1. What are the possible ways to cause fermentation?
Improper operation of the culture process of the culture medium is not completely sterilized. The fermentation equipment (such as fermentation tanks and pipelines) is not tightly sealed. The air sterilization is not clean.
2. Since the bacteria are inevitable, what should we do?
It is necessary to improve the level of production technology, strengthen the management of the production process, grasp the links of various susceptible bacteria, and prevent the occurrence of fermentation bacteria as much as possible; and once the bacteria are infected, it is necessary to find out the cause of the pollution as soon as possible and take corresponding measures. Effective measures to reduce the damage caused by fermentation bacteria to small Zui.
3. What are the different types of bacteria in the different fermentation processes?
Fermentation of penicillin Fermentation of streptomycin from A. faecalis fermentation of Bacillus brevis, Pseudomonas tetracycline Diplococcus, Bacillus, bacillus glutamic acid fermentation Phage citric acid fermentation Penicillium
4. The effect of dyeing bacteria on fermentation in different production stages:
In the seed culture period, the bacteria are harmed to the whole fermentation process. The bacteria in the early stage of fermentation: seriously interfere with the growth and reproduction of the production bacteria. In the middle of the fermentation, the bacteria are interfered with: the metabolism of the production bacteria is affected, and the production of the product is affected.
5. The effect of different bacterial causes on fermentation:
Seed-borne bacteria: will lead to the continuous expansion of the range of bacteria, resulting in significant losses in production.
Air-borne bacteria: the fermentation of a large area of ​​bacteria culture medium or equipment is not thorough: generally does not have continuity, so that single (batch) fermenter fermentation failure equipment leakage: a greater chance of infection
6. Analysis of abnormal fermentation phenomenon and cause of infection:
(1) Abnormal changes in dissolved oxygen When the bacteria are aerobic microorganisms, the change in dissolved oxygen falls in a short period of time until it is close to zero, and cannot rise for a long time;
When the bacteria are non-aerobic microorganisms, the production bacteria inhibit growth due to contamination, which reduces oxygen consumption and increases dissolved oxygen.
(2) Abnormal changes in CO2 emissions. When contaminated with bacteria, sugar consumption is accelerated, CO2 content is increased, and after phage contamination, sugar consumption is slowed down and CO2 content is reduced. Therefore, the bacteria can be judged based on the abnormal change in the CO2 content.
(3) Other anomalies:
Such as poor growth of bacteria, abnormal changes in pH, abnormal increase in foam during fermentation, abnormal color change of fermentation broth, abnormal drop in metabolite content, abnormal prolongation of fermentation cycle, abnormal increase in viscosity of fermentation broth, etc. bacteria.
7. Analysis of the causes of infection:
(1) Analysis of the cause of infection from a large number of fermenting bacteria: a large number of fermentation tanks in the fermenter: air system part of the fermenter (or tank group) bacteria: the early stage may be contaminated with seeds, or sterilization is not complete, in the middle and late may It is the intermediate feeding system or the continuous fermentation of individual fermenters caused by problems in the piping system: equipment problems (such as valve leakage or tank corrosion and wear), the bacteria caused by corrosion and wear of the equipment will appear in each batch. The phenomenon of fermentation time of fermentation is moving forward. Individual fermenters accidentally inoculate bacteria: the reason is more complicated, because various bacterial infection pathways may cause.
