CN115844930B - Ginkgo leaf processing method and product obtained by same - Google Patents
Ginkgo leaf processing method and product obtained by same Download PDFInfo
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Abstract
The invention provides a ginkgo leaf processing method and a product obtained by the ginkgo leaf processing method. The ginkgo leaf processing method comprises the following steps: 1) Adding water into folium Ginkgo, and adding microorganism for anaerobic fermentation; 2) After fermentation, the fermentation broth is left to stand and then subjected to solid-liquid separation. 3) Adding sorbitol into the separated solid, and freeze-drying to obtain freeze-dried powder. The ginkgo leaf processing method provided by the invention abandons the traditional Chinese medicine processing method, adopts a microbial fermentation mode to decompose toxic components in ginkgo leaves, does not leave organic solvents in products, and is more environment-friendly in preparation process.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine processing method by utilizing microbial fermentation.
Background
The folium Ginkgo is dry leaf of Ginkgo biloba (also called as Ginkgo tree) belonging to the family Ginkgo. Usually, the leaves are collected in autumn when the leaves are Shang Lu, and the leaves are dried in time.
The ginkgo leaf has the effect of astringing lung, relieving asthma, promoting blood circulation to remove blood stasis, relieving pain and the like. Can be used for treating cough and asthma due to lung deficiency, coronary heart disease, angina pectoris, hyperlipidemia, and blood coagulation. Ginkgo leaf preparations also have a certain effect of improving memory. The flavonoid glycoside, amino acid and amino acid in ginkgo leaf synthesized collagen component has the functions of beautifying human body, inhibiting melanin growth and maintaining skin luster and elasticity.
Ginkgo contains toxic ginkgolic acid, and poisoning symptoms include muscle twitch, pupil dilation, etc. Ginkgolic acid is water-soluble and can not be directly drunk by ginkgo She Paoshui. The existing ginkgo leaf extract is mostly extracted by using an organic solvent, for example, patent application CN115154492A discloses a preparation method of the ginkgo leaf extract, reflux-extracting ginkgo leaves by using ethanol solution, and collecting extract; concentrating the extract into extract, heating the extract to dissolve in water, circularly stirring with cold water, standing for 2-4 hours, separating supernatant, eluting with soft water through a resin column, then resolving with ethanol solution, deacidifying the resolved solution through a deacidification resin column, and collecting deacidification liquid; concentrating deacidification liquid into extract, drying and pulverizing the extract to obtain extract powder.
The extraction method in the prior art inevitably leads to organic solvent residues in the product, and the extraction process route is longer,
disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a ginkgo leaf processing method which utilizes microbial fermentation to process ginkgo leaves into fermentation products with low toxicity.
The second object of the present invention is to propose the product obtained by said processing method.
The technical scheme for realizing the aim of the invention is as follows:
a processing method of ginkgo leaves comprises the following steps:
1) Adding microorganism into folium Ginkgo for anaerobic fermentation; the microorganism is selected from one or more of Saccharomyces, bifidobacterium, and Lactobacillus;
2) After fermentation, standing the fermentation liquor, and then carrying out solid-liquid separation;
3) Adding sorbitol into the separated solid, and freeze-drying to obtain freeze-dried powder.
The ginkgo leaf is collected in autumn and is crushed to 0.5-1 mm in size.
Wherein, in the step 1), the feed liquid ratio of the water added into the ginkgo leaf is 1: (2-3) (kg: liter). Adding water into folium Ginkgo, and autoclaving to obtain basic fermentation broth.
The autoclaving conditions may be 121℃for 15-30 min.
Further, in the step 1), the sum of ginkgo leaf and water is taken as a basic fermentation liquid, 30-50 g/L of sugar and 3-5 g/L of soluble starch are added into each liter of basic fermentation liquid, and 100U/g of cellulase is added into each gram of ginkgo leaf.
Preferably, the microorganism is lactobacillus, the strain is firstly cultured on MRS liquid culture medium to obtain seed liquid, the seed liquid is inoculated into fermentation liquid, and the inoculation amount is 1-5% (v/v).
The culture mode of the seed liquid is preferably shake flask culture, the culture temperature is 25-28 ℃, and the rotation speed is 150-200 r/min. The culture time is 8-12 h.
More preferably, in step 1), the cultured seed liquid is transferred into a fermenter containing a fermentation liquid at an inoculum size of 3 to 5% (v/v), and the fermentation conditions are as follows: the pot temperature is kept at 25-28 ℃, the pot pressure is 0.5-0.6 kg/cm, and the stirring speed is 150-180 rpm. For example, the tank temperature is kept at 25℃and the tank pressure is kept at 0.5kg/cm, and the stirring rate is 150rpm.
Wherein the fermentation time of the step 1) is 42-60 hours. More preferably for 48 hours.
