CA1194437A - Method for producing single and/or mixed strain concentrates of bacteria - Google Patents
Method for producing single and/or mixed strain concentrates of bacteriaInfo
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- CA1194437A CA1194437A CA000419949A CA419949A CA1194437A CA 1194437 A CA1194437 A CA 1194437A CA 000419949 A CA000419949 A CA 000419949A CA 419949 A CA419949 A CA 419949A CA 1194437 A CA1194437 A CA 1194437A
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Abstract
ABSTRACT
An improved method which differentiates or separates heterogeneous populations of fast and slow acid producing strains of bacteria by growth of the strains under closely controlled unique conditions so as to allow the selection of a colony of one or the other strains is described Preferably a gelled, solid growth medium containing in admixture: (1) milk protein, a milk protein derivative or a milk protein substitute; (2) an acid pH sensitive color change indicator;
and (3) a buffering agent is used. The colonies have a contrasting color within and around them because of the effect of the acid produced by the bacteria on the indicator. The growth of the bacteria is under anaerobic or near anaerobic conditions in order to achieve certainty in the colony selection for fast or slow acid production. The bacteria can also be mixed with phage which inhibit or kill the members of a heterogeneous or homogeneous population of bacteria on the medium and grown to produce phage resistant colonies. The relatively large colonies which exhibit enhanced acid production and proteolysis of the milk protein on the plating container are selected for commercial use in preparing fermented products, particularly fermented foods.
An improved method which differentiates or separates heterogeneous populations of fast and slow acid producing strains of bacteria by growth of the strains under closely controlled unique conditions so as to allow the selection of a colony of one or the other strains is described Preferably a gelled, solid growth medium containing in admixture: (1) milk protein, a milk protein derivative or a milk protein substitute; (2) an acid pH sensitive color change indicator;
and (3) a buffering agent is used. The colonies have a contrasting color within and around them because of the effect of the acid produced by the bacteria on the indicator. The growth of the bacteria is under anaerobic or near anaerobic conditions in order to achieve certainty in the colony selection for fast or slow acid production. The bacteria can also be mixed with phage which inhibit or kill the members of a heterogeneous or homogeneous population of bacteria on the medium and grown to produce phage resistant colonies. The relatively large colonies which exhibit enhanced acid production and proteolysis of the milk protein on the plating container are selected for commercial use in preparing fermented products, particularly fermented foods.
Description
3~
RJP/paa 1/18/83 1255E 24767 .
IMPROVED METHOD FOR PRODUCING SINGLE AND/OR
MIXED STRAIN CONCENTRATES OF BACTERIA
BACKGROUND OF THE INVENTION
1. Field of the Invention .. . . _ _ The present invention relates to an impro~ed me-thod device for differentiating or separating heterogeneous populations of fast and slow acid producing strains of bacteria to produce single strains or clones. In particular, the present invention relates to a method wherein special growth media and conditions are utilized to achieve the differentiatiQn an~ wherein the differentiated and selected strains are preferably provided as cultures to producers of ~ermented products.
RJP/paa 1/18/83 1255E 24767 .
IMPROVED METHOD FOR PRODUCING SINGLE AND/OR
MIXED STRAIN CONCENTRATES OF BACTERIA
BACKGROUND OF THE INVENTION
1. Field of the Invention .. . . _ _ The present invention relates to an impro~ed me-thod device for differentiating or separating heterogeneous populations of fast and slow acid producing strains of bacteria to produce single strains or clones. In particular, the present invention relates to a method wherein special growth media and conditions are utilized to achieve the differentiatiQn an~ wherein the differentiated and selected strains are preferably provided as cultures to producers of ~ermented products.
2. Prior Art .
The principal prior art is described in:
McKay et al., Applied Microbiology, Vol. 23, pages 1090-10~6 (1972);
Limsowtin, G. K. and _e~ B. E., New Zealand Journal of Dairy Science & Technology, Volume 11, pages 65 and 66 (1976);
Limsowtin, G~ K., et al., New Zealand Journal of Dairy .
Science Technology 13, pages 1 to 8 (1978);
R J Marshall et al., Dairy Research, Vol. 43, pages -449 to 458 (1976); and Rull, R. R., rrhe Australian Journal of Dairy Technology, pages 65 and 77 (June 1977)~
McKa~ et al. describe the problem of the 105s oE
lactose fermPnting ability in lactic acid producing cultures in RJP/paa 1/18/83 1255E 24767 a broth medium. A non-milk agar containing bromocresol purple as an indicator is described for separating colonies which product acid (yellow) from non-acid producing strains (white~
under aerobic conditionsO There is no attempt at selection of phage insensitive mutants or recognition of ~he problem. Thus, McKay was studying loss of lactose fermentation and used the non-rnilk agar medium containing an acid-base indicator to detect non-lactose fermenting (lac ) cells. 1~he selection of lac cells on the McKay medium would not be a worthwhile approach to isolating cells which would yield fast acid producing cultures in milk. Another important determinant for fast acid production in milk is proteolysis (prt)O The McKay medium only distinguishes between lac and lac and lac prt and lac prt cells appear the same on his rnedium, yet the former would be slow in milk while the latter fast.
The problem not solved by McKay et al. is -to distinguish between lac~ prt and lac+ prt~ cells, particularly since the large majority of slow acid-producing ~ariant cells in milk cultures are lac+ prt .
Limsowtin et al. (1976) describe a glycerophosphate buffered, nonfat milk-basedl agar medium (GMA) for the differentiation of fast and slow milk coagulating lactic streptococci. Aerobic (air) growth conditions were used for the growth of ~he bacteria. The medium has been found to be impractical to use since it produced uncertain diEferentiation of fast and slow acid producing cultures of certain Streptococcus crernoris or Streptococcus lactis and i~ was dificult to see white or translucent streptococcal colonies on the white bac;~ground of this medium. I~hus, two strains known to be fast acid producing strains produced colonies which had RJP/paa 1/18/83 1255E 24767 only a 0~5 mm colony diameter thereby erroneously indicating that they were all slow acid producersO Oblique illumination was used to obtain the published photographs, however visual selection is difficulto Marshall et al. describe other ~ .
phosphate buffered media for the selection process under aerobic conditions where selection is difficult. In selecting star~er strains for commercial fermenkations, par~icularly for making fermented dairy products, the method for differentiating and selecting the strains must be completely reliable because of the large volumes of milk or other food being fermented.
Hull describes a method wherein the method of Limsowtin et al. can be used by producers of cultures to provide phage resistant, fast acid producing bacterial cultures to producers of fermented dairy products. By this process a portion of the fermented product or a by-product (whey) provides a source for phage which are mixed on the plating medium with the bacteria for differentiation and selection of phage resistant strains. New phaye resistant strains are provided to the producers of fermented dairy products and older strains are dropped as phage appear before they have a chance to propagate and to vitally infect the older strains~ The problem is that the Limsowtin et al. method is not reliable or certain enough to make the method suggested by Hull commercially feasible for the culture producers.
References which discuss the use of buffered plating media include:
Barach, J. T~, I'Improved Enumeration of Lactic Acid Streptococci on Elliker Agar Containing Phosphate," _p~
~nviron. Microbiol. 38:173-174 (1979);
RJP/paa 1/18/83 1255E 24767 Douglas, JO~ "A Critical Review of the Use of Glycerophosphates in Microbiological Media," Lab. Pract.
20:414-416, 42~ (1971);
Huggins, A~ R~, and W. E. Sandine, I'Selection and Characterization of Phage-Insensitive Lactic Streptococci," J.
Dairy Sci. 62:70 (1979);
Hunter, Go J. E., "A Simple Agar Medium for the Growth of Lactic Streptococci: The Role of Phosphate in the Medium,"
J~ Dairy Res. 14:283-290 (1946);
Keogh, B. P., "Appraisal of Media and Methods for Assay of Bacteriophages of Lactic Streptococci, Ap~l. Environ~
Microbiol. 40:798-802 (1980), Lee, S. Y. et al., "An Agar Medium for Differential Enumeration of Yogurt Starter Bacteria," J. Milk Food Techno1.
37:272-276 (1974); the Limsowtin (1976) article;
Mullan, W. M. A. "Lactic Streptococcal Bacteriophage Enumeration; A Review of Factors A~fecting Plaque Formation,"
Dairy Ind. Int. 44(7):11-15 (1979);
Reddv, M. S., et al., "Agar Medium for Differential Enumeration of Lac~ic Streptococci," Appl. Microbiol.
2~:947-952 (1973);
ReiterJ B., "Lysogenic Strains of Lactîc Streptococci," Nat~re 1640667-668 (1949~;
Shankar 7 Po A. ~ et al., 'IA Note on the Suppression of Lactobacillus bulgaricus in Media Containing B-glycerophosphate and Application of Such Media to SelectiYe Isolation of Streptococcus thermophilus from Yoghurt," _ Soc. Dair~
Technol. 30:28-30 (1977);
She~, D. I., "Effect of Calcium on the Developmenk of Streptococcal Bacteriophages," Nature 164:492-493 ~1949);
____
The principal prior art is described in:
McKay et al., Applied Microbiology, Vol. 23, pages 1090-10~6 (1972);
Limsowtin, G. K. and _e~ B. E., New Zealand Journal of Dairy Science & Technology, Volume 11, pages 65 and 66 (1976);
Limsowtin, G~ K., et al., New Zealand Journal of Dairy .
Science Technology 13, pages 1 to 8 (1978);
R J Marshall et al., Dairy Research, Vol. 43, pages -449 to 458 (1976); and Rull, R. R., rrhe Australian Journal of Dairy Technology, pages 65 and 77 (June 1977)~
McKa~ et al. describe the problem of the 105s oE
lactose fermPnting ability in lactic acid producing cultures in RJP/paa 1/18/83 1255E 24767 a broth medium. A non-milk agar containing bromocresol purple as an indicator is described for separating colonies which product acid (yellow) from non-acid producing strains (white~
under aerobic conditionsO There is no attempt at selection of phage insensitive mutants or recognition of ~he problem. Thus, McKay was studying loss of lactose fermentation and used the non-rnilk agar medium containing an acid-base indicator to detect non-lactose fermenting (lac ) cells. 1~he selection of lac cells on the McKay medium would not be a worthwhile approach to isolating cells which would yield fast acid producing cultures in milk. Another important determinant for fast acid production in milk is proteolysis (prt)O The McKay medium only distinguishes between lac and lac and lac prt and lac prt cells appear the same on his rnedium, yet the former would be slow in milk while the latter fast.
