157

Jaumal afFood Protectioll, Val. 44, No.2, Pages 157-163(f'ebruary 1981)

Copyright© 1981, International Association of Milk, Food, and Environmental Sanitarians

Thermophilic Organisms in Food

Spoilage: Flat-Sour Aerobes
KEITHA.ITO
Natio11al Food Processors Assaciatio11, 1950 Sixth Street, Berkeley, Califomia 94710

(Received for publication May 28, 1980)

ABSTRACT stand. Additionally, they were troubled by the term

Bacillus stearothennophilus is the typical organism causing thermophilic because a comparison of the growth temp-
flat sour spoilage in low acid foods. It was first named by Donk. erature range of 9 Bacillus spores showed that growth
The spores are ubiquitous in nature, being isolated in areas temperatures cannot be used to separate the species.
from the arctic to the deserts as well as from foods. The They suggested that the term "thermophilic" be used to
organism is difficult to characterize in its growth, germination denote cultures capable of growing at 55 C but never to
and sporulation requirements, and is difficult to destroy with describe species. They also found that although the
heat or chemicals. The organism is not pathogenic.
maximum temperature of growth varied widely, its use
might be ofvalue to separate B. stearothermophilus from
other Bacillus species. All 87 strains they tested grew at
The flat-sour aerobes are two in number, one Bacillus 65 C (water bath determination), whereas none of the
stearothermophilus, typical of flat-sour spoilage of low other species tested did. Table 1 shows some selected
acid foods and the other Bacillus coagulans, typical of characteristics of B. stearothermophilus. They are
flat-sour spoilage of acid foods. The latter organism is selected from Bergeys Manual, 8th ed. (6) and
considered in another paper in this series, and so this Agriculture Handbook No. 427 (31).
discussion will be concerned only with B. stearothermo- TABLE 1. Selected characteristics ofB. stearothermophilus.
philus. The discussion will be limited to classification,
source, metabolism and biochemistry of spores, resist- Character Description
ance and pathogenicity. 0.6 to 1.0 1-1 by 2.0 to 3.5 1-1
1. Vegetative rods
Although there are indications that the existence of the 2. Sporangia Swollen
organism was known early in the history of food canning, 3. Spores Mature spores
the organism was not named until1920. At that time P. -1.0 to 2 1-1 by 1.5 to 2.2 1-1
J. Donk of the NCA Washington Research Laboratories -Oval and subterminal to terminal
published a paper (22) giving the cultural characteristics 4. Gram reaction Variable
and thermal resistance of an organism isolated from 5. Growth temperature Maximum 65 to 75 C
spoiled "Standard Maine Style" corn. He also recovered Minimum 30to45 C
this same organism from spoiled string beans and corn 6. Growth on proteose
on the cob. He proposed the name Bacillus stearother- peptone acid agar None
mophilus for this unknown species. 7. Production acetyl-
methyl carbinol Negative
CLASSIFICATION 8. Growth in
Different workers characterized thermophilic bacteria Anaerobic agar Negative
7o/oNaCI Negative
in different ways. One definition which seemed to have
.02 o/o Azide Negative
some acceptance by microbiologists was the definition Negative
.001 o/o Lysozyme
given by Cameron and Esty (1 0); cultures growing at 55 C Acid (pH 4.8 to 5.8)
V-PBroth
but not at 37 C were classified as obligate thermophiles, Acid from glucose Positive
and those growing at both 55 and 37 C as facultative Hydrolysis of starch Positive
thermophiles. Gordon and Smith (.32) in 1949 made a
detailed study of 87 cultures of B. stearothermophilus SOURCE OF SPORES
and published an amended description of B. stearother- Nature
mophilus Donk. They felt the name to be cumbersome, Spores appear to be ubiquitous in nature. Fields (25)
but based on an extensive literature survey found no reported investigating soil from four different tempera-
information which would predate the naming of the ture areas: tropic, sub-tropic, desert and temperate, and
organism by Donk and thus they felt the name must found thermophilic aerobes in all areas investigated. He

JOURNAL OF FOOD PROTECTION. VOL 44, FEBRUARY 1981

158
found that spore counts were correlated with pH of the
soil. McBee and McBee (44) looked for and found
thermophilic bacteria in arctic soils and water. B.
stearothermophilus was identified among the organisms
isolated (45). In contrast to this environment, Marsh and
Larsen (41) looked for the thermophiles in a more likely
environment, the hot springs of Yellowstone National
Park. They found 21 of their isolates to conform closely
to the description of B. stearothermophilus.