Wherein, in the step 2), after fermentation is finished, standing the fermentation liquor for 10-12 hours, and then carrying out solid-liquid separation, wherein the solid-liquid separation method comprises centrifugal separation, and the conditions of the centrifugal separation are as follows: the rotating speed is 6000r/min, and the time is 5-10 min.
And the fermentation liquor can be naturally settled after standing, and part of clear liquor is poured out before centrifugation, so that the liquid quantity required to be centrifuged is reduced.
The freeze-dried powder obtained by adopting the freeze-drying mode has lactobacillus conidium, and is beneficial to intestinal health.
Sorbitol is a lyoprotectant. In step 3), 0.5 to 2g of sorbitol are added per kg of separated solids. For example, 1g sorbitol is added per kg of separated solids.
In step 3), the freeze-drying comprises two stages of pre-freezing and vacuum freeze-drying,
wherein the prefreezing is prefreezing for 2 hours at-80 ℃;
the conditions of the vacuum freeze drying are as follows: pressure 130 x 10 -3 mbar～135×10 -3 And freeze-drying for 12-18 h at-50 ℃ to-55 ℃ under the condition of mbar.
The ginkgo leaf freeze-dried powder is obtained by the processing method.
The ginkgo leaf freeze-dried powder obtained by the invention has the effects of astringing lung, relieving asthma, promoting blood circulation by removing blood stasis and relieving pain. Can be used for preparing medicines for treating coronary heart disease, angina pectoris, hyperlipemia and blood coagulation. The ginkgo leaf freeze-dried powder also has the effects of adjusting the flora in the intestinal tract and improving the gastrointestinal comfort of a user.
The invention has the beneficial effects that:
the ginkgo leaf processing method provided by the invention abandons the traditional Chinese medicine processing method, adopts a microbial fermentation mode to decompose toxic components in ginkgo leaves, does not use organic solvents, does not leave organic solvents in products, and is more environment-friendly in preparation process.
The invention optimizes the strain beneficial to human body, so that the components in ginkgo leaves are decomposed and the intestinal environment is further improved.
The freeze-dried powder prepared by the method disclosed by the invention has the advantages that the total flavonol glycoside in the raw materials is reserved to a large extent, the freeze-dried powder is convenient to store and transport, and convenience is brought to the subsequent pharmaceutical process.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. In the examples, all means used are conventional in the art unless otherwise specified.
The parts in the examples refer to parts by mass unless otherwise specified. The proportions used are mass ratios.
The total flavonol glycoside content (%) is determined by high performance liquid chromatography. The reference substances are quercetin, kaempferide and isorhamnetin,
total flavonol glycoside content= (quercetin content+kaempferide content+isorhamnetin content) ×2.51
The ginkgolic acid content (mg/kg) was measured by liquid chromatography. Calculated as the sum of the peak areas of ginkgolic acid C13:0, ginkgolic acid C15:1 and ginkgolic acid C17:1.
The raw material ginkgo leaf used in the examples is a dry leaf having a moisture content of about 12% and a total flavonol glycoside content of 1.41%.
Test example:
the test is to add microorganism into ginkgo leaf for anaerobic fermentation to remove toxic substances in ginkgo leaf. The microorganism selected should be an acidophilic and healthy species.
The test strains adopt dry powder purchased in the market, and the dry powder is respectively as follows: 1-yeast (Angel Yeast), 2-bifidobacterium (micro Kang Yisheng (Suzhou) Co., ltd.), and 3-lactobacillus (Zhongdao Marine biotechnology (Ningbo Co., ltd.).
In the test example, the strain is cultured by a shake flask culture method, the temperature of shake flask culture is 25-28 ℃, and the rotating speed is 150r/min. The incubation time was 10h. The amount of the strain added into each shaking flask is 1g/L.
Based on the sum of ginkgo leaf and water, 30g/L sugar, 5g/L soluble starch and 100U/g cellulase are added into each liter of basic fermentation liquid.
The culture conditions of each test are 25-28 ℃ and 150-200 r/min.
Sampling at different time points after inoculation, counting the bacterial content in the fermentation liquid, taking the fermentation liquid for detection before starting fermentation and after reaching a platform phase, and calculating the reduction rate of the ginkgo folic acid. After reaching the plateau phase, the fermentation broth was centrifuged, and the total flavonol glycoside content (%) in the precipitated fraction was detected. The results are shown in the following table.
Table 1: fermentation comparison of strains
| Project | 1-Yeast | 2-Bifidobacterium | 3-Lactobacillus |
| Time to reach plateau (h) | 46 | 48 | 42 |
| Rate of decrease in ginkgo folate (%) | 90.2 | 88.9 | 95.6 |
| Total flavonol glycoside content (%) | 0.76％ | 0.59％ | 0.80％ |
The effect of microorganism on decomposing ginkgo folic acid, the fermentation time and the content of total flavonol glycoside after fermentation are comprehensively compared, and lactobacillus is screened as a proper strain.
