146

Journal of Food Protection, Vol. 49, No. 2, Pages 146-153 (February 1986)
Copyright10 Internationa! Association of Milk, Food, and Environmental Sanitarians

Toxicological Safety Evaluation of a

Bacillus acidopullulyticus Pullulanase
M. STAVNSBJERG1*, R. K. HJORTKJAER 1 , V. BILLE-HANSEN1, B. F. JENSEN 1 ,
R. J. GREENOUGH 2 , M. McCONVILLE 2 , M. HOLMSTROEM 2 and K. P. HAZELDEN2

Novo Industri A/S, Research and Development Division, 2880 Bagsvaerd, Denmark and Inveresk Research International Ltd., Musselburgh EH21
7UB, Scotland

(Received for publication February 28, 1985)

ABSTRACT of starches used for this purpose contain 75 to 85%

amylopectin. Amylopectin is a highly branched polysac-
Promozyme®, an amylopectin debranching enzyme produced
by Bacillus acidopullulyticus, was studied to evaluate its safety charide consisting of linear chains of 1,4-alpha-linked D-
in the food industry. A dietary subchronic toxicity study incor- glucose residues, joined together by 1,6-alpha-glucosidic
porating fertility and teratogenicity studies was performed in 1- linkages. Hydrolysis of 1,6-alpha links is often called de-
month-old rats at concentrations of 0.5, 1.5 and 5% Prom- branching, and use of a specific amylopectin debranching
ozyme. No adverse effects were seen at the 0.5 and 1.5% dose enzyme can improve the efficiency of the saccharification
levels, and at the 5% dose level only minor or equivocal signs reaction. Debranching enzymes, such as isoamylase and
of toxicity were recorded. With the exception of a moderate pullulanase, have been known for many years, but their
reduction in body weight gain the F I A litters at the 5% dose use in the glucose syrups industry is far from widespread.
level, no effects were found in the fertility study, and Prom- Pullulanase from Klebsiella pneumoniae, Streptomyces
ozyme was not teratogenic. In a 13-wk oral toxicity study in
spp. and Bacillus cereus var. mycoides and isoamylases
dogs, no adverse effects resulted from 0.5 g/kg/d, whereas mild
from Pseudomonas amyloderamosa and Cytophaga spp.
gastrointestinal disturbances were seen clinically at 1.5 and 5.0
g/kg/d. In dogs given 5.0 g/kg/d, terminal investigations have been described. None of these is able to function
showed increased kidney weights and mineralized casts in renal under the conditions prevailing in industry in the normal
cortical tubules. This was probably due to the high content of saccharification processes. A pullulanase suitable for in-
ash (phosphorus) in the test material. Lack of mutagenic poten- dustrial processing and for production of food has been
tial was confirmed in bacterial mutagenic assays with Sal- obtained from a Bacillus sp. of a new taxonomic group
monella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 known as Bacillus acidopullulyticus (5). The trade name
and TA 100) and in an in vivo cytogenetic study in rat bone of this microbial food enzyme is Promozyme.
marrow cells after a single dose and daily dosing for 5 d of
This paper reports results of studies done to establish
up to 8 g/kg/d. In an acute inhalation study with 4 h of expo-
sure of rats, no death occurred at the highest dose level used, the safety of Promozyme when used as a processing aid
i.e., 2 mg/L. The test material was non-irritating to skin and in the food industry. The studies elucidate the consumer
did not produce eye injury in rabbits. A skin sensitization study safety and industrial occupational health effects of the use
in guinea pigs revealed no indication that the enzyme is a sen- of this enzyme. The microorganism was tested for
sitizer. The pathogenic potential of the enzyme-producing B. pathogenicity and production of antibiotics, and the en-
acidopullulyticus was investigated by single intraperitoneal and zyme product was examined with regard to general oral
subcutaneous administrations to rats and mice; the microor- toxicity (acute and subchronic), effects on reproduction,
ganism was found to be nonpathogenic (LD 50 >10 10 cells/kg). mutagenic potential, inhalation toxicity, irritancy to skin
Tests of culture broths revealed that the microorganism does and eye, and skin sensitization. A preliminary report of
not produce antibiotics. Results indicated that production and
these studies has been published previously (2).
the intended use of Promozyme can be regarded as safe for
plant workers and consumers.

