Fermentation technology: comprehensive analysis and prevention measures of contamination

What are the possible pathways that can cause fermentation contamination?
Improper operation during the cultivation process of bacterial strains
Incomplete sterilization of culture medium
Fermentation equipment (such as fermentation tanks and pipelines) is not tightly sealed
Unclean air sterilization
Since bacterial infection is inevitable, what should we do?
To improve the level of production technology, strengthen the management of the production process, grasp the various links that are prone to bacterial contamination, and prevent the occurrence of fermentation bacterial contamination as much as possible; And once bacterial contamination occurs, the cause of the contamination should be identified as soon as possible, and corresponding effective measures should be taken to minimize the losses caused by fermentation bacterial contamination.
What are the most harmful types of miscellaneous bacteria in different fermentation processes?
Fermentation of Penicillin by Bacillus subtilis
Fermentation of Streptomycin by Bacillus subtilis and Pseudomonas aeruginosa
Fermentation of tetracycline by Streptococcus, Bacillus, and Clostridium perfringens
Fermentation bacteriophage of glutamic acid
Fermentation of citric acid by Penicillium
4. The impact of bacterial contamination on fermentation at different production stages:
Bacterial contamination during seed cultivation period: extremely harmful to the entire fermentation process
Pre fermentation bacterial contamination: severely interfering with the growth and reproduction of production bacteria
Mid fermentation bacterial contamination: interferes with the metabolism of production bacteria and affects the generation of products
Late stage bacterial infection during fermentation: relatively minor impact
5. The impact of different bacterial infection reasons on fermentation:
Seed contamination: It will lead to the continuous expansion of the contaminated area, causing significant losses to production.
Air borne bacteria: causing extensive bacterial contamination during fermentation
Incomplete sterilization of culture medium or equipment: generally lacks continuity, resulting in fermentation failure of individual (batch) fermentation tanks
Equipment leakage: There is a high probability of bacterial contamination
6. Analysis of fermentation abnormalities and bacterial contamination reasons:
(1) Abnormal changes in dissolved oxygen
When the mixed bacteria are aerobic microorganisms, the change in dissolved oxygen decreases in a short period of time until it approaches zero, and
Unable to recover for a long time;
When the mixed bacteria are non aerobic microorganisms, and the production bacteria inhibit their growth due to contamination, resulting in a decrease in oxygen consumption and dissolved oxygen
Raise.
(2) Abnormal changes in CO2 emissions from exhaust
When contaminated with miscellaneous bacteria, sugar consumption accelerates and CO2 content increases. After bacteriophage contamination, sugar consumption slows down and CO2 content decreases. Therefore, bacterial contamination can be determined based on abnormal changes in CO2 content.
(3) Other abnormal phenomena:
Such as poor growth of bacteria, abnormal change of pH value, abnormal increase of foam during fermentation, abnormal change of color of fermentation liquid, abnormal decline of metabolite content, abnormal prolongation of fermentation cycle, abnormal increase of viscosity of fermentation liquid, etc.
7. Analysis of bacterial infection reasons:
(1) Analyzing the reasons for bacterial infection based on the scale of bacterial infection
Large scale fermentation tanks contaminated with bacteria: air system
Partial fermentation tanks (or tank groups) may be contaminated with bacteria: in the early stage, it may be due to seed contamination or incomplete sterilization, while in the middle and later stages, it may be caused by problems with the intermediate feeding system or oil pipeline system
Individual fermentation tanks are continuously contaminated with bacteria: equipment problems (such as valve leakage or tank corrosion and wear) may cause the contamination time of each batch of fermentation to shift forward due to equipment corrosion and wear
Individual fermentation tanks may accidentally become infected with bacteria: the reasons are quite complex, as various infection pathways can cause it.
