CN112852898B - Lipstatin fermentation method and fermentation medium - Google Patents
Lipstatin fermentation method and fermentation medium Download PDFInfo
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Abstract
In order to overcome the problems of low fermentation unit, high cost of fermentation raw materials, complex and difficult control process, inapplicability to large-scale industrial production and the like in the conventional lipstatin fermentation method, the invention provides a simple, convenient and efficient lipstatin fermentation method and a fermentation culture medium, which comprise the following steps: plate separation and slant culture, slant shake flask fermentation screening of strains, shake flask seed liquid preparation, first-stage seeding tank culture, second-stage seeding tank culture and fermentation tank culture. The lipstatin fermentation method and the fermentation medium obtained by the combined optimization strategies of screening high-yield strains through shaking flask fermentation, optimizing various levels of culture media, improving fermentation process technology and the like have the advantages of high fermentation unit, low fermentation raw material cost, simple and convenient fermentation process control, no need of adding exogenous toxic precursors and the like, and have very important significance for improving the lipstatin fermentation yield and product quality and reducing the production cost.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a lipstatin fermentation method and a fermentation medium.
Background
Lipstatin is a natural product produced by Streptomyces toxytricini (Streptomyces toxytricini) with good lipase inhibiting activity, and orlistat, the hydrogenated derivative thereof, is currently the most popular weight-loss drug worldwide. Orlistat, as a gastrointestinal lipase inhibitor, is bound to form covalent bonds with the lipase active serine site in the stomach and small intestinal lumens to inactivate lipase and thus not decompose fat in food into absorbable free fatty acid and monoacylglycerol, thereby reducing caloric intake, controlling weight gain, achieving the purposes of controlling weight, lowering blood sugar, lowering blood pressure, regulating fat, and the like. Orlistat is the only over-the-counter drug approved by FDA, EMA, and NMPA in the united states for the treatment of obesity, and is the only weight loss drug approved by NMPA for sale and use in china.
The prior production process of orlistat has two technical routes, namely a fermentation semi-synthesis method and a chemical total synthesis method, and the chemical total synthesis route has the disadvantages of long synthesis steps, complex reaction operation, harsh reaction conditions, low separation and purification yield, great environmental pollution and no accordance with the development trend of green chemical industry low-carbon economy. Currently, orlistat is industrially prepared mainly through a fermentation semisynthesis technology, firstly, streptomyces toxytricini is fermented to obtain a fermentation liquid rich in lipstatin, then, lipstatin is obtained through separation and purification, and the target component orlistat can be efficiently obtained through one-step pressure hydrogenation. Although the semi-synthetic orlistat needs to be fermented, separated, purified, synthesized and the like, and the technical route and the process parameter control are complex, compared with the chemical full-synthesis method, the method is more environment-friendly and more suitable for the clean production requirement.
Since orlistat is used as a weight-reducing drug to enter the clinic, the work of improving the biosynthesis mechanism analysis, strain genetic breeding, fermentation medium screening and fermentation process optimization of lipstatin fermentation units always belongs to the research and development hot spot in the field of biological fermentation, and a large number of papers are published without returning to the hotness till now. There are many patent reports on the fermentation production of lipstatin at home and abroad. US patent US4598089, US2005089978A1 adopt a non-supplement batch fermentation process, and the maximum fermentation unit is only 1.7-1.8 g/L. The international patent W02007134836A1 and the European patent EP0803576 adopt a fed-batch fermentation process for promoting the synthesis of lipstatin by adding precursors such as linoleic acid, caprylic acid and leucine in the fermentation process, and the maximum fermentation unit is 3.0-3.2 g/L. The US20110014664A1 adopts a fed-batch fermentation process of simultaneously feeding linoleic acid and oleic acid, and controls the residual concentration of the linoleic acid and the oleic acid in the fermentation liquor within a proper range, so that the fermentation unit of the lipstatin can be increased to more than 6 g/L. According to the Chinese patent CN103320481B, linoleic acid and leucine precursors are added in the fermentation process, and the dissolved oxygen is combined to control and regulate the metabolism of the strain, so that the yield of lipstatin is increased, and the fermentation unit can reach 7-8 g/L. According to the Chinese patent CN103555610A, by carrying out combined optimization on the formula proportion of the fermentation medium, the lipstatin fermentation unit is increased to about 10g/L, but the lipstatin fermentation unit only stays at the shaking flask fermentation stage, and no further industrial application report is seen. In summary, the current fermentative production of lipstatin still faces the following problems: 1) the fed-batch fermentation process relates to precursors such as linoleic acid, leucine and the like, the problems of insufficient supply of the precursors and unbalanced precursor are important bottlenecks in the high-efficiency biosynthesis of lipstatin, in addition, an exogenous precursor substrate has a toxic effect on thalli, and the residual concentration of the precursors in fermentation liquor must be strictly controlled in a proper range, so that the precursor feeding strategy of the fed-batch fermentation process is complex and difficult to control; 2) in the fermentation process, the linoleic acid and leucine precursors have large dosage and high price, so that the cost of the fermentation raw materials is high; 3) the unit yield of the non-supplement batch fermentation process is low, and the large-scale industrial application cannot be realized; 4) the fermentation unit needs to be continuously broken through and improved.
Disclosure of Invention
The invention aims at solving the technical problems of low fermentation unit, high cost of fermentation raw materials, complex and difficult control of the fermentation process and unsuitability for large-scale industrial production in the existing lipstatin fermentation method, and provides a lipstatin fermentation method and a fermentation culture medium which have the advantages of high fermentation unit, low cost of the fermentation raw materials, simple and convenient control of the fermentation process, no need of adding exogenous toxic precursors and are suitable for industrial production.