MATERIALS AND METHODS

In the starch industry, enzymes are used to catalyze Test material

the hydrolysis of starch to glucose syrups. The majority Test material, pullulanase 5P247, ("tox-batch") was produc-
ed according to the procedure used to prepare Promozyme.
However, the purification procedure followed was designed to
'Novo Industri A/S. result in a more crude preparation. This was done to ensure
2
lnveresk Research International Ltd. that commercial product that would be prepared by improved

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 TOXICOLOGICAL SAFETY OF BACILLUS PULLULANASE 147

procedures which would yield a more purified preparation B. acidopullulyticus strain was nonpathogenic to rats and
would also be covered by the safety studies performed on the mice. Results of the antibiotic activity tests indicate that
tox-batch. The following procedure was used to prepare the tox-
the B. acidopullulyticus strain does not produce antibio-
batch: (a) the fermentation broth was cooled to 5 to 10°C; (b)
tics.
bacterial cells and extraneous material were removed by de-
sludger centrifugation; (c) the protein residues from the growth
medium were precipitated by adjusting pH to 3.8 with phos- Oral toxicity studies in rodents, including fertility and tet-
phoric acid; (d) the precipitate was collected by centrifugation; ratogenicity studies
(e) the pH of the supernatant fluid was adjusted to pH 6.0 with Acute toxicity. Acute toxicity studies in mice and rats
NaOH; and (f) the supernatant fluid was filtered, concentrated indicated that pullulanase was well-tolerated up to and in-
by evaporation, and spray dried. cluding 20 and 10 g/kg, respectively, which were the
The tox-batch was characterized by chemical and micro- highest dose levels tested.
biological tests. The following results were obtained: (a) en- Inhalation toxicity. During exposure to pullulanase, the
zyme activity, 554 PUN/g (One Pullulanase unit (PUN) is de- test animals showed a slight reduction in respiratory rate.
fined as the amount of enzyme required to produce 1 u,mole
Clear nasal secretion and red nasal encrustation were ob-
of reducing sugar per min under standard conditions); (b) pro-
served after 30 min and 3 h of exposure, respectively.
tein (Kjeldahl of acetone precipitate), 9.8% (wt/wt); (c) nitro-
gen-containing substances other than protein (i.e., peptides, Following exposure, the animals also exhibited a subdued
amino acids) determined as [N,-Np (acetone precipitate)] x appearance. There were no deaths, and no abnormalities
6.25, 6.8% (wt/wt); (d) protein [Kjeldahl of trichloroacetic acid were detected during the subsequent 14-d observation
(TCA) precipitate], 2.3% (wt/wt); (e) nitrogen-containing sub- period or at the postmortem examination. Because no
stances other than protein (i.e., peptides, amino acids) deter- death occurred, the LD 5 0 exceeded 2.14 mg/L.
mined as [Nt-Np (TCA)] x 6.25, 14.3% (wt/wt); (f) water con- Subacute toxicity. Decreased food intake and concomit-
tent, 7.1% (wt/wt); (g) total carbohydrates (Antron), 27.7% (wt/ ant slower body weight development were seen in the
wt); (h) fats (Soxhlet), <0.1% (wt/wt); (i) ash content, 35.9%
males fed 10% pullulanase, and both males and females
(wt/wt); (j) Na, 3.3% (wt/wt); (k) K, 8.7% (wt/wt); (1) Mg,
fed 10% pullulanase had increased water intake. Micro-
0.8% (wt/wt); (m) Ca, 0.8% (wt/wt); (n) CI, 0.5% (wt/wt); (o)
P0 4 , 22.1% (wt/wt); (phosphorus = 7.26%); and (p) S0 4 , scopic examination revealed decreased mineralization of
