CN109576318B - Method for extracting non-viable bacteria from fermentation liquor - Google Patents
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Abstract
The invention relates to a method for extracting non-viable bacteria from fermentation liquor, belonging to the technical field of fermentation. The optimized content of resin in the fermentation medium is 5-40 g/L; the method for producing the non-viable bacteria element by fermentation comprises the steps of inoculating the seed liquid of streptomyces into the fermentation culture medium for liquid fermentation, and extracting the non-viable bacteria element from the fermentation liquid after the fermentation culture is carried out for 96-168 hours. The fermentation medium and the fermentation method greatly improve the fermentation unit of the non-viable bacteria element, and are suitable for the large-scale production of the non-viable bacteria element.
Description
The application is a divisional application of the invention patent application on application date 2015.11.05, application number 201510744556.6, title "fermentation medium and fermentation method for improving yield of inactive biotin".
Technical Field
The invention relates to the technical field of fermentation, in particular to a method for extracting non-viable bacteria from fermentation liquor.
Background
Nonactin (Nonactin) is the simplest member of the large family of macrocyclic polyether ionophore antibiotics known as macrocyclic tetralactones. These macrocyclic tetralactones include nonactin, monomycin, dinactin, triactin and tetranectin. The non-viable bacteria is an ionophore, can combine metal ions such as ammonium ions, calcium ions, potassium ions and the like, and can transport cations to pass through biological membranes and artificial membranes, so that the non-viable bacteria can be used for manufacturing ammonium electrode plasma selective electrodes, microelectrodes and sensors. In addition, the non-viable bacteria also has antibacterial activity; anti-tumor activity; and anti-tumor multi-drug resistance; and is an effective antiviral agent, and may be effectively developed into an anti-AIDS drug.
At present, the international reported approaches for obtaining the non-viable bacteria include:
chemical total synthesis: the complete synthesis of the non-viable bacteria element can be realized, but because of the complexity of the structure of the non-viable bacteria element, the synthesis steps are many, the total yield is very low, and the preparation of the non-viable bacteria element by chemical complete synthesis is not practical.
Identification of anti-generic products viaSaccharomyces cerevisiaeThe bioassay, which is an antibiotic in macromolecular drug spectrum, a potential and mode of action, Med Mycol, 2013, 51: 280-289, reports that streptomyces fermentation is utilized to obtain inactive biotin, 7.2L fermentation medium finally obtains 2.7mg of inactive biotin, and the yield is only 0.38 mg/L. The fermentation medium used was: 2 g/L of yeast extract, 5g/L of wort, 2 g/L of glucose and 7.0 of pH; the fermentation conditions were 250rpm and the culture was carried out at 28 ℃ for 7 days. The literature, Streptomyces, a new cytoxic compound of the methyl pyridine class from a marine-derived Streptomyces sp. KORDI-3238. J. Antibiot, 2006, 59: 234-. The fermentation medium used was: 1g/L beef extract, 1g/L yeast extract, 2 g/L acid hydrolyzed casein, 5g/L glucose and 5g/L malt extract; the fermentation conditions were 200 rpm and the culture was carried out at 27 ℃ for 7 days.
At present, the production cost of the non-viable bacteria is higher, the fermentation period is long, the yield is low, the fermentation result is not ideal, and the industrialization is difficult to realize. The medium and fermentation process must be changed to increase the level of fermentation.
Disclosure of Invention
The invention aims to provide a fermentation medium and a fermentation method for improving the yield of non-viable bacteria, which are used for solving the technical problems of low yield of the non-viable bacteria in the fermentation production in the prior art and the like.
The technical scheme for realizing the invention is as follows:
a fermentation medium for increasing the yield of inactive bacteria, wherein the fermentation medium contains resin.
Preferably, the resin content in the fermentation medium is 5-40 g/L;
further preferably, the resin content in the fermentation medium is 10-30 g/L;
even more preferably, the fermentation medium has a resin content of 20 g/L;
the content is measured in terms of the actual liquid content of the fermentation vessel (e.g. fermenter or shake flask).
Preferably, the resin is one or more of HP-20 resin, XAD-16 resin, XAD-8 resin, D202 resin, XD-5 resin and the like; further preferred are HP-20 resin and XAD-16 resin.
The fermentation medium also contains a carbon source.
