CN109468250B - Method for increasing content of chlorogenic acid in honeysuckle distillation residual liquid - Google Patents
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Abstract
The invention discloses a method for improving the content of chlorogenic acid in honeysuckle distillation residual liquid, and belongs to the field of biotransformation. The method comprises the following steps: the preservation number is CCTCC NO: inoculating the Bacillus subtilis hx0210 of M2018803 into LB culture medium, culturing at 30 deg.C and 150r/min for 16-18h to obtain Bacillus subtilis hx 0210; mixing the bacillus subtilis hx0210 bacterial liquid, the honeysuckle distillation residual liquid and the LB culture medium according to the volume ratio of 4:8:3, and fermenting at 30 ℃ and 150rpm for 6 hours. The bacillus subtilis hx0210 used in the invention can greatly improve the content of chlorogenic acid in a fermentation culture; the invention utilizes the high-concentration organic wastewater discharged by the distillation section in the production process of the honeysuckle flower distillate, reduces pollution and resource waste, and provides a new channel for producing chlorogenic acid.
Description
Technical Field
The invention belongs to the field of biotransformation, and particularly relates to a method for improving the content of chlorogenic acid in honeysuckle distillation residual liquid.
Background
Honeysuckle (Lonicera japonica Thunb) is a dried flower bud belonging to a Caprifoliaceae plant, is a green natural product with wide application, is homologous in medicine and food, is an important Chinese medicinal material in China, and can be used as both medicine and food. Honeysuckle has the functions of clearing away heat and toxic materials, dispelling wind and heat, resisting bacteria and diminishing inflammation and the like, so the honeysuckle is called as 'national treasure Yizhihua'. The honeysuckle contains organic acid compounds such as protocatechuic acid, isochlorogenic acid, chlorogenic acid and the like, wherein the chlorogenic acid is a representative component of the organic acid, and the honeysuckle has the capabilities of diminishing inflammation, inhibiting bacteria, removing free radicals, resisting oxidation, resisting viruses and the like due to the existence of the chlorogenic acid.
At present, more researches on honeysuckle flowers and leaves are carried out at home and abroad than the researches on honeysuckle flowers and leaves, and the honeysuckle distillation residues are less utilized. The honeysuckle distillation residual liquid is high-concentration organic wastewater discharged from a distillation working section in the production process of the commercially available honeysuckle distillate, and in industrial production, the honeysuckle distillation residual liquid is usually directly discarded, wherein the honeysuckle distillation residual liquid contains high-content bioactive components with important pharmacological effects, such as chlorogenic acid, flavone, saponin, tannin and the like, and the direct discharge of the honeysuckle distillation residual liquid not only causes resource waste, but also generates wastewater pollution.
Chlorogenic acid is widely applied in the industries of medical industry, food, cosmetics and the like, and has large demand and high price on the market. Chlorogenic acid is produced by utilizing honeysuckle distillation residues through microbial conversion, so that more economic benefits can be created, and the discharge of industrial wastewater can be reduced.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for improving the content of chlorogenic acid in honeysuckle distillation residual liquid and bacillus subtilis hx0210 used in the method.
The purpose of the invention is realized by the following technical scheme:
the Bacillus subtilis is classified and named as Bacillus subtilis hx0210, and the preservation number is CCTCC NO: m2018803, deposited at the China center for type culture Collection (address: university of Wuhan, China) on 11/19/2018.
The bacillus subtilis can be used for producing chlorogenic acid; particularly, the distillation residual liquid of honeysuckle is used as a raw material for producing chlorogenic acid, namely the raw material for producing chlorogenic acid preferably comprises the distillation residual liquid of honeysuckle.
A method for improving the content of chlorogenic acid in honeysuckle distillation residual liquid comprises the following steps:
(1) inoculating the bacillus subtilis hx0210 into a culture medium for activation culture to obtain a bacillus subtilis hx0210 bacterial liquid.
(2) Mixing the bacillus subtilis hx0210 bacterial liquid, the honeysuckle distillation residual liquid and a culture medium for fermentation culture.
Preferably, the activation culture condition in the step (1) is culture at 30 ℃ and 150r/min for 16-18 h.