Example 1
A processing method of ginkgo leaves comprises the following steps:
1) The ginkgo leaves are crushed into 0.5 to 1 mm. The feed liquid ratio of water is 1:2.5 (kg: liter), adding water, sterilizing in an autoclave at 121deg.C for 15min to obtain basic fermentation broth.
Inoculating lactobacillus on MRS liquid culture medium, wherein the strain amount added in the MRS liquid culture medium is 1g/L. Culturing by a shake flask culture method to obtain seed liquid, wherein the temperature of shake flask culture is 25-28 ℃ and the rotating speed is 150r/min. The culture time is 12 hours, and seed liquid is obtained. Seed liquid was inoculated into the fermentation broth in an amount of 5% (v/v).
Based on the sum of ginkgo leaf and water, 30-50 g/L sugar, 5g/L soluble starch and 100U/g cellulase are added into each liter of basic fermentation liquid. And adding lactobacillus seed solution into the obtained fermentation liquor for anaerobic fermentation. The pot temperature was kept at 25℃and the pot pressure was 0.5kg/cm, and the stirring rate was 150rpm.
The fermentation time was 48 hours.
2) After fermentation, standing the fermentation broth for 10 hours, pouring out part of supernatant, and then carrying out solid-liquid separation by a centrifugal method under the following centrifugal conditions: the rotating speed is 6000r/min, and the time is 5min.
In this example, the bacteria content in the fermentation broth was 20 hundred million/mL, and the centrifugation conditions were: 10000rpm for 15 minutes, the concentration of the obtained concentrated bacterial liquid is 150 hundred million/mL.
The content of ginkgolic acid in the precipitate is further reduced by centrifugally separating the fermentation liquid. The water content of the fermentation precipitate obtained by centrifugation in this example was 15%.
3) Adding sorbitol dissolved in water (to prepare about 10% aqueous solution by mass fraction) in a proportion of 1g sorbitol per kg of separated fermentation precipitate; freeze-drying, including pre-freezing and vacuum freeze-drying,
wherein the prefreezing is prefreezing for 2 hours at-80 ℃;
the conditions of the vacuum freeze drying are as follows: pressure 133X 10 -3 mbar～135×10 -3 mbar, lyophilization at-50℃for 12h.
Ginkgolic acid with ginkgolic acid content of 10ppm and total flavonol glycoside content of 0.86% in the freeze-dried powder.
Example 2
A processing method of ginkgo leaves comprises the following steps:
1) The ginkgo leaves are crushed into 0.5 to 1 mm. The feed liquid ratio of water is 1:2.5 (kg: liter), adding water, sterilizing in an autoclave at 121deg.C for 15min to obtain basic fermentation broth.
Lactobacillus was cultured on MRS liquid medium to obtain seed solution, and the culture method was the same as in example 1.
Based on the sum of ginkgo leaf and water, 50g/L sugar, 5g/L soluble starch and 100U/g cellulase are added into each liter of basic fermentation liquid. And adding lactobacillus seed solution into the obtained fermentation liquor for anaerobic fermentation.
The pot temperature was kept at 25℃and the pot pressure was 0.5kg/cm, and the stirring rate was 150rpm.
The fermentation time was 42 hours.
2) After fermentation, standing the fermentation broth for 10 hours, pouring out part of supernatant, and then carrying out solid-liquid separation by a centrifugal method under the following centrifugal conditions: the rotating speed is 6000r/min, and the time is 5min.
In this example, the bacteria content in the fermentation broth was 18 hundred million/mL, and the centrifugation conditions were: 10000rpm for 15 minutes, the concentration of the obtained concentrated bacterial liquid is 140 hundred million/mL.
The content of ginkgolic acid in the precipitate is further reduced by centrifugally separating the fermentation liquid. The water content of the fermentation precipitate obtained by centrifugation in this example was 15%.
3) Lyophilization was performed as in example 1.
The content of ginkgo folic acid in the freeze-dried powder is not more than 10ppm, and the content of total flavonol glycoside is 0.83%.
Example 3
A processing method of ginkgo leaves comprises the following steps:
1) The ginkgo leaves are crushed into 0.5 to 1 mm. The feed liquid ratio of water is 1:2.5 (kg: liter), adding water, sterilizing in an autoclave at 121deg.C for 15min to obtain basic fermentation broth.
Lactobacillus was cultured on MRS liquid medium to obtain seed solution, and the culture method was the same as in example 1.
