Developing A Chemical Effectively Harsh Against C. Botulinum But Inert On Other Substances
Background
Food contamination refers to the presence of pathogens in food, that when ingested causes foodborne disease such as botulism. Botulism is an acute neurologic disorder that causes potentially life-threatening neuroparalytic. This disease is caused by ingestion of a neurotoxin produced by Clostridium botulinum bacterium. C. botulinum is a gram-positive rod-shaped anaerobic bacterium that can produce endospores. This bacterium is motile and can able to produce several different types of neurotoxins that when ingested causes illnesses of fatal diseases. Neurotoxins are poisonous chemicals that affect the central nervous system and thus cause paralysis which results in respiratory failure and might lead to death. Clostridium botulinum is commonly found in soil worldwide and feeds on decaying organic matter, this bacterium is not toxic when ingested and it is commonly consumed in fruits, seafood, and vegetables.
However, when C. botulinum reproduces and grows under special conditions such as low or absence of oxygen and low pH (pH < 5. 4, acidic), it produces neurotoxins. These conditions in which the production of neurotoxins is favored are usually found in many canned foods, hence people consuming canned food are more likely to suffer from botulism. The conditions in which C. botulinum grow best inhibits the growth of many bacteria thus preventing food spoilage. Bacteria grow best in pH that is near neutrality or more alkaline and many of them grow best irrespective of the oxygen content thus course food spoilage.
So in either case, spoilage of contamination of food will always occur. Many food companies are focussing on making a profit thus they make food products that have extended shelf life, hence paying more attention to factors that inhibit food spoilage ignoring factors that favour food contamination. It is difficult to treat or kill C. botulinum in food because it consists of endospores that are resistant to heat. Fortunately, extreme levels of heat may disrupt and act against these endospores. The problem is that high levels of heat damage the constituents of food (proteins, vitamins, trace elements, etc. ), therefore heat is not really a good sterilization method for microorganisms with endospores. Other sterilization methods like radiation cause chemical reactions that alter the food and thus alter the chemical makeup of food, its nutritional content and sensory qualities. In addition, there is botulism antitoxin bivalent also known as equine. This antitoxin is used to treat botulism, however, the treatment has unwanted side effects that include: nausea; rash; a headache; irregular heartbeat; inflammation of the joints; swollen lymph glands; unconsciousness and etc. Due to these problems, it is clear that there is no best prevention nor cure for botulisms. Fortunately, this proposal aims to produce a C. botulinum antitoxin addictive that is not just active against the neurotoxins produced by C. botulinum but also active against the cell and the endospores as well and still remain inert to other substances especially food composition.
The Clostridium botulinum antitoxin addictive does not have any effect on the food or its constituents, it has been tested to be 99. 9% inert to the food content and only destructive against C. botulinum strains.
Method
Developing antitoxin addictive that will be added during food preparation to fight off botulism, the additive is safe for adults and infants as it is inert to food. As mentioned before, this is not a cure for botulism poisoning since there is a treatment or cure for it already. This is a preventative method of dealing with Clostridium botulinum as it starts to grow on foods (mostly canned foods). The antitoxin will be mixed with the usual additives that are used in the prolonging of canned foods. The antitoxins function will be to destroy or eliminate Clostridium botulinum toxin in its early stages of developing. An Antitoxin is a type of antibody that gets rid of toxins by micro-organisms. Since this is a toxin that we want to get rid of, an antitoxin which will be in form of powder will be produced and target specifically target C. clostridium in food during food preparation as it will be added as addictive. This antitoxin addictive will be able to combat all seven types of botulinum toxin types (From A-G).
For experimental purposes, the production and experimental test of the antitoxin addictive will be done on mice then once the experiment succeeds, the antitoxin addictive powder can be used during food preparation and then the food can be tested to be relatively C. clostridium antitoxin free. For antitoxin production, the toxin needs to be obtained from its source, C. botulinum which is found in the soil. C. botulinum produces neurotoxin only when it has form endospores which are found everywhere and can survive harsh environmental conditions. Methods used to produce C. botulinum antitoxin addictive:
Apparatus to be used
Distilled water; clean test tubes containing peptone water; vortexes; Bunsen burner; CIB agar; Inoculating loop; incubator; Gram stain reagents; microscope slides; microscope; emersion oil; timer; biochemical test reagents; mice; myeloma cells; sterile syringe, all this apparatus will be obtained from CPUT laboratory and soil in which Botulinum will be obtained from intensive deserted area.
The process of isolating C. botulinum strains from the soil
- 100mL of distilled water will be transferred into a sterile beaker.
- 0. 8 gram of the soil sample will be weighed out and transferred into the beaker containing distilled water and vortex.
- A serial dilution will be carried out by transferring 1mL of the liquid homogenate sample (basically the homologous mixture of the soil and distilled water) into a tube labeled 10-1 that contains 9 mL of 0,1% peptone water, using a 1000 µL Gilson micropipette. The 10-1 labeled tube will be vortexes and 1 ml (1000 µL) will then be transferred into the next tube (labeled 10-2) containing 9 mL of 0,1% peptone water. The dilution series will be carried out by diluting 1 mL of the previous diluting tube (and vortex ting the previous tubes) into the next. This dilution series will be conducted in 6 tubes labeled according to the dilutions conducted, ranging from 10-1 to 10-2.