7.5% (wt/wt). Total organic substance (TOS) was defined as the cartilage in the epiphysis of the femur and nephrocal-
100% - (A + W + D)%, where A is ash content, W is cinosis in both sexes fed 10% pullulanase (rats fed 1%
water content and D is the content of diluents. In the tox-batch, and 5% pullulanase were not microscopically examined).
TOS was 57%. In the commercial preparation, "Promozyme These effects were ascribed to both the high content of
200L", TOS was 10% and diluents and water were 90%, in- phosphorus in the test material resulting in an unfavorable
cluding approved additives, i.e., maximum 0.1% potassium sor- calcium:phosphorus (Ca:P) ratio and a high absolute level
bate and maximum 0.1% sodium benzoate. of phosphorus.
For the pathogenicity studies, viable cells from the fermenta- Subchronic toxicity incorporating fertility and
tion were washed and suspended in M-9 buffer to a total count teratogenicity. In Phase I (fertility study), no differences
of ca. 5 x 109/CFU/ml. that could be attributed to dosing with pullulanase were
Methods found between the control and treated F 0 rats. The F 1 A
Studies done to establish GRAS (Generally Recognized As litter body weight showed a moderate reduction in gain
Safe) status for pullulanase are shown in Table 1. The animal in rats receiving 5% pullulanase (Table 3). No dose re-
studies were based on rats [either the Wistar strain (Moel-
lated differences were seen among the other parameters
legaards Breeding Center Ltd., Denmark) or the Sprague-Daw-
investigated.
ley CD strain (Charles River, U.K.)], NMRI mice, albino rab-
bits [Danish Landstrain (Novo, Denmark)], guinea pigs [Dunkin In Phase II (toxicity study), males and females receiv-
Hartley strain (Hacking and Churchill Ltd., U.K.)] and Beagle ing 5% pullulanase showed a moderate reduction in body
dogs [purpose bred in the U.K.]. The animals were fed com- weight gain (Table 4). With the exception of a decrease
mercial standard diets (Brood Stock Feed for Rats and Mice in female eosinophils at Week 7, no intergroup differ-
- R3 - Astra Ewos, Ewos Brood Stock Feed for Rabbits and ences in the hematological parameters were evident in
Guinea Pigs, Special Diet Services Rat and Mouse (modified) either sex at Week 7 or 13. The clinical chemistry re-
No. 1 or No. 3 Diet and FD1, and Special Diet Services Dog vealed an elevation in alkaline phosphatase in both sexes
Diet). In the combined reproduction study, batches of the diet at Weeks 7 and 13, but this elevation was statistically
with relatively high calcium and low phosphorus contents were significant (P<0.01) only in the females at Week 13.
selected to minimize the effects of the high phosphorus content
Other parameters showing statistically significant differ-
in the test material. The scheme of the combined reproduction
ences were a decrease in aspartate aminotransferase in
toxicity study is shown in Figure 1.
males at Week 7, a decrease in total protein in males
at Week 13, and a decrease in lactate dehydrogenase in
RESULTS
females at Week 13. However, none of these changes
Pathogenicity and production of antibiotics was regarded as a significant toxicological effect. A
Clinical and autopsy findings, and estimated LD 5 0 slight decrease in urinary volume and pH was seen in
values are shown in Table 2. The few adverse findings both males and females at Weeks 7 and 13 (Table 5).
compared with the high dosages used indicate that the Postmortem examination revealed a dose-related increase