Preferably, the volume ratio of the bacillus subtilis hx0210 bacterial liquid to the honeysuckle distillation residual liquid in the step (2) is 1: 2; further preferably, the volume ratio of the bacillus subtilis hx0210 bacterial liquid to the honeysuckle distilled residual liquid to the culture medium is 4:8: 3.
Preferably, the fermentation culture conditions in step (2) are 30 ℃ and 150rpm for 6 h.
Preferably, the culture medium in the steps (1) and (2) is LB culture medium.
Preferably, the method for increasing the content of chlorogenic acid in the distilled residual liquid of honeysuckle comprises the following steps:
(1) inoculating the bacillus subtilis hx0210 into an LB culture medium, and culturing at 30 ℃ and 150r/min for 16-18h to obtain a bacillus subtilis hx0210 bacterial liquid.
(2) Mixing the bacillus subtilis hx0210 bacterial liquid, the honeysuckle distillation residual liquid and the LB culture medium according to the volume ratio of 4:8:3, and fermenting at 30 ℃ and 150rpm for 6 hours.
Compared with the prior art, the invention has the following advantages and beneficial effects: the bacillus subtilis hx0210 can greatly improve the content of chlorogenic acid in a fermentation culture; the invention utilizes the high-concentration organic wastewater discharged by the distillation section in the production process of the honeysuckle flower distillate, reduces pollution and resource waste, and provides a new channel for producing chlorogenic acid.
Drawings
Figure 1 is a graph of the standard curve of chlorogenic acid.
FIG. 2 is a graph showing the effect of fermentation temperature on chlorogenic acid production.
FIG. 3 is a graph of the results of the effect of fermentation time on chlorogenic acid production.
FIG. 4 is a graph showing the effect of inoculum size on chlorogenic acid production.
FIG. 5 is a graph of the results of the effect of rotational speed on chlorogenic acid production.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
Filtering the distilled residual liquid of flos Lonicerae naturally stored for more than 20 days to obtain clear stock solution, sucking 1mL of stock solution with a pipette in a superclean bench, transferring into a test tube filled with 9mL of sterile water, and shaking thoroughly to obtain 10-1Diluting the residual liquid of honeysuckle flower to obtain 10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Diluting the honeysuckle distillation raffinate. Preparing PDA culture medium, coating PDA plates with 10% concentration-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9After the dilution is cultured for 72h at 37 ℃, and after a colony grows out, a single colony is selected and streaked on a new PDA culture medium until a purified single colony is obtained.
And screening for multiple times according to the process to obtain the bacillus subtilis with high chlorogenic acid yield, which is named as bacillus subtilis hx 0210.
The bacillus subtilis hx0210 is round, dirty and white, has incomplete edges, slightly bulges in the middle, is rough and opaque in surface, is not easy to pick up, forms wrinkles and takes a rod shape when observed under an oil microscope.
The Bacillus subtilis hx0210 is preserved in a China center for type culture collection (address: China, Wuhan university) in 2018, 11 and 19 months, is classified and named as Bacillus subtilis hx0210, and has a preservation number of CCTCC NO: m2018803.
EXAMPLE 2 determination of chlorogenic acid production
(1) Drawing standard curve of chlorogenic acid
6.20mg of chlorogenic acid standard substance is accurately weighed, the volume is determined to be 100mL volumetric flask by RO water, and the solution is shaken up for standby. Sucking 0mL, 1mL, 2mL, 3mL, 4mL and 5mL of chlorogenic acid standard solution into a 10mL volumetric flask by using a pipette, drawing a standard curve by using a group without the addition of the chlorogenic acid standard solution as a blank, using the concentration (C) of the chlorogenic acid standard solution as an abscissa and using the absorbance (A) of the chlorogenic acid standard solution with different concentrations as an ordinate, and solving a corresponding regression equation.
The absorbance of the chlorogenic acid standards at different concentrations was measured at a wavelength of 320nm and the results are shown in Table 1.
TABLE 1 absorbance of chlorogenic acid standards of different concentrations
The standard curve is shown in FIG. 1, and the standard curve equation of chlorogenic acid is A-58.721C-0.1199, R20.9994. The linear relation between the concentration of the chlorogenic acid standard substance and the absorbance value is good within the range of 0.0062-0.0310 mg/mL.