(2) Analyze from the time of bacterial infection
Early infection during fermentation: seed contamination, incomplete sterilization of culture medium and equipment, dead corners in equipment or pipelines
Mid to late stage bacterial infection: intermediate feeding, equipment leakage, unreasonable operation
(3) Analyze from the type of bacteria:
Heat resistant Bacillus subtilis: dead corners or incomplete sterilization
Cocci, yeast: may be brought from condensed water or air of steam
Light green colony (Gram negative bacteria): Leakage in the cooling pipe or jacket of the fermentation tank
Mold: Incomplete sterilization or lax aseptic operation
Reasons for equipment leakage: When equipment leaks, the occurrence time of each batch of bacteria is often gradually advanced
Corrosion of equipment by corrosive substances in fermentation broth
B abrasion
Poor processing of C equipment
Prevention of coil leakage:
After placing the fermentation tank, it is necessary to carefully clean it
Control the quality of cooling water and reduce the chloride ion content in it
Regularly inspect and test the coil for leaks, and promptly detect any gaps
Leakage testing method: air pressure test, water pressure test
Prevention of Leakage in Air Distribution Pipes
④ Prevention of tank leakage
⑤ Leakage of pipe fittings
(4) Pipeline connection:
Threaded connection flange connection welding
(5) Reasons for air filtration system:
Cause analysis: Air inlet, production environment, air filter
Characteristics of bacterial contamination: The presence of bacteria in the air filtration system can cause batch by batch contamination of fermentation tanks and tanks. In this case, sterile sample testing of the air filtration system is necessary
8. Inspection and judgment of bacterial contamination
Aseptic test method
Microscopic examination method
Broth culture method
Plate streaking culture or inclined plane culture inspection method
④ Observation method for abnormal phenomena in fermentation process
If the broth changes color (from red to yellow) or becomes cloudy three times in a row, or if abnormal bacterial colonies are found in plate culture three times in a row, it can be judged as bacterial infection.
9. Rescue and treatment of bacterial contamination:
(1) Seed cultivation period bacterial contamination: should be sterilized and discarded, and seed tanks, pipelines, etc. should be carefully inspected and thoroughly sterilized
(2) Pre fermentation bacterial infection: The consumption of nutrients is not high, and it should be quickly re sterilized; Additionally, supplement necessary nutrients and re inoculate for fermentation.
(3) Infection during the middle and late stages of fermentation: Appropriate fungicides or antibiotics can be added, as well as normal fermentation broth, to inhibit the growth rate of miscellaneous bacteria; If the content of the product reaches a certain value, as long as it is clearly contaminated with bacteria, it can also be placed in a can.
(4) Equipment treatment after fermentation: The empty tank should be heated and sterilized to above 120 ℃ for 30 minutes before use. It can also be treated by methods such as formaldehyde fumigation or soaking in formaldehyde solution for more than 12 hours
10. Pathways and prevention of bacterial contamination:
(1) Seed borne bacteria and their prevention and control:
Reason analysis: Seed preservation, seed cultivation equipment, seed replacement, and inoculation of seed culture medium
Preventive measures:
Check the preserved bacterial strains used for contamination.
Check the triangular bottles and other containers used for inoculating contaminated seed jars.
Check the sterilization operations performed on triangular bottles, cotton plugs, culture media, etc.
Check the working condition of the mushroom pot and verify the temperature and pressure gauges. When the sterilization pot is working, the air inside the pot must be completely drained.
Check the aseptic condition of the sterile room, especially the inoculation box. Check the disinfectant used in the vaccination box.
Check the operational skills of the operators.
Perform microbiological testing on miscellaneous bacteria or bacteriophages.
(2) Airborne carriers and their prevention and control:
Preventive measures:
Oil seeps into the air: Use a compressor without oil lubrication instead.
Perforation of tubular cooler.
The filter medium is loose and the air is short circuited.
The filtering medium is wetted by water.
Filter media aging
(3) Bacterial contamination caused by equipment leakage or "dead corners" and its prevention and control:
Equipment leakage mainly refers to the formation of tiny holes in fermentation tanks, sugar replenishment tanks, cooling coils, pipeline valves, etc. due to chemical corrosion, electrochemical corrosion, abrasion between the metal at the spreading mouth and the sediment in the raw materials, poor processing and manufacturing, and other reasons, resulting in leakage and bacterial contamination.
'Dead corners' include a certain part of fermentation equipment or connecting pipelines, as well as a certain part of culture medium or other materials.
Leakage and bacterial infection of coil and prevention:
Cause analysis: Due to temperature differences (internal cooling water temperature, external sterilization temperature), rapid temperature changes, low pH of fermentation broth, severe chemical corrosion, and other reasons.
Preventive measures: Careful cleaning, leakage inspection, or reducing the content of Cl-1 in cooling water can be taken in production to prevent and control it.