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

148 STAVNSBJERG ET AL.

TABLE 1. Diagram of safety tests.

Dose levels or
maximum concentration
Safety aspect Type of test Source material for test design obtained for exposure
Pathogenicity (rats and Acute test with single inocula- Methods developed by Novo. lntraperitoneally 10" cells/kg
mice) and production of tion No international guideline is Subcutaneously
antibiotics recommended.

Determination of antibacterial FAO/WHO (1982): Specifica-

activity tions for Identity and Purity
of Carrier Solvents, Emulsifiers
and Stabilizers, Enzyme Prep-
arations, Flavouring Agents,
Food Colours, Sweetening
Agents and Other Food Addi-
tives. Joint FAO/WHO Expert
Committee on Food Additive,
Food Agriculture Organization
of the United Nations, Rome.

Acute toxicity Acute oral toxicity OECD Guidelines for Testing 20 g/kg (mice)
of Chemicals (1981) Paris. lfj g/kg (rats)

Acute inhalation toxicity, 4-h OECD Guidelines for Testing 2.14 mg/L
exposure of Chemicals (1981) Paris.

Dietary toxicity studies Subacute toxicity (4 wk dose Methods designed from general 0, 1, 5 and 10%
in rodents, including range and target organ finding guidelines for 4-wk toxicity
fertility and teratogenicity study) testing in rodents.
studies
Subchronic toxicity incorporat- Food Safety Council (1980): 0, 0.5, 1.5 and 5%
ing fertility and teratogenicity Proposed System for Food
Safety Assessment. V.O.
Wodicka, L. Goldberg, and
C. J. Carr (eds.) Food Safety
Council, Washington, DC.

Toxicity studies in dogs Maximum tolerated dose Method designed by the test 5 g/kg/d
study facility.

Subchronic toxicity, 90-d Conventional design using 4 0, 0.5, 1.5 and 5 g/kg/d
dietary exposure groups of 3 male and 3 female
dogs.

Mutagenic evaluation Gene mutation in vitro Ames, McCann & Yamasaki 9.1 mg/L
(1977). Handbook of
Mutagenicity Testing Proce-
dures, Amsterdam.

Chromosome aberration in Ad Hoc Committee on Chromo- 1, 3.2 and 8 g/kg

vivo some Methodologies in Muta-
gen Testing, Toxicol. Appl.
Pharmacol. 22:269-275, 1972.

Irritation Skin irritation (4-h exposure) in Code of Federal Regulations

rabbits (1979): Title 16, § 1500.41
and 1500.42, adapted to OECD
Eye irritation in rabbits Guidelines.

Sensitization Delayed contact hypersensitivity E. V. Buehler (1965): Arch.

in guinea pigs Dermat., vol. 91.

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 TOXICOLOGICAL SAFETY OF BACILLUS PULLULANASE 149

in the number of females with calcified casts in kidneys.

Also, severity of the lesions increased with dose level
(Table 6). Minor changes in organ weights were ob-
served, but only the increase in relative kidney weights
in the females correlated with any findings at his-
topathological examination (Table 7). These findings
were ascribed to the unfavorable Ca:P ratio and the abso-
lute level of phosphorus in the diet.
In Phase III (teratogenicity study), the F 0 female rats
receiving 5% pullulanase had a slight reduction in body
weight gain during gestation when compared to control
Developmental/Behavioral ~
rats. There were no effects on pregnancy performance,
Assessments
on fetal and placental weight or on the incidence of fetal
abnormalities. Further, there were no differences in nec-
ropsy findings in the F 0 animals between control animals
and those groups receiving pullulanase.
Laboratory
Phase II
nvesti'ation
(13 Week Oral toxicity studies in dogs
Toxicity)
Maximum tolerated dose study. One male and one
female Beagle dog were dosed daily for 4 wk at weekly
Laboratory
investigations
dose levels of 1, 4, 8 and 5 g of pullulanase/kg/d. The
main finding was a dose-related incidence of emesis and
loose fecal production beginning at 2 g of pullulanase/kg/
d. Further, one male and one female Beagle dog were
Figure 1. General study plan of the dietary subchronic toxicity dosed daily for 14 d with pullulanase at a dose level of
study incorporating fertility and teratogenicity studies to 5 g/kg/d. Again, incidences of emesis and loose feces
evaluate the safety of B . acidopullulyticus pullulanase (Prom- were the prominent reactions to the compound.
ozyme).
Subchronic toxicity. Emesis was frequently observed in
all dogs receiving 5 g of pullulanase/kg/d. Other clinical
signs observed in this group were loose feces, salivation
after dosing, and increased urinary output. Dogs receiv-
ing 1.5 g of pullulanase/kg/d vomited infrequently, but
diarrhea and loose feces were frequently seen among the
females in this group. No adverse clinical signs were
TABLE 2. Pathogenicity testing o / B . acidopullulyticus.
Dosage LD 50
Route of (CFU/kg (CFU/kg
Animal species inoculationa body weight) Clinical findings Autopsy findings body weight)
Mice 0 (Control) NAD b NAD b
(6 males per 10 7 NAD NAD
group) i.p. 10 9 NAD NAD 10lo<LD5O<10"
10
10 Diarrhoea Peritonitis
10" Depression, deaths NAD
0 (Control) NAD NAD
s.c. 108 NAD NAD 10 I O <LD 5 0
10 9 NAD NAD
10 10 NAD NAD
Rats 0 (control) NAD NAD
(6 males per 10 7 NAD NAD
group) i.p. 10 9 NAD NAD 10"<LD5O
10 10 NAD Peritonitis
10" Depression, diarrhoea, Peritonitis
deaths
0 (Control) NAD NAD
s.c. 10 8 NAD NAD 1010<LD50
10 9 NAD NAD
10 10 NAD NAD
a
i.p., intraperitoneally; s . c , subcutaneously.
b
NAD, no abnormality detected.

