DE1467742A1 - Process for the manufacture of antibiotics - Google Patents

Description

The previous invention "relates to the manufacture of Setracycline. by fermentation, especially certain new ones Mutant strains of Streptoiayces aureofaciens exhibiting the trait have, ^ tetracycline to the exclusion of chlortetracycline independent of the fluoride ion content of the perniation medium to eriiaugo-i. . -.

It has been known for some time that microorganisms of the species Streptoisyces aureofaciens, which produce setracycline, also produce ghlortetracycline at the same time. The simultaneous production of Chlortstracyclin iat disadvantageous when to be tetracycline the product to Ilauptprodukt "While generally smaller by eng medicines Standard presence!.! Close of ChI locations tr acy elin in drug- pure 2etracyclin may be present, is the presence of more considerable Kengen to Ohlortetracyclin disadvantageous. Moreover, the failure of these two antibiotics in any way

ORfGfNAt.

809810/1319

New © Unterlaaen

which considerable amounts in the fermentation mash pose difficult problems of separation in the purification or Extraction process. It is of course possible to extract the antibiotics from the fermentation mash and through selective cleaning processes to separate the antibiotics. However, the cleaning procedures are to be carried out the separation of the antibiotics is not without difficulty and usually results in a loss of total antibiotic activity, moreover, in cases where tetracycline is the main product of fermentation, chlortetracycline can be used as Contamination is considered and is usually discarded as it is usually not present in sufficient quantity about the cost of a separate cleaning process to it to justify bringing it up to the drug standard. So far, the removal of chloride ion and the use of chlorination inhibitors are the agents available to suppress the formation of chlortetracycline.

The present invention is based on the discovery that certain new mutant strains of Streptomyces aureofaciens in the fermentation tetracycline with the complete exclusion of chlortetracycline produce «These new mutant strains of Streptomyces aureofaciens are thus chloride ion ignoring strains, since they are independent of the chloride ion concentration of the medium only produce tetracycline.

The new mutant strains of the present invention are strains of the species S. aureofaciens. The beschriebe- nen below S. aureofaciens tribe are all direct descendants of

Chlortetracycline producing S. aureofaciens isolated from soil A-377, which is described in U.S. Patent 2,482,055 and at Northern Regional Research Laboratories, Peoria, Illinois and assigned index number NRRL-2209. These new mutant strains of S. aureofaciens were made by treating the A-377 strain with mutagenic agents such as ultraviolet radiation, nicotine and nitrogen mustard. Typical mutant strains of S. aureofaciens that are insensitive to chlorine ions are, were called T-135, T-l4o, T-1 ^ 3, T-225, S-2308 and S-2589. Live cultures of these new mutants - fl

strains were at the American Type Culture Collection (ATCC) deposited in Washington, D.C, USA and given the following numbers:

Strain ATCC no. ■

T-135 13,908

T-140 13,909

T-143 13,910

T-225 13,911.

In general, the new mutant strains are the pre-. present invention characteristic of the species S. aureofaciens, but differ from previously described Strains of S. aureofaciens not only in the pigmentation, but also in the colors of the whole obtained with it Crop mashing. The colors of the entire harvest mash obtained by fermentation with the new mutant strains according to the vprlie- ·

809810/1319

lowing invention all show a value of; ΔΕ "at 420 πιμ, which is greater than that of AR" at 430 mu. '. The "attached curves Pig. 1 Ma Pig. 8 are reflection spectrograms of 1 cm Glaasel-l-esv which have been measured with the whole intact obtained with the new mutant strains of S. aureofaciens, of the present invention The reflection spectrogram of a material is' a permanent 'record that makes it unnecessary to keep a sample. ·' * In addition, the units in which the curve is expressed are understood and recognized throughout the world. In Pig. 1 to 8, the Wavelength of light in mp. Plotted as absfiese against reflection as ordinate. The wavelength of light is internationally recognized as a round length unit to which all other length units are referred. These reflex spectrograms were made with a .} Standard spectrophotometer using magnesium - carbonate was added as a reference material _ '-; the Buohstabe R denotes the reflectance at a Ewert ■'.. ·, particular wavelength, then b For example, Rjjqq is the reflection value at a wavelength of 500 mu. The character AR denotes the '_; vertical distance between the reflection curve (if both the values for ^ reflection and the wavelength are plotted on a linear scale) and a straight line passing through the '. ■ Curve point · drawn at 400 mu and 550. Let »This graphical determination of the values for AR at. 420 mu and 430 au can easily be durohgtftüirt / Further confer in each pail ■ the new mutin strains of the present invention of the

809810/1319 : \ - \

velvet Ernteniaiaelie such a color that AR at 420 mu is greater than ΔΒ. at 430 mu.

The mathematical determination of the fourth of ΔΚ. at 420 mu and 430 ημ can also be performed using the following equation will: . · "

ΔΗ at 420 icfi = 20 (R ₅₅₀ J + 130 (R ₄₀₀ ) -150 ΔΕ at 420 Hu = 30 (

. where R ₄₀₀ * ^ 420 » ^£ 4" 0 ¹ ^ ^Γι 550
V / ellenlängen 400 εμ, 420 ηιμ, - 450 nu and 550 mu represent. This mathematisclis Eostimxauns does not depend on the scale used for the ^ reflection or the wavelength.

A "sown harvest maize" is the untreated mash » obtained when the fermentation progressed to the point is where the biosynthesis of a main product is prak-. table is finished. In general the antibiotic increases Permentationasiaiselia activity no longer significant, if-the fermentation lasted about 140 to 160 hours Has. -

In order to show the visual sarbic differences between the new mutant strains of S. aureofaciens that produce only tetracycline, these strains were grown on corn steep agar and the following observations were made.

