阅读说明：本技术 一种硫酸新霉素发酵生产中的流加补料方法 (Feeding method in neomycin sulfate fermentation production ) 是由 刘铭 郑新 曾勇 李文豪 肖金华 王翀 舒玉婷 廖鹏 于 2020-12-16 设计创作，主要内容包括：一种硫酸新霉素发酵生产中的流加补料方法,该方法包括以下步骤：步骤1：在补料管道上按照进料方向依次设置流量计和气动隔膜阀；步骤2：确定每次取样的时间间隔H；步骤3：确定总补料量S,计算补料速率V；步骤4：设定补料周期t；步骤5：确定补料瞬间速率V-瞬；步骤6：计算t-(开启)；步骤7：计算时间t-(关闭)；步骤8：完成硫酸新霉素发酵生产中的流加补料。本发明提供的一种硫酸新霉素发酵生产中的流加补料方法,去掉了计量罐及其大量管道阀门,通过气动隔膜阀、手动隔膜阀及排污阀、电磁流量计来实现对发酵罐的精准流加补料,操作简单,无灭菌死角,降低了补料系统染菌的风险,降低了补料系统灭菌的次数。(A fed-batch method in neomycin sulfate fermentation production comprises the following steps: step 1: a flow meter and a pneumatic diaphragm valve are sequentially arranged on the material supplementing pipeline according to the feeding direction; step 2: determining the time interval H of each sampling; and step 3: determining the total feed supplement amount S, and calculating the feed supplement rate V; and 4, step 4: setting a material supplementing period t; and 5: determination of the feed transient Rate V Instant heating device (ii) a Step 6: calculating t Is opened (ii) a And 7: calculating the time t Close off (ii) a And 8: finishing the feeding in the neomycin sulfate fermentation production. According to the fed-batch method in neomycin sulfate fermentation production, provided by the invention, a metering tank and a large number of pipeline valves thereof are removed, accurate fed-batch of a fermentation tank is realized through a pneumatic diaphragm valve, a manual diaphragm valve, a blowdown valve and an electromagnetic flowmeter, the operation is simple, no sterilization dead angle is caused, the risk of contamination of a feed-batch system is reduced, and the sterilization frequency of the feed-batch system is reduced.)

1. A fed-batch method in neomycin sulfate fermentation production is characterized by comprising the following steps:

step 1: directly communicating a feeding pipeline (1) with a neomycin sulfate fermentation tank (2), and sequentially arranging a flowmeter (3) and a pneumatic diaphragm valve (4) on the feeding pipeline (1) according to a feeding direction;

step 2: determining the time interval H of each sampling;

and step 3: sampling fermentation liquor in a neomycin sulfate fermentation tank (2) by a sampling person at intervals of a sampling time interval H to detect indexes such as reducing sugar, ammonia nitrogen and the like, determining the total feeding amount S of the sampling time interval H by combining the metabolism condition of the fermentation liquor, and calculating the feeding rate V of the sampling time interval, wherein the calculation formula is as follows:

feeding rate V is total feeding quantity S/sampling time interval H;

s, the unit is L; h, in hours;

and 4, step 4: setting a material supplementing period t in the sampling interval H;

and 5: determination of the feed transient Rate V_{Instant heating device}

When the pneumatic diaphragm valve (4) is opened, the instantaneous flow measured by the flowmeter (3) is the instantaneous velocity V of feeding_{Instant heating device}；

V_{Instant heating device}The unit is L/h;

step 6: calculating the opening time t of the pneumatic diaphragm valve (4) in one feeding period t_{Is opened}The calculation formula is t_{Is opened}(feed rate V x feed period t) ÷ instantaneous feed rate V_{Instant heating device}；

t_{Is opened}In seconds;

and 7: calculating the closing time t of the pneumatic diaphragm valve (4) in one feeding period t_{Close off}The calculation formula is

t_{Close off}Feeding period t-t_{Is opened}；

t_{Close off}In seconds;

and 8: according to the feeding period t determined in the step 4 and the time t of opening the pneumatic diaphragm valve (4) in one feeding period t calculated in the step 6_{Is opened}Within this sampling interval H, do notAnd (3) repeatedly opening and closing the pneumatic diaphragm valve (4) until the total supplement amount S of a sampling time interval H is completely supplemented into the neomycin sulfate fermentation tank (2), namely, the fed-batch supplement in the neomycin sulfate fermentation production is completed.