Based on the sum of ginkgo leaf and water, 30g/L sugar, 5g/L soluble starch and 100U/g cellulase are added into each liter of basic fermentation liquid. And adding lactobacillus seed solution into the fermentation liquor for anaerobic fermentation.
The pot temperature was kept at 25℃and the pot pressure was 0.5kg/cm, and the stirring rate was 150rpm.
The fermentation time was 60 hours.
2) After fermentation, standing the fermentation liquor for 10 hours, and then carrying out solid-liquid separation by a centrifugal method, wherein the centrifugal conditions are as follows: the rotating speed is 6000r/min, and the time is 5min.
In this example, the bacteria content in the fermentation broth was 20 hundred million/mL, and the centrifugation conditions were: 10000rpm for 15 minutes, the concentration of the obtained concentrated bacterial liquid is 150 hundred million/mL.
The content of ginkgolic acid in the precipitate is further reduced by centrifugally separating the fermentation liquid. The water content of the fermentation precipitate obtained by centrifugation in this example was 15%.
3) Lyophilization was performed as in example 1.
The content of ginkgo folic acid in the freeze-dried powder is not more than 10ppm, and the content of total flavonol glycoside is 0.88%.
Although the invention has been described in detail hereinabove, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1. A processing method of ginkgo leaves is characterized by comprising the following steps:
1) Adding water into ginkgo leaf, wherein the feed liquid ratio of the water added into ginkgo leaf is 1: 2-3 kg: adding water, sterilizing under high pressure, and adding microorganism for anaerobic fermentation; taking the sum of ginkgo leaves and water as a basic fermentation liquid, adding 30-50 g/L of sugar and 3-5 g/L of soluble starch into each liter of the basic fermentation liquid; adding 100U/g of cellulase into each gram of ginkgo leaf; the microorganism is lactobacillus, firstly, strain is cultured on MRS liquid culture medium to obtain seed liquid, the seed liquid is inoculated into fermentation broth, and the inoculation amount is 1-5% v/v; fermenting for 42-60 hours;
2) After fermentation, standing the fermentation liquor, and then carrying out solid-liquid separation;
3) Adding sorbitol into the separated solid, and freeze-drying to obtain freeze-dried powder; wherein, each kilogram of separated solid is added with 0.5-2 g of sorbitol;
the lyophilization includes two stages of pre-freezing and vacuum freeze drying,
wherein the prefreezing is prefreezing for 2 hours at-80 ℃;
the conditions of the vacuum freeze drying are as follows: pressure 130 x 10 -3 mbar~135×10 -3 And (3) performing freeze drying for 12-18 h at the temperature of-50 ℃ to-55 ℃ under the condition of mbar.
2. The method for processing ginkgo leaves according to claim 1, wherein the seed liquid is cultured in a shaking flask at a temperature of 25-28 ℃ at a speed of 150-200 r/min for 8-12 h.
3. The method according to claim 1, wherein in step 1), the cultured seed liquid is inoculated into a fermentation tank containing a fermentation liquid at an inoculum size of 1-5% v/v, the tank temperature is kept at 25 ℃, the tank pressure is kept at 0.5kg/cm, and the stirring rate is 150rpm.
4. The method for processing ginkgo leaves according to claim 1, wherein in the step 2), after fermentation is completed, the fermentation liquid is left for 10-12 hours, and then solid-liquid separation is performed, wherein the method for solid-liquid separation comprises centrifugation, and the conditions of the centrifugation are as follows: the rotating speed is 6000r/min, and the time is 5-10 min.
5. The ginkgo leaf freeze-dried powder obtained by the processing method according to any one of claims 1 to 4.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104771419A (en) * | 2015-04-20 | 2015-07-15 | 龙岩学院 | Preparation method of ginkgo leaf fermentation preparation |
| CN106822196A (en) * | 2017-03-23 | 2017-06-13 | 山东天智绿业生物科技有限公司 | A kind of method for extracting Ginkgo biloba polysaccharide and ginko leaves flavone simultaneously from ginkgo leaf |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104771419A (en) * | 2015-04-20 | 2015-07-15 | 龙岩学院 | Preparation method of ginkgo leaf fermentation preparation |
| CN106822196A (en) * | 2017-03-23 | 2017-06-13 | 山东天智绿业生物科技有限公司 | A kind of method for extracting Ginkgo biloba polysaccharide and ginko leaves flavone simultaneously from ginkgo leaf |
Non-Patent Citations (2)
| Title |
|---|
| 适合银杏叶发酵的菌株筛选及发酵效果;林标声;吴江文;江火香;戴爱玲;杨小燕;;江苏农业科学(第04期);315-317 * |
| 银杏叶发酵研究进展;杨芳;赵崇妍;屈青松;刘自尧;史新元;;中国现代中药(第08期);1034-1038 * |
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