BATH

3098 10/13 19

■ 1)

Color observationu ο ^ c >·> r ^ B. ρ. υ re of aci ens i

AP4

6! Age incubation at 26.5 ° C

Stem single Eolor ioii

Hate enwaclistuni

ΐ-135 Dark lcaiarbsn (Dark luggage tan) Maple color ! -140 Orange rust-colored (Orange rust) Maple-colored (Kaple) 1-143 Eichenbrarai (θεώ brown) Light brown (Light brcwa ! -225 Dark tan (Dark luggage tan) Yellow, maple-colored

(Yellow maple)

1) 3 colors according to "Color Harmony 3 £ anuc £}. ^N , third edition, Container Corporation of iliaerica.

Parbbeob & chtunken '% S, aureofaeienst

AP6-I, iaiquolla.c; ar: 6 days incubation at 26 _% 5 ° C

Strain single colonies

Kaas growth

2-135 tan (Luggage tan)

I-HO Orange rust-colored (Orange rust)

! -143 Dark tan (Dark luggage tan)

! -225 Luggage tan ^

Light spice brown

Maple (Maple) Maple (Maple) Maple (Maple)

1) Colors according to "Color Harmony XfanuGpL", third edition, ■ Container Corporation of America.

BATH ORIGINAL

809810/1319

for

Sucrose 10 _g . 0 »25 g KH ^ PO ₄ 2 β "■. £ Maiaquell \ rasaer ν 4th & Agar according to Bacto (· ϋ <** · ^ * * a * V 20th fill up with water 1000 a!

as for AP4-Afar,>

β g per liter of kaisgueli water.

The aeueu Mutantenstäaaae of S _f sar © oXacieas der-Tor-. Invention, which excluded Ietracyolia produ-;

adorn "* show the same general characteristic auiy- '

- · ■ ■ ■■ ■ ' ^: ■ - -. '. . . ■ v those strains that contain both chlorotetracycline and

y xuid tuitersoheidea develop with one another in the same general way as the tetracycline-producing and chlortetracycline-producing strains differ, and this has been described in a number of scientific publications. The following data serve for further differentiation ϋίΰτ new Hutantenstäiame τοη S.aureofaoiena of the present invention τοη the previously known Stäaaaetn τοη fl.aureofacienn, such as the original taimii A-377 »4er al» SSBIr-2209 obtained. . . , ·. '"

ly

809810/1318

1487942

The new StrtptomfCtS aureofaciena .Mutantenstämme ■ were of S.AKr # ofacie "S A-377 (HRRIr-2209) by observing Wachstumseharakteristiea in various media en at incubate at 26.5 ⁰ C dif- / ferenziert * '·." · '- "' ■ '·''■" -

1 '' Slyeerin Asparagine Pleiao Extract Agar

Glycerin - - -— «———— i _t o

1-A8paragin - · —-—- 0.0!

Jleiach extract - ^ - ^ ~ ....... o, 2

Agar according to Baeto ————...-., - ,, -j _t 5

Make up to —-— 100.0 with saturated water

pH adjusted to ~ ■■ -7.0 with 50 $ KOH

pH value after sterilization - ~~~~~ - .. _r .. ■■ 7.2.

♦ ': - V · "; ■

ft-o.Aa iu7ia.i-fl.

aureofaciena

Grows""

Sporulation

! Piffusible
Pigment;

back

' O

ί-135 good, hyallnj apigmented-up ! P (topaz) * * *

J -HO good, hyaline; apig mentiort M S (topaa) '

"* 43 good, liyallRi

l0ilfS3? T3 (

luggage

auageseie & aet

/."■•377 good

j knows

sohwaoh,

; White

p "to aiem"

Iicli well, know _t il "bd

"bia

very

biherfarbea VrQ ₁ de ^ a. (light fawa "to Ί)

saJxwassh Ida i well know tais (light

kslae

no

none

getting brewed (2opaz * becoming oak brown)

Hyaline, Sopaa, eiohenbraua becoming (ajopaz / oak brown becoming)

Well

very bright

light amber hyaline, dark brown (light

pretty good

light yellow

dark brown mahogany A

yellow to
yellow

Colors according to Color Sarraany lamual,
Container Corporation of Amerioa.

β *

"'Jf

2. Stepping out

Dextrin ~ - ■

BTaITO ₃

MgSO ..

PeSO ^ .7H ₂ O ■

Agar after. Bacto

distilled ^ Wagper , pH after. Sterilize

t _r 0 Ji 0.2 Si 0.1 * 0.05 ft 0.05 5t 0.001g 1.5 ft, 100.0 # 7 »2

Streptomycea aureofaciena

Staina

Growth.

Iiufthyphen

Sporulation

Diffusible a
pigment

back

ί-135 weak, thin »half translucent!

weak »white

no

none

white, translucent.

t-140

I-U3

weak, thin »semi-translucentj White"

awake, thin »half

translucent;

white ¹

weak, thin, half dttwitstsfretoteiea © ■ pec Iv «Eifl.1 '

A-377 good

none

weak »white

weak »white

no

no

no

abundant »mouse- * very rich gray (mouse gray) borrowed to leaded rattj
wasaerhellö Obear "
surface spheres
(water-white surface . ■ [ _s globules)

none

none

none

Traces; pale yellow

white, translucent

knows »shining through *

white, translucent "

apigmented »pink traces.

colors according to Color Harmony Manual, 3rd edition, Container Corporation of America.

3. Αϊ.