- Then the tubes will be incubated at 37 for 24 hours. After the incubation period, the 10-6 sample will be aseptically plated in a CBI plate will is selective and only promote C. botulinum growth
- The CBl plate will be incubated for a further 24 hours in 37. After the incubation period, the colonies that will grow on the plate will be identified using gram staining techniques to ensure pure culture.
- When pure culture is obtained, the biochemical test will be carried out to identify the c. botulinum trains. The molecular test may be carried out as well if the financial budget is not tight but it is not necessary.
Production of antibodies using immunology techniques
- Once the c. botulinum strains are obtained, it is more likely that they are concentrated with the toxin, hence the spores were exposed to harsh condition during isolation.
- Then the C. botulinum toxins will be attenuated, by exposing them to gamma rays then the attenuated toxins will be injected into mouse, the mouse immune system will then be activated (immunized) due to the presence of these antigen (C. botulinum toxin) the immune system will produce a group of B-lymphocytes which differentiate into plasma and memory cells.
- Spleen (blood) from the mouse will be isolated from the immunized mouse and transfer it into clean test tubes. Since the serum contains different plasma cells, myeloma cells will be added to the serum and thus fuse with the plasma cells and form hybridomas. Myeloma cells are cancerous cells, they lost their ability to stop multiplying, and hence they will produce many copies of the plasma cells. Since the serum is heterogeneous, it will be centrifuged to isolate the plasma cells active against C. botulinum strains.
- Once a homogenous mixture is obtained, the plasma cells meanwhile produce antibodies against the C. botulinum strains.
- The product will then be recovered as it contains some impurities (i. e. proteins, amino acid, nucleic acids, blood platelets, etc) so the antibodies solution will be purified by affinity chromatography. Affinity Chromatography to Purify the recovered product
- The solution will be injected into affinity chromatography, and based on affinity chromatography, the components of the solution will interact with the stationary phase composed of ligands. A wash buffer that is inert to antibodies will be added to the column to get rid of all the unwanted impurities. Then a second wash buffer will be added to break the interaction between the ligand and the stationary phase thus allowing antibodies to be eluted out of the column.
- Then the product will be crystallized by addition of HCl and dried into a powder.
- The antitoxin powder will then be packaged for commercial purposes.
- How much will the overall production (facility and equipment) of the Clostridium botulinum antitoxin cost and what would be the actual price of the product?
- Will the Clostridium botulinum antitoxin be appropriate for use by all patients?
- Will the product be ingested orally, or will it be injected into the patients?
- Should the antitoxin be ingested, won’t it cause or lead to any health complications?
- What are the control measures to be implemented should the antitoxin not yield results as expected or should there be a need for it to be discontinued/difficulties to be expected
- What could be the expected production challenges?
- Why is the pharmacist the best professional to fill this need? Consider the number of pharmacists needed to provide the antitoxin to aid in its availability and if there are any other major costs which will be added
- What will be the financial impacts on patients, society?
Downstream processing recovering the product
Research Questions
Delineation
The team will only be focusing on producing C. botulism antitoxin additive that will be effectively harsh against C. botulinum but inert on other substances, especially on foodstuff. The focus is on the C. botulinum antitoxin additive production that can be added to food whenever food is prepared. This additive will destroy all the C. botulinum strains that might be present in food and it can be added to any type of food as it is tasteless and inert to the food constituents. This experiment only includes the production of the additive and nothing other than this will be discussed.
Expected Outcomes
The significance of this research is to find possible ways of fighting off food-borne diseases that are due to food contamination, and further prevent food contamination where possible. The Clostridium botulinum antitoxin will kill C. botulinum bacteria, whilst being inert on food and humans, thus eliminating the risk of food contamination and later food-borne diseases. Food will be safely consumed and the rate of deaths due to food-borne diseases will drastically decrease in Africa. The preparation process of this antitoxin will need several technicians to work with, thus creating job opportunities for bioprocess engineers to showcase their skills after they have graduated. Several employment opportunities will be opened, for skills to be sharpened and new products produced for the continents’ sake.
Conclusion
The production of C. botulinum antitoxin additives will provide safety in food consumption, leading to very low risks of food-borne diseases in humans and thus reasonably minimize death rates due to food contamination.
Summary
The microorganism C. botulinum is a bacterium that is normally found in the soil. The presence of this microorganism in food leads to food poisoning illnesses, known as botulism. The nutrients in food support microbial growth which causes the food to be susceptible to bacterial spoilage and contamination. As the C. botulinum grow best in canned foodstuffs, it produces neurotoxins that cause potentially life-threatening neuroparalysis. However, it is difficult to get rid of this micro-organism in food because it produces endospores that require extreme conditions to destroy but these extreme conditions alter the texture of the food. This research proposal is to develop a C. botulinum antitoxin additive that will kill this microbe instantly, but still be inert in substances like foodstuffs. The production process of these antitoxins will be held at CPUT laboratory; where a specific procedure will be carefully followed, taking into consideration all possible challenges and limitations. The final product will assist in preventing botulism that would be caused by the contamination of clostridium botulinum in food. Food will be safely consumed, and the risk of humans being infected with botulism will drastically decrease, thus leading to minimal death rates due to food contamination in Africa.
