WO1989000204A1 - A medium and method for yeast detection in non-alcoholic beverages - Google Patents
A medium and method for yeast detection in non-alcoholic beverages Download PDFInfo
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- WO1989000204A1 WO1989000204A1 PCT/US1988/001269 US8801269W WO8900204A1 WO 1989000204 A1 WO1989000204 A1 WO 1989000204A1 US 8801269 W US8801269 W US 8801269W WO 8900204 A1 WO8900204 A1 WO 8900204A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Definitions
- This invention relates to a selective growth medium for a number of different yeast genera to the exclusion of bacteria and fungi.
- This invention also relates to a two-component base system of dehydrated medium constituents and an antibiotic additive. It further relates to a method of rapid detection of yeast contamination in non-alcoholic beverages.
- Microorganisms are ubiquitous; they may be found wherever the nutritional and environmental conditions are favorable for their proliferation; that is, wherever sufficient carbon, oxygen, nitrogen, and carbon sources are available, and the correct temperature and pH are provided. Their niche can include food and beverage products, the colonization of which can be inconsequential if the microorganisms present are the normal flora of the product. Alternatively, the contamination of ingestible products with various undesirable or pathogenic microorganisms can result in detrimental changes in organoleptic properties, e.g. taste, texture, and/or visual appeal of the product, or in its spoilage. Such contamination can result in wasteful losses to food and beverage suppliers.
- Microorganisms consist of a heterogeneous population of simple, mostly unicellular organisms including prokaryotes, e.g. bacteria and molds, and lower eucaryotes, e.g. yeasts.
- the most significant spoilers of non-alcoholic beverages are usually the yeasts, the most prevalent of which belong to the genera Saccharomyces. Torulopsis.
- Candida. Hansenula. Pichia. and Rhodotorula These organisms can synthesize off-flavor or toxic metabolites, and may also have the ability to alter otherwise unacceptable substrates such that pathogenic bacteria can flourish.
- Yeast contamination can be manifested in beverages as sediment or floating particles; ropiness or a slimy, stringy consistency; haziness; increased pressure; foaming; and changes in color, smell, and taste.
- This screening procedure typically consists of culturing a small sample of the beverage in a medium, such as M-Green Broth (Microbiological and Particulate Analysis of Beverages; PM605, Millipore Corp., Bedford, MA (1981), pp. 8.), which readily supports the growth of a number of different types of microorganisms.
- M-Green Broth Microbiological and Particulate Analysis of Beverages; PM605, Millipore Corp., Bedford, MA (1981), pp. 8.
- These growth media contain the essential nutrients for microbial reproductive life including a carbon source, e.g. fructose, dextrose, or peptone; a nitrogen source, e.g.
- fruit pulps, peptone, amino acids, or ammonium salts may contain a vitamin source such as beef or yeast extract, and an additional nutrient source such as casein digest or casamino acids.
- Yeast has a slower growth rate in most media than do bacteria and molds, which causes an unfavorable balance in competition for nutrients. As a result, yeast proliferation is frequently retarded to the point of suppression, leading to erroneous reporting of minimal, or even nonexistent yeast contamination.
- Attempts to provide a more favorable growth environment for yeasts have included using media having an acidic pH, such as Difco orange serum broth (Difco Manual. (Tenth Ed.) 1984. pp. 630-632).
- supplements containing antibiotics and/or other compounds which specifically interfere with normal metabolic processes in prokaryotes have been incorporated into screening media to allow for the selective growth of yeasts. Examples of such media include those disclosed in U.S. Patents Nos.
- anti-prokaryotic antibiotics and/or metabolic poisons are not sufficient to enable more rapid proliferation of yeast.
- Specific nutrients, and in particular multiple-vitamin supplements, such as yeast extract, have been found to have growth-promoting qualities, but the identity
- a selective growth medium according to the invention for the rapid detection of yeasts in non-alcoholic beverages contains carbon, nitrogen, vitamin, and nutrient sources, an osmophilic constituent for providing a buffered environment, and an antibiotic supplement to inhibit growth of prokaryotic organisms.
- the carbon source consists essentially of dextrose;
- the vitamin source preferably includes vitamin K and yeast extract and can also contain inositol;
- the preferred nitrogen source consists essentially of about two parts inorganic nitrogen, such as ammonium sulfate, and about one part organic nitrogen, such as asparagine.
- the preferred nutrient source includes casein digest and 1-cysteine or casamino acids, and one desirable osmophilic constituent includes ammonium sulfate and sodium chloride.
- the invention also provides an improved growth medium for yeasts, and which comprises a carbon source, a nitrogen source, a nutrient source, a vitamin source, and antibiotics to inhibit growth of prokaryotic organisms.
- the point of novelty includes a high concentration of dextrose as the carbon source, and vitamin K as a nutrient supplement.