1 * 67 * 42

Corn spring water -: 0.4- f »

Sucrose ———- ■ _ $ 1.0

MgSO ₄ -TH ₂ O. · $ 0.025

^KH 2 ^P0 4 """■""*'~" ~ ' - ° » ² st

(M ₄ ) ₂ HP0 ₄ ~ - _: r 0.2 j

Agar according to Bacto. - ~ 2.0 #

with. Tap water, fill up to --—— 100.0. #

pH value after sterilization ——. ·. 6.5

809810/1319

Streptonyoes aureofaoiena

tribe 77achatum 3iufthyplia
ν * Sporular
tion Soluble
pigment back T-135 moderate ' sparse, plentiful
becoming; beaver-
colors (beaver) ¹ moderate bia
plentiful orange-brown light brown to chocolate
shop brown OO ■ '
O
co Ϊ-UO wet moderate; beaver colored
bia chocolate
colors (beaver bia
chocolate) moderate orange-brown Maroon to
deep brown. ε i / o 18 ! P-U 3 plentiful. plentiful; pale-
greyish-brown
(fawn ¹ ) bia beaver-
Colours moderate to
plentiful bright orange
Brown deep brown to dark
Brown - »
IO plentiful very plentiful, *
chocolate colored very rich
lich bright orange
Brown dark pink brown bia
Ebony brown A-377 excellent plentiful; pale
greyish-brown
(fawn) ¹ very rich
lich light yellow bia
Amber
Colours light tan

Colors according to Color Harmony Manual, 3 «edition, Container Corporation of America.

ι σ »

AP6:.: _ Ir v ^- ..ll agar

Iiaisquellwa3ser .__....___._-_, 6 g

Sucrose 10 g

MgSO ₄ .7H ₂ O 0.25 g

• irpr -pn ___ _,,. - ■ ".,", ..- ". ■.""9 o ·

"i PT ^ fI ___—., ,, ■, ι., ■ -. η. ι» -. »· .. .η.r .. 9 C

/ 2 ^ο " ¹ * 4 —— ~ *""" * ■ e>

Agar according to Baato ^ - 20 g.

Fill up with Leitungsraaaer to 1000 ml.

pH value according to. Sterilize 6.5 »

80 98J0 / 13 J 9

StreptoEiycea atireofaoiens

tribe

Waohaturn

Iiufthyphen Sporulation

Soluble pigment

Rear

• «155 plentiful

moderately to plentiful} pale greyish brown (fawn) 'to greyish brown (mist brown!) moderately to.
plentiful

orange to orange-brown

'. chestnut brown to dark brown

3M4O plenty

OO O CO

2-143 abundant ■

T-225 plentiful

A-37? excellent

moderate to plentiful: very rich beaver colored (bearer ¹ ) borrowed to pale grayish brown (fawn) - ·

weak bia moderate; beige ¹ to gray brown (mist brown)

plentiful; chocolate-colored Λ (chocolate)

moderately bia abundant;

pale greyish brown

(fawn ¹ ) pretty
Well

orange to orange-brown

orange-brown

very rich orange-brown borrowed

very rich orange-brown borrowed

chestnut brown to dark brown

chocolate brown

ebeiihoi ■; λ '(Ebony ν: ο ....)

orange to orange · yellow.

** colors according to Color Harmony Manual, 3rd edition,. Container Corporation of America. _f

5. Q * Corn steep agar

Maiaquellw & seer Sucrose -— «-

fill up with water

pH value after sterilization -

■ ¹ OV \ - 0.25 g ^f ν ² '■ «. · · ■ 30 g 1000 ^: ml.

6.5.

ρ-. "- growth Str.eptoHaßje's, aureofaeiena Sporular
tion •. · ■ Soluble
pigment • ■ ^ »» - . tribe plentiful Aerial hyphae plentiful deep orange
Brown Pdickaeite · '' 2-135 ■ very rich plentiful; pale
greyish-brown
(fawn ¹ ) bia beaver-
Colours · plentiful deep orange
Brown copper brown bia
deep brown Φ ί
j T-HO ' very plentiful very plentiful,
pale grayish brown
(fawn ') to beaver-
Colours very rich
lich deep orange
Brown copper brown to
deep brown α? -Η3 very plentiful
beige 'to gray
brown (mist brown) deep brown bia
chocolate brown

Ϊ-225 very plentiful

very plentiful, * chocolate-colored

very rich amber colors

A-377 excellent! pale yellow
(pale yellow ¹ )

very plentiful; dark brown

very rich orange-brown
borrowed

orange to orange-yellow

! »Grains according to Color Harmony Manual» 3 «edition Container Corporation of America.

/ t

β. r

medium

Streptorayces rmroofaciens ^s

Strain Ϊ-135 strain 1-140

Nutrient agar bad ms fairly good growth; white to pale yellow (pale yellow ^). No air hyphens. Back; white to pale

Glucose / good Y, '£ ch3tu :: i; hyaline; Asparagine- apigiaenticrt to olaß-J ¹ MsChorange (p: il £ .oran%; o '). extract- No air hyphae. Sück agar string apigmented to pale orange.

Waksman's
Agar

Purple"
milk
(Purple
Milk) ^l

X potato slant ~

agar

good growth, semi-opaque j white to pale orange (pale orange '). air hyphae: sparse, becoming plentiful; white to pale grayish brown (fawn). Sporulation: sparse, becoming plentiful. Back! dark tan to dark spicy brown. (dark luggage tan to dark spice brown) "amber, soluble pigment.

"Pale yellow growth margin. partial peptonization _t pH 7.1 poor growth; ^ to pale yellow}. No air hyphae. Reverse: white to pale yellow.

good growth; .hyalin, apigmented to pale orange (pale orange '). Weak aerial hyphae; White. Back apigmented to pale orange.

good growth »semi-opaque; white to * pale orange (pale orange) _t becoming orange with age. Air hyphens sparse, becoming rich, white to beaver-colored. Sporulation: sparse, becoming very profuse. Back; beige to copper brown. Traces of light. "Amber soluble pigment. '

pale yellow growth margin partly peptonia, pH 7.0.

very plentiful, moist, very plentiful, moist, lump-shaped growth; lump-shaped growth, chestnut brown to dark, light, spicy brown ·. Barngawürzbraun, rust brown ·, red (Barn red) soluble soluble pigment, white Pigment.Schwache

Air hyphenation.

