U.S. patent application number 14/119071 was filed with the patent office on 2014-04-24 for method for killing microorganism.
This patent application is currently assigned to Kabushiki Kaisha Yakult Honsha. The applicant listed for this patent is Masakazu Ikeda, Masahiko Ito, Keiko Kasaha, Tadashi Sato. Invention is credited to Masakazu Ikeda, Masahiko Ito, Keiko Kasaha, Tadashi Sato.
Application Number | 20140113350 14/119071 |
Document ID | / |
Family ID | 47217185 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113350 |
Kind Code |
A1 |
Ikeda; Masakazu ; et
al. |
April 24, 2014 |
METHOD FOR KILLING MICROORGANISM
Abstract
Provided is a technique for enabling a liquid of microbial cells
having an enzymatic activity to be easily stored and used. The
technique is a method for killing a microorganism while maintaining
the enzyme titer of a microbial cell liquid, characterized by
including adjusting the pH of a liquid of microbial cells having an
enzymatic activity, and then performing a heating treatment of the
liquid, and also the technique is a method for killing a
microorganism while maintaining the enzyme titer of a microbial
cell liquid, characterized by including adding a carbohydrate to a
liquid of microbial cells having an enzymatic activity, and then
performing a heating treatment of the liquid.
Inventors: |
Ikeda; Masakazu; (Tokyo,
JP) ; Sato; Tadashi; (Tokyo, JP) ; Kasaha;
Keiko; (Tokyo, JP) ; Ito; Masahiko; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ikeda; Masakazu
Sato; Tadashi
Kasaha; Keiko
Ito; Masahiko |
Tokyo
Tokyo
Tokyo
Tokyo |
|
JP
JP
JP
JP |
|
|
Assignee: |
Kabushiki Kaisha Yakult
Honsha
Tokyo
JP
|
Family ID: |
47217185 |
Appl. No.: |
14/119071 |
Filed: |
May 18, 2012 |
PCT Filed: |
May 18, 2012 |
PCT NO: |
PCT/JP12/62746 |
371 Date: |
November 20, 2013 |
Current U.S.
Class: |
435/207 |
Current CPC
Class: |
C12N 9/2471 20130101;
C12N 1/06 20130101; C12Y 302/01023 20130101 |
Class at
Publication: |
435/207 |
International
Class: |
C12N 9/38 20060101
C12N009/38 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2011 |
JP |
2011-113170 |
May 20, 2011 |
JP |
2011-113171 |
Claims
1. A method for killing a microorganism while maintaining an enzyme
titer of a microbial cell liquid, the method comprising adjusting
the pH of a liquid of microbial cells having an enzymatic activity,
and then performing a heating treatment of the liquid, wherein an
enzyme titer of the liquid of microbial cells is maintained.
2. The method of claim 1, wherein the pH of the liquid of microbial
cells is adjusted to 3.5 to 6.5.
3. The method of claim 1, wherein the heating treatment is
performed at 40 to 50.degree. C. for at least 1 hour.
4. The method of claim 1, wherein the enzymatic activity is
.beta.-galactosidase activity.
5. The method of claim 1, wherein the microbial cells are yeast
cells.
6. The method of claim 5, wherein the yeast is Sporobolomyces
singularis.
7. The method of claim 1, wherein the enzyme titer after performing
the heating treatment is 80% or more of the enzyme titer before
performing the heating treatment.
8. The method of claim 1, further comprising adding a carbohydrate
to the liquid of microbial cells (i) before adjusting the pH, (ii)
after adjusting the pH but before performing the heating treatment,
or both (i) and (ii).
9. The method of claim 8, wherein the carbohydrate is added in an
amount of 0.2 to 30 mass/vol %.
10. The method of claim 1, wherein the pH is adjusted using a
carbonate.
11. A killed microbial cell liquid, wherein an enzyme titer of a
microbial cell liquid before killing microbial cells is
maintained.
12. The killed microbial cell liquid of claim 11, wherein the
microbial cells have .beta.-galactosidase activity.
13. The killed microbial cell liquid of claim 11, wherein the
microbial cells are yeast cells.
14. The killed microbial cell liquid of claim 13, wherein the yeast
is Sporobolomyces singularis.
15. The killed microbial cell liquid of claim 11, wherein the
enzyme titer after killing the microbial cells is 80% or more of
the enzyme titer before killing the microbial cells.
16. The killed microbial cell liquid of claim 11, obtained by
adjusting the pH of a liquid of microbial cells having an enzymatic
activity, and then performing a heating treatment of the liquid,
wherein an enzyme titer of the liquid of microbial cells is
maintained.
17. The killed microbial cell liquid of claim 16, wherein the pH of
the microbial cell liquid is adjusted to 3.5 to 6.5.
18. The killed microbial cell liquid of claim 16, wherein the
microbial cell liquid is heated at 40 to 50.degree. C. for at least
1 hour.
19. The killed microbial cell liquid of claim 16, wherein a
carbohydrate is added to the liquid of microbial cells (i) before
adjusting the pH, (ii) after adjusting the pH but before performing
the heating treatment, or both (i) and (ii).
20. The killed microbial cell liquid of claim 19, wherein the
carbohydrate is added in an amount of 0.2 to 30 mass/vol %.
21. The killed microbial cell liquid of claim 16, wherein the pH is
adjusted using a carbonate.
22. A dry killed microbial cell powder obtained by drying the
killed microbial cell liquid of claim 11.
23. An enzyme composition comprising the killed microbial cell
liquid of claim 11.
24. A method for killing a microorganism while maintaining an
enzyme titer of a microbial cell liquid, the method comprising
adding a carbohydrate to a liquid of microbial cells having an
enzymatic activity, and then performing a heating treatment of the
liquid, wherein an enzyme titer of the liquid of microbial cells is
maintained.
25. The method of claim 24, wherein the heating treatment is
performed at 40 to 55.degree. C. for at least 1 hour.
26. The method of claim 24, wherein the enzymatic activity is
.beta.-galactosidase activity.
27. The method of claim 24, wherein the microbial cells are yeast
cells.
28. The method of claim 27, wherein the yeast is Sporobolomyces
singularis.
29. The method of claim 24, wherein the carbohydrate is added to
the liquid of microbial cells in an amount of 0.2 to 30 mass/vol
%.
30. The method of claim 24, wherein the carbohydrate is at least
one carbohydrate selected from the group consisting of lactose,
glucose, maltose, a galactooligosaccharide, and a dextrin.
31. The method of claim 24, wherein the enzyme titer after
performing the heating treatment is 80% or more of the enzyme titer
before performing the heating treatment.
32. A killed microbial cell liquid, comprising a carbohydrate,
wherein an enzyme titer of a microbial cell liquid before killing
microbial cells is maintained.
33. The killed microbial cell liquid of claim 32, wherein the
microbial cells have .beta.-galactosidase activity.
34. The killed microbial cell liquid of claim 32, wherein the
microbial cells are yeast.
35. The killed microbial cell liquid of claim 34, wherein the yeast
is Sporobolomyces singularis.
36. The killed microbial cell liquid of claim 32, comprising the
carbohydrate in an amount of 0.2 to 30 mass/vol %.
37. The killed microbial cell liquid of claim 32, wherein the
carbohydrate is at least one carbohydrate selected from the group
consisting of lactose, glucose, galactose, a lactose isomer, a
galactooligosaccharide, maltose, a maltooligosaccharide, and a
dextrin.
38. The killed microbial cell liquid of claim 32, wherein the
enzyme titer after killing the microbial cells is 80% or more of
the enzyme titer before killing the microbial cells.
39. The killed microbial cell liquid of claim 32, obtained by
adding the carbohydrate to the liquid of microbial cells having an
enzymatic activity, and then performing a heating treatment of the
liquid, wherein an enzyme titer of the liquid of microbial cells is
maintained.
40. The killed microbial cell liquid of claim 39, wherein the
heating treatment is performed at 40 to 55.degree. C. for at least
1 hour.
41. A dry killed microbial cell powder obtained by drying the
killed microbial cell liquid of claim 32.
42. An enzyme composition comprising the killed microbial cell
liquid of claim 41.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for killing a
microorganism, more particularly relates to a method for killing a
microorganism while maintaining the enzyme titer of a liquid of
microbial cells having an enzymatic activity.
BACKGROUND ART
[0002] There are many microorganisms that have a useful enzymatic
activity, and such microorganisms are widely used in the production
of functional food materials such as carbohydrates, amino acids,
and phospholipids. Among these, there are known many microorganisms
which can be used in the production of carbohydrate materials,
particularly oligosaccharides, and for example, it has been
reported that a galactooligosaccharide is produced by utilizing the
.beta.-galactosidase activity of yeast belonging to the genus
Sporobolomyces singularis (PTL 1).
[0003] Generally speaking, a microorganism is killed by a heating
treatment and then stored etc, however, in the case where a
microorganism having an enzymatic activity as described above is
subjected to a heating treatment, whilst the microorganism is
killed, the enzymatic activity is significantly decreased, which is
practically useless. Further, as a technique for killing a
microorganism while maintaining the enzymatic activity, there has
been reported a technique for killing a microorganism while
maintaining the enzymatic activity in which a transformed
microorganism is treated with a chemical such as an alcohol at 25
to 35.degree. C. (PTL 2). However, this technique has a problem
that the use thereof after killing the microorganism is limited
because a genetically modified microorganism is used or the alcohol
or the like used in the treatment for killing the microorganism
remains.
CITATION LIST
Patent Literature
[0004] PTL 1: JP-B-5-58714 [0005] PTL 2: JP-A-2002-355028
SUMMARY OF INVENTION
Problems to be Solved by the Invention
[0006] Accordingly, an object of the invention is to provide a
technique for enabling a liquid of microbial cells having an
enzymatic activity to be easily stored and used.
Means for Solving the Problems of the Invention
[0007] The inventors of the present invention made intensive
studies of the conditions for killing a microorganism by a heating
treatment in order to solve the above problems, and as a result,
they found that the microorganism can be killed while maintaining
the enzyme titer by adjusting the pH before performing the heating
treatment for killing the microorganism in a liquid of microbial
cells having an enzymatic activity or by allowing a carbohydrate to
be present when performing the heating treatment, and thus
completed the invention. Further, they found that the microbial
cell liquid in which the microorganism has been killed as described
above maintains the enzyme titer of the microbial cell liquid
before killing the microorganism, and thus completed the
invention.
[0008] That is, the invention is directed to a method for killing a
microorganism while maintaining the enzyme titer of a microbial
cell liquid, characterized by including adjusting the pH of a
liquid of microbial cells having an enzymatic activity, and then
performing a heating treatment of the liquid.
[0009] Further, the invention is directed to a killed microbial
cell liquid, characterized in that the enzyme titer of the
microbial cell liquid before killing the microorganism is
maintained.
[0010] Still further, the invention is directed to a method for
killing a microorganism while maintaining the enzyme titer of a
microbial cell liquid, characterized by including adding a
carbohydrate to a liquid of microbial cells having an enzymatic
activity, and then performing a heating treatment of the
liquid.
[0011] Yet still further, the invention is directed to a killed
microbial cell liquid, characterized in that a carbohydrate is
contained, and the enzyme titer of the microbial cell liquid before
killing the microorganism is maintained.
Advantages of the Invention
[0012] According to the method for killing a microorganism while
maintaining the enzyme titer of a microbial cell liquid of the
invention, the microorganism can be killed while maintaining the
enzyme titer of a liquid of microbial cells having an enzymatic
activity. Further, according to this method, the thermal stability
of the enzyme can be increased, and the microorganism can be killed
while maintaining the enzyme titer under wider temperature
conditions. Therefore, the microbial cell liquid after killing the
microorganism can be easily stored and used. That is, in the case
where the microorganism is alive, some sort of metabolite is
produced by the microorganism during storage to deteriorate the
quality of the microbial cell liquid, and also the storage
stability of the enzyme titer may be affected. Meanwhile, in the
case where the microorganism has been killed, such a problem does
not arise, and therefore, the killed microbial cell liquid has
excellent storage stability with respect to the enzyme titer.
[0013] Further, the killed microbial cell liquid of the invention
is configured such that the microorganism has been killed in a
state where the enzymatic activity is maintained, and therefore can
be easily stored and used.
[0014] Further, by drying the above-described killed microbial cell
liquid to form a dry killed microbial cell powder, it can be stably
stored and used for a longer time.