The purpose of the invention is realized by the following technical scheme: a lipstatin fermentation process comprising the steps of:
a. plate separation and slant culture: naturally separating spores of a lipstatin production strain in a plate culture medium, and culturing for 10-14 days; selecting a single colony which is well separated and has plump spores, inoculating the single colony to a slant culture medium, culturing for 7-9 days, and refrigerating the slant of the strain which grows well;
b. strain slant shake flask fermentation screening: inoculating the strain slant into a shake flask seed culture medium for culturing for 25-35 h; then inoculating 5-10% of the inoculum size into a shake flask fermentation medium for culturing for 7-9 days, measuring the titer by using HPLC after the fermentation is finished, selecting a strain inclined plane with the shake flask titer being more than or equal to 8000 mu g/ml, and continuously refrigerating for fermentation production;
c. preparing a seed solution in a shake flask: inoculating the strain slant with the shake flask titer being more than or equal to 8000 mug/ml into a shake flask seed culture medium, and culturing for 25-35 h to obtain shake flask seed liquid;
d. first-level seeding tank culture: inoculating the shake flask seed solution into a first-stage seeding tank for culture according to 1-2 per mill of the volume of a first-stage seeding tank culture medium, wherein the tank pressure is 0.04-0.05 MPa, Dissolved Oxygen (DO) is controlled to maintain 70-80%, and the culture period is 20-30 h;
e. secondary seed tank culture: inoculating the cultured culture solution of the primary seeding tank to a secondary seeding tank according to 8-12% of the volume of the culture medium of the secondary seeding tank for culture, controlling Dissolved Oxygen (DO) under the pressure of 0.04-0.05 MPa, keeping the DO at 40-60%, and culturing for 10-20 h;
f. culturing in a fermentation tank: transferring the cultured secondary seed culture solution to a fermentation tank culture medium by 8-12% of inoculum size for fermentation culture, wherein the tank pressure is 0.04-0.05 MPa, the dissolved oxygen is controlled by coupling in stages by alternately adjusting the stirring rotation speed to be 75-150 r/min and the ventilation volume to be 0.8-1.6 VVM, the fermentation period is 7-9 days, and the fermentation unit is 10000-13000 ug/ml;
wherein the culture temperature is 27-28 ℃.
Optionally, the step d of "controlling Dissolved Oxygen (DO)" includes:
dissolved oxygen is controlled by alternately adjusting the stirring speed of 200-350 rpm and the ventilation of 1-2 VVM in a coupling manner, so that DO is maintained at 70-80%.
Optionally, the step e of "controlling Dissolved Oxygen (DO)" includes:
dissolved oxygen is controlled by alternately adjusting the stirring speed to 90-150 rpm and the ventilation volume to 0.8-1.6 VVM in a coupling manner, so that DO is maintained at 40-60%.
Optionally, the step f of controlling dissolved oxygen by alternately adjusting the stirring speed at 75-150 r/min and the ventilation volume at 0.8-1.6 VVM in a staged coupling manner comprises the following steps:
(1) after fermentation started and before DO rebound: when the dissolved oxygen is reduced for 0-24 h of fermentation, firstly adjusting the rotating speed and then adjusting the air volume, and alternately performing the steps, wherein the air volume is adjusted upwards by 0.15-0.2 VVM each time, the air volume is 1.6VVM at most, the rotating speed is adjusted upwards by 2-4 Hz each time, the rotating speed is 150r/min at most, and the DO is controlled to be more than or equal to 30%; fermenting for 24-32 h: controlling DO to be more than or equal to 20%, keeping the highest stirring speed and the highest air flow unchanged when the DO is less than 10%, and cooling to 26.5-27 ℃;
(2) and (3) after DO rebounds until fermentation is finished: fermenting for about 28-36 hours, rapidly increasing DO to 70-90%, rapidly reducing air volume and tank pressure, regulating rotating speed, controlling DO at 40-60%, continuously maintaining for 10-20 hours, and controlling pH not to exceed 7.30, wherein pH is raised immediately; when the pH value continuously rises and is higher than 7.30, the pH value is continuously controlled by reducing the air quantity, the rotating speed and the tank pressure and further reducing the dissolved oxygen level, so that the pH value is ensured to fall; from 10 to 20 hours after DO rebounding to the middle and later stages of fermentation, the pH tends to be stable and is in a slow descending trend, the DO is controlled to be 20 to 30 percent by gradually and alternately increasing the air quantity and the rotating speed, the maximum air quantity and the maximum rotating speed are not higher than the maximum values of the air quantity and the rotating speed before DO rebounding, and the pH is controlled to be between 6.8 and 7.2 to stably operate;
(3) and (3) starting water supplement after DO drops to zero in the middle and later stages of fermentation, starting first water supplement after DO drops to zero, supplementing water once every 24 hours, wherein the volume of the supplemented water is 4-6% of the volume of the fermentation liquor, and the fermentation period is 7-9 days.
The invention also provides a culture medium suitable for the lipstatin fermentation method, and the culture medium sequentially comprises a plate and slant culture medium, a shake flask seed culture medium, a shake flask fermentation culture medium, a first-stage seeding tank culture medium, a second-stage seeding tank culture medium and a fermentation tank culture medium.
Optionally, the plate and the slant culture medium comprise the following components: 3-5 g/L of glucose, 3-5 g/L of yeast extract, 8-12 g/L of malt extract, 1-3 g/L of calcium carbonate and 18-20 g/L of agar powder;
the pH value of the plate and the slant culture medium before sterilization is 6.5-7.0.