(2) Determination of chlorogenic acid yield in sample
And (3) sucking 1mL of the sample to be detected into a 100mL volumetric flask by using a pipette, and adding RO water to dilute to a scale mark to obtain the sample liquid to be detected. And (3) determining the absorbance of the sample to be detected under the condition that the maximum absorption wavelength of the chlorogenic acid standard substance is 320nm by taking RO water as a blank, calculating the content of the chlorogenic acid in the sample to be detected according to a standard curve, and repeating the test for three times. The chlorogenic acid yield was calculated by formula (1):
E＝(Csample liquid·V－CMother liquor·V)·1000(1)
In the formula: e: the yield of chlorogenic acid is mu g/mL; cSample liquid: the mass concentration of chlorogenic acid in three fermentation broth samples in parallel experiments is mg/mL; cMother liquor: the mass concentration of chlorogenic acid in the mixed liquor which is not fermented by the bacterial liquid and corresponds to the three parallel samples is mg/mL; v: volume of liquid fermentation broth, mL.
Example 3 Single factor experiment
Taking flos Lonicerae distillation raffinate (Hubei Chutian Shu pharmaceutical industry Co., Ltd.) and sterilizing at high temperature of 121 deg.C in autoclave for 20min for subsequent experiment.
Inoculating the bacillus subtilis into an LB culture medium, and culturing for 18h in a shaking table at the rotating speed of 150r/min and the temperature of 30 ℃ to obtain activated bacillus subtilis liquid.
The liquid fermentation liquid container is set to be a 500mL conical flask according to the dissolved oxygen amount, the total fermentation system is set to be 150mL, and the liquid fermentation system comprises bacillus subtilis liquid, honeysuckle distillation residual liquid and LB culture medium, wherein the LB culture medium is 30 mL. Putting the fermentation system into a shaking table for cultivation according to a certain inoculation ratio (the bacteria liquid: the honeysuckle distilled residual liquid, V: V), taking out the fermentation liquid after the cultivation is finished, centrifuging, taking the supernatant, measuring the absorbance, obtaining the concentration of the chlorogenic acid according to a standard curve regression equation of the chlorogenic acid, and obtaining the yield of the chlorogenic acid through a chlorogenic acid yield calculation formula. The influence of each level on the yield of chlorogenic acid under the factors of bacillus subtilis strain, fermentation temperature (DEG C), fermentation time (h), inoculum size (V: V), rotating speed (r/min) and the like is respectively inspected through a single-factor test.
(1) Influence of different Bacillus subtilis strains on chlorogenic acid yield
Respectively taking a bacillus subtilis (commercially available, CCTCC AB 90008) bacterial solution and a bacillus subtilis hx0210 bacterial solution, adding 30mL of LB culture medium, 40mL of bacterial solution and 80mL of honeysuckle distillation residual liquid into a 500mL conical flask according to the inoculation amount of the bacterial solution and the honeysuckle distillation residual liquid of 1:2(V: V), and putting the conical flask into a shaking table at the rotating speed of 150r/min and the temperature of 30 ℃ for culturing for 6 hours. Taking out the fermentation mixed liquor after 6h, centrifuging, taking the supernatant, measuring the absorbance, calculating to obtain the yield of the chlorogenic acid, and performing 3 groups of experiments in parallel to investigate the influence of two bacillus subtilis strains on the yield of the chlorogenic acid under the same condition. The results are shown in Table 2, the yield of chlorogenic acid after fermentation with Bacillus subtilis hx0210 is much higher than that of Bacillus subtilis (commercially available).
TABLE 2 Absorbance and chlorogenic acid yield after fermentation of two Bacillus subtilis strains
(2) Influence of fermentation temperature on chlorogenic acid yield
According to the inoculation amount of bacillus subtilis hx0210 bacterial liquid and honeysuckle distillation residual liquid of 1:2(V: V), 30mL of LB culture medium, 40mL of bacterial liquid and 80mL of honeysuckle distillation residual liquid are added into a 500mL conical flask, and the mixture is put into a shaking table at the rotating speed of 150r/min and the temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively for culture for 6 hours. Taking out the fermentation mixed liquor after 6h, centrifuging, taking the supernatant, measuring the absorbance, calculating to obtain the yield of the chlorogenic acid, and carrying out 3 groups of experiments in parallel to investigate the influence of the fermentation temperature on the yield of the chlorogenic acid. The results are shown in table 3 and fig. 2.