1 ." Colors according to Color Harmony Manual, 3rd edition, Container Corporation of America,

BATH ORIGINAL

8 0 9 8 10/1 3.1

/ f

! ntorryn r> ^ r.-.ir eo'.V. e

medium

Strain T-H3

Strain 2-225

Hook agar

bad growth: woiss weak "to fairly,
. "to pale orange yellow (light good growth white biö v orange-yellow ^) No air-golden brown · (golden brown ')" hyphen back: White. Ms No aerial hyphae ■> light orange-gerb back Seites translucent +..' ·

white Ma air brick brown ■ ',

(adobe brown). No, sol- ^

. lichee pigment. .:. ', ·

glucose
Asparagine
meat- .
extract agar

Good T / achatu: *

ample vigilance.

tfuxes r / acns / cu: *: ^ j ^ x & n, rexcnxienes wacneiium.

pale yellow to orange " ^1. Air- Very abundant aerial hyphae} hyphae:". weak, v / white. beaver-colored (beaver ·) * '■. ■■■ :.

Reverse j hvalin: "pale - Sr> orulations - very rich - '·

Back side: hyaline; pale yellow to copper brown to deep brown.

Waksman * s good growth i light tan agar colored to topaz (topaz ¹ ). Aerial hyphae: pretty good Ms plenty * beige-brown to pale grayish-brown (fawn). Sporulation: bad, becoming very profuse. Back: dark tan (dark luggage • tan) to deep brown. Light brown to amber colored, soluble pigment.

Potato slant

Agar

very profuse, moist, nodular growth; beaver colored 'to light spicy brown. Dense white aerial hyphae on isolated Flocks. Rusty brown, soluble pigment.

Purple- "'pale yellow (pale yellow) milk growth _{margin. Φ} Little distinctive (purple-characterizing pH change milk) · ■ or visible peptonization · pH 6.7.

Sporulation: very profuse. Rear ;. dark * aubergine-colored (dark eggplant). Very light amber soluble pigment. \

abundant growth. Abundant aerial hyphae! taupe brown · to chocolate · '■' shop colors. Backside »·· ebony-colored. Light amber, stone colored, soluble ;; _; Pigment »."'· .-. ·· - < ^: - *%

ruisu

very abundant, moist, smooth, nodular ·>. Growth; orange-rust- ;, ι ben (orange rust ¹ ) bis, -Vl egg-brown, with increasing-. | the age of chocolate .- .; j becoming colors. Orange- r | brown, soluble ■ pigment *

heavy '^ pale yellow ·) growth ■' ■ '.;.? edge. Peptonization by rope, pH 6.78. No ;;.; soluble pigment · ", ^: ν

colors according to Color Harmony Manual, 3rd edition Container Corporation of America.

809810/1319 * ■ - ^ i

146774

StTeρtomyces anreo facicns

Kedium

St * TTiTn A-377

Nutrient agar good growth - no aerial hyphae. Pale gel "be3 'soluble pirinont. Iiüekai t e: oil au j el ο.

Glucose Good 7 / aehstua. Aerial hyphae white, asparagine with increasing spore formation, flesh increasingly gray, extract traces: yellow-orange soluble agar pigment. Reverse side: light yellow to pink-orange.

Y / aksman's Good Wachatura. air hyphen aieni-agar: borrowed good, becoming plentiful: white to taupe-brown (taupe-brown '). Light yellow, soluble pigment. Küekoeite: Camel to air brick brown (camel to adobe brown).

Potato very r ^ eIiIi.; H.: S, fsuchtes,

Q λΙι Τ * ϊ3 IT «" 1 ίί * ί ~ "frs * - \ ^Γ " · ■ f '\ ί "/" * ~ "· ■" · * Ήΐ * ",'" ί TOC »

agar Waahatuia, holl-brown-yellow (light brovm-yellow ') to beige to cedar-colored, - No soluble tiginent.

Purple-slightly yellow milky to pale yellow growth margin. (Purple v '/ enig characteristic pH change MIk) or visible peptonization in 14 days. pH 6.45.

Parts according to Color Harmony Manual, 3rd edition, Container Corporation of America.

9 8 YY0Ü3Y 9

BAD ORiGfNAL

ti

7 «MiTkroalcopl see observations

Sjre ptomj ce S autreο fa-, 1.ona

Sbamm 'U-135 Bbamiii T-HO

! »Tedium

My eel

Üporen Liyoel

Spurs

AP ₄ corn

q.uell-

winding, running, branched. 0 0.3-1.0 a steroidally wound, until smooth running, # 0.8-1.5 μ vcrav / eigt. 0 0.3-1.0 µ

spheroidal bia ovate. 0 0.8-1.5 u

winding, continuous, branched. 0 0.3-1.0 µ

spheroidally wound,

until eifor- continuously,

ctig. ve x 'branches

0 1.0-1.2 µ 0 0.8-1.0 µ

spheroidal to ovoid. 0 1.0-1.2 ρ

./akaiaan'a
Agar

sinuous, continuous, verswoi ^ u » 0 0.8-1.0 u sinuous, bia eiför- continuous,: .iiC · branched. 0 1.2-1.5 u 0 0.8-1.0 u

spheroidal to ovoid. 0 1.0-1.2 u.

'..' • sdiuift

llillj "" ■ ■ "'" · * ' * ^ '»γ * it: .- u Q-Teicχ ο? -i -., .. ri μ ■) .- -.-

My c el

Spore strain ΐ-225 Myeel spores

Asparagine

Fleisoh

extract-

gev / unden, continue:!, branched. 0 0.8-1.0 u

3ph.eroidal wound, steroidal

bia elfor- continuously bia ovoid.

TTiIz * "distorted. 0 1.2-1.5». 0 1.0-1.2 μ. 0 0.8-1.0 μ.

