CN111349550B - Device and method for fermenting aerobic strains at high flux and application of device and method - Google Patents

Abstract

The invention relates to the technical field of strain breeding, and discloses a device and a method for fermenting aerobic strains with high flux and application thereof, wherein the device for fermenting aerobic strains with high flux comprises a high-flux screening pore plate and a pore plate cover, at least one baffle plate perpendicular to the side wall is arranged on the inner side wall of a micropore of the high-flux screening pore plate, and the relationship between the length A of the baffle plate and the diameter D of the micropore of the high-flux screening pore plate satisfies the following requirements: 0 < A.ltoreq.2/7D, preferably: a is more than or equal to 1/35D and less than or equal to 1/7D; the method comprises the step of inoculating the aerobic bacteria into the device filled with the culture medium to perform fermentation culture on the aerobic bacteria, and the method and the device can effectively solve the problems of low strain screening flux, low screening efficiency, high evaporation rate, poor pellet shape and poor fermentation effect of the conventional shake flask fermentation aerobic strains.

Description

A device and method for high-throughput fermentation of aerobic strains and their applications

technical field

The invention relates to the technical field of strain breeding, in particular to a device and method for high-throughput fermentation of aerobic bacteria and their application.

Background technique

High-throughput screening technology refers to the method of using deep-well plates as culture or reaction medium at the DNA level or cell level, replacing manpower with automated equipment to process tens of millions of samples, and obtaining data sensitively and quickly. Sensitive and other characteristics. At present, the research on high-throughput parallel cell culture instruments mainly focuses on two aspects: 1), improving the throughput of culture by minimizing the volume of the reactor, that is, using miniature (milliliter or even microliter) bioreactors; 2), based on cell Rich experience in the cultivation process, in the high-throughput cultivation process, simulate the conditions of industrial-grade reactors as much as possible, that is, realize the process control and parameter control of micro-reactors.

In the traditional strain breeding process, strains with better fermentation effects were selected through shake flask primary screening and re-screening, and then small-scale fermenter tests were carried out. For the screening of aerobic strains, shake flask culture is easy to implement, and has a high oxygen transfer rate, which can provide the oxygen required for the growth and metabolism of aerobic strains, and is widely used in the early research of fermentation.

With the development of biotechnology, the construction and breeding of strains and the related research on the scale-up of fermentation engineering need to achieve high throughput to improve efficiency. Due to the low throughput and high evaporation rate of shake flasks, it consumes a lot of manpower and material resources. It takes a long time and cannot meet the current work needs. Although the high-throughput screening orifice plates currently on the market can achieve high-throughput to improve efficiency, due to the particularity of the growth and metabolism of aerobic bacteria, and The demand for the detection of metabolites of aerobic bacteria (the total volume of the current orifice plate is relatively small), the high-throughput screening orifice plate cannot meet the needs of the growth and metabolism of aerobic bacteria and the detection requirements of aerobic bacteria .

Therefore, there is an urgent need in the art to solve the problems of low strain screening throughput, low screening efficiency, high evaporation rate, poor morphology of bacterial spheres and poor fermentation effect that exist in traditional shake flask fermentation of aerobic strains.

Contents of the invention

The purpose of the present invention is to overcome the problems of low strain screening throughput, low screening efficiency, high evaporation rate, poor shape of bacteria balls and poor fermentation effect in traditional shake flasks that exist in the prior art when fermenting aerobic strains. The problem is to provide a device and method for high-throughput screening of aerobic bacteria and their application. The device and method can effectively solve the problems of low strain screening throughput, low screening efficiency, and High evaporation rate, poor shape of bacteria balls and poor fermentation effect.

In order to achieve the above object, the first aspect of the present invention provides a device for high-throughput fermentation of aerobic strains, said device for high-throughput fermentation of aerobic strains includes a high-throughput screening orifice plate and an orifice cover, said At least one baffle plate perpendicular to the side wall is provided on the inner sidewall of the microwell of the high-throughput screening orifice plate, wherein the distance between the length A of the baffle plate and the diameter D of the microwell of the high-throughput screening orifice plate is The relationship satisfies: 0<A≤2/7D, preferably: 1/35D≤A≤1/7D.