- a base system for the preparation of a selective growth medium for yeasts, to the exclusion of other microorganisms, consists essentially of two components: a mixture of dry medium ingredients which includes a carbon source, a nitrogen source, a nutrient source, a vitamin source, and an osmophilic constituent to maintain a buffered environment; and an antibiotic supplement.
- a preferred antibiotic supplement for practice on the invention includes at least two of the group consisting of chloramphenicol, chlortetracycline-HCl, and streptomycin.
- the antibiotic supplement is added to the mixture following the hydration, sterilization, and cooling of the mixture, and prior to the inoculation of an aliquot of a non-alcoholic beverage in which contaminating yeasts may be proliferating.
- the medium can further include, as an optional element, a growth indicator which changes color as the pH of the medium changes due to the metabolic activity of the yeasts growing therein.
- a method for the rapid detection of contaminating yeasts in non-alcoholic beverages includes the steps of (i) hydrating a pre-mixed dry medium including carbon, nitrogen, nutrient and vitamin sources, and an osmophilic constituent to maintain a buffered environment; (ii> sterilizing and cooling the hydrated medium; (iii) inoculating an antibiotic supplement which includes at least two of the group consisting of chloramphenicol, c lortetracycline-HCl, and streptomycin to prevent growth of prokaryotic organisms; (iv) inoculating and incubating an aliquot of non-alcoholic beverage in the medium under environmental conditions favorable for yeast growth; and (v) detecting and scoring the yeasts growing therein.
- the foregoing features enable the invention to provide a growth medium which encourages yeast proliferation while selectively inhibiting growth of other microorganisms.
- This invention also provides a rapid, facile, and economical method of screening for yeast contamination in non-alcoholic beverages.
- the two components of the base system comprising the growth medium can easily be prepared, packaged, and distributed to the location of beverage production for convenient and expedient on-the-site testing.
- the selective growth medium according to the present invention includes the essential carbon, hydrogen, oxygen, and nitrogen-containing nutrients, and required osmophilic constituents for the sustenance of microbial life in general. It also includes nutrients and growth promoting constituents such as specific vitamins for the stimulation of yeast growth, in particular.
- the carbon source provides a substrate for yeast fermentation.
- This source can be a carbohydrate composed of various sugar residues, and is usually a disaccharide such as dextrose which is composed of a glucose and a fructose residue, or a monosaccharide such as fructose or glucose. Because the majority of soft drinks contain dextrose or high fructose corn syrup as the sweetener, dextrose is preferred as the sole, or primary, carbon source.
- the concentration of dextrose in the medium is in the range about of 10 to 15 grams per liter, and a preferred concentration is about 12 grams per liter.
- a preferred practice of the invention employs water as the hydrogen source, and air present naturally in the medium as the oxygen source.
- Yeasts are faculative anaerobes which are able to exist on essentially whatever level of oxygen is present in the medium container or is dissolved in the medium itself. Therefore, an additional oxygen source is not essential.
- SUBSTI UTE SHEET Most organisms are capable of utilizing organic nitrogen-containing compounds, such as proteins and a ino acids, as nitrogen sources.
- the present medium contains both proteins and amino acids, and preferably contains asparagine at a concentration in the range of about from 1.0 to 2.5 grams per liter medium, and preferably at about 1.5 grams per liter medium.
- the preferred inorganic nitrogen source is an ammonium salt.
- the ammonium salt of choice is ammonium sulfate in an amount ranging about from 2.0 to 5.0 grams per liter medium, and specifically at about 3.5 grams per liter medium.
- the present medium provides both organic and inorg-anic nitrogen sources at concentrations which take into account these metabolic limitations.
- the growth medium include an osmophilic constituent, or a combination
- Magnesium and/or sodium salts, and more particularly, magnesium sulfate substantially in the range of about from 0.2 to 0.7 grams per liter, and sodium chloride substantially in the range of about from 1.0 to 1.5 grams per liter can be added for this purpose.
- magnesium sulfate and sodium chloride are included in the present medium.
- Certain vitamins are potent growth stimulators for yeast (S.A. Koser, Vitamin Requirements of Bacteria and Yeasts. 1968 Charles C. Thomas Co., Springfield, MA., pp. 478). These vitamins have been typically added to growth media in the form of yeast extract, which is known to be rich in water-soluble B and C vitamins, and in vitamin E.
- a practice according to the present invention includes yeast extract in a range of about from 1.0 to 3.0 grams per liter medium, and preferably at about 2.25 grams per liter medium.
- Neat vitamin K is included at a concentration substantially in the range of about from 0.005 to 0.03 grams per liter medium, and preferably at about 0.01 grams per liter.
- Inositol, a B-complex vitamin can also be included at a concentration substantially in the range of about from 0.025 to 0.075 grams per liter medium, and preferably at about 0.05 grams per liter.
- Other vitamin sources may also be included.