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

150 STAVNSBJERG ET AL.

TABLE 3. Fertility study in rats (Fj generation) fed pullulanase (SP 247).
Male ineonates Female neonates
Group 1 2 3 4 1 2 3 4

0.5% 1.5% 5.0% 0.5% 1.5% 5.0%

Day of Control Pullulanase Pullulanase Pullulanase Control Pullulanase Pullulanase Pullulanase
lactation in diet in diet in diet in diet in diet in diet

1 5.91±0.91a 5.94±0.57 a 6.24±0.68 a 5.99±0.78 a 5.61+0.92 a 5.61±0.52 a 5.78±0.73 a 5.61±0.76 a

4 8.16± 1.80 8.36± 1.32 8.54± 1.10 8.41 ±1.25 7.79± 1.67 7.97+1.17 8.06± 1.14 7.81 ±1.27
12 20.71 ±2.81 20.40±2.73 20.86±2.88 19.31 ±2.13 19.79±2.81 19.13±3.06 19.67±2.95 17.96±2.21 b
21 38.69±6.35 37.99±5.64 39.04 ±5.69 35.72±4.47 36.87±6.22 35.59±5.80 37.07±5.85 33.48±4.55

Weight gain (g) 2.25 2.42 2.30 2.42 2.18 2.36 2.28 2.20
Day 1-4
% of difference — 7.6 2.2 7.6 — 8.3 4.6 0.9
from controls
Weight gain (g) 12.55 11.99 12.32 10.90 12.00 11.16 11.61 10.15
Day 4-12
% of difference — -4.5 -1.8 -13.1 — -7.0 -3.3 -15.4
from controls

Weight gain (g) 17.98 17.64 18.19 16.41 17.08 16.46 17.40 15.52
Day 12-21
% of difference — -1.9 1.2 -8.7 — -3.6 1.9 -9.2
from controls
''Group mean body weight (g) ± standard deviation of F, generation litters during lactation.
b
P : s0.05, significantly different from control using Student's t-test.

TABLE 4. Growth rates of rats (F, generation) fed pullulanase (SP 247) during a 14-wk toxicity study.
Treatment period Dose group/Dose level (% pullulanase in diet)"
(Weeks from 5<J 6<J IS 8<J 5? 6? 7$ 8?
selection) 0 0.5 1.5 5.0 0 0.5 1.5 5.0
97b,c
0 119 b 121 b 128 b 112 b 110 b 103 b 114 b
1 183 185 188 173 149 141 152 133 c
2 238 241 239 218 175 164 178 156d
3 283 282 280 260 c 192 178 195 171 d
4 325 318 319 292 c 212 196 214 186 e
5 355 344 345 322 c 226 211 230 201 d
6 367 367 374 331 c 231 220 241 208 d
7 388 388 397 355 c 244 233 257 220 d
8 410 406 413 370 c 256 241 264 227 e
9 426 419 427 383 c 259 245 265 233 d
10 448 438 448 403 c 267 255 278 245 c
11 466 453 460 417 c 275 261 284 250 c
12 476 462 473 426 c 282 264 289 254 d
13 478 467 480 426 c 281 271 295 256 c
14 495 479 490 437 d 286 271 296 258 d
Weight gain (Week 0-14) 376 358 362 325 176 168 182 161
% of controls — 95 96 85 — 95 103 91
a
Fifteen rats were fed in each dose group.
b
Body weight; group mean values (g).
C
P<0.05, significantly different from control using Student's t-test.
d
P < 0 . 0 1 , significantly different from control using Student's t-test.
e
P < 0 . 0 0 1 , significantly different from control using Student's t-test.

