated, does 3 groups of experiments in parallel, investigates influence of the fermentation temperature to chlorogenic acid yield.
As a result as shown in Table 3 and Fig. 2.
Absorbance and chlorogenic acid yield at a temperature of 3 different fermentations of table
When fermentation temperature is less than or equal to 30 DEG C, with the raising of fermentation temperature, chlorogenic acid yield increases.When temperature reaches
At 30 DEG C, yield highest, constant temperature is increased to 40 DEG C of this section of section, and the yield of chlorogenic acid is on a declining curve.This may be
Because 30 DEG C are the optimum temperature of bacillus subtilis, as temperature continues growing, pyrolytic product or it is unsuitable for withered
The growth of careless bacillus, so that the ability of microorganism conversion declines.Therefore, it can be seen that the optimum temperature of fermentation is 30 DEG C.
(3) influence of the fermentation time to chlorogenic acid yield
By bacillus subtilis hx0210 bacterium solution: honeysuckle vinasse is the inoculum concentration of 1:2 (V:V), and 30mL LB is trained
Support base, 40mL bacterium solution, 80mL honeysuckle vinasse are added in 500mL conical flask, be put into that revolving speed is 150r/min, temperature is
4h, 6h, 8h, 10h, for 24 hours are cultivated in 30 DEG C of shaking table respectively.Fermentation mixed liquor is taken out later, centrifuging and taking supernatant simultaneously surveys absorbance,
By the way that chlorogenic acid yield is calculated, 3 groups of experiments are done in parallel, investigate influence of the fermentation time to chlorogenic acid yield.As a result such as table 4
With shown in Fig. 3.
Absorbance and chlorogenic acid yield under the 4 different fermentations time of table
With the growth of fermentation time, chlorogenic acid yield is also constantly increasing, and when fermentation time is 6h, has reached peak
Value, later as time increases, the yield of chlorogenic acid are all to continue reduction.Therefore, it can be seen that the best duration of fermentation is answered
For 6h.
(3) influence of the inoculum concentration to chlorogenic acid yield
By bacillus subtilis hx0210 bacterium solution: honeysuckle vinasse is respectively 2:1,1:1,1:2,1:3,1:4 (V:V)
Inoculum concentration, by 30mL LB culture medium, the mixed liquor of 120mL bacillus subtilis hx0210 bacterium solution and honeysuckle vinasse
It is added in 500mL conical flask, is put into the shaking table that revolving speed is 150r/min, temperature is 30 DEG C and cultivates 6h.Hair is taken out after 6h
Ferment mixed liquor, centrifuging and taking supernatant simultaneously surveys absorbance, by the way that chlorogenic acid yield is calculated, does 3 groups of experiments in parallel, investigates inoculum concentration
Influence to chlorogenic acid yield out.As a result as shown in table 5 and Fig. 4.
Absorbance and chlorogenic acid yield under 5 different vaccination amount of table
With the reduction of inoculum concentration ratio, it is most at 1:1 (V:V) that chlorogenic acid yield, which is the trend of reduction after first increasing,
Big value is (79.70 ± 4.77) μ g/mL.Then, with the reduction of inoculum concentration ratio, the yield of chlorogenic acid is lower and lower, may
It is because of the reduction with inoculum concentration ratio, bacillus subtilis hx0210 bacterium solution is less, cannot timely be converted, separately
Outside, nutriment is inadequate, consumes the chlorogenic acid of generation, so, chlorogenic acid yield reduces.Therefore, it can be seen that optimal connect
Kind amount is 1:1 (V:V).
(5) influence of the revolving speed to chlorogenic acid yield
By bacillus subtilis hx0210 bacterium solution: honeysuckle vinasse is the inoculum concentration of 1:2 (V:V), and 30mL LB is trained
It supports base, 40mL bacterium solution, 80mL honeysuckle vinasse to be added in 500mL conical flask, being put into revolving speed is respectively 0r/min, 50r/
Min, 100r/min, 150r/min, 200r/min, temperature are to cultivate 6h in 30 DEG C of shaking table.Fermentation mixed liquor is taken out after 6h,
Centrifuging and taking supernatant simultaneously surveys absorbance, by the way that chlorogenic acid yield is calculated, does 3 groups of experiments in parallel, investigates revolving speed to chlorogenic acid out
The influence of yield.As a result as shown in table 6 and Fig. 5.