- yeasts grow more slowly in most media that do prokaryotic microorganisms such as
- ITUTE SHffil bacteria and fungi
- Antibiotics such as chloramphenicol, chlortetracycline-HCl, streptomycin, neomycin, penicillin, dihydrostreptomycin, streptomycin, and compounds such as potassium tellurite, bismuth sulfite, monotetrazolium compounds, copper sulfate, sodium propionate, and diphenyl are preferred for this purpose. These compounds have the ability to selectively interfere with various metabolic processes specific for bacteria, fungi, or prokaryotic organisms in general.
- the medium can include two of the three antibiotics listed in each of the above variations.
- each illustrated variation of the iedia includes dissolving the ingredients listed above, exclusive of the antibiotics, in water such that the final volume after hydration is one liter. .
- the solution is poured into an autoclavable flask or container, i.e. one able to withstand sterilization temperatures. Alternatively, the solution is aliquoted out into a number of smaller containers. The solution is then autoclaved for about 15 minutes at about 120 degrees Centigrade, and cooled at room temperature to about room temperature. A filter-sterilized antibiotic supplement is then added under sterile conditions, and the solution mixed thoroughly. The medium is now ready for use.
- an 18 ml sample of the test solution is poured into a Yeast and Mold Swab Kit Sampler (The Millipore Corp., Bedford, MA 01730), and the Sampler paddle is inserted.
- the Sampler is then placed horizontally onto a flat surface with its membrane facing down, for a period of time sufficient for uniform wetting of the membrane, e.g., for one minute.
- the paddle is then removed, and the Sampler case emptied of excess medium.
- the Sampler is incubated with the membrane facing downward at 28-32 degrees Centigrade for 48-72 hours.
- the resulting yeast colonies are scored using an illuminated magnifier or microscope.
- liquid samples of beverage can be tested using the Beverage Monitor method (The Millipore Corp., Bedford, MA 01730).
- Each variation of the above- media containing, agar is prepared under sterile conditions and kept in liquid phase with moderate heat. Approximately 2.5 ml of beverage sample is put into a sterile petri dish. Approximately 25 ml of liquid medium is added and the resulting solution mixed with dish cover replaced. The plates are allowed to solidify at room temperature. They are then incubated at 37°C for 72 to 96 hours, after which time yeast colonies are visually scored.
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Abstract
A selective growth medium for the rapid detection of yeast genera contains carbon, nitrogen, vitamin and nutrient sources, and includes chloramphenicol and chlortetracycline-HCl to inhibit growth of prokaryotic organisms, and inorganic salts to promote an osmophilic environment. A two-component base system for the preparation of the selective growth medium has a mixture of dehydrated medium constituents and an antibiotic supplement. A method for testing for yeast contamination of non-alcoholic beverages is also disclosed.
Description
A MEDIUM AND METHOD FOR YEAST DETECTION IN NON-ALCOHOLIC BEVERAGES
Field of' the Invention
This invention relates to a selective growth medium for a number of different yeast genera to the exclusion of bacteria and fungi. This invention also relates to a two-component base system of dehydrated medium constituents and an antibiotic additive. It further relates to a method of rapid detection of yeast contamination in non-alcoholic beverages.
Background of the Invention
Microorganisms are ubiquitous; they may be found wherever the nutritional and environmental conditions are favorable for their proliferation; that is, wherever sufficient carbon, oxygen, nitrogen, and carbon sources are available, and the correct temperature and pH are provided. Their niche can include food and beverage products, the colonization of which can be inconsequential if the microorganisms present are the normal flora of the product. Alternatively, the contamination of ingestible products with various undesirable or pathogenic microorganisms can result in detrimental changes in organoleptic properties, e.g. taste, texture, and/or visual appeal of the product, or in its spoilage. Such contamination can result in wasteful losses to food and beverage suppliers.
Microorganisms consist of a heterogeneous population of simple, mostly unicellular organisms including prokaryotes, e.g. bacteria and molds, and lower eucaryotes, e.g. yeasts. The most significant spoilers of non-alcoholic beverages are usually the yeasts, the most prevalent of which belong to the genera Saccharomyces. Torulopsis. Candida. Hansenula. Pichia. and Rhodotorula. These organisms can synthesize off-flavor or toxic metabolites, and may also have the ability to alter otherwise unacceptable substrates such that pathogenic bacteria can flourish. Yeast contamination can be manifested in beverages as sediment or floating particles; ropiness or a slimy, stringy consistency; haziness; increased pressure; foaming; and changes in color, smell, and taste.
In order to avoid the distribution of contaminated soft drinks and fruit beverages, preventative testing for the presence of microorganisms is usually performed before aliquoting. This screening procedure typically consists of culturing a small sample of the beverage in a medium, such as M-Green Broth (Microbiological and Particulate Analysis of Beverages; PM605, Millipore Corp., Bedford, MA (1981), pp. 8.), which readily supports the growth of a number of different types of microorganisms. These growth media contain the essential nutrients for microbial reproductive life including a carbon source, e.g. fructose, dextrose, or peptone; a nitrogen source, e.g. fruit pulps, peptone, amino acids, or ammonium salts; water as a hydrogen source; and air or another oxygen
source air. In addition, they may contain a vitamin source such as beef or yeast extract, and an additional nutrient source such as casein digest or casamino acids.