Infection and prevention of bacteria in the "dead corners" of air distribution pipes:
Cause analysis: Due to the influence of mixing and ventilation, it is prone to abrasion and perforation
Preventive measures: Take measures such as frequent replacement of air distribution pipes or careful washing
Leakage and infection of fermentation tanks and prevention measures:
Cause analysis:
Localized chemical corrosion or abrasion occurs, resulting in perforation and leakage.
It is easy for dirt to accumulate around the welding joints of the tank.
Poor production of fermentation tanks.
Various interfaces on the head of the fermentation tank.
The repair welding position of the fermentation tank is improper.
Preventive measures:
Applying anti-corrosion coating to the inner wall of the tank, strengthening cleaning, and regularly removing dirt are effective measures to eliminate bacterial infections.
The use of stainless steel or composite steel can effectively overcome this drawback and paying attention to cleaning can avoid bacterial contamination.
④ Unreasonable installation or configuration of pipelines creates "dead corners":
Cause analysis:
Often gather some pipelines into one overall pipeline. Most pipelines are connected by flanges.
Preventive measures:
The use of separate exhaust, drainage, and sewage pipes can effectively prevent the occurrence of bacterial infections.
The processing, welding, and installation of flanges should comply with sterilization requirements and minimize or eliminate measures such as connecting flanges as much as possible
⑤ Bacterial infection and prevention caused by leakage of pipe fittings:
(4) Bacterial contamination caused by incomplete sterilization of production materials:
*Cause analysis:
Starch based raw materials are prone to clumping when heated too quickly or mixed unevenly, causing the center of the clumps to become "sandwiched" and making it difficult for steam to enter and kill bacteria
The solid content in the culture medium, such as bran and soybean cake, is relatively high. When feeding, it splashes onto the tank wall or various supports inside the tank, which can easily form a pile up. Due to slow heat transfer during the sterilization process, some miscellaneous bacteria are also difficult to kill.
*Preventive measures:
Before heating up, stir and mix evenly, and add a certain amount of amylase for liquefaction; When there are large particles present, they should be screened and removed before sterilization;
For culture media with high solid content such as bran and soybean cake, pre mixing ingredients outside the tank and then transferring them to the fermentation tank for sterilization is more effective.
*Specific measures:
The purchased raw materials should be dry, and the stored raw materials should be kept dry.
When preparing various culture media, the crushed raw materials should be soaked in cold water first, and the mixing tank should be thoroughly stirred to prevent clumping.
The crushed raw material particles should be small enough, as large particles can affect the sterilization effect.
④ If the production raw materials are severely contaminated by spores, after making the culture medium, they can be kept at 30 ℃ for several hours until the spores are ready
Sterilize after germination.
(5) Improper operation causing bacterial infection and its prevention and control:
*Cause analysis:
During sterilization, due to improper operation, the air inside the tank was not completely expelled, resulting in a "false pressure" displayed on the pressure gauge.
When foam is produced and serious foaming occurs, foam can rise to the tank top or even escape, so that foam can top the tank.
During the continuous sterilization process, the sterilization temperature and residence time of the culture medium did not meet the sterilization requirements.
④ Improper sterilization method for probes or components.
*Preventive measures:
Open the exhaust valve and exhaust all cold air before sterilization and heating up.
Add defoamer for defoaming treatment.
During the continuous sterilization process, avoid excessive fluctuations in steam pressure and strictly control the sterilization temperature.
The probe or component ensures thorough sterilization.
(6) Phage contamination and its prevention and control:
Preventive measures:
Carefully preserve the production strains to ensure they are not contaminated.
It is strictly prohibited to discharge live bacterial cells.
Strengthen equipment management.
Strengthen environmental sanitation work.
Strengthen the monitoring of sterile air, airborne bacteria, and bacteriophages.
*The methods used after infection with bacteriophages:
Breeding resistant strains.
Rotate the use of strains with different specificities.
Add chemical drugs (such as 2-4 ppm chloramphenicol, 0.1% sodium tripolyphosphate, and 0.6% lemon for glutamic acid fermentation)
Sodium or ammonium salts, etc.
④ Re sterilize the culture medium before inoculation (bacteriophages are not heat-resistant and can be killed within 5 minutes at 70-80 ° C)