Optionally, the shake flask fermentation medium comprises the following components: 15-25 g/L of glycerol, 35-45 g/L of low-temperature soybean meal, 85-95 g/L of soybean oil and 8-10 g/L of powdered soybean lecithin;
the pH value of the shake flask fermentation medium before sterilization is 7.0-7.5.
Optionally, the shake flask seed culture medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal and 4.0-6.0 g/L of yeast extract;
the pH value of the shake flask seed culture medium before sterilization is 6.5-7.0.
Optionally, the first-stage seeding tank culture medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal, 4.0-6.0 g/L, PPG2000 g/0.2-0.4 g/L of yeast extract polypropylene glycol;
the pH value of the primary seeding tank culture medium before sterilization is 6.5-7.0.
Optionally, the secondary seeding tank medium comprises the following components: 25-35 g/L of glycerol, 25-35 g/L of low-temperature soybean meal, 4.0-6.0 g/L of yeast extract and 10-20 g/L of soybean oil or sunflower oil;
the pH value of the secondary seed tank culture medium before sterilization is 6.5-7.0.
Optionally, the fermenter medium comprises the following components: 20-30 g/L of glycerol, 80-100 g/L of soybean oil or sunflower oil, 15-20 g/L of concentrated soybean phospholipid, 71.0-3.0 g/L of dispersant Remulls 217, 30-40 g/L of low-temperature soybean meal, 5-15 g/L of corn gluten powder and 0.1-0.2 g/L of zinc sulfate heptahydrate;
the pH value of the fermentation tank culture medium before sterilization is 7.0-7.5.
Compared with the prior art, the invention has the beneficial effects that: according to the lipstatin fermentation method and the lipstatin fermentation medium, which are obtained by the combined optimization strategies of single colony shake flask fermentation screening of high-yield strains, precursor self-supply, self-balancing fermentation medium optimization, fermentation process optimization and the like, the complex operations of adding expensive linoleic acid and leucine exogenous precursors and the like are not needed, and the consumption cost of raw materials can be reduced by more than 20% compared with that of a precursor fed-batch fermentation process; meanwhile, the fermentation process is convenient and simple to control, the fermentation level is obviously improved, and large-scale production can be effectively realized; the method for producing lipstatin by fermentation with the culture medium and the fermentation process is suitable for single-tank fermentation at a scale of 10m3The fermentation unit can be as high as 10-13 g/L.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to embodiments and examples. It should be understood that the detailed description and examples, while indicating the preferred embodiment of the invention, are given by way of illustration only.
One embodiment of the invention discloses a lipstatin fermentation method, which comprises the following steps:
a. plate separation and slant culture: preparing a culture dish plate and a test slant culture medium, sterilizing for later use, performing gradient dilution on a lipstatin production strain spore glycerol tube which is preserved by liquid nitrogen at ultralow temperature by using normal saline, performing plate separation in the plate culture medium, selecting a single bacterial colony which is better in separation, plump in spore and uniform in color and luster after the culture is finished, inoculating the single bacterial colony to the slant culture medium for continuous culture, and refrigerating the slant of the strain which grows well for later use. The plate separation and slant culture conditions are as follows: the culture temperature is 27-28 ℃, the humidity is 40-60%, the plate separation culture period is 10-14 days, the slant culture period is 7-9 days, and the slant refrigeration temperature is 4-8 ℃.
b. Strain slant shake flask fermentation screening: and c, preparing a shake flask seed culture medium and a shake flask fermentation culture medium, then sterilizing for later use, inoculating the strain slant refrigerated in the step a to the shake flask seed culture medium, inoculating the strain slant to the shake flask fermentation culture medium according to the inoculation amount of 5-10% after the culture is finished, carrying out shake flask fermentation, placing the bottle after the fermentation is finished to detect the titer of lipstatin, and selecting the strain slant with the shake flask titer of more than or equal to 8000g/ml for continuous refrigeration and large-scale fermentation production. The shake flask seed culture conditions are as follows: the culture temperature is 27-28 ℃, the rotation speed of a shaking table is 250-275 r/min, the amplitude is 5 +/-1 cm, and the culture period is 25-35 h. The shake flask fermentation culture conditions are as follows: the culture temperature is 27-28 ℃, the rotating speed of a shaking table is 250-275 r/min, the amplitude is 5 +/-1 cm, and the culture period is 7-9 days.
c. Preparing shake flask seed liquid: and c, sterilizing the prepared shake flask seed culture medium for later use, inoculating the strain slant with the titer of more than or equal to 8000 mu g/ml obtained by shake flask fermentation and screening in the step b into the shake flask seed culture medium for culture, and preparing shake flask seed liquid. The shake flask seed culture conditions are as follows: the culture temperature is 27-28 ℃, the rotating speed of a shaking table is 250-275 r/min, the amplitude is 5 +/-1 cm, and the culture period is 25-35 h.
d. First-level seeding tank culture: and c, sterilizing the culture medium in a first-stage seeding tank, cooling to room temperature, maintaining the pressure with sterile air, and inoculating the shake flask seed liquid which is cultured in the step c and has the thallus concentration of 20-30%, the pH value of 6.0-7.0 and no other bacteria pollution into the first-stage seeding tank for culture under the protection of flame, wherein the inoculation amount is 1-2 per mill of the volume of the culture medium in the first-stage seeding tank. The first-level seeding tank culture conditions are as follows: the volume of the culture tank is 100L, the temperature of the culture tank is 27-28 ℃, the pressure of the culture tank is 0.04-0.05 MPa, dissolved oxygen is controlled by alternately adjusting the stirring speed to be 200-350 rpm and the ventilation volume to be 1-2 VVM in a coupling manner, DO is maintained at 70-80%, and the culture period is 20-30 h.