The second aspect of the present invention provides a method for fermenting aerobic bacteria. The aerobic bacteria are inoculated into the above-mentioned device filled with culture medium to ferment and cultivate the aerobic bacteria.

The third aspect of the present invention provides the application of the above-mentioned device or fermentation method in high-throughput screening of aerobic strains.

Through the above technical scheme, the device and method for high-throughput screening of aerobic bacteria according to the present invention are used to ferment and cultivate aerobic bacteria. The device and method can effectively solve the problems existing in traditional shake flask fermentation of aerobic bacteria Low strain screening throughput, low screening efficiency, high evaporation rate, poor shape of bacteria balls and poor fermentation effect. For example, in Example 1, the screening throughput of Aspergillus niger can reach 132 colonies, the liquid evaporation rate is only 1.5%, and the acidity is 15.67%.

Description of drawings

Fig. 1 is the top view of high-throughput screening orifice plate in embodiment 1

Figure 2 is a schematic diagram of the opening of the fastening device;

Fig. 3 is the micrograph of the Aspergillus niger obtained by fermentation culture in Example 1.

Explanation of reference signs

1 high-throughput screening plate 11 microwell 12 baffle

2 fastening device 21 lock cover 22 lock buckle 23 lock hook

detailed description

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

In the present invention, unless stated otherwise, the orientation word "upper" refers to the position where the sandwich-type orifice cover is in direct contact with the air, and "down" refers to the position where the sandwich-type orifice cover is in direct contact with the air. The position where the orifice plate is in direct contact.

The first aspect of the present invention provides a device for high-throughput fermentation of aerobic strains. The device for high-throughput fermentation of aerobic strains may include a high-throughput screening orifice plate 1 and an orifice cover. The high-throughput At least one baffle 12 perpendicular to the side wall is provided on the inner sidewall of the microhole 11 of the volume screening orifice plate 1, wherein the length A of the baffle is between the diameter D of the microwell of the high-throughput screening orifice plate The relationship satisfies: 0<A≤2/7D, preferably: 1/35D≤A≤1/7D.

In the present invention, the relationship between the thickness T of the baffle plate 12 and the diameter D of the microholes of the high-throughput screening orifice plate satisfies: 0<T≤1/7D, preferably 1/35D≤T≤3/35D .

In the present invention, the shape of the microholes of the high-throughput screening orifice plate is not specifically limited, and can be existing, for example, square holes (rectangular or square) or circular holes, when the shape of the microholes is In the case of a square hole, the diameter D refers to the diameter D of the inscribed circle of the square hole.

In the present invention, the specific shape of the baffle 12 is not limited, for example, it may be a rectangle or a square, which can be set by those skilled in the art according to specific situations.

According to a specific embodiment of the present invention, the baffle plate 12 is a rectangle, and the side of the shorter side of the rectangle is connected to the bottom wall of the microwell 11 of the high-throughput screening orifice plate 1, so The side of the longer side of the baffle plate 12 can be vertically connected to the side wall of the microwell 11 of the high-throughput screening orifice plate 1, and the other side of the longer side is not connected to the side wall of the high-throughput screening orifice plate 1. The side walls of the microwells 11 of the orifice plate 1 are in contact.

In addition, according to another specific embodiment of the present invention, the rectangle is only connected to the bottom wall of the microwell 11 of the high-throughput screening orifice plate 1 through the shorter side, and the longer sides are not connected to each other. It is in contact with the side wall of the microwell 11 of the high-throughput screening orifice plate 1 .

In the present invention, the relationship between the height H of the baffle plate 12 and the depth S of the microwells 11 of the high-throughput screening orifice plate 1 can satisfy: 0<H≤S, preferably 1/2S≤H≤S.

The inventors of the present invention found in research that when the relationship between the length A of the baffle and the diameter D of the microhole of the high-throughput screening orifice satisfies: 1/35D≤A≤1/7D, the baffle The relationship between the thickness T of the plate 12 and the diameter D of the microholes of the high-throughput screening orifice plate satisfies: 1/35D≤T≤3/35D, the height H of the baffle plate 12 and the high-throughput screening orifice plate 1 The relationship between the depth S of the micropores 11 can satisfy: in the case of 1/2S≤H≤S, the evaporation rate can be further reduced, the fermentation effect can be improved, and a better shape of bacteria balls can be obtained.