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 TOXICOLOGICAL SAFETY OF BACILLUS PULLULANASE 151

TABLE 5. Urinalysis results during Week 13 for rats (F, generation) fed pullulanase (SP 247) during a 14-wk toxicity study.
Dose level Volume
Sex (% Pullulanase) pH SGa (ml)
0 Mean 8.2 1.043 2.3
Control SD 0.7 0.006 1.0
Male
5.0 Mean 6.7 1.043 1.8
SD 0.9 0.003 1.2

0 Mean 7.7 1.040 1.4

Control SD 1.0 0.005 0.6
Female
5.0 Mean 6.9 1.045 1.0
SD 1.4 0.011 0.4
a
SG, specific gravity.

TABLE 6. Incidence of kidney histopathological lesions in female rats (Fj generation) fed pullulanase (SP 247) during a 14-wk
toxicity study.
Dose group/Dose level
7
Kidney lesion 0.5% 1.5% 5.0%
Control Pullulanase Pullulanase Pullulanase
in diet in diet in diet
No. of rats examined 10 15 15 10
Calcified casts ( + ) 11
(++) 3
(+ + +)
(+ + + + )
Calcified casts with
dilated tubules - 1 2 10

Total No. with casts 4 9 14 10

Percentage 40 60 93 100

seen for dogs receiving 0.5 g of pullulanase/kg/d or the

TABLE 7. Relative organ weight of female rats (F, generation) vehicle. Body weight changes throughout the study were
fed pullulanase (SP 247) during a 14-wk toxicity study. normal and food consumption was maximal for all dogs.
Body A possible treatment-related increase in white blood cell
Dose weight count was seen in dogs given 5 g of pullulanase/kg/d
(%) (g) Kidneys" (Table 8). Postmortem examination revealed a seemingly
Number 15 15 dose-related increase in mild, interstitiel and focal lym-
0.0 Mean 281 0.41 phoid reactions, and presence of mineralized casts in
SDb 34 0.05 renal cortical tubules. Organ weight analysis showed a
Number 15 15 significant increase in absolute and relative kidney
0.5 Mean 266 0.39 weights in animals given 5 g of pullulanase/kg/d (Table
SD 24 0.03 9).
Number 15 15
1.5 Mean 291 0.39 Mutagenic evaluation
SD 29 0.04 Gene mutation in vitro. Pullulanase was not mutagenic
Number 15 15 when tested up to a concentration of 9.1 mg/ml of incu-
5.0 Mean 254c 0.44d bation mixture.
SD 20 0.03 Chromosome aberrations in vivo. There was no evi-
a
% of body weight. dence in either the acute or subacute study, to indicate
b that pullulanase induced chromosomal aberrations in rat
SD, standard deviation.
C
P<0.01, significantly different from control using Student's t- bone marrow cells, whereas treatment with the positive-
test. control substances, i.e., ethyl methanesulphonate and
d
P<0.05, significantly different from control using Student's t- cyclophosphamide, resulted in small and extensive yields
test. of chromosome damage, respectively.

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

152 STAVNSBJERG ET AL.

TABLE 8. Hematology during Week 12 of dogs (six per group) fed pullulanase during a 13-wk oral toxicity study.
Dose Hba RBCb PCVC MCHd MCVe MCHCf Retig WBCh Neut1 Lympi Monok Eos1
(g/kg/d) (g/dl) X10l2/L (%) (pg) (fl) (g/dl) (%) (X109/L) (X109/L) (X109/L) (X109/L) (X109/L)
Control Mean 14.6 5.4 44 27 81 33 1.4 9.7 5.7 3.3 0.5 0.2
SD 0.8 0.3 2 1 3 1 0.3 0.8 0.8 0.5 0.1 0.1
0.5 Mean 13.6 5.1 41 27 81 33 1.4 9.6 5.8 3.2 0.4 0.2
SD 1.0 0.3 3 1 2 1 0.7 2.0 1.7 0.7 0.2 0.2