Foods
Sugar and starch. As early as 1926, sugar was observed
to carry thermophilic bacteria. Cameron and Williams in
1928 (11) published the results of several years of study
relating the thermophilic flora of sugar to canning and
subsequent potential hazard. They also investigated the
potential for contamination and spore build up in a
sugar refining operation. In 1936, Cameron (8) published
a procedure for detecting thermophilic bacteria,
including flat-sour, in sugar. When this was combined
with the standards given by Cameron (7), a valuable tool
was available to assist in the control of thermophilic
spoilage of canned foods. These standards, though
modified, are still operative today. There are still
thermophiles present in sugar, as evidenced by Scarr's
(56) investigation in a sugar refinery in 1968. Her
findings appear to corroborate those of Fields (24) who,
in 1970, indicated that the quality of sugar had improved
regarding number ofthermophiles present.
Cameron (9), in 1937, pointed out that starch could
also be an ingredient which could cause problems in
thermophilic spoilage. Clark and Tanner (14), in 1937,
proposed a method for examining starch for thermo-
philic organisms and used the same standards as for
sugar. The use of these standards has continued to this
day and has assisted in minimizing the thermophilic
spoilage in canned foods.
Milk. Thermophiles can be found in milk. Prickett (50)
found that the growth of thermophiles in milk produced
undesirable change in product quality. Breed et at. (4), in
1929, looked at the significance of thermophilic spores in
pasteurized milk, and found that it was difficult to
obtain pasteurized milk which was always free of
thermophiles.
Other foods. Richmond and Fields (52) investigated
the distribution of thermophiles in various food
ingredients. They examined sugar, starch, flour, spices,
milk powder, yeast, cocoa, gelatin, dried soup and green
split peas. They found B. stearothermophilus in only
sugar and turmeric. Yesair and Williams (66) examined
30 spices for thermophilic aerobes and found them in 14
with ground black pepper containing the highest
number.
Processing plant
B. stearothermophilus can grow and spoil low-acid
canned foods, such as corn, peas, asparagus, etc. It can
enter the plant in the soil, in food ingredients and on
foods. Once in the plant it can multiply where an
JOURNAL OF FOOD PROTECTION. VOL. 44. FEBRUARY 1981
ITO
environment suitable to its growth can be found, such as
in warm holding tanks, blanchers, warm filler bowls, etc.
Areas of particular problems have been blanchers and
pumpkin wilters which are difficult to clean and sanitize.
METABOLISM AND BIOCHEMISTRY OF SPORES
Spore activation and germination
The requirements for spore activation are dependent
upon species or organism. Usually heat or chemicals are
used for spore activation. Among the first workers to use
heat were Curran and Evans (20). They found that
10 min at 95 C activated spores when heated in distilled
water and skim milk. Finley and Fields (28) investigated
heat activation in water and phosphate buffer. They
found activation occurred in water at temperatures over
100 C. One suspension of B. stearothermophilus 1518
required 3 min at 115 C for maximal activation, while
others required 7 to 10 min at 110 C. Strains of an
unknown thermophile labeled "M" required 9-15 min at
110 C for activation. They found that heating in M/120
Sorenson's phosphate buffer (pH 7.1) rather than
distilled water greatly affected recovery of the heated
spores, with recovery being significantly decreased by
heating in phosphate buffer. Cook and Brown (16) found
activation temperatures ranging from 13 h at 100 C to
1-2 min at 121 C when spores were heated in water. They
also found that spores heated in phosphate buffer
showed reduced activation. Cook and Gilbert (17) found
heat activation in water required 8 min at 115 C, and a
slightly longer time, 12 min, was required when heating
was conducted in a yeast suspension.
On occasion chemicals have been used successfully to
activate bacterial spores. Mefford and Campbell (46)
used furfural to activate thermophilic aerobes. They
found furfural at 10" 6 dilution, when used to treat
unheated spores for 30 min, gave equivalent activation to
heating. Lewis et al. (38) succeeded in activating the
spores by treatment at pH 1.5 for 80 min at 25 C.