In the present invention, the length A of the baffle 12 refers to the length of the baffle 12 along the diameter direction of the microwell 11 of the high-throughput screening orifice plate 1 .

In the present invention, the depth S refers to the distance from the bottom wall of the microwell 11 of the high-throughput screening orifice plate 1 to the orifice.

In the present invention, the height H refers to the length of the baffle plate 12 along the depth S direction.

In the present invention, the thickness T refers to the length in a direction perpendicular to the length A of the baffle 12 .

In the present invention, the orifice plate cover is a sandwich-type orifice plate cover, and the sandwich-type orifice plate cover sequentially includes a stainless steel orifice plate, a filter membrane and a silica gel orifice plate from top to bottom.

In the present invention, the filter membrane is not specifically limited, for example, it may be a polytetrafluoroethylene filter membrane, filter paper or hollow fiber filter membrane.

In the present invention, the high-throughput screening orifice plate 1 can be selected from a 6-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate, preferably a 6-hole plate (as shown in FIG. 1 ).

In the present invention, the well plate cover can be selected from a 6-well plate cover, a 24-well plate cover, a 48-well plate cover, a 96-well plate cover or a 384-well plate cover, preferably a 24-well plate cover.

In the present invention, the orifice plate cover can be selected from a 6-well plate cover, a 24-well plate cover, a 48-well plate cover, a 96-well plate cover or a 384-well plate cover, which means the holes of the stainless steel orifice plate in the orifice plate cover The number of holes is 6 holes, 24 holes, 48 holes, 96 holes or 384 holes.

In the present invention, the number of holes of the silica gel orifice plate in the orifice plate cover can be greater than, less than or equal to the number of holes of the orifice plate cover, preferably, the holes of the silica gel orifice plate in the orifice plate cover The number is equal to the number of holes in the stainless steel orifice plate. According to a specific embodiment of the present invention, the number of holes of the silica gel orifice plate in the orifice plate cover is equal to the number of holes of the stainless steel orifice plate, and each microwell on the high-throughput screening orifice plate 1 11 can correspond to the four holes in the silica gel orifice plate.

In the present invention, the diameter of the holes on the orifice plate cover (stainless steel orifice plate) can be 1-4mm, and the diameter of the silica gel orifice plate can be 1-3mm. Particularly, when the orifice plate cover (stainless steel orifice plate) The diameter of the hole on the top is 2.5-4mm, and the diameter of the silica gel orifice plate is 1-2mm, which can further reduce the liquid evaporation without affecting the fermentation effect of aerobic bacteria.

According to a preferred embodiment of the present invention, the high-throughput screening orifice plate 1 is a 6-orifice plate (as shown in Figure 1), the number of holes in the stainless steel orifice plate is 24 holes, and the number of holes in the silica gel orifice plate The number of holes is 24 holes.

In the present invention, the total volume of the high-throughput screening orifice 1 can be 30-360mL, particularly, in the present invention, when the high-throughput screening orifice 1 is a 6-orifice plate (as shown in Figure 1) can provide a better growth environment for aerobic bacteria, improve the screening throughput of aerobic bacteria, and improve screening efficiency. Preferably, the total volume of the high-throughput screening orifice plate 1 can be 240-300mL .

In the present invention, the screening throughput refers to the number of bacterium colonies ( That is, the high-throughput screening plate on the shaker and the amount of one culture of the shaker flask).

In the present invention, the device may also include a clamp, which is used to fix the high-throughput screening orifice plate 1 on a shaker, and fix the orifice plate cover on the high-throughput screening orifice plate 1 superior.

As shown in Fig. 2, according to a preferred embodiment of the present invention, the clamp may include a base, a fastening device 2 and a fixing mechanism for fixing the base on the shaker, wherein the fastening device is used to secure the The orifice plate cover is fixed on the high-throughput screening orifice plate, and in the present invention, the high-throughput screening orifice plate 1 can be placed in the base.

In the present invention, the base is not specifically limited. The base may include two side walls or four side walls. When there are two side walls, the two side walls are parallel to each other. When there are four side walls, The positions where the four side walls are connected to each other are perpendicular to each other.