1.5 Mean 13.8 5.2 41 27 79 34 1.5 11.4 6.9 3.8 0.4 0.3
SD 1.2 0.5 3 1 4 1 0.6 1.6 1.3 0.5 0.2 0.1
5.0 Mean 13.2m 5.1 39m 26m 77m 34 1.1 13.0" 7.6m 4.7" 0.6 0.1
SD 0.8 0.2 2 1 5 1 0.3 0.9 1.0 0.6 0.2 0.2
a
HB, hemoglobin
b
RBC, red blood cell count. DISCUSSION AND CONCLUSIONS
C
PCV, packed cell volume
d
MCH, mean cell hemoglobin. Human consumption of enzyme preparations is calcu-
e
MCV, mean cell volume lated only on the basis of the amount of total organic
f
MCHC, mean cell hemoglobin concentration. substance (TOS) in the preparation, because the rest of
g
Reti, reticulocyte count.
h the preparation (i.e., ash, water and diluents) is fully
WBC, white blood cell count.
characterized and known to be safe for consumption at
'Neut, neutrophil count.
'Lymp, lymphocyte count. the levels involved. The use of Promozyme in the pro-
k
Mono, monocyte count. duction of glucose and high maltose syrups and beer re-
'Eos, eosinophil count. sults in maximum residues of 400 mg enzyme-TOS/kg
m based on syrup dry matter and 25 mg enzyme-TOS/kg
P<0.05, significantly different from control using Student's t-test.
"P<0.001, significantly different from control using Student's t-test. based on beer. In calculating the human consumption of
TABLE 9. Organ weights of dogs fed pullulanase during a 13- enzyme-TOS, the following points should be considered:
wk oral toxicity study. (a) the enzyme-TOS residue levels stated above are
"worst case" theoretical values with no account of the
Dose Body weight Kidneysa
(g/kg/d) (kg) diminishing effect any subsequent purification steps
would have on the residues of enzyme-TOS in the final
Control Mean 11.1 0.25
foods; (b) the greatest portion of enzyme-TOS is com-
SD 1.5 0.02
posed of proteins and carbohydrates from traditional food
0.5 Mean 11.3 0.27 sources used in the fermentation; and (c) based on the
SD 1.4 0.04 average consumption of glucose and high maltose syrups
1.5 Mean 11.5 0.27 (/) and beer (7), the total exposure of the consumer from
SD 0.7 0.03 these two sources is estimated to be 0.318 mg enzyme-
5.0 Mean 11.1 0.31 b TOS per kg body weight per day (60 kg person), if all
SD 2.1 0.05 syrups and beer are produced by the use of Promozyme.
a
% of body weight. Because Promozyme is not always used to prepare these
b
P<0.001, significantly different from control using Student's products, this figure is obviously over-estimated.
t-test. The toxicological investigations needed for evaluation
of microbial enzymes are under discussion (3,4,6); how-
ever, it is generally agreed that the evaluation of safety
should be based on knowledge of the source microor-
Irritation ganism and animal testing on the enzyme preparation.
A primary skin irritation score of 0 was obtained for The long history of safe use of Bacillus spp. for produc-
pullulanase. Hence, pullulanase was classified as a "non- tion of food enzymes and the safety testing reported in
irritant" to skin and was considered to have no potential this paper provide a reasonable base for the safety evalua-
for eye injury. tion of Promozyme. The teratogenicity, fertility and
mutagenicity tests revealed no adverse effects of pul-
Sensitization lulanase. In the 3-month toxicity studies, no adverse ef-
None of the animals in the group receiving pullulanase fects were seen in rats given 1.5% pullulanase in the diet
showed any reactions on examination at 24 and 48 h or in dogs given 0.5 g of pullulanase/kg/d. Calculating
post-application. In the positive-control group, formalin- the amount of Promozyme in the test material on the
positive reactions were seen in six of ten animals. In a basis of TOS and the amount of Promozyme to be con-
concurrent irritation study, none of the animals showed sumed by humans using the estimates described above,
signs of irritation. This sensitization test indicated that the provides a safety margin of approximately 1400 and 900,
enzyme is not a sensitizer in guinea pigs. respectively. Moreover, it is unknown if the effects seen

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 TOXICOLOGICAL SAFETY OF BACILLUS PULLULANASE 153

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