BRIEF DESCRIPTION OF DRAWINGS
[0015] FIG. 1 is a view showing a residual titer ratio after
performing a heating treatment by maintaining an Ss concentrate
liquid having a lactose concentration of 2 mass/vol % at 45.degree.
C. or 50.degree. C. for 5 hours in Example 9.
[0016] FIG. 2 is a view showing a residual titer ratio after
performing a heating treatment by maintaining an Ss concentrate
liquid having a lactose concentration of 2 mass/vol % at 45.degree.
C. or 50.degree. C. for 18 hours in Example 9.
[0017] FIG. 3 is a view showing a residual titer ratio after
performing a heating treatment by maintaining an Ss concentrate
liquid having a lactose concentration of 5 mass/vol % at 45.degree.
C. or 50.degree. C. for 5 hours in Example 9.
[0018] FIG. 4 is a view showing a residual titer ratio after
performing a heating treatment by maintaining an Ss concentrate
liquid having a lactose concentration of 5 mass/vol % at 45.degree.
C. or 50.degree. C. for 18 hours in Example 9.
[0019] FIG. 5 is a view showing a residual titer ratio after
adjusting the pH of an Ss culture solution using a sodium hydroxide
solution, a potassium hydroxide solution, or a sodium carbonate
solution at various concentrations in Example 11.
DESCRIPTION OF EMBODIMENTS
[0020] The method for killing a microorganism while maintaining the
enzyme titer of a microbial cell liquid according to the invention
(hereinafter referred to as "the present inventive method") can be
performed by a method including adjusting the pH of a liquid of
microbial cells having an enzymatic activity, and then, performing
a heating treatment of the liquid (hereinafter referred to as "the
present inventive method 1") or a method including adding a
carbohydrate to a liquid of microbial cells having an enzymatic
activity, and then performing a heating treatment of the liquid
(hereinafter referred to as "the present inventive method 2").
[0021] In the present inventive method, the microorganism to be
killed is not particularly limited as long as it is a microorganism
such as a bacterium, yeast, or a fungus, and an enzyme is bound to
cell walls or an enzyme is intracellularly and/or extracellularly
produced, that is, it is a microorganism having an enzymatic
activity. Examples of such a microorganism having an enzymatic
activity include bacteria belonging to the genus Streptococcus, the
genus Lactobacillus, the genus Bacillus, the genus Bifidobacterium,
etc.; and yeast belonging to the genus Sporobolomyces, the genus
Bullera, the genus Kluyveromyces, the genus Lipomyces, the genus
Candida, the genus Cryptococcus, the genus Sterigmatomyces, the
genus Bensingtonia, the genus Ballistosporomyces, the genus
Fellomyces, the genus Filobasidium, the genus Sirobasidium, the
genus Tilletiopsis, the genus Itersonilia, the genus Tilletia, the
genus Saccharomyces, the genus Schizosaccharomyces, the genus
Hansenula, the genus Rhodotorula, the genus Debaryomyces, the genus
Pichia, and the genus Torulopsis. Further, examples of the enzyme
include carbohydrate degrading enzymes such as amylase, sucrase,
.alpha.-galactosidase, .beta.-galactosidase, glucose isomerase,
.alpha.-glucosidase, .beta.-glucosidase, .beta.-fructofuranosidase,
.alpha.-mannosidase, .beta.-mannosidase, and xylanase.
[0022] Among the above-described microorganisms having an enzymatic
activity, a microorganism having .beta.-galactosidase activity is
preferred. Examples of such a microorganism include yeast belonging
to the genus Sporobolomyces, the genus Bullera, the genus
Kluyveromyces, the genus Lipomyces, the genus Candida, the genus
Cryptococcus, the genus Sterigmatomyces, the genus Bensingtonia,
the genus Ballistosporomyces, the genus Fellomyces, the genus
Filobasidium, the genus Sirobasidium, the genus Tilletiopsis, the
genus Itersonilia, the genus Tilletia, the genus Saccharomyces, the
genus Schizosaccharomyces, the genus Hansenula, the genus
Rhodotorula, the genus Debaryomyces, the genus Pichia, and the
genus Torulopsis; and bacteria belonging to the genus
Streptococcus, the genus Lactobacillus, the genus Bacillus, and the
genus Bifidobacterium.
[0023] Further, among the above-described microorganisms having
.beta.-galactosidase activity, yeast or a bacterium is preferred,
and in particular, yeast is more preferred. Specific examples of
the yeast having .beta.-galactosidase activity include particularly
Sporobolomyces singularis, Sterigmatomyces elviae, Cryptococcus
laurentii, Rhodotorula lactosa, Sirobasidium magnum, and Lipomyces
lipofer. Specific examples of the bacterium having
.beta.-galactosidase activity include particularly Streptococcus
thermophilus, Lactobacillus bulgaricus, Streptococcus lactis,
Lactobacillus salivarius, Lactobacillus leichmannii, Lactobacillus
helveticus, Bacillus brevis, Bacillus stearothermophilus,
Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium
longum, and Bifidobacterium adolescentis.
[0024] As the yeast having .beta.-galactosidase activity which is
particularly preferably used, Sporobolomyces singularis can be
exemplified, and Sporobolomyces singularis JCM 5356 (ATCC 24193),
which is one of the examples thereof, is available for a fee from
RIKEN BioResource Center (2-1 Hirosawa, Wako-shi, Saitama-ken,
351-0198, Japan), ATCC (10801 University Boulevard Manassas, Va.,
20110, USA), or the like.
[0025] Further, as another example of Sporobolomyces singularis,
yeast obtained as a .beta.-galactosidase high-producing mutant
microorganism by a production method described in JP-A-2003-325166
can be exemplified. Among these, as a specific example of yeast
obtained by the steps (a) to (c) in the above-described patent
literature using the above-described Sporobolomyces singularis JCM
5356 as a parent strain, Sporobolomyces singularis ISK-#4D4,
ISK-#5A5, and ISK-##2B6 can be exemplified, and these strains were
deposited on Apr. 10, 2002 in International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology (Chuo 6, Higashi 1-1-1, Tsukuba-shi, Ibaraki-ken,
305-8566, Japan) under accession Nos. FERM P-18818, FERM P-18819,
and FERM P-18817, respectively.
[0026] Among the present inventive methods for killing a
microorganism described above, the method including adjusting the
pH of a liquid of microbial cells having an enzymatic activity, and
then performing a heating treatment of the liquid (the present
inventive method 1) will be described. In the implementation of the
present inventive method 1, first, a culture solution is obtained
by culturing a microorganism having an enzymatic activity in a
medium or the like according to a common procedure. This culture
solution may be used as the microbial cell liquid as it is, or a
liquid obtained by appropriately performing washing or
concentration using a centrifuge, a membrane concentration device,
or the like may be used as the microbial cell liquid. The solid
content in this microbial cell liquid is not particularly limited,
and specifically, a solid content of 0.5 to 10% can be exemplified.
Incidentally, the "solid content" in this specification refers to a
solid content of the cells in the microbial cell liquid, and for
example, in the case where medium components are contained in the
microbial cell liquid, the solid content derived from the medium
components is not included in the "solid content" in this
specification.
[0027] Subsequently, the pH of this microbial cell liquid is
adjusted. The range of the pH is not particularly limited, however,
from the viewpoint that the enzyme titer can be maintained higher
in a wider temperature range, the range of the pH is preferably
from 3.5 to 6.5, more preferably from 4.2 to 6.3, further more
preferably from 4.5 to 5.7. A substance to be used for adjusting
the pH is not particularly limited, and any of an acid, a base, and
a salt can be used. Specifically, hydrochloric acid, acetic acid,
sulfuric acid, an aqueous solution of sodium hydroxide, potassium
hydroxide, sodium carbonate, or the like, or any of a variety of
buffers such as a sodium phosphate buffer can be used as needed,
however, from the viewpoint that the titer after the pH adjustment
is not decreased, it is preferred to use a carbonate, and it is
more preferred to use one or more carbonates selected from the
group consisting of sodium carbonate, potassium carbonate, ammonium
carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate,
and ammonium hydrogen carbonate.
[0028] Incidentally, from the viewpoint that the thermal stability
of the enzyme is further improved and the enzyme titer can be
maintained higher, it is preferred that a carbohydrate is added to
the microbial cell liquid before adjusting the pH and/or a
carbohydrate is added to the microbial cell liquid after adjusting
the pH and before performing the heating treatment. The
carbohydrate to be used here is not particularly limited, and any
of a monosaccharide, a disaccharide, a tri- or higher
oligosaccharide, and a polysaccharide can be used. Examples of the
monosaccharide include glucose, galactose, fructose, and mannose,
examples of the disaccharide include lactose, lactose isomers,
maltose, sucrose, and trehalose, examples of the tri- or higher
oligosaccharide include various oligosaccharides such as
galactooligosaccharides, maltooligosaccharides, and
fructooligosaccharides, and examples of the polysaccharide include
dextrins and starch. Among these carbohydrates, from the viewpoints
of an effect of maintaining the enzyme titer and cost, at least one
carbohydrate selected from the group consisting of lactose,
glucose, maltose, a galactooligosaccharide, and a dextrin is
preferred, and in particular, it is preferred to use at least one
carbohydrate selected from the group consisting of lactose,
glucose, maltose, and a galactooligosaccharide. The amount of the
carbohydrate to be added to the microbial cell liquid is not
particularly limited, however, the lower limit of the addition
amount of the carbohydrate is, for example, preferably 0.2 mass/vol
% (hereinafter simply expressed in "%") or more, more preferably
0.5% or more, further more preferably 2% or more with respect to
the amount of the microbial cell liquid. Further, when or how the
carbohydrate is added to the microbial cell liquid is not
particularly limited, and for example, a method in which a
separately prepared concentrated solution of the carbohydrate is
added can be exemplified, however, when the amount of a
carbohydrate solution to be added to the microbial cell liquid is
too large, a problem may arise in some cases that the concentration
of the microbial cells in the microbial cell liquid is decreased,
and therefore, the enzyme titer per unit weight of the microbial
cell liquid is decreased. Accordingly, the upper limit of the
addition amount of the carbohydrate is preferably 30% or less, more
preferably 15% or less, further more preferably 10% or less. In
view of this, the amount of the carbohydrate to be added to the
microbial cell liquid is preferably from 0.2 to 30%, more
preferably from 0.5 to 15%, further more preferably from 0.5 to
10%, particularly preferably from 2 to 10%.
[0029] In the case where the carbohydrate to be added to the
microbial cell liquid can be a substrate for the enzyme (for
example, in the case where lactose is added to a liquid of
microbial cells having .beta.-galactosidase activity), there may be
cases where part or most of the carbohydrate is subjected to an
enzymatic reaction during a period from when the carbohydrate is
added to when the heating treatment is completed, however, the
effect of the addition of the carbohydrate is exhibited regardless
of the degree of reaction (the degree of degradation or the degree
of polymerization), and therefore, there is no problem at all from
the viewpoint of stabilization of the enzyme titer. For example,
after the carbohydrate is added, even if the microbial cell liquid
is kept as it is, or is subjected to a cooling treatment or a
heating treatment, there is no problem at all from the viewpoint of
stabilization of the enzyme titer. In addition, in such a case,
another carbohydrate produced by subjecting the added carbohydrate
to the enzymatic reaction is contained in the microbial cell liquid
in some cases, however, even if such a carbohydrate is present,
there is no problem at all from the viewpoint of stabilization of
the enzyme titer. Examples of the carbohydrate to be produced by
subjecting the carbohydrate added to the microbial cell liquid to
the enzymatic reaction include monosaccharides, disaccharides, tri-
or higher oligosaccharides, and polysaccharides, and examples of
the monosaccharide include glucose, galactose, fructose, and
mannose, examples of the disaccharide include lactose, lactose
isomers, maltose, sucrose, and trehalose, examples of the tri- or
higher oligosaccharide include various oligosaccharides such as
galactooligosaccharides and fructooligosaccharides, and examples of
the polysaccharide include dextrins and starch. Specifically, in
the case where the carbohydrate to be added to the liquid of the
microbial cells having a carbohydrate degrading enzyme (for
example, .beta.-galactosidase) activity is lactose, since lactose
is subjected to the enzymatic reaction, as the carbohydrate
contained in the microbial cell liquid, glucose, galactose,
lactose, a lactose isomer, and a galactooligosaccharide can be
exemplified; as the carbohydrate contained in the microbial cell
liquid in the case where the carbohydrate to be added is maltose,
glucose, maltose, and a maltooligosaccharide can be exemplified;
and as the carbohydrate contained in the microbial cell liquid in
the case where the carbohydrate to be added is a dextrin, glucose,
maltose, a maltooligosaccharide, and a dextrin can be exemplified.