The problem not solved by McKay et al. is -to distinguish between lac~ prt and lac+ prt~ cells, particularly since the large majority of slow acid-producing ~ariant cells in milk cultures are lac+ prt .
Limsowtin et al. (1976) describe a glycerophosphate buffered, nonfat milk-basedl agar medium (GMA) for the differentiation of fast and slow milk coagulating lactic streptococci. Aerobic (air) growth conditions were used for the growth of ~he bacteria. The medium has been found to be impractical to use since it produced uncertain diEferentiation of fast and slow acid producing cultures of certain Streptococcus crernoris or Streptococcus lactis and i~ was dificult to see white or translucent streptococcal colonies on the white bac;~ground of this medium. I~hus, two strains known to be fast acid producing strains produced colonies which had RJP/paa 1/18/83 1255E 24767 only a 0~5 mm colony diameter thereby erroneously indicating that they were all slow acid producersO Oblique illumination was used to obtain the published photographs, however visual selection is difficulto Marshall et al. describe other ~ .
phosphate buffered media for the selection process under aerobic conditions where selection is difficult. In selecting star~er strains for commercial fermenkations, par~icularly for making fermented dairy products, the method for differentiating and selecting the strains must be completely reliable because of the large volumes of milk or other food being fermented.
Hull describes a method wherein the method of Limsowtin et al. can be used by producers of cultures to provide phage resistant, fast acid producing bacterial cultures to producers of fermented dairy products. By this process a portion of the fermented product or a by-product (whey) provides a source for phage which are mixed on the plating medium with the bacteria for differentiation and selection of phage resistant strains. New phaye resistant strains are provided to the producers of fermented dairy products and older strains are dropped as phage appear before they have a chance to propagate and to vitally infect the older strains~ The problem is that the Limsowtin et al. method is not reliable or certain enough to make the method suggested by Hull commercially feasible for the culture producers.
References which discuss the use of buffered plating media include:
Barach, J. T~, I'Improved Enumeration of Lactic Acid Streptococci on Elliker Agar Containing Phosphate," _p~
~nviron. Microbiol. 38:173-174 (1979);
RJP/paa 1/18/83 1255E 24767 Douglas, JO~ "A Critical Review of the Use of Glycerophosphates in Microbiological Media," Lab. Pract.
20:414-416, 42~ (1971);
Huggins, A~ R~, and W. E. Sandine, I'Selection and Characterization of Phage-Insensitive Lactic Streptococci," J.
Dairy Sci. 62:70 (1979);
Hunter, Go J. E., "A Simple Agar Medium for the Growth of Lactic Streptococci: The Role of Phosphate in the Medium,"
J~ Dairy Res. 14:283-290 (1946);
Keogh, B. P., "Appraisal of Media and Methods for Assay of Bacteriophages of Lactic Streptococci, Ap~l. Environ~
Microbiol. 40:798-802 (1980), Lee, S. Y. et al., "An Agar Medium for Differential Enumeration of Yogurt Starter Bacteria," J. Milk Food Techno1.
37:272-276 (1974); the Limsowtin (1976) article;
Mullan, W. M. A. "Lactic Streptococcal Bacteriophage Enumeration; A Review of Factors A~fecting Plaque Formation,"
Dairy Ind. Int. 44(7):11-15 (1979);
Reddv, M. S., et al., "Agar Medium for Differential Enumeration of Lac~ic Streptococci," Appl. Microbiol.
2~:947-952 (1973);
ReiterJ B., "Lysogenic Strains of Lactîc Streptococci," Nat~re 1640667-668 (1949~;
Shankar 7 Po A. ~ et al., 'IA Note on the Suppression of Lactobacillus bulgaricus in Media Containing B-glycerophosphate and Application of Such Media to SelectiYe Isolation of Streptococcus thermophilus from Yoghurt," _ Soc. Dair~
Technol. 30:28-30 (1977);
She~, D. I., "Effect of Calcium on the Developmenk of Streptococcal Bacteriophages," Nature 164:492-493 ~1949);
____
3~
RJP/paa 1/1~/83 1255E 24767 .
Stainer, R. Y. "The Microbiol World, p. 37-40, 68 (1976); and ~ erzaghi, B. E., and W. E. Sandine, "Improved Medium for Lactic Streptococci and their Bacteriophages," Ap~l.
Microbiol. 29:807-813 (1975).
Hunter demonstrated that with addition of sodium phosphate (3307mM) to a simple plating medium, more growth and larger colonies resulted. Buffering provided by the phosphate allowed more cell divisions before the hydrogen ion concentration became inhibitory to further growth.
Improved growth of lactic streptococci results when disodium-B-glycerophosphate (62mM) is incorporated into a complex medium by Terzaghi and Sandine. This medium is also lactose-limiting which enables the final pH to remain well above pH 500 and avoid any acid injury to the cells~ Douglas claimed one of the advantages of glycerophosphates over inorganic phosphates was that microbiological media could be formulated with high phosphate concentrations without precipitating out many essential metals. This feature i5 especi~lly useful in pla~uing lactic bacteriophages where Ca~2 is required for optimum formation of plaques (articles by Reiter; Shew).
Sodium-B-glycerophosphate is also utilized in two different plating media designed to diEferentiate "fast" and "slow" colonies of lactic streptococci (articles by ~uggins and Sandine; Limsowtin). These buffered media enable fast acid producing colonies to grow to a larger si~e so that they can be distinguished from smaller slow coloniesO
Shankar and Davies found B-glycerophosphate inhibited some strains of L~ bul~aricus and more recently there have been 3~
RJP/paa 1/18/83 1255E 24767 reports of inhibition of a few strains of lactic streptococci (articles by Keogh; Mullan).
Barach improved the enumeration of lactic streptococci by as much as 7. 75 times by incorporating 3UmM diammonium phosphate into unbuffered Elliker agar.
Insoluble CaC03 has been added to agar media to help preserve neutral conditions for isolation and cultivation of acid-producing bacteria. Production of acid is localized around colonies and can be detected by clear halos resulting from acid-solubilization of CaC03 immediately surrounding the colonies as described by Stanier. Such a buffering system is used in a plating medium for differential enumeration of lactic strep~ococci by Reddy, et al.~ and in another for differentiating colonies of S _ hermophilus from I. bulgaricus by Leer et al.
OBJECTS
_, ~ . .
It is therefore an object of the present in~Jention to provide a method wherein heterogeneous populations of fast and slow acid producing strains of bacteria are readily and reproducibly differentiated or separated as to acid producing ability~ Further it i5 an object of the present invention to provide a method wherein phage insensitive strains can be reliably selected. Further still it is an object of the present invention to provide the selected strains as c~ltures or concentrates of phage insensiti~Je strains of bacteria as a single strain or as a mixture of several such single strains.
These and other objects will become increasingly apparent by reference to the following description and to the drawingO
-- 6 ~
In _e Drawing The drawing is a photograph showing clearly differen-tiated fast (f) and slow (s) acid producing single strain bacterial colonies produced by the method and plating device of the present invention using an indicator and anaerobic fermen-tation conditions.
General Descri.ption The present invention relates to an improved method for the differentiation of heterogeneous populations of fast acid producing strains of a species of bacteria from slow acid producing strains of the same bacteria which comprises providing a gelled, solid bacterial growth medium containing milk protein, a milk protein derivative or a milk protein substitute and containing a p~l sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7 and growing the heterogeneous populations of a strain or strains of acid producing bacteria on ~he medium to produce single strain acid producing bacterial colonies, wherein the colonies are of varying si~es and have a contrasting color from the growth medium around and within the colonies because of the reaction of the acid in the colonies with the indicator A bacterial plating device is used for differen-tiating and selecting fast acid producing strains of a species of bacteria in a heterogeneous population with slow acid produ~ing strains or variants of the same bac-teria. A closed container is used containing a gelled, solid growth medium for the bacteria in admixture with milk protein, a milk protein derivative or a milk protein substitute and a pH sensitive indicator which changes color upon contact with acid in the pH
range between about 4 and 7. Preferably anaerobic or near anaerobic conditions are provided in the space.
The present invention also relates to an improved method for the differentiation of heterogeneous popu-lations of fast acid producing strains of a species of bacteria from slow acid producing strains by visual observation of color differences amony colonies grown on a gelled, solid bac-terial growth medium by providing a medium that includes milk protein, a milk protein derivative or a milk protein substitute, a pH
sensitive indicator which changes color upon contact with acid in the p~ range between about 4 and 7 even in the absence of milk proteolysis, and a substantial non~oxic, insoluble buffering agent.
The method of the present inventi.on also relates to the production of a concentrate o:E a single strain or a mixture of single strains of a species of bacteria of the gen-era Streptococcus or Lactobacillus of the type used :Eor produc-... . . . ~
ing lactic acid in foods by -fermentation. The selected bacter ium is characterized by being a fast acid producer and by hav-ing been grown anaerobically on a gelled solid growth medium, wherein the medium includes a pH sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7, a bufferiny agent and a milk protein, a milk protein derivative or a rnilk protein substitute, and by being able to grow in the presence of a phage which kills or impairs the growth of parent strains of the same species to produce the single strains which are then selected and regrown in a second RJP/paa 1/18/83 1255E 24767 fluid growth medium to produce the concentrates which contains at least about 1 x 106 cells per ml.
The present invention also relates to the method for selecting phage resistant strains of acid producing bacteria and concentrating the cells for use in producing fermented food products which comprises: growing heterogeneous or homogeneous populations of a strain or strains of the acid producing bacteria anaerobically or nearly anaerobically in the presence of phage on a solid growth medium so as to produce colonies wherein fast acid producing strains produce relatively larger colonies than slow acid producing strains; selecting a large colony; and growing the cells in a fluid growth medium to at least about 106 cells per ml to provide a concentrate of cells.
