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WO1988004319A1 - Substance et procede stimulant la croissance de bacteries anaerobies - Google Patents

Substance et procede stimulant la croissance de bacteries anaerobies Download PDF

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Publication number
WO1988004319A1
WO1988004319A1 PCT/US1987/003290 US8703290W WO8804319A1 WO 1988004319 A1 WO1988004319 A1 WO 1988004319A1 US 8703290 W US8703290 W US 8703290W WO 8804319 A1 WO8804319 A1 WO 8804319A1
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WO
WIPO (PCT)
Prior art keywords
membrane fragments
sterile membrane
nutrient medium
medium
oxygen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1987/003290
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English (en)
Inventor
Howard I. Adler
James C. Copeland
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxyrase Inc
Original Assignee
Oxyrase Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxyrase Inc filed Critical Oxyrase Inc
Publication of WO1988004319A1 publication Critical patent/WO1988004319A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

Definitions

  • the present invention relates generally to promoting the growth of anaerobic bacteria and in particular relates to the use of sterilized mitochondrial derivatives for the production of anaerobic conditions in media to promote the growth of anaerobes.
  • the medium in which the anaerobe is to be grown In order to grow anaerobes, it is necessary for the medium in which the anaerobe is to be grown to be substantially free of oxygen. Oxygen can be removed fairly efficiently from such media by sparging with high purity nitrogen or other inert gas. However, these liquid media are subject to foaming so that this process presents a number of mechanical difficulties. Further, after sparging is stopped, the medium is easily recontaminated with oxygen.
  • reducing agents most of these agents ⁇ tt ⁇ e strong reducing agents and any residual agent or its by-products in the media tends to inhibit the subsequent growth of anaerobes in the media. filso, the reducing agent which is consumed during the initial removal of oxygen is not available to act upon oxygen which might later find its way into the system.
  • the principle object of this invention is to provide a material with similar oxygen-consuming properties, but derived from a distinctly different, non-bacterial source. More specifically, this invention provides oxygen consuming enzyme systems from mitochondria obtained from a variety of higher non-bacterial organisms, as well as a method for using such systems to grow anaerobic bacteria.
  • the oxygen-consuming enzyme systems of this invention, obtained -from mitochondria exhibit properties which make them useful for increasing the range of anaerobic bacteria that can be grown using the membrane fragments obtained from bacteria disclosed in U.S. 4,476,224.
  • Other objects and advantages of the invention will become apparent from the following descriptions of various embodiments and examples of the invention.
  • sterile membrane fragments of this invention can be employed without foaming problems. They are non-toxic and the presence of these membranes has no adverse effect upon the growth of the anaerobic bacteria in the media, even when used at high levels, such as a ten-fold excess. Further the presence of the membrane fragments in the media provides the media with the capacity to reduce additional oxygen which later may enter the system so that extreme methods of sealing the system are not required.
  • Yeast, fungal cells, algae and protozoa, having mitochondria membranes containing an electron transfer system which reduces oxygen to water are grown under suitable conditions of active aeration and a temperature which is conducive to the growth of the cells, usually about 20°C to 45°C in a broth media.
  • mitochondria may be obtained from cells of animal or plant origin.
  • the cells are collected by centrifugation or filtration, and are washed with distilled water. 3.
  • a concentrated suspension of the cells is treated to break up the cell walls and mitochondria. This is accomplished by known means, for example by ultrasonic treatment or by passing the suspension several times through a French pressure cell at 20,000 psi.
  • the cellular debris is removed by low speed centrif ⁇ gation or by microfiltration (cross-flow filtration).
  • the supernatant or filtrate is subjected to high speed centrifugation (175,000Xg at 5°C) or ultrafiltration.
  • the cells of step 2 are suspended in a buffer containing 1.0M sucrose and are treated by means which break up the cell walls or membranes but leave the mitochondria intact. This is accomplished by known means, for example, by ultrasonic treatment, passage through a French pressure cell at low pressure, enzymatic digestion or high speed blending with glass beads.