2. The fed-batch method in the fermentative production of neomycin sulfate according to claim 1, characterized in that: in the step 1, a first manual diaphragm valve (5) and a second manual diaphragm valve (6) are respectively arranged on the material supplementing pipeline (1) at two ends of the pneumatic diaphragm valve (4), and a sewage discharge valve (7) is respectively arranged on the first manual diaphragm valve (5) and the second manual diaphragm valve (6).

3. The fed-batch method in the fermentative production of neomycin sulfate according to claim 1, characterized in that: in step 2, the time interval H of each sampling is determined by the growth and metabolism rules of neomycin sulfate and the operability of sampling detection, and is usually 4 to 8 hours.

4. The fed-batch method in the fermentative production of neomycin sulfate according to claim 3, characterized in that: and 4, step 4: and setting the feeding period t in the sampling interval H as 20-300 seconds.

5. The fed-batch method in the fermentative production of neomycin sulfate according to claim 1, characterized in that: the flowmeter (3) in the step 1 is an electromagnetic flowmeter and is arranged at the bottom end of the vertical material supplementing pipeline (1), and the electric wire of the flowmeter is connected to a metal flange of the material supplementing pipeline (1).

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a fed-batch method in neomycin sulfate fermentation production.

Background

At present, a fed-batch feeding method (a feeding method for continuously feeding some nutrient substances to a fermentation system in a batch fermentation process) is widely applied to the antibiotic fermentation industry, but the fed-batch feeding method is not applied to domestic fermentation production of neomycin sulfate, and the main reason is that a large amount of carbon and nitrogen sources with higher substrate concentration are required to be fed as nutrient substances (such as sugar materials) in the neomycin sulfate fermentation process, the substrate concentration is as high as 40 percent, the substrate has certain viscosity, if the fed-batch feeding method is adopted, the feeding rate is low, the sugar materials easily block pipeline valves, inconvenience is brought to overhaul, and sterilization dead corners are easily caused.

For how to realize fermentation and feeding of neomycin sulfate, the prior art generally adopts a metering tank with a plurality of valves to manually feed fermentation liquor in a fermentation tank, the structure of the adopted device is shown in figure 1, nutrient substances such as sugar, ammonium sulfate and the like required by the fermentation liquor in a future period are calculated at intervals according to measured indexes, the required nutrient substances are firstly pressed into the metering tank (the metering tank is provided with a marked graduated tube), and then the metering tank is fed into the fermentation tank at one time.

The manual feeding mode is completely dependent on people in operation and control, uncertain risks exist, accurate metering and control cannot be achieved, operation is complicated, labor intensity is high, manual feeding is one-time feeding, too much one-time feeding amount can cause too many strains to grow in a large quantity, dissolved oxygen is rapidly reduced, pH is unstable, fermentation environment fluctuation is caused, normal metabolism of hyphae is influenced, and a large amount of toxic metabolites are accumulated to inhibit conversion and synthesis of neomycin.

In order to solve the problem caused by the manual feeding, the sanxia pharmaceutical limited company in Yichang city has also developed an automatic metering cup feeding system for feeding, and based on the manual feeding (fig. 1), a pneumatic diaphragm valve, a liquid level electrode and a control system are added, and a liquid level electrode is added to a metering tank to meter the volume and a pneumatic diaphragm valve is used to control the feeding and discharging, and the principle is that the nutrient substances to be fed are converted into corresponding time according to the feeding rate according to the volume of the metering tank, and then the nutrient substances are fed into a fermentation tank at equal intervals, for example, 2000L of sugar is fed for 4 hours, the volume of the metering tank is 20L, and then 1 hour is fed for 25 tanks, as shown in fig. 2.

