Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a process for produc-
ing lactase from a heat-toleran~ organism belonging to
the genus Bacillus.
There has been considerable interest in recent
years in developing methods for reducing lactose level3
in milk and milk-derived products. This interest has been
.
heightened by recent evidence that a large percentage of
the human population suffers from a lactase deficiency which
is either an inherited trait or a result of the aging pro-
cess. Such lactase deficiencies lead to intestinal dis-
order3 when dietary levels of lactose are high. Moreover,
a similar laatose intolerance has also been observed in
certain domestic animals.
The hydrolysis of lactose3 in milk and milk-
derived products to produce glucose and galactose is an
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attractive goal not only becau3e it would solve the lactose
intolerance problem but it would also increase the sweet-
ness of the products and reduce so-called sandy textures
in certain milk-derived products caused by laotose crystal-
lization. Those working in the field have long appreciatedthe desirability of effecting this bydrolysis by the use Qf
lactase. In spite of khe fact that lacta~e occurs rather
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widely in nature and is produced by many microorganisms,
the use o~ lactase in the commercial production of milk
and milk-derived produat6 has been very limited. One
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reason for the limited commercial use of lactase is that
many of the common lactases such as those derived from
yeast exhibit optimum enzyme activity at temperatures
which are also conducive to bacterial growth. Accordingly,
there has been increasing interest in finding a lactase
having a high degree of hea~ stability. Such a heat stable
lactase would permit lactose hydrolysis to be carried out
under conditions that are unfavorable for growth for certain
bacteria which are commonly present in milk or milk-deriv~d
products. One such lactase derived from Streptomyces
coelico~lor has recently been described in United States
Patent No. 3,816,259. It is not clear at this time, how-
ever, whether lactase from S. coelicolor can be used with
impunity because certain members of that species have been
reported to produce antibiotics.
The production of lactase by members of the
genus Bacillus has been previously reported. P.J. Anema
has reported in Biochim. Biophys. Acta 89 (3), 495-502
(1964) the isolation of lactase from B. subtllis. Lactase
from B. me~atexium was described by S.R. Rohlfing and
I.P. Crawford in J. Bacteriology 92 (4), 1258-9 (1966~.
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Neither o these organisms, however, is regarded as heat-
tolerant and the lactase produced by them must generally
be used at temperatures below about 50C. in order to re-
tain u~eful enzyme ackivity for extended periods of time.
In accordance with the present invention thereis provided a proceas for produci~g a lactase having im-
~ proved thermal stability which comprises cultivating an
; organism belonging to the genus Bacillus in a nutrient
medium containing lactose and recovering the lacta~e thereby
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1~554~6
produced, the organism being one which exhibits optimum
growth at a temperature of at least about 45C.
A preferred organism for use in accordance with
this invention has been isolated from a soil sample.
Culturing the organism in a suitable nu~rient medium con-
taining lactose produces the desired lactase intracellular-
ly. Characterization of this culture has been carried out
and it has been identified as falling within the species
Bacillus coagulans according to the classification given
in Bergey's Manual of Determinative Bacteriology, 7th
Edition. This organism is included in the culture collec-
tion of the U.S.D.A. Northern Regional Research Laboratory
under the designation NRRL Ei-8100. The taxonomic properties
of this strain are shown in Table 1. ~ -
TABLE I
A. Morphological characteristics
1. Vegetative RDds: Less than 0.9 micron in diameter
varying in length generally up to
5.0 - 6.0 microns. Some fila-
ments. Not in chains. Gram
positive, staining uniformly.
Motile. :~
2. Sporangia: Generally not swollen but
occasional swollen sporangia may
be found.
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3. Spores: 0.9 by 1.2 to 1.5 microns, ellip-
~oidal, subterminal to terminal.
B. Cultural Characteristics
1. Gelatin agar streak plate -- No hydrolysis.
2. Agar colonies -- Opaque, small, round. Not
distinctive.
3. Agar slants -- Scant to moderate growth. ~lat,
~mooth, opaque.
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4. Glucose agar slant -- Growth heavier than on
nutrient agar. Smooth white.
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5. Glucose asparagine agar slants -- No growth at 24
hours. Moderate
growth at 48 hours.
6. Proteose peptone acid agar slants -- Good growth,
better than on
nutrient agar.
7. Soybean agar slants -- Growth moderate, slightly
heavier than on nutrient agar.
`' 8. Stock culture agar slants -- Growth scant at 24 hours,
as good as nu~rient agar
at 48 hours.
9. Broth -- Growth poor after 24 hours.
10. Sodium chloride broth -- No growth in 7% sodium
chloride.
}5 ll. Milk agar streak plate -- No hydrolysis.
; 12. Potato -- Scant, dry, wrinkled.
C. Physi~logical Characteristics
l. Using peptone as the nitrogen source, the organism
produced acid but no gas from glucose, lactose,
~arab:inose, xylose, mannitol and maltose. Neutral
reaction from sucrose and~glycerol.
2. The pH of glucose broth is 5.0 or less in seven days.
3. Citrates not utilize~.
4. Tomàto yeast milk curdled in 24 hours at 45C.
25~ 5. Nltrites not produced from nitrates~
6. Voges-Proskauer tes~ i9 negative. Ph o~ Voges-
Proskauer broth is 4.2.