Germination requirements of B. stearothermophilus
have been investigated. Fields and Finley (26) investi-
gated germination with monosaccharides, disaccharides
and polysaccharides in .0083 M phosphate buffer
(pH 7.1). With the monosaccharides, glucose and
fructose, positive responses were noted at 0.001 and
0.1 M glucose and .01 M fructose. With the disaccha-
rides, sucrose and maltose, positive responses were noted
only at 0.1 M sucrose. With the polysaccharides,
dextrose and starch, a positive response was noted with
2% dextrose and 2% starch with one spore suspension
but not with the other spore suspensions tested. This was
typical in that different results were noted among various
strains and spore suspensions tested.
Fields and Frank (2 7) investigated germination in
sodium dipicolinate. They followed the germination by
measuring percent decrease of initial absorbance. The
germinating solution was 4 mM sodium dipicolinate.
Germination was found to occur over a very narrow
range of pH with an optimum at pH 5.5. Germination
 FLAT -SOUR AEROBES
occurred over a wide temperature range of 20 to 70 C
with an optimum of between 40 and SO C. They found
that neither EDTA nor L-alanine induced germination
over a wide pH range. Diep et al. (21) used B.
stearothennophilus BOOS to study germination. They
found germination occurred with L-alanine (10 mM).
After 1 h of incubation at 45 C, 99% of the spores had
germinated. Optimum germination occurred between
pH 6 to 7 with reduced germination, about SSo/o,
occurring at pH 5.0 and pH 6.0. Their results are in
contrast to that of Titus (59), who found only L-histidine
to be an effective germinant. O'Brien and Campbell (48)
found that addition of 12 #JM of L-alanine/ml did not
stimulate germination, and Campbell et al. (12) noted
that L-glutamic acid and L-lysine were effective
germinants. Thus it would appear that no single
chemical will facilitate germination of all strains or
suspensions of B. stearothennophilus.
Pearce and Wheaton (49) noted the phenomenon of
autosterilization of thermophilic aerobes in low-acid
canned foods. They inoculated flat-sour aerobes into low
acid canned foods, applied a thermal process, cooled the
containers and stored them at 21 C, a temperature below
the growth range of the added thermophile. They found
that as time progressed, the spores lost their viability in
the product. This phenomenon is probably due to heat
activation of the spores, followed by germination, but not
outgrowth. In time, the germinated spores lose viability,
accounting for lowered recovery.
Growth requirements
Long and Williams (39) investigated the cultural
conditions which affected growth temperature of seven
strains of B. stearothennophilus. They found a tryptose
basamin glucose broth provided satisfactory nutrients for
6 of the 7 strains tested and allowed growth at 37 as well
as 55 C. Additionally, a surface to volume ratio of 52:1
was the most effective in promoting good growth at 37 C,
suggesting that good growth was correlated with good
oxygen supply. Long and Williams (40) confirmed that
an adequate oxygen supply was needed for vegetative cell
growth and spore formation at 37 and 55 C. However,
they found aeration at 55 C and higher was not required
for spore formation and may in fact be inhibitory.
Downey (23) found that cell mass yield in a complex
medium was a function of the oxygen concentration
during growth. Optimum growth yield occurred at or
near the oxygen concentration available in the meso-
philic temperature range (143-240 ,an). If oxygen
concentration increased above this point, growth was
retarded.
Hill and Fields (34) found that absence of oxygen
influenced generation time. Growth still occurred, but
they found the rough population had a longer generation
time under anaerobic and limited oxygen supply than did
the smooth population. Both populations had a longer
generation time at 45 C with unlimited oxygen than at
65 C. They also found (35) that growth was influenced by
the pH ofthe growth medium. No growth occurred at pH
JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY 1981
159
5 and 9 in nutrient broth. When they used pH 7 as the
optimum, they found a shorter lag at pH 6 and 8 with the
rough variant than with the smooth, indicating less
sensitivity to the acid and alkaline environments by the
rough variant. They also found in this study that the
smooth variant required arginine, histidine, isoleucine,
methionine, valine, biotin and thiamine for growth. The
rough variant required methionine and biotin for growth.