e. Secondary seed tank culture: and d, sterilizing the culture medium in a secondary seeding tank, cooling to room temperature, maintaining the pressure by using sterile air, and transferring the culture solution of the cultured thallus in the step d, which has the concentration of 20-25%, the pH of 6.0-7.0 and no other bacteria pollution, into the secondary seeding tank for culture, wherein the inoculation amount is 8-12% of the volume of the culture medium of the secondary seeding. The secondary seed tank culture conditions are as follows: the volume of the culture tank is 1000L, the temperature of the culture tank is 27-28 ℃, the pressure of the culture tank is 0.04-0.05 MPa, dissolved oxygen is controlled by alternately adjusting the stirring speed to 90-150 rpm and the ventilation volume to be 1-2 VVM in a coupling mode, DO is maintained at 40-60%, and the culture period is 10-20 h.
f. Culturing in a fermentation tank: and sterilizing the fermentation culture medium in a fermentation tank, cooling to room temperature, maintaining the pressure by using sterile air, and transferring the cultured secondary seed culture solution with the thallus concentration of 25-35%, the pH of 6.0-6.5 and no other bacteria pollution into a fermentation medium tank for culture, wherein the inoculation amount is 8-12% of the volume of the fermentation culture medium. The culture conditions of the fermentation tank are as follows: the volume of the fermentation tank is 10m3The temperature of a culture tank is 27-28 ℃, the pressure of the culture tank is 0.04-0.05 MPa, dissolved oxygen is controlled by alternately adjusting the stirring speed to be 75-150 r/min and the ventilation volume to be 0.8-1.6 VVM in a staged coupling manner, the fermentation period is 7-9 days, and the dissolved oxygen is controlled by the coupling of fermentation culture as follows:
(1) after fermentation started and before DO rebound: respectively adjusting the initial temperature to be 27-28 ℃, the air flow to be 0.8-0.9 VVM, the stirring rotation speed to be 70-80 r/min, the tank pressure to be 0.04-0.05 Mpa, and the dissolved oxygen to be corrected to be 100%; when the fermentation starts to 24 hours, firstly adjusting the rotating speed and then adjusting the air volume when the dissolved oxygen is reduced, and alternately performing the steps, wherein the air volume is adjusted upwards by 0.15-0.2 VVM each time, the air volume is 1.6VVM at most, the rotating speed is adjusted upwards by 2-4 HZ each time, the rotating speed is 150r/min at most, and the dissolved oxygen is controlled to be more than 30%; fermenting for 24-32 h: controlling the dissolved oxygen to be more than or equal to 20%, keeping the highest stirring speed and the highest air flow unchanged when the dissolved oxygen is less than 10%, and cooling to 26.5-27.0 ℃ for culture.
(2) And (4) after DO rebounds, ending fermentation: and (3) fermenting for 28-36 h until DO rapidly rises to 70-90%, rapidly reducing the air volume and the tank pressure and regulating the rotating speed when the pH is closely raised, controlling the DO to be 40-60% (dissolved oxygen is very sensitive to the pH, controlling the pH by reducing the dissolved oxygen, and the pH cannot rise rapidly after the dissolved oxygen is reduced), controlling the pH not to exceed 7.30, and continuing the process for 10-20 h. And if the pH value continuously rises to be higher than 7.30, further reducing dissolved oxygen by adjusting the air quantity and the rotating speed to control the pH value and ensure that the pH value is reduced. And (3) from 10-20 hours after DO rebounding to the middle and later stages of fermentation, the pH tends to be stable and is in a slow descending trend, dissolved oxygen is controlled to be 20-30% by gradually and alternately increasing the air quantity and the rotating speed, the maximum air quantity and the maximum rotating speed are not higher than the maximum values of the air quantity and the rotating speed before DO rebounding, and the pH is controlled to be 6.8-7.2 for stable operation. And starting first water supplement after dissolved oxygen drops to zero, supplementing water once every 24 hours, wherein the water supplement volume is 4-6% of the fermentation broth volume, and the fermentation period is 7-9 days.
Further preferably, in an embodiment of the present invention, the step b includes the steps of: and c, preparing a shake flask seed culture medium and a shake flask fermentation culture medium, then sterilizing for later use, inoculating the refrigerated strain inclined plane in the step a to the shake flask seed culture medium, inoculating the cultured strain inclined plane to the shake flask fermentation culture medium according to the inoculation amount of 5% for shake flask fermentation, placing the bottle after the fermentation is finished to detect the titer of lipstatin, and selecting the strain inclined plane with the shake flask titer of more than or equal to 8000g/ml for continuous refrigeration and large-scale fermentation production. The shake flask seed culture conditions are as follows: the culture temperature is 27-28 ℃, the rotating speed of a shaking table is 250-275 r/min, the amplitude is 5 +/-1 cm, and the culture period is 25-35 h. The shake flask fermentation culture conditions are as follows: the culture temperature is 27-28 ℃, the rotation speed of a shaking table is 250-275 r/min, the amplitude is 5 +/-1 cm, and the culture period is 7-9 days.
In one embodiment of the present invention, the fermentation unit or shake flask valence effect refers to the content of lipstatin in the fermentation broth, and the measurement of the fermentation unit or shake flask valence effect can be performed by a measurement method conventional in the art.