The present invention particularly relates to the method for providing phage resistant strains of acid producing bacteria to producers of fermented food produc~s which comprises growing heterogeneous or homogeneous populations of a strain or strains of an acid producing bacteria anaerobically or nearly anaerobically in the presence of phage from a sample of the food product or a by-product of the food product obtained from a producer of the fermented food product on a growth medium which differentiates slow acid producing strains from fast acid producing strains of the bacteria by producing relatively larger colonies of cells of fast acid producing strains in the presence of phage; selecting cells in a large colony having a diameter of at least about 1 mm and growing the cells in the larger colony to at least about 106 cells per ml to provide a culture of ce:Lls; and providing the producer which supplied the sample with the culture for use in prsducing fermented food productsO
The method for differentiation and selection produces a eoneentrate of improved homologous cells of a single strain which is from a clone of a single member of the species and which has the most desirable acid producing properties as well as preferably phage insensitivity. The term "strain" or "variant" as used herein means a member of a single species of bacteria which has a common source or parent with other members anc~ whieh generally has almost all of the same fermen-tation characteristics with other members, but which can have a weak-ness in the ability to produce acid or in the case of mi]k fermentations have a poor proteolytic ability or have phage sensitivity. A single species of bacteria may have many strains of the same bacterium which differ by one or more fer-mentation characteristics and thus form a "heterogeneous popu-lation'l. The general fermentation eharacteristics are deter-mined in relation to sugars and other assimilable carbon sources for the species as listed in Bergey's Manual, Eighth Edition (1974). There are commercially available devices for determining -fermentation characteristies on sugars and other substrates using microassay techniques. The API series Erom Analytab Products, Inc., in Carle Place, N.Y., is particularly suitable~
The ~erms "slow acid producing" and "fast acid pro-ducing" are used in the milk fermentation industry in relation to lactie acid produeing bacteria. Slow lactic acid producing strains are those whieh fail to eoagulate milk in 18 hours at 21C using a 1~ by volume inoculum (Citti, J.E., et al~
"Cornparison of slow and fast acid producing streptococcus lac-tis," J. Dairy Sci~ 48.14-18 (1965~). Fast lactic acid produc-ing strains develop 'che relatively larger colonies.
The baeteria are grown so that there are between about 30 and 300 colonies per petri plate or between about 0~5 and 5 3~
RJP/paa l/:L8/83 1255E 24767 per square centimeter of gelled growth medium. PreEerably there are less than 100 colonies per plate (about 1.6 per square cm of medium). The reason for these preferred colony densities is that there can be too few or too many colonies on the plate outside of the broad ranges for reliable results.
The fast acid producing colonies are at least about 1 mm in diameter.
Many acid indicators can be used in the present invention. One such indicator is litmus which changes from blue to red upon contact with acid. Litmus makes proteolysis of the milk protein in the medium by the bacteria readily visible in a ring (p) around the fringes of the fast acid producing colonies (f) as shown in Figure 1. Milk proteolysis is necessary in most fermentations. Other indicators can be used, such as bromcresol purple (pH range 5.2 to 6.8) and bromthymol blue (pH range 6~0 to 7.6) which do not rely on milk proteolysis.
Anaerobic growth conditions provide unexpectedly superior results in terms of the diferentiation of fast and slow acid producing colonies. The anaerobic conditions can be provided in the confined space by a vacuum or by providing a reducing or rare gas in the confined space. The preferred gases are nitrogen, hydrogen or other nontoxic gases alone or mixed with carbon dioxide which is assimilated by the lactic acid producing bacteria. Preferably between about 5~ and 50 by volume carbon dioxide is used with the balance being hydrogen or nitrogen. Other useful non-oxidizing ~ases include the rare gases, such as neon and krypton and particularly argon.
An important improvement of the present invention is the use of the acid indicator combined with anaerobic growth RJP/paa 1/18/83 1255E 24767 conditions which provides a synergistic resul~ ih the differentia~ion and selection and in the homogeneity of ~he single strain of cells produced. Unexpectedly it has been found that significantly i.mproved differentiation and selection of fast acid producing strains can be achieved with this combinakion.
The growth medium includes milk protein, a milk protein derivative, preferably nonfa~ milk or a casein digest, or a milk protein substitute in an amount between about 5 and 15 percent by weight of the gelled solid medium (W/V).
Another preferred ingredient is a buffering agent, particularly a compound that is substantially nontoxic to the bacteria in the medium and that is an alkali metal carbonate, phosphate, hydroxidel oxide/ or an organic sulfonate D The organic sulfonates include alkali metal salts of piperazine-N~
N'-bis~2-ethane-sulfonic acid (PIPES), morpholinopropane sulfonic acid (~IOPS) and 2(n-morpholino)ethane sulEonic acid (MES) as described in the abstract of the American Dairy Society Association Meetings June 24 to 27 (1979). Disodium glycerophosphate is a suitable buffering agent when present in an amount between about 0.5 and 5 percent by weight based upon the volume of the gelled solid growth medium (W/V). But, substantially nontoxic! insoluble buffering agen~s enable even better visual differentiation, by color, of "fast" and "slow"
colonies, when the buffering agent is present :in an amoun~ of between 0.1 and 1 percent, preferably about 0~25 percent.
Specific insoluble buffering agents are phosphatesJ including ammonium phosphates, carbonates and hydroxides of magnesium and carbonates and hydroxides of calcium and zinc oxide.
Outstanding results are obtained using phosphates oE magnesium, particularly trimagnesium phosphate.
The agars are preferably Davist.m. agar which is produced by the Davis Gelatine Company, Christ Church, New Zealand or Bacto-Agart m from Difco, Detroit, Michigan.
Agar substitutes and other gelled solids such as gelatin are also available. Preferably the plating device has a trans-parent window on the confined space so that the growth of the bacteria can be observed. A conventional petri dish containing the gelled solid growth medium sealed with a confined space around the medium is generally used.
After grow~h of the bacteria 7 the relatively large colony is picked from the gelled solid medium and transferred to a second fluid growth medium including assimilable carbohy-drate and nitrogen sources and is grown to at least abou~
1 x 106 cells per ml and preferably to 108 to 109 cells per ml. In this manner, large numbers of bacterial cells can be produced as a concentrate wherein virtually every individual cell has the same fast acid producing capability. This can be demonstrated by replating the cells on the gelled solid growth medium.
The cells can be held as a concentrated, refrigerated milk culture where milk is the fluid growth medium containing about 1 x 106 to 1 x 109 cells per ml or can be concen-trated further to above about 1 x 109 to 1 x 1012 cells per ml by removing some of the growth medium~ The single strains can be mixed with other single strains produced by -the rnethod. The thus concentrated bacteria can be frozen for storage and/or shipment preferably with a freezing stabilizing agent such as glycerol in an amount up to 20 percent by volume or they can be lyophilized. The bacteria can also be stabilized as described in U.S~ Paten-t No. 4~282/255.
RJP/paa 1/18/~3 1255E 24767 The bacteria which can be differentiated are preferably species selected from the genera Streptococcus and Lactobacillus and are used for lactic acid production in food products by fermentation. Included are ~lS, Stre~tococcus lactis, ~ cus lactis subspecies -diacetylactis, Streptococcus_~ ermophilus~ Lactobacillus bul~aricus, Lactobacillus acidophilus~ Lactobacillus caseii, Lactobacillus lactis, and Lactobacillus helveticusO These are . _ _ bacteria which are sensitive to phages.
The homologous or heterogeneous phages for the particular strain of bacteria are preferably present when they are grown by the plating method and thus the bacteria produced by the method are thus also phage insensitive. The method can also be repeated using different races of phages when the host bacterium is sensitive to more than one phage. In these ways, cells resistant to more than one phage can be generated.
Phayes occur in whey from cheese making. Whey can be used to continuously produce phage insensitive strains by repeated periodic exposure to these phages. Other sources of phage from the producers oE fermented products can also be used. This method prevents failures resulting in the loss of hundreds or thousands of gallons of milk in making cultured dairy products where the bacteria are phage sensitive.
SPECIFIC DEscRIprrIoN
In the following Examples 1-3, the bufering agent was disodium glycerophosphate; the indicator was litmus; and anaerobic growth conditions were used~
L ~
RJP/paa 1/18/83 1255E 24767 Example 1 The composition of the growth med,ium, referred to as fast-slow differential agar (FSDA), was as shown in Table 1.
Table 1 Percent Grams/liter ~Y_~ gh~
Nonfat milk powder (NFM) 100.0 g 76.9%
Davist m agar (New Zealand) 10.~ g 7.7%
Disodium glyce.rophosphate 19.0 g 14.6%
Bactolitmust m (litmus) 1.0 g 0.8%
100 . 0%
The medium was prepared by dispersing lOg of Davis agar in 550 ml of double distilled wa~er in a 2 liter flask by steaming for 30 minutes. To the melted agar, 1.0 g of Bactolitmust m and 19 g of disodium glycerophosphate were added and mixed until dissolved. In a separate 1 liter flask, 100 g of the NF~ powder was dissolved in 450 ml of double distilled water. The two mixtures were then autoclaved separately for 17 minutes and rapidly cooled to 55C in a water bath, combined, and poured into petri plates and flamed to eliminate bubbles as needed. The p~ates were dried by inverting overnight at room temperature~
Single colony strains were obtained by spreading or streaking a mixture of a heterogeneous population of a specific species o~' bact~ria on the agar and growing the mixtureO An atmosphere of hydrogen or hydrogen and carbon dioxide was generated using Gas Pakt m gas generating envelopes (Bio-Quest, Cockeysville, MD) in the confined space over the growth medium in petri plates which were seal.ed in an air RJP/paa 1/18/83 1255E 24767 evacuated Gas Pakt m jar. Typical colonies produced within 24 to 36 hours at 30C were as shown in Figure 1, wherein the large fast acid producing colonies (f) were readily distinguishable from the smaller slow acid producing colonies (s). The large colonies (f) measured 1 to 3 mm in diameter and the small colonies (s) between 0.2 to ~5 mm in diameter or less. The large colonies were selected and had a clear proteolysis ring (p).