Preferably, the carbon source content in the fermentation medium is 4.0-15.0 g/L.
Further preferably, the carbon source content in the fermentation medium is 6.0-10.0 g/L.
Preferably, the carbon source is one or more of oatmeal, glycerol, glucose, corn starch, maltodextrin and the like.
The fermentation medium also contains a nitrogen source.
Preferably, the content of the nitrogen source in the fermentation medium is 0.2-3.5 g/L.
Further preferably, the content of the nitrogen source in the fermentation medium is 0.5-1.5 g/L.
Preferably, the nitrogen source is one or more of soybean meal powder, cottonseed meal powder, corn steep liquor hydrolysate, acid hydrolyzed casein, yeast extract/powder and the like.
Preferably, the carbon source is oatmeal, glycerol and glucose; the nitrogen source is acid hydrolyzed casein and yeast extract/powder.
Further, the fermentation medium also comprises inorganic salts used in conventional fermentation media.
The preferable range of the dosage of the inorganic salt is 0.5-2.5 g/L; the inorganic salt is preferably calcium carbonate;
specifically, the fermentation medium contains: 15-25g/L of resin, 2.5-7.5g/L of oatmeal, 0.5-5g/L of glycerol, 0.1-2.5g/L of glucose, 0.1-2.5g/L of yeast extract, 0.1-1.0g/L of acid hydrolyzed casein and 0.5-2.5g/L of calcium carbonate.
In a preferred embodiment of the invention, the fermentation medium comprises: 10.0g/L HP-20 resin, 10.0g/L XAD-16 resin, 5.0g/L oatmeal, 2.5g/L glycerol, 0.5g/L glucose, 0.5g/L yeast extract, 0.2 g/L acid hydrolyzed casein and 1.0g/L calcium carbonate.
In another preferred embodiment of the invention, the fermentation medium contains: 25.0 g/L HP-20 resin, 7.5g/L oatmeal, 5g/L glycerin, 2.5g/L glucose, 2.5g/L yeast extract, 1g/L acid hydrolyzed casein and 2.5g/L calcium carbonate.
In a further preferred embodiment of the invention, the fermentation medium contains: 15.0g/L of XAD-16 resin, 2.5g/L of oatmeal, 0.5g/L of glycerol, 0.1g/L of glucose, 0.1g/L of yeast extract, 0.1g/L of acid hydrolyzed casein and 0.5g/L of calcium carbonate.
The pH of the fermentation medium is 6.8-7.5, preferably pH 7.2.
The fermentation medium can be prepared and sterilized by a conventional method; for example, sterilization is carried out at 121 ℃ for 30-60 min.
The invention also provides a method for producing the non-viable bacteria element by fermentation, which comprises the steps of inoculating the seed liquid of streptomyces into the fermentation culture medium for liquid fermentation, and extracting the non-viable bacteria element from the fermentation liquid after the fermentation culture.
Preferably, the fermentation culture time is 96-168 hours; the fermentation culture temperature is 28-32 ℃; the pH value is 6.8-7.5 during the fermentation culture process.
The seed liquid of the streptomyces can be prepared by a conventional method.
For example, the slant culture of Streptomyces is inoculated into a seed culture medium, and cultured at 28-30 ℃ and a rotation speed of 180-.
The seed culture medium of the streptomyces can adopt a culture medium which is conventional in the field.
Preferably, the streptomyces seed culture medium contains: 10.0g/L of wort, 4.0 g/L of yeast extract and 4.0 g/L of glucose; its pH value is 7.3; sterilization is generally carried out at 121 ℃ for 30 min.
The method for extracting the inactive bacillus from the fermentation liquor can adopt a conventional method.
Preferably, the method for extracting the non-viable bacteria from the fermentation broth comprises the following steps:
filtering the fermentation liquor to obtain two parts of hypha and filtrate; lyophilizing the mycelium (generally for 5-7 days), extracting with methanol for 1-3 times, extracting with ethyl acetate for 1-3 times, and extracting with n-hexane for 1-2 times to obtain mycelium extractive solution; extracting the filtrate with ethyl acetate of the same volume for 1-3 times to obtain filtrate extract; mixing the mycelium extract and the filtrate extract, and concentrating under reduced pressure to obtain an extract; and (3) performing column chromatography on the extract by using silica gel (230-400 meshes), performing gradient elution by using 0%, 50%, 80%, 100% ethyl acetate-n-hexane and 100% methanol respectively, collecting 50% and 80% ethyl acetate-n-hexane eluents, crystallizing at room temperature, and recrystallizing by using acetone to obtain the (non-viable bacteria essence refined product).
