Ep0087920a1.pdf

Europaisches P a t e n t a m t European Patent Office

© Publication number:

087

A1

Office europeen des brevets

EUROPEAN

0

PATENT

APPLICATION

© Application number: 83300952.5 ~ (22) Date of filing: 23.02.83

© Int. CI.3: C 12 P 1 9 / 4 2 C 12 N 1 / 2 0 //C12R1/01

© Priority: 26.02.82 JP 29064y82 26.02.82 JP 29065/82 26.02.82 JP 29066/82

© Applicant: NIPPON OIL CO. LTD. 3-12, 1-chome Nishishinbashi Minato-ku Tokyo(JP)

© Date of publication of application: 07.09.83 Bulletin 83/36

© Inventor: Kojima, Ichiro 4-2-22, Shonan Takatori Yokosuka-shi Kanagawa-ken(JP)

© Designated Contracting States: DE FR GB

© Inventor: Komiya, Kouji 931-77, Kuden-cho Totsuka-ku Yokohama-shi Kanagawa-ken(JP) © Inventor: Sato, Hiroshi 2-235, Kosugi-cho Nakahara-ku Kawasaki-shi Kanagawa-ken(JP) © Inventor: Oguchi, Yutaka 1-28-17, Higashitamagawa Setagaya-ku Tokyo(JP) © Representative: Myerscough, Philip Boyd et al, J.A.Kemp &Co. 14, South Square Gray's Inn London, WC1R5EU(GB)

© Process for producing vitamin B12 by the fermentation technique, and vitamin B12-producing microorganism.

o CM C) rs 00 o

A process for producing vitamin B12 by the fermentation technique which comprises cultivating a vitamin B12producing microorganism belonging to the genus Propionibacterium in a culture medium containing a carbon source and a nitrogen source, and collecting vitamin B12 accumulated in the cells of the microorganism; characterized in that (1) cultivation is carried out while adding a base to maintain the pH at 5 to 7.5, and (2) cultivation is carried out while adding a carbon source portionwise to the cultivation system nearly within 10 minutes of adding the base, (3) provided that when a vitamin B12-producing microorganism having resistance to propionic acid is used as the microorganism, the portionwise addition of the carbon source in (2) can be omitted; and a propionic acid-resistant, vitamin B12-producing microorganism of the genus Propronibactenum.

9 2 0

This for by

invention vitamin

producing an

industrially

relates

resistance

to

aforesaid a

comprises

Propionibacterium carbon

and

source

in a

an

alkali

system

so

that

system

is

maintained

about

nitrogen

adding

the

system

nearly

in

that

the

the

carbon

at

having

used

invention

in

the

the

to

source

the

time

same

cultivation

the the

of

about

5

while

out the

to

the

as

cultivation

range

portionwise

while

out

carried

is

micro-

the

to

a

collecting

of

times in

genus

and

carried

pH

B12-

containing

is

to

fermentation

a vitamin

cultivation, a

relates

the

by

cells

cultivation the

at

be

can

source, the

during

(2)

microorganism

medium

in

suitable

and

also

B12

a culture

at

7.5,

invention

this

cultivation

adding

to

The

belonging

B12 a c c u m u l a t e d characterized organism; the

output

cultivating

vitamin

(1)

increased

vitamin

microorganism

producing

process

process.

producing

which

technique

improved

markedly

which

acid,

specifically,

for

process

an

B12-producing

propionic

More

a

to

operation.

easy

improved

in

B12

a vitamin

to

relates

cultivation of

addition

the

alkali, (3)

having

microorganism used of

as the

the

that

provided

resistance

source

This

(2)

in

prppionic

process; said

and and

which

also

to

can a

be

relates

used

process

addition

omitted.

be

in in

for

to

vitamin

a

resistance

microorganism having B12-producing a c i d w h i c h is n o t d e s c r i b e d pionic literature

Bl2-producing acid is

portionwise

can

also

invention

to

the

microorganism,

carbon

a vitamin

when

the

the

to

prior

aforesaid

producing

the

microorganism. A process

for

producing

vitamin

pro-

B12

has

afore-

been

previously

known

which

comprises

vitamin

Bl2-producing

microorganism,

vitamin

Bl2-producing

microorganism

carbon

and

source

a culture

in

Propionibacterium,

nitrogen

a

cultivating for of

in t h e m i c r o b i a l B12 a c c u m u l a t e d U.S. P a t e n t No. 2 , 9 5 1 , 0 1 7 ) . example, order

to

the

the

improve

aforesaid

inventors

present

of

B12

using

belonging

to

tion

of

the

acts

as

an

vitamin

a vitamin

the

in

factor

important

vitamin

by

suitable

to

changes

to

maintain

the

at

vitamin

the

an

of

performing

amount to

cultivation, and of

the

Comparative vitamin

vitamin

by

by

B12-producing

the

or

cultivation

the more

by in

adding

cultivation

system

shown

of

large

the in

the

the

all

initial

hereinbelow

alkali,

advantage

as

adding the

as

about

at

industrial

as

so

while

and

addition

with

alkali

system system

the

to

medium

also the

B12-producing

Propionibacterium, obtained

discovery

case

carbon of

stage by

Examples

Examples.

has

B12

the

of

during

according

as

be

cells

system

cultivation

will

amount

cultivation

produced

culture

the

an

portionwise

times

system

adding

cultivation,

time

concentra-

while

the

be

the

in

to

during

as

It

led

in

microorganism

microbial

cultivation

two

the

the

the

same

that

cultivation

pH of

can

in

the

pH of

source

B12

source

the

7.5

carbon

nearly

to

the

about

5 to

fermentation

changes

cultivation

times in

the

investigations

performing

at

on

in

B12 a c c u m u l a t e d cultivation. Further

found

the

in

the

and

B12-producing

source

(for

in

B12

Propionibacterium,

genus

carbon

by

cells

investigations

vitamin

process,

technique

that

of

output

manufacturing

production

the

made

a

collecting

vitamin

The

genus

containing

and

source,

a

example

the

medium

a

been

that

found

fermentation

hydrolyzing

of

microorganism

of

of

the

the

spent

molasses

spent

the

production

technique

microorganism

a hydrolyzate

in

using genus

molasses

which

genus

a

the

vitamin

is

Propionibacterium as

a carbon

amount

of

vitamin

which

source vitamin

in

contained

to

in

and by

copresence

also

been

instead

of

effect

found

the

of

particularly replaced

by It

of

spent

results and of

the is

cost of

that

vitamin

and

increases

takes

B12 a c c u m u l a t e d , not i n t e r f e r e d

be

can the

increasing

if

even of

It

utilized

aforesaid

one-half,

the

a part,

fructose

is

glucose. been

acts

of

molasses

the

of

low

industrial

The

present

of

vitamin

component

of

with

entire

the

value

of

fructose whose

source of

cost

production further

technique, the

the

utilizability

carbon

a

as

fermentation

the

better

the

with

hydrolyzate

gives

cost

other

the

cooperatively

coupled

one-third

that

found

this,

by

the

interfered

not

molasses

B12

vitamin

fructose

some

about

molasses

hydrolyzate.

because

hydrolyzate

fructose

in

glucose

presumably and

that

of

at

glucose,

genus

is

available

of

the

the

of

effect

molasses

hydrolyzate

cells

increasing

further

has

of

amount

the

more.