In the present invention, the fastening device 2 may include a lock cover 21, a lock catch 22 and a lock hook 23, the lock catch 22 is arranged on the lock cover 21, and the lock cover 21 is arranged on the side of the base On the wall, the locking hook 23 is arranged on the orifice plate cover, and the fastening device fixes the orifice plate cover on the high-throughput screening orifice plate through locking of the lock and the locking hook.

In the present invention, the fixing mechanism is not specifically limited, and may be an existing one that can be used for fixing. For example, the fixing mechanism may be bolts or welding.

In the present invention, regarding a specific implementation of the clamp, the base is fixed on the shaker by bolts, and the base includes two side walls parallel to each other, and each of the two side walls is provided with a The lock cover 21 in the fastening device 2, the lock cover 21 is provided with a lock catch 22, the two sides corresponding to the two side walls of the orifice cover and the base are provided with the fastening device 2, the lock cover 21 of the fastening device 2 is hinged on the side wall of the base, the lock catch 22 is hinged on the lock cover 21, and the lock catch 22 can be snapped into the lock hook 23 , the schematic diagram when the fastening device 2 is loosened is shown in FIG. 2 .

The second aspect of the present invention provides a method for fermenting aerobic bacteria, which may include inoculating the aerobic bacteria into the microwells 11 of the above-mentioned high-throughput screening orifice plate 1 filled with medium, Fermentation of aerobic bacteria.

In the present invention, the aerobic bacteria can be any aerobic bacteria, preferably selected from Rhizopus, Mucor, Neurospora, Aspergillus or Penicillium, more preferably Aspergillus.

In the present invention, the Aspergillus can be selected from Aspergillus flavus, Aspergillus niger or Aspergillus fumigatus, and according to a preferred embodiment of the present invention, the Aspergillus is Aspergillus niger.

In the present invention, in the high-throughput screening orifice plate 1, based on each milliliter of culture medium, the inoculation amount of the aerobic bacteria makes the number of spores of aerobic bacteria be 50,000-400,000 spores, according to the present invention A preferred embodiment, based on per milliliter of culture medium, the inoculum size of the aerobic bacteria makes the number of spores of aerobic bacteria be 150,000 to 300,000 spores. measured by counting.

In the present invention, before the aerobic bacteria are inoculated into the medium, the medium needs to be sterilized. In the present invention, the conditions for the sterilization are not specifically limited, and can be existing and can be used for culturing Based sterilization, for example, the sterilization conditions may be: sterilization temperature of 121-125° C. and pressure of 0.1-0.15 MPa for 20-30 minutes.

According to the fermentation method of aerobic strains of the present invention, there is no special requirement on the composition of the medium, as long as it can be used for the fermentation medium of aerobic strains. Preferably, when the aerobic strain is Aspergillus niger, the culture medium contains enzymolyzed products obtained from enzymatic hydrolysis of starchy raw materials, and preferably the amount of enzymolyzed products obtained from enzymatically hydrolyzed starchy raw materials accounts for 80-100% by weight of the amount. Generally, the product obtained by enzymatic hydrolysis of starchy raw materials is called liquefaction liquid, and the liquefaction liquid is separated from solid and liquid to obtain enzymatic hydrolysis residue and liquefied supernatant liquid. Usually, the liquefied supernatant liquid can be used to prepare the culture medium, or the liquefied supernatant liquid can be combined with The liquefied solution is mixed and used to prepare the culture medium. Therefore, in the present invention, the enzymatic hydrolysis product obtained from the enzymatic hydrolysis of starch raw materials includes the above-mentioned liquefied supernatant liquid obtained through solid-liquid separation, also includes liquefied liquid without solid-liquid separation, and also includes a mixture of the above two. The culture medium is preferably obtained by mixing the liquefied liquid and the liquefied supernatant with water or not mixing with water, wherein the total sugar of the liquefied liquid and the liquefied supernatant is 10-20%, and the weight of the liquefied liquid and the liquefied supernatant can be (0.1- 0.3):1.

In the present invention, the starch liquefaction supernatant can be prepared by various methods, for example, it can be prepared by the following method: pulverize the starchy raw material, perform enzymolysis on the pulverized product, and then solidify the product obtained by enzymolysis. liquid separation to obtain liquefied clear liquid and enzymolysis residue, and the solid-liquid separation condition makes the solid content of the enzymolysis residue 45-55% by weight, preferably 49-51% by weight.

