ALPHA-AMYLASE FROM BACILLUS SUBTILIS
1. EXPLANATION
Enzymes used for the hydrolysis of starch, generally called
amylases, have a long history of use by the food industry. The
amylase catalyzes the hydrolysis of 1,4 alpha-glucosidic linkages in
common polysaccharide. Bacterial (Bacillus subtilis) alpha-
amylase has been in common use to control the viscosity of chocolate
syrup since 1929 and in the brewing industry since 1936. The enzyme
preparation derived from these various Bacillus strains is usually
added directly to the food to be processed and then removed from the
final product by filtration. This preparation, BAN - Bacterial
Amylase Nova, an alpha-amylase produced by submerged fermentation of
a selected strain of Bacillus subtilis Strain F (ATCC 23350 DSM
7), has not been previously evaluated by the Joint FAO/WHO Expert
Committee on Food Additives. Not evaluated in this document but
previously reviewed by the Committee (Appendix 1 ref 27) is mixed
microbial carbohydrase and protease from Bacillus subtilis.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No information available.
2.2 Toxicological studies
2.2.1 Acute Toxicity studies
2.2.1.1 Rat
Groups of 10 male and 10 female rats (Wistar strain) were given
by oral gavage a salt-free batch (PPY 1316) of BAN (aqueous
suspension) at either 0, 4 or 10 g/kg b.w. None of the rats died or
showed any signs of toxicity. The author concluded that the LD50
of this preparation was above 10 g/kg b.w. (Aarup, 1983).
2.2.2 Short term studies
2.2.2.1 Rat
Groups of 10 male and 10 female rats (Wistar strain) were
exposed to a salt-free batch (PPY 1316, enzyme activity 4780 KNU/g)
at dietary levels of 0, 1.5, and 10% for 14 days. The rats were
observed at least once daily for any signs of toxicity, food and
water consumption was recorded weekly, body weights were recorded
prior to dosing and on days 1, 8, and 15, and blood collected at
termination. The animals were sacrificed after 15 days and
examined. There was increased water intake in high dose males,
slightly decreased food utilization and elevated serum urea nitrogen
concentrations in treated males, a decrease in serum urea nitrogen
concentrations in treated females, and decreased liver weights in
the high dose males and females. The author concluded that these
changes were not clinically important and that the NOEL was above
10% in the diet (9.87 g/kg b.w./day) (Stavnsbjerg, 1984).
Groups of 10 male and 10 female rats (Sprague-Dawley strain)
were exposed to a salt-free batch (PPY 1316, enzyme activity 4780
KNU/g) at dietary levels of 0, 0.5, 1.5, and 5.0% for 4 weeks. The
rats were observed at least once daily for any signs of toxicity,
food consumption and body weights recorded weekly, blood was
collected for haematology and clinical chemistry and urinalysis was
performed during week 4. The animals were sacrificed after 4 weeks,
autopsied, and histopathological examination of the organs
performed. In animals of both sexes receiving the high dose there
was a reduction in food consumption and a slight reduction in body
weight gain. Females showed mild dose-related increases in liver
weights but no differences were found in gross or histopathology of
any organ. The authors concluded that these changes were not
treatment-related and that the NOEL was above 5% in the diet (4.0
g/kg b.w./day) (Everett & Perry, 1983).
2.2.3 Special study on acute toxicity of beta-glucanase
and alpha-amylase
2.2.3.1 Rat
Groups of 6 male and 6 female rats (Wistar strain) were given
by oral gavage test compound DB 150, which contained beta glucanase
(200 BGU/g) as well as alpha-amylase (130 KNU/g) at either 0, 750 or
1500 KNU/kg b.w./day divided in two parts, or 2250 KNU/kg bw/day,
divided in three parts for 30 days. The rats were observed at least
once daily for any signs of toxicity, food consumption and body
weights recorded twice weekly, water consumption was recorded
weekly, and blood collected for haematology and clinical chemistry
and urine collected for urinalysis. Two animals died due to
aspiration of the test substance; no other rats died. There were
signs of gastric irritation and a slight increase in the liver,
kidneys and spleen weights relative to body weight in female rats.
The author concluded that the maximal tolerated dose was 2250 KNU/kg
bw/day (Modeweg-Hansen, 1983).
3. COMMENTS
Although the Committee had available only acute, 2-week and 4-
week rat studies in which levels up to 10% in the diet elicited no
adverse effects, the Committee was able to draw upon the previous
evaluation of mixed microbial carbohydrase and protease from B.
subtilis (strain not identified) (Annex I, ref. 27).
4. EVALUATION
In accordance with the earlier findings, the Committee
allocated an ADI "not specified" to this preparation.
5. REFERENCES
AARUP, V. (1983). Acute toxicity of BAN tox-batch PPY 1316 given
once orally to rats. Unpublished report No. 832329 from Inveresk
Research International, Musselburgh, Scotland. Submitted to WHO by
Novo Industri A/S Novo Alle, Bagsvaerd, Denmark.
EVERETT, D.J. & PERRY, C.J. (1983). Bacterial amylase NOVO (BAN): 4
week toxicity study in rats (oral administration by diet).
Unpublished report No. 430289 from Inveresk Research International,
Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S Novo
Alle, Bagsvaerd, Denmark.
MODEWEG-HANSEN, L. (1983). Oral toxicity of Bacillus subtilis
carbohydrase given daily to rats for 30 days. Unpublished report
from Inveresk Research International, Musselburgh, Scotland.
Submitted to WHO by Novo Industri A/S Novo Alle, Bagsvaerd, Denmark.
STAVNSBJERG, M. (1984). Dose range finding study in rats (by
dietary administration for 14 days). Unpublished report No. Ph-
840621 from Inveresk Research International, Musselburgh, Scotland.
Submitted to WHO by Novo Industri A/S Novo Alle, Bagsvaerd, Denmark.
See Also:
Toxicological Abbreviations
alpha-AMYLASE FROM BACILLUS SUBTILIS (JECFA Evaluation)