AP, LIais l

, LIa
'lll-

gev / unden, continuous, branched, 0 0.8-1.2 μ

apiieroidal

bi3 öifor-

aig.

^ 0.8-1.5 gev / unden, spheroidally continuously to ovoid branched. 0 1.0-1.2 and L. 0 0.8-1.0 Ji,

ii 0 PY 3 1 o / 13

medium

Streptomyoea aureofaciena strain A-377 ■

liycel spores

Glycerine /
Asparagine
I ¹ Ie is chexfcraktagar

sinuous

i'ori running end v / aigt d ~ 1 _r 0 μ

^Λ Ρ "L

fiai a quell- gey / mid ^ a

.Spheroidal to egg-shaped.
0 1.0-1.5 u.

until a-

> '1.2-1.5 p.

The bodiiigiin ^ on of fermentation aind im ali _d '; e * - nieinen the same - ..' ic i "ür the current economic process for the production of chlorotetracycline by Jermenbabion." Dan-hei!. · T ₃ "iaa" IPernontationsmedLim Contains the usual tiährstox'fe mid! .liner al sub a tank. Suitable nutrients include starch, dextrose, cane sugar, glucose, uölacii-jo, Ga.jubohneniielil, leek solids, RsLa ₁ ? Leiach.e: cbrakte, peptones, urea, liaiaq, uelllüasigkeit, ipestillatio-naniaischen · ', fishmeal substances . NnorgraiisohÄ salaen include, for example, calcium carbonate, anaionic sulfate, non-chloride, and the salts of the various trace elements v.'is luangan, cobalt, sin! C, copper, iron and dev ^ ic-ic-han.

The other general conditions for which P: 3r ~ mentation like J

BATH

8 0 9 3 1 0 / S -v; il

Time, speed of filling, production of the inoculuma, Sterilization, inoculation and the like conventional and similar to JcÖiwen ^ u those for the division of chlortetracycline used aein as described in the US ^ atentschrift 2,482,055.

Obtaining the tetracyclines from the permentation fluids i-at hcrlcörr-lich and needs no explanation, since numerous processes for the production of tetracycline also fermentation fluids have already been published are .

The following examples serve for better Understanding the invention without limiting it.

example 1 Production of an inoculum of S. aureofaciena strain S-2308

Saves of S.aureofaeiens strain S-2308 were made by Washed agar slants with sterile distilled water, which resulted in a suspension with 60-80 × 10 spores / ml «One part of 0.33 ffil of this suspension was used to make a <| 8 "shaking cylinder to inoculiscen the 8 ml of a Contained medium that was manufactured according to the following recipe

Sucrose 30 g

Ammonium sulfate 2 g

Calcium carbonate 7 g

Corn spring washer .................., 16.5 ml.

Tap water, make up to .... 1000 ml.

8 0 9 810/131 9

'. Tor dec ¹ Irtoculierur *; became the medium in the autoclave. 20 minutes uniac a pressure of 1.05 lcg / ca (15 pai) eteriliaiert »whereby the pH value from 6.3 to 6.9 ragged. The inooulierte shaker tube was then 24 hours at ⁰ 2S C on a reciprocating Schüttelmaachine with 116 oscillations per ί; ceuliert _.- Γ- ± j \. The inocula of the other new mutant strains of the present invention are 'produced identically'. \ ".: · -V ^:: '""· .-.

- "" - ■ - · - '■■ · Example 2 :.'"■." ^: - '. ^; '■ · ■ -'"■

. '* ' ■ - . ·· '■ - ■ - · X''■■■' ·. · "■ · '• Chloragtin-I-geat for differentiation from getraoyolin .

; ·. "· Ν and chlorotetraoycline '■ ·'" - - '■';

The standard solution A was prepared by dissolving 100 g of chloramine T in 900 ml of 0.1 tfv £ a ~ P0, (38 g of β ^ ΡΟ ^. 12H ₂ O / 1,000 ml of distilled water). A working solution B was prepared by mixing 180 ml of the stock solution A with 820 ml of an aqueous solution of ethylenediaminetetraacetic acid Uatriumaai (5 g per 1,000 ml of distilled water). To carry out the actual seat, 0.5 ml of the permeation mash to be examined was mixed with 20 ml of working solution B in a test tube. This is then shaken intermittently for 30 to 60 minutes. At the end of this shaking time, the test tube is examined for its color. Mashing with. high concentrations of tetracycline turn wine red (positive reaction), while mashes with high concentrations of chlorotetraoycline produce a

BAOORfOfNAL < 809810/1319, -. ^ Jk

deep berastone color. This chloramine-T-iEest is a further means to differentiate the new mutant strains of the forward lying invention AEEN previously known strains of S.aureofaciens. .

'". Example ^... ^: ·

Sirber nitrate test for CMLoriaion .1

■. f. . · ■

To a 10 lal portion of the total peraentation mash, 10 ml of 0.2 ml: 1. KlIö, 0.5 g of chloride-free DiatomSnerde-Pilterli aid are added. The combined materials are mixed thoroughly and then filtered. The filtrate obtained in this way is filtered again as often as is necessary to obtain a clear piltrate. Then 2 ml of 0.01KW AgSO 2 are added to a 2 ml part of the clear Piltrata in an Eea-tube. The mixture is "observed for one minute. The formation of AgCl, observed either as turbidity or as a white precipitate" indicates the presence of free, unbound chloride ion throughout. Mash sample. On. . . >

Example 4

getracycline production by fermentation in shaking flasks A. Voli — chlt> ridIia3rtlgea - ^ / Iedium · ■ ' , /

A permeation medium with full chlorine content for the production of tetracycline was produced as follows;