TABLE 3 Absorbance and chlorogenic acid yields at different fermentation temperatures
When the fermentation temperature is less than or equal to 30 ℃, the yield of the chlorogenic acid is increased along with the increase of the fermentation temperature. When the temperature reaches 30 ℃, the yield is the highest, and the yield of the chlorogenic acid is in a descending trend when the temperature is continuously increased to 40 ℃. This is probably because 30 ℃ is the optimum temperature for Bacillus subtilis, and as the temperature continues to increase, the products are decomposed at high temperature or are not suitable for the growth of Bacillus subtilis, so that the microbial transformation ability decreases. Thus, it can be seen that the optimum temperature for fermentation is 30 ℃.
(3) Influence of fermentation time on chlorogenic acid yield
According to the inoculation amount of bacillus subtilis hx0210 bacterial liquid and honeysuckle distillation residual liquid of 1:2(V: V), 30mL of LB culture medium, 40mL of bacterial liquid and 80mL of honeysuckle distillation residual liquid are added into a 500mL conical flask, and the conical flask is put into a shaking table with the rotation speed of 150r/min and the temperature of 30 ℃ for cultivation for 4h, 6h, 8h, 10h and 24h respectively. Then taking out the fermentation mixed liquor, centrifuging to take the supernatant and measuring the absorbance, obtaining the yield of the chlorogenic acid by calculation, and carrying out 3 groups of experiments in parallel to investigate the influence of the fermentation time on the yield of the chlorogenic acid. The results are shown in table 4 and fig. 3.
TABLE 4 Absorbance and chlorogenic acid yield at different fermentation times
The yield of chlorogenic acid is continuously increased along with the increase of the fermentation time, the peak value is reached until the fermentation time is 6 hours, and the yield of chlorogenic acid is continuously reduced along with the increase of the fermentation time. Thus, it can be seen that the optimal duration of the fermentation should be 6 h.
(3) Effect of inoculum size on chlorogenic acid yield
According to the inoculation amounts of the bacillus subtilis hx0210 bacterial liquid and the honeysuckle distillation residual liquid which are respectively 2:1, 1:2, 1:3 and 1:4(V: V), 30mL of LB culture medium, 120mL of the mixed liquid of the bacillus subtilis hx0210 bacterial liquid and the honeysuckle distillation residual liquid are added into a 500mL conical flask, and the conical flask is put into a shaking table with the rotating speed of 150r/min and the temperature of 30 ℃ for cultivation for 6 hours. Taking out the fermentation mixed liquor after 6h, centrifuging, taking the supernatant, measuring the absorbance, calculating to obtain the yield of the chlorogenic acid, and carrying out 3 groups of experiments in parallel to investigate the influence of the inoculation amount on the yield of the chlorogenic acid. The results are shown in table 5 and fig. 4.
TABLE 5 Absorbance and chlorogenic acid yields at different inoculum sizes
The chlorogenic acid yield, which is a tendency to increase first and then decrease with decreasing ratio of the inoculum, was at a maximum at 1:1(V: V) and was (79.70. + -. 4.77). mu.g/mL. Then, the yield of chlorogenic acid is lower and lower with the reduction of the ratio of the inoculation amount, probably because the bacillus subtilis hx0210 has less bacterial liquid and cannot be transformed in time with the reduction of the ratio of the inoculation amount, and in addition, the yield of chlorogenic acid is reduced because the generated chlorogenic acid is consumed due to insufficient nutrient substances. Therefore, it can be seen that the optimal inoculation amount is 1:1(V: V).