In one embodiment of the present invention, the dissolved oxygen refers to a percentage value of oxygen concentration in the fermentation broth during the fermentation process to oxygen concentration in the fermentation broth at the beginning of the fermentation (i.e. DO value). The measurement of dissolved oxygen in the fermentation broth can be carried out by a measurement method conventional in the art.
In one embodiment of the invention, strain shake flask fermentation screening is carried out before shake flask seed liquid is prepared, shake flask fermentation is carried out under specific culture conditions, strains with poor properties and low shake flask price efficiency are eliminated, only strains with the price efficiency of more than or equal to 8000g/ml are selected, and the strains are kept in inclined plane refrigeration for standby and used for subsequent large-scale fermentation production, and the inclined plane shake flask fermentation screening of the strains lays a foundation for improvement of lipstatin fermentation units.
In one embodiment of the invention, the dissolved oxygen is controlled by alternately adjusting the stirring speed and the ventilation rate in the primary seeding tank culture stage, the secondary seeding tank culture stage and the fermentation tank culture stage. In the fermentation process of the streptomyces toxytricini, dissolved oxygen is an important index, the growth of thalli is not facilitated due to overhigh dissolved oxygen, and substances such as peroxide, superoxide and the like are easily formed by excessive oxygen to destroy cell components, so that the growth of thalli is destroyed; too low dissolved oxygen may result in slow microbial metabolism and slow lipstatin synthesis due to insufficient oxygen supply. The requirements of thalli on dissolved oxygen are different in different fermentation stages, the dissolved oxygen is coupled and controlled in stages in a mode of alternately adjusting the stirring rotating speed and the ventilation volume, on one hand, the fermentation unit of lipstatin is improved, on the other hand, a material supplementing mode is not needed, the cost is saved, and meanwhile, the automatic large-scale industrial production can be realized.
In one embodiment of the present invention, the shake flask fermentation medium comprises the following components: 15-25 g/L of glycerol, 35-45 g/L of low-temperature soybean meal, 85-95 g/L of soybean oil and 8-10 g/L of powdered soybean phospholipid.
In one embodiment of the present invention, the shake flask seed medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal and 4.0-6.0 g/L of yeast extract;
in one embodiment of the invention, the primary seeding tank medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal and 4.0-6.0 g/L, PPG2000 g/0.2-0.4 g/L of yeast extract polypropylene glycol;
in one embodiment of the invention, the secondary seeding tank medium comprises the following components: 25-35 g/L of glycerol, 25-35 g/L of low-temperature soybean meal, 4.0-6.0 g/L of yeast extract and 10-20 g/L of soybean oil or sunflower oil;
in one embodiment of the invention, the fermenter medium comprises the following components: 20-30 g/L of glycerol, 80-100 g/L of soybean oil or sunflower oil, 15-20 g/L of concentrated soybean phospholipid, 71.0-3.0 g/L of dispersant Remulls217, 30-40 g/L of low-temperature soybean meal, 5-15 g/L of corn gluten powder and 0.1-0.2 g/L of zinc sulfate heptahydrate
Preferably, the low-temperature soybean meal powder meets the requirements of more than two levels in quality indexes and classification standards of GB/T21494-2008 low-temperature edible soybean meal;
the soybean oil meets the primary requirements of the quality indexes of the finished product soybean oil in GB/T1535-2017 soybean oil;
the sunflower oil meets the first-level requirement of the quality index of Chinese patent product sunflower oil GB/T1535-2017 sunflower oil;
the physical and chemical indexes of the concentrated soybean phospholipid meet the quality regulation in 2.2 clause of GB 28401-2012 food additive phospholipid;
the dispersant is Remuls2177 super dispersant produced by Shanghai Luqi chemical technology, Inc., is prepared by deep processing of natural plant-derived grease through a special process and related carriers, and has extremely excellent emulsifying and dispersing performances.
In one embodiment of the invention, single soybean oil or sunflower oil is used as a main carbon source and a linoleic acid precursor source, low-temperature soybean meal powder and corn gluten powder are used as a main nitrogen source and a leucine precursor source, and natural products are used as a carbon source and a nitrogen source, so that the requirements of environmental protection and sanitation are met, green production can be realized, the production cost is reduced, linoleic acid and leucine are prevented from being added, the operation process in the fermentation process is simplified, and the method is suitable for large-scale industrial production.
In another embodiment of the present invention, the carbon source may also be selected from one or more of peanut oil, rapeseed oil, corn oil, cottonseed oil, linseed oil, safflower seed oil and palm oil.
In another embodiment of the present invention, the nitrogen source may also be selected from one or more of high temperature soybean meal, low temperature soybean meal, high temperature soybean meal, whole soybean meal, and yeast powder.
The present invention will be further illustrated by the following examples.
Example 1
a. Plate separation and slant culture: preparing a spore culture medium according to the following proportion: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L, calcium carbonate 2g/L, agar powder 18g/L, adding alkali to adjust pH to 6.75, sterilizing at 115 deg.C for 30min, and making into plate culture medium and slant culture medium. Gradiently diluting Pristatin production strain spore glycerol tubes preserved at liquid nitrogen ultralow temperature with sterile normal saline, coating the mixture on the plate culture medium, culturing for 12 days at the temperature of 28 ℃ and the humidity of 40-50%, selecting single colonies with good separation, abundant spores and uniform color, inoculating the single colonies to a slant culture medium, and continuously culturing for 8 days; selecting the slant surfaces of the slant surfaces from the test tube slants which grow mature, are all full of uniform, white and compact bacterial layers, and storing the slant surfaces in a refrigerator at 4 ℃.