809810/1310

(BH ₄ ) ₂ S0 ₄ .. 5 S

CaCO ₃ .... 7-9 g

1.5 g

0.4-0.6 g

MaSO ₄ .4H ₂ O 0.05 g

- ZnSO ₄ .7H ₂ O 0.1 g

Corn steep water 20-30 g

Cottonseed flour 0-2. G

_Ä starch., ............, 55 g

P 'c · ^; · ■■■! "

Sehweiaae-aehmalzöl - 20 ml

■ - J

Fill up with tap water to 1000 ml

B. Medium with "limited chloride content

A permentation medium with limited chloride content; for the production of tetracycline was produced as follows ι

(HH ₄ ) ₂ SO ₄ 6.83 s

CaCO ^ 7-9 g

MnS0 ₄ .4H ₂ 0 .......... 0.05 g

ZnSO ₄ -TH ₂ O 0.1 g

liaia spring water 20-20 - g

Cottonseed flour 0-2 g

Strength .; ... 55 g

'BcwaillohmalzoT 20 ml

fill up with tap water to ......... 1000 ml ·

80 98 10/1319

Itinf 25 ^ l l-: xic.ilt3 both media were put in. 15 pei) sterilized. After the Steriliaicron ^ edea? Aar of the 25 ml. Portions in various ways with 1.0 ml of a .as in the ""

Example 1 produced Inokuluma -inoculated. The! Fermentation was in the first 18 hours "at 28 ⁰ C and then from the end of the 18th Ms. to 142 hours at 24 ⁰ C fcei pH 6 ~?
carried out on a rotating shaker at -180 rpm. Under these conditions, the in
given results. . '-.:.:' -

809810/1319

! 9 I.

Test .

Strain Me- Chlortetra-! Tetracycline Chloramine- Silver-Uo. dium cyclin _t J ₅ meg './ ml. . Test · ...; ■ nitrate test ²

2-135; A. <5O ⁵ 2460 + '' ■■ '.. ■ +' ■. '· B. <50 2510 + f-HO A. <50 2210 + ; '■' .- ■ + - '- · ■ " B. <50 2200 + '- ■ 1-143 « A. <50 , 3820 - + - · ■■ ". '■ + · B. <50 3470 + 1-225 A " ^ 5O 3890 ■. + ■■. "■ '^; - ■■ - +"': B. <50 3990 . · '■ ^: '". + ■ · -: S-2308 A '. <^ 0 " 6580 + • ■ '"'" + ' B- . -C50 6520

- \. * Chloramin-T-Oiest, according to Example 2. ';

2 "Silver! Step-sest according to example 3" - '

3 * The test method based on yiietracycline is only ■ · '· ■ ·. "Sensitive down to 50 mcg / ml. The.. ·

· Cultures, however, showed on chromatography

"■ ·" "· .. no chlorotetracycline on paper strips.» ■? Γ

809810/1319

Tetraoyciinherstellur · "hoi the fermentation in shaking '■

■ Effect of the aura on high prifl concentration

The permeation medium A of Example 4 was ^:. with a UH ₄ Cl content of 0.0, 0.1, 0.2, 0.5 and 1.5 g / l and a constant content of 5.0. g / 1 (HH ^ gSO, produced "The fermentation was carried out as described in Example 4", the results given in Table 2 being shown.. · ^; · ■ ". ·

BH ₄ Cl Table II ¹ - test 3430 Silver- I. Stem Ge—
No. - stop
g / l Chlorotetra- [! Tetracycline
cyclin
mcg / ml mcg / ml 2770 , ■ nitrate ".
'3? Est ¹ T-225 0.0 <50 ² 2900 ; + · ■ -.- '· ' ■ '' _ '■ 0.1 <50 2940 ... ₊ , .-;., V 0.2 <50 3390
-— "3390— ~ Τ" ~ . 0.5 " <50 5760 : ■ · "■ + ..: ■> .-." ■ 1.5; <50 4580. "T -... - · - + S-2308 0.0 <50 5080 ■■ '■ + -. . ■:. ■■ ■■ ·· ■ '. 0.1. <50 5440. : - + '"■ ■ · -. 0.2 <50 6080 ' 0.5 <50 1.5 <50 ♦ ■

Silver nitrate test according to example 3

The test method for ^ ü ^ äffcetracyolin is only sensitive down to below 50 mcg / ml. However, the cultures showed none in the chromatography on paper strips

9810/1319 ": -.:, ·

• '. · ■ · Example 6 ·,. _ '·' ■■

Reaction of the tetracycline producing strains
■ on the ultraviolet point of view -

. The new mutant strains of the present invention can easily be derived from previously known chlortetracycline-producing strains of S. aureofaciens (as well as from established strains which primarily contain other known! Tetracyclines such as 6-desmethyltetracycline, 7-chloro-6-desmethyl- · »tetracycline and 7-ChlQr-5 $ "(11; ^) - dehydrotetraeyolin. Generate) '·. By comparing the waves greater under ultraviolet light

i O

.; length, especially at 3660 A, differentiated fluorescence generated will. A particularly suitable medium for growth * - and fluorescence observations is Qq Sporulation Agar, the is made as follows:

; ,. .MgSO ₄ '.7H ₂ O .0.25 g

KH ₂ PO ₄ 0.4 g

; "(M ₄ ) ₂ HP0 ₄ '0.4 g

Sucrose ....... "... 15. G

'Staley nutrient medium CHF 22 9 g

fill up with tap water to 1000 mT.

The pH of this medium was adjusted to 6.6 by means Ěîńęâŕ [SO ^ KOH solution strength and placed dannYi7,5 ml portions of the medium brought in.25 x 150 mm culture tubes Glaä and - closed · the tubes with cotton plugs. The culture tubes containing the medium were heated at 120 ^° C. for 20 minutes

809810/1319

sterilized, left in an inclined position to give as large a surface as possible and refrigerated in the refrigerator until use. Spores of S. aureofaciens strain A-377 were washed from an agar slant with sterile, distilled water, resulting in a suspension with 60-80 × 10 spores / ml. 0.1 ml portions of this suspension were used to inoculate the glasses prepared as described above with Qg sporulation agar. After inoculating the Qg-AgapSchrägpräparate were 26.5 - 27.5 incubated ^0C. After I ² J days, the spore

education generally ended. The same procedure was used in the ν Case of all other investigated microorganisms used, which are listed in Table III below.