Wherein, the streptomycete is preferably streptomycete griseus subspecies (S.griseus)Streptomyces griseus subsp.Griseus) The Collection number is CGMCC4.181, and it is commercially available from China General Microbiological Culture Collection Center (CGMCC).
The invention has the beneficial effects that:
the invention solves the defects of high production cost and non-ideal fermentation level of the non-viable bacteria, and provides a fertilizer beneficial to grayStreptomyces griseus subspecies (Streptomyces griseus subsp. Griseus) A fermentation medium for producing non-viable bacteria (Nonactin) by high-efficiency fermentation and a corresponding fermentation method. The improvement of the yield of the non-viable bacteria element is promoted by skillfully adding the resin into the fermentation culture medium, and the fermentation unit of the non-viable bacteria element is greatly improved by optimizing the fermentation culture medium and optimizing the fermentation parameters such as culture temperature, pH and the like, so that the method is suitable for the large-scale production of the non-viable bacteria element. Experiments show that under the culture condition provided by the invention, 1.6g of non-viable bacteria can be obtained from 20L of fermentation medium, the yield is as high as 80 mg/L, and the separation and purification process is simple and can be used for industrial production of the non-viable bacteria.
Drawings
FIG. 1 shows the absence of viable bacteria in Experimental example 11An H-NMR spectrum;
FIG. 2 shows the absence of viable bacteria in Experimental example 113C-NMR spectrum.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Each component in the medium formulation in the following examples is a commercially available product.
The experimental strain is Streptomyces griseus subspecies (Streptomyces griseus subsp. Griseus) CGMCC4.181, available from China General Microbiological Culture Collection Center (CGMCC).
The HPLC detection conditions for the viable bacteria-free content of the fermentation liquid in the following examples and comparative examples are as follows:
the instrument comprises the following steps: waters 2690 high performance liquid chromatograph;
a chromatographic column: phenomenex Gemini 5 μm NX-C18110, 250 x 4.6 mm;
mobile phase: methanol A and water B. The flow rate is 1 mL/min; the gradient settings are as follows:
| time (min) | A% | B% |
| 0 | 40 | 60 |
| 20 | 90 | 10 |
| 25 | 40 | 60 |
| 30 | 40 | 60 |
A detector: PL-ELS 2100 evaporative light scattering detector.
Example 1
Inoculating a slant culture of an experimental strain streptomyces griseus subspecies CGMCC4.181 to a seed culture medium, wherein the culture conditions are as follows: culturing at 30 deg.C and 200 rpm for 2 days; obtaining a seed solution; inoculating the seed solution into 40 2L shake flasks containing 500 mL of fermentation medium at 2.5% (V/V) for fermentation at 30 deg.C and 200 rpm for 5 days. After the fermentation is finished, a sample is taken for measurement (HPLC), and the viable bacteria-free titer is 142 mg/L.
The seed culture medium comprises the following components: 10.0g/L of wort, 4.0 g/L of yeast extract, 4.0 g/L of glucose and the balance of water; its pH value is 7.3; sterilizing at 121 deg.C for 30 min.
The fermentation medium comprises the following components: 10.0g/L of HP-20 resin, 10.0g/L of XAD-16 resin, 5.0g/L of oatmeal, 2.5g/L of glycerol, 0.5g/L of glucose, 0.5g/L of yeast extract, 0.2 g/L of acid hydrolyzed casein, 1.0g/L of calcium carbonate and the balance of water; its pH value is 7.2; sterilizing at 121 deg.C for 60 min.