b. Strain slant shake flask fermentation screening: preparing a shake flask seed culture medium and a fermentation culture medium according to the following proportions for shake flask screening, wherein the shake flask seed culture medium comprises: low-temperature soybean meal powder 20g/L, yeast extract 5g/L, glycerin 20g/L, alkali is added to adjust the pH value to 6.80, and the filling amount of each bottle of a 500mL triangular flask is 50 mL; shake flask fermentation medium: 20g/L of glycerol, 40g/L of low-temperature soybean meal, 70g/L of soybean oil and 7g/L of powdered soybean phospholipid, adding alkali to adjust the pH value to 7.25, and filling the mixture into a 500mL triangular flask with the volume of 80mL per bottle. Inoculating the strain slant refrigerated at 4 ℃ to a seed bottle under the aseptic condition, wherein the slant number and the seed bottle number are in one-to-one correspondence; placing the seed bottles in a constant-temperature shaking culture at 27-28 ℃, carrying out shaking culture at the rotating speed of a shaking table of 250r/min for 30h, then placing the seed bottles in an aseptic condition, transferring each seed bottle to 2 fermentation bottles according to the inoculation amount of 5-10%, placing the inoculated fermentation bottles in a constant-temperature shaking culture at 27-28 ℃, carrying out shaking culture at the rotating speed of the shaking table of 275r/min, placing the fermentation bottles after 8 days of culture, detecting the titer of lipstatin, selecting a slope with the titer of the shaking bottle being more than or equal to 8000 mu g/ml, and placing the slope in a refrigerator at 4 ℃ for continuous storage for later use.
c. Preparing a seed solution in a shake flask: preparing a shake flask seed culture medium according to the following proportion, wherein the low-temperature soybean meal powder is 20g/L, the yeast extract is 5g/L, the glycerol is 20g/L, the pH value is adjusted to 6.75 by adding alkali, and the filling amount of each bottle of a 500mL triangular flask is 50 mL. Preparing the strain slant with the shake flask titer more than or equal to 8000ug/ml into spore suspension by using normal saline, inoculating the spore suspension into a shake flask seed culture medium, culturing at 27-28 ℃ and at a shaking table rotation speed of 250r/min for 30h to obtain shake flask seed liquid.
d. First-level seeding tank culture: performing first-stage seeding tank culture by adopting a 100L tank, and preparing a liquid culture medium used by the seeding tank according to the following proportion: the method comprises the following steps of preparing a first-grade seed tank culture solution, namely, low-temperature soybean meal powder 20g/L, yeast extract 5g/L, glycerol 20g/L, PPG2000 polypropylene glycol 0.3g/L, adding alkali to adjust the pH value to 6.80, adjusting the volume to 75L after actual digestion, inoculating 100ml of seed solution into a seed tank in a proportion of about 1 per mill for culture when the temperature is reduced to 28 ℃, controlling dissolved oxygen by alternately adjusting the stirring speed of 200-350 rpm and the ventilation quantity of 1-2 VVM in a coupling manner under the conditions that the temperature is 28 ℃ and the tank pressure is 0.04MPa, maintaining DO at 70-80%, and culturing for 26 hours to obtain the first-grade seed tank culture solution.
e. Secondary seed tank culture: using 1m3Performing secondary seeding tank culture in a tank, and preparing a liquid culture medium used by the seeding tank according to the following proportion: 30g/L of low-temperature soybean meal, 5g/L of yeast extract, 30g/L of glycerol and 10g/L of sunflower oil, adding alkali to adjust the pH value to 6.80, and the volume after digestion is 0.75m3When the temperature is reduced to 28 ℃, completely transplanting the primary seed liquid into a secondary seed tank for culture, controlling dissolved oxygen by alternately adjusting the stirring speed to 90-150 rpm and the ventilation volume to 1-2 VVM under the conditions that the temperature is 28 ℃ and the tank pressure is 0.04MPa, maintaining DO at 40-60%, and culturing for 15h to obtain the secondary seed tank culture liquid.
f. Culturing in a fermentation tank: by using 10m3Carrying out fermentation culture in a tank, and preparing a liquid culture medium for the fermentation tank according to the following proportion: 25g/L of glycerol, 35g/L of low-temperature soybean meal, 10g/L of corn gluten powder, 90g/L of sunflower oil, 18g/L of concentrated soybean phospholipid, Remuls 21772, 21772 g/L of dispersing agent, 0.15g/L of zinc sulfate heptahydrate, alkali adjustment to pH value of 7.25, and sterilization at 121 ℃ for 40 minutes. After digestion, the volume is 7.5m3, when the temperature of the culture medium is reduced to 28 ℃, the cultured secondary seeding tank strain is inoculated into a fermentation tank for culture, the inoculum size is about 10 percent, and the dissolved oxygen is controlled by coupling in stages through alternately adjusting the stirring speed to be 75-150 r/min and the ventilation volume to be 0.8-1.6 VVM, the method comprises the following steps:
(1) after fermentation started and before DO rebound: respectively adjusting the initial temperature of 28.0 ℃, the air flow of 0.8VVM, the stirring speed of 75r/min, the tank pressure of 0.04MPa and the DO to be 100 percent; fermenting for 0-24 h, adjusting the rotating speed first and then adjusting the air volume when the dissolved oxygen is reduced, and alternately performing the steps, wherein the air volume is adjusted to 0.15VVM each time, the air volume is 1.6VVM at most, the rotating speed is adjusted to 3HZ each time, the rotating speed is 150r/min at most, and the DO is controlled to be more than or equal to 30%; fermenting for 24-32 h: controlling DO to be more than or equal to 20%, keeping the stirring speed and the air flow unchanged at the highest, and cooling to 27.0 ℃ for culture when the DO is less than 10%.