It is speculated that the problems encountered by the prior art with aerobic growth of the bacteria on GMA were due to the higher oxygen tension relative to liquid NFM. The effect of anaerobic incubation on colony appearance was remarkable. Not only was excellent colony differentiation achieved, but the incubation period required at 30C was about one-half of the time re~uired when incubated under aerobic conditions in air.
Subsequently, 20 differen~ active strains were differentiated and fast acid producing strains selected in the manner of Example 1 including: Sc A2, Sl C2, Sc H2, Sc HP, Sc 205, Sl c10, Sl ML8, Sc 104, Sc 286, Sc 287, Sc 288/
Sc 289A, Sc 289C, Sc 290, Sc 290A, Sc 291, Sc 292~ Sc 134~ Sc 108 and Sl E where "Sc" represents Streptococcus cremoris and "S1" represents Strep~ococcus lactis. These strains were from the culture collection of Oregon State University, Corvallis, Oregon and samples are freely available to the public without charge~
All of these species produced fast acid strain colonies (f) within 24 to 36 hours at 30C using the improved plating methodO Slow acid producing colonies (s) were apparent in different proportions in many strains~
RJP/paa 1/18/83 1255E 24767 When fast and slow acid producing strains were isolated, cultured separately in NFM for 16 hr, and again plated separa~el~ on FSDA using the anaerobic method of the invention, both types yielded all of the same single or homologous colonies produced by the method. Concentrated cultures of fast acid producing strains (f) produced from the selected strains usually were significantly more active in acid production than the parent cultures that consisted of a heterogeneous population of fast and slow strains, which was unexpected. The differentiation and selection of phage resistant strains by the method was also very unexpected.
The following Example 2 describes the differentiation and selection of phage resistant strains.
Example 2 Using strains Sc A2, Sc H2, S1 C2~ and Sc 104, active phage-insensitive strains were isolated on FSDA when incubated aerobically. These strains were isolated by directly plating the host with an excess of phage by spreading on FSDA
as in Example 1~ E`ollowing 2 to 4 days incubation at 30C, fast and slow acid producing colonies were apparent. Ten fast acid producing colony strains were picked from each plate and subcultured in NFM as a second fluid growth medium to about 1 x cells per ml at 21C and at 30C, with and without added phage, and the phage insensitivity ~as confirmed. Preliminary characterization of the fast acid producing strains lndicated that they adsorbed phage but without subsequent DNA
penetration. Similar variants that adsorb phage withou~
subsequent plaque formation were produced as had been reported by Limsowtin et al., N~Z. Jr. Dairy Sci. Technol., 13:1~ 1978.
-RJP/paa 1/18/83 1255~ 24767 The first three strains grew aerobically on GL~A; however thiswas not true of Sc 104 which had to be grown anaerobically.
This demonstrates the reported difficulty that some investigators have had in selecting phage-insensitive mutants that have adequate acid production as described by Limsowtin et al., N.Z. J. Dairy Sci. Technol., 13-1297 1978.
As phag~s appeared for fast acid producing strains, isolates of phage-insensitive strains were obtained and used in place of the original phage-insensitive parent strains in Cheddar cheese making. Whey was used as the phage source.
Strain Sl ML8 produced by the method was a classic phage-resistant mutant that did not absorb phages, as determined by conventional phage absorption experiments.
The finding of improved colony growth and differentia ion using the method of Example 2 was the same for other phage-host combinakions. Another alternative is a modification of that reported by Xull, Aust. J~ Dairy TechnolO
32, 65 ~1977) called "Whey Adaptation". This involved culturing the host strain with a whey sample containing phages and then streaking this infected culture and selecting resistant colonies on FSDA. The method of selection and diferentiation appeared to be universal for acid producing bacteria, Example 3 shows the growth of the bacteria under aerobic conditions~
~ ple 3 Example 1 was repeated under aerobic conditions using the FSDA medium. Recognition of typical fast acid producing colonies was elther poor or inapparent for 12 (43%~ out of 28 RJP/paa 1/18/83 1255E 24767 single strains incubated aerobically. In general, colony growth was markedly retarded under aerobic incubation for all strains. Consequently, differentiation of fast and slow acid producing colonies was either unr~liable or inapparen~ as all colonies were small (0.5 mm in diameter) and appeared as slow acid producers.
This explains why the method in the original report by Limsowtin and Terzaghi (1976) was unsuccessful in the differentiation of fast and slow colonies. They reported that some fast acid-producing strains gave rise to only slow appearing colonies on glycerophosphate milk agar incubated aerobically. They did not investiga~e the use of anaerobic incubation to overcome this problem. The problem was that milk-based media such as glycerophosphate milk agar and FSDA
would not effectively differentiate the fast and slow colonies because of growth inhibition under aerobic conditions~
In the following Example 4, the b~ffering agent was an insoluble alkali metal compound, trimagnesium phosphate7 the indicator was bromcresol purple~ and anaerobic growth conditions were used. Tests were also conducted using FSDA
medium for comparison.
_x~
Trimagnesium phosphate was substitu~ed for disodium glycerophosphate in a plating medium Eor differentiation of "fast" and "slowi' acid-producing colonies oE lactic streptococci developed by Limsowtin and Terzaghi and later improved by Huggins and Sandine, "Selection and Characterization of Phage-Insensitive Lactic Streptococci," JO
Dairy Sci. 62:70 (1979). This medium, entitled FSDA-I L, was 3~ , RJP/paa l/18/83 1255E 24767 prepared in three separate components. Component A was made by adding 20.0 ml of a 0.25% (w/v) bromcresol purple solution and 10.0 g Davis agar to 400 ml of distilled water. Three drops of an anti-foaming agent, Pourite (Scientific Products) were also added to this component to reduce entrapped air bubbles in the medium when pouring plakes. Incorporation of anti Eoaming agent obviated the need to flame plates during pouring.
Component B was prepared by dissolving lO0 g NDM in 500 ml of distilled waterO Component C was made by adding 5.0 g trimagnesium phosphate (Stauffer Chemical Co.) to lO0 ml of distilled water~ Component C was generally prepared in multiple units and stored at room temperature. Components A
and C were autolaved for 15 min~ a~ 121C. Component ~ was autoclaved for only lO min. at 121C7 The three components were tempered to approximately 55C in a water bath at which time components B and C were aseptically added to A. The complete medium was then mixed on a magnetic stirrer and ~urther cooled to 45C in a water bath. Plates were poured and allowed to air dry for 48 h at xoom temperature. Occasional swirling of the flask was necessary to maintain Mg3(po~)2 in suspension while pouring plates~ Streaked or spread plates of FSDA-II were incubated at 30C for 48 h under arlaerobic conditions. After incubation, there was excellent visual contrast of yellow colonies on a blue background.
A comparison between E~SDA-II and FSDA was made by spread plating S. lactis C2 (lac~, prt+) and the three plasmid-charac~erized slow variants of S. lactis C2, LM0210 (lac ~ prt ), LM0220 (lac t prt ) and LM0231 (lac , prt ) (provided by L, L~ McKay) on the two media. The C2 parent and the three variants were grown in litmus milk (1L%
o~
RJP/paa 1/18/83 1255E 24767 w/w3 at 21C for 24 h~ The following mixtures of freshly grown cultures were made: 0.1 ml (lac+, prt~) + 0.25 ml (lac~, prt ) -~ 0.5 ml (lac , prt+) + 0O5 ml (lac , prt );
0.25 ml (lac+, prt+) + 0O5 ml (lac+, prt ); 0.~5 ml (lac+, prt ) + 0.5 ml (lac , prt )O Each of the three mixtures was serially diluted and spread-plated on FSDA and FSDA-II.
Streptococcus thermophilus strains 404, 410, 440, 19987, L12l S122 and C3; ~ strains 404 and 448; and Lactobacillus helveticus strain6 L112, 15807 and 450 were all streaked from freshly coagulated litmus milk cultures onto FSDA and FSDA-II. The pla~es were incuba-ted at 37C for 30 h under anaerobic conditions. Qualitative comparisons of growth by these thermophilic strains were made on FSDA and FSDA- I I .
Halos resulting from solubilized Mg3(po4)2 were not detectable on FSDA-II because of the opaqueness contributed by the NDM solidsu However, as with FSDA, differentiation between fast and slow acid-producers was possible on FSDA-IIo Differentiation between lac , prt+ and lac , prt was not possible on FSDA-II as with FSDA. Three distinct colony types were evident on FSDA-II. The S. lactls C2 parent appeared as a full-colored yellow colony against an opaque baby-blue background. The lac~ prt colonies were less brightly colored and resembled a yellow doughnut~ Colonies of +
lac / prt and lac , prt were indistinguishable from each other but clearly differentiated from the other two colony types. They appeared as colorless, translucent colonies; best visualized by observing their projection above the surface oE
the plating medium.
RJP/paa 1/18/83 1255E 24767 In a comparative platiny of lac , prt and lac , prt on both FSDA and F5DA-II media, ~isual observation of the FSDA plate revealed only a single colony type. A precise measurement of colony size enabled differentiation between the two colony types (lac~, prt being slightly larger than lac , prt ). Howe~ler, with FSDA-II it was visually apparent that two distinct colony types were present.
A comparative plating of all four phenotypes mixed toyether was conducted. On FSDA, two distinctly different colony types were evident, and again precise measurement would have allowed differentiation of an intermediate-sized colony representing the lac , prt variant. On FSDA-II, three distinctly different colony types were clearly di~tinguishable without any measurementO The lac , prt+ was a bright, fully-colored yellow colony with a darkened halo; the lac~
prt was not as brightly colored and was without a halo. The lac , prt+ and lac , prt appeared as colorless colonies.
When lac~, prt and lac+, prt were plated on both media, two colony types were discernible on both FSDA and FSDA-II.
