JPH01503407A - How to monitor fermentation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to monitor fermentation The present invention relates to monitoring fermentation in any fermentation process performed.

background The difficulties in monitoring fermentation are widely acknowledged. Is fermentation progressing properly? Please check whether the inoculum is intact and whether the fermentation is free from contaminants (contaI). In an attempt to determine whether the Ijnants) are contaminated with sewage, , so far efforts have been made regarding the identification of contaminants. This is usually Culture samples from fermentation broth to identify any contaminating bacteria. It consisted of Of course, culturing takes time, so fermentation Continue without waiting for the results, get the appropriate measurements and save on fermentation. Sometimes it is done. In some fields, things like this happen frequently. , huge amounts of money are being wasted.

Therefore, it is possible to monitor fermentation in a relatively short period of time, obtain appropriate measurement values, and avoid unnecessary fermentation. In contrast to prior art methods of monitoring fermentation, The method according to the invention comprises an inoculum, a desired fermentation product and It concerns the monitoring of products selected from the group consisting of important contaminants; The method is accomplished with a labeled specific antibody against the product to be monitored.

The method of monitoring fermentation in a fermentation broth according to the invention comprises The total number of particles in the A total of 11,111 L of the above sample was added to the inoculum, the desired fermentation product and any significant contaminants. an excess amount of a labeled specific antibody against a product selected from the group consisting of a dye; Mix and label using the number of particles consisting of the product bound to the labeled specific antibody This comparison can be used to determine whether the fermentation is contaminated or not. It is characterized by indicating whether dyeing particles are present in the fermentation broth.

The products that can be monitored by the method of the invention fall into three main categories: the cells, e.g. the inoculum used, the extracellular fermentation products and the intracellular fermentation products (e.g. For example, when using recombinant DNA technology, the desired fermentation product is expressed within the cell. It can be divided into

When the product to be monitored is a cell, the specific antibody used is directed against a cell surface antigen. and connect them.

When the product being monitored is an intracellular fermentation product, detergents or chemicals may be used. to disrupt the cells and add a labeled specific antibody that binds to the product. liberate things.

Important contaminants may be identified, for example, if the bacteria are expected to occur commonly and/or It is important because its presence is tolerated only up to a certain concentration.

The specific antibody used is labeled according to known methods, and the label used is Already used in the field of enzyme markers, radioisotopes or immunoassays It can be any type of fluorescent marker.

Brief description of the drawing The drawings schematically illustrate the elements in a continuous monitoring line used in a preferred embodiment of the invention. It is expressed in terms of

Brief description of the drawing The drawing consists of the following elements:

Fermentation tank 1, first particle separator 2, and second pump 3, first mixer a first device 6 for indicating the value of the optical density, a second reservoir 7, a third pump 8, second mixing coil 9, fourth reservoir 10, fifth pump 11 , fourth mixing coil 12, fourth pump 13, valve 14, FAC5 or Low site fluorimeter 15, first reservoir 16, first pump 17, first a second particle separation device 18, a second device 19 for indicating the value of optical density, a third storage 20 , the fourth pump 21 and the third mixing coil 22° Detailed description of the invention According to one embodiment of the present invention, a method for monitoring fermentation in a fermentation broth is as follows. This can be done at various times during the fermentation.

Samples from the fermentation broth taken from one or several parts of the fermentation tank. , a sonic separator (sonal 5 eparator) or a supervisor as necessary. Filters with appropriate porosity according to the particle size of the product to be detected Pass through a particle separator as necessary. The total number of particles in the sample is A device that indicates the value of optical density, such as a spectrophotometer, where that value is present in the sample. is a measure of the total number of particles. Then the product being monitored Add an excess amount of labeled specific antibody to the sample and bind to the labeled specific antibody. The number of particles consisting of the product is counted using the label used. how this is How this is done is explained below in connection with preferred embodiments of the invention.

The measured number of particles consisting of the product being monitored is defined as the measured total number of particles in the sample. Compare with numbers. By performing this comparison, it is possible to determine whether particles that contaminate fermentation are present in the sample. In other words, it can be determined whether or not it exists in the fermentation broth.

Thus, by considering the results of the above comparisons made at various times during fermentation, fermentation can be monitored.

The products that can be monitored by the method of the invention fall into three main categories: the cells, e.g. the inoculum used, the extracellular fermentation products and the intracellular fermentation products (e.g. For example, when using recombinant DNA technology, the desired fermentation product is expressed within the cell. It can be divided into

When the product to be monitored is a cell, the specific antibody used is directed against the selected surface of the cell. It binds to surface antigens.