(2) And (3) after DO rebounds until fermentation is finished: fermenting for 34h, rebounding DO, rapidly rising to 85% within 10min, rapidly rising pH, rapidly reducing air volume and tank pressure, and adjusting rotation speed, controlling DO at 40-50%, continuing the process for 16h, and keeping the highest pH value not more than 7.25. After DO rebounds for 16 hours, the pH tends to be stable and slowly reduced, the air quantity and the rotating speed are gradually and alternately increased, the dissolved oxygen is controlled to be 20-30%, and the pH is controlled to be about 7.0 to stably run. After the DO is fermented for 97h, zero is removed, water is supplemented for 97h, 121h and 145h once respectively, the volume of the supplemented water is 5% of the volume of the fermentation liquor each time, the fermentation period is 167h, and the tank release titer is 12968 g/L.
Example 2
The culture medium and the culture conditions of plate separation and slant culture, slant shake flask fermentation screening of strains, shake flask seed culture, primary seeding tank culture and secondary seeding tank culture are the same as example 1, the formula of the fermentation tank culture medium and the culture conditions before dissolved oxygen rebound are the same as example 1, the dissolved oxygen DO rebound time is 29h, the dissolved oxygen is controlled by adjusting the air volume and the rotating speed according to the method in example 1 after rebound, the dissolved oxygen begins to drop to zero after fermentation for 89h, water is supplemented once for 89h, 113h and 137h respectively, the water supplementing volume is 5% of the fermentation liquid volume each time, the fermentation period is 161h, and the tank release potency is 13570 ug/ml.
Example 3
The culture medium and the culture conditions of plate separation and slant culture, slant shake flask fermentation and screening of strains, shake flask seed culture and first-stage seeding tank culture are the same as example 1, sunflower oil in the culture medium is replaced by soybean oil with the same amount in the culture process of a second-stage seeding tank and a fermentation tank, the culture conditions of the second-stage seeding tank are the same as example 1, the culture conditions before the rebound of dissolved oxygen in the fermentation tank are the same as example 1, the rebound time of dissolved oxygen DO is 32h, the dissolved oxygen is controlled by adjusting the air volume and the rotating speed according to the method in example 1 after the rebound, the fermentation is carried out until 116h, the dissolved oxygen begins to drop to zero, water is supplemented for 116h, 140h and 164h respectively, the water supplementing volume is 5% of the fermentation broth volume every time, and the titer of 11890ug/ml is released in 187h of the fermentation period.
Example 4
The culture medium and the culture conditions of plate separation and slant culture, slant shake flask fermentation and screening of strains, shake flask seed culture and first-stage seeding tank culture are the same as example 1, sunflower oil in the culture medium is replaced by soybean oil with the same amount in the culture process of a second-stage seeding tank and a fermentation tank, the culture conditions of the second-stage seeding tank are the same as example 1, the culture conditions before the rebound of dissolved oxygen in the fermentation tank are the same as example 1, the rebound time of the dissolved oxygen is 28h, the dissolved oxygen is controlled by adjusting the air volume and the rotating speed according to the method in example 1 after the rebound, the fermentation is carried out until 120h, the dissolved oxygen begins to drop to zero, water is supplemented once for 120h, 144h and 168h respectively, the water supplementing volume is 5% of the volume of the fermentation liquor, the fermentation period is 190h, and the discharge titer is 12360 ug/ml.
The results of the embodiment show that the combined optimization strategies of high-yield strain single colony shake flask fermentation screening, optimization of culture media at all levels, fermentation process and the like are adopted, so that the complex operations of adding expensive linoleic acid and leucine exogenous precursors and the like are not needed, the fermentation process is convenient and simple to control, the fermentation level is obviously improved, the large-scale production can be effectively realized, and the fermentation unit can reach 10-13 g/L.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A lipstatin fermentation process, comprising the steps of:
a. plate separation and slant culture: naturally separating spores of a lipstatin production strain in a plate culture medium, and culturing for 10-14 days; selecting a single colony which is well separated and has abundant spores, inoculating the single colony to a slant culture medium, culturing for 7-9 days, and refrigerating a slant of a strain which grows well;
b. strain slant shake flask fermentation screening: inoculating the strain slant into a shake flask seed culture medium for culturing for 25-35 h; inoculating 5-10% of the inoculum size into a shake flask fermentation medium for culturing for 7-9 days, measuring the titer after the fermentation is finished, selecting a strain inclined plane with the shake flask titer being more than or equal to 8000 mu g/ml, and continuously refrigerating for fermentation production;
c. preparing shake flask seed liquid: inoculating the strain slant with the shake flask titer being more than or equal to 8000 mug/ml into a shake flask seed culture medium, and culturing for 25-35 h to obtain shake flask seed liquid;
d. first-stage seeding tank culture: inoculating the shake flask seed solution into a first-stage seeding tank for culture according to 1-2 per mill of the volume of a first-stage seeding tank culture medium, wherein the tank pressure is 0.04-0.05 MPa, Dissolved Oxygen (DO) is controlled to maintain 70-80%, and the culture period is 20-30 h;