Fast-slow differentiation on F5DA was dependent on size differences. Crowded plates or dehydrated plates (surface concentration of agar) make SiZQ differentia~ion somewhat difficult. Injured cells also appear initially as slow colonies on FSDA but with additional incubation time they slo~ly transform into typical fast colony typesO
Differentiation on F5DA-II is based primarily on color differences and not size. This enables ~Jisual differentiation without the need for colony size measurement~ Be~ause of the RJP/paa 1/18/~3 1255E 24767 lower phosphate concentration in FSDA-II (0.5% w/v) compared to nearly 2~ (w/v) in FSDA, injured cells are more likely to reco~rer and develop their true colony types on FSDA-II within the 48 h incubation period.
All the S. thermophilus st~ains examined grew equally well on ~SDA or FSDA-II4 However, ~ aricus 404 and 448 and L. helveticus 450 did not grow nearly as well on FSDA as on FSDA-II and MRS agar (control mediumr Difco). And L~
helveticus L112 and 15807, while growing poorly on ~SDA-II, were completely inhi~ited on FSDA.
There are numerous applications for the improved method and plating device~ The most important is the direct selection of phage-insensitive fast acid producing strains.
~he improved method and plating device can also be used as a tool to directly study various chemicals or conditions influencing the appearance of variants in bacterial strains.
Commercially the method is advantageous in the isolation, selection, and screening of fast lactic acid producing starter strains, particularly Cottage and Cheddar cheese and buttermilk producing strainsO Thus a Cheddar cheese plant in Olympia/
Washington, has been on the same culture developed for them for about 2 years, producing about 20,000 pounds of cheese a day or over 5 mil]ion pounds. A plant in Tillamook, Oregon, now has over 1200 consecutive 38,000 pound vats (over 4 million pounds of cheese) using a culture developed for their use. The FSDA
agar is used to isolate phage-resistant fast acid-producing mutants whenever viruses appear for any of the 6 strains in the multiple strain starter cultures~ We are convinced that all cheese plants can operate this well when this method is used to keep the cultures active in plants~ The cheese is of excellent RJP/paa 1/18/83 1255E 24767 quality and these cultures eliminate public health problems because vf slow vatsO The FSDA agar facilitates selective manipulation oE starter strains which has not been possible previously.
Tests indicate that the addition of optimum amounts (1000 units per petri plate and 0.5~ by weight) of catalase and pyruvate, respectively; are substitutes for the anaerobic incubation and addition of ferrous sulfate appears to be effective. These additives degrade the hydrogen peroxide that colonies of laçtic streptococci produce when grown aerobically on milk-based media. This minimizes auto-inhibition of cell growth due to hydrogen peroxide accumulation. ~owever, these additives are not ~uite as good as anaerobic incubation. Other additives which simulate anaerobic conditions by stimulating the growth of the bacteria under aerobic or anaerobic conditions can be used.
RJP/paa 1/1~/83 1255E 24767 .
Stainer, R. Y. "The Microbiol World, p. 37-40, 68 (1976); and ~ erzaghi, B. E., and W. E. Sandine, "Improved Medium for Lactic Streptococci and their Bacteriophages," Ap~l.
Microbiol. 29:807-813 (1975).
Hunter demonstrated that with addition of sodium phosphate (3307mM) to a simple plating medium, more growth and larger colonies resulted. Buffering provided by the phosphate allowed more cell divisions before the hydrogen ion concentration became inhibitory to further growth.
Improved growth of lactic streptococci results when disodium-B-glycerophosphate (62mM) is incorporated into a complex medium by Terzaghi and Sandine. This medium is also lactose-limiting which enables the final pH to remain well above pH 500 and avoid any acid injury to the cells~ Douglas claimed one of the advantages of glycerophosphates over inorganic phosphates was that microbiological media could be formulated with high phosphate concentrations without precipitating out many essential metals. This feature i5 especi~lly useful in pla~uing lactic bacteriophages where Ca~2 is required for optimum formation of plaques (articles by Reiter; Shew).
Sodium-B-glycerophosphate is also utilized in two different plating media designed to diEferentiate "fast" and "slow" colonies of lactic streptococci (articles by ~uggins and Sandine; Limsowtin). These buffered media enable fast acid producing colonies to grow to a larger si~e so that they can be distinguished from smaller slow coloniesO
Shankar and Davies found B-glycerophosphate inhibited some strains of L~ bul~aricus and more recently there have been 3~
RJP/paa 1/18/83 1255E 24767 reports of inhibition of a few strains of lactic streptococci (articles by Keogh; Mullan).
Barach improved the enumeration of lactic streptococci by as much as 7. 75 times by incorporating 3UmM diammonium phosphate into unbuffered Elliker agar.
Insoluble CaC03 has been added to agar media to help preserve neutral conditions for isolation and cultivation of acid-producing bacteria. Production of acid is localized around colonies and can be detected by clear halos resulting from acid-solubilization of CaC03 immediately surrounding the colonies as described by Stanier. Such a buffering system is used in a plating medium for differential enumeration of lactic strep~ococci by Reddy, et al.~ and in another for differentiating colonies of S _ hermophilus from I. bulgaricus by Leer et al.
OBJECTS
_, ~ . .
It is therefore an object of the present in~Jention to provide a method wherein heterogeneous populations of fast and slow acid producing strains of bacteria are readily and reproducibly differentiated or separated as to acid producing ability~ Further it i5 an object of the present invention to provide a method wherein phage insensitive strains can be reliably selected. Further still it is an object of the present invention to provide the selected strains as c~ltures or concentrates of phage insensiti~Je strains of bacteria as a single strain or as a mixture of several such single strains.
These and other objects will become increasingly apparent by reference to the following description and to the drawingO
-- 6 ~
In _e Drawing The drawing is a photograph showing clearly differen-tiated fast (f) and slow (s) acid producing single strain bacterial colonies produced by the method and plating device of the present invention using an indicator and anaerobic fermen-tation conditions.
General Descri.ption The present invention relates to an improved method for the differentiation of heterogeneous populations of fast acid producing strains of a species of bacteria from slow acid producing strains of the same bacteria which comprises providing a gelled, solid bacterial growth medium containing milk protein, a milk protein derivative or a milk protein substitute and containing a p~l sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7 and growing the heterogeneous populations of a strain or strains of acid producing bacteria on ~he medium to produce single strain acid producing bacterial colonies, wherein the colonies are of varying si~es and have a contrasting color from the growth medium around and within the colonies because of the reaction of the acid in the colonies with the indicator A bacterial plating device is used for differen-tiating and selecting fast acid producing strains of a species of bacteria in a heterogeneous population with slow acid produ~ing strains or variants of the same bac-teria. A closed container is used containing a gelled, solid growth medium for the bacteria in admixture with milk protein, a milk protein derivative or a milk protein substitute and a pH sensitive indicator which changes color upon contact with acid in the pH
range between about 4 and 7. Preferably anaerobic or near anaerobic conditions are provided in the space.
The present invention also relates to an improved method for the differentiation of heterogeneous popu-lations of fast acid producing strains of a species of bacteria from slow acid producing strains by visual observation of color differences amony colonies grown on a gelled, solid bac-terial growth medium by providing a medium that includes milk protein, a milk protein derivative or a milk protein substitute, a pH
sensitive indicator which changes color upon contact with acid in the p~ range between about 4 and 7 even in the absence of milk proteolysis, and a substantial non~oxic, insoluble buffering agent.
The method of the present inventi.on also relates to the production of a concentrate o:E a single strain or a mixture of single strains of a species of bacteria of the gen-era Streptococcus or Lactobacillus of the type used :Eor produc-... . . . ~
ing lactic acid in foods by -fermentation. The selected bacter ium is characterized by being a fast acid producer and by hav-ing been grown anaerobically on a gelled solid growth medium, wherein the medium includes a pH sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7, a bufferiny agent and a milk protein, a milk protein derivative or a rnilk protein substitute, and by being able to grow in the presence of a phage which kills or impairs the growth of parent strains of the same species to produce the single strains which are then selected and regrown in a second RJP/paa 1/18/83 1255E 24767 fluid growth medium to produce the concentrates which contains at least about 1 x 106 cells per ml.
The present invention also relates to the method for selecting phage resistant strains of acid producing bacteria and concentrating the cells for use in producing fermented food products which comprises: growing heterogeneous or homogeneous populations of a strain or strains of the acid producing bacteria anaerobically or nearly anaerobically in the presence of phage on a solid growth medium so as to produce colonies wherein fast acid producing strains produce relatively larger colonies than slow acid producing strains; selecting a large colony; and growing the cells in a fluid growth medium to at least about 106 cells per ml to provide a concentrate of cells.
The present invention particularly relates to the method for providing phage resistant strains of acid producing bacteria to producers of fermented food produc~s which comprises growing heterogeneous or homogeneous populations of a strain or strains of an acid producing bacteria anaerobically or nearly anaerobically in the presence of phage from a sample of the food product or a by-product of the food product obtained from a producer of the fermented food product on a growth medium which differentiates slow acid producing strains from fast acid producing strains of the bacteria by producing relatively larger colonies of cells of fast acid producing strains in the presence of phage; selecting cells in a large colony having a diameter of at least about 1 mm and growing the cells in the larger colony to at least about 106 cells per ml to provide a culture of ce:Lls; and providing the producer which supplied the sample with the culture for use in prsducing fermented food productsO
The method for differentiation and selection produces a eoneentrate of improved homologous cells of a single strain which is from a clone of a single member of the species and which has the most desirable acid producing properties as well as preferably phage insensitivity. The term "strain" or "variant" as used herein means a member of a single species of bacteria which has a common source or parent with other members anc~ whieh generally has almost all of the same fermen-tation characteristics with other members, but which can have a weak-ness in the ability to produce acid or in the case of mi]k fermentations have a poor proteolytic ability or have phage sensitivity. A single species of bacteria may have many strains of the same bacterium which differ by one or more fer-mentation characteristics and thus form a "heterogeneous popu-lation'l. The general fermentation eharacteristics are deter-mined in relation to sugars and other assimilable carbon sources for the species as listed in Bergey's Manual, Eighth Edition (1974). There are commercially available devices for determining -fermentation characteristies on sugars and other substrates using microassay techniques. The API series Erom Analytab Products, Inc., in Carle Place, N.Y., is particularly suitable~
The ~erms "slow acid producing" and "fast acid pro-ducing" are used in the milk fermentation industry in relation to lactie acid produeing bacteria. Slow lactic acid producing strains are those whieh fail to eoagulate milk in 18 hours at 21C using a 1~ by volume inoculum (Citti, J.E., et al~
"Cornparison of slow and fast acid producing streptococcus lac-tis," J. Dairy Sci~ 48.14-18 (1965~). Fast lactic acid produc-ing strains develop 'che relatively larger colonies.