Although above-mentioned automatic measurement cup formula feed supplement mode has reduced intensity of labour, the manual operation risk has been reduced, also avoided the too much adverse effect that brings of disposable feed supplement to a certain extent, but this set of feed supplement system has related to a large amount of pipeline valves, form the sterilization dead angle easily, cause the risk of contracting bacteria to whole feed supplement system more easily, it is inconvenient to maintain, and because the nutrient substance concentration that neomycin sulfate fermentation was mended into is high, viscidity, for example, sugar material, can often glue on the liquid level electrode, influence the signal transmission of liquid level electrode, cause the inaccurate or signal delay of measurement and delay the scheduling problem of feed supplement time.

Disclosure of Invention

The invention aims to solve the technical problem of providing a fed-batch method in neomycin sulfate fermentation production, which removes a metering tank and a large number of pipeline valves thereof, realizes accurate fed-batch of a fermentation tank through a pneumatic diaphragm valve, a manual diaphragm valve, a blow-down valve and an electromagnetic flowmeter, has simple operation and no sterilization dead angle, reduces the risk of contamination of a feed-batch system, and reduces the sterilization times of the feed-batch system; after the method disclosed by the invention is adopted, the fermentation environment is more stable, the hypha metabolism is more vigorous, the conversion rate of useful products is higher, and the titer is obviously increased.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a fed-batch method in neomycin sulfate fermentation production comprises the following steps:

step 1: directly communicating a feeding pipeline with a neomycin sulfate fermentation tank, and sequentially arranging a flowmeter and a pneumatic diaphragm valve on the feeding pipeline according to a feeding direction;

step 2: determining the time interval H of each sampling;

and step 3: sampling fermentation liquor in a neomycin sulfate fermentation tank by a sampling person at an interval of sampling time H to detect indexes such as reducing sugar, ammoniacal nitrogen and the like, determining the total supplement quantity S of the sampling time interval H by combining the metabolic condition of the fermentation liquor, and calculating the supplement rate V of the sampling time interval, wherein the calculation formula is as follows:

feeding rate V is total feeding quantity S/sampling time interval H

S, the unit is L; h, in hours;

and 4, step 4: setting the feeding period t in the sampling interval H to be 20-300 seconds;

and 5: determination of the feed transient Rate V_{Instant heating device}

When the pneumatic diaphragm valve is opened, the instantaneous flow measured by the flowmeter is the instantaneous velocity V of feeding_{Instant heating device}

V_{Instant heating device}In the unit of L/h

Step 6: calculating the opening time t of the pneumatic diaphragm valve in the one-time material supplementing period t, wherein the calculation formula is

t start ═ (feed rate V × feed period t) ÷ instantaneous feed rate V_{Instant heating device}

t is turned on in seconds;

and 7: calculating the closing time t of the pneumatic diaphragm valve in the one-time feeding period t, wherein the calculation formula is

t off-t on feed period

t is off in seconds;

and 8: and (4) opening the valve of the pneumatic diaphragm valve according to the feeding period t determined in the step (4) and the time t of opening the pneumatic diaphragm valve in the feeding period t for one time calculated in the step (6), and continuously and repeatedly opening and closing the pneumatic diaphragm valve in the sampling time interval H until the total feeding amount S of one sampling time interval H is completely supplemented into the neomycin sulfate fermentation tank, namely the fed-batch feeding in the neomycin sulfate fermentation production is completed.

Preferably, in step 1, a first manual diaphragm valve and a second manual diaphragm valve are respectively arranged on the feeding pipeline at two ends of the pneumatic diaphragm valve, and a blowdown valve is respectively arranged on the first manual diaphragm valve and the second manual diaphragm valve.

Preferably, in step 2, the time interval H between each sampling is determined by the growth and metabolism rules of neomycin sulfate and the operability of the sampling test, and is usually 4-8 hours.