  • step 7 The cellular debris from step 6 is removed by differential centrifugation or filtration. ⁇ .
  • the supernatant or retentate from step 7 is passed through a French Press at 20,000 psi to break the mitochondria into small pieces.
  • Mitochondrial debris from step 7 is removed by centrifugat ion at 12,000Xg for approximately 15 rninutes or by microfiltration.
  • the supernatant or filtrate from step 9 is subjected to high speed centrifugation (175,000Xg at 5oC) or ultrafiltration.
  • the pellet or retentate from step 5 (crude mitochondrial fragments) or the pellet or retentate from step 10 (purified mitochondrial membrane fragments) are resuspended in a buffer solution at a pH of about 7.0 to about 7.5.
  • a preferred buffer solution is 0.02M solution of N-2-hydroxyethylpiperazine-N'-2- ethane sulfonic acid (HEPES).
  • the membrane fragments in the buffer solution are then passed under pressure through a filter having openings of a size which will retain any intact microorganisms to effect sterilization. Openings of about 0.2 microns are satisfactory.
  • the sterilized suspension is then preferably used promptly or stored for use at about -20°C or it may be freeze dried.
  • a small amount of the sterile membrane fragment suspension is added to a liquid medium which is to be used for the growth of the anaerobic bacteria (about 25 to 3000 mg of fragments per liter of medium).
  • the medium is permitted to stand for a short period of time at a temperature of from about 10°C to about 60°C until the oxygen is consumed. This action takes up to about 20 to 30 minutes, depending upon the concentration of the sterile membrane fragments and the temperature. fit concentrations of about 500 mg/l and temperatures of about 35°C, removal is effected in from about 2-8 minutes.
  • an inoculum of anaerobic bacteria is introduced into the medium.
  • the inoculated medium is then incubated for the growth period at the proper temperature for the bacteria which are to be grown.
  • the air space above the liquid medium in its container is flooded with an inert gas such as nitrogen. This minimizes the amount of oxygen that must be removed by the membrane syster ⁇ and prolongs the life of the oxygen-consuming system. This also gives ass ⁇ rance that, if there is an accidental leak of air into the system, the system will consume that air and insure that the growth of the anaerobic bacteria will not be retarded.
  • the membrane preparation is preferably added to the medium in a molten state at approximately 45°C at a level of about 25 to 3000 mg of fragments per liter of medium.
  • the medium is then poured into Petri dishes, or the like, in which it is held at a temperature of from about 10oC to about 60°C until the oxygen is consumed, usually in a period of time less than 20 to 30 minutes. The time of removal depends upon the temperature and concentration of the sterile membrane fragments, as pointed out above.
  • the medium is then inoculated with the anaerobe to be grown and incubated at the proper temperature for growth.
  • the Petri dishes should preferably be maintained in an atmosphere of inert gas, such as nitrogen, but good results can be obtained on rapidly growing anaerobes without such a precaution since the membrane system is capable of consuming reasonable amounts of oxygen from any air which may leak into the dish.
  • inert gas such as nitrogen
  • a synthetic media it may be necessary to add a small amount of a hydrogen donor which does not interfere with the growth of the eelected anaerobic bacteria.
  • Suitable hydrogen donors are sodium lactate, sodium succinate, alpha-glycerol phosphate, alpha-keto gluterate or sodium formate.
  • Most natural media do not require the addition of a hydrogen donor, but with some media, particularly synthetic media, the addition of the hydrogen donor is necessary for the sterile membrane fragments to perform their oxygen removing function.
  • anaerobic bacteria representing 8 different genera will flourish in media which has had its oxygen removed by the sterile membrane system of this invention. Because of the fact that the sterile membrane fragments are in the form of particles which do not penetrate the cell walls the system does not adversely affect the anaerobes being grown. This is in contrast to a system in which a chemical reducing agent is used to accomplish the removal of dissolved oxygen when the residual reducing agent or its by-product may penetrate the cell walls. Thus, because the anaerobic bacteria are being grown under more natural conditions, they flourish at a greater rate than in media which has been treated with such reducing agents.
  • a nutrient broth is inoculated with Saccharomyces cervisiae (ATCC 18790).
  • the nutrient broth employed is Malt Extract Broth sold by Difco Laboratories, Detroit, Michigan.