Yeast has a slower growth rate in most media than do bacteria and molds, which causes an unfavorable balance in competition for nutrients. As a result, yeast proliferation is frequently retarded to the point of suppression, leading to erroneous reporting of minimal, or even nonexistent yeast contamination. Attempts to provide a more favorable growth environment for yeasts have included using media having an acidic pH, such as Difco orange serum broth (Difco Manual. (Tenth Ed.) 1984. pp. 630-632). In addition, supplements containing antibiotics and/or other compounds which specifically interfere with normal metabolic processes in prokaryotes have been incorporated into screening media to allow for the selective growth of yeasts. Examples of such media include those disclosed in U.S. Patents Nos. 4,140,580, and 3,360,440, Difco Laboratories Bacto BiGGY Agar, Candida BCG Agar Base, Chlamydospore Agar, Pagano Levin Base, Sabouraud Culture Media, and YM Broth (Difco Manual Tenth Ed., (1984). Detroit," MI) . .
However, anti-prokaryotic antibiotics and/or metabolic poisons, alone, are not sufficient to enable more rapid proliferation of yeast. Specific nutrients, and in particular multiple-vitamin supplements, such as yeast extract, have been found to have growth-promoting qualities, but the identity
'
of which vitamins specifically encourage yeast growth has not been clearly defined. In addition, the available concentration of required nutrients, such as the carbon source, to induce more rapid yeast growth has not been determined.
Therefore, it is an object of this invention to provide a selective growth media which encourages the rapid growth of yeasts to the exclusion of other microorganisms.
It is also an object of the invention to provide a base preparation of yeast media consisting of a mixture of required and dehydrated nutrients and an antibiotic supplement which inhibits the growth of prokaryotic organisms.
Further, it is an object of the invention to provide a rapid method of detecting yeast contamination in non-alcoholic beverages.
Summary of the Invention
A selective growth medium according to the invention for the rapid detection of yeasts in non-alcoholic beverages contains carbon, nitrogen, vitamin, and nutrient sources, an osmophilic constituent for providing a buffered environment, and an antibiotic supplement to inhibit growth of prokaryotic organisms. The carbon source consists essentially of dextrose; the vitamin source preferably includes vitamin K and yeast extract and can also contain inositol; and the preferred nitrogen
source consists essentially of about two parts inorganic nitrogen, such as ammonium sulfate, and about one part organic nitrogen, such as asparagine. The preferred nutrient source includes casein digest and 1-cysteine or casamino acids, and one desirable osmophilic constituent includes ammonium sulfate and sodium chloride.
The invention also provides an improved growth medium for yeasts, and which comprises a carbon source, a nitrogen source, a nutrient source, a vitamin source, and antibiotics to inhibit growth of prokaryotic organisms. The point of novelty includes a high concentration of dextrose as the carbon source, and vitamin K as a nutrient supplement.
Further, a base system, according to the invention, for the preparation of a selective growth medium for yeasts, to the exclusion of other microorganisms, consists essentially of two components: a mixture of dry medium ingredients which includes a carbon source, a nitrogen source, a nutrient source, a vitamin source, and an osmophilic constituent to maintain a buffered environment; and an antibiotic supplement. A preferred antibiotic supplement for practice on the invention includes at least two of the group consisting of chloramphenicol, chlortetracycline-HCl, and streptomycin. The antibiotic supplement is added to the mixture following the hydration, sterilization, and cooling of the mixture, and prior to the inoculation of an aliquot of a non-alcoholic beverage in which
contaminating yeasts may be proliferating. The medium can further include, as an optional element, a growth indicator which changes color as the pH of the medium changes due to the metabolic activity of the yeasts growing therein.
A method according to the invention for the rapid detection of contaminating yeasts in non-alcoholic beverages includes the steps of (i) hydrating a pre-mixed dry medium including carbon, nitrogen, nutrient and vitamin sources, and an osmophilic constituent to maintain a buffered environment; (ii> sterilizing and cooling the hydrated medium; (iii) inoculating an antibiotic supplement which includes at least two of the group consisting of chloramphenicol, c lortetracycline-HCl, and streptomycin to prevent growth of prokaryotic organisms; (iv) inoculating and incubating an aliquot of non-alcoholic beverage in the medium under environmental conditions favorable for yeast growth; and (v) detecting and scoring the yeasts growing therein.
The foregoing features enable the invention to provide a growth medium which encourages yeast proliferation while selectively inhibiting growth of other microorganisms. This invention also provides a rapid, facile, and economical method of screening for yeast contamination in non-alcoholic beverages. The two components of the base system comprising the growth medium can easily be prepared, packaged, and distributed to the location of beverage production for convenient and expedient on-the-site testing.