When observed under ultraviolet light (3660 8) during the incubation period of ο-14 days, the vegetative Growth of the tetracycline-producing strains brlllant-goldenorange, while the agar body became more and more brilliant orange-yellow to yellow. During this growth phase, chlorine

Tetracycline producing strains have a faint fluorescence similar Kind of show. At the same time, the upper part of the agar slant, where the thickness of the medium is smallest, fluoresces green-yellow at f Tetracycline producing strains and golden yellow for chlortetracycline producing strains. When the sloping crops become ripe, In the case of the tetracycline-producing strains, the yellow fluorescence of the agar body remains when irradiated with ultraviolet Light of 3660 S exist, while with the chlorine tetra-

809810/1319

cyelijBr-producing strains blafi blue w ^ rdear.

A permentation medium for tetracycline production was made according to the following recipe:

'. (M ₄ ) ₂ SO ₄ .............. 5.58 / g

CaCO, 7-10 g

₄ ^ ₁ techii.rein) 0.08 '. g / -

'HaiacLUellv / asser.,. ».. 20-30" g

Strength-. 47.5 ^: g. - '.

■ MaiBEiehl 14.5 g. ..

'-' ··· Pork Lard Oil .20-32 bQ.

fill up with tap water to 1000 ml.

, The permentation method was carried out according to the instructions dea example 4 carried out, the in labeile III below

listed microorganisms and those in table ■ ..-. an- III below / given results were obtained «

8 0 9810/1319

1 * 67742

III

■ Cklortetra-
cyclin
mcg / ml ig l'etracyciin
. mcg / ml Q Agar slurry culture S.aureo- -, 50 at $ 3660: faciens
Tribe Mo <5O ¹ 620 A-377 '.' · ■ <5ö BO Ϊ-135 <5O 5670 ■. " m '00. 4140- ' . # <50 664Ö ■■■ · ■■■■ Φ-225 <50 S770 S-230S ■ ■ ■: _.
1 «The ifritfiisg se ^ f Cnlortetraö ^ alin is
*' - to. 5® w & s / ^ k öEtpiiiiiäXi.ßl £ »you Κβ ^ ΐβο? niar liia it down
shows - ieäach, tea.

the

ais fafiers tea »germ

ä ^ reii

from. S. aureofsciena strain 2-135 were grown from an agarseciiräglcultur with sterile, distilled water to form a. Suspension of 60-80 χ 10 spores / ml washed »A 0.33 ml portion of this suspension was used to inoculate a 20 cm (8") shaker cylinder,

309810/1319

■ • ". · '■ · ■"

the 8 ml of an Iladiuss eiotMelfe, daa after the. in Example 1 given recipe was prepared . You subsequent preparation of the new InocuXa used liutantenstämme of S.aureofaciens happened "oh the process of Example 1 was prepared a Permentationsiaedium the following composition:

t ζ -,? £ i i?

KhSO « ilQ ^ t techniseä Eeitt) ....... G, 0ä-G _r 15 g

..... ..— ».- 20-30 g

Corn flour ......... "..." ... ■ ..... «14- _e " 5 p

fill up to 1000 ml with ieitttitgsweejssr.

ileaea Eeäitsm. in eiaea AutoklaTea 20 BS »s.tea
'- ■■ ■ 2 ■.

eiaem Ssiicfe tcsl T _r Q5 feg / eas (15 psi)

aligaate rope ^ foa 25 ml in. 25ö eü. i £ L SeilsEi made as described above. Inaculum inolculates. Perraentation was carried out for 18 hours at 23 ° C and then -i. ^ Oh-4e-to s * 142 hours at 24 ° C on a rotating shaker machine with 180 revolutions per Kinttte durolijofuhrt. I3oi the other microorganisms used, which are listed in '! ^ SIIe IY. "Aind, wurdr _; claa same Yerxahr & n banutst ,.

BATH ORIGINAL

8098-0 / 1319 -. -

, Me testing for tetracycline was performed by the standard Hiscox method: a, while chlortetracycline testing was performed by a fluorinated method. The results are shown in Table IV. The reflection curves for the geearote harvest mash were obtained using a General Electric CD.Spektrophotometera, and are shown in fig. 1 to Fig. € of the drawings. The samples were measured in a 1 cm glass cuvette, which was attached in the reflective position on the back of the sphere, magnesium cerbonate being used as the reference substance. The self (mirror) reflection of the cell was taken into account and a range from 400 ιψ to 700 τψ examined. The values for AR at 420 μm and at 430 μm were determined and are given in Table IV below. .

Tabel IV AR ** at 430 ma 420 ΐημ 18th S. aureo— test 27;. · ■. ". 18th faciens
Trunk Ko. Chlortetracycline
Weight # Tetracycline
Weight # 32 -9 T-135 none * 100. 59 3 T-140 Il ■ * 100 72 12th 2-143 none * 100, 63 36 1-225- none * 100 94 S-2308 none * 100 S-2589 - none * 100

* The cultures showed in the chromatograph on paper- do not touch chlorotetracycline.

** The scales for reflection and wavelength in Pig. 1 to Pig. 8 do not run linearly with the applied quantities. This is due to the special display type of the spectrophotometer and in no way affects the calculation of the AR values.