The starchy raw material can be various starch-containing raw materials known in the art that can be used for enzymolysis and fermentation to prepare citric acid, for example, can be selected from one or more of corn, potatoes (such as cassava) and wheat, Preferably, the starchy raw material is corn.

In the present invention, in the high-throughput screening orifice plate 1, the total consumption of the medium can be 30-120 g, and the medium is equally distributed to each microwell of the high-throughput screening orifice plate 1 11 in.

In the present invention, the fermentation culture is carried out under the condition of stirring, and the conditions of the fermentation culture may include: the temperature is 30-40°C, the time is 36-96h, and the stirring speed is 200-400rpm. Preferably, the fermentation The cultivation conditions may include: the temperature is 35-40° C., the time is 50-80 hours, and the stirring speed is 200-300 rpm.

The third aspect of the present invention provides an application of the above-mentioned device or fermentation method in high-throughput screening of aerobic strains.

In the present invention, using the device or method to ferment the aerobic strains can effectively reduce the evaporation rate of the liquid in the high-throughput screening orifice plate 1, and the obtained aerobic strains form a cluster of chrysanthemums shape, the shape of the bacterium ball is better, compared with the traditional shake flask culture, the mycelium length of the aerobic strains fermented by the device and method is shortened by 5-10%, and the length of the obtained mycelium is shorter, which helps The air circulation between the aerobic strains can provide an excellent fermentation environment for the aerobic strains even during the fermentation process of the aerobic strains.

The present invention will be described in detail below by way of examples.

In the following examples,

Screening throughput refers to the one-time culture capacity of high-throughput screening orifice plates or shake flasks that can be placed on a shaker of the same size, and 22 colonies can be placed on the same shaker plate (length×width=60cm×40cm) High-throughput screening orifice plate or 40 shake flasks, then the screening throughput of the high-throughput screening orifice plate is 132 (the high-throughput screening orifice plate is a 6-well plate, and each microwell is inoculated with one colony), and the number of shake flasks The screening throughput was 40 (one colony was inoculated per shake flask).

Filling factor (%)=(total volume (mL)/volume (mL) of the reactor added to the material of the reactor) × 100%;

Liquid evaporation rate (%)=(the total weight (g) of the material that is added to the reactor before fermentation-the total weight (g) of the material in the reactor after the end of fermentation/the total weight (g) that is added to the high-throughput screening orifice plate before the fermentation The gross weight of material (g)) * 100%, described reactor comprises high-throughput screening orifice plate and shake flask, and described material comprises culture medium and Aspergillus niger spore suspension;

The acidity parameter is measured by NaOH titration method, measured with reference to the national standard GBT 8269-2006;

Conversion rate (%)=(the concentration of fermented liquid * the weight of the volume of fermented liquid/total sugar) * 100%, wherein, the concentration of gained fermented liquid is referred to as acidity;

The shape of the bacteria ball is measured by a microscope (Shanghai Tiansheng Instrument Co., Ltd., model is) XSP-16C computer-based biological microscope. is excellent; the shape of the chrysanthemum ball is clustered and chrysanthemum-like, but there are broken hyphae fragments, the shape of the mycelium is good; The grade of form is poor;

Aspergillus niger was purchased from China Microorganism Culture Collection Center, and the strain type was Aspergillus niger CO827.

Example 1

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

(1) Add the culture medium to the microwells of the high-throughput screening well plate of the 6-well plate (the volume of each microwell is 50mL, and the specifications of the baffles in the microwells meet: A=2/35D, H=S , T=1/35D, the number of baffles is 4 (equally spaced and vertically arranged)), and sterilized at 0.15MPa, 121°C for 30min, cooled to 35°C, and set aside, wherein the medium is corn liquefaction liquid and corn liquefaction supernatant, the total sugar is 16.15% by weight, and the filling amount of the culture medium in each microwell is 10mL;

(2) Aspergillus niger (Aspergillus niger spore suspension) is inoculated into the medium of step (1), and the inoculation amount of every milliliter of medium is 200,000 spores;