Since it is preferred to use at least one carbohydrate selected
from the group consisting of lactose, glucose, maltose, a
galactooligosaccharide, and a dextrin as the carbohydrate to be
added to the microbial cell liquid, it is preferred that in the
microbial cell liquid to which such a carbohydrate was added, such
a carbohydrate and a carbohydrate produced by subjecting such a
carbohydrate to the enzymatic reaction are contained, specifically,
at least one carbohydrate selected from the group consisting of
lactose, glucose, galactose, a lactose isomer, a
galactooligosaccharide, maltose, a maltooligosaccharide, and a
dextrin is contained. Further, since it is more preferred to use at
least one carbohydrate selected from the group consisting of
lactose, glucose, maltose, and a galactooligosaccharide as the
carbohydrate to be added to the microbial cell liquid, it is more
preferred that in the microbial cell liquid, at least one
carbohydrate selected from the group consisting of lactose,
glucose, galactose, a lactose isomer, a galactooligosaccharide,
maltose, and a maltooligosaccharide is contained.
[0030] After adjusting the pH as described above, a heating
treatment is performed. The heating treatment method is not
particularly limited, and a method using a continuous plate heat
exchanger, a method of heating a tank containing the microbial cell
liquid by heating steam or hot water in a batchwise process, or the
like can be applied, however, in order to control the temperature
so as to prevent the inactivation of the enzyme as much as
possible, a method of heating a tank containing the microbial cell
liquid by heating steam or hot water in a batchwise process is
preferred. The heating treatment in a batchwise process is not
particularly limited as long as the conditions for the heating
treatment can kill the microorganism while maintaining the enzyme
titer, however, it is performed at preferably 40 to 50.degree. C.,
more preferably 40 to 48.degree. C., further more preferably 40 to
46.degree. C. Further, in the case where a carbohydrate is added to
the microbial cell liquid before adjusting the pH and/or a
carbohydrate is added to the microbial cell liquid after adjusting
the pH and before performing the heating treatment in a batchwise
process, the heating treatment is performed at preferably 40 to
60.degree. C., more preferably 40 to 55.degree. C., further more
preferably 40 to 50.degree. C. Further, the heating time is also
not particularly limited, but is preferably 1 hour or more, more
preferably 6 hours or more. However, if the heating time is too
long, the enzyme titer is decreased in some cases, and therefore,
the heating time is preferably 24 hours or less, more preferably 20
hours or less, further more preferably 18 hours or less. In view of
this, the heating time is preferably from 1 to 24 hours, more
preferably from 1 to 20 hours, further more preferably from 1 to 18
hours, still further more preferably from 6 to 24 hours, yet still
further more preferably from 6 to 20 hours, particularly preferably
from 6 to 18 hours. Further, in the case where a carbohydrate is
added before performing the heating treatment, the heating time is
preferably from 1 to 24 hours, more preferably from 4 to 20 hours,
further more preferably from 5 to 18 hours. Incidentally, the
phrase "killing a microorganism" in the present inventive method 1
refers to the reduction of the live microbial cells by the heating
treatment to 1000 cfu/mL or less, and it is desirable to reduce the
live microbial cells to preferably 100 cfu/mL or less, more
preferably 10 cfu/mL or less.
[0031] By performing the heating treatment as described above, the
microorganism in the microbial cell liquid is killed while
maintaining the enzyme titer of the microbial cell liquid. Here,
the phrase "maintaining the enzyme titer" refers to that the enzyme
titer after performing the heating treatment (after killing the
microorganism) is 80% or more, preferably 90% or more, more
preferably 95% or more of the enzyme titer before performing the
heating treatment (before killing the microorganism).
[0032] The microbial cell liquid after the heating treatment (a
killed microbial cell liquid) can be stored for a long time under
low-temperature conditions of 25.degree. C. or lower, preferably
from 0 to 5.degree. C. Then, the microbial cell liquid after the
heating treatment can be used in various applications using the
enzymatic activity.
[0033] In addition, the microbial cell liquid after the heating
treatment can be further dried by a known drying method such as
spray drying or lyophilization and formed into a killed microbial
cell dry powder. At this time, in order to prevent a decrease in
enzyme titer due to the drying step or storage after drying, it is
also possible to add a carbohydrate to the microbial cell liquid
after the heating treatment. The carbohydrate to be used is not
particularly limited, and any of a monosaccharide, a disaccharide,
a tri- or higher oligosaccharide, and a polysaccharide can be used.
Examples of the monosaccharide include glucose, galactose,
fructose, and mannose, examples of the disaccharide include
lactose, lactose isomers, maltose, sucrose, and trehalose, examples
of the tri- or higher oligosaccharide include various
oligosaccharides such as galactooligosaccharides,
maltooligosaccharides, and fructooligosaccharides, and examples of
the polysaccharide include dextrins and starch. Among these
carbohydrates, from the viewpoints of an effect of maintaining the
enzyme titer, ease of drying, and cost, it is preferred to use at
least one carbohydrate selected from the group consisting of
lactose, maltose, and a dextrin, and in particular, it is preferred
to use lactose and/or maltose, and it is more preferred to use
lactose. The amount of the carbohydrate to be added is not
particularly limited, and the lower limit of the addition amount of
the carbohydrate is, for example, preferably 0.1% or more, more
preferably 0.5% or more, further more preferably 1% or more with
respect to the amount of the microbial cell liquid. On the other
hand, if the amount of the carbohydrate to be added to the
microbial cell liquid is too large, the enzyme titer per unit
weight of the dry microbial cell powder is decreased when the
microbial cell liquid is formed into a dry powder, and therefore,
the upper limit of the addition amount of the carbohydrate is
preferably 30% or less, more preferably 15% or less, further more
preferably 10% or less, still further more preferably 5% or less,
yet still furthermore preferably 3% or less. In view of this, the
amount of the carbohydrate to be added to the microbial cell liquid
is preferably from 0.1 to 30%, more preferably from 0.5 to 15%,
further more preferably from 0.5 to 10%, still further more
preferably from 1 to 10%, yet still further more preferably from 1
to 5%, particularly preferably from 1 to 3% with respect to the
amount of the microbial cell liquid. The method for adding the
carbohydrate is not particularly limited, however, for example, a
method in which a separately prepared concentrated solution of the
carbohydrate is added can be exemplified. The degree of drying is
not particularly limited, however, for example, the drying is
performed until the water content in the dry powder is reduced to
about 10% or less. By performing this drying, longer time storage
as compared with the case of a liquid, for example, 1 year or
longer storage can be achieved while maintaining the enzyme titer
at 80% or more of the enzyme titer immediately after drying.
Further, the dry killed microbial cell powder can be used in
various applications using the enzymatic activity.
[0034] As for the conditions for the above-described spray drying,
the inlet and outlet temperatures of a drying chamber may be in a
range in which the enzyme is not significantly inactivated, and
further, the rotation speed of an atomizer, the feeding amount of a
stock solution, etc. hardly affect the final enzyme titer, although
a dry microbial cell powder having slightly different properties as
a product is obtained depending on such conditions, and therefore,
it is not necessary to pay much attention to such conditions.
Specifically, the inlet temperature of a drying chamber can be, for
example, from 70.degree. C. to 200.degree. C., preferably from
110.degree. C. to 180.degree. C., and the outlet temperature of a
drying chamber can be, for example, from 50.degree. C. to
120.degree. C., preferably from 70.degree. C. to 90.degree. C.
Further, the rotation speed of an atomizer can be, for example,
from 10,000 to 30,000 rpm, and the feeding amount of a stock
solution can be, for example, from 0.2 to 200 kg/hour. The spray
drying can also be performed using a spraying system such as a
two-fluid nozzle other than an atomizer. Incidentally, from the
viewpoint that continuous production can be performed industrially,
it is preferred to use a spray drying method.
[0035] The thus obtained microbial cell liquid after the heating
treatment and dry killed microbial cell powder can also be
incorporated alone or in combination with each other to form an
enzyme composition. It is also possible to add a diluent, an
excipient, a surfactant, a preservative, or the like to this enzyme
composition within a range that does not affect the enzymatic
activity. Further, also this enzyme composition can be used in
various applications using the enzymatic activity.
[0036] Next, among the above-described present inventive methods
for killing a microorganism, a method including adding a
carbohydrate to a liquid of microbial cells having an enzymatic
activity, and then performing a heating treatment of the liquid
(the present inventive method 2) will be described. In the
implementation of the present inventive method 2, first, a culture
solution is obtained by culturing a microorganism having an
enzymatic activity in a medium or the like according to a common
procedure. This culture solution may be used as the microbial cell
liquid as it is, or a liquid obtained by appropriately performing
washing or concentration using a centrifuge, a membrane
concentration device, or the like may be used as the liquid of
microbial cells having an enzymatic activity. The solid content in
this microbial cell liquid is not particularly limited, and
specifically, a solid content of 0.5 to 10% can be exemplified. As
described above, the "solid content" in this specification refers
to a solid content of the cells in the microbial cell liquid, and
for example, in the case where medium components are contained in
the microbial cell liquid, the solid content derived from the
medium components is not included in the "solid content" in this
specification.
[0037] Subsequently, a carbohydrate is added to this microbial cell
liquid. The carbohydrate to be used here is not particularly
limited, and any of a monosaccharide, a disaccharide, a tri- or
higher oligosaccharide, and a polysaccharide can be used. Examples
of the monosaccharide include glucose, galactose, fructose, and
mannose, examples of the disaccharide include lactose, lactose
isomers, maltose, sucrose, and trehalose, examples of the tri- or
higher oligosaccharide include various oligosaccharides such as
galactooligosaccharides, maltooligosaccharides, and
fructooligosaccharides, and examples of the polysaccharide include
dextrins and starch. Among these carbohydrates, from the viewpoints
of an effect of maintaining the enzyme titer and cost, at least one
carbohydrate selected from the group consisting of lactose,
glucose, maltose, a galactooligosaccharide, and a dextrin is
preferred, and in particular, it is preferred to use at least one
carbohydrate selected from the group consisting of lactose,
glucose, maltose, and a galactooligosaccharide. The amount of the
carbohydrate to be added to the microbial cell liquid is not
particularly limited, however, the lower limit of the addition
amount of the carbohydrate is, for example, preferably 0.2% or
more, more preferably 0.5% or more, further more preferably 2% or
more with respect to the amount of the microbial cell liquid.
Further, when or how the carbohydrate is added to the microbial
cell liquid is not particularly limited, and for example, a method
in which a separately prepared concentrated solution of the
carbohydrate is added can be exemplified, however, when the amount
of a carbohydrate solution to be added to the microbial cell liquid
is too large, a problem may arise in some cases that the
concentration of the microbial cells in the microbial cell liquid
is decreased, and therefore, the enzyme titer per unit weight of
the microbial cell liquid is decreased. Accordingly, the upper
limit of the addition amount of the carbohydrate is preferably 30%
or less, more preferably 15% or less, further more preferably 10%
or less. In view of this, the amount of the carbohydrate to be
added to the microbial cell liquid is preferably from 0.2 to 30%,
more preferably from 0.5 to 15%, further more preferably from 0.5
to 10%, particularly preferably from 2 to 10%.
[0038] After adding a carbohydrate to the microbial cell liquid as
described above, a heating treatment is performed. The heating
treatment method is not particularly limited, and a method using a
continuous plate heat exchanger, a method of heating a tank
containing the microbial cell liquid by heating steam or hot water
in a batchwise process, or the like can be applied, however, in
order to control the temperature so as to prevent the inactivation
of the enzyme as much as possible, a method of heating a tank
containing the microbial cell liquid by heating steam or hot water
in a batchwise process is preferred. The heating treatment in a
batchwise process is not particularly limited as long as the
conditions for the heating treatment can kill the microorganism
while maintaining the enzyme titer, however, it is performed at
preferably 40 to 60.degree. C., more preferably 40 to 55.degree.