' "
Description of Embodiments
The selective growth medium according to the present invention includes the essential carbon, hydrogen, oxygen, and nitrogen-containing nutrients, and required osmophilic constituents for the sustenance of microbial life in general. It also includes nutrients and growth promoting constituents such as specific vitamins for the stimulation of yeast growth, in particular.
The carbon source provides a substrate for yeast fermentation. This source can be a carbohydrate composed of various sugar residues, and is usually a disaccharide such as dextrose which is composed of a glucose and a fructose residue, or a monosaccharide such as fructose or glucose. Because the majority of soft drinks contain dextrose or high fructose corn syrup as the sweetener, dextrose is preferred as the sole, or primary, carbon source. The concentration of dextrose in the medium is in the range about of 10 to 15 grams per liter, and a preferred concentration is about 12 grams per liter.
A preferred practice of the invention employs water as the hydrogen source, and air present naturally in the medium as the oxygen source. Yeasts are faculative anaerobes which are able to exist on essentially whatever level of oxygen is present in the medium container or is dissolved in the medium itself. Therefore, an additional oxygen source is not essential.
SUBSTI UTE SHEET
Most organisms are capable of utilizing organic nitrogen-containing compounds, such as proteins and a ino acids, as nitrogen sources. The present medium contains both proteins and amino acids, and preferably contains asparagine at a concentration in the range of about from 1.0 to 2.5 grams per liter medium, and preferably at about 1.5 grams per liter medium.
During certain periods of their growth cycle, yeasts have been found to utilize nitrogen from inorganic sources, such as nitrates or ammonium salts, more efficiently than from organic sources. In addition, it has been determined that particular metabolic processes enable yeasts to utilize the lower oxidative potential in ammonium salts more efficiently than to utilize the higher oxidative potential in nitrates. Therefore, the preferred inorganic nitrogen source is an ammonium salt. In particular, the ammonium salt of choice is ammonium sulfate in an amount ranging about from 2.0 to 5.0 grams per liter medium, and specifically at about 3.5 grams per liter medium.
The present medium provides both organic and inorg-anic nitrogen sources at concentrations which take into account these metabolic limitations. Preferably, two parts by weight inorganic nitrogen to one part by weight organic nitrogen are included in the medium.
It is desirable that the growth medium include an osmophilic constituent, or a combination
BSTITUTE SHEET
of compounds which buffer solutions such that the proper pH and intracellular pressures are maintained. Magnesium and/or sodium salts, and more particularly, magnesium sulfate substantially in the range of about from 0.2 to 0.7 grams per liter, and sodium chloride substantially in the range of about from 1.0 to 1.5 grams per liter can be added for this purpose. Preferably, about 0.5 grams magnesium sulfate and about 1.3 grams sodium chloride per liter are included in the present medium.
Certain vitamins are potent growth stimulators for yeast (S.A. Koser, Vitamin Requirements of Bacteria and Yeasts. 1968 Charles C. Thomas Co., Springfield, MA., pp. 478). These vitamins have been typically added to growth media in the form of yeast extract, which is known to be rich in water-soluble B and C vitamins, and in vitamin E. A practice according to the present invention includes yeast extract in a range of about from 1.0 to 3.0 grams per liter medium, and preferably at about 2.25 grams per liter medium. Neat vitamin K is included at a concentration substantially in the range of about from 0.005 to 0.03 grams per liter medium, and preferably at about 0.01 grams per liter. Inositol, a B-complex vitamin can also be included at a concentration substantially in the range of about from 0.025 to 0.075 grams per liter medium, and preferably at about 0.05 grams per liter. Other vitamin sources may also be included.
Because yeasts grow more slowly in most media that do prokaryotic microorganisms such as
ITUTE SHffil"
bacteria and fungi, it is desirable to include in the growth medium compounds which selective inhibit the growth of these microorganisms to the exclusion of yeasts. Antibiotics such as chloramphenicol, chlortetracycline-HCl, streptomycin, neomycin, penicillin, dihydrostreptomycin, streptomycin, and compounds such as potassium tellurite, bismuth sulfite, monotetrazolium compounds, copper sulfate, sodium propionate, and diphenyl are preferred for this purpose. These compounds have the ability to selectively interfere with various metabolic processes specific for bacteria, fungi, or prokaryotic organisms in general.
Experiments have shown that no one antibiotic or compound adequately controls bacterial proliferation during routine analysis. Therefore, a combination of antibiotics and/or metabolic poisons are often utilized for this purpose. It is preferable in the practice of this invention that at least two of the group consisting of chloramphenicol, chlortetracycline-HCl and streptomycin, all of which selectively inhibit prokaryotic protein synthesis, be added in the range of about from 0.35 to 0.75 grams each per liter. It is deemed further preferable that the medium include about 0.5 grams per liter each of at least two of the group consisting of chloramphenicol, chlortetracycline-HCl, and streptomycin. These compounds degrade upon autoclaving, or at sterilization temperatures, and are thus added as a filter-sterilized solution to a cooled, post-autoclaved solution of medium constituents.