(3) Put a 24-well plate cover (the number of holes on the silica gel plate is 24, the diameter of the stainless steel plate is 2.5 mm, and the diameter of the silica gel plate is 1 mm) with a sandwich-type orifice plate cover on the high-throughput screening orifice plate;

(4) The high-throughput screening orifice plate is fixed on the shaker by a clamp, and the orifice cover is fixed on the high-throughput screening orifice plate (the fastening device of the clamp is shown in Figure 2), and the fermentation is carried out. The conditions of the fermentation culture were as follows: the temperature was 35° C., the time was 72 hours, and the stirring speed was 300 rpm.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured respectively, and the results are shown in Table 1.

Example 2

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

(1) Add the culture medium to the microwells of the high-throughput screening well plate of the 6-well plate (the volume of each microwell is 40mL, and the specifications of the baffles in the microwells meet: A=3/35D, H=3 /4S, T=2/35D, the number of baffles is 4 (equally spaced and vertically arranged)), and sterilized at 0.12MPa, 123°C for 25min, cooled to 37°C, and set aside, wherein, the culture medium is For corn liquefaction liquid and corn liquefaction clear liquid, the total sugar is 18% by weight, and the loading amount of the culture medium in each microwell is 10 mL;

(2) Aspergillus niger (Aspergillus niger spore suspension) is inoculated into the medium of step (1), and the inoculation amount of every milliliter of medium is 220,000 spores;

(3) Put the 24-well plate cover (the number of holes on the silica gel plate is 24, the diameter of the stainless steel plate is 3 mm, and the diameter of the silica gel plate is 1.5 mm) with a sandwich-type orifice plate cover on the high-throughput screening orifice plate;

(4) The high-throughput screening orifice plate is fixed on the shaker by a clamp (as shown in Figure 2), and the orifice cover is fixed on the high-throughput screening orifice plate (the fastening device of the clamp is shown in Figure 2 Shown), carry out fermentation culture, the condition of fermentation culture is: temperature is 37 ℃, time is 72h, and stirring speed is 250rpm.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured respectively, and the results are shown in Table 1.

Example 3

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

(1) Add the culture medium to the microwells of the high-throughput screening well plate of the 6-well plate (the volume of each microwell is 45mL, and the specifications of the baffles in the microwells meet: A=1/14D, H=1 /2S, T=3/35D, the number of baffles is 4 (equally spaced and vertically arranged)), and sterilized at 0.13MPa, 124°C for 22min, cooled to 40°C, and set aside, wherein, the culture medium is For corn liquefaction liquid and corn liquefaction clear liquid, the total sugar is 16.5% by weight, and the filling amount of the culture medium in each microwell is 10 mL;

(2) Aspergillus niger is inoculated (Aspergillus niger spore suspension) is planted in the substratum of step (1), and the inoculation amount of every milliliter of substratum is 180,000 spores;

(3) Put a 24-well plate cover (the number of holes on the silica gel plate is 24, the diameter of the stainless steel plate is 3.5 mm, and the diameter of the silica gel plate is 2 mm) with a sandwich-type orifice plate cover on the high-throughput screening orifice plate;

(4) The high-throughput screening orifice plate is fixed on the shaker by a clamp, and the orifice cover is fixed on the high-throughput screening orifice plate (the fastening device of the clamp is shown in Figure 2), and the fermentation is carried out. The conditions of the fermentation culture are as follows: the temperature is 40° C., the time is 72 hours, and the stirring speed is 200 rpm.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 4

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that in step (1), the total sugar is 20% by weight, and step (2) inoculates Aspergillus niger (Aspergillus niger spore suspension) into the medium of step (1), every The inoculum size per milliliter of medium is 250,000 spores.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 5

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that the volume of each microwell is 35 mL.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 6

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that the sandwich type orifice cover of the 24-well plate cover (the number of holes on the silica gel orifice cover is 24) is replaced by a 96-well plate cover (the number of holes on the silica gel orifice cover is 24). 96) sandwich orifice cover.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 7

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that the high-throughput screening orifice plate of the 6-well plate is replaced by the high-throughput screening orifice plate of the 24-well plate, specifically:

