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