or

spent

nearly

cheaper

of

of

is

to

up

the

show

hydrolyzate,

fructose

in

investigations the

aforesaid

the

times

two

useful

increase

markedly

hydrolyzate

increasing

the

has

the

can

is

utilizing

microorganism

B12-producing Further

of

accumulated

B12

Propionibacterium

part

difficult

of

process

this

invention. production of

the

genus

vitamin

cultivation

B12 f a i l s . cultivation, when

the

of

propionic

amount

cultivation the

and

builds of

system

vitamin

the

sometimes

the

up

shown in

formed and

propionic exceeds

B12-producing

that

acid

vitamin

cultivation in

amount, in

accumulated

a certain

limit,

microorganism

the

during

the

increases

the stops

of

production

have

acid

of

growth

microorganism

Investigations

gradually

system

the

Propionibacterium,

B12-producing the

during

in

that

technique B12 by t h e f e r m e n t a t i o n vitamin B12-producing microorganisms

conventional

using

found

inventors

the is

and

the growth

inhibited

until

its

finally

vitamin

B12 c e a s e s . The p r e s e n t in

investigations in

the

of

the

which

acid

pionic in

an

can

of

a

vitamin

the

genus

the

strain

and

two

times

more

improved

The this

following

this

At

the for

the

above

and

will

natural

been

found

be

can

the

caltivated parental

cultivation in

B12

an

output

(1)

(1)

vitamin

a vitamin

above, (2)

and

invention

producing

genus

by

and

condition

this

using

B12

by t h e B12 p r o d u c e d the c u l t i v a t i o n is

the

of

pro-

combination

a

for

conditions

object

technique

invention

time,

to

microorganism

also

special

vitamin

under

out

an

of

to

strain

any

discovery

created

parental

has

vitamin

of

that

process

microorganism

be

conditions

for

the

output.

vitamin

treatment It

produce

under

is

fermentation

of

can

preferably It

an

need

to

resistance

B12-producing

carried

preferably

led

can

output

improved

producing

microorganism

strain.

parental

of

capable

treatment.

the

conditions, more

have

having

cultivation

same

without

or

trouble

fermentation

an

Propionibacterium

aforesaid

the

in

B12

mutation-inducing

mutation-inducing under

strain

improved

artificial

that

vitamin

give

being

markedly

strain

aforesaid

the

by

B12

investigations

and

subjecting of

vitamin

microorganism

a

the

their

B12-producing microorganisms and t o d e v e l o p a Propionibacterium,

These that

continued

remove

of

production

vitamin

using

genus

process

to

the

and

stops

inventors

order

production

technique of

growth

to B12

above.

p r o v i d e by

the

B12-producing

Propionibacterium.

other

objects

become

more

and

advantages

apparent

from

the

description. In

B12-producing

the

process

of

microorganism used.

this of of

bacterium

is

producing

microorganism

include

shermanii

IFO

IFO

Fermentation,

12391 Osaka,

Examples and

Japan;

invention, the

genus

such

a vitamin

Propioni-

a vitamin

B12-

Propionibacterium

12426 freely

[Institute

for

distributable

known

and

strains]; 12424

for

[Institute

Fermentation, strain].

known

distributable able

Osaka,

They

known

strains

deposited

the

aforesaid

deposit

under

IFO

Propionibacterium freudenreichii

in

freely

freely

distribut-

aforesaid

depository

are

the

Japan;

numbers.

Other

usable

strains include Propionibacterium B12-producing NOC 1 1 0 1 1 h a v i n g thereto shermanii the imparted

vitamin

property

of

Research

Institute,

Technology, NOC

Institute, the

strain

deposited and

11C13

having acid

propionic

[FERM

The of

the

the

is

strain

are

the

the

of

properties

has

resistance

greater

biological of

the

IFO

parental

12424

propionic these

example, 8th

NOC

IFO

12391 The

stand.

microbiological

parental

12391

NOC

The

Propionibacterium it

strains

as

has

it

that The

the

same

11012

strain

except

acid.

propionic

the

when

shermanii

the

IFO

parental

are

strain

Bergy's

the

11013

acid.

edition.

of

of

rate

Propionibacterium

that

in

its

the

except

parental

that

of

properties

freudenreichii

to

those

except

of

shermanii

Propionibacterium

the

as

that

known

to

of

same

to

the

deposited

the

Propionibacterium those

as

same

left

is

system

under

Treaty].

shermanii

Propionibacterium

cultivation

strain

are

than

faster

Technology,

freudenreichii

properties

strain

parental

sedimentation

and

resistance

Budapest

microbiological

the

Science

thereto

Propionibacterium shermanii known

to

internationally

BP-87;

under

shermanii

Research

Propionibacterium

imparted

internationally

resistance

Fermentation

Industrial

of

and

internationally

Propionibacterium

BP-86;

Agency Treaty],

Budapest NOC

[FERM

acid

Science

deposited

thereto

imparted

having

propionic Japan;

Treaty],

Fermentation

BP-85;

Industrial

strain

the

Budapest

11012

of

Agency

Japan;

the

under

[FERM

sedimenting

easily

those

microknown

freudenreichii

greater

resistance

to

microbiological

properties

of

are

Manual

known, of

and

described,

Determinative

for

Bacteriology,

shermanii the

The

above-mentioned

NOC

11011 strain

parental

12391

ultraviolet in

resistance be

to

the

inducing

strain

wherein

the

culture

medium and

which

by

and

a to

being

strain,

treatment

being at

a carbon

above

the

minumum

natural

IFO

(for

12426

example, NOC

shermanii which

have

the

genus

propionic

(for been

vitamin

acid

can

be

(for

described

above.

of

vitamin

under for

the

and

natural

same be

example,

example,

IFO

12391

freudenreichii

Propionibacterium

FERM BP-85

having

created

the

to

that

strain),

hereinabove. microorganism

and

resistance subjecting

by

strain parental B12- p r o d u c i n g b a c t e r i u m as e x e m p l i f i e d above strain

vitamin

B12-producing

treated

the

of

may,

cited

treatment

of

amount

stain

example,

mutation-inducing

of

growth

times

strain),

Propionibacterium

liquid

concentration

Propionibacterium

12424

11011

already The

shermanii

strains),

the

1.5

strain

parental

IFO

the

a

mutation-inducing

until

about

treatment

a nitrogen

concentration

repeated

mutation-

in

source,

inhibits

of

surviving

cultivated

parental B12 p r o d u c e d by s a i d cultivation conditions.

and

the

the

said

Propionibacterium

artificial

an

acid, acid

least

strain

parent

can

comprises

mutation-inducing is

containing

propionic

The

shown

above

which

process

subjecting

strain

propionic

reaches

is

exemplified

B12-producing

treated

parental

B12

it

hereinbelow.

as

a natural

to

acid

propionic at

example

treatment,

treated

source

acid

Propionibacterium

genus

of

IFO with

procedure

example

1 given

propionic vitamin

a

subjecting

shermanii

microorganism B12-producing genus P r o p i o n i b a c t e r i u m h a v i n g

for

produced,

from

derived

vitamin the

to

One

Example

The

be

can

a mutant-forming

irradiation.

Referential

belonging

BP-85)

Propionibacterium

utilizing

by

(FERM

Propionibacterium

of

the

to

an

then

genus

of

to a vitamin Propioni-

artificial subjecting

mutation-inducing

the treatment

Means treatment

artificial

for

known

are

invention.

per of Examples

irradiation

of

cobalt inducing and

such

and

such

as

2-aminopurine.

nitrosoguanidine,

irradiation

treatment,

such

be

selected.