(5) Influence of rotational speed on chlorogenic acid yield
According to the inoculation amount of bacillus subtilis hx0210 bacterial liquid and honeysuckle distillation residual liquid of 1:2(V: V), 30mL of LB culture medium, 40mL of bacterial liquid and 80mL of honeysuckle distillation residual liquid are added into a 500mL conical flask, and the conical flask is put into a shaking table at the rotating speed of 0r/min, 50r/min, 100r/min, 150r/min and 200r/min respectively and the temperature of 30 ℃ for culture for 6 hours. Taking out the fermentation mixed liquor after 6h, centrifuging, taking the supernatant, measuring the absorbance, calculating to obtain the yield of the chlorogenic acid, and carrying out 3 groups of experiments in parallel to investigate the influence of the rotating speed on the yield of the chlorogenic acid. The results are shown in table 6 and fig. 5.
TABLE 6 Absorbance and chlorogenic acid yield at different rotation speeds
The yield of chlorogenic acid is increased continuously with the increase of the rotating speed until the yield reaches the peak value when the rotating speed is 150rpm, and then the yield of chlorogenic acid is reduced inversely with the increase of the rotating speed. Thus, it can be seen that the optimum rotational speed is 150 rpm.
Example 5 orthogonal experiments
As a result of the one-factor experiment, the optimum values of the fermentation time (A), the inoculation amount (B), the fermentation temperature (C) and the rotation speed (D) were 6 hours, 1:1(V: V), 30 ℃ and 150rpm, respectively. Taking bacillus subtilis hx0210 as a fermentation strain, determining three levels of each factor according to the result of a single-factor experiment, wherein the three levels are L9(34) The three-level four-factor (table 7) test was performed in the orthogonal experimental design of the horizontal design. The orthogonal test design and results are shown in Table 8, and the results of the ANOVA are shown in Table 9.
TABLE 7 orthogonal test factors and levels
TABLE 8 orthogonal test design and results and range analysis
TABLE 9 analysis of variance of orthogonal test results
Note: "-" indicates no significance for the result (P > 0.05).
As can be seen from the range analysis results in Table 8, the influence of each factor on the yield of chlorogenic acid is in the following order: c is more than A and more than D and more than B, namely, the fermentation temperature is more than the fermentation time and more than the rotating speed of a shaking table and more than the inoculation amount.
Table 9 analysis of variance also shows that the influence sequence of each factor on the yield of chlorogenic acid is C > A > D > B, but the influence of each factor is not obvious, and the optimal fermentation process condition is A2B3C1D2Namely fermentation time of 6h, inoculation amount of 1:2(V: V), temperature of 25 ℃, rotation speed of 150rpm, and chlorogenic acid yield of (191.67 +/-39.25) mu g/mL.
The optimal fermentation process conditions are used for carrying out parallel experiments for 3 times, the yield of the chlorogenic acid is (193.03 +/-47.60) mu g/mL, and is higher than the highest yield of the chlorogenic acid (191.67 +/-39.25) mu g/mL in the table 8, and the fermentation conditions of the biotransformation of the honeysuckle distillation residue liquid determined by orthogonal design are optimal.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
1. A bacillus subtilis, characterized in that: classified name is Bacillus subtilis (A)Bacillus subtilis) hx0210, with a preservation number of CCTCC NO: m2018803.
2. Use of the bacillus subtilis of claim 1 for the production of chlorogenic acid, characterized in that: the raw material for producing chlorogenic acid comprises flos Lonicerae distillation residue.
3. A method for improving the content of chlorogenic acid in honeysuckle distillation residual liquid is characterized by comprising the following steps: the method comprises the following steps:
(1) inoculating the bacillus subtilis of claim 1 into an LB culture medium, and culturing at 30 ℃ and 150r/min for 16-18h to obtain a bacillus subtilis liquid;
(2) and (2) mixing the bacillus subtilis liquid obtained in the step (1), the honeysuckle distillation residual liquid and the LB culture medium according to the volume ratio of 4:8:3, and fermenting at 30 ℃ and 150rpm for 6 hours.
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Effective date of registration: 20210728 Address after: 438699 Luotian Economic Development Zone, Huanggang City, Hubei Province Patentee after: HUBEI CHUTIANSHU PHARMACEUTICAL Co.,Ltd. Address before: 438000 146 Xingang two road, Huanggang Development Zone, Hubei Patentee before: HUANGGANG NORMAL University |
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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 |