The following illustrative preferred examples of a growth medium and methods of detecting yeast set forth further features of the invention.
INGREDIENT FUNCTION g/1 MOLARITY WT % dextrose C 12.50 0.069 48.43 ammonium sulfate Inorganic N 3.50 0.026 13.56 casamino acids yeast extract vitamin K casein digest magnesium sulfate sodium chloride chloramphenicol chlortetracycline-HC streptomycin
Alternatively/ the medium can include two of the three antibiotics listed in each of the above variations.
The preferred preparation of each illustrated variation of the iedia includes dissolving the ingredients listed above, exclusive of the antibiotics, in water such that the final volume after hydration is one liter. .The solution is poured into an autoclavable flask or container, i.e. one able to withstand sterilization temperatures. Alternatively, the solution is aliquoted out into a number of smaller containers. The solution is then autoclaved for about 15 minutes at about 120 degrees Centigrade, and cooled at room temperature to about room temperature. A filter-sterilized antibiotic supplement is then added under sterile conditions, and the solution mixed thoroughly. The medium is now ready for use.
SUBSTITUTE SHEET
In a typical use of the medium, an aliquot of a non-alcoholic beverage to be tested is inoculated under sterile conditions into the medium.
In one illustrative instance, an 18 ml sample of the test solution is poured into a Yeast and Mold Swab Kit Sampler (The Millipore Corp., Bedford, MA 01730), and the Sampler paddle is inserted. The Sampler is then placed horizontally onto a flat surface with its membrane facing down, for a period of time sufficient for uniform wetting of the membrane, e.g., for one minute. The paddle is then removed, and the Sampler case emptied of excess medium. After reinsertion of the paddle, the Sampler is incubated with the membrane facing downward at 28-32 degrees Centigrade for 48-72 hours. The resulting yeast colonies are scored using an illuminated magnifier or microscope.
Alternatively, liquid samples of beverage can be tested using the Beverage Monitor method (The Millipore Corp., Bedford, MA 01730).
SUBSTITUTE SH
EXAMPLE II
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Vari ation ( f )
Each variation of the solid medium described above may also be prepared with two of the three antibiotics listed.
Each variation of the above- media containing, agar is prepared under sterile conditions and kept in liquid phase with moderate heat. Approximately 2.5 ml of beverage sample is put into a sterile petri dish. Approximately 25 ml of liquid medium is added and the resulting solution mixed with dish cover replaced. The plates are allowed to solidify at room temperature. They are then incubated at 37°C for 72 to 96 hours, after which time yeast colonies are visually scored.
Alternatively, approximately 25 ml of the warm liquid medium is placed into a sterile petri dish. The plates are allowed to solidify at room temperature with a closed lid. A portion of the beverage sample to be test is then sterilely streaked onto the solid medium plates. Plates are incubated
SUBSTITUTE SHEET
at 37°C for 72 to 96 hours, after which time yeast colonies are scored.
It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in carrying out the above method and in the composition set forth without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.
It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.
Having described the invention, what is claimed as new and secured by Letters Patent is:
Claims
1. A selective growth medium for the rapid detection of yeasts comprising
(a) a carbon source,
(b) a nitrogen source,
(c) a vitamin source,
(d) an antibiotic supplement and for inhibiting the growth of prokaryotic organisms,
(e) an osmophilic constituent for maintaining a normal intracellular pressure osmophilic environment, and
(f) a nutrient source.
2. A medium according to claim 1, wherein the antibiotic supplement comprises at least two of the group consisting of chloramphenicol, chlortetracycline-HCl and streptomycin.
3. A medium according to claim 1, wherein the weight ratio of said carbon source to said nitrogen source is in a range from about 2:1 to about 3:1.
4. A medium according to claim 1, wherein said nitrogen source consists essentially of about two parts inorganic constituent and about one part organic constituent.
5. A medium according to claim 4, wherein said carbon source consists essentially of dextrose, said inorganic nitrogen source consists essentially of ammonium sulfate, and said organic nitrogen source consists essentially of asparagine.
6. A medium according to claim 4, wherein said carbon source consists essentially of dextrose, said inorganic nitrogen source consists essentially of ammonium sulfate, and said organic nitrogen source consists essentially of casamino acids. •
7. A medium according to claim 1 further comprising a growth indicator which changes color as the pH of said medium changes due to metabolic activity therein.
8. A medium according to claim 1, wherein said vitamin source consists essentially of neat vitamin K substantially in the range of from about 0.005 to about 0.03 grams per liter, and yeast extract substantially in the range of from about 1.0 to about 3.0 grams per liter.
9. A medium according to claim 8, further comprising inositol substantially in the range of from about 0.025 to 0.075 grams per liter.
10. A medium according to claim 1, wherein said osmophilic constituent consists essentially of magnesium sulfate substantially in the range of from about 0.2 to about 1.0 grams per liter, and sodium chloride substantially in the range of from about 0.5 to about 2.0 grams per liter.