When the product being monitored is an intracellular fermentation product, detergents or chemicals may be used. to disrupt the cells and add a labeled specific antibody that binds to the product. liberate things.

The specific antibody used is labeled according to known methods, and the label used is Already used in the field of enzyme markers, radioisotopes or immunoassays It can be any type of fluorescent marker.

A preferred embodiment of the present invention is the fermentation in fermentation broth carried out continuously in the following manner. It consists of a method for monitoring fermentation.

Fermentation in fermentation broth can be carried out under suitable conditions in any conventional fermentation tank (1). It will be done.

Samples are taken continuously from one or several locations in the fermentation tank (1), and the sample flow The sonic separation device or (can be changed depending on the particle size of the particles you want to pass through) ) in the first particle separator (2), such as a filter with suitable porosity; If necessary, it is passed by a second pump (3) that pumps liquid in the direction of the line of sight. . If necessary, the sample flow is transferred to a second pump (3) placed before or after the first pump. Pass through the mixing coil (4) or (5).

The sample stream is then transferred to a first device (6) that indicates the value of the optical density (e.g. ultraviolet spectrophotometer). total). Use this value to determine the amount of particles needed to maintain a constant number of particles in the sample stream. Determine the amount of diluent to use. The diluent is determined by the optical density value (rcad-ing). The amount determined by added to the flow. This amount is determined by a computer based on the optical density value. This third pump (8) is controlled by a signal from the computer. can be controlled.

The diluted sample stream is passed through a second mixing coil (9) and the inoculum, desired binding to a product selected from the group consisting of fermentation products and significant contaminants; A liquid containing a labeled specific antibody (measured constant particles present in the flow) from the fourth reservoir (10) by the fifth pump (11) in excess (based on the number) Add to diluted sample stream. This addition of labeled specific antibody to the sample stream can be controlled by a computer with the help of a value about the total number of particles The computer then delivers an appropriate amount of said liquid containing the labeled specific antibody to the sample stream. A fifth pump (11) is controlled. The sample flow is then subjected to a second mixing and and a delay coil (12) to combine the product to be monitored with a labeled specific antibody. The latter specific antibodies are present in excess, making it difficult for the organism being monitored to interact. All particles of the product will be bound to the labeled antibody.

The sample flow is sent to a fourth pump (13) which maintains the monitoring line at constant pressure. , if the pressure becomes excessive, it will activate when the pressure in the monitoring line exceeds a preset value. An appropriate amount of sample flow is discharged through the valve (14). The pressure in the monitoring line is The computer that controls the fourth pump (13) and valve (14) also maintains a constant Retained.

Therefore, the sample flow discharged from the fourth pump (13) is 1 per unit time. has a constant number of particles and is monitored per unit time with the help of the label used. The number of particles consisting of the product can be determined, and the measured number of particles present can be measured. can be compared to a fixed total number. This comparison indicates that particles contaminating the fermentation are present in the sample stream. , i.e., in the fermentation broth. In this way, Measures to control fermentation, such as aborting or freezing the fermentation broth as appropriate Or you can get a measure for processing.

Depending on the label used, the number of particles consisting of the product being monitored can vary between several known types. It can be measured by

When the label used is a radioactive isotope, free labeled specific antibody and biological The sample stream containing both the labeled specific antibody bound to the product is chromatographically or preferably the sample stream is separated by continuous chromatography. Analyze this sample stream for free or bound labeled antibody by separating do. Next, either the separated free labeled antibody or the bound labeled antibody ( What is labeled is a measure of the number of particles) determined by the device that indicates the radioactivity value .

If the label used is an enzyme, free label-specific antibody and product-bound A sample of the sample stream containing both labeled specific antibodies can be sampled using an enzyme-linked antibody, e.g. Labeled antibodies free or bound by nosorbent assay (ELISA) Analyze about.

This is preferred if the label used is a fluorophore, and free For example, a sample of a sample stream containing both a label-specific antibody and a label-specific antibody bound to a product is e.g. by fluorescence microscopy or flow cytofluorimeter or fluorescence activation Continuously sort free or bound labeled antibodies by automated cell sorter (FACS). Let's analyze it.

In the most preferred embodiment of the invention, a fluorescence activated cell sorter (FACS) is used to Continuously monitor the sample flow as described below.