(1) Add the culture medium to the microwells of the high-throughput screening well plate of the 6-well plate (the volume of each microwell is 12.5mL, and the specifications of the baffles in the microwells meet: A=2/35D, H= S, T=1/35D, the number of baffles is 4 (equally spaced and vertically arranged)), and sterilized at 0.15MPa, 121°C for 30min, cooled to 35°C, and set aside, wherein the medium is corn Liquefaction liquid and corn liquefaction supernatant liquid, the total sugar is 16.15% by weight, and the loading amount of the culture medium in each microwell is 2.5mL;

(2) Inoculate Aspergillus niger (Aspergillus niger spore suspension) into the culture medium of step (1), and the inoculum amount per milliliter of culture medium is 200,000 spores.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 8

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of embodiment 1, the difference is that the baffle plate in the microhole satisfies: A=2/7D, H=1/3S, T=1/7D, and the number of baffle plate is 4 (equidistant vertical arrangement)).

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Example 9

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that the diameter of the stainless steel orifice plate of the 24-well plate cover is 2 mm, and the diameter of the silica gel orifice plate is 2 mm.

The screening flux, evaporation rate, acidity, conversion rate and morphology of bacterial spheres were measured, and the results are shown in Table 1.

Example 10

This example is used to illustrate the device and method for high-throughput screening of aerobic bacteria provided by the present invention

According to the method of Example 1, the difference is that the stirring speed is 400 rpm.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Comparative example 1

This comparative example is used to illustrate the device and method for aerobic bacteria in the prior art

According to the method of Example 1, the difference is that Aspergillus niger is fermented by shake flasks, specifically,

(1) Add the medium to 40 250mL shake flasks, sterilize at 0.15MPa, 121°C for 30min, cool to 35°C, and set aside. is 16.15% by weight, and the loading amount of the culture medium in the shake flask is 40mL;

(2) Aspergillus niger is inoculated (Aspergillus niger spore suspension) is planted in the substratum of step (1), and the inoculum size of every milliliter of substratum is 200,000 spores;

(3 The shaking flask is fixed on the shaker by the clamp, and Aspergillus niger is fermented and cultivated, the conditions of the fermentation and culture are: the temperature is 35°C, the time is 72h, and the stirring speed is 300rpm.

The screening flux, evaporation rate, acidity, conversion rate and spheroid morphology were measured, and the results are shown in Table 1.

Table 1

As can be seen from Examples 1-10, Comparative Example 1 and Table 1, under the same fermentation conditions, compared with the fermentation method (Comparative Example 1) of traditional aerobic strains, the high-throughput fermentation of the present invention is better The device and method of oxygen bacteria effectively improve the screening efficiency and fermentation effect (higher acidity) of Aspergillus niger, reduce the evaporation rate, and the shape of the bacteria ball is good;

In particular, as can be seen from Examples 1-3, when the relationship between the length A of the baffle and the diameter D of the microhole of the high-throughput screening orifice plate satisfies: 1/35D≤A≤1/7D, the The relationship between the thickness T of the baffle plate 12 and the diameter D of the microholes of the high-throughput screening orifice plate satisfies: 1/35D≤T≤3/35D, the height H of the baffle plate 12 and the high-throughput screening orifice plate The relationship between the depth S of the microwell 11 of 1 can satisfy: 1/2S≤H≤S, and in the case of a high-throughput screening orifice plate with a 6-hole plate, the evaporation rate can be further reduced and the fermentation effect can be improved. Obtain better bacterial morphology.

The preferred embodiments of the present invention have been described in detail above, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the content disclosed in the present invention. All belong to the protection scope of the present invention.

Claims (16)