80 98 10/1319

1: ^ 1 game 8 ... y

Characterization »inea Tetr acyeli n-ergeugenden Stammeg of S.aure o £ acie ws by Rg fleKiongspelctrographie the . · ;. entire Ern.tema i »ohe in the synthetic medium

Spores of S aureofaciens Staera S-2589 were with

sterile distilled fiber to form a suspension with 60-80 χ 10 {spores per ml from an agara slab culture

^. washed. A 0.33 nl portion of this suspension was used

W. '· ■ · ■ · .. ■ ■ · ·

^ turns to inoculate a 20 cm (8 ") shaker cylinder eu,

the 1 ^ i8 ml of one according to the recipe in example 1 \ berge- »■

presented medium. ' '\

It became a retirement medium of Composition 'made j -.;' : "· ■

(JIH ₄ J ₂ SO ₄ ,., 8.0. G

,., Cornstarch. ;. ". 55» O 'g;"'.

15gCl ₂ .6H ₂ 0 2 ^ 0 g. ,

. . 'feS0 ₄ .7H ₂ Q 0.06 g. \

ZnSO ₄ .7H ₂ O ο .............. ' 0.10 g;

■. ' mso ₄ .H ₂ o .......... —....... w .... Cv o »o5 ^ g ^v .

'. CoCl ₂ UH ₂ P ' ^: \ 0 ^ 005 g ζ . ".: .-

! "Methionine ...." "0, UO · g

,;. ir-hiatidine 0 * 80 'g

. fill up with distilled H ₂ O to «·· 1000 ml«

8098143/1319

A combination of pork lard oil and mineral oil (made from pork lard oil and mineral oil U-SP quality 5s1) was added to this medium, namely 0.63 ml per 25 ml of the medium. To. Sterilize this medium for 20 minutes. In the autoclave at a pressure of 1.05 kg / cm (15 psi) aliquots of 25 ml in 250 ml Erlenmeyer flasks were inoculated with 1.0 ml portions of the inoculum prepared as described above. The fermentation was carried out for 18 hours * ei la 28 ⁰ C and then from the 19th "to 142j hour, ⁰ C on a ro- X animal forming Schüttelmaechine performed in 24-180 TJpKT.

When tested, the total harvest maize should show a content of 8060 micrograms tetracycline per ml according to the standard Hiscox test method, the culture not showing any chlorotetracycline on paper chromatography. Using a General Electric Co. spectrophotometer, a total yeast mash curve was obtained as shown in FIG. reproduced'iat. The sample was in a 1 cm glass cuvette, which was in the reflective position on the back of the sphere, and magnesium carbonate became g

Used as reference material. The self-reflection of the. Cell was considered and a range from 400 mu to 700 au examined * The fourth for AR at 420 mu was 118 and for ΔΕ at 430 mp was 87. ' ₍

809810 / 131S

ϊ ** ■ '■ ""

frt is »ic 1 & ^?

Characterization »i no Τ * ΪΥ * & γ & 1η * ~ $ Υζβυ.ββηα6Ώ. Aureofaciens tribe fl urch 1t "ft" jct ons spectrography of the entire Ej-nteraaiach.ei n j & tTOCkn§tem. M olkenroaium.

Spores from 3.8-urtofaeiena strain S-2589 were infected with sterile distilled water to form a suspension with 60-80 χ 10, spores per ml from an agar slant culture '■' -

washed. χ »· 0.33 ml of this suspension was used for
WL inoculate a 20 cn (8 ") cylinder with 8 ml of a naon
Example 1 produced medium used.

Eb was a peraontation medium made of the following composition? ■ - ^:

Dried whey ·. 15 * 5 g

Cornmeal. ' 42.7 g

Corn starch 30.8 g

CaCO ₅ 11.5 g

• (M ₄ ) ₂ S0 ₄ 8.5 g

HH ₄ Cl '2.0 g

. MnSO. (70 jS, technically pure) O ₉ HOg

CoOl ₂ .6H ₂ O ο 0.005 g

Lactic acid 0.650 g.

Fill up with line S7iasaer to ......... 1000 ml.

0.75 ml of lard oil was added to these hedium added per 25 ml of the medium after 20 minutes Sterilizing the llediura in an autoclave at one pressure 1.05 kg / cm (15 psi) aliquots of 25 ml

BATH ORIGINAL 809 8 10/1319.

inoculated in 250 al Erlenimeyericolöen with 1 _} 0 "al parts of the Xnoculum prepared as above. The fermentation was' ^! 18 hours at 28 * ^e east and then from the 19th to the 142 »hour at 24 ⁰ C on a rotating shaker with

180 rpm. '.

The entire Exatemal -sche showed on examination 6070 Micrograms tetraeylin per nil according to atm standard Hiscox test method, the culture showing no chlorotetracycline on paper chromatography. By means of a genera! Electric Co, Spectrophotometer, the reflection curve of the entire harvest mash was obtained, which is reproduced in Pig.8. the The sample was in a 1 cm glass cuvette in the reflection position on the back of the sphere, magnesium was carbonate used as a suction substance. The self-reflection of the cell, was taken into account and the range of. 400 mu examined up to 700 mu. 5 value for ΔΕ at 420 mu was 129, that for ΔΗ at 430 mu was 66.

8098 10/1319

Claims (1)

«Ver-vi« iiule t .. the calibration through the feeling, the gaßßnieu harvest time to give a color au, ttti * wil ^ ur with the ^{ruler 1} applied Reficuciou8kv.rve dor vertical distance between the Iteflexionskurvt- ' and fcinnr straight line, which the flexion curve at ^ 00 tnjuvna ί> 30 nyt eohneidet, at ^ 120 ψ ( greater than at H $ Q m ^ and through the free gate ability, in Qg spor- ulatione & even after 14-day incubation at & 6.5 bie S? _# 5 ° C with the action of Ultravicilett-Lifehi · of .5660 A oine golbe Pluoreβ ztmz VAx result, BADORJGINAL 809810/1319. _ Documents (Article 7S1Abs.2Nr.1Satz3de8Anderungsgeä.v.4.9.t967i