These studies were carried out at 55 C. Atkinson et al. (2)
used B. stearothennophilus 1.503 and found that at 60 C,
pH 7.0 and with sucrose as a carbon source, only the
amino acids serine and arginine were used in appreciable
quantities. However, with glycerol as the carbon source,
glutamate, aspartate and threonine were used in addition
to serine and arginine. With both carbon sources, biotin
was required. Rowe et al. (54) also used B. stearothermo-
philus 1.503 to develop defined and minimal liquid and
solid growth media. They used a defined medium
containing 20 amino acids at a concentration equivalent
to a casitone medium, vitamins, salts and glucose as the
carbon source. Incubation was at 60 C. Using this basic
medium, they found a requirement for manganese.
Manganese became growth-limiting at a concentration of
about 2.5 x 10" 9M. They also found that phosphate
concentrations higher than 5 x 1Q·3M inhibited colony
formation on the solid medium. They found the
organism to require glutamate, histidine, isoleucine,
methionine and valine for optimal growth with glucose as
the carbon source. Good growth was obtained with
glucose, fructose, sucrose and glycerol as the carbon
source. Growth occurred with starch as the carbon
source, but no growth occurred with tricarboxylic acid
cycle intermediates, acetate or lactate. Campbell and
Williams (11) employed a synthetic medium containing
amino acids, vitamins and glucose as the carbon source.
Using B. stearothennophilus 1503, they found a
requirement for valine, nicotinic acid and biotin at 55 C.
Thus for the same organism, investigations by three
different groups have shown three different growth
requirements. It may be that the differences are
accounted for in the manganese and phosphate in the
growth medium.
Coultate and Sundaram (19), using B. stearothermo·
phi/us nondiastaticus, found the molar growth yield is
not maximum at the temperature optimum for growth
rates. This is not due to a high turnover rate of proteins
used to repair or resynthesize thermally denatured
macromolecules but may be due to other factors such as
inefficient coordination between oxidative and non-
oxidative phases of metabolism, causing incomplete
utilization of the carbon source.
Sporo.lation
Obtaining large quantities of heat resistant spores of
B. stearothermophilus is not easy. Long and Williams
(40) found that aeration at 37 C increased sporulation,
whereas it decreased sporulation at 55 C. Kim and
Naylor (37) allowed vegetative growth to occur in TYG
broth medium (1 o/o tryptone, O.So/o Difco yeast extract,
160
0.2% K 2HP0 4, pH 7.2) then transferred the organism to
sporulation agar (Nutrient broth 0.8%, Difco yeast
extract 0.4%, MnCI 2 • 4H 20, 10 ppm, 2% agar, pH 7.2).
Growth was at 52 to 53 C. Thompson and Thames (60)
developed a sporulation medium consisting of tryptone,
manganese sulfate and phosphate buffer, pH 6.8. They
obtained maximum spore yields using inocula grown
anaerobically in a broth medium containing tryptone
(O.So/o), glucose (0.1 %) and beef-extract (0.3%), and then
transferring to the sporulation medium. Sporulation
proceeded the aeration obtained by bubbling air at less
than 1.3 liters per min. Higher air flow rates suppressed
sporulation. Incubation was at 60 C. Manganese at 15 to
30 ppm, stimulated sporulation and concentrations
greater than 30 ppm depressed spore yields. Yao and
Walker (64) reported on a liquid medium consisting of
0.8% nutrient broth, phosphate buffer and 2 !Jglml of
Mn++-, ca++- and NO;, pH 6.8 to 7.2. Incubation was at
55 C and aeration at 1.2 to 1.5 liters of air per minute.
Sporulation was completed in 18 to 24 h. Rotman and
Fields (53) studied factors affecting sporulation of rough
and smooth variants of B. stearothermophilus. Sporula-
tion was carried out at 55 C, using nutrient agar, nutrient
broth and trypticase soy agar (TSA). They found that on
nutrient agar calcium had no effect on the smooth
variant but inhibited sporulation of the rough variant.
Cobalt reduced sporulation of the smooth but at 6 ppm
and higher, enhanced sporulation of the rough variant.
Manganese reduced sporulation of the smooth but at
1 ppm greatly enhanced sporulation of the rough variant.
Sporulation was unsuccessful on TSA. Sporulation
occurred in aerated nutrient broth fortified with yeast
extract (0.4o/o) and manganese or cobalt with the smooth
variant. The rough variant did not sporulate under the
conditions tested.