From the fourth pump (13) where the total number of particles present per unit time is known A sample flow of 100 mL is supplied to the FAC5 (15) at a constant pressure. FACS allows liquid to flow at a constant rate. It has a nozzle that forms droplets and passes this liquid layer through one or several laser beams. The prior radiation from the particle (light ea+1 ssion) from an angle of e.g. 90'' to the laser beam, and Light scattering from another angle, e.g. laser Particle size recorded from an angle of 150' from the light beam and measured by this light scattering Evaluate.

The sample flow from the fermentation broth is continuously monitored through the viewing window of the FACS to determine e.g. (read i ngs) and the relative particle size (horizontal axis) versus relative fluorescence intensity (vertical axis) of the particles. axis) into a coordinate system.

In this way, by following the evolution of the product monitored through the viewing window, fermentation development can be continuously monitored. The product being monitored depends on its particle size and fluorescence. Therefore, non-fluorescent contaminants that can be seen through a viewing window in one location are It is seen near the horizontal axis according to the degree.

The signal from FAC5 can be used to issue a warning signal or a signal to control the state of fermentation. can be transferred to a computer that can

Controls with the same or different type of fluorescent marker used for the specific antibody Particles can also be incorporated into the monitoring line. Control particles are the first pump (17) to a part of the particles, preferably the first, somewhat forward of the fourth pump (13). After the separation device (2) and before the first mixing coil (4 or 5), a monitoring lamp is installed. The sample stream is fed into the sample stream.

The particle size of the control particles is monitored and the particle size consists of the product bound to the labeled specific antibody. The granularity of the child is chosen to be different. Therefore, the control particles are can be seen in certain places. The control particles move away from that particular location. This happens when there is some kind of failure in the monitoring line, for example when the pressure drops.

When the product to be monitored is a cell, e.g. an inoculum, a fermentation tank (+) A second pump (3) transfers the sample flow from the (4 or 5) directly to the first device (6) which indicates the value of the optical density. Can be sent. Diluent from the second reservoir (7) to the third pump (8) Therefore, it may or may not be added to the sample stream. When not adding diluent, The amount of cells produced in the fermentation broth is monitored by FAC5 to ensure optimal harvest. The timing can be determined.

When the product to be monitored is an extracellular fermentation product, the The sample stream is first passed through a first particle separator (2), e.g. remove.

When the product being monitored is an intracellular fermentation product, the sample flow is transferred to a second pump ( 3), optionally the first particle separator ( 2) and/or through the first mixing coil (4 or 5), the optical density The detergent or chemical is passed through the first device (6) showing the value of After the chemical has been added to the sample stream by the third pump (8), this sample stream is transferred to the second pump. The cells are destroyed by passing through a kissing coil (9). This sample stream is then subjected to sound in a suitable second particle separator, such as a wave separator or filter (18). After passing as needed to remove cell debris from the sample stream, the fifth pump (1 1) Determine the amount of labeled specific antibody added to the sample stream from the fourth reservoir by Particles present in the sample stream are determined by a second device that indicates the value of the optical density used to Measure the total number of.

The particle size of the product bound to the labeled specific antibody is too small for FAC8 classification of particles. In some cases, it may also contain particles having specific antibodies against the product to be monitored. The third reservoir (20) is located before or after the labeled specific antibody addition site. A fourth pump (21) for transporting the particles into the sample stream is supplemented to the above monitoring line. In this case, a third mixing coil (22) can be added to the specific antibody. Insert the monitoring line between the addition site of the particle with the specific antibody and the labeled specific antibody, respectively. Enter.

The entire monitoring line can be automated by computer, and Fermentation conditions can be controlled by signals.

In a suitable application of the method according to the invention, the fermentation in the fermentation broth is carried out from the inoculum. It is possible to monitor continuously up to the end.

First, at the start of fermentation, the evolution of the inoculum is tracked and the fermentation broth is free of contaminants. Monitor by checking that it does not contain. Next, fermentation is carried out to produce the desired fermentation product The fermentation broth should be monitored to ensure that it is free of contaminants. sample flow Since the product may not be diluted, the amount of product produced can be tracked to determine the optimal harvest time. can be determined.

Using the fermentation monitoring method according to the invention, the inoculum, desired fermentation products and important contaminants can be monitored. Several products selected from the group consisting of Advantageously, as long as the fluorescence is different, they can be monitored simultaneously by FACS. Ru. Antibodies against certain important expected and/or common contaminants (along with knowledge of their granularity) provide a warning signal that contaminants are present as well as provide early confirmation and measurement of the concentration of this contaminant. Comparable to currently recommended methods for terminating, freezing, or otherwise disposing of can be done.