Filtering the fermentation liquor to obtain mycelium and filtrate; lyophilizing mycelium for 7 days, extracting with 1L methanol for 3 times, 1L ethyl acetate for 3 times, and 1L n-hexane for 2 times to obtain mycelium extractive solution; extracting the filtrate with ethyl acetate of the same volume for 3 times to obtain filtrate extract; and mixing the hypha extracting solution and the filtrate extracting solution, and concentrating under reduced pressure to obtain about 16 g of extract. Subjecting the extract to column chromatography with silica gel (230-400 mesh), performing gradient elution with 0%, 50%, 80%, 100% ethyl acetate-n-hexane and 100% methanol respectively 1L, collecting 50% and 80% ethyl acetate-n-hexane eluents, crystallizing at room temperature, and recrystallizing with acetone to obtain refined product without viable bacteria 1.6 g; the yield thereof was found to be 80 mg/L.
Example 2
Inoculating a slant culture of an experimental strain streptomyces griseus subspecies CGMCC4.181 to a seed culture medium, wherein the culture conditions are as follows: culturing at 30 deg.C and 200 rpm for 2 days; obtaining a seed solution; inoculating the seed solution into 40 2L shake flasks containing 500 mL of fermentation medium at 3% (V/V) for fermentation at 28 deg.C and 250rpm for 6 days. After the fermentation is finished, sampling (HPLC) is carried out for determination, and the viable bacteria free titer is 125 mg/L.
The seed medium was the same as in example 1.
The fermentation medium comprises the following components: 25.0 g/L HP-20 resin, 7.5g/L oatmeal, 5g/L glycerin, 2.5g/L glucose, 2.5g/L yeast extract, 1g/L acid casein hydrolysate, 2.5g/L calcium carbonate and the balance of water; its pH value is 7.2; sterilizing at 121 deg.C for 60 min.
Extracting the non-viable bacteria from the fermentation liquor by the same method as the example 1 to obtain 1.5g of a non-viable bacteria refined product; the yield thereof was found to be 75 mg/L.
Example 3
Inoculating a slant culture of an experimental strain streptomyces griseus subspecies CGMCC4.181 to a seed culture medium, wherein the culture conditions are as follows: culturing at 30 deg.C and 200 rpm for 2 days; obtaining a seed solution; inoculating the seed liquid into 40 2L shake flasks containing 500 mL of fermentation medium at an inoculation amount of 2% (V/V) for fermentation at 28 deg.C and 250rpm for 6 days. After the fermentation is finished, sampling (HPLC) is carried out for determination, and the viable bacteria free titer is 98 mg/L.
The seed medium was the same as in example 1.
The fermentation medium comprises the following components: 15.0g/L of XAD-16 resin, 2.5g/L of oatmeal, 0.5g/L of glycerol, 0.1g/L of glucose, 0.1g/L of yeast extract, 0.1g/L of acid hydrolyzed casein, 0.5g/L of calcium carbonate and the balance of water; its pH value is 7.2; sterilizing at 121 deg.C for 60 min.
Extracting the non-viable bacteria from the fermentation liquor by the same method as the example 1 to obtain 1.2 g of a non-viable bacteria refined product; the yield thereof was found to be 60 mg/L.
Comparative example 1:
a live-free fermentation broth was prepared in the same manner as in example 1, except that the fermentation medium contained no HP-20 resin and no XAD-16 resin. After the fermentation is finished, sampling (HPLC) is carried out for determination, and the viable bacteria free titer is 4 mg/L.
Comparative example 2:
a live-free fermentation broth was prepared in the same manner as in example 2, except that the fermentation medium did not contain HP-20 resin. After the fermentation is finished, sampling (HPLC) is carried out for determination, and the inactive biotin titer is 3 mg/L.
A live-free fermentation broth was prepared in the same manner as in example 3, except that the fermentation medium contained no XAD-16 resin. After the fermentation is finished, sampling (HPLC) is carried out for determination, and the viable bacteria free titer is 1 mg/L.
Experimental example 1
The viable bacteria-free extract prepared in example 1 is colorless needle-shaped crystals; structure of no viable bacteria element1H-NMR and13C-NMR was confirmed; further confirmed by HRMS, which detected [ M + Na]+ 759.4293 peak of quasi-molecular ion, and calculated molecular weight of non-viable bacteria [ M + Na ]]+759.4295 anastomosis; it is composed of1H-NMR and13the C-NMR spectra are shown in figure 1 and figure 2 respectively.