The baeteria are grown so that there are between about 30 and 300 colonies per petri plate or between about 0~5 and 5 3~
RJP/paa l/:L8/83 1255E 24767 per square centimeter of gelled growth medium. PreEerably there are less than 100 colonies per plate (about 1.6 per square cm of medium). The reason for these preferred colony densities is that there can be too few or too many colonies on the plate outside of the broad ranges for reliable results.
The fast acid producing colonies are at least about 1 mm in diameter.
Many acid indicators can be used in the present invention. One such indicator is litmus which changes from blue to red upon contact with acid. Litmus makes proteolysis of the milk protein in the medium by the bacteria readily visible in a ring (p) around the fringes of the fast acid producing colonies (f) as shown in Figure 1. Milk proteolysis is necessary in most fermentations. Other indicators can be used, such as bromcresol purple (pH range 5.2 to 6.8) and bromthymol blue (pH range 6~0 to 7.6) which do not rely on milk proteolysis.
Anaerobic growth conditions provide unexpectedly superior results in terms of the diferentiation of fast and slow acid producing colonies. The anaerobic conditions can be provided in the confined space by a vacuum or by providing a reducing or rare gas in the confined space. The preferred gases are nitrogen, hydrogen or other nontoxic gases alone or mixed with carbon dioxide which is assimilated by the lactic acid producing bacteria. Preferably between about 5~ and 50 by volume carbon dioxide is used with the balance being hydrogen or nitrogen. Other useful non-oxidizing ~ases include the rare gases, such as neon and krypton and particularly argon.
An important improvement of the present invention is the use of the acid indicator combined with anaerobic growth RJP/paa 1/18/83 1255E 24767 conditions which provides a synergistic resul~ ih the differentia~ion and selection and in the homogeneity of ~he single strain of cells produced. Unexpectedly it has been found that significantly i.mproved differentiation and selection of fast acid producing strains can be achieved with this combinakion.
The growth medium includes milk protein, a milk protein derivative, preferably nonfa~ milk or a casein digest, or a milk protein substitute in an amount between about 5 and 15 percent by weight of the gelled solid medium (W/V).
Another preferred ingredient is a buffering agent, particularly a compound that is substantially nontoxic to the bacteria in the medium and that is an alkali metal carbonate, phosphate, hydroxidel oxide/ or an organic sulfonate D The organic sulfonates include alkali metal salts of piperazine-N~
N'-bis~2-ethane-sulfonic acid (PIPES), morpholinopropane sulfonic acid (~IOPS) and 2(n-morpholino)ethane sulEonic acid (MES) as described in the abstract of the American Dairy Society Association Meetings June 24 to 27 (1979). Disodium glycerophosphate is a suitable buffering agent when present in an amount between about 0.5 and 5 percent by weight based upon the volume of the gelled solid growth medium (W/V). But, substantially nontoxic! insoluble buffering agen~s enable even better visual differentiation, by color, of "fast" and "slow"
colonies, when the buffering agent is present :in an amoun~ of between 0.1 and 1 percent, preferably about 0~25 percent.
Specific insoluble buffering agents are phosphatesJ including ammonium phosphates, carbonates and hydroxides of magnesium and carbonates and hydroxides of calcium and zinc oxide.
Outstanding results are obtained using phosphates oE magnesium, particularly trimagnesium phosphate.
The agars are preferably Davist.m. agar which is produced by the Davis Gelatine Company, Christ Church, New Zealand or Bacto-Agart m from Difco, Detroit, Michigan.
Agar substitutes and other gelled solids such as gelatin are also available. Preferably the plating device has a trans-parent window on the confined space so that the growth of the bacteria can be observed. A conventional petri dish containing the gelled solid growth medium sealed with a confined space around the medium is generally used.
After grow~h of the bacteria 7 the relatively large colony is picked from the gelled solid medium and transferred to a second fluid growth medium including assimilable carbohy-drate and nitrogen sources and is grown to at least abou~
1 x 106 cells per ml and preferably to 108 to 109 cells per ml. In this manner, large numbers of bacterial cells can be produced as a concentrate wherein virtually every individual cell has the same fast acid producing capability. This can be demonstrated by replating the cells on the gelled solid growth medium.
The cells can be held as a concentrated, refrigerated milk culture where milk is the fluid growth medium containing about 1 x 106 to 1 x 109 cells per ml or can be concen-trated further to above about 1 x 109 to 1 x 1012 cells per ml by removing some of the growth medium~ The single strains can be mixed with other single strains produced by -the rnethod. The thus concentrated bacteria can be frozen for storage and/or shipment preferably with a freezing stabilizing agent such as glycerol in an amount up to 20 percent by volume or they can be lyophilized. The bacteria can also be stabilized as described in U.S~ Paten-t No. 4~282/255.
RJP/paa 1/18/~3 1255E 24767 The bacteria which can be differentiated are preferably species selected from the genera Streptococcus and Lactobacillus and are used for lactic acid production in food products by fermentation. Included are ~lS, Stre~tococcus lactis, ~ cus lactis subspecies -diacetylactis, Streptococcus_~ ermophilus~ Lactobacillus bul~aricus, Lactobacillus acidophilus~ Lactobacillus caseii, Lactobacillus lactis, and Lactobacillus helveticusO These are . _ _ bacteria which are sensitive to phages.
The homologous or heterogeneous phages for the particular strain of bacteria are preferably present when they are grown by the plating method and thus the bacteria produced by the method are thus also phage insensitive. The method can also be repeated using different races of phages when the host bacterium is sensitive to more than one phage. In these ways, cells resistant to more than one phage can be generated.
Phayes occur in whey from cheese making. Whey can be used to continuously produce phage insensitive strains by repeated periodic exposure to these phages. Other sources of phage from the producers oE fermented products can also be used. This method prevents failures resulting in the loss of hundreds or thousands of gallons of milk in making cultured dairy products where the bacteria are phage sensitive.
SPECIFIC DEscRIprrIoN
In the following Examples 1-3, the bufering agent was disodium glycerophosphate; the indicator was litmus; and anaerobic growth conditions were used~
L ~
RJP/paa 1/18/83 1255E 24767 Example 1 The composition of the growth med,ium, referred to as fast-slow differential agar (FSDA), was as shown in Table 1.
Table 1 Percent Grams/liter ~Y_~ gh~
Nonfat milk powder (NFM) 100.0 g 76.9%
Davist m agar (New Zealand) 10.~ g 7.7%
Disodium glyce.rophosphate 19.0 g 14.6%
Bactolitmust m (litmus) 1.0 g 0.8%
100 . 0%
The medium was prepared by dispersing lOg of Davis agar in 550 ml of double distilled wa~er in a 2 liter flask by steaming for 30 minutes. To the melted agar, 1.0 g of Bactolitmust m and 19 g of disodium glycerophosphate were added and mixed until dissolved. In a separate 1 liter flask, 100 g of the NF~ powder was dissolved in 450 ml of double distilled water. The two mixtures were then autoclaved separately for 17 minutes and rapidly cooled to 55C in a water bath, combined, and poured into petri plates and flamed to eliminate bubbles as needed. The p~ates were dried by inverting overnight at room temperature~
Single colony strains were obtained by spreading or streaking a mixture of a heterogeneous population of a specific species o~' bact~ria on the agar and growing the mixtureO An atmosphere of hydrogen or hydrogen and carbon dioxide was generated using Gas Pakt m gas generating envelopes (Bio-Quest, Cockeysville, MD) in the confined space over the growth medium in petri plates which were seal.ed in an air RJP/paa 1/18/83 1255E 24767 evacuated Gas Pakt m jar. Typical colonies produced within 24 to 36 hours at 30C were as shown in Figure 1, wherein the large fast acid producing colonies (f) were readily distinguishable from the smaller slow acid producing colonies (s). The large colonies (f) measured 1 to 3 mm in diameter and the small colonies (s) between 0.2 to ~5 mm in diameter or less. The large colonies were selected and had a clear proteolysis ring (p).
It is speculated that the problems encountered by the prior art with aerobic growth of the bacteria on GMA were due to the higher oxygen tension relative to liquid NFM. The effect of anaerobic incubation on colony appearance was remarkable. Not only was excellent colony differentiation achieved, but the incubation period required at 30C was about one-half of the time re~uired when incubated under aerobic conditions in air.
Subsequently, 20 differen~ active strains were differentiated and fast acid producing strains selected in the manner of Example 1 including: Sc A2, Sl C2, Sc H2, Sc HP, Sc 205, Sl c10, Sl ML8, Sc 104, Sc 286, Sc 287, Sc 288/
Sc 289A, Sc 289C, Sc 290, Sc 290A, Sc 291, Sc 292~ Sc 134~ Sc 108 and Sl E where "Sc" represents Streptococcus cremoris and "S1" represents Strep~ococcus lactis. These strains were from the culture collection of Oregon State University, Corvallis, Oregon and samples are freely available to the public without charge~
All of these species produced fast acid strain colonies (f) within 24 to 36 hours at 30C using the improved plating methodO Slow acid producing colonies (s) were apparent in different proportions in many strains~
RJP/paa 1/18/83 1255E 24767 When fast and slow acid producing strains were isolated, cultured separately in NFM for 16 hr, and again plated separa~el~ on FSDA using the anaerobic method of the invention, both types yielded all of the same single or homologous colonies produced by the method. Concentrated cultures of fast acid producing strains (f) produced from the selected strains usually were significantly more active in acid production than the parent cultures that consisted of a heterogeneous population of fast and slow strains, which was unexpected. The differentiation and selection of phage resistant strains by the method was also very unexpected.
The following Example 2 describes the differentiation and selection of phage resistant strains.