7. Hydrolysis of starch - Positive.
8. Catalase - Po~itive.
30 ~ 9. No growth in nitrate medium under anaerobic condi-
tions. Growth in glucose broth under anaerobic con-
ditions produces a pH less than 5.2 in seven days.
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~ 10. Aerobic, facultatively anaerobic.
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11. Minimum temperature for growth is 25C. Maximum
temperature or growth is 60C. Optimum growth
occurs at 45-50C.
- As indicated unfler the physiological characteristics
listea in Table l, the Bacillus organism disclosed herein
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exhibits optimum growth at temperatures of about 45-50C~
and is, therefore, regarded as a heat-tolerant organism in
comparison with other Bacilli which display optimum growth
at about 37C. A heat-tolerant Bacillus organism is de-
fined herein as one which exhibits optimum growth at temp-
eratures of about 45C. and above.
E X A M P L E
____
The pH optimum of the lactase produced by this
organism was determined by assaying whole cells in the
presence of a-nitrophenyl-~-galactoside (O~PG) as the sub-
strate. The procedure used was essentially that of
J. Lederberg as described in J. Bacteriology 60, 381 tl950).
Cells were first treated with toluene, a phosphate buffer
was used and the assay temperature was 37C. hactase
activity was observed in the range o~ about pH 4.5 to 8.0
with optimum activity occurring at about pH 6Ø
Stabili~y of the lactase enzyme produced by
B. coagulans was evaluated by using ONPG as the substrate
in a modification of the Lederberg procedure. For this
evaluation washed cells were suspended in 0.05M pho~phate
buffer at pH 7.0 in the presence o~ the ONPG substrate.
The suspenslon was then maintained at 60C. for ~our days
with ~amples being periodically withdrawn for determining
the lactase activity remaining. The temperature for this
~i 25 stability test was selected as approximating the temperature
levels used in low temperature pasteurization. Results of `
the test are sbown in Table 2.
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TABLE 2
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Lactase Activity as Per Cent
Time in Hours of Original Activity
100 ~
21 92
51
71 36
99 20
The lactase produced by Bacillus coagulans
exhibits useful enzyme activity up to about 70C. Optimum ~-
activity appears to occur at temperatures of 60 to 65C. -.
The particular temperature selected for effecting lactose - : :
. . . .hydrolysis by the use of this enzyme will depend somewhat :
on the substrate medium involved. Generally speaking,
however, temperatures between about 45 and 65C. are pre- :
!~ ferred.
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~3;~ A typical medium for cultivating Bacillus co-
agulans to produce lactase is as follows:
Proteose peptone 1.0% ~ :
: 20 Yeast extract 1.0%
Potassium dihydrogen phosphate 0.8%
: Lactose (Rterilized separately) 2.0
pH 6.0
Cultureis are incubated on a rotary shaker for 48 hours at
:25 45C. At the end of the incubation period toluene is added
to the broth ~0.5% on a volume per volume basis) and the
mixture is agitated for 30 minutes. Cell~ are then recover-
ed by floccuLation techniques described in United States
Patent No. 3,821,086 and the floaculated cell aggregate
thereby obtained is dried at 55C. Lacta~e activity of khe
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dried aggregate particles is 38.5 units per gram where a -
unit is defined as the quantity of enzyme necessary to pro
duce one micromole of dextrose per minu~e under the assay
conditions. The assay method used for this determination
is that of Weetall et al~, as published in Biotechnology
and Bioengineering 16, 295 (1974).
The effectiveness of the lactase produced by
Bacillus coa~ulans was demonstrated by the hydrolysis of
lactose in a sweet whey feed stock. The organism was
cultivated and the cells were recovered as described above. ~ -
The dried aggregate particles wsre sieved and 5 grams of
the 16-20 mesh portion were hydrated in a S0 per cent
lactose solution buffered at pH 7.0 with a 0.05M phosphate -
buffer. The hydrated particles were then packed into a
small glass column that was maintained at a ~emperature of
60C~ Through this packed column was passed continuously
an aqueous solution containing 70 grams of a commercial
,~ dried sweet whey powder per liter, the solution being
buffered with 0.05M phosphate at p~I 7Ø The feed solukion
~ 20 contained approximately 5 per cent by weight lactose based
-~ on the lactose content of the sweet whey and 100 milligrams
.
per liter of methyl p-hydroxybenzoate was added as a pre-
~~ ~ servative. Flow rate through the column was maintained at
'~ 375 milliliters per day and the degree of lactose hydrolysis
was monitored daily by routine analysis. Initial degree of
lactose hydrolysis was found to be 90 per cent. After 3
weeks of continuous operation the degree of lactose hydrolysis
had decreased to 80 per cent.
; It will be appreciated tha~ the lactasq produced in
accordance with this lnvention mav be used for either batch
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or continuous treatment of lactose substrate media. More- :
over, the lactase may be utilized by direct use of the cells
or it may be used in the form of cell-free enzyme by apply-
ing techniques known to those skilled in the art.
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