e. secondary seed tank culture: inoculating the cultured culture solution of the primary seeding tank to a secondary seeding tank according to 8-12% of the volume of the culture medium of the secondary seeding tank for culture, controlling Dissolved Oxygen (DO) under the pressure of 0.04-0.05 MPa, keeping the DO at 40-60%, and culturing for 10-20 h;
f. culturing in a fermentation tank: transferring the cultured secondary seed culture solution to a fermentation tank culture medium by 8-12% of inoculation amount for fermentation culture, wherein the tank pressure is 0.04-0.05 MPa, and dissolved oxygen is controlled by alternately adjusting the stirring rotation speed to be 75-150 r/min and the ventilation volume to be 0.8-1.6 VVM in a staged coupling manner, and the method comprises the following steps:
(1) after fermentation started and before DO rebound: when the dissolved oxygen is reduced for 0-24 h of fermentation, firstly adjusting the rotating speed and then adjusting the air volume, and alternately performing the steps, wherein the air volume is adjusted to be 0.15-0.2 VVM each time, the air volume is 1.6VVM at most, the rotating speed is adjusted to be 2-4 HZ each time, the rotating speed is 150r/min at most, and the DO is controlled to be more than or equal to 30%; fermenting for 24-32 h: controlling DO to be more than or equal to 20%, keeping the highest stirring speed and the highest air flow unchanged when the DO is less than 10%, and cooling to 26.5-27 ℃;
(2) and (3) after DO rebounds until fermentation is finished: fermenting for about 28-36 hours, rapidly increasing DO to 70-90%, rapidly reducing air volume and tank pressure, regulating rotating speed, controlling DO at 40-60%, continuously maintaining for 10-20 hours, and controlling pH not to exceed 7.30, wherein pH is raised immediately; when the pH value continuously rises and is higher than 7.30, the dissolved oxygen is further reduced by reducing the air volume, the rotating speed and the tank pressure to control the pH value and ensure that the pH value is reduced; after DO rebounds for 10-20 hours to the middle and later stages of fermentation, the pH tends to be stable and is in a slow descending trend, DO is controlled to be 20-30% by gradually and alternately increasing the air volume and the rotating speed, the maximum air volume and the maximum rotating speed are not higher than the maximum values of the air volume and the rotating speed before DO rebounds, and the pH is controlled to be 6.8-7.2 to stably operate;
(3) starting water supplement after DO drops to zero in the middle and later stages of fermentation, starting the first water supplement after the DO drops to zero, supplementing water once every 24 hours, wherein the water supplement volume is 4-6% of the volume of the fermentation liquor, the fermentation period is 7-9 days, and the fermentation unit is 10000-13000 ug/ml;
the culture temperature is 27-28 ℃.
2. The lipstatin fermentation method according to claim 1, wherein the step d of "controlling Dissolved Oxygen (DO)" comprises:
dissolved oxygen is controlled by alternately adjusting the stirring speed to 200-350 rpm and the ventilation volume to 1-2 VVM in a coupling manner, so that DO is maintained at 70-80%.
3. The lipstatin fermentation method of claim 1, wherein the step e of controlling Dissolved Oxygen (DO) comprises:
dissolved oxygen is controlled by alternately adjusting the stirring speed to 90-150 rpm and the ventilation volume to 0.8-1.6 VVM in a coupling manner, so that DO is maintained at 40-60%.
4. The lipstatin fermentation process of claim 1, wherein the plate and slant medium comprises the following components: 3-5 g/L of glucose, 3-5 g/L of yeast extract, 8-12 g/L of malt extract, 1-3 g/L of calcium carbonate and 18-20 g/L of agar powder;
the pH value of the plate and the slant culture medium before sterilization is 6.5-7.0.
5. The lipstatin fermentation process of claim 1, wherein the shake flask fermentation medium comprises the following components: 15-25 g/L of glycerol, 35-45 g/L of low-temperature soybean meal, 85-95 g/L of soybean oil and 8-10 g/L of powdered soybean phospholipid;
the pH value of the fermentation medium before sterilization is 7.0-7.5.
6. The lipstatin fermentation process according to claim 1, wherein the shake flask seed medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal and 4.0-6.0 g/L of yeast extract;
the pH value of the shake flask seed culture medium before sterilization is 6.5-7.0.
7. The lipstatin fermentation process of claim 1, wherein the primary seedtank medium comprises the following components: 15-25 g/L of glycerol, 15-25 g/L of low-temperature soybean meal and 4.0-6.0 g/L, PPG2000 g/0.2-0.4 g/L of yeast extract polypropylene glycol;
the pH value of the primary seeding tank culture medium before sterilization is 6.5-7.0.
8. The lipstatin fermentation process of claim 1, wherein the secondary seedtank medium comprises the following components: 25-35 g/L of glycerol, 25-35 g/L of low-temperature soybean meal, 4.0-6.0 g/L of yeast extract and 10-20 g/L of soybean oil or sunflower oil;
the pH value of the secondary seeding tank culture medium before sterilization is 6.5-7.0.
9. The lipstatin fermentation process of claim 1, wherein the fermentor medium comprises the following components: 20-30 g/L of glycerol, 80-100 g/L of soybean oil or sunflower oil, 15-20 g/L of concentrated soybean phospholipid, 71.0-3.0 g/L of dispersant Remuls 2170, 30-40 g/L of low-temperature soybean meal, 5-15 g/L of corn gluten powder and 0.1-0.2 g/L of zinc sulfate heptahydrate;
the pH value of the fermentation tank culture medium before sterilization is 7.0-7.5.
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| CN106497995A (en) * | 2016-12-13 | 2017-03-15 | 东南大学成贤学院 | A kind of method of multistage fermenting and producing Lipstatin |
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| CN102268466A (en) * | 2011-07-23 | 2011-12-07 | 鲁南新时代生物技术有限公司 | Method for fermentation production of lipstatin and culture medium components thereof |
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