When monitoring fermentation according to the present invention by FAC'S, unknown unknown Possible ways to separate and recover contaminants in pure form from cultured organisms and control the situation can be tested quickly.

The method according to the invention provides that the inoculum is Used to check the purity of the inoculum by labeled specific antibodies and preferably by FACS. It will be clear to those skilled in the art that this embodiment can be It is within the surrounding area.

Using the method of monitoring fermentation in fermentation broth according to the invention, continuous Fermentation conditions can be controlled by methods.

Submission of translation of written amendment (Article 184-8 of the Patent Law) February 6, 1999 Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Display of patent application PCT/SE 87100343 2. Name of the invention How to monitor dephotofermentation 3. Patent applicant Address: 1A4, Asbvagen, Sorrentouna, Sweden, Agent (Postal code 100) 3-2-3 Marunouchi, Chiyoda-ku, Tokyo [Telephone Tokyo (211) 2321 Oshiro 5. Date of submission of written amendment October 6, 1988 6. List of attached documents Fermentation in the fermentation broth according to the present invention from page 1, line 32 to page 2, line 14 of the specification The monitoring method is to determine the total number of particles in the sample from the fermentation broth, compared to the total number of particles present. It is measured by a device that indicates the value of the optical density, which is a measure, and the said sample is a product selected from the group consisting of a desired fermentation product and a significant contaminant (i.e. Mixing with an excess amount of labeled specific antibody against the particulate fermentation component) The number of particles consisting of the product (i.e., the fermentation component) bound to the specific antibody The number of particles measured is compared to the total number of particles measured using the label, and this comparison determines the Characterized by indicating whether particles contaminating the fermentation are present in the fermentation broth The scope of the claims 16. A method for monitoring fermentation in a fermentation broth, the method comprising monitoring fermentation in a sample from the fermentation broth. The total number of particles in the The samples were analyzed for the inoculum, the desired fermentation product and any significant contaminants (c onLam+nant) for particulate fermentation components selected from the group consisting of from the fermentation components bound to the labeled specific antibody. The number of particles is compared with the total number of particles measured using the label used, This comparison will indicate whether particles are present in the fermentation broth that could contaminate the fermentation. A method characterized by:

3. The label used is an enzyme marker, and the fermentation component is bound to the labeled specific antibody. The number of particles consisting of 3. The method according to claim 1 or 2, wherein the method is measured by SA).

4. The label used is a radioactive isotope, and the sample is chromatographically Separate the amount of free labeled antibody or bound labeled antibody and calculate the radioactivity value. from the fermentation components bound to the labeled specific antibody by measuring with the device shown. The method according to claim 1 or 2, characterized in that the number of particles is .

5. The label used is a fluorescent substance, and it can be used for fluorescence microscopy, flow cytofluorescence, etc. Label specific antibodies by cell sorter or fluorescence activated cell sorter (FAC3). Claim 1 or 2 characterized by the number of particles consisting of bound fermentation components. How to put it on.

6. The label used is a fluorescent substance, and it is placed in one or several places in the fermentation tank (1). Continuously collect fermentation broth from the parts, This sample stream is passed through a suitable first particle separator (2) as required. control particles with a fluorescent marker according to the first pump (17). in addition to the sample stream from the reservoir (16); Sample flow by a second pump (3), before (4) or after the second pump (3) (5) through the first mixing coil (4, 5) disposed in The sample flow is then a measure of the total number of particles present in the sample flow and added to the sample flow. The optical density value used to determine the amount of diluent, detergent or chemical passing through the first device (6) shown; A determined amount of diluent, detergent or chemical is delivered to the second pump by a third pump (8). The sample stream is added to the sample stream from the reservoir (7) of the ) as necessary, The sample flow is measured by a second, which indicates the value of optical density, which is a measure of the total number of particles present in the sample flow. passing through the device (19) as necessary, Select from the group consisting of inoculum, desired fermentation product and important contaminants as required. Particles having a specific antibody that binds to the selected particulate fermentation component are transferred to a fourth port. sample stream from the third reservoir (20) by means of a pump (21). passing the feed stream through a third mixing coil (22); particles selected from the group consisting of an inoculum, a desired fermentation product and a significant contaminant; The excess label-specific antibody that binds to the fermentation components is transferred to the fifth pump (1■). Thus, after adding to the sample stream from the fourth reservoir (lO), the sample stream is subjected to a fourth mixing process. and a delay coil (12) so that the sample flow exceeds a preset value. The monitoring line is maintained at a constant pressure by a valve (14) that drains a portion of the sample flow when The sample discharged from the fourth pump (13) is A firefly filter separates the particles present in the sample stream by relative particle size and relative fluorescence. By passing through a photoactivated cell sorter (FACS) (15), Continuously monitor the sample flow from the fermentation broth by FACS (15) , the method according to claim 1 or 2.