(2) Analyze the early stage of fermentation from the time of bacterial infection: seed bacteria, medium and equipment are not completely sterilized, equipment or pipelines have dead angles, late stage dyeing: intermediate feeding, equipment leakage, unreasonable operation ( 3) Analysis from the type of bacteria:
Heat-resistant Bacillus: dead-horn or sterilized incomplete cocci, yeast: may be light green colonies brought from steam condensate or air (Gram-negative bacilli): cooling tube or jacket leak of fermenter Mold: Incomplete sterilization or aseptic operation is not strict (4) Reasons for leakage of equipment: When equipment leaks, the time of occurrence of each batch of bacteria is gradually advanced. Corrosion of corrosive substances in the fermentation liquid c equipment processing failure (5) coil leakage prevention:
After the tank is placed, the fermenter should be carefully cleaned to control the quality of the cooling water, reduce the chloride ion content, periodically check and test the coil, and find the leak test method in time: pressure test, water pressure test, air distribution tube leakage, prevention of tank seepage Leakage prevents the connection of the leaking pipe of the pipe:
Threaded connection Flange connection Welding (6) Air filtration system reasons:
Reason analysis: air inlet, production environment, air filter. Characteristics of bacteria: The bacteria in the air filtration system will cause the fermentation tank to be dyed in the batch and the tank can be infected. At this time, the air filtration system should be tested aseptically.
8. Inspection and judgment of infected bacteria 1 Aseptic test method 2 Microscopic examination (microscopic examination)
3 broth culture method 4 plate streak culture or slant culture test abnormal phenomenon of fermentation process observation method If the broth has three times of color change reaction (from red to yellow) or turbidity, or plate culture for three consecutive times found abnormal colonies The appearance of it can be judged as bacterial infection.
9. Rescue and treatment of contaminated bacteria:
(1) Inoculation of bacteria in the seed culture period: it should be discarded after sterilization, and the seed tanks, pipelines, etc. should be carefully inspected and thoroughly sterilized. (2) In the early stage of fermentation, the bacteria are consumed: nutrients are not consumed much, and should be quickly resterilized. In addition, supplement the necessary nutrients and re-inoculate for fermentation.
(3) In the middle and late stages of fermentation: appropriate bacteria or antibiotics and normal fermentation broth can be added to inhibit the growth rate of the bacteria; if the content of the product reaches a certain value, it can be put into the tank as long as it is clear .
(4) Treatment of equipment after fermentation: After being heated and sterilized in an empty tank, it can be used until it is 120 ° C or more and 30 minutes later. Can also be treated by formaldehyde fumigation or soaking in formaldehyde solution for more than 12h.
10. Ways and prevention of contamination of bacteria:
(1) Seed carrier and its control:
Cause analysis: Seed preservation Seed culture equipment Seed replacement and inoculation Seed culture medium Control measures:
Check the stored bacteria used for contamination.
Check the vessels such as the triangular bottles used for inoculation of contaminated seed cans.
Check the sterilization of the flask, cotton plug, medium, etc.
Check the working condition of the natural bacteria pot, check the temperature gauge and pressure gauge. The air in the pot must be drained when the sterilizer is working.
Check the sterility of the sterile room, especially the inoculation box. Check the bactericidal fumigant used in the inoculation box.
Check the operator's operating technique.
Microbiological testing of bacteria or phage.
(2) Airborne bacteria and their control:
Prevention:
Oil seeps into the air: use a non-oily compressor.
The tube cooler is perforated.
The filter media is loose and the air is shorted.
The filter media is wetted by water.
Filter media aging.
(3) Leakage or "dead angle" caused by equipment and its prevention and treatment:
Leakage of equipment mainly refers to fermenters, sugar-filling tanks, cooling coils, pipeline valves, etc., due to chemical corrosion, electrochemical corrosion, turbidity of the metal between the booth and the sediment in the raw materials, poor processing and other reasons to form a small leak Leakage and infection occurred after the hole.
"Dead angle" includes a part of the fermentation equipment or connecting pipe and a part of the medium or other materials.
Leakage and prevention of coils:
Analysis of the cause: Due to the temperature difference (internal cooling water temperature, external sterilization temperature), the temperature changes abruptly, or the pH of the fermentation liquid is low, and the chemical corrosion is serious.
Control measures: measures such as careful cleaning, inspection of leakage, or reduction of Cl-1 content in cooling water can be taken for prevention and control.