For

properly

irradiated

be

can

minutes. and

the

with

treated 100

mg/liter

for

30

the

present

surviving

after

treatment

is

treatment

wherein

and

source

to

the

medium

a nitrogen

which

acid

propionic

produced

times

1.5

strain

parental

The for

the

kind

of

g/liter a

the of

culture

centration

of

above

of

same

medium. containing minimum

of

growth the

reaches

acid the treatof acidat

by t h e B12 p r o d u c e d cultivation conditions. concentration

inhibitory depending

used, In

amount

propionic

strain

differs

strain

the

propionic

until

vitamin

in

concentration

created

growth

strain

a carbon

only

mutation-inducing

out

the

the

strain

parental

medium

be

can

cultivated

not

the

B12-producing

minimum

culture

properly

treated

is

minimum

natural

under

parental

with

mutation-inducing

also

inhibits

by

that

the

strain but

the

carried

vitamin

resistant, least

The

repeatedly B12

2

inducing

strain

natural

a

source

at

vitamin

rays

for

the

can

a concentration

containing

above

is

time,

mutation-inducing

treated

a concentration

ment

and

be

parental

artificial

subjected

strain.

of

invention,

in

parental

the

treatment

also

in

mutation-

minutes.

the

culture

liquid

the

example,

the

can

(e.g.,

for

erg/mm2

amount

nitrosoguanidine

In

a

for

time,

rays

ultraviolet

300

of

the

as

treating

For

selected.

a dose

such

agents,

dose

example,

conditions

Treating

inducing agent

in

the

as

such

hydroxylamine

conditions

Treating

rays

artificial

an

this

include

means

radioactive

with

in

utilized

mutation-inducing

treatment

agent

be

can

treating

X-rays

rays,

and

60),

and

se

artificial

ultraviolet

as

mutation-inducing

the

but

present

propionic growth

is acid

upon about

the 10

invention, in

inhibitory

a con-

for

concentration, g/liter

of

culture

25

g/liter

of

repetitions

ment to

is

of

of

the

creation

of

to

is

natural

30 15

number treat-

mutation-inducing this

and

20,

lead

can

vitamin

acid-resistant,

a propionic

about

to

The

utilized.

about

5 to

about

about

preferably

medium

about

usually

the

medium,

culture

10

about

example,

w h i c h w i l l p r o d u c e v i t a m i n B12 strain B12-producing in t h e d e s i r e d cultivation amount. The r e p e a t e d is carried

out

propionic

by

acid-containing

cultivation, propionic them

acid-containing then

there, second

and

inoculating

propionic

third in

culture

the

cultivation One

vitamin

colonies

of

the

cultivated taining

20

treated

strains

at

an

determined are

culture

medium

in

5 days. the

same In

5 days. During

the

this

and

in

610

for 25

20

the of

a

the

as the

be

carried

number

of

propionic

after

the

treatment

are

medium

liquid

con-

acid

for

the

order

of

good

amount

of

growth

the nm.

the

The

second

for

5 days.

selected time of

g/liter

treated

cultivated

way,

repetition

in

propionic

containing

Likewise, way

time

of

cultivated

also

surviving

measuring

by

for

progressively

producing

selected

density

strains for

are

separately

cultivation

mutation-inducing of

medium medium

may

acid

strain

first

g/liter

optical

of

treated the

for

on

microorganism B12-producing For e x a m p l e , 100 detail.

some

artificial

aforesaid

growth

in

a

above

a

prepared

increases.

embodiment

below

given

have

propionic

cycles

acid-resistant, is

of

content

increasing

the

first

grown

liquid

cultivation

which

media

in

a

cultivating

liquid

them

acid-containing

repeated

and

well

on

the

in

separately

colonies

repeating

grown

medium

cultivating

and

The

procedure.

a

acid-containing

and

time,

in

liquid

collecting

propionic

prepared

out

them

well medium

liquid

inoculating

the

the

colonies

collecting

cultivation cultivation,

are

third can a

fifty a

liquid

propionic

strains the

in

Fifty

be

mutant

acid

selected time

for

repeated. having

resistance

to

The

cultivation

can

be

medium

a

medium

liquid

resulting

mutant

resistant

strain.

can

be

be

this

both

of

g/liter

accumulated. the

finally

strain in

a acid

propionic acid.

propionic

no

a

as

separated

culture

in

used

20

and

example

containing

can

Known

for

grown,

containing

derived

until

repeated

equivalently

liquid and

is

is

acid

propionic

The acid-

propionic

media

and

cultivation

conditions

invention

for

cultivating

vitamin

of t h e g e n u s P r o p i o n i microorganisms B12-producing bacterium the a f o r e s a i d acidincluding propionic vitamin

resistant,

microorganisms. Bl2-producing culture medium w h i c h c a n be u s e d in

The invention and

if

contains

source

are

Specific They

glycerol. of

Examples nitric

the

acid

salts,

meat

extract,

fish

residues,

and

Examples medium

ponents

such

such

phosphates,

calcium salts,

zinc

aluminum

tion

with

cultivation about

37°C,

vitamin

cultivation, N2 gas

salts,

molybdenum

or

C02

temperature the

such

can

com-

minerals iron salts

out

under

desirable.

Preferably, cultiva-

and

aeration-agitation

gas

can

be

is,

for

example,

about

time

for

cultivation

The

employed. is,

and acid.

pantothenic

carried

is

salts,

salts, copper

as

be

this

and

the

potassium

cobalt

salts,

cultivation

and

of

B12-constituting salts,

vitamins

residue,

soybean

urea,

other

salts,

casein,

extract,

yeast

magnesium

conditions,

stationary

ammonium

are

and

combinations.

components

and

salts,

anaerobic

suitable

of

manganese

salts, The

in

5,6-dimethylbenzimidazole,

as

salts,

acid

wastes.

include

culture

as

tartaric

fermentation the

and

acids,

acid,

liquor,

steep

carbon

fructose,

peptone,

corn

B12the

glucose,

source

nitrogen

sourse

are

used

be

of

organic

sugars,

lactic

can

vitamin

Examples

examples

galactose,

mannose,

etc.

components,

a nitrogen

vitamins,

carbohydrates,

alcohols.

and

source

minerals,

required,

constituting

carbon

this

25 C

to

example,

about

3 days

about

to

vitamin

is

least

containing

under

the

the

alkali

of

cultivation below)

the at

about

in

the

6 to

about

carbon

to

the

in

out (1)

that

during

maintained preferably

is

of

the

alkali); B12-

to

propionic

the

as

micro-

of

the

carbon

used

for

pH a d j u s t m e n t

calcium

source

potassium and

hydroxide,

used

either

singly

carbon

source

added

be

may

system alkali

the

vitamin

hydroxide,

carbonate,

adding

10 m i n u t e s

the

used

alkali

sodium

They

of

addition

Examples include

of

resistance

having

while

out

cultivation

the

when

omitted.

ammonia.

that 7.5,

about

addition

hereinabove

sodium

values

or

a mixture.

above

is

the

The portionwise is

group Sugars

at

50

about

added

in

(1).

of

kind

can

consisting as

about to

least

such

the

70

about

alkali

of

amount

preferably

preferably

it

the

to

up

be

above

the

addition

the

can

The

of

time

a

portionwise

(2)

hydroxide, as

at

provided

the in

aqueous

as

microorganism

organism, set

time

than

described

as

to

about

portionwise

later

while pH

is

source or

source

the

system

5 to

at

which so

carried

same

out

system

is

(3) acid

adjusted

cultivation

or

producing

be

about

medium

and

7;

(simultaneously earlier

(at

the

the

at

nearly

of

genus

(3):

to

times

cultivation

range

(2) the

the

the

a nitrogen

carried

cultivation

invention

of

is

can

system

the

to

cultivation, a pH

suitable

at

an

shown

(1)

cultivation

adding

this

a culture

and

source

conditions

following (1)

in

cultivated

carbon

a

of

process

microorganism

B12-producing

Propionibacterium

the

days.