C., further more preferably 40 to 50.degree. C. Further, the
heating time is also not particularly limited, but is preferably 1
hour or longer, more preferably 4 hours or longer, further more
preferably 5 hours or longer. However, if the heating time is too
long, the enzyme titer may be decreased, and therefore, the heating
time is preferably 24 hours or shorter, more preferably 20 hours or
shorter, further more preferably 18 hours or shorter. In view of
this, the heating time is preferably from 1 to 24 hours, more
preferably from 4 to 20 hours, further more preferably from 5 to 18
hours. The phrase "killing a microorganism" in the present
inventive method 2 refers to the reduction of the live microbial
cells by the heating treatment to 1000 cfu/mL or less, and it is
desirable to reduce the live microbial cells to preferably 100
cfu/mL or less, more preferably 10 cfu/mL or less.
[0039] By performing the heating treatment as described above, the
microorganism in the microbial cell liquid is killed while
maintaining the enzyme titer of the microbial cell liquid. Here,
the phrase "maintaining the enzyme titer" refers to that the enzyme
titer after performing the heating treatment (after killing the
microorganism) is 80% or more, preferably 90% or more, more
preferably 95% or more of the enzyme titer before performing the
heating treatment (before killing the microorganism).
[0040] In the case where the carbohydrate to be added to the
microbial cell liquid can be a substrate for the enzyme (for
example, in the case where lactose is added to a liquid of
microbial cells having .beta.-galactosidase activity), there may be
cases where part or most of the carbohydrate is subjected to an
enzymatic reaction during a period from when the carbohydrate is
added to when the heating treatment is completed, however, the
effect of the addition of the carbohydrate is exhibited regardless
of the degree of reaction (the degree of degradation or the degree
of polymerization), and therefore, there is no problem at all from
the viewpoint of stabilization of the enzyme titer. For example,
after the carbohydrate is added, even if the microbial cell liquid
is kept as it is, or is subjected to a cooling treatment or a
heating treatment, there is no problem at all from the viewpoint of
stabilization of the enzyme titer. In addition, in such a case,
another carbohydrate produced by subjecting the added carbohydrate
to the enzymatic reaction is contained in the microbial cell
liquid, however, even if such a carbohydrate is present, there is
no problem at all from the viewpoint of stabilization of the enzyme
titer. Examples of the carbohydrate to be contained in the
microbial cell liquid include monosaccharides, disaccharides, tri-
or higher oligosaccharides, and polysaccharides, and examples of
the monosaccharide include glucose, galactose, fructose, and
mannose, examples of the disaccharide include lactose, lactose
isomers, maltose, sucrose, and trehalose, examples of the tri- or
higher oligosaccharide include various oligosaccharides such as
galactooligosaccharides and fructooligosaccharides, and examples of
the polysaccharide include dextrins and starch. Specifically, in
the case where the carbohydrate to be added to the liquid of the
microbial cells having a carbohydrate degrading enzyme (for
example, .beta.-galactosidase) activity is lactose, since lactose
is subjected to the enzymatic reaction, as the carbohydrate
contained in the microbial cell liquid, glucose, galactose,
lactose, a lactose isomer, and a galactooligosaccharide can be
exemplified; as the carbohydrate contained in the microbial cell
liquid in the case where the carbohydrate to be added is maltose,
glucose, maltose, and a maltooligosaccharide can be exemplified;
and as the carbohydrate contained in the microbial cell liquid in
the case where the carbohydrate to be added is a dextrin, glucose,
maltose, a maltooligosaccharide, and a dextrin can be exemplified.
Since it is preferred to use at least one carbohydrate selected
from the group consisting of lactose, glucose, maltose, a
galactooligosaccharide, and a dextrin as the carbohydrate to be
added to the microbial cell liquid, it is preferred that in the
microbial cell liquid to which such a carbohydrate was added, such
a carbohydrate and a carbohydrate produced by subjecting such a
carbohydrate to the enzymatic reaction are contained, specifically,
at least one carbohydrate selected from the group consisting of
lactose, glucose, galactose, a lactose isomer, a
galactooligosaccharide, maltose, a maltooligosaccharide, and a
dextrin is contained. Further, since it is more preferred to use at
least one carbohydrate selected from the group consisting of
lactose, glucose, maltose, and a galactooligosaccharide as the
carbohydrate to be added to the microbial cell liquid, it is more
preferred that in the microbial cell liquid, at least one
carbohydrate selected from the group consisting of lactose,
glucose, galactose, a lactose isomer, a galactooligosaccharide,
maltose, and a maltooligosaccharide is contained.
[0041] The microbial cell liquid after the heating treatment (a
killed microbial cell liquid) can be stored for a long time under
low-temperature conditions of 25.degree. C. or lower, preferably
from 0 to 5.degree. C. Then, the microbial cell liquid after the
heating treatment can be used in various applications using the
enzymatic activity.
[0042] In addition, the microbial cell liquid after the heating
treatment can be further dried by a known drying method such as
spray drying or lyophilization and formed into a dry killed
microbial cell powder. The degree of drying is not particularly
limited, however, for example, the drying is performed until the
water content in the dry powder is reduced to about 10% or less. By
performing this drying, longer time storage as compared with the
case of a liquid, for example, 1 year or more storage can be
achieved while maintaining the enzyme titer at 80% or more of the
enzyme titer immediately after drying. Further, the dry killed
microbial cell powder can be used in various applications using the
enzymatic activity.
[0043] As for the conditions for the above-described spray drying,
the inlet and outlet temperatures of a drying chamber may be in a
range in which the enzyme is not significantly inactivated, and
further, the rotation speed of an atomizer, the feeding amount of a
stock solution, etc. hardly affect the final enzyme titer, although
a dry microbial cell powder having slightly different properties as
a product is obtained depending on such conditions, and therefore,
it is not necessary to pay much attention to such conditions.
Specifically, the inlet temperature of a drying chamber can be, for
example, from 70.degree. C. to 200.degree. C., preferably from
110.degree. C. to 180.degree. C., and the outlet temperature of a
drying chamber can be, for example, from 50.degree. C. to
120.degree. C., preferably from 70.degree. C. to 90.degree. C.
Further, the rotation speed of an atomizer can be, for example,
from 10,000 to 30,000 rpm, and the feeding amount of a stock
solution can be, for example, from 0.2 to 200 kg/hour. The spray
drying can also be performed using a spraying system such as a
two-fluid nozzle other than an atomizer. Incidentally, from the
viewpoint that continuous production can be performed industrially,
it is preferred to use a spray drying method.
[0044] The thus obtained microbial cell liquid after the heating
treatment and dry killed microbial cell powder can also be
incorporated alone or in combination with each other to form an
enzyme composition. It is also possible to add a diluent, an
excipient, a surfactant, a preservative, or the like to this enzyme
composition within a range that does not affect the enzymatic
activity. Further, also this enzyme composition can be used in
various applications using the enzymatic activity.
EXAMPLES
[0045] Next, the invention will be more specifically described with
reference to Examples, however, the invention is by no means
limited to these Examples. Incidentally, in the following Examples,
the .beta.-galactosidase titer, the solid content, the residual
water content in the dry microbial cell powder, a particle size
distribution, the live cell count of Sporobolomyces singularis, and
the sugar composition were measured or analyzed by the following
methods.
(1) Method for Measurement of .beta.-galactosidase Titer
(a) Preparation of Test Liquid
[0046] About 2.5 g to about 6.0 g of a microbial cell liquid was
accurately weighed in a 50-mL volumetric flask, and brought to a
constant volume with a 50 mM sodium phosphate-citric acid buffer
solution (pH 4.0) (hereinafter referred to as "buffer solution"),
and then, sufficiently dissolved or suspended therein, whereby a
test liquid was prepared. Further, in the case where the microbial
cell liquid contained a carbohydrate (lactose), about 2.5 g of the
microbial cell liquid was accurately weighed in a 50-mL centrifugal
tube, and washing was performed by suspending the liquid in the
buffer solution, followed by centrifugation (20,000 G, 15 mins),
whereby the carbohydrate was removed. After this washing procedure
was performed 3 times, the washed material was transferred to a
50-mL measuring flask, brought to a constant volume with the buffer
solution, and well suspended therein, whereby a test liquid was
prepared. Further, in the case where a test sample was a dry
microbial cell powder (hereinafter referred to as "dry product"),
about 150 to 350 mg of the dry product was washed in the same
manner as described above, and thereafter a test sample was
prepared.
(b) Measurement
[0047] In a 100-mL volumetric flask, 0.3766 g of
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG) was weighed,
dissolved in the buffer solution and brought to a constant volume,
whereby a 12.5 mM ONPG solution was prepared. In a test tube, 0.8
mL of this ONPG solution was placed, and the test tube was
maintained in a thermoregulated water bath at 30.degree. C. for 5
minutes. Thereto, 0.2 mL of the test liquid was added and mixed
well, and a reaction was allowed to proceed at 30.degree. C. for 10
minutes. Then, 4 mL of a 0.25 M sodium carbonate solution was added
to stop the reaction (a test system). Separately, in a test tube,
0.8 mL of the ONPG solution and 4 mL of a 0.25 M sodium carbonate
solution were placed, and further 0.2 mL of the test liquid was
added thereto and mixed well (a blank system). Each of the test
system and the blank system was centrifuged (2,000 G, 10 mins, 15
to 20.degree. C.), and the absorbance of the resulting supernatant
was measured at a wavelength of 420 nm, and then, the number of
units was calculated according to the following formula. The amount
of the enzyme required for releasing 1 .mu.mol of o-nitrophenol
(ONP) in 1 minute under the above-described reaction conditions was
determined as 1 U.
Activity value * = A 1 - A 2 0.91 .times. 1 0.2 .times. 1 10
.times. B A 1 : absorbance of test liquid A 2 : absorbance of blank
B : dilution ratio * : U / g [ Math . 1 ] ##EQU00001##
(2) Solid Content
[0048] 5 to 10 g or 5 to 10 mL of a microbial cell liquid before
and after performing a treatment of killing a microorganism was
accurately weighed on an aluminum dish, followed by drying at
105.degree. C. for 16 hours. From the weights of the microbial cell
liquid before and after drying by this procedure, the solid content
(mass %) was calculated. Further, in spray drying, the solid
content in a stock solution to be dried and in a dry product to be
used when the .beta.-galactosidase titer per solid content is
calculated were also obtained under the same conditions.
Incidentally, when the microbial cell liquid was a culture solution
(when medium components were contained), the microbial cell liquid
was accurately weighed in a centrifugal tube, and washing was
performed by centrifugation to remove the medium components, and
then, the entire amount of the washed microbial cells were
transferred onto an aluminum dish, and the microbial cells were
dried in the same manner as described above, and then, the solid
content was calculated.
(3) Residual Water Content
[0049] The residual water content in the dry product obtained by
spray drying was measured using an infrared aquameter manufactured
by Kett Electric Laboratory under the conditions of 105.degree. C.
for 15 minutes.
(4) Particle Size Distribution
[0050] The particle size distribution of the dry product was
measured by a dry process using a laser diffraction particle size
distribution analyzer (HELOS & RODOS system) manufactured by
Sympatec, Inc.
(5) Live Cell Count of Sporobolomyces singularis
[0051] Lactose (2.5%), yeast extract (0.5%), monopotassium
phosphate (0.1%), magnesium sulfate (0.05%), and agar (1.5%) were
dissolved, and the pH of the resulting solution was adjusted to 5.0
with 2 N hydrochloric acid. Then, the solution was sterilized by
autoclaving (121.degree. C., 10 mins), and a flat plate (.phi. 90
mm) was prepared. On this plate, 100 .mu.L of a sample dissolved
and diluted with physiological saline was plated, and cultured at
25.degree. C. for about 1 week. The resulting colonies were counted
and the obtained value was determined as the live cell count of
Sporobolomyces singularis.
(6) Analysis of Sugar Composition
[0052] The sugar composition was analyzed using HPLC under the
following conditions.
[HPLC Conditions]
[0053] Column: Shodex SUGAR KS-802 (Showa Denko K.K.)
[0054] Solvent: pure water
[0055] Flow rate: 0.5 mL/min
[0056] Temperature: 80.degree. C.
