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    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)