the

to

According the

10

be one of

the

about

200g,

carbon

properly carbon

to

carbohydrates,

glucose,

per

fructose

to but

selected usgars and

(2)

more

gram

source

selected, source

300g,

in

equivalent be

added

preferably from and

converted

the alcohols. sugar

are added

the

of

not

about

ably of

the

suited the of

tion

the

(1). of

in

and

hampered,

or

at

of

addition the

for

system

to

30

g/liter

source

in

about

the

in

(2)

be

performed

For

for

by

relation

in

pump

equipped

is

designed

pH

of

the

cultivation

this

carbon time

same

inven-

source the

as

addi-

cultivation

words,

it

may

be

effected

later

time.

earlier

or

time of

of

the

alkali

in

adding

the

carbon is is

produced

E12

achieve

tc

If

(1)

microorganism

vitamin

the

improved

alkali

carton

the

of

relationship. the an

that

it

system

shifts

to

in

and

(2) it

nearly

supplying

carton

For

example,

source

source it

can

supplying supplying

alkaly

pH c o n t r o l

means

which

a change

in

the

the

of

the

from

a

and

when

an

acidic

the

is

the

an

detects

and

a carbon

source

of

automatic

when

cultivation

a carbon motion

(1)

alkali

(1)

addition

in

source an

in

effecting

practice,

operating

addition

for

employee

industrial

by

to

system

be

the

operating

with so

of

the

can

of

co-acting

pump

the

pH-adjusting

alkali

pump

in

the

analytical

source.

process of

an

to

of

means

effected

supplying

carbon

the

impossible

time.

the

by

alkali

the

amount

addition

preferably pump

cultivation

invention.

of

same

at

growth

it

this

portionwise at

prefer-

concentration

carbon

pH-adjusting

from

Various the

of

some

the

the

making

process

other

the

(2),

reduced,

cultivation

determined

addition

In

adding

residual be

nearly

differs

definitely source

concentra-

more

the

the

respective

pH-adjusting

in

time

the

practice

simultaneously, the

the

in

5 g/liter

can

the

effected

the

system

of

portionwise

is

(2)

the

g/liter,

in

source

system for

In in

60

that

example,

about

at

amount

cultivation

tion,

such

be

to

source

medium). The

method

(for

carbon

carbon

source

about

g/liter

maintained

culture

the

carbon

exceed

residual

be

can

50

the

preferably

residual

does

system

of

amount

is

portionwise

tion

of

The

preferred.

side

pH

pH

predetermined be

it

the

change

to

according

pump

value, make

which

utilized

in

of

the

the

alkali

Other

pH. the

possible

addition

simultaneous

actuates

supply

means

can

substantially

alkali

and

the

carbon

source. Preferred this

invention

carbon

are

hydrolysis fructose

of

example

of

product and

carbon

manufacturing

industry.

ing

sugarcane

or

and

collecting of

contents and

salts

becomes

invention, fructose of

and

(preferably cheap

glucose an

adding

or

sulfuric

be

molasses liquid The it

its

or

becomes

mixture

about

inverting

invertase

60

time 10

Or an

about

is

to

the

to

added

be

can

hydrolysis an

mixture

a

a part

fructose

can

source. be

performed acid

hydrochloric of

the

that

so

and

induce

by

spent

the

of

pH

the

the

heating

hydrolysis.

selected;

for

example,

minutes. can

invertase. in

as

suitably 30

about

enzyme,

carbon

to

molasses,

and

the

4,

present

spent

of

about

blackish

replacing

liquid

120°C

the

weight)

dilution

1 to

be

method,

such

syrupy

can

such

molasses

a

sugar

of

of

acid

viscosity

In

by

spent

its

time.

by

sugar

and

a syrupy

as

aqueous

about

at

treating is

to

are

obtained 50%

more

utilized

inorganic acid

increase

desired

about

inverted

materials, no

the

crystals,

sugar

sugar

material

a sugar-making

product

any

Hydrolysis by

concentrat-

this

glucose

can

of

Molasses at

by

to

up

a procedure

liquor

a hydrolysis

fructose

advantageous the

finally

left

produced

containing

of

as

organic

obtained.

by-product

A

by-product

a

resulting

until

higher,

economically brown

are

mother

the

and

glucose, glucose.

industrially

juice

non-sugar

in

and

molasses

When

beet

the

in

source.

molasses

Spent

and

fructose

an

(2)

in

fructose-containing

fructose

spent is

glucose

the

a

of

containing

source

added

sources

fructose, mixture

a

source,

a carbon

carbon

amount

be

effected

For of

example,

about

0.05

by

using enzyme to

about

0.5%

based

the

the

on

molasses of

a pH for

6 and

3 to

60°C.

about

The

2

According the

vitamin

bacterium

carbon

a

conditions

(1)

suitable

during

the

system

is

about

to

the

of

be

can 24

about

at 20

properly

to

chosen,

hours. of

process

the

to

and

7.5, to

a

this

invention,

nitrogen

(1)

while

cultivation the

of

pH

at

a pH

(2)

while

in

of

addition

adding

the

the

alkali

an

that

so

cultivation of

range the

about

carbon

almost

system the

under

source

system

adding

cultivation

the

the

as

and

i.e.

(2),

maintained

time

time

to

cultivation,

portionwise same

treatment

temperature

source

and

times

about

5 to

inverting

and

strain of t h e g e n u s P r o p i o n i B12-producing is c u l t i v a t e d in a c u l t u r e medium at l e a s t

containing at

a

molasses,

spent

to

treating

about

example

the

subjected

are

about

of

weight

alkali.

source the

at

vitamin

Then,

in t h e m i c r o b i a l cells in t h e c u l t u r e B12 a c c u m u l a t e d broth are c o l l e c t e d . vitamin Thus, B12 can be p r o d u c e d by The

the

fermentation

aforesaid

condition

(2)

is

organism

used

the

as

an be

can

vitamin

acid-resistant,

propionic

in

technique

improved omitted

output. when

a micro-

B12-producing

micro-

vitamin B.p-producing

organism. Collection by

broth

by

centrifugal and

separating

vitamin

B12 c a n be cells from the

microbial

the

separating

of

for

separation, vitamin

recovering

carried culture and

example, from

B12

out

the

microbial

cells. of

vitamin

B12

from

be

carried

out

by

Separation its

purification For

the

in

cells

can

example, the

form

dimethylbenzimidazole obtained the

the

or dark

membranes by

an

with

of

a

it

by

a

solvent

coenzyme),

means as

an

from

B12 ( 5 , 6 crushed cells

microbial means are

and

means.

separated

vitamin

physical such

cells

various

be

to

collected

ultrasonicating a

is

coenzyme

cobamide the

crushing

cellular

milling in

by

when

the

cells such

or

as

extracted

alcohol

(e.g.,

ethanol

methanol, it

is

to

be

the

from

or

or

pyridine.

the

of

hydroxocobalamin

in

separated the

cells,

When

isopropanol) form of

extract

the

vitamin

coenzyme

as a b o v e a r e e x p o s e d t o l i g h t t o c o n v e r t B12 o b t a i n e d is d e s i r e d When i t it to h y d r o x o c o b a l a m i n . to s e p a r a t e it

in

cells

of

the

form

are

extracted

the

presence

and

potassium

of

from

cyanocobalamin the

with

solvent

such

sodium

as

the

cells,

aforesaid

salt

a cyanide

the

in cyanide

cyanide. another

to

According

a vitamin

embodiment,

for impurities, containing liquid B12-containing obtained crushed liquid by c r u s h i n g ample a c e l l cellular

membranes the

extracting

cells

above-exemplified

is

is

liquid surface from

a Cl-C6

embodiment

about

styrene

example

a

alkyl-substituted ethylstyrene,

propylstyrene)

and

ing

to

whereby

in

which

its

or

B12-containing

resin m2/g

700

having

and

derived

derivative

such

(e.g.,

dimethylstyrene

acid,

a

functional

derivative

unsaturated

an

or of

ester

alkyl

expressed

by

the

an

follow-

formula

R represents

alkyl

group

having

bond,

and

is

example

di-

triisopropenyl propenyl t12

an

polycarboxylic

wherein

for

by

the

subjected

vitamin

functional

methylstyrene, aromatic

is

a copolymer

least

at

for

with

adsorbents,

using

aforesaid

with

divinylbenzene,

derivative, as

the

contacted of

this

to

used,

area

obtained

product

solvent

the

purified.