[0057] Detector: Differential refractometer
Example 1
Killing of Microorganism:
(1) Culturing
[0058] Sporobolomyces singularis YIT 10047 (ISK-##2B6, hereinafter
referred to as "Ss") was aerobically cultured at 27.degree. C. for
4 days in a medium (pH 5) containing glucose (5%), yeast extract
(1.0%), monopotassium phosphate (0.1%), and magnesium sulfate
(0.05%). This culture solution was centrifuged (10000 G, 30 mins)
to obtain wet cells, and sterilized tap water was added thereto and
the wet cells were well suspended therein. The resulting suspension
was centrifuged under the same conditions, and the obtained wet
cells were suspended in a small amount of tap water such that the
solid content was about 5%, and the thus obtained suspension was
used as an Ss concentrate liquid.
(2) Killing of Microorganism <Examination of pH and Heating
Temperature>
[0059] The pH of the Ss concentrate liquid (solid content: 5.0%)
obtained in the above (1) was adjusted stepwise from 3.5 to 6.3
with a 5 N sodium hydroxide solution. The thus obtained liquids and
the Ss concentrate liquid with an unadjusted pH of 3.1 were
subjected to a heating treatment by maintaining the liquids at
35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., or
55.degree. C. for 18 hours. The results of the live cell count of
Ss before and after the heating treatment and the ratio of the
.beta.-galactosidase titer after the heating treatment to the
.beta.-galactosidase titer before the heating treatment (residual
titer ratio) are shown in Table 1.
TABLE-US-00001 TABLE 1 Before heating 35.degree. C. 40.degree. C.
45.degree. C. 50.degree. C. 55.degree. C. Live cell Residual Live
cell Residual Live cell Residual Live cell Residual Live cell
Residual Live cell count titer count titer count titer count titer
count titer count (cfu/mL) ratio (%) (cfu/mL) ratio (%) (cfu/mL)
ratio (%) (cfu/mL) ratio (%) (cfu/mL) ratio (%) (cfu/mL) pH 3.1
5.2E+09 97.6 5.0E+01 92.2 <10 68.5 <10 22.6 <10 -- --
(unadjusted) pH 3.5 3.7E+09 99.4 <10 102.6 <10 89.4 <10
42.6 <10 -- -- pH 4.0 1.2E+10 -- -- 96.3 <10 82.9 <10 25.8
<10 0.9 <10 pH 4.2 3.9E+09 97.6 1.7E+03 102.5 <10 97.5
<10 63.3 <10 -- -- pH 4.7 4.9E+09 97.7 4.4E+04 100.2 <10
97.5 <10 70.0 <10 -- -- pH 5.0 1.5E+10 -- -- 99.5 <10 94.8
<10 53.1 <10 1.2 <10 pH 5.8 9.0E+09 -- -- 101.1 <10
98.3 <10 59.0 <10 2.1 <10 pH 6.3 6.0E+09 -- -- 97.5 <10
94.1 <10 54.4 <10 1.0 <10 --: not tested
[0060] In the case where the heating treatment was performed after
adjusting the pH to 3.5 or 4.0, the Ss was killed at a heating
temperature of 40 to 45.degree. C., and the residual titer ratio
was 80% or more at 45.degree. C., and 90% or more at 40.degree. C.
In the case where the pH was adjusted to 3.5, the Ss was killed and
the residual titer ratio was 90% or more even at 35.degree. C. In
the case where the heating treatment was performed after adjusting
the pH to 4.2, 4.7, 5.0, 5.8, or 6.3, the Ss was killed and the
residual titer ratio was 90% or more at a heating temperature of 40
to 45.degree. C. On the other hand, in the case where the heating
treatment was performed without performing the pH adjustment (pH
3.1), the Ss was killed at a heating temperature of 40.degree. C.,
and the residual titer ratio at 40.degree. C. was 90% or more, but
the value thereof was 92.2%, and therefore, the residual titer
ratio was lower as compared with the case where the pH adjustment
was performed. Further, the residual titer ratio was lower than 80%
when the heating temperature was 45.degree. C., and therefore, it
was presumed that the temperature conditions in which the
microorganism can be killed and the enzyme titer can be maintained
are narrower by 5.degree. C. or more as compared with the other pH
conditions.
(3) Killing of Microorganism <Detailed Examination of Heating
Temperature>
[0061] The pH of an Ss concentrate liquid (solid content: 5.6%)
obtained in the same manner as in the above (1) was adjusted to
4.0, 4.5, 4.9, 5.7, or 6.5 with a 5 N sodium hydroxide solution.
The thus obtained liquids were subjected to a heating treatment by
maintaining the liquids at 44.degree. C., 46.degree. C., 48.degree.
C., 50.degree. C., or 52.degree. C. for 5 hours or 18 hours. The
results of the live cell count of Ss before and after the heating
treatment and the residual titer ratio are shown in Table 2 (5-hour
heating treatment) and Table 3 (18-hour heating treatment).
TABLE-US-00002 TABLE 2 Before heating 44.degree. C. 46.degree. C.
48.degree. C. 50.degree. C. 52.degree. C. Live cell Residual Live
cell Residual Live cell Residual Live cell Residual Live cell
Residual Live cell count titer count titer count titer count titer
count titer count (cfu/mL) ratio (%) (cfu/mL) ratio (%) (cfu/mL)
ratio (%) (cfu/mL) ratio (%) (cfu/mL) ratio (%) (cfu/mL) pH 4.0
3.9E+10 95.1 <10 95.6 <10 87.4 <10 67.3 <10 37.2 <10
pH 4.5 7.0E+09 -- -- 100.6 <10 95.8 <10 88.0 <10 -- -- pH
4.9 2.7E+11 98.8 1.7E+02 99.7 <10 95.1 <10 91.8 <10 60.3
<10 pH 5.7 2.2E+10 99.7 4.5E+02 97.6 <10 92.1 <10 94.1
<10 55.8 <10 pH 6.5 1.1E+10 99.1 2.4E+03 93.9 <10 87.5
<10 73.1 <10 44.9 <10
TABLE-US-00003 TABLE 3 Before heating 44.degree. C. 46.degree. C.
48.degree. C. 50.degree. C. Live cell Residual Live cell Residual
Live cell Residual Live cell Residual Live cell count titer count
titer count titer count titer count (cfu/mL) ratio (%) (cfu/mL)
ratio (%) (cfu/mL) ratio (%) (cfu/mL) ratio (%) (cfu/mL) pH 4.0
3.9E+10 94.5 <10 87.3 <10 70.0 <10 46.3 <10 pH 4.5
7.0E+09 -- -- 91.0 <10 80.3 <10 65.5 <10 pH 4.9 2.7E+11
100.3 <10 96.1 <10 84.5 <10 72.3 <10 pH 5.7 2.2E+10
96.9 <10 101.4 <10 81.6 <10 76.9 <10 pH 6.5 1.1E+10
96.7 <10 94.0 <10 77.0 <10 57.6 <10
[0062] In the 5-hour heating treatment, in the case where the
heating treatment was performed after adjusting the pH to 4.0, the
Ss was killed at a heating temperature of 44 to 48.degree. C., and
the residual titer ratio was 80% or more at 48.degree. C., and 90%
or more at 44 to 46.degree. C. In the case where the heating
treatment was performed after adjusting the pH to 4.5, the Ss was
killed at a heating temperature of 46 to 50.degree. C., and the
residual titer ratio was 80% or more at 50.degree. C., and 90% or
more at 46 to 48.degree. C. In the case where the heating treatment
was performed after adjusting the pH to 4.9 or 5.7, the Ss was
killed and the residual titer ratio was 90% or more at a heating
temperature of 44 to 50.degree. C. In particular, at a heating
temperature of 46 to 50.degree. C., the live cell count of Ss was
10 cfu/mL or less. In the case where the heating treatment was
performed after adjusting the pH to 6.5, the Ss was killed at a
heating temperature of 44 to 48.degree. C., and the residual titer
ratio was 80% or more at 48.degree. C., and 90% or more at 44 to
46.degree. C. In particular, at a heating temperature of 46 to
48.degree. C., the live cell count of Ss was 10 cfu/mL.
[0063] Further, in the 18-hour heating treatment, in the case where
the heating treatment was performed after adjusting the pH to 4.0,
the Ss was killed at a heating temperature of 44 to 46.degree. C.,
and the residual titer ratio was 80% or more at 46.degree. C., and
90% or more at 44.degree. C. In the case where the heating
treatment was performed after adjusting the pH to 4.5, 4.9, or 5.7,
the Ss was killed at a heating temperature of 44 to 48.degree. C.,
and the residual titer ratio was 80% or more at 48.degree. C., and
90% or more at 44 to 46.degree. C. In the case where the heating
treatment was performed after adjusting the pH to 6.5, the Ss was
killed and the residual titer ratio was 90% or more at a heating
temperature of 44 to 46.degree. C.
(4) Killing of Microorganism <Examination of Heating
Time>
[0064] The pH of an Ss concentrate liquid (solid content: 5.0%)
obtained in the same manner as in the above (1) was adjusted to 4.5
with a 5 N sodium hydroxide solution. The thus obtained liquid was
subjected to a heat treatment by maintaining the liquid at
40.degree. C. or 45.degree. C. The results of the live cell count
of Ss measured over time during the heating treatment are shown in
Table 4.
TABLE-US-00004 TABLE 4 Maintaining time Heating temperature (hour)
40.degree. C. 45.degree. C. 0 1.3E+09 3.0E+09 1 1.5E+08 5.0E+01 2
9.0E+06 <10 3 2.2E+06 <10 4 9.0E+04 <10 5 6.0E+03 <10 6
1.7E+02 <10 9 <10 <10 13 <10 <10 18 <10
<10
[0065] The live cell count of Ss was decreased over time at either
of the heating temperatures of 40.degree. C. and 45.degree. C., and
in the case where the heating treatment was performed at 40.degree.
C., the live cell count was decreased to 1000 cfu/mL or less when
performing the heating treatment for 6 hours, and decreased to 10
cfu/mL or less when performing the heating treatment for 9 hours.
Further, in the case where the heating treatment was performed at
45.degree. C., the live cell count was decreased to 1000 cfu/mL or
less when performing the heating treatment for 1 hour, and
decreased to 10 cfu/mL or less when performing the heating
treatment for 2 hours.
(5) Killing of Microorganism <in Combination with
Lactose>
[0066] By using an Ss concentrate liquid (solid content: 5.6%)
obtained in the same manner as in the above (1), three types of
samples were prepared as follows: a sample in which the pH of the
Ss concentrate liquid was adjusted to 4.5 with a 5 N sodium
hydroxide solution; a sample in which lactose was added at 1% to
the Ss concentrate liquid (the pH adjustment was not performed);
and a sample in which lactose was added at 1% to the Ss concentrate
liquid, and then, the pH thereof was adjusted to 4.5. The thus
obtained samples were subjected to a heating treatment by
maintaining the samples at 50.degree. C. for 18 hours (test I).
Another test was performed in the same manner as described above
for the case where lactose was added at 2% (test II). The residual
titer ratio of each of the Ss suspensions is shown in Table 5.
Incidentally, the live cell count of Ss after the heating treatment
was 10 cfu/mL or less in all the cases.
TABLE-US-00005 TABLE 5 Only pH Only addition pH adjustment +
adjustment of lactose addition of lactose Test I 61.8 72.0 87.9
Test II 62.9 84.3 94.4
[0067] In both the tests I and II, the residual titer ratio in the
case where the pH adjustment and the addition of lactose were
combined was higher than that in the case where a single treatment
of either of the pH adjustment and the addition of lactose was
performed. It was found that an effect of stabilizing the titer by
the pH adjustment was exhibited even in the presence of lactose,
and moreover, the residual titer ratio was increased in the
presence of lactose.
Example 2
Preparation of Dry Product:
[0068] The pH of an Ss concentrate liquid (solid content: 5.3%)
obtained in the same manner as in Example 1 was adjusted to 4.5
with a 5 N sodium hydroxide solution. The thus obtained liquid was
subjected to a heating treatment by maintaining the liquid at
40.degree. C. for 18 hours. To 7.8 L of this killed Ss concentrate
liquid, 2.6 L of a 8, 20, or 40% lactose solution was added,
followed by mixing well, whereby stock solutions to be dried were
prepared such that the solid content of Ss was about 4%, and the
lactose content was 2, 5, or 10%. These stock solutions and the
killed Ss concentrate liquid to which lactose was not added were
dried using a pilot apparatus for spray drying (PRODUCTION MINOR,
GEA Process Engineering, Inc.) under conditions that the inlet
temperature was 120.degree. C., the outlet temperature was about
80.degree. C., the rotation speed of the atomizer was 12500 rpm,
and the processing amount of the stock solution was 4 kg/hr,
whereby favorable dry products were obtained. The measurement
results of the residual titer ratio (the ratio of the titer per
solid content in the dry product to the titer per solid content in
the stock solution to be dried), the average particle size, and the
residual water content of each of these dry products are shown in
Table 6.