11. A medium according to claim 1, wherein said nutrient source consists essentially of casein digest substantially in the range of from about 0.5 to about 2.5 grams per liter, 1-cysteine substantially in range of from about 0.005 to about 0.03 grams per liter, and 1-tryptophan substantially in range of from about 0.1 to about 0.3 grams per liter.
12. A medium according to claim 1, wherein
(a) said carbon source consists essentially of dextrose in an amount substantially between from about 5.0 to about 15.0 grams per liter,
(b) said nitrogen source consists essentially of ammonium sulfate in an amount substantially between from about 2.0 to about 5.0 grams per liter, asparagine in an amount substantially between from about 1.0 to about 2.0 grams per liter, and 1-tryptophan in an amount substantially between from about 0.1 to about 0.3 grams per liter,
(c) said vitamin source includes yeast extract in an amount substantially between from about 0.005 to about 0.030 grams per liter, and vitamin K in an amount substantially between from about 1.0 to about 3.0 grams per liter. (d) said antibiotic supplement consisting essentially of at least two of the group consisting of chloramphenicol in an amount substantially between from about 0.35 to about 0.75 grams per liter, chlortetracycline-HCll in an amount substantially between from about 0.35 to about 0.75 grams per liter and streptomycin in an amount substantially between from about 0.35 to about 0.75 grams per liter,
(e) said osmophilic constituent includes magnesium sulfate in an amount substantially between from about 0.2 to about 2.0 grams per liter, and sodium chloride in an amount substantially between from about 0.5 to about 2.0 grams per liter, and
(f) said nutrient source includes casein digest in an amount substantially between from about 0.5 to about 2.5 grams per liter, and 1-cysteine in an amount substantially between from about 0.005 to about 0.030 grams per liter.
13. A medium according to claim 12 further comprising inositol in an amount substantially between from about 0.025 to 0.075 grams per liter.
14. A medium according to claim 1, wherein
(a) said carbon source consists essentially of about 12.5 grams per liter dextrose,
(b) said nitrogen source consists essentially of about 3.5 grams per liter ammonium sulfate and about 0.2 grams per liter 1-tryptophan,
(c) said vitamin source includes about 2.25 grams per liter yeast extract and about 0.01 grams per liter vitamin K, (d) said antibiotic supplement consisting essentially of about 0.5 grams per liter chlortetracycline-HCll and about 0.5 grams per liter chloramphenicol,
(e) said osmophilic constituent includes about 0.5 grams per liter magnesium sulfate and about 1.3 grams per liter sodium chloride,
(f) said nutrient source includes about 1.25 grams per liter casein digest and about 0.01 grams per liter 1-cysteine.
15. A medium according to claim 14, further comprising about 0.05 grams per liter inositol.
16. A growth medium for the detection of yeasts having a carbon source, a nitrogen source, a nutrient source, and antibiotics which inhibit the growth of prokaryotic organisms, and further comprising
(a) dextrose in an amount substantially between from about 12.0 to about 15.0 grams per liter as said carbon source, and
(b) vitamin K in an amount substantially between from about 0.005 to about 0.03 grams per liter.
17. A growth medium according to claim 16 further comprising inositol in an amount substantially between from about 0.025 to about 0.075 grams per liter.
18. A dehydrated base system for the preparation of a selective growth medium for yeasts present in a non-alcoholic beverage, wherein the growth medium is prepared by hydrating, sterilizing, and cooling the base system, and by adding to the hydrated base system an antibiotic supplement after cooling, said base system consisting essentially of
(a) a mixture of dehydrated ingredients which includes carbon, nitrogen, nutrient, and vitamin sources, and an osmophilic constituent, said carbon source consisting essentially of from about 10 to about 15 grams per liter dextrose, and said vitamin source consisting essentially of from about 0.005 to about 0.03 grams per liter vitamin K and from about 1.0 to about 3.0 grams per liter yeast extract, and
(b)an antibiotic supplement including at least two of the group consisting of chloramphenicol, chlortetracycline-HCl, and streptomycin for addition to said hydrated, sterile, cool mixture prior to the innoculation of an aliquot of the non-alcoholic beverage which said yeasts may be contaminating.
19. A base system according to claim 18, wherein
(a) said nitrogen source consists essentially of two parts inorganic component and one part organic component by weight,
(b) said nutrient source consists essentially of from about 0.5 to about 2.5 grams per liter casein digest, from about 0.005 to about .03 grams per liter 1-cysteine, and from about 0.1 to about 0.3 grams per liter 1-tryρtophan, and (c) said osmophilic constituent consists essentially of from about 0.2 to about 2.0 grams per liter magnesium sulfate and from about 0.5 to about 2.0 grams per liter sodium chloride.