According adsorbent

extract

an

crushed

treatment

be

can

B12

its

or

or

extracting

adsorption-elution vitamin

above,

as

ex-

on

or

n

C3-C10

unsaturated

2 or

3, alkenyl

resin,

and

cause

eluting

adsorption the

such

esters

1,2,4-benzenetricarboxylate to

double

a carbon-carbon

tri-C3-C10

terephthalate the

a

adsorbed

as

and

diiso-

of

vitamin

vitamin

B12 w i t h an fractions. batchwise

The

or

Adsorption of

about

The

for

aqueous

ethanol,

eluents

can is

be

an

the

include

lower

acids

as

sodium

acid

The

eluent

the

kinds

and

and

alcohols

are

alcohol

cited.

out

at

room

carried

etc.

The

to

required

less

desired, example,

6-20% can

or

Heating although

it

operating

the

lower having for

example

isopropanol be

cooling is

of

not

carried be

may

particularly is

temperature

60°C.

about

may

of

50%,

operation

the

kind

alcohols

and

to

according

solutions

ethanol

sodium

phosphate.

the

about

such

ammonium

carbonate,

hydrous

and

acetic

and

selected

than

eluting

temperature.

Acrive as

of

15-40%

For

necessary.

and

eluents

alkalies

potassium

Aqueous

acids,

the

acid,

impurities,

the

ethanol

phosphate

and

of

2%

selected

of

acid,

sodium

as

conOrdinary

alcohols,

phosphoric

properly

preferred,

if

out 30

be

amounts

methanol,

be

about

also

content

can

such

eluent

methanol,

ammonium

phosphate

resin,

as

as

low

by,

example,

examples

hydrochloric

salts

can

adsorbent

25-50%

and

sodium

acetate,

an

such

hydroxide,

hydroxide,

such

fractions.

methanol,

an

be

performed

For

lower

Specific

alcohols

isopropanol,

of

a

the

eluate

having

eluent. of

at

isopropanol.

solution

salts.

boric

the

as

and

desired,

be

5% a q u e o u s

consisting

group and

acid,

used

if

may

a pH

After

active

give

at

7,

30°C.

may,

1% a q u e o u s

and

method.

about

alcohols

example,

aqueous

alkalies

to

hydrous

using

centrations,

resin

treatment

washing

example,

from

the

performed

example,

about

to

eluated

then

for

out,

10°C

about

be

can

preferably

treatment,

and

eluent

8,

eluate

chromatographic

carried

about

of

adsorption

for

a column be

can

temperature

active

collecting

adsorption-elution

by

5 to

washed,

and

eluent,

eluted be

recrystallization, According

fractions

subjected

to

are

collected,

and

concentraticn,

etc. to

still

another

embodiment

in

which

adsorbent

an

liquid

[determined

diameter

"Porous

of

31-73

published of

at

least

250

R,

for

volume

0.6

ml/g

of

vitamin

and

adsorbed

B12 w i t h fractions.

As

Diaion

procedure, (tradenames also

can

for

Japan)

Ltd., with

be

after be

therefor

same

out

as

there

can

be

activated

suitable

the

same

above and

HP-50

Chemical

Co.,

Such

resins

divinylbenzene such

as

same

here-

described

as

carried

optionally treatment

with

and

operations

and

operations

described

is

of

out

also

can

the

under the

first-

this

inven-

to

respect

the

recovered if

and

means

may

process

from

desired, also

be

extraction

employed

adsorption

carbon,

cellulose,

and

column

of

the

microbial

can

then

used. with with

be

For

cells purified.

example, adsorption

phenol,

ion-exchange

chromatography

in

combinations. The

invention

elution

elution

the

above,

purifying

or

HP-40

derivative

treatment

practice

B12

Other

resins

the

available.

the

be

and as

the

vitamin

described

with

in

embodiment. In

tion,

by

conditions

mentioned

and

may

washing

adsorption,

carried

the

eluting

collecting

HP-30,

functional

adsorption

The

inabove.

adsorption

hereinabove.

exemplified

conditions

and

copolymerizing

by

its

or

by

used

above

example

cause

Mitsubishi

commercially

produced

The

resin of

products

to

eluent

HP-20,

HP-10,

are

styrene

those

adsorbent

the

Kondo,

Company,

for

ml/g,

followed

an

pages

at l e a s t about 1200 A a n d

to

ml/g

resin,

vitamin eluted

active

the

on

B12

1.2

about

to

up

0.6

than

more

at

preferably 200

about

pore

Renichi

by

Gihodo

by

R,

frequent

described

written

200

about example

of

a most

1973

5,

a divinylbenzene/

method

the

Material",

September

pore

with

having

by

on

Japan] about

resin

copolymer

styrene

B12-containing in t h e a f o r e s a i d

described

contacted

be

may

vitamin

the

used,

impurities

containing

embodiment

a

is

in

following greater

Examples

detail.

illustrate

the

present

1

Example

Six of

25g

ing

of

of

mg of

of

lOg

IFO

shermanii

This

medium.

and

of

16g

2

10

ug

of

strain

seed

its

and

blowing the

culture

for

supplying be

motion

The

concentration

broth

in

added

broth,

and

days.

At

used

of

day

reached was

solution

a

of

the

this

3.2 650 used

ml, was

of

and 650

of

liquor,

CuSO4·5H2O, calcium into

fed

in

of

the

out

the

culture 30°C

at

the

controlling

sodium solution

of

pH

of

was

relation

sodium

remaining 10

glucose in

while

A pump

hydroxide.

to

hydroxide.

in

the On

g/liter.

culture the

5,6-dimethylbenzimidazole 10

of

mg/liter was

the

continued

the

amount

of

the

The

amount

of

5N s o d i u m

the

amount

of

the

ml.

5-liter

a

milliliters

carried

cultivation

liters.

B

started.

about

at

time,

under

medium

steep

mg of

was

was

glucose

amount

pg

supplying

cultivation,

then

culture

automatically

for

pump

an

corn

10

was

(W/V)

maintained

was

fourth was

of

culture

cultivated

inoculated

5N

operated

the

of

Sixty was

using

a 60%

ml

30°C.

a

automatically

broth

to

designed

broth

and

the

was

of

water

cultivation

gas

N2

of

cultivation

The

in

50

and

sterilized.

and

a 200

in

Propionibacterium

at

MnSO4·4H2O,

liter

per

and

KH2PO4, 1 . 5 g of N a 2 H P O 4 1 2 H 2 O , 10 mg o f mg of C o ( N O 3 ) 2 · 6 H 2 O ,

(NH4)6Mo7O24·4H2O

fermentor medium

30

of

5 mg

pantothenate above

80g

of

pg

of

0.4g

MgSO4·7H2O,

ZnSO4·7H2O,

liters

glucose,

(NH4)2SO4,

of

0.5g

of

lOg

4 days

for

ZnSO47H2O,

10

put

and

strain

microorganism

Separately, containing

was

of

0.5g

pantothenate

inoculated

was

conditions

stationary

water

of

mg

CuSO4·5H2O,

sterilized,

12391

10

of

3g

liquor,

Na2HPO4·12H2O,

calcium

of

liter

CaC03 per flask Erlenmeyer

of

A contain-

medium

steep

of

of

pg

5 mg

(NH4)6Mo7O244H2O,

corn

Co(NO3)26H2O, 50

MnSO44H2O,

a culture

1.5g

KH2P04,

30

MgSO47H2O,

of of

20g

glucose,

0.4g

NH4NO3, 5 mg

milliliters

The

amount

culture broth

culture 60% of

3

for

hydroxide

glucose

vitamin

B12

by

of

liter

per

In

leichmannii

IFO

3376.

vitamin

was

obtained

Comparative added

2-liters

to

ed The

in

the

There

the

culture

mixture

was

broth

into of was

culture

an

induction the

on

an

after,

the

amount

of

and

the

The

amount

the of

broth

was

IFO

3376.