TABLE-US-00006 TABLE 6 Product 1 Product 2 Product 3 Product 4
Concentration in Ss 4 4 4 4 stock solution to be Lactose 2 5 10 0
dried (%) Total solid content 6 9 14 4 Rotation speed of atomizer
(rpm) 12500 12500 12500 12500 Feeding amount of stock solution
(kg/hr) 4.0 4.0 4.0 4.0 Inlet temperature of drying
chamber(.degree. C.) 120 120 120 120 Outlet temperature of drying
chamber (.degree. C.) 80 80 80 80 Dry product Average particle size
(.mu.m) 19.6 21.5 24.5 18.3 Residual water content (%) 4.7 4.6 5.2
4.9 Residual titer ratio (%) 92.1 96.9 99.8 97.8
[0069] In the case where the concentration of lactose in the stock
solutions to be dried was from 0 to 10%, dry products in which the
residual titer ratio after drying was 90% or more could be
obtained. Incidentally, the live cell count of Ss in any of these
dry products was 10 cfu/mL or less, and the residual titer ratio
before and after the heating treatment was 90% or more in all the
cases.
Example 3
Test for Production of Oligosaccharides:
(1) Preparation of Suspension of Dry Product
[0070] Among the dry products obtained in the above-described
Example 2, each of Product 1 and Product 2 in an amount
corresponding to 45 U was weighed, and 10 mL of ion exchanged water
was added thereto to suspend the dry product.
(2) Production Reaction of Oligosaccharides
[0071] To 800 mL of a 60% lactose solution, the entire amount of
each of the suspensions of the dry products prepared in the above
(1) or an Ss concentrate liquid obtained in the same manner as in
Example 1 in an amount corresponding to 45 U was added and mixed,
and a reaction was allowed to proceed at 65.degree. C. for 22 hours
at pH 6. The results obtained by examining the sugar composition at
this time are shown in Table 7.
TABLE-US-00007 TABLE 7 Ss concentrate Product 1 Product 2 liquid
Sugar Tetra- or higher 1.0 1.0 0.9 composition oligosaccharide (%)
Trisaccharide 30.1 30.0 29.6 Disaccharide 57.8 58.0 58.7 Glc 10.8
10.7 10.5 Gal 0.3 0.3 0.4
[0072] There is no significant difference in sugar composition to
be produced or reaction rate between each of the suspensions of the
dry products and the Ss concentrate liquid (live cell suspension),
and it was found that the dry product can be used in the production
of oligosaccharides.
Example 4
Killing of Microorganism:
(1) Culturing
[0073] An Ss concentrate liquid (solid content: 5.8%) was obtained
in the same manner as in (1) of Example 1.
(2) Killing of Microorganism <Examination of Concentration of
Lactose and Heating Temperature>
[0074] The Ss concentrate liquid obtained in the above (1) and a
lactose solution were mixed at 3:1, whereby Ss concentrate liquids
containing lactose at 0.2 to 15% were prepared. The thus obtained
liquids and the Ss concentrate liquid to which lactose was not
added were subjected to a heating treatment by maintaining the
liquids at 35.degree. C., 40.degree. C., 45.degree. C., 50.degree.
C., or 55.degree. C. for 18 hours. The results of the measurement
of the live cell count of Ss before and after the heating treatment
and the ratio of the .beta.-galactosidase titer after the heating
treatment to the .beta.-galactosidase titer before the heating
treatment (residual titer ratio) are shown in Table 8.
TABLE-US-00008 TABLE 8 Before heating 35.degree. C. 40.degree. C.
45.degree. C. 50.degree. C. 55.degree. C. Re- Re- Re- Re- Re- Live
sidual Live sidual Live sidual Live sidual Live sidual Live cell
titer cell titer cell titer cell titer cell titer cell count ratio
count ratio count ratio count ratio count ratio count (cfu/mL) (%)
(cfu/mL) (%) (cfu/mL) (%) (cfu/mL) (%) (cfu/mL) (%) (cfu/mL) Con-
0% (without 1.7E+10 98.3 6.0E+07 97.0 4.0E+01 73.8 <10 28.2
<10 -- -- centra- addition) tion of 0.2% 7.0E+09 101.3 4.0E+07
101.3 <10 81.8 <10 37.6 <10 -- -- lactose 0.5% 9.0E+09
101.6 8.0E+07 103.1 <10 92.1 <10 55.4 <10 -- -- (w/v) 1%
1.3E+10 101.5 1.3E+08 100.0 <10 100.8 <10 75.9 <10 -- --
2% 1.3E+10 99.7 3.0E+07 105.3 <10 106.7 <10 90.7 <10 -- --
5% 1.0E+10 -- -- 105.8 <10 105.0 <10 101.5 <10 60.4 <10
10% 1.4E+10 -- -- 105.4 <10 103.7 <10 103.0 <10 92.7
<10 15% 7.0E+10 -- -- 104.4 <10 104.5 <10 102.2 <10
98.5 <10 --: not tested
[0075] In the case where the heating treatment was performed after
adding lactose at 0.2%, the Ss was killed at a heating temperature
of 40 to 45.degree. C., and the residual titer ratio was 80% or
more at 45.degree. C., and 90% or more at 40.degree. C. In the case
where the heating treatment was performed after adding lactose at
0.5 to 1%, the Ss was killed at 40 to 45.degree. C., and the
residual titer ratio was 90% or more. In the case where the heating
treatment was performed after adding lactose at 2 to 5%, the Ss was
killed and the residual titer ratio was 90% or more at a heating
temperature of 40 to 50.degree. C. In the case where the heating
treatment was performed after adding lactose at 10 to 15%, the Ss
was killed and the residual titer ratio was 90% or more at a
heating temperature of 40 to 55.degree. C. On the other hand, in
the case where the heating treatment was performed without adding
lactose, the Ss was killed at a heating temperature of 40.degree.
C., and the residual titer ratio at 40.degree. C. was 90% or more,
but the residual titer ratio was lower than 80% at 45.degree. C.,
and therefore, it was presumed that the temperature conditions in
which the microorganism can be killed and the enzyme titer can be
maintained are narrower by 5.degree. C. or more as compared with
the case where lactose is added.
(3) Killing of Microorganism <Examination of Type of
Carbohydrate>
[0076] An effect of stabilizing the enzymatic activity when a
microorganism was killed was compared among a dextrin (NSD #300,
San-ei Sucrochemical Co., Ltd.), a tri- or higher oligosaccharide
fraction of a galactooligosaccharide, lactose, maltose, and
glucose. An Ss concentrate liquid (solid content: 5.6%) obtained in
the same manner as in the above (1) and a 8 or 20% carbohydrate
solution were mixed at 3:1, whereby Ss concentrate liquids
containing a carbohydrate at 2% or 5% were prepared. The thus
obtained liquids were subjected to a heating treatment by
maintaining the liquids at 45.degree. C. or 50.degree. C. for 18
hours. The results of the ratio of the .beta.-galactosidase titer
after the heating treatment to the .beta.-galactosidase titer
before the heating treatment (residual titer ratio) are shown in
Table 9. The live cell count of Ss before the heating treatment was
10.sup.8 cfu/mL, however, after the heating treatment, the live
cell count of Ss was decreased to 10 cfu/mL or less, and therefore,
the Ss was killed in the case of any carbohydrate at any
concentration.
TABLE-US-00009 TABLE 9 Concentration of carbohydrate (w/v) and
heating temperature 2% 5% 45.degree. C. 50.degree. C. 45.degree. C.
50.degree. C. Type of Dextrin NSD 300 93.9 48.6 98.8 68.8
carbohydrate Tri- or higher oligosaccharide 102.5 81.5 108.1 97.8
fraction of galactooligosaccharide Lactose 103.5 83.4 103.6 101.1
Maltose 101.3 91.0 104.8 98.2 Glucose 104.4 88.5 107.3 99.4
[0077] In the case where the carbohydrate was not added, the
residual titer ratio was 72.7% at a heating temperature of
45.degree. C., and 29.5% at a heating temperature of 50.degree. C.
On the other hand, in the case where any of the carbohydrates was
allowed to coexist, the residual titer ratio was higher, and except
the case where the dextrin was allowed to coexist at a heating
temperature of 50.degree. C., the residual titer ratio was improved
to 80% or more.
(4) Killing of Microorganism <Examination of Heating
Temperature>
[0078] An Ss concentrate liquid (solid content: 5.0%) obtained in
the same manner as in the above (1) and a 20% lactose solution were
mixed at 3:1, whereby an Ss concentrate liquid containing lactose
at 5% was prepared. The thus obtained liquid was subjected to a
heating treatment by maintaining the liquid at 40.degree. C. or
45.degree. C. The results of the live cell count of Ss measured
over time during the heating treatment are shown in Table 10.
TABLE-US-00010 TABLE 10 Maintaining time Heating temperature (hour)
40.degree. C. 45.degree. C. 0 1.7E+09 3.0E+09 1 1.1E+07 <10 2
9.0E+04 <10 3 6.0E+03 <10 4 2.1E+01 <10 5 <10 <10 6
<10 <10 9 <10 <10 13 <10 <10 18 <10 <10
[0079] The live cell count of Ss was decreased over time at either
of the heating temperatures of 40.degree. C. and 45.degree. C., and
in the case where the heating treatment was performed at 40.degree.
C., the live cell count was decreased to 1000 cfu/mL or less when
performing the heating treatment for 4 hours, and decreased to 10
cfu/mL or less when performing the heating treatment for 5 hours.
Further, in the case where the heating treatment was performed at
45.degree. C., the live cell count was decreased to 10 cfu/mL or
less when performing the heating treatment for 1 hour.
(5) Killing of Microorganism <Examination of Sugar
Composition>
[0080] An Ss concentrate liquid (solid content: 5.3%) obtained in
the same manner as in the above (1) and a 8, 20, or 40% lactose
solution were mixed at 3:1, whereby Ss concentrate liquids
containing lactose at 2%, 5%, or 10% were prepared. The thus
obtained liquids were subjected to a heating treatment by
maintaining the liquids at 40.degree. C. for 18 hours. The sugar
composition after the heating treatment was analyzed under the
above HPLC conditions. The results are shown in Table 11. Further,
the results obtained by measuring the titer before the heating
treatment and calculating a residual titer are also shown in Table
11. The live cell count of Ss before the heating treatment was
10.sup.8 cfu/mL, however, after the heating treatment, the live
cell count of Ss was decreased to 10 cfu/mL or less, and therefore,
the Ss was killed in all the cases.
TABLE-US-00011 TABLE 11 Sugar composition (%) Concentration of Tri-
or higher Di- Mono- Residual titer lactose (w/v) oligosaccharide
saccharide saccharide ratio (%) 2% 0.0 5.3 94.7 104.1 5% 0.2 12.4
87.4 105.6 10% 6.1 42.7 51.3 104.4
[0081] In the Ss concentrate liquid (live cell suspension)
containing lactose, lactose is subjected to the
.beta.-galactosidase activity during the heating treatment,
however, it was found that the effect of stabilizing the titer is
maintained regardless of the degree of the reaction. Further, it
was confirmed that even if a monosaccharide (glucose or galactose)
or a galactooligosaccharide produced by subjecting the added
lactose to an enzymatic reaction exists in the Ss concentrate
liquid, there is no problem in terms of stabilization of the enzyme
titer, and it was also confirmed that a monosaccharide or a
galactooligosaccharide can be used as the carbohydrate.
Example 5
Preparation of Dry Product:
[0082] An Ss concentrate liquid (solid content: 5.3%) obtained in
the same manner as in Example 1 and a 8, 20, or 40% lactose
solution were mixed at 3:1, whereby stock solutions to be dried
were prepared such that the solid content of Ss was about 4%, and
the lactose content was 2, 5, or 10%. The thus obtained liquids
were subjected to a heating treatment by maintaining the liquids at
40.degree. C. for 18 hours. These killed Ss concentrate liquids
were dried using a pilot apparatus for spray drying (PRODUCTION
MINOR, GEA Process Engineering, Inc.) under conditions that the
inlet temperature was 120.degree. C., the outlet temperature was
about 80.degree. C., the rotation speed of the atomizer was 12500
rpm, and the processing amount of the stock solution was 4 kg/hr,
whereby favorable dry products were obtained. The residual titer
ratio (the ratio of the titer per solid content in the dry product
to the titer per solid content in the stock solution to be dried),
the average particle size, and the residual water content of each
of these dry products were measured. The results are shown in Table
12.
