Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors

Abstract

Aims: To further improve the soy‐based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale‐up the Clostridium tetani process into small fermentors. Methods and Results: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l‐tyrosine and l‐cysteine could be eliminated from our soy‐based medium without effect. Seven carbon sources were compared with glucose in the soy‐based medium, but none was found to be superior to glucose. The process was successfully scaled‐up into 250‐ml bottles, 1‐l bottles and 1‐l fermentors. Conclusions: Quite remarkably, when comparing the tetanus production process in our soy‐based medium with the traditional animal/dairy‐containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l‐tyrosine and l‐cysteine components could be eliminated, making the medium (Hy‐Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale‐up from test tubes into 1‐l fermentors allows us to predict that further scale‐up into large fermentors will be successful. Significance and Impact of the Study: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow’s disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable‐based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests

“…With our experience in developing a soy-based process for preparation of tetanus toxin (16)(17)(18)(19), we decided to address this need and attempt to develop a high-level production process for C. difficile toxin totally lacking animal and dairy products. The successful result is described in this paper.…”

Production of tetanus toxin by using media substantially free from meat and blood

“…Because TT is a highly purified and concentrated antigen that is administered to humans, there is a much greater awareness and concern with potential contaminates like bovine spongiform encephalopathy (BSE). In 2007, a paper by Fang et al outlined a soy based medium that was successfully used to manufacture TT [3]. Based on these studies, it is clear that clostridia strains can be readily grown in media free from animal products.…”

“…The composition of the fermentation medium used for tetanus toxin production was kept same as seed medium that is MMM medium [3]. In the present work, we have studied the yield and quality of toxin during scale-up of the process as well as the efficiency of MMM medium as compared to conventional meat-based medium for tetanus toxin production.…”

“…tetani (Harvard strain), is a Gram-positive, anaerobic bacillus, being employed to produce tetanus toxin. This toxin is further converted to toxoid using either formaldehyde or glutaraldehyde by destroying the activity of the peptide toxin due to the generation of interpeptide crosslinks [3]. Tetanus toxin is converted to TT by formaldehyde by losing its immunogenic property.…”

Fed‐batch production of tetanus toxin by Clostridium tetani

“…Among the most recent works involving the production of tetanus toxin by this microorganism, the use of gaseous nitrogen to strip gases formed during fermentation 4 as well as the study of alternative cultivation media is noteworthy, for example, based on soy. 5,6 In the cultivation of C. tetani, a rise in concentrations of glucose leads to higher cell concentration 7 as a result of higher energy and carbon availability; however, protein toxin synthesis may suffer catabolic repression, a fact that was already detected in C. difficilis by Osgood et al 8 It is also well known that, when glucose concentration decreases throughout batch cultivation, tetanus toxin is formed and released, 9 indicating that the effect of repression ceases. Thus, supplying the carbon source by fed-batch process could be of great significance to avoid catabolite repression by glucose and then to increase the biosynthesis of tetanus toxin during the idiophase.…”