Preferably, the flowmeter in step 1 is an electromagnetic flowmeter, and is installed at the bottom end of the vertical feed supplement pipeline, and the electric wire of the flowmeter is connected to a metal flange of the feed supplement pipeline.

The fed-batch method in neomycin sulfate fermentation production provided by the invention overcomes the defect that the fed-batch cannot be realized by the previous neomycin sulfate fermentation, overcomes the problems that an automatic metering cup type feed supplement system is complex in structure, is easy to cause sterilization dead angles, is inconvenient to maintain, is easy to interfere with electrodes and the like, removes a metering tank and a large number of pipeline valves thereof, realizes accurate fed-batch of a fermentation tank through a pneumatic diaphragm valve, a manual diaphragm valve, a blow-down valve and an electromagnetic flowmeter, greatly simplifies the structure and feed supplement steps, and reduces the cost; the operation is simple, no sterilization dead angle exists, the risk of contamination is reduced, the sterilization frequency of a feed supplement system is reduced, two ends of each pneumatic diaphragm valve are respectively provided with one manual diaphragm valve, the manual diaphragm valves are prevented from being closed in time when the pneumatic diaphragm valves are out of order and need to be overhauled in production, the whole feed supplement pipeline and a fermentation tank are prevented from being polluted, the safety factor is higher, the manual diaphragm valves are respectively connected with one blow-down valve, the condition that steam is unblocked when the feed supplement pipeline or the fermentation tank is extinguished is ensured, no sterilization dead angle exists between the valves, the electromagnetic flowmeter is installed at the bottom end of the vertical feed supplement pipeline, an electric wire is connected onto a metal flange, the electromagnetic interference resistance is effective, the electromagnetic flowmeter is not influenced by fluid density.

After the neomycin sulfate fermentation is carried out by adopting the method disclosed by the invention, the dissolved oxygen in the tank is more stable, the hypha metabolism is more vigorous, the conversion rate of the neomycin serving as a useful product is higher, and the tank-placing titer is obviously increased.

Drawings

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

FIG. 1 is a schematic diagram of manual feeding in the background of the invention;

FIG. 2 is a schematic diagram of an automatic measuring cup type feeding system in the background art of the present invention;

FIG. 3 is a schematic diagram of the connections of the flow meters and valves of the process of the present invention;

FIG. 4 is a graph of dissolved oxygen in accordance with a second embodiment of the present invention, wherein the abscissa represents the fermentation period in units of h and the ordinate represents dissolved oxygen.

Detailed Description

Example one

A fed-batch method in neomycin sulfate fermentation production comprises the following steps:

step 1: the feeding pipeline 1 is directly communicated with the neomycin sulfate fermentation tank 2, and a flowmeter 3 and a pneumatic diaphragm valve 4 are sequentially arranged on the feeding pipeline 1 according to the feeding direction.

Step 2: the time interval H for each sampling is determined.

And step 3: sampling fermentation liquor in a neomycin sulfate fermentation tank 2 by a sampling person at an interval of sampling time H to detect indexes such as reducing sugar, ammoniacal nitrogen and the like, determining the total feeding amount S of the sampling time interval H by combining the metabolic condition of the fermentation liquor, and calculating the feeding rate V of the sampling time interval, wherein the calculation formula is as follows:

feeding rate V is total feeding quantity S/sampling time interval H

S, the unit is L; h, in hours.

And 4, step 4: too short time of the feeding period t can cause frequent switching of the pneumatic diaphragm valve 4, the service life of the pneumatic diaphragm valve 4 is influenced, the stability of the whole flow feeding system is influenced, too long time of the feeding period t can not reach the effect of flow feeding, and the step 4: setting the feeding period t in the sampling interval H to be 20-300 seconds;

for example: the sampling time interval H is 4 hours, the total feeding amount S is 3600L, the feeding rate V is the total feeding amount S/the sampling time interval H is 3600L/4H is 900L/H, the feeding period t is 10S, and the instantaneous feeding rate V is_{Instant heating device}3600L/h, the pneumatic diaphragm valve 4 is opened within one feeding period t for a time t_{Is opened}(feed rate V x feed period t) ÷ instantaneous feed rate V_{Instant heating device}＝(900L/h×10S)÷362.5S, the pneumatic diaphragm valve 4 is closed for time t in a feeding period t_{Close off}Feeding period t-t_{Is opened}Although the feeding effect is good, the pneumatic diaphragm valve 4 is switched 360 times within 1 hour, and the switching is too frequent when the feeding effect is 10S-2.5S-7.5S.