8. selected from the group consisting of inoculum, desired fermentation product and important contaminants. Claim 6 or 7, wherein several types of particulate fermentation components are simultaneously monitored. The method described in.

international search report Ri, E.

Claims (1)

[Claims] 1. A method for monitoring fermentation in a fermentation broth, the method comprising: The total number of particles is determined by a device that indicates the value of optical density, which is a measure of the total number of particles present. The samples were analyzed for inoculum, desired fermentation products and important contaminants (co (ntaminant) in excess of the product selected from the group consisting of The number of particles consisting of the product bound to the labeled specific antibody is determined by The number of particles measured using the label used is compared with the total number of particles measured, and from this comparison, Characterized by indicating whether particles contaminating the fermentation are present in the fermentation broth How to do it. 2. measuring the total number of particles in the sample from the fermentation broth by an ultraviolet spectrophotometer; A method according to claim 1. 3. The label used is an enzymatic marker, and the product bound to the label-specific antibody The number of particles is determined by enzyme-linked immunosorbent assay (ELISA). ) The method according to claim 1 or 2, wherein the method is measured by: 4. The label used is a radioisotope and the sample is separated by chromatography. The amount of separated free labeled antibody or bound labeled antibody is expressed as a radioactivity value. consisting of the product bound to the labeled specific antibody, as measured by a device that 3. A method according to claim 1 or 2 for determining particle number. 5. The label used is a fluorescent substance, and fluorescence microscopy, flow cytofluorimetry, etc. Attach to labeled specific antibodies using a cell sorter or fluorescence-activated cell sorter (FACS). According to claim 1 or 2, the number of particles consisting of the combined product is determined. the method of. 6. The label used is a fluorescent substance and is Continuously collect fermentation broth from the This sample stream is passed through a suitable first particle separator (2) as required. control particles with a fluorescent marker according to the first bomb (17). in addition to the sample stream from the reservoir (16); The sample flow is directed by the second bomb (3), either before (4) or after the second bomb (3). (5) through the first mixing coil (4, 5) disposed in The sample flow is then a measure of the total number of particles present in the sample flow and added to the sample flow. The optical density value used to determine the amount of diluent, detergent or chemical passing through the first device (6) shown; A determined amount of diluent, detergent or chemical is delivered to the second tank by a third bomb (8). The sample stream is added to the sample stream from the reservoir (7) of the particle separator (18). ) as necessary, The sample flow is measured by a second, which indicates the value of optical density, which is a measure of the total number of particles present in the sample flow. passing through the device (19) as necessary, Select from the group consisting of inoculum, desired fermentation product and important contaminants as required. Particles with specific antibodies that bind to the selected product are placed in a fourth bomb (21 ) to the sample stream from the third reservoir (20) and optionally transfer the sample stream to the third reservoir (20). passing through the mixing coil (22) of a product selected from the group consisting of an inoculum, a desired fermentation product and a significant contaminant; The excess labeled specific antibody that binds to the substance is transferred to the fourth bomb (11) by the fifth bomb (11). After adding the sample stream from the reservoir (10), the sample stream is passed through a fourth mixing and delay controller. (12), and the sample flow is stopped when the pressure exceeds a preset value. A fourth bottle holds the monitoring line at constant pressure by means of a valve (14) which vents a portion of the flow. (13), and the sample stream discharged from the fourth bomb (13) is Fluorescence-activated cell that sorts particles present in a sample stream by intensity and relative fluorescence By passing through a sorter (FACS) (15), Continuous monitoring of sample flow from fermentation broth by FACS (15), claims The method according to item 1 or 2. 7. According to claim 6, the monitoring line is controlled by a computer. the method of. 8. selected from the group consisting of inoculum, desired fermentation product and significant contaminants. Method according to claim 6 or 7, in which several products are monitored simultaneously. . 9. Fermentation buoy according to any one of the preceding claims, for controlling the state of fermentation The use of fermentation monitoring methods during the process. 10. Claims 6, 7 or 7, wherein the fermentation conditions are continuously controlled. Use of the method for monitoring fermentation in a fermentation broth according to any one of clauses 8.