TABLE-US-00012 TABLE 12 Product 5 Product 6 Product 7 Concentration
in Ss 4 4 4 stock solution to Lactose 2 5 10 be dried (%) Total
solid 6 9 14 content Rotation speed of atomizer (rpm) 12500 12500
12500 Feeding amount of stock solution 4.0 4.0 4.0 (kg/hr) Inlet
temperature of drying chamber 120 120 120 (.degree. C.) Outlet
temperature of drying chamber 80 80 80 (.degree. C.) Dry product
Average particle 22.9 22.4 26.8 size (.mu.m) Residual water 7.4 7.5
5.6 content (%) Residual titer 94.0 96.0 97.2 ratio (%)
[0083] In the case where the concentration of lactose in the stock
solutions to be dried was from 2 to 10%, dry microbial cell
products in which the residual titer ratio after drying was 90% or
more could be obtained. Incidentally, the live cell count of Ss in
any of these dry products was 10 cfu/mL or less, and the residual
titer ratio before and after the heating treatment was 90% or more
in all the cases.
Example 6
Test for Production of Oligosaccharides:
(1) Preparation of Suspension of Dry Product
[0084] Among the dry products obtained in the above-described
Example 5, each of Product 5 and Product 6 in an amount
corresponding to 45 U was weighed, and 10 mL of ion exchanged water
was added thereto to suspend the dry product.
(2) Production Reaction of Oligosaccharides
[0085] To 800 mL of a 60% lactose solution, the entire amount of
each of the suspensions of the dry products prepared in the above
(1) or an Ss concentrate liquid obtained in the same manner as in
Example 1 in an amount corresponding to 45 U was added and mixed,
and a reaction was allowed to proceed at 65.degree. C. for 22 hours
at pH 6. The results obtained by examining the sugar composition at
this time are shown in Table 13.
TABLE-US-00013 TABLE 13 Ss concentrate Product 5 Product 6 liquid
Sugar Tetra- or higher 1.0 1.0 0.9 composition oligosaccharide (%)
Trisaccharide 30.3 30.3 29.6 Disaccharide 57.2 57.4 58.7 Glc 11.0
11.0 10.5 Gal 0.4 0.4 0.4
[0086] There is no significant difference in sugar composition to
be produced between each of the suspensions of the dry products and
the Ss concentrate liquid (live cell suspension), and it was found
that the dry product can be used in the production of
oligosaccharides.
Example 7
Killing of Microorganism:
(1) Culturing
[0087] An Ss concentrate liquid (solid content: 5.2%) was obtained
in the same manner as in (1) of Example 1.
(2) Killing of Microorganism <Examination of Concentration of
Lactose and Heating Temperature>
[0088] The Ss concentrate liquid obtained in the above (1) and a
lactose solution were mixed at 3:1, whereby Ss concentrate liquids
containing lactose at 2 to 5% were prepared. The thus obtained
liquids and the Ss concentrate liquid to which lactose was not
added were subjected to a heating treatment by maintaining the
liquids at 50.degree. C. for 18 hours. The results of the ratio of
the .beta.-galactosidase titer after the heating treatment to the
.beta.-galactosidase titer before the heating treatment (residual
titer ratio) are shown in Table 14. Incidentally, the live cell
count of Ss after the heating treatment was 10 cfu/mL or less in
all the cases.
TABLE-US-00014 TABLE 14 Concentration of lactose (w/v) Residual
titer ratio (%) 2% 85.2 3% 95.8 4% 98.2 5% 102.0
[0089] The residual titer ratio was higher than 80% in the case
where the concentration of lactose was from 2 to 5%. Further, it
was found that in the case where the concentration of lactose was
3% or more, the residual titer ratio was higher than 90%.
Example 8
Killing of Microorganism:
(1) Culturing
[0090] An Ss concentrate liquid (solid content: 5.4%) was obtained
in the same manner as in (1) of Example 1.
(2) Killing of Microorganism <in Combination with
Lactose>
[0091] By using the Ss concentrate liquid obtained in the above
(1), three types of samples were prepared as follows: a sample in
which the pH of the Ss concentrate liquid was adjusted to 4.5 with
a 2 N sodium hydroxide solution; a sample in which lactose was
added at 1% to the Ss concentrate liquid (the pH adjustment was not
performed); and a sample in which lactose was added at 1% to the Ss
concentrate liquid, and then, the pH thereof was adjusted to 4.5.
The thus obtained samples were subjected to a heating treatment by
maintaining the samples at 45.degree. C. for 18 hours (test III).
Another test was performed in the same manner as described above
for the case where lactose was added at 2% (test IV). The residual
titer ratio of each of the Ss suspensions is shown in Table 15. The
live cell count of Ss after the heating treatment was 10 cfu/mL or
less in all the cases.
TABLE-US-00015 TABLE 15 Only pH Only addition pH adjustment +
adjustment of lactose addition of lactose Test III 97.2 100.2 102.9
Test IV 98.3 102.0 103.1
[0092] In both the tests III and IV, the residual titer ratio in
the case where the pH adjustment and the addition of lactose were
combined was higher than that in the case where a single treatment
of either of the pH adjustment and the addition of lactose was
performed. It was found that an effect of stabilizing the titer by
the pH adjustment was exhibited even in the presence of lactose,
and moreover, the residual titer ratio was increased in the
presence of lactose.
(3) Killing of Microorganism <in Combination with
Lactose>
[0093] By using the Ss concentrate liquid obtained in the above
(1), three types of samples were prepared as follows: a sample in
which the pH of the Ss concentrate liquid was adjusted to 4.5, 5.0,
or 5.5 with a 2 N sodium hydroxide solution; a sample in which
lactose was added at 2% to the Ss concentrate liquid (the pH
adjustment was not performed); and a sample in which lactose was
added at 2% to the Ss concentrate liquid, and then, the pH thereof
was adjusted to 4.5, 5.0, or 5.5. The thus obtained samples were
subjected to a heating treatment by maintaining the samples at
50.degree. C. for 5 hours or 18 hours. The residual titer ratio of
each of the Ss suspensions is shown in Table 16. Incidentally, the
live cell count of Ss after the heating treatment was 10 cfu/mL or
less in all the cases.
TABLE-US-00016 TABLE 16 50.degree. C. Live cell count Maintaining
time 5 hr Maintaining time 18 hr before heating Residual titer Live
cell count Residual titer Live cell count Treatment conditions
(cfu/ml) ratio (%) (cfu/ml) ratio (%) (cfu/ml) 2% lactose 9.2E+10
100.1 <10 89.7 <10 pH 4.5 4.6E+10 88.0 <10 65.5 <10 pH
4.5 + 2% lactose 7.3E+10 102.9 <10 97.1 <10 pH 5.0 1.1E+11
91.8 <10 72.3 <10 pH 5.0 + 2% lactose 1.0E+11 103.6 <10
99.5 <10 pH 5.5 6.1E+09 94.1 <10 76.9 <10 pH 5.5 + 2%
lactose 4.6E+10 100.8 <10 99.5 <10
[0094] In all the cases, the residual titer ratio in the case where
the pH adjustment and the addition of lactose were combined was
higher than that in the case where a single treatment of either of
the pH adjustment and the addition of lactose was performed.
Further, in the case where the heating maintaining time was 18
hours, by adjusting the pH to 4.5 to 5.5, the residual titer ratio
was 90% or more even when the concentration of lactose was 2%.
Example 9
Killing of Microorganism:
(1) Culturing
[0095] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (2%), yeast extract (0.4%),
monopotassium phosphate (0.05%), and magnesium sulfate (0.025%).
This culture solution was centrifuged (10000 G, 30 min) to obtain
wet cells, and sterilized tap water was added thereto and the wet
cells were well suspended therein. The resulting suspension was
centrifuged under the same conditions, and to the obtained wet
cells, a lactose solution and sterilized water were added, whereby
Ss concentrate liquids containing lactose at 2% or 5% and having a
cell density of 2.5%, 4.5%, or 6.5% (w/v) were prepared.
(2) Killing of Microorganism <Examination of Concentration of
Lactose and Heating Temperature>
[0096] The Ss concentrate liquids obtained in the above (1) were
subjected to a heating treatment by maintaining the liquids at
45.degree. C. or 50.degree. C. for 5 hours or 18 hours. The
measurement of the live cell count of Ss before and after the
heating treatment and the ratio of the .beta.-galactosidase titer
after the heating treatment to the .beta.-galactosidase titer
before the heating treatment (residual titer ratio) are shown in
FIGS. 1 to 4.
[0097] At any cell density, the residual titer ratio after killing
the microorganism was 80% or more, and a difference in residual
titer ratio was not observed. In particular, in the case where the
concentration of lactose was 5%, the residual titer ratio after
killing the microorganism was 90% or more at any cell density.
Example 10
Killing of Microorganism:
(1) Culturing
[0098] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (4%), yeast extract (0.8%),
monopotassium phosphate (0.1%), and magnesium sulfate (0.05%),
whereby an Ss culture solution (solid content: 2.5%) was
obtained.
(2) Killing of Microorganism
[0099] The pH of the Ss culture solution obtained in the above (1)
was adjusted stepwise from 3.5 to 5.0 with a 2 N sodium hydroxide
solution. The thus obtained solutions and the Ss culture solution
in which the pH adjustment was not performed were subjected to a
heating treatment by maintaining the solutions at 40.degree. C. or
45.degree. C. for 5 hours or 18 hours. The results of the live cell
count of Ss after the heating treatment and the ratio of the
.beta.-galactosidase titer after the heating treatment to the
.beta.-galactosidase titer before the heating treatment (residual
titer ratio) are shown in Table 17.
TABLE-US-00017 TABLE 17 Culture lot No. Lot. 1 Lot. 2 Lot. 3 Titer
after completion of culturing (U/g) 2.29 2.52 2.37 Unadjusted pH
3.10 3.17 3.53 Conditions Residual Live cell Residual Live cell
Residual Live cell Temperature Time titer count titer count titer
count pH (.degree. C.) (h) ratio (%) (cfu/ml) ratio (%) (cfu/ml)
ratio (%) (cfu/ml) Treatment Unadjusted 40 5 65.9 >1.0E+02 -- --
95.2 4.3E+04 of killing 18 64.1 <10 -- -- 94.2 <10
microorganism 45 5 41.0 <10 -- -- 85.0 <10 18 31.3 <10 --
-- 77.9 <10 3.5 40 5 -- -- 86.3 4.6E+04 -- -- 18 -- -- 85.8
<10 -- -- 45 5 -- -- 72.4 <10 -- -- 18 -- -- 64.0 <10 --
-- 4.0 40 5 94.2 >1.0E+02 95.0 4.6E+06 99.0 1.2E+06 18 96.2
<10 95.7 <10 98.7 7.0E+00 45 5 92.9 <10 90.0 <10 98.5
<10 18 80.3 <10 79.3 <10 94.2 <10 4.5 40 5 95.4
>1.0E+02 96.5 9.9E+06 99.7 2.0E+06 18 98.1 <10 99.9 <10
102.6 <10 45 5 96.9 <10 98.3 <10 99.8 <10 18 88.4
<10 91.9 <10 99.7 <10 5.0 40 5 95.4 >1.0E+02 99.5
1.5E+07 100.3 1.5E+07 18 98.1 <10 101.5 <10 99.0 1.7E+02 45 5
99.9 <10 101.7 <10 100.7 <10 18 90.8 <10 94.9 <10
100.8 <10
[0100] In any of the culture solutions, as the pH during the
treatment of killing the microorganism was higher, the residual
titer ratio was improved. Further, in any of the culture solutions,
the conditions that met the requirements of 80% or more of the
residual titer ratio and 10 cfu/mL or less of the live cell count
of Ss were such that the pH was adjusted to 4.0 or higher and the
heating treatment was performed at 45.degree. C. for 5 hours.