This

was

produced

410g

per

Cultivation in

IFO

There3.0 400

was

of

the

determined mg of

liters, ml.

resulting a

by

Lactobacillus 78

The

3 days. was

used

was

leichmannii

vitamin

B12

used.

glucose

carried

the

amount

of

the

the

amount

of

sodium

of

amount

out

the of

broth

60%

that

12391.

7 days

culture

29

in

used

instead

When a

broth

hydroxide

glucose

in

out

the

same

way

Propionibacterium

IFO

vitamin was

carried was

for

was

culture

broth.

time

liter it

12424

shermanii

bacterium

The

broth

for

this

at

only

was

1 except

Example

freudenreichii

amount

culture

culture

continued

5,6-

2

Example as

growth,

the

using

of

for

to

per

that

hydroxide.

added

mg when

means

blowing

cultivation,

of

inoculat-

controlling

sodium

2 days

hydroxide

method

bioassay

customary

while

of

broth

26

of

the

started.

day

was

B12

of

automatically 5N

in

a 5-liter

was

30°C

at

was

1 was

Example

using

mg/liter

vitamin

into

milliliters in

and

sodium

was

used.

B shown

medium

out

sixth

culture of

mg o f

solution

charged

period

cultivation

amount

culture

10

of

amount

glucose

cultivation

broth

the

dimethylbenzimidazole in

its

fermentor

therefore,

of

glucose

used

as

carried

was

the

138

Sixty

and

fermentor,

cultivation

N2 gas t h e pH and

strain

seed

(W/V)

sterilized.

and

fermentor

a 60%

of

the

and

1,

Example same

of

ml

words,

410g

determined

1

Example 650

mg when

Latobacillus

using

other per

43

was

method

bioassay

a customary B12

broth

culture

the

the

5-liter

of

Propioni-

cultivation fermentor,

was

3.1

liters,

used

was

600

ml.

The

used

was

also

600

solution

and

of t h e per l i t e r B12 o b t a i n e d by a mg when i t was d e t e r m i n e d

ml.

IFO

leichmannii

ml

600 to

2

ample

1,

and

fermentor

and

in

while

blowing

sodium added

the

the

was

continued

the

culture

sodium

broth

it

that

NOC

liter

of

automatically period

The

culture

was

B12

broth of

amount of

amount

amount

broth

IFO

was

cultivation

the

the

a customary

by

the

5N

using

culture

the

time,

ml.

the

vitamin

of 16

was

method

bioassay 3376.

produced

mg

This

means

from

380g

used.

3 Example

shermanii

IFO

12391.

out

for

the

cultivation

7 days

5N s o d i u m 60%

in

product

customary

vitamin was

65

bioassay

of

When

B12

cultivation

3.3

was used it

liters.

660

ml,

per was

using

and

also

was

obtained

method

same

way

shermanii

was

the

fermentor,

was

mg when

the

Propionibacterium

the

a 5-liter used

in

out

Propionibacterium

instead

solution

glucose of

that

product

hydroxide

carried

was

1 except used

ed

30°C

at

induction

and

leichimannii

was

amount

this

420

11012

the

At

was

Cultivation as

out and

Then,

used

was

Example in

of

mg/liter

liters,

mg of

cultivation

product

an

seed

a

12424

its

and

was

2.9

determined

glucose

there

of

IFO

fermentor

was

per

46

broth

cultivation

10

B12

only

a 5-liter

carried

cultivation.

Lactobacillus

using

ml

60

was

the

3 days.

hydroxide

vitamin

of

for

Ex-

in

5,6-dimethylbenzimidazole

of of

day

shown

into

inoculated,

the

growth,

sixth

was

freudenreichii

Since

amount

an

solution

medium

Then,

into of

pH

glucose

charged

cultivation

N2 gas

for

in

when

was

was

hydroxide.

2 days

on

culture

2,

The

controlling of

the

sterilized.

started.

was

(W/V)

mixture

Example

vitamin

used.

glucose

a 60%

Propionibacterium

strain,

mg of

90

2 of

the

that

means of

380g

of

liters

added

Lactobacillus

using

This

per

Example

Comparative

shown

3376.

produced

was

B12

method

biaoassay

customary

liter

carried

amount

of

The

amount

of

the

amount

of

660 of

determined

ml.

The

the by

Lactobacillus

cultivata

IFO

leichimannii vitamin

was

B12

Comparative 2

added

to

ample

1 and

This

produced

ml

of of

the

mixture

the

Propionibacterium

Example

3 was 30°C

and

automatically 5N

broth

while

blowing

two

for

in

sixth

the

on

cultivation

day

the

amount

of

the

the

amount

of

sodium

broth

vitamin

of

amount

40

was

method

bioassay 3376.

mg when

This

produced

of

416g

per

NOC

11011

used

was IFO

for

in

7 days

culture

broth

sodium

solution of was

bioassay 3376.

shown

in

there

fermentor

the

culture

was

an

broth

induction

5,6-dimethylbenzimidazole

At

the

this

time,

was

3.0

liters,

used

was

400

ml.

per

liter

of

the

determined of

mg

culture

Then,

3 days.

broth

the

of

mg/liter

for

by

and The culture

a customary

leichimannii

vitamin

IFO

B12

was

the

same

used.

a

of

method This

630

used

B12

obtained

using

means

that

the

and

glucose

mg when

cultivation

fermentor, was

it

was

the

shermanii

was

liter

determined mg of

of

by

a

the

B12

culture

customary

leichimannii

vitamin

the

The

ml. of

the 5N

of

amount 630

performed

of

amount

also

per

was

amount

The

ml.

Lactobacillus 154

way

Propionibacterium

the

liters,

used

vitamin 48

of

When

3.2

in

out

Propionibacterium

instead

5-liter

was

carried

that

12391.

hydroxide

amount brcth

of

pH

cultivation.

was

1 except

Example

shermanii

60%

the

glucose

Cultivation in

a seed

4

Example as

11012

the

of

120

that

means

into

Lactobacillus

using

of

carried

10

was

broth

was

obtained it

5-liter

Ex-

cultivation

hydroxide

B12

a

NOC

of

culture

in

its

Since

continued

was

B shown

was

and

growth,

amount

an

ml

glucose

fermentor

N2 gas

controlling

days

60

the

The

hydroxide.

added

was

in

of

into

charged

shermanii

sodium

of

period

medium

started.

at

using

culture Then,

inoculated

out

used.

glucose

solution

sterilized.

was

mg o f

215

(W/V)

was

strain,

cultivation

of

4l6g

per

60%

a

liters and

that

means

3

Example 660

fermentor

3376.

was

IFO

produced

630 added

in

Example

ml 2

to

while

the

hydroxide.