1. the application of a device for high-throughput fermentation of Aspergillus niger in improving the fermentation effect of Aspergillus niger, characterized in that, the device for high-throughput fermentation of Aspergillus niger comprises a high-throughput screening orifice plate and an orifice cover, the The inner wall of the microhole of the high-throughput screening orifice plate is provided with a baffle perpendicular to the side wall, the microhole is a circular hole, the number of the baffle is 4, and the baffle is arranged vertically at equal distances ; Wherein, the relationship between the length A of the baffle and the diameter D of the microhole of the high-throughput screening orifice satisfies: 1/35D≤A≤1/7D; The relationship between the height H of the baffle and the depth S of the microholes of the high-throughput screening orifice plate satisfies: 1/2S≤H≤S; The relationship between the thickness T of the baffle and the diameter D of the microholes of the high-throughput screening orifice plate satisfies: 1/35D≤T≤3/35D; Wherein, the high-throughput screening orifice plate is a 6-orifice plate; The total volume of the high-throughput screening orifice plate is 240-300mL. 2. The application according to claim 1, wherein the orifice plate cover is a sandwich-type orifice plate cover, and the sandwich-type orifice plate cover comprises a stainless steel orifice plate, a filter membrane and a silica gel orifice plate sequentially from top to bottom. 3. The application according to claim 1, wherein the well plate cover is selected from a 6-well plate cover, a 24-well plate cover, a 48-well plate cover, a 96-well plate cover or a 384-well plate cover. 4. The application according to claim 3, wherein the well plate cover is a 24 well plate cover. 5. The application according to any one of claims 1-4, wherein the device further comprises a clamp, which is used to fix the high-throughput screening orifice plate on a shaker, and place the well The plate cover is fixed on the high-throughput screening orifice plate. 6. The application according to claim 5, wherein the fixture comprises a base, a fastening device and a fixing mechanism for fixing the base on the shaking table, wherein the fastening device is used to fix the orifice plate cover on The high-throughput screening well plate. 7. The application according to claim 6, wherein the fastening device comprises a lock cover, a lock catch and a lock hook, the lock catch is arranged on the lock cover, and the lock cover is arranged on the base On the side wall, the locking hook is arranged on the orifice plate cover, and the fastening device fixes the orifice plate cover on the high-throughput screening orifice plate through locking of the locking hook and the locking hook. 8. a fermentation method of aspergillus niger, it is characterized in that, described method comprises inoculating described aspergillus niger into the micropore of the high-throughput screening orifice plate of the device of the high-throughput fermentation aspergillus niger that is filled with substratum, To ferment and cultivate Aspergillus niger; Wherein, the device for the high-throughput fermentation of Aspergillus niger comprises a high-throughput screening orifice plate and an orifice cover, and the inner wall of the microwell of the high-throughput screening orifice plate is provided with a baffle perpendicular to the side wall, so The microholes are circular holes, the number of the baffles is 4, and the baffles are equidistantly arranged vertically; Wherein, the relationship between the length A of the baffle and the diameter D of the microhole of the high-throughput screening orifice plate satisfies: 1/35D≤A≤1/7D; The relationship between the height H of the baffle and the depth S of the microholes of the high-throughput screening orifice plate satisfies: 1/2S≤H≤S; The relationship between the thickness T of the baffle and the diameter D of the microholes of the high-throughput screening orifice plate satisfies: 1/35D≤T≤3/35D; Wherein, the high-throughput screening orifice plate is a 6-orifice plate; The total volume of the high-throughput screening orifice plate is 240-300mL. 9. The method of claim 8, wherein the well plate cover is selected from a 6-well plate cover, a 24-well plate cover, a 48-well plate cover, a 96-well plate cover or a 384-well plate cover. 10. The method of claim 9, wherein the well plate cover is a 24 well plate cover. 11. The method according to any one of claims 8-10, wherein the device further comprises a clamp for fixing the high-throughput screening orifice plate on a shaker and placing the well The plate cover is fixed on the high-throughput screening orifice plate. 12. The method according to claim 11, wherein the fixture comprises a base, a fastening device and a fixing mechanism for fixing the base on the shaking table, wherein the fastening device is used to fix the orifice plate cover on The high-throughput screening well plate. 13. The method according to claim 12, wherein the fastening device comprises a lock cover, a lock catch and a lock hook, the lock catch is arranged on the lock cover, and the lock cover is arranged on the base On the side wall, the locking hook is arranged on the orifice plate cover, and the fastening device fixes the orifice plate cover on the high-throughput screening orifice plate through locking of the locking hook and the locking hook. 14. The method according to claim 8, wherein the fermentation culture is carried out under the condition of stirring, and the stirring speed is 200-400rpm. 15. The method according to claim 14, wherein the stirring speed is 200-300 rpm. 16. Application of the fermentation method described in any one of claims 8-15 in high-throughput screening of Aspergillus niger.

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