Absorbance and chlorogenic acid yield under 6 different rotating speeds of table
With the increasing of revolving speed, chlorogenic acid yield also constantly increases, and when revolving speed is 150rpm, yield reaches peak value,
Later, with the increase of revolving speed, chlorogenic acid yield declines instead.Therefore, it can be seen that optimum speed is 150rpm.
5 orthogonal experiment of embodiment
According to the result of experiment of single factor it is found that fermentation time (A), inoculum concentration (B), fermentation temperature (C) and revolving speed (D)
Optimum value, respectively 6h, 1:1 (V:V), 30 DEG C, 150rpm.Using bacillus subtilis hx0210 as fermenting microbe, according to list
The result of Factor Experiment determines three levels of each factor, in L9(34) level design Orthogonal Experiment and Design in carry out three horizontal
The test of four factors (table 7).Orthogonal Experiment and Design and it the results are shown in Table 8, the results of analysis of variance is shown in Table 9.
7 orthogonal test factor of table and level
8 Orthogonal Experiment and Design of table and result and range analysis
9 orthogonal experiments variance analysis of table
Note: "-" indicates not significant (P > 0.05) to result.
By the range analysis result of table 8 it is found that influence order of each factor to chlorogenic acid yield are as follows: C > A > D > B, i.e.,
Fermentation temperature > fermentation time > shaking speed > inoculum concentration.
9 variance analysis of table also indicates that influence order of each factor to chlorogenic acid yield is C > A > D > B, but each factor
Influence it is not significant, obtaining best technological condition for fermentation is A2B3C1D2, i.e. fermentation time 6h, inoculum concentration 1:2 (V:V), temperature
25 DEG C, revolving speed 150rpm, chlorogenic acid yield is (191.67 ± 39.25) μ g/mL.
Using best technological condition for fermentation parallel test 3 times, the yield of chlorogenic acid is (193.03 ± 47.60) μ g/mL, high
In table 8 Content of Chlorogenic Acid maximum output (191.67 ± 39.25) μ g/mL, show honeysuckle vinasse determined by orthogonal design
The fermentation condition of bioconversion is optimal.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of bacillus subtilis, it is characterised in that: classification naming is Bacillus subtilis hx0210 withered grass gemma
Bacillus hx0210, deposit number are CCTCC NO:M 2018803.
2. application of the bacillus subtilis described in claim 1 in production chlorogenic acid.
3. application according to claim 2, it is characterised in that: production chlorogenic acid is raw materials used residual comprising honeysuckle distillation
Liquid.
4. a kind of method for improving honeysuckle vinasse Content of Chlorogenic Acid, characterized by the following steps:
(1) bacillus subtilis described in claim 1 is inoculated into culture medium and carries out activation culture, obtain bacillus subtilis
Bacterium bacterium solution；
(2) bacillus subtilis bacterium solution, honeysuckle vinasse, culture medium are mixed and carries out fermented and cultured.
5. according to the method described in claim 4, it is characterized by: the condition of activation culture described in step (1) be 30 DEG C,
150r/min cultivates 16-18h.
6. according to the method described in claim 4, it is characterized by: bacillus subtilis hx0210 bacterium solution, gold and silver in step (2)
The volume ratio of flower vinasse is 1:2.
7. according to the method described in claim 4, it is characterized by: bacillus subtilis hx0210 bacterium solution, gold and silver in step (2)
Spend vinasse, the volume ratio of culture medium is 4:8:3.
8. according to the method described in claim 4, it is characterized by: the condition of fermented and cultured described in step (2) be 30 DEG C,
150rpm fermentation 6h.
9. according to the method described in claim 4, it is characterized by: culture medium described in step (1), (2) is LB culture medium.
10. according to the method described in claim 4, it is characterized by comprising following steps:
(1) bacillus subtilis described in claim 1 is inoculated into LB culture medium, 30 DEG C, 150r/min culture 16-18h
Obtain bacillus subtilis bacterium solution；
(2) by bacillus subtilis bacterium solution, honeysuckle vinasse, LB culture medium by volume 4:8:3 ratio mix, 30
DEG C, 150rpm ferment 6h.
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| CN109468250B (en) | 2020-12-01 |
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Denomination of invention: A Method for Increasing Chlorogenic Acid Content in Flos Lonicerae Distillation Residue Effective date of registration: 20221017 Granted publication date: 20201201 Pledgee: Hubei Luotian Rural Commercial Bank Co.,Ltd. Pledgor: HUBEI CHUTIANSHU PHARMACEUTICAL Co.,Ltd. Registration number: Y2022420000350 |