Since

for

in the

sixth

cultivation

10

the

the

amount

of

sodium

This

3376. per

it

freudenreichii bacterium

NOC

carried

the

amount of

liters,

used

was

390

ml.

of

the

and The culture

a customary

by

leichimannii

vitamin

was

B12

IFO produced

out

IFO

12391.

of

for

7 days

culture

the

5N s o d i u m

60%

of

the

The

amount

of

vitamin

customary

broth

was

bioassay

leichimannii B12

IFO was

42

used

the

instead

When

in

the

a 5-liter

broth

hydroxide

amount

in

out

same

way

Propionibacterium

was

the

culture

that

11013

shermanii

was

vitamin

time,

2.0

carried

was

1 except

Example

amount

the

was

mg of

on

used.

glucose

Cultivation in

broth

5

Example as

84

that

2

added

was

this

liter

sodium of

period

At

determined

was

5N

using

Thereafter,

per

30°C

at

automatically

culture

Lactobacillus

using

means

of

398g

obtained

B12

Example

out and

broth

strain,

cultivation

its

3 days.

hydroxide

mg when

method

bioassay

in

shown

and

the

of

a 5-liter

a seed

of

induction

broth

culture

vitamin 29

into

fermentor

for

continued

was

was

ml

cultivation.

the

of

of

broth

B shown

carried

was

an

mg/liter

amount of

medium

11011

culture

was

glucose

5,6-dimethylbenzimidazole

the

amount

NOC

the

the

there

of

day

of

pH

60

fermentor,

into

gas

growth,

amount

an

the

of

charged

Then,

cultivation

N2

controlling days

in

The

blowing

was

shermanii

inoculated

started.

was

culture

the

mixture

the

solution

(W/V)

of

liters

Propionibacterium was

a 60%

sterilized.

and

fermentor 5,

4

of

1 and

used.

glucose

Example

Comparative was

of

398g

per

3.3

was used

was

solution

glucose

of

Propioni-

cultivation fermentor, liters. 680

and

ml,

used

The

was

680

of t h e per l i t e r B12 o b t a i n e d by a mg when i t was d e t e r m i n e d

method 3376.

produced

using

This per

Lactobacillus

means 428g

that of

139

glucose

mg

of

used.

ml.

Comparative

680 added

was

Example

and

and

shown its

fermentor culture was

Example

induction on

cultivation the

amount

and

the

The

amount

of

by

the of

amount of

cultivation

IFO was

B12 6

Example

containing of

corn

steep

of

1.5g

of

50

µg

of

calcium

at

fructose,

liquor,

put

The

30°C

containing of

ml

5g

liquor,

of 16g

Na2HPO4·12H2O,

of O.5g

5g

(NH4)2SO4, of

the

mg o f used.

glucose, of

5 mg

of

A 20g

KH2PO4, 30 mg o f MnSO4.4H2O, mg

of

IFO

12391

carried

was

liters

fructose,

of

of CaC03 p e r l i t e r flask and s t e r i l i z Erlenmeyer

stationary 2

ml.

(NH4)6Mo7O24·4H2O, 5

lOg

cultivation

under

430

medium

0.4g

shermanii

Propionibacterium

Separately, steep

and

a 200

in

of

ug

liters,

MgSO4·7H2O,

ZnSO4·7H2O,

10

3.0

glucose

of

12.5g of

time,

72

a culture

NH4NO3,

0.5g

of

10mg

of

3g

pantothenate

inoculated. days

of

of

this

determined

was

that of

428g

per

the

Lactobacillus

using

means

milliliters

CuSO4·5H2O,

was and

ed,

This

Na2HPO412H2O,

Co(NO3)2.6H2O,

water

method

10

Then,

liter

it

there

of

was

per

the

5,6-

amount

used

B12 o b t a i n e d was 24 mg when

of

pH

cultivation.

was

the

growth,

an

hydroxide

fermentor

Since

was

3376.

12.5g

the

product

produced

Sixty

for in

NOC

the

into

cultivation

bioassay

of

cultivation

At

sodium

broth

in

gas

N2

in

a 5-liter

a

3 days.

product

leichimannii

of

for

vitamin

a customary

vitamin

of

continued

ml

The

2 days

day

was

into

hydroxide.

added

sixth

B shown

controlling

was

the

medium

freudenreichii

blowing

of

period

dimethylbenzimidazole mg/liter

60

started.

sodium

5N

using

glucose

inoculated

was

while

of

charged

Then

automatically

broth

an

culture

was

5,

was

30°C

at

and

solution

Propionibacterium

in

out

the

mixture

cultivation

carried

of

(W/V)

sterilized.

strain,

11013

a 60%

of

the

and

1,

a seed

ml

2 liters

to

fermentor

5

Example

out

was for

4

conditions. of of

a culture glucose,

C.4g

MgSO4.7H2O,

of 30

medium 80g

KH2PO4, mg

of

of

B

corn 1.5g

10

Co(NO3)2·6H2O, 50

of

pg

CUSO4·5H2O,

calcium

mg of

mg

and

fermentor

of

ZnSO4·7H2O,

10

pg

was

sterilized.

Then,

was

inoculated

cultivation

was

started.

The blowing

the

controlling sodium

of

pH

containing

fructose

was

response

to

hydroxide. glucose

for

of

The

concentration the

5 g/liter. of

mg/liter

tion

the

and

its

30°C

at

while

5N

using an

aqueous and

automatically

in

for

sodium

supplying

each

of

At

and

maintained

day

of

cultivation,

in

an

amount

then

and

broth,

fructose

was

added

was

aforesaid

glucose

broth

3 days.

for

of

fourth

culture

continued

was

of

culture

5,6-dimethylbenzimidazole 10

each

10

automatically

broth

a pump

On t h e

out

and

operate

motion

the

of

supplying

(W/V)

to

in

carried

culture

the

remaining

about

30%

ml

fermentor

fermentor

the

designed

60

the

was

A pump

hydroxide.

solution

at

the

into

gas

N2

and

a 5-liter

into

charged

in

cultivation

MnS04·4H20,

(NH4)6Mo7O24.4H2O

pantothenate

strain

seed

of

of

5 mg

this

the

of cultiva-

the

time,

amount

of

the

amount

of

5N

sodium

amount

of

the

solution 30% o f containing aqueous 30% of g l u c o s e was 640 ml. The a m o u n t

fructose of was

fructose

was

166

mg

and

of

202g

Cultivation NOC

in

6 except

Example

11012

was

used

shermanii

IFO

12391.

out

for

the

culture

sodium aqueous

of

ml.

The

culture

broth

a customary

by

B12

640

the

leichmannii

vitamin of

was

the

was

IFO

bioassay

3376.

produced

This

per

202g

glucose.

7

Example as

determined

and

liters,

used

liter

per

Lactobacillus

using that

means

it

3.2

was

hydroxide

obtained

B12

mg when

52

method of

and

vitamin

broth

culture

7 days

in

broth

hydroxide solution

was that

instead When

a 5-liter

carried

in

the

same

Propionibacterium of

way

shermanii

Propionibacterium

the

cultivation

fermentor,

was

3.2

liters,

used

was

650

containing

out

ml.

30%

and The (W/V)

the the

was

amount amount

amount each

carried

of

of

of of the

fructose

and

glucose

also

was

per l i t e r B12 o b t a i n e d when i t was d e t e r m i n e d Lactobacillus

using that

240

fructose Example

and

of

was

3376.

produced

75

was

mg

method

bioassay

IFO

This

means

205g

of

per

glucose.

lamps to

microorganism Then, in

20

days

20

g/liter

a

growth

strain)

to

a

grown

at

a height

subject

it

to

a

microorganism

inhibitory

minimum colonies

40

was

culture were

cm f r o m

the

medium

by

above for

shown

collected.