Tandon and Gollakota (58) studied growth of B.
stearothermophilus 1518 rough variant in a chemically
defined liquid medium. The medium contained glucose,
eight amino acids, vitamins, and mineral salts at
pH 6.75. Sporulation was obtained at 60 C. Glutamate
was needed for good sporulation and lysine was needed
for good cell growth. Sporulation occurred within 24 h
after transfer into the final sporulation flask. Aeration
was obtained using a reciprocating shaker bath. At the
start of sporulation, pH was 7.1 and increased to 8.4 by
completion of sporulation. Yazdany and Lashkari (65)
modified the medium of Thompson and Thames (60) to
obtain a high yield of spores at pH 7.7 to 8.7. Yazdany
and Lashkari (65) found when the medium was initially
adjusted to pH 7.0 vegetative cell growth reduced the pH
to 5.5, with few spores being produced. Adjustment of
the medium to an initial pH 8.5 was followed by a
reduction in pH to about 7.0 after vegetative cell growth,
and sporulation followed. They found sporulation to be
inhibited below pH 5.0 and above 9.0. The sporulation
medium consisted of peptone (0.3%), tryptone (0.25%),
yeast extract (0.4%), beef extract (0.25%), K 2HP0 4 (0.2o/~
and MnSO 4 (10 !Jglml).
JOURNAL OF FOOD PROTECTION, VOL 44. FEBRUARY 1981
ITO
We have obtained good sporulation on agar at 55 C,
using nutrient agar with 1.52 g of Tris buffer/L. The
buffer raises and maintains the pH above 7.0.
Spore recovery
Cook and Gilbert (18) investigated the effect on
recovery of heated spores of incubation temperature,
media composition, diluent and incubation time. They
found recovery of heated spores was best at 45-50 C.
Recovery of activated spores was best at 50-SS C, but as
heating time increased, maximum recovery was obtained
at the lower temperature. Maximum recovery was ob-
tained within 3 days at SO C. Highest recovery was ob-
tained on antibiotic assay medium (AAM), pH 7.3 with
0.1 o/o (w/v) added starch. Dextrose tryptone agar with
0.1 o/o added starch, pH 7.3, gave lower recovery. They
found water to be the best diluent for spore recovery after
the spores were subjected to severe heat.
RESISTANCE
Heat
Growth medium. B. stearothermophilus spores are
very heat resistant. The resistance is affected by growth
conditions. Williams and Robertson (63) showed that as
incubation temperature increased to near the maximum,
heat resistance of the spores produced increased. Cook
and Gilbert (18) also found increasing resistance with
increasing sporulation temperature. They also found that
the heat resistance was not correlated with manganese
sulfate in the sporulation medium except at high
concentrations (1000 ppm). Mayou and Jezeski (42)
found that heat resistance was greater with spores grown
on nutrient agar fortified with milk than those grown on
nutrient agar with 40 ppm of manganese sulfate.
Heating medium. The medium in which spores are
heated also affects resistance of the organism. Williams
and Hennessee (62) observed that spores heated in
disodium phosphate buffer showed an apparent increase
in resistance with decreasing molal concentration of
phosphate from M/15 to M/120. This inhibition appears
due to the phosphate added to the recovery medium
when the heating medium phosphate concentration was
greater than M/120. Cook and Gilbert (18), using M/15
and M/40 phosphate buffer, found no significant
difference between the two. Gauthier et al. (29) studied
the effect of various concentrations of Butterfield
phosphate buffer added to four parenteral solutions on
survival of B. stearothermophilus. They found the effect
of the phosphate buffer dependent upon the parenteral
solution composition and the buffer composition. They
concluded that the type of ions and their concentration
affected the spore's destruction.
Cook and Gilbert (18) also looked at the effect of
buffer composition and pH of the heating medium. In
comparing Mcilvaine's citric acid-phosphate buffer at
pH 7.0 with Sorensen's phosphate buffer at pH 7.0, they
found slightly greater resistance when heating in
Sorensen's buffer. They also found that as pH increased,
the resistance increased with a maximum at pH 7.0.