Under the same condition, when the feeding period t is set to 3600S, the pneumatic diaphragm valve 4 is opened within one feeding period t for a time t_{Is opened}(feed rate V x feed period t) ÷ instantaneous feed rate V_{Instant heating device}Closing time t of the pneumatic diaphragm valve 4 in one feeding period t is 900S (900L/h is multiplied by 3600S) ÷ 3600L/h_{Close off}Feeding period t-t_{Is opened}3600S-900S-2700S, the pneumatic diaphragm valve 4 only needs to be opened and closed once within 1 hour, 900L of materials are supplemented once within 900S of the opened pneumatic diaphragm valve 4, and then the pneumatic diaphragm valve 4 is closed for 2700S, which obviously cannot achieve the effect of feeding materials;

therefore, the feeding period t in the present sampling interval H is usually set to 20 to 300 seconds.

And 5: determination of the feed transient Rate V_{Instant heating device}

When the pneumatic diaphragm valve 4 is opened, the instantaneous flow measured by the flowmeter 3 is the instantaneous velocity V of feeding_{Instant heating device}

V_{Instant heating device}The unit is L/h.

Step 6: calculating the time t for opening the pneumatic diaphragm valve 4 in one feeding period t_{Is opened}The calculation formula is

t_{Is opened}(ii) a feeding rate V x a feeding period t/a feeding transient rate V_{Instant heating device}

t_{Is opened}In seconds;

and 7: calculating the closing time t of the pneumatic diaphragm valve 4 in one feeding period t_{Close off}The calculation formula is

t_{Close off}Feeding period t-t_{Is opened}

t_{Close off}In seconds;

and 8: according to the feeding period t determined in the step 4 and the time t of opening the pneumatic diaphragm valve 4 in the feeding period t calculated in the step 6_{Is opened}And continuously and repeatedly opening and closing the pneumatic diaphragm valve 4 within the sampling time interval H until the total feed supplement amount S of one sampling time interval H is completely supplemented into the neomycin sulfate fermentation tank 2, namely the fed-batch feed supplement in the neomycin sulfate fermentation production is completed.

In the step 1, a first manual diaphragm valve 5 and a second manual diaphragm valve 6 are respectively arranged on the material supplementing pipeline 1 at two ends of the pneumatic diaphragm valve 4, and a blowdown valve 7 is respectively arranged on the first manual diaphragm valve 5 and the second manual diaphragm valve 6.

In step 2, the time interval H of each sampling is determined by the growth and metabolism rules of neomycin sulfate and the operability of sampling detection, and is usually 4 to 8 hours.

The flowmeter 3 in the step 1 is an electromagnetic flowmeter, is arranged at the bottom end of the vertical material supplementing pipeline 1, and is connected with a metal flange of the material supplementing pipeline 1 through an electric wire.

The numerical calculation process of the above embodiment is as follows:

the sampling time interval H is 4 hours, the total feeding amount S is 3600L, the feeding rate V is the total feeding amount S/the sampling time interval H is 3600L/4H is 900L/H, the feeding period t is 60S, and the instantaneous feeding rate V is_{Instant heating device}3600L/h, the pneumatic diaphragm valve 4 is opened within one feeding period t for a time t_{Is opened}(feed rate V x feed period t) ÷ instantaneous feed rate V_{Instant heating device}The pneumatic diaphragm valve 4 is closed for a time t within one feeding period t (900L/h × 60S) ÷ 3600L/h ═ 15S_{Close off}Feeding period t-t_{Is opened}And (5) 60S-15S-45S, the pneumatic diaphragm valve 4 opens 15S and closes 45S again in one feeding period t, and opens 15S and closes 45S again in the next feeding period t until the feeding is completed in a sampling interval time H, and then the feeding of the next shift is started to realize the continuous feeding.