15W

treatment.

cultivated

was

concentration

2 minutes two

obtained

(which

11012)

from

12391

mutation

medium

acid

propionic

minimum

IFO

shermanii

culture

for

irradiated

was

NOC

shermanii

placed

the

plate

of

acid-resistant

propionic

light

Propionibacterium

sterilizing

a

(Propionibacterium

Ultraviolet

the

a customary

of

Derivation strain

and

by

broth

culture

8

(1)

onto

the

vitamin

of

amount

of

B12

205g

per

The

leichimannii

vitamin

of

mg

ml.

650

adding the

the in

for

parent

Table

1,

The in

5 days 20

a

the

minimum

cultivation did

not

Two ml

a 500

shown

pressure

broth

of

each the

at of

120°C the

culture

mutant

(Propionibacterium

sterilized

medium

under

an

stationary

the

pH was

20%

NACO3

amount and

conditions. a day.

Table

3 was

strain in

steaming

The

NOC the

at

culture

the to

for

2,

and

11012)

was

the

aforesaid

30°C

During

adjusted

by

Table

in

placed

cultivated

shown in

which

collected.

minutes.

2 ml

This

B12 p r o d u c t i v i t y medium a culture

of

shermanii of

to

colonies

and were

cultivated

intermittently once

2.

Table

sterilized

10

for

medium

in

in

in

parental

5 days

inoculated

in

vitamin

and

flask,

Erlenmeyer

strain)

milliliters

hundred

adding

minimum

the

times,

evaluating

composition

under

shown

by

for

parental

inhibition

growth

for

the

ten

repeated

was

Test the

medium

culture

undergo

(2) having

for

concentration

inhibitory

obtained

(above

acid

propionic

cultivated

were

medium

culture

liquid of

g/liter

colonies

resulting

for 7

days

cultivation,

about

7 by

using

Vitamin 0.3

ml

acetate

of

the

at

more

and

by

hot

stable

and

water

strain.

B12 that

are

produced by

was

3376

the

of

(propionic shown

in

the

parental

is the

as

a

acid-resistant

Table

4.

mutants strain.

The

were

using

the to

cell

a

Lactobacillus

B12-requiring standard. and

strains

amounts

twice

an

minutes

15 it

strain

parent

of

from

B12

vitamin

a

used

was

for

by

To

solution

converted

bioassayed

for

KCN

vitamin

ml

4.5

boiled

was

which

results

by

1 ml

extract

Cyanocobalamin

mutants PAr)

It

IFO The

as

mixture to

added

and

follows:

as

were

simultaneously

CN-form.

leichmannii

five

the

85°C

than

4.7)

(pH

determined

broth

culture

buffer

(lg/liter),

was

B12

as

of large

the indicated

vitamin as

9

Example

acid-resistant

Propionic bacterium

freudenreichii,

the

parental

IFO

12424

vitamin

in

B12

seen

the

that

the

mutants

the

parental

the were

Referential

the

way

of

results

amounts twice

Example

(FERM

following

as

in

as

of

vitamin

large

as

from

freudenreichii

Example

8,

strains

shown

are

derived

were

these

(Propioni-

in B12

that

and

the

were

Table

5.

It

produced

by

produced

by

strain.

The 11011

same the

11013)

Propionibacterium

productivities and

evaluated, is

strain

NOC

strains

mutant,

BP-85),

1 Propionibacterium used

in

Example

shermanii

3 was

obtained

NOC by

procedure.

A culture

broth

of

Propionibacterium

shermanii

IFO

12391

was

and

dish, placed tube

having

Then,

2 ml

an of

ultraviolet

of

8 ml

of

This

procedure

broth

the

to

inoculated

in

a plate

2% of

cultivation

colonies

thus

obtained tubes

each

containing

culture which

medium showed

separated,

having

A,

good

and

an

sedimentation

named

NOC

culture

outside

cultivated 11011

1 to

10

supplied.

culture

for of

of

strain.

medium One

A,

hundred

transferred the

5 days.

the

was

medium

diameter ml

7

and

cultivation

respectively

containing

and

The

a g a r to the c u l t u r e a t 30°C f o r 2 w e e k s .

were

culture

Every

times.

repeated

by

and

26

test

a

sterilized.

additionally

by

followed

test

A was

in

collected,

was

the

of

with

the

to

30°C.

at

lamps

mm and

17

added

obtained adding

Petri

a

placed

irradiated

broth

repeated

by

100

stand

medium

obtained

to

was

culture

was

1 was of

broth

above

as

in

milliliters

Eight

Example

culture

culture

and

cm.

diameter

the

finally

diluted

40

ml

6.5

15W u l t r a v i o l e t

outside the

of

two

in

allowed

8 ml

days,

with

A shown

light

and

amount

an

of

a height

medium

medium,

in

irradiated

at

culture

put

cells

of

17

mm

sterilized Strains were

1.

for

A process

fermentation vitamin

which

technique

in

a carbon

and

source

accumulated B12 characterized organism;

vitamin

cultivation

(1) a base

maintain

to

the

cultivation

(2) carbon

source

within

10

the

in

that

cells

is

carried

pH

at

is

carried

5 to

portionwise

to

the

before

or

after,

that

when

minutes,

medium

to

a the

containing

and

source,

in

a

cultivating

belonging

a culture

a nitrogen

by

B12

comprises

microorganism

B12-producing

Propionibacterium

genus

vitamin

producing

collecting

the

micro-

out

while

adding

7.5,

and

out

while

of

adding

cultivation of

a

system the

adding

base, (3)

provided having

microorganism used the

as

in

source

A process the

gram

of

carbon the

base

A process

carbon

source

the

A process

carbon

source

A process

carbon

source

and

glucose.

of

to

50g

the 300g

amount per

(1). to

claim

in

is

at

(2)

1 or

least

2 wherein

one

of

the

hydro-

alcohols. in

(2)

to

claim

3 wherein

is

fructose

to

claim

is

a mixture

the

a fructose-

or

source. according

added

in

a carbon

or

1 wherein

is

according

added

5.

glucose,

(2)

according

carbon

and

in

and

sugars

containing

added in

is

omitted.

claim

added

acid addition

portionwise

be

can

B12-Producing

propionic

to

added

4.

(2)

to

according

source

3. carbons,

resistance

microorganism,

carbon

2. of

the

a vitamin

(2)

source

3 wherein

the

of

fructose

containing

fructose

claims

A process a c c o r d i n g t o a n y o n e of t h e p r e c e d i n g w h e r e i n t h e amount of t h e c a r b o n s o u r c e added

in

is

6. (2)

remaining

such in

that

the

the

amount

cultivation

of

the

system

carbon

does

not

source exceed

60

g/liter

of

the

culture

7.

A process

claims

wherein

source

in

(2)

is

supplying

the

carbon

of

for

supplying

a pump

A process

claims

wherein

is and

selected 12426,

from

propionic

Freudenreichii

10.

the

by in

of

operating relation

alkali to

the

any

in one

preceding

the

carbon for

a pump to

the

motion

(1). of

the

preceding

vitamin

B12-producing microorganism Propionibacterium shermanii IFO 1 2 3 9 1 shermanii

acid-resistant

freudenreichii NOC 1 1 0 1 1 ,

IFO

12424,

propionic

shermanii

NOC 1 1 0 1 2 ,

Propionibacterium

NOC 1 1 0 1 3 .

A microorganism

Propionibacterium

vitamin

acid-resistant,

microorganism

freudenreichii

of

one

addition

Propionibacterium

A propionic

producing

source

Propionibacterium

acid-resistant

9.

out

according the

any

portionwise

carried

Propionibacterium and

to

according the

8.

medium.

of

the

genus

according

shermanii

NOC 1 1 0 1 3 .

B12Propionibacterium.

to

claim

NOC 1 1 0 1 2

or

9 which

is

Propionibacterium