  • the inoculated broth is maintained at 24°C and is actively aerated. The growth is continued until it is in the late logarithmic phase.
  • the broth containing the Saccharomyces cervisiae cells is centrifuged at 4,000Xg to harvest the yeast cells.
  • the cells are washed with distilled water and centrifuged. This washing and centrifuging are repeated.
  • the harvested cells are cooled to 0-2 oC and suspended in two volumes of a buffer containing 1.0M sucrose, 0.02M tris-(hydroxymethyl)amino methane (Tris) and 0.0001 sodium ethylene diamine tetra acetate (EDTA) at pH 7.4.
  • Tris tris-(hydroxymethyl)amino methane
  • EDTA sodium ethylene diamine tetra acetate
  • the eupernatant is centrifuged at 2400Xg for 20 minutes to remove unbroken cells and debris.
  • the mitochondria are harvested from the supernatant by centrif ⁇ ging at 20,000Xg for 15 minutes.
  • the mitochondria are then resuspended in 0.02M HEPES buffer at pH 7.5 (CalBiochem-Behring Corporation, LaJolla, California) and passed three times through a French pressure cell at 20,000 psi.
  • the suspension of the membrane fragments in the buffer solution is then passed through a 0.22 micron filter under pressure to produce the sterile membrane fragments to be used to produce anaerobiosis in the media to be used for growing anaerobes.
  • the suspension of membrane fragments is stored at about -20°C.
  • the dry weight of the solids in the suspension is about 30 rng/ml which is determined by desiccating samples over phosphorous pentoxide in a vacuum.
  • Example I Ten microliters of the sterile membrane fragment preparation of Example I is added per rnilliliter of synthetic medium which is supplemented by the addition of sodium lactate to a final concentrat ion of 0.25M.
  • the synthetic medium consists generally of inorganic salts, ethanol and sodium acetate. It is prepared from reagent grade chemicals. A sample of medium containing the membrane fragments is held at 37°C to remove oxygen and is inoculated with Clostridium kluyveri. The inoculated medium is incubated at 34°C for 96 hours. fit the end of that time a luxuriant growth (approximately 10/9 cells per ml) is observed.
  • the sterile membrane fragment suspension of Example I is added to molten agar at 45°C at a level of 10 microliters of suspension per rnilliliter of agar.
  • the agar is poured into a Petri dish and held to obtain anaerobic conditions. It is inoculated with Clostridium kluyveri and incubated in the presence of an inert gas atmosphere at a temperature of 34°C for a period of 96 hours. Colonies with a diameter of approximately 2 millimeters are observed at that time.
  • a hydrogen donor which is compatible with the bacteria being cultured should be employed to supplement the medium.
  • the hydrogen donor e.g. sodium lactate, sodium succinate, alpha-glycerol phosphate, or sodium formate should be employed at a level of the order of 0.15 to 0.25M.
  • Anaerobic bacteria which can be successfully grown in nutrient media treated by the sterile membrane system described herein are:
  • sterile membrane-containing media may be used in clinical laboratories to stimulate growth of anaerobic bacteria from human patients.
  • Sterile membrane containing media may be used to increase the survival of anaerobes in medium used to transport samples from the patient to the laboratory and also for the determination of antibiotic sensitivity patterns of anaerobic bacteria. It also has use in producing the anaerobic conditions required in many industrial fermentation processes.
  • the use of sterile membrane fragments, as described above, produce little or no toxic side effects when used in arnounts much greater than those required to achieve oxygen-free conditions. Small quantities of the membrane fragments reduce a medium that is initially saturated with oxygen to an anaerobic condition and maintain that condition even though small amounts of air are introduced.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Sustainable Development (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Substance et procédé stimulant la croissance de bactéries anaérobies, comprenant un milieu nutritif contenant un donneur d'hydrogène et des fragments stériles de membrane mitochondriale possédant un système de transfert d'électrons qui réduit l'oxygène en eau. L'oxygène dissous dans le milieu est extrait en ajoutant les fragments stériles de membrane au milieu nutritif et en maintenant ce milieu à une température comprise entre 10°C et 60°C jusqu'à l'achèvement de l'extraction de l'oxygène dissous.
PCT/US1987/003290 1986-12-05 1987-12-04 Substance et procede stimulant la croissance de bacteries anaerobies Ceased WO1988004319A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93819086A 1986-12-05 1986-12-05
US938,190 1986-12-05