20. A method for the rapid detection of contaminating yeasts in non-alcoholic beverages, said method comprising the successive steps of
(a) providing a premixed dry medium base including carbon, nitrogen, nutrient, and vitamin sources, and an osmophilic constituent to maintain normal intracellular pressure, and wherein said carbon source consists essentially of from about 10 to about 15 grams per liter dextrose, and said vitamin source consists essentially of from about 0.005 to about 0.03 grams per liter vitamin K and from about 1.0 to about 3.0 grams per liter yeast extract, and said nitrogen source consists essentially of two parts inorganic component and one part organic component by weight,
(b) hydrating said premixed medium base,
(c) sterilizing and cooling said hydrated medium,
(e) adding an antibiotic supplement to prevent growth of contaminating prokaryotic organisms,
(f) inoculating said medium with an aliquot of non-alcoholic beverage,
(g) incubating said aliquot in said medium under environmental conditions conducive for yeast growth, and for a time sufficient for the detection of said yeast growth, and
(f) detecting and scoring said yeast.
21. The method of claim 20, wherein said base-providing step further includes casein digest from about 0.5 to about 2.5 grams per liter as said nutrient source, from about 0.005 to about 0.03 grams per liter 1-cysteine, and from about 0.1 to about 0.3 grams per liter 1-tryptophan as said nutrient source, and magnesium sulfate from about 0.2 to about 2.0 grams per liter, and sodium chloride from about 0.5 to about 2.0 grams per liter as said osmophilic constituent.
22. The method of claim 20, wherein said base-providing step further includes casein digest from about 0.5 to about 2.5 grams per liter as said nutrient source, casamino acids from about 2.0 to about 5.0 grams per liter as said organic nitrogen source, and magnesium sulfate from about 0.2 to about 2.0 grams per liter and sodilum chloride from about 0.5 to about 2.0 grams per liter as said osmophilic constituent.
23. The method of claim 20, wherein said adding of said antibiotic supplement includes selection of at least two of the group consisting of chloramphenicol, chlortetracycline-HCl, and streptomycin.
24. The method of claim 20, wherein said base-providing step further includes inositol from about 0.025 to about 0.075 grams per liter as said vitamin source.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6857387A | 1987-07-01 | 1987-07-01 | |
| US068,573 | 1987-07-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1989000204A1 true WO1989000204A1 (en) | 1989-01-12 |
Family
ID=22083414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1988/001269 WO1989000204A1 (en) | 1987-07-01 | 1988-04-11 | A medium and method for yeast detection in non-alcoholic beverages |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1989000204A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0430151A1 (en) * | 1989-11-30 | 1991-06-05 | Becton, Dickinson and Company | Selective fungal medium |
| US5368717A (en) * | 1990-11-26 | 1994-11-29 | The Regents Of The University Of California, Office Of Technology Transfer | Metallization of electronic insulators |
| ES2093568A1 (en) * | 1995-06-08 | 1996-12-16 | Univ Salamanca | Culture media and process for identifying yeasts |
| KR100548767B1 (en) * | 2005-03-30 | 2006-02-02 | 이장원 | Butyl sealing material alleviated its primary adhesive |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57166995A (en) * | 1981-04-06 | 1982-10-14 | Sankin Kogyo Kk | Liquid medium for diagnosis of candidasis |
| JPH06229999A (en) * | 1993-02-04 | 1994-08-19 | Nippon Kayaku Co Ltd | Column for liquid chromatography |
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1988
- 1988-04-11 WO PCT/US1988/001269 patent/WO1989000204A1/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57166995A (en) * | 1981-04-06 | 1982-10-14 | Sankin Kogyo Kk | Liquid medium for diagnosis of candidasis |
| JPH06229999A (en) * | 1993-02-04 | 1994-08-19 | Nippon Kayaku Co Ltd | Column for liquid chromatography |
Non-Patent Citations (3)
| Title |
|---|
| Difco Manual of Dehydrated Culture, Media and Reagents, Ninth edition, 1977, Difco Laboratories (Detroit, Michigan, US), pages 250-253 * |
| Patent Abstracts of Japan, vol. 11, no. 206, (C-433)(2653), 3 July 1987; & JP-A-6229999 (EIKEN KAGAKU K.K.) 7 February 1987 * |
| Patent Abstracts of Japan, vol. 7, no. 11, (C-145)(1156), 18 January 1983; & JP-A-57166995 (SANKIN KOGYO K.K) 14 October 1982 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0430151A1 (en) * | 1989-11-30 | 1991-06-05 | Becton, Dickinson and Company | Selective fungal medium |
| US5368717A (en) * | 1990-11-26 | 1994-11-29 | The Regents Of The University Of California, Office Of Technology Transfer | Metallization of electronic insulators |
| ES2093568A1 (en) * | 1995-06-08 | 1996-12-16 | Univ Salamanca | Culture media and process for identifying yeasts |
| KR100548767B1 (en) * | 2005-03-30 | 2006-02-02 | 이장원 | Butyl sealing material alleviated its primary adhesive |
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