PROTEASE (Streptomyces fradiae) Explanation This enzyme preparation has not been previously reviewed by the Joint FAO/WHO Expert Committee on Food Additives. Each production batch is commenced with a selected colony of S. fradiae (strain No. 2019 in the mycological collection of the Museum National d'Histoire Naturelle, Laboratoire de Cryptogamie, Paris). The incubation medium in the industrial fermenter in the final stage (approximately 60 hours) of mycelium production is (wt/vol%): soya flour, 1; corn starch, 3; corn steep liquour, 0.5; K2HPO4, 0.1; Fe2(SO4)3, 0.0015; MgSO4, 0.005; CaCl2, 0.05. The medium is filtered, concentrated and protein is salted out. Crude protease is redissolved and processed with further salting out to yield "pure" (i.e., concentrated) protease, with an activity of about 12 000 Anson Units/mg. The diluted (industrial) enzyme is prepared by mixing with lactose to give a final activity of 1000 Anson Units/mg. The industrial product is free of amylase, invertase and lipase activity. A solution of 100 mg/ml of the industrial product had no antibiotic activity against a battery of test organisms. BIOLOGICAL DATA TOXICOLOGICAL STUDIES Short-term studies Rat Wistar rats distributed in 3 groups each containing 12 males and 12 females were fed S. fradiae protease at levels of 0, 0.1 and 0.2% in the diet for 185 days. The treated diets contained 2 million and 4 million Anson Units of enzyme activity, respectively, per kilogram of foodstuff. There were no significant differences between groups in growth rate or food intake. One male control rat died with a pancreatic tumour. One female rat in the 0.2% test group had a gastric lesion that was revealed in routine post mortem examination at the end of the trial period. No other lesions were detected. There were no significant differences between groups in respect of weights of liver, kidneys, testes and pancreas except that, in females only in the group fed at the higher level (0.2%), the weights of the liver, kidneys and pancreas were significantly less than those in the control group, being about 90% of control; however, the ranges overlapped and the means of the test group were well within the range of the control group for each organ. Mean values for serum total nitrogen and serum urea were presented for females in all three groups and for males in the 2 test groups (but not the control group); serum urea was elevated to 13% above control in females in the high test group; in all other cases the values were below those for the female control group (Ferrando, 1971). Pig European and Landrace pigs of about 50 days of age were divided into male control, female control, male test and female test groups, each containing 10 pigs; littermates of each sex were divided between control and test groups. The feedstuff of test groups contained 200 000 Anson Units of protease activity per kilogram. The trial lasted 119 days. Feed intake, body weight increase and efficacy of conversion did not differ significantly between control and test groups. The pigs were slaughtered at the end of the trial and carcasses, organs and intestine from all the pigs were rated and classified as fit for sale by a veterinary surgeon; there were no significant differences between control and test groups on the ratings of quality (Istituto de Farmacologia, Española, 1978). Chicken Day-old chicks of Arbon Acres and Vantress types were fed with growing mixtures in flour or granule form for 56 days. Test feed-stuff contained 800 000 Anson Units of protease activity per kilogram. Observations were made on 200 chicks in the laboratory and 13 000 chicks under farm conditions, each divided into test and control groups. At the end of the trial period, mortality was less in the treated than in the control farm chicks. No adverse effects were observed (Istituto de Farmacologia, Española, 1968). Comments No adverse effects have been noted in short-term trials with S. fradiae protease incorporated in feedstuff at levels (Anson Units/kg) of 4 000 000 in rats, 200 000 in pigs, and 800 000 in chicks. However, observations were limited to macroscopic post mortem examination and serum urea and nitrogen assays in rats and to standard meat-inspection procedures in pigs and chicks. The level of use of the protease in, for instance, beer appears to be about 1000 Anson Units per litre. The Committee allocated a temporary ADI "not specified" pending the submission of the ongoing feeding studies, including histopathological observations, and information about the occurrence in nature of the microorganism from which the enzyme is obtained. EVALUATION Estimate of temporary acceptable daily intake for man Not specified.* FURTHER WORK OR INFORMATION Required by 1984 (1) Submission of the results of a feeding study, including histopathological observations. (2) Information about the occurrence of Streptomyces fradiae in nature. * The statement "ADI not specified" means that, on the basis of the available data (toxicological, biochemical, and other), the total daily intake of the substance, arising £rom its use or uses at the levels necessary to achieve the desired effect and from its acceptable background in food, does not, in the opinion of the Committee, represent a hazard to health. For this reason, and for the reasons stated in individual evaluations, the establishment of an acceptable daily intake (ADI) in mg/kg bw is not deemed necessary. REFERENCES Ferrando, R. (1971) Compte rendu des essais de recherche de toxicité à moyen terme de la protease "2000" lot or 405 B (2ème essai). Appendix D 1 in Appendices to Technical Information on Streptomyces fradiae protease manufactured by Compañia Española de la Penicilina y Antibioticos, S.A., Madrid, Spain. Technical Report No. 2, 10 May 1979. Unpublished report submitted to WHO Istituto de Farmacologia, Española (1968) Tests on chicks. Appendix D 3 in Appendices to Technical Information on Streptomyces fradiae protease manufactured by Compañia Española de la Penicilina Antibioticos, S.A., Madrid, Spain. Technical Report No. 2, 10 May 1979. Unpublished report submitted to WHO Istituto de Farmacologia, Española (1978) Estudio de Fradiase en crecimiento de ganado porcino en cria industrial. Appendix D 2 in Appendices to Technical Information on Streptomyces fradiae protease manufactured by Compañia Española de la Penicilina Antibioticos, S.A., Madrid, Spain. Technical Report No. 2, 10 May 1979. Unpublished report submitted to WHO
See Also: Toxicological Abbreviations