Publications (1)

Publication Number Publication Date
WO1988004319A1 true WO1988004319A1 (fr) 1988-06-16

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Application Number Title Priority Date Filing Date
PCT/US1987/003290 Ceased WO1988004319A1 (fr) 1986-12-05 1987-12-04 Substance et procede stimulant la croissance de bacteries anaerobies

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AU (1) AU1050388A (fr)
CA (1) CA1290271C (fr)
WO (1) WO1988004319A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0489849A4 (en) * 1989-08-29 1992-10-21 James C. Copeland A method, composition and device for removing oxygen from solutions containing alcohols and/or acids
EP0520757A3 (en) * 1991-06-24 1993-11-10 Becton Dickinson Co Material for promoting growth of bacteria
EP0427813B1 (fr) * 1989-03-07 1994-05-11 COPELAND, James C. Appareil et procede d'extraction en continu d'oxygene present dans des courants de fluides
WO1999002650A1 (fr) * 1997-07-11 1999-01-21 Oxoid Limited Enrichissement selectif et detection de micro-organismes
US7374905B2 (en) * 2000-11-08 2008-05-20 Oxyrase, Inc. Medium composition, method and device for selectively enhancing the isolation of anaerobic microorganisms contained in a mixed sample with facultative microorganisms

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4476224A (en) * 1982-05-10 1984-10-09 Adler Howard I Material and method for promoting the growth of anaerobic bacteria

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4476224A (en) * 1982-05-10 1984-10-09 Adler Howard I Material and method for promoting the growth of anaerobic bacteria

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochemistry, issued 1979 (Academic Press, Inc., New York, USA), (METZLER) see page 571. *
Biotechnology and Bioengineering Symposium, No. 11, issued May 1981, (John Wiley & Sons, Inc., New York, USA), (ADLER et al.), "A Novel Approach to the Growth of Anaerobic Microorganisms", pages 533-540, see particularly Summary (page 533) and page 534 and 539. *
Journal of Bacteriology, Volume 147, No. 2, issued August 1981 (Williams and Wilkins, Co., Baltimore, Maryland, USA), (ADLER et al.), "Cytoplasmic Membrane Fraction That Promotes Septation in an Escherichia coli Ion Mutant", pages 326-332, see particularly pages 326 and 329. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427813B1 (fr) * 1989-03-07 1994-05-11 COPELAND, James C. Appareil et procede d'extraction en continu d'oxygene present dans des courants de fluides
EP0489849A4 (en) * 1989-08-29 1992-10-21 James C. Copeland A method, composition and device for removing oxygen from solutions containing alcohols and/or acids
EP0520757A3 (en) * 1991-06-24 1993-11-10 Becton Dickinson Co Material for promoting growth of bacteria
WO1999002650A1 (fr) * 1997-07-11 1999-01-21 Oxoid Limited Enrichissement selectif et detection de micro-organismes
WO1999002649A1 (fr) * 1997-07-11 1999-01-21 Oxoid Limited Support de recuperation et de culture de micro-organismes
US7374905B2 (en) * 2000-11-08 2008-05-20 Oxyrase, Inc. Medium composition, method and device for selectively enhancing the isolation of anaerobic microorganisms contained in a mixed sample with facultative microorganisms

Also Published As

Publication number Publication date
CA1290271C (fr) 1991-10-08
AU1050388A (en) 1988-06-30

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