? "dead angle" of the air distribution tube and prevention:
Analysis of the causes: Abrasion and perforation affected by agitation and ventilation. Prevention and control measures: measures such as frequent replacement of air distribution pipes or careful washing. Leakage and control of fermenters:
Cause Analysis:
Local chemical corrosion or abrasion occurs, resulting in perforation leakage.
It is easy to accumulate dirt around the can welding site.
Poor production of fermenters.
Each interface on the fermenter head.
The repairing position of the fermenter was improperly welded.
Prevention:
It is a measure to effectively eliminate the bacteria by applying anti-corrosion paint on the inner wall of the tank, strengthening the cleaning and regularly removing dirt.
The use of stainless steel or composite steel can effectively overcome this drawback and pay attention to cleaning can avoid bacterial infection.
4 The installation of the pipeline or the configuration of the pipeline is unreasonable to form a "dead angle":
Cause Analysis:
Some lines are often pooled into one total line. Most of the pipes are flanged.
Prevention:
The use of separate exhaust, drainage and drain pipes can effectively prevent the occurrence of bacteria.
Flange processing, welding and installation should comply with the requirements of sterilization to minimize or eliminate the connection of flanges, etc. 5 leakage of pipe fittings to form bacteria and control:
(4) Infected bacteria caused by incomplete sterilization of raw materials:
*Cause Analysis:
Starch material is easy to agglomerate when the temperature rises too fast or the mixture is not uniform, so that the center of the mass is “sandwichedâ€, and the steam is not easy to enter. The content of solids such as bran and soybean cake is reduced in the medium of killing bacteria. Many of them are splashed on the tank wall or various brackets in the tank during feeding, and it is easy to form a pile. These deposits are not easy to be killed due to slow heat transfer during the sterilization process.
*Prevention:
Before heating, mix and mix evenly by stirring, and add a certain amount of amylase to liquefy; when large particles are present, they should be removed by sieve and then sterilized;
For the medium with a high content of solids such as bran and soybean cake, it is more effective to use the pre-dosage outside the tank and then transfer it to the fermenter for solid tank sterilization.
*specific measure:
The purchased raw materials should be dried and the storage materials should be kept dry.
When preparing each medium, the pulverized raw materials are first wetted with cold water, and the mixing tank is stirred to sufficiently prevent agglomeration.
The pulverized raw material granules should be small enough that large granules will affect the bactericidal effect.
If the production material is seriously contaminated by spores, the medium can be kept at 30 ° C for several hours, and the spores are germinated and then sterilized.
(5) Improper operation causes the infection and its prevention:
*Cause Analysis:
When the sterilization was unreasonable, the air in the tank was not completely eliminated, causing the pressure gauge to display "false pressure".
When the foaming of the foam is severe, the foam can rise to the top of the tank or even escape, so that the foam can be topped.
During continuous sterilization, the temperature at which the medium was sterilized and its residence time did not meet the requirements for sterilization.
Improper sterilization of the probe or component.
*Prevention:
Open the exhaust valve before sterilizing and drain the air.
Defoaming agent is added for defoaming treatment.
During continuous sterilization, avoid excessive fluctuations in steam pressure and strictly control the sterilization temperature.
The probe or component is guaranteed to be sterilized thoroughly.
(6) Phage pollution and its prevention and treatment:
Prevention:
Carefully preserve the production strains to ensure they are not contaminated.
It is strictly forbidden to discharge live cells.
Strengthen equipment management.
Strengthen environmental health work.
Strengthen the monitoring of sterile air, space bacteria and phage.
* The method used after infection of phage:
Breeding resistant strains.
Rotation uses strains with different specificities.
Add chemical drugs (such as glutamic acid fermentation can add 2 ~ 4ppm chloramphenicol, 0.1% sodium tripolyphosphate, 0.6% sodium citrate or ammonium).
Re-sterilize the culture solution and inoculate (the phage is not heat-resistant, 70~80?C can be killed in 5 minutes)
Other methods
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