Nuclear magnetic resonance spectrum data of the product without viable bacteria:
1H-NMR (CDCl3， 500 MHz) δ 4.97 (1H， m， H-8)， 4.01 (1H， m， H-3)， 3.85 (1H， m， H-6)， 2.50 (1H， m， H-2)， 1.98 (1H， m， H-5)， 1.92 (1H， m， H-4)， 1.76 (2H， m， H-7)， 1.61 (1H， m， H-4)， 1.49 (1H， m， H-5)， 1.23 (3H， d， J=6.3 Hz， 8-CH3)， 1.08 (3H， d， J=7.0 Hz， 2-CH3)。
13C-NMR (CDCl3， 125 MHz) δ 174.4 (s， C-1)， 80.2 (d， C-3)， 76.6 (d， C-6)， 69.3 (d， C-8)， 45.4 (d， C-2)， 42.5 (t， C-7)， 31.6 (t， C-5)， 28.3 (t， C-4)， 20.7 (q， 8-CH3)， 13.0 (q， 2-CH3)。
although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (5)
1. A method for extracting inactive biotin from a fermentation broth, comprising: filtering the fermentation liquor to obtain mycelium and filtrate; freeze-drying the mycelium, extracting with methanol for 1-3 times, extracting with ethyl acetate for 1-3 times, and extracting with n-hexane for 1-2 times to obtain mycelium extractive solution; extracting the filtrate with ethyl acetate of the same volume for 1-3 times to obtain filtrate extract; mixing the mycelium extract and the filtrate extract, and concentrating under reduced pressure to obtain an extract; performing column chromatography on the extract by using silica gel, performing gradient elution by using 0%, 50%, 80%, 100% ethyl acetate-n-hexane and 100% methanol respectively, collecting eluent of 50% and 80% ethyl acetate-n-hexane, crystallizing at room temperature, and recrystallizing by using acetone to obtain the extract;
the preparation method of the fermentation liquor comprises the following steps: inoculating the seed liquid of the streptomycete into a fermentation medium for liquid fermentation, and obtaining the fermentation liquid after fermentation culture;
the fermentation medium contains: 10.0g/L of HP-20 resin, 10.0g/L of XAD-16 resin, 5.0g/L of oatmeal, 2.5g/L of glycerol, 0.5g/L of glucose, 0.5g/L of yeast extract, 0.2 g/L of acid hydrolyzed casein and 1.0g/L of calcium carbonate;
or the fermentation medium contains: 25.0 g/L HP-20 resin, 7.5g/L oatmeal, 5g/L glycerin, 2.5g/L glucose, 2.5g/L yeast extract, 1g/L acid casein hydrolysate, and 2.5g/L calcium carbonate;
or the fermentation medium contains: 15.0g/L of XAD-16 resin, 2.5g/L of oatmeal, 0.5g/L of glycerol, 0.1g/L of glucose, 0.1g/L of yeast extract, 0.1g/L of acid hydrolyzed casein and 0.5g/L of calcium carbonate.
2. The method of claim 1, wherein the fermentation broth is produced by fermentation of streptomyces; the streptomycete is streptomycete griseus subspecies (Streptomyces griseus subsp. Griseus) And the preservation number is CGMCC 4.181.
3. The method of claim 1, wherein the fermentation medium has a pH of 6.8 to 7.5.
4. The method according to claim 1, wherein the fermentation broth is prepared by a method comprising: the fermentation culture time is 96-168 hours; and/or the presence of a gas in the gas,
the fermentation culture temperature is 28-32 ℃; and/or the presence of a gas in the gas,
the pH value is 6.8-7.5 during the fermentation culture process.
5. The method according to any one of claims 1 to 4, wherein the fermentation broth is prepared by a method comprising: the streptomyces seed culture medium contains: 10.0g/L of wort, 4.0 g/L of yeast extract and 4.0 g/L of glucose; its pH value is 7.3.
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Also Published As
| Publication number | Publication date |
|---|---|
| CN105368892A (en) | 2016-03-02 |
| CN105368892B (en) | 2018-12-18 |
| CN109371073A (en) | 2019-02-22 |
| CN109371073B (en) | 2021-11-05 |
| CN109576319B (en) | 2022-02-01 |
| CN109576319A (en) | 2019-04-05 |
| CN109628520B (en) | 2022-02-01 |
| CN109576318A (en) | 2019-04-05 |
| CN109628520A (en) | 2019-04-16 |
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