Example 2 Using strains Sc A2, Sc H2, S1 C2~ and Sc 104, active phage-insensitive strains were isolated on FSDA when incubated aerobically. These strains were isolated by directly plating the host with an excess of phage by spreading on FSDA
as in Example 1~ E`ollowing 2 to 4 days incubation at 30C, fast and slow acid producing colonies were apparent. Ten fast acid producing colony strains were picked from each plate and subcultured in NFM as a second fluid growth medium to about 1 x cells per ml at 21C and at 30C, with and without added phage, and the phage insensitivity ~as confirmed. Preliminary characterization of the fast acid producing strains lndicated that they adsorbed phage but without subsequent DNA
penetration. Similar variants that adsorb phage withou~
subsequent plaque formation were produced as had been reported by Limsowtin et al., N~Z. Jr. Dairy Sci. Technol., 13:1~ 1978.
-RJP/paa 1/18/83 1255~ 24767 The first three strains grew aerobically on GL~A; however thiswas not true of Sc 104 which had to be grown anaerobically.
This demonstrates the reported difficulty that some investigators have had in selecting phage-insensitive mutants that have adequate acid production as described by Limsowtin et al., N.Z. J. Dairy Sci. Technol., 13-1297 1978.
As phag~s appeared for fast acid producing strains, isolates of phage-insensitive strains were obtained and used in place of the original phage-insensitive parent strains in Cheddar cheese making. Whey was used as the phage source.
Strain Sl ML8 produced by the method was a classic phage-resistant mutant that did not absorb phages, as determined by conventional phage absorption experiments.
The finding of improved colony growth and differentia ion using the method of Example 2 was the same for other phage-host combinakions. Another alternative is a modification of that reported by Xull, Aust. J~ Dairy TechnolO
32, 65 ~1977) called "Whey Adaptation". This involved culturing the host strain with a whey sample containing phages and then streaking this infected culture and selecting resistant colonies on FSDA. The method of selection and diferentiation appeared to be universal for acid producing bacteria, Example 3 shows the growth of the bacteria under aerobic conditions~
~ ple 3 Example 1 was repeated under aerobic conditions using the FSDA medium. Recognition of typical fast acid producing colonies was elther poor or inapparent for 12 (43%~ out of 28 RJP/paa 1/18/83 1255E 24767 single strains incubated aerobically. In general, colony growth was markedly retarded under aerobic incubation for all strains. Consequently, differentiation of fast and slow acid producing colonies was either unr~liable or inapparen~ as all colonies were small (0.5 mm in diameter) and appeared as slow acid producers.
This explains why the method in the original report by Limsowtin and Terzaghi (1976) was unsuccessful in the differentiation of fast and slow colonies. They reported that some fast acid-producing strains gave rise to only slow appearing colonies on glycerophosphate milk agar incubated aerobically. They did not investiga~e the use of anaerobic incubation to overcome this problem. The problem was that milk-based media such as glycerophosphate milk agar and FSDA
would not effectively differentiate the fast and slow colonies because of growth inhibition under aerobic conditions~
In the following Example 4, the b~ffering agent was an insoluble alkali metal compound, trimagnesium phosphate7 the indicator was bromcresol purple~ and anaerobic growth conditions were used. Tests were also conducted using FSDA
medium for comparison.
_x~
Trimagnesium phosphate was substitu~ed for disodium glycerophosphate in a plating medium Eor differentiation of "fast" and "slowi' acid-producing colonies oE lactic streptococci developed by Limsowtin and Terzaghi and later improved by Huggins and Sandine, "Selection and Characterization of Phage-Insensitive Lactic Streptococci," JO
Dairy Sci. 62:70 (1979). This medium, entitled FSDA-I L, was 3~ , RJP/paa l/18/83 1255E 24767 prepared in three separate components. Component A was made by adding 20.0 ml of a 0.25% (w/v) bromcresol purple solution and 10.0 g Davis agar to 400 ml of distilled water. Three drops of an anti-foaming agent, Pourite (Scientific Products) were also added to this component to reduce entrapped air bubbles in the medium when pouring plakes. Incorporation of anti Eoaming agent obviated the need to flame plates during pouring.
Component B was prepared by dissolving lO0 g NDM in 500 ml of distilled waterO Component C was made by adding 5.0 g trimagnesium phosphate (Stauffer Chemical Co.) to lO0 ml of distilled water~ Component C was generally prepared in multiple units and stored at room temperature. Components A
and C were autolaved for 15 min~ a~ 121C. Component ~ was autoclaved for only lO min. at 121C7 The three components were tempered to approximately 55C in a water bath at which time components B and C were aseptically added to A. The complete medium was then mixed on a magnetic stirrer and ~urther cooled to 45C in a water bath. Plates were poured and allowed to air dry for 48 h at xoom temperature. Occasional swirling of the flask was necessary to maintain Mg3(po~)2 in suspension while pouring plates~ Streaked or spread plates of FSDA-II were incubated at 30C for 48 h under arlaerobic conditions. After incubation, there was excellent visual contrast of yellow colonies on a blue background.
A comparison between E~SDA-II and FSDA was made by spread plating S. lactis C2 (lac~, prt+) and the three plasmid-charac~erized slow variants of S. lactis C2, LM0210 (lac ~ prt ), LM0220 (lac t prt ) and LM0231 (lac , prt ) (provided by L, L~ McKay) on the two media. The C2 parent and the three variants were grown in litmus milk (1L%
o~
RJP/paa 1/18/83 1255E 24767 w/w3 at 21C for 24 h~ The following mixtures of freshly grown cultures were made: 0.1 ml (lac+, prt~) + 0.25 ml (lac~, prt ) -~ 0.5 ml (lac , prt+) + 0O5 ml (lac , prt );
0.25 ml (lac+, prt+) + 0O5 ml (lac+, prt ); 0.~5 ml (lac+, prt ) + 0.5 ml (lac , prt )O Each of the three mixtures was serially diluted and spread-plated on FSDA and FSDA-II.
Streptococcus thermophilus strains 404, 410, 440, 19987, L12l S122 and C3; ~ strains 404 and 448; and Lactobacillus helveticus strain6 L112, 15807 and 450 were all streaked from freshly coagulated litmus milk cultures onto FSDA and FSDA-II. The pla~es were incuba-ted at 37C for 30 h under anaerobic conditions. Qualitative comparisons of growth by these thermophilic strains were made on FSDA and FSDA- I I .
Halos resulting from solubilized Mg3(po4)2 were not detectable on FSDA-II because of the opaqueness contributed by the NDM solidsu However, as with FSDA, differentiation between fast and slow acid-producers was possible on FSDA-IIo Differentiation between lac , prt+ and lac , prt was not possible on FSDA-II as with FSDA. Three distinct colony types were evident on FSDA-II. The S. lactls C2 parent appeared as a full-colored yellow colony against an opaque baby-blue background. The lac~ prt colonies were less brightly colored and resembled a yellow doughnut~ Colonies of +
lac / prt and lac , prt were indistinguishable from each other but clearly differentiated from the other two colony types. They appeared as colorless, translucent colonies; best visualized by observing their projection above the surface oE
the plating medium.
RJP/paa 1/18/83 1255E 24767 In a comparative platiny of lac , prt and lac , prt on both FSDA and F5DA-II media, ~isual observation of the FSDA plate revealed only a single colony type. A precise measurement of colony size enabled differentiation between the two colony types (lac~, prt being slightly larger than lac , prt ). Howe~ler, with FSDA-II it was visually apparent that two distinct colony types were present.
A comparative plating of all four phenotypes mixed toyether was conducted. On FSDA, two distinctly different colony types were evident, and again precise measurement would have allowed differentiation of an intermediate-sized colony representing the lac , prt variant. On FSDA-II, three distinctly different colony types were clearly di~tinguishable without any measurementO The lac , prt+ was a bright, fully-colored yellow colony with a darkened halo; the lac~
prt was not as brightly colored and was without a halo. The lac , prt+ and lac , prt appeared as colorless colonies.
When lac~, prt and lac+, prt were plated on both media, two colony types were discernible on both FSDA and FSDA-II.
Fast-slow differentiation on F5DA was dependent on size differences. Crowded plates or dehydrated plates (surface concentration of agar) make SiZQ differentia~ion somewhat difficult. Injured cells also appear initially as slow colonies on FSDA but with additional incubation time they slo~ly transform into typical fast colony typesO
Differentiation on F5DA-II is based primarily on color differences and not size. This enables ~Jisual differentiation without the need for colony size measurement~ Be~ause of the RJP/paa 1/18/~3 1255E 24767 lower phosphate concentration in FSDA-II (0.5% w/v) compared to nearly 2~ (w/v) in FSDA, injured cells are more likely to reco~rer and develop their true colony types on FSDA-II within the 48 h incubation period.
All the S. thermophilus st~ains examined grew equally well on ~SDA or FSDA-II4 However, ~ aricus 404 and 448 and L. helveticus 450 did not grow nearly as well on FSDA as on FSDA-II and MRS agar (control mediumr Difco). And L~
helveticus L112 and 15807, while growing poorly on ~SDA-II, were completely inhi~ited on FSDA.
There are numerous applications for the improved method and plating device~ The most important is the direct selection of phage-insensitive fast acid producing strains.
~he improved method and plating device can also be used as a tool to directly study various chemicals or conditions influencing the appearance of variants in bacterial strains.
Commercially the method is advantageous in the isolation, selection, and screening of fast lactic acid producing starter strains, particularly Cottage and Cheddar cheese and buttermilk producing strainsO Thus a Cheddar cheese plant in Olympia/
Washington, has been on the same culture developed for them for about 2 years, producing about 20,000 pounds of cheese a day or over 5 mil]ion pounds. A plant in Tillamook, Oregon, now has over 1200 consecutive 38,000 pound vats (over 4 million pounds of cheese) using a culture developed for their use. The FSDA
agar is used to isolate phage-resistant fast acid-producing mutants whenever viruses appear for any of the 6 strains in the multiple strain starter cultures~ We are convinced that all cheese plants can operate this well when this method is used to keep the cultures active in plants~ The cheese is of excellent RJP/paa 1/18/83 1255E 24767 quality and these cultures eliminate public health problems because vf slow vatsO The FSDA agar facilitates selective manipulation oE starter strains which has not been possible previously.