Anderson and Friesen (J) heated spores in acetate and
 FLAT-SOUR AEROBES
tris buffer from pH 3.0 to 9.0. Using acetate buffer, they
found the resistance increased as the pH increased from
3 to 7.0. The resistance in tris buffer from pH 7-9 was
similar, and possibly increased slightly as the pH
increased. However, the spores heated in tris had an
apparent lower resistance at the same pH than those
heated in acetate buffer, confirming the importance of
heating medium upon the apparent resistance of the
organism.
Reed et al. (SI) determined the resistance of B.
stearothermophilus 1518 in phosphate buffer, asparagus,
green beans, corn, peas, pumpkin, shrimp, spinach and
sweet potatoes. They found z values ranging from 15.6 to
23.2 F and F 250 ranging from 21 to 44 min for inoculum
levels of 10,000 to 28,500/ml. Mayou and Jezeski (42)
found B. stearothermophilus had a D 250 = 2.4 min in
reconstituted nonfat dry milk (10%), and 3.5 min in skim
milk, pH 6.5. The resistance of bacteria is affected by the
Aw of the heating environment. Murrell and Scott (47)
found the spores to have maximum resistance at Aw
0.2-0.4. They found only about a 20-fold increase in
resistance for B. stearothermophilus as the Aw was
decreased from 1.0 to 0.2. Collier and Townsend US)
determined the resistance of B. stearothermophilus in
superheated steam. They reported an F 350 =0.708 min
com pared to a resistance in saturated steam of
F 250 = 9.6 min. Alderton and Snell (I) found that at
Aw = 0.28, the survivor curves were curvilinear and thus
the D-value concept was not applicable at this
intermediate water activity for B. stearothermophilus.
Harnlilv et al. (JJ) determined the resistance of the spore
at different Aw in water, aqueous solutions ofNaCl, LiCI,
glucose and glycerol and in whole egg powder, fish
protein concentrate and wheat flour. Maximum resist-
ance in water vapor was at Aw = 0.21 and in glycerol at
Aw = 0.33. Little change in resistance was noted in NaCI
and glucose with decreases in Aw over the range tested.
The spores in LiCl had a minimum resistance at
Aw = 0.5 with increases at higher and lower Aw. The
resistance of the spores in egg powder, fish protein
concentrate and wheat flour was maximum at Aw =0.33.
Germicides
Just as the spore is resistant to destruction by heat,
the spore resists destruction with chemicals. The effect of
some of these chemicals on the germicidal resistance of
the spore is shown in Table 2. The result of our own work
TABLE 2. Germicidal resistance q{B. stearothermophilius.
Germicide Resistance Reference
5o/ophenol 50% kill in 5 days (57)
5%white coal tar disinfectant 60%kill in 5 days (57)
2%chlorhexidine digluconate No kill in 2 days (57)
1% HCI in 70% EtOH 106 kill in 5 h (57)
0.2% chlorocresol No kill (5)
.0 l% benzalkonium chloride No kill (5)
500 ppm peracetic acid 1Q6 kill in 15 min (.10)
D = 1.5 min (til)
25.8%H 202
10 URNAL OF FOOD PRO TEC110N, VOL. 44, FEBRUARY 1981
161
with other germicides at 25 C, pH 6.5 is shown in
Table 3.
TABLE 3. Germicidal resismnce of B. stearothermophilus
Germicide Resistance
500 ppm iodophor 99.99% kill in >60 min
200 ppm chlorine 99.99%kill in 9 min
25 ppm chlorine dioxide 99.99%kill in 12 min
3.5 ozone 99.9%kill in 9 min
PATHOGENICITY
Breed et al. (S} felt that thermophilic bacteria could
not be pathogenic as they would not grow at
temperatures found in the human body. Sattar et al. (55)
investigated use of B. stearothermophilus var. Ottawa 70
as a biological tracer. They challenged mice to a spore
suspension given intraperitoneally or as an aersol. The IP
challenge was with 0.2 ml of spores at three different
concentrations, 10 3, 10 5, and 107 spores/0.2 mi. They
estimated that the mice inhaled about 5 x 103 spores
during this exposure. All mice were observed for 3 weeks.
They concluded that B. stearothermophilus was not
capable of producing an active infection in the mice
tested (3-week-old male Swiss albino, 25 g).
In conclusion, B. stearothermophilus spores are
ubiquitous, non-pathogenic, difficult to destroy with heat
or chemicals and difficult to characterize with regard to
growth, germination and sporulation requirements.
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