Example two

After the method is adopted in the fermentation process of neomycin sulfate in the second branch factory of the Sanxia pharmaceutical Co., Ltd, dissolved oxygen in the tank is more stable, hypha metabolism is more vigorous, and biological potency is obviously increased, 3 batches of fermentation tanks with the same or similar tank placing period are respectively selected in the first branch factory of the Sanxia pharmaceutical Co., Ltd for comparison experiments, the fermentation tank batches F353/0696, F356/0698 and F358/0700 are selected in the first branch factory, and the fermentation tank batches F309/247, F312/248 and F303/249 are selected in the second branch factory, wherein the comparison results are as follows:

first, comparison of dissolved oxygen in a tank: selecting a period of 60-84 h from the experimental tanks for dissolved oxygen comparison, wherein the period is vigorous in metabolism, large in material supplement amount and more obvious in dissolved oxygen fluctuation, and as is obvious from the graph of FIG. 4, the dissolved oxygen in the tanks rapidly slides down after each material supplement in the experimental tanks of F353/0696, F356/0698 and F358/0700 in a branch factory in 60-84 h; after the experiment tank batches F309/247, F312/248 and F303/249 are fed in 60-84 h by adopting the method of the invention in the secondary plant, the dissolved oxygen in the tank does not fluctuate sharply, and the dissolved oxygen in the tank is relatively stable.

(II) metabolic comparison: after the method is adopted in the neomycin sulfate fermentation feeding process in the Sanxia pharmaceutical second division factory, the metabolism is more vigorous, the sugar supplement amount is obviously increased, the culture medium volume of the fermentation tank of the first division factory is 75T, the culture medium volume of the fermentation tank of the second division factory is 115T, for the purpose of more intuitive comparison, the fermentation medium volume of the first division factory is converted into 115T, and under the condition that the culture medium volumes of the fermentation tanks are the same, manual measuring cup type feeding experimental tanks adopted by the first division factory are compared, wherein the manual measuring cup type feeding experimental tanks adopted by the first division factory are F353/0696, F356/0698 and F358/358 0, and the metabolic conditions of the experimental tanks adopted by the second division factory are compared:

table 1 shows the amount of sugar supplement in the metabolism of a batch of the one-plant experimental tanks and the amount of sugar supplement in the metabolism after the conversion (the amount of sugar supplement after the conversion is equal to the amount of sugar supplement)

X 115 ÷ 75), table 2 is the metabolism sugar supplement amount of the two-division factory experimental tank batch;

as is apparent from tables 1 and 2, the average sugar supplement amount of the two-branch plant experimental tank batch is increased by about 30%, the metabolic sugar supplement amount is greatly increased, the tank discharge volume is greatly increased, and under the condition that the tank discharge titer is not changed, the yield is greatly increased, namely the tank discharge titer x the tank discharge volume.

TABLE 1

TABLE 2

(III) titer comparison: after the method is adopted in the fermentation and feeding process of neomycin sulfate in the Sanxia pharmaceutical binary factory, the tank discharge titer is obviously increased:

table 3 shows that the manual metering cup type feeding experiment tank batches F353/0696, F356/0698 and F358/0700 are adopted for discharging the tank titer in one plant experiment tank batch;

table 4 shows the tank discharge titer of the experimental tank batches F309/247, F312/248 and F303/249 in the two-branch plant by adopting the method of the invention;

as is apparent from tables 3 and 4, the average can discharging titer of the two-branch plant experimental tank lot is improved by about 14%, the can discharging titer is obviously increased, and under the condition that the can discharging volume is not changed, the yield is increased by about 14%, and the yield is equal to the can discharging titer multiplied by the can discharging volume.

TABLE 3

TABLE 4

The above-described embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and features in the embodiments and examples in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.