Further, also in the case where the pH was adjusted to 5.0 and the
heating treatment was performed at 45.degree. C. for 18 hours, the
above-described requirement of the residual titer ratio was
met.
Example 11
Examination of pH Adjusting Agent:
(1) Culturing
[0101] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (4%), yeast extract (0.8%),
monopotassium phosphate (0.1%), and magnesium sulfate (0.05%),
whereby an Ss culture solution (solid content: 2.8%) was
obtained.
(2) pH Adjustment
[0102] To the Ss culture solution obtained in the above (1), a 0.5,
2, or 5 N sodium hydroxide solution or potassium hydroxide
solution, or a 10 or 20% sodium carbonate solution was added
dropwise, whereby the pH of the solution was adjusted to 5.0. The
ratio of the .beta.-galactosidase titer after the pH adjustment to
the .beta.-galactosidase titer before the pH adjustment (residual
titer ratio) is shown in FIG. 5.
[0103] In the case where sodium hydroxide or sodium hydroxide was
used as the pH adjusting agent, the titer was decreased by 7 to 8%
immediately after the pH adjustment. On the other hand, in the case
where sodium carbonate was used as the pH adjusting agent, the
titer was hardly decreased.
Example 12
Examination of pH Adjusting Agent:
(1) Culturing
[0104] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (5%), yeast extract (1%),
monopotassium phosphate (0.1%), and magnesium sulfate (0.05%),
whereby an Ss culture solution (solid content: 2.5%) was
obtained.
(2) Killing of Microorganism
[0105] The pH of the Ss culture solution obtained in the above (1)
was adjusted stepwise from 4.0 to 5.0 with a 2 N sodium hydroxide
solution or a 20% sodium carbonate solution. The thus obtained
solutions were subjected to a heating treatment by maintaining the
solutions at 40.degree. C. or 45.degree. C. for 5 hours or 18
hours. The live cell count of Ss after the heating treatment and
the ratio of the .beta.-galactosidase titer after the heating
treatment to the .beta.-galactosidase titer before the heating
treatment (residual titer ratio) are shown in Table 18.
TABLE-US-00018 TABLE 18 pH adjusting agent when killing
microorganism 2N NaOH 20% Na.sub.2CO.sub.3 Conditions Residual Live
cell Residual Live cell Temperature Time titer count titer count pH
(.degree. C.) (h) ratio (%) (cfu/ml) ratio (%) (cfu/ml) Treatment
4.0 40 5 99.7 6.7E+06 99.7 4.1E+06 of killing 18 100.9 <10 101.2
<10 microorganism 45 5 94.7 <10 95.9 <10 18 86.5 <10
87.1 <10 4.5 40 5 99.7 9.3E+09 99.6 5.8E+06 18 102.4 >1.0E+02
101.7 >1.0E+02 45 5 100.4 <10 99.8 <10 18 94.6 <10 93.5
<10 5.0 40 5 101.6 9.2E+06 103.7 7.8E+06 18 103.1 >1.0E+02
100.0 >1.0E+02 45 5 104.2 <10 102.1 <10 18 98.9 <10
97.5 <10
[0106] A difference was not observed between the pH adjusting
agents in terms of the residual titer ratio before and after the
treatment of killing the microorganism and the live cell count of
Ss.
Example 13
Preparation of Dry Product:
(1) Preparation of Ss Concentrate Liquid
[0107] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (4%), yeast extract (0.8%),
monopotassium phosphate (0.1%), and magnesium sulfate (0.05%). This
culture solution was centrifuged (10000 G, 30 mins), and a cell
concentrate liquid was collected. Then, the cells were washed by
adding sterilized tap water to the cell concentrate liquid and well
suspending the cells. The resulting suspension was centrifuged
under the same conditions, and adjustment was performed such that
the solid content was about 5%, and the thus obtained material was
used as an Ss concentrate liquid (solid content: 5.1%).
(2) Preparation of Ss Culture Solution
[0108] Ss was aerobically cultured at 27.degree. C. for 4 days in a
medium (pH 5) containing glucose (4%), yeast extract (0.8%),
monopotassium phosphate (0.1%), and magnesium sulfate (0.05%),
whereby an Ss culture solution was obtained.
(3) Killing of Microorganism in Ss Concentrate Liquid (pH
Adjustment)
[0109] The pH of the Ss concentrate liquid obtained in the above
(1) was adjusted to 4.8.+-.0.2 with a 20% sodium carbonate
solution. The thus obtained Ss concentrate liquid was subjected to
a heating treatment by maintaining the liquid at 45.degree. C. for
7 hours. The results of the live cell count of Ss after the heating
treatment and the ratio of the .beta.-galactosidase titer after the
heating treatment to the .beta.-galactosidase titer before the
heating treatment (residual titer ratio) are shown in Table 19.
Further, immediately before drying, the Ss concentrate liquid after
killing the microorganism and a 24% lactose solution were mixed at
5:1, whereby a stock solution to be dried containing lactose at 4%
was prepared.
(4) Killing of Microorganism in Ss Concentrate Liquid (Coexistence
of Lactose)
[0110] The Ss concentrate liquid obtained in the above (1) and a
24% lactose solution were mixed at 5:1, whereby an Ss concentrate
liquid containing lactose at 4% was prepared. The thus prepared Ss
concentrate liquid was subjected to a heating treatment by
maintaining the liquid at 50.degree. C. for 7 hours. The results of
the live cell count of Ss after the heating treatment and the ratio
of the .beta.-galactosidase titer after the heating treatment to
the .beta.-galactosidase titer before the heating treatment
(residual titer ratio) are shown in Table 19. The Ss concentrate
liquid after killing the microorganism was directly used as a stock
solution to be dried.
(5) Killing of Microorganism in Ss Concentrate Liquid (Combination
of pH Adjustment and Coexistence of Lactose)
[0111] The Ss concentrate liquid obtained in the above (1) and a
24% lactose solution were mixed at 10:1, whereby an Ss concentrate
liquid containing lactose at 2.2% was prepared. Further, the pH of
the thus prepared Ss concentrate liquid was adjusted to 4.8.+-.0.2
with a 20% sodium carbonate solution. The thus obtained Ss
concentrate liquid was subjected to a heating treatment by
maintaining the liquid at 50.degree. C. for 7 hours. The results of
the live cell count of Ss after the heating treatment and the ratio
of the .beta.-galactosidase titer after the heating treatment to
the .beta.-galactosidase titer before the heating treatment
(residual titer ratio) are shown in Table 19. Further, immediately
before drying, the Ss concentrate liquid after killing the
microorganism and a 24% lactose solution were mixed at 11:1,
whereby a stock solution to be dried containing lactose at 4% was
prepared.
(6) Killing of Microorganism in Ss Concentrate Liquid (pH
Adjustment)
[0112] The pH of the Ss culture solution obtained in the above (2)
was adjusted to 4.8.+-.0.2 with a 20% sodium carbonate solution.
The thus obtained Ss culture solution was subjected to a heating
treatment by maintaining the solution at 45.degree. C. for 7 hours.
The results of the live cell count of Ss after the heating
treatment and the ratio of the .beta.-galactosidase titer after the
heating treatment to the .beta.-galactosidase titer before the
heating treatment (residual titer ratio) are shown in Table 19.
Further, the Ss culture solution after killing the microorganism
was centrifuged (16000 G), and wet cells were collected. Then,
sterilized tap water was added thereto and the wet cells were well
suspended therein and adjusted such that the solid content was
about 5%, whereby an Ss concentrate liquid in which the
microorganism was killed (solid content: 5.2%) was obtained.
Immediately before drying, this Ss concentrate liquid in which the
microorganism was killed and a 24% lactose solution were mixed at
5:1, whereby a stock solution to be dried containing lactose at 4%
was prepared.
(7) Spray Drying
[0113] The stock solutions to be dried obtained in the above (3) to
(6) were dried using a pilot apparatus for spray drying (PRODUCTION
MINOR, GEA Process Engineering, Inc.) under conditions that the
inlet temperature was 120.degree. C., the outlet temperature was
80.degree. C., the rotation speed of the atomizer was 15000 rpm,
and the processing amount of the stock solution was about 4.5
kg/hr, whereby favorable dry products were obtained. The residual
titer ratio, the average particle size, and the residual water
content of each of these dry products were measured. The results
are shown in Table 19.
TABLE-US-00019 TABLE 19 a b c d Stock solution Concentrate liquid
Culture solution Titer (U/kg) 5500 2450 Method for killing
microorganism pH adjusting Lactose coexis- pH/lactose com- pH
adjusting method tence method bination method method Step of
Treatment Initial pH 4.8 .+-. 0.2 -- 4.8 .+-. 0.2 4.8 .+-. 0.2
killing conditions Concentration of lactose (%) -- 4 2 -- micro-
Temperature (.degree. C.) 45 50 50 45 organism Maintaining time
(hr) 7 7 7 7 pH (actual Before killing microorganism 4.9 3.9 5.0
4.7 value) After killing microorganism 5.1 4.7 5.2 4.7 Liquid
Residual titer ratio (%) 99.9 103.7 102.2 92.7 subjected to Live
cell count of Ss (cfu/mL) <10 <10 <10 <10 treatment of
killing microorganism Step of Addition of stabilizing agent
(lactose)/ 4 -- 2 4 spray additionally added amount (%) drying
Stock solution Solid content Ss cells 4.3 4.3 4.3 4.6 to be dried
concentration Stabilizing agent 4 4 4 4 (w/v %) (lactose) Total
solid content 8.3 8.3 8.3 8.6 Titer (U/kg) 4680 4760 4720 4690
Operating Rotation speed of atomizer (rpm) 15000 conditions Feeding
amount of stock solution 4.5 4.5 4.4 4.6 (kg/hr) Temperature Inlet
120 (.degree. C.) Outlet 80 Dry product Average particle size
(.mu.m) 21.1 18.4 19.5 20.0 Residual water content (%) 4.8 6.0 5.2
4.7 Titer (U/g) 54.2 49.7 52.6 55.0 Residual titer ratio (%) 96.2
91.3 95.3 95.8
[0114] In all the conditions for killing the microorganism,
favorable dry products in which the residual titer ratio was 90% or
more and the live cell count of Ss was less than 10 cfu/ml were
obtained.
Example 14
Storage Test:
[0115] The dry products (Products 1 to 3) prepared in Example 2 and
the dry products (Products 5 to 7) prepared in Example 5 were
stored at 5.degree. C. or 25.degree. C. for 360 days. The ratio of
the .beta.-galactosidase titer after the storage to the
.beta.-galactosidase titer before the storage (residual titer
ratio) was determined. The results are shown in Table 20.
TABLE-US-00020 TABLE 20 Example 2 (pH adjustment) Example 5
(addition of lactose) Product 1 Product 2 Product 3 Product 5
Product 6 Product 7 (lactose 2%) (lactose 5%) (lactose 10%)
(lactose 2%) (lactose 5%) (lactose 10%) Residual 5.degree. C. 100.2
101.0 98.0 95.7 95.4 93.5 titer 25.degree. C. 93.3 95.4 95.7 87.3
90.2 92.8 ratio (%)
[0116] It was found that even if the dry products were stored at
5.degree. C. or 25.degree. C. for 360 days, the residual titer
ratio was 80% or more.
Example 15
Killing of Microorganism:
[0117] A microorganism was killed in the same manner as in Example
1 except that Sporobolomyces singularis JCM 5356 (ATCC 24193) was
used in place of Ss, and as a result, substantially the same
results were obtained with respect to the residual titer ratio and
the like. For example, the residual titer ratio when the heating
treatment was performed at pH 5.0 and 45.degree. C. was 80% or
more.
Example 16
Killing of Microorganism <Examination of Sugar
Composition>:
[0118] A microorganism was killed in the same manner as in Example
4 except that Sporobolomyces singularis JCM 5356 (ATCC 24193) was
used in place of Ss, and as a result, substantially the same
results were obtained with respect to the residual titer ratio and
the like. For example, the residual titer ratio when the heating
treatment was performed at a lactose concentration of 5% and
45.degree. C. was 80% or more.
INDUSTRIAL APPLICABILITY
[0119] According to the present inventive method, a microorganism
can be killed while maintaining the enzyme titer of a liquid of
microbial cells having an enzymatic activity. Due to this, the
microbial cell liquid treated by the present inventive method is
easy to handle and also can be stored, and therefore can be
advantageously used in the industry using the enzymatic
activity.
* * * * *