Tests indicate that the addition of optimum amounts (1000 units per petri plate and 0.5~ by weight) of catalase and pyruvate, respectively; are substitutes for the anaerobic incubation and addition of ferrous sulfate appears to be effective. These additives degrade the hydrogen peroxide that colonies of laçtic streptococci produce when grown aerobically on milk-based media. This minimizes auto-inhibition of cell growth due to hydrogen peroxide accumulation. ~owever, these additives are not ~uite as good as anaerobic incubation. Other additives which simulate anaerobic conditions by stimulating the growth of the bacteria under aerobic or anaerobic conditions can be used.
Claims (22)
1. A method for obtaining, from a heterogeneous population of acid producing strains of a species of bac-teria, a fast acid producing strain that is suitable for use in the production of a fermented food product, the method comprising differentiating strains which are fast acid producing from strains of the same population that are slow acid producing by (a) providing a solid bacterial growth medium on which fast acid producing strains grow to relatively larger colonies of cells than slow acid produ-cing strains; (b) dispursing a heterogeneous population of strains of acid producing bacteria on the medium so that isolated single strain colonies of acid producing bacteria can grow from individual cells of the heterogeneous popula-tion; (c) growing the bacteria substantially anaerobically on the medium to produce single strain colonies, wherein the colonies are of varying sizes, relatively large colonies being formed by strains which are fast acid producing and relatively small colonies being formed by strains which are slow acid producing; and (d) collecting cells from a large colony to establish a homogeneous culture of a fast acid producing strain suitable for use in the production of a fermented food product.
2. A method for obtaining, from a heterogeneous population of acid producing strains of a species of bacteria, a fast acid producing strain that is suitable for use in the production of a fermented food product, the method comprising differentiating strains which are fast acid producing from strains of the same population that are slow acid producing by (a) providing a solid bacterial growth medium on which fast acid producing strains grow to relatively larger colonies of cells than slow acid produ-cing strains, the medium containing milk protein, a milk protein derivative or a milk protein substitute and con-taining a pH sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7;
(b) dispursing a heterogeneous population of strains of acid producing bacteria on the medium so that isolated single stain colonies of acid producing bacteria can grow from individual cells of the heterogeneous population; (c) growing the bacteria substantially anaerobically on the medium to produce single strain colonies, wherein the colonies have a contrasting color from the growth medium around and within the colonies because of the reaction of the acid in the colonies with the indicator and the colonies are of varying sizes, relatively large colonies being formed by strains which are fast acid producing and relatively small colonies being formed by strains which are slow acid producing; and (d) collecting cells from a large colony to establish a homogeneous culture of a fast acid producing strain suitable for use in the production of a fermented food product.
(b) dispursing a heterogeneous population of strains of acid producing bacteria on the medium so that isolated single stain colonies of acid producing bacteria can grow from individual cells of the heterogeneous population; (c) growing the bacteria substantially anaerobically on the medium to produce single strain colonies, wherein the colonies have a contrasting color from the growth medium around and within the colonies because of the reaction of the acid in the colonies with the indicator and the colonies are of varying sizes, relatively large colonies being formed by strains which are fast acid producing and relatively small colonies being formed by strains which are slow acid producing; and (d) collecting cells from a large colony to establish a homogeneous culture of a fast acid producing strain suitable for use in the production of a fermented food product.
3. The method of claim 2 wherein the bacteria are grown in the presence of a phage which can infect the population.
4. The method of claim 3 wherein the bacteria are lactic acid producing and wherein the phage are derived from whey samples from Cottage cheese or cheese making.
5. The method of claim 2 wherein the indicator is selected from the group consisting of litmus, brom-cresol purple, bromthymol blue, and mixtures thereof.
6. The method of claim 5 wherein anaerobic conditions are provided by hydrogen or nitrogen or by a mixture of hydrogen or nitrogen and carbon dioxide in a confined space.
7. The method of claim 2 wherein the growth medium contains a buffering agent to partially neutralize acid produced by the bacteria
8. The method of claim 7 wherein the buffering agent is disodium glycerophosphate.
9. The method of claim 7 wherein the buffering agent is a substantially nontoxic, insoluble alkali metal carbonate, phosphate, hydroxide, oxide, or zinc oxide
10. The method of claim 9 wherein the buffering agent is a phosphate, ammonium phosphate, carbonate, hydroxide or oxide of magnesium or a carbonate or hydroxide of calcium or zinc oxide.
11. The method of claim 7 wherein the buffering agent is trimagnesium phosphate.
12. The method of claim 2 wherein the bacteria are of the genera Streptococcus or Lactobacillus and are used for lactic acid production in milk by fermentation.
13. The method of claim 12 wherein the bacteria axe selected from Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus caseii, Lactobacillus lactis, or Lactobacillus helveticus.
14. A method for obtaining, from a heterogeneous population of acid producing strains of a species of bacteria, a fast acid producing strain that is suitable for use in the production of a fermented food product, the method comprising differentiating strains which are fast acid producing from strains of the same population that are slow acid producing by (a) providing a solid bacterial growth medium on which fast acid producing strains grow to relatively larger colonies of cells than slow acid producing strains, the medium containing (1) milk protein, a milk protein derivative or a milk protein substitute, (2) bromcresol purple, a pH sensitive indicator which changes color upon contact with acid, and (3) trimagnesium phosphate, a buffering agent; (b) dispursing a heteroge-neous population of strains of acid producing bacteria on the medium so that isolated single strain colonies of acid producing bacteria can grow from individual cells of the heterogeneous population; (c) growing the bacteria substantially anaerobically on the medium to produce single strain colonies, wherein the colonies have a contrasting color from the growth medium around and within the colonies because of the reaction of the acid in the colonies with the indicator and the colonies are of varying sizes, relatively large colonies being formed by strains which are fast acid producing and relatively small colonies being formed by strains which are slow acid producing; and (d) collecting cells from a large colony to establish a homogeneous culture of a fast acid producing strain suitable for use in the production of a fermented food product.
15. A solid bacterial growth medium for the differentiation of heterogeneous populations of fast acid producing strains of a species of bacteria from slow acid producing strains of the same bacteria, the medium con-taining: milk protein, a milk protein derivative, or a milk protein substitute; bromocresol purple; and trimag-nesium phosphate.
16. A method for obtaining, from a heterogeneous population of acid producing strains of a species of bacteria, a phage resistant, fast acid producing strain that is suitable for use in the production of a fermented food product, the method comprising differentiating strains which are fast acid producing in the presence of phage from strains of the same population that are slow acid producing in the presence of phage, the differentia-ting being accomplished by (a) providing a solid bacterial growth medium on which fast acid producing strains grow to relatively larger colonies of cells than slow acid produ-cing strains; (b) dispursing a heterogeneous population of strains of acid producing bacteria on the medium so that isolated single strain colonies of acid producing bacteria can grow from individual cells of the the heterogeneous popula-tion: (c) growing the bacteria substantially anaerobically on the medium in the presence of phage from a sample of a food product or a by-product of the food product to produce single strain colonies, wherein the colonies are of varying sizes, relatively large colonies being formed by strains which are fast acid producing and relatively small colonies being formed by strains which are slow acid producing; (d) collecting cells from a large colony having a diameter of at least about 1 mm; and (e) growing the cells from the large colony to at least about 106 cells per ml to establish a homogeneous culture of a fast acid producing strain suitable for use in the production of a fermented food product.
17. The method of claim 16 wherein the bacteria are lactic acid producing and wherein the food is a dairy product with whey as the by-product which is a source of the phage.
18. The method of claim 16 wherein the growth medium is a solid growth medium which contains a pH
sensitive indicator which changes color upon contact with acid in the pH range between about pH 4 and 7, thereby making the bacteria more readily visible for selection.
sensitive indicator which changes color upon contact with acid in the pH range between about pH 4 and 7, thereby making the bacteria more readily visible for selection.
19. The method of claim 16 wherein the bacteria are grown substantially anaerobically in the presence of hydrogen, hydrogen and carbon dioxide, nitrogen, or a rare gas as the nonoxidizing gas.
20. The method for selecting phage resistant strains of acid producing bacteria fox use in producing fermented food products which comprises: (a) growing heterogeneous or homogeneous populations of a strain or strains of the acid producing bacteria substantially anaerobically with a vacuum or with a nonoxidizing gas in the presence of phage on a gelled, solid growth medium so as to produce colonies wherein fast acid producing strains produce relatively larger colonies than slow acid produ-cing strains; (b) selecting a large colony; and (c) growing the cells in a fluid growth medium to at least about 106 cells per ml to provide a concentrate of cells.
21. The method of claim 20 wherein the solid and the fluid growth media contains milk protein, a milk protein derivative, or a milk protein substitute and agar.
22. The method for selecting phage resistant strains or acid producing bacteria which comprises: (a) growing heterogeneous or homogeneous populations of a strain or strains of the acid producing bacteria in the presence of phage on a gelled, solid bacterial growth medium containing milk protein, a milk protein derivative, or a milk substitute and containing a pH sensitive indicator which changes color upon contact with acid in the pH range between about 4 and 7 so as to produce colonies; and (b) selecting a strain which is resistant to the phage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000419949A CA1194437A (en) | 1983-01-21 | 1983-01-21 | Method for producing single and/or mixed strain concentrates of bacteria |
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| Application Number | Priority Date | Filing Date | Title |
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| CA000419949A CA1194437A (en) | 1983-01-21 | 1983-01-21 | Method for producing single and/or mixed strain concentrates of bacteria |
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| CA1194437A true CA1194437A (en) | 1985-10-01 |
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| WO2023211926A3 (en) * | 2022-04-25 | 2023-12-07 | The Regents Of The University Of Colorado A Body Corporate | System and methods to measure cell viability in high throughput via continuous geometry |
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1983
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| WO2023211926A3 (en) * | 2022-04-25 | 2023-12-07 | The Regents Of The University Of Colorado A Body Corporate | System and methods to measure cell viability in high throughput via continuous geometry |
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