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Detection of Acid-Producing Bacteria Nachweis Von Säureproduzierenden Bakterien Détection De Bactéries Produisant Des Acides

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Detection of Acid-Producing Bacteria Nachweis Von Säureproduzierenden Bakterien Détection De Bactéries Produisant Des Acides (19) TZZ ¥ _T (11) EP 2 443 249 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/04 (2006.01) G01N 33/84 (2006.01) 19.11.2014 Bulletin 2014/47 (86) International application number: (21) Application number: 10790013.6 PCT/US2010/038569 (22) Date of filing: 15.06.2010 (87) International publication number: WO 2010/147918 (23.12.2010 Gazette 2010/51) (54) DETECTION OF ACID-PRODUCING BACTERIA NACHWEIS VON SÄUREPRODUZIERENDEN BAKTERIEN DÉTECTION DE BACTÉRIES PRODUISANT DES ACIDES (84) Designated Contracting States: (74) Representative: Isarpatent AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Patent- und Rechtsanwälte GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Friedrichstrasse 31 PL PT RO SE SI SK SM TR 80801 München (DE) (30) Priority: 15.06.2009 US 187107 P (56) References cited: 15.03.2010 US 314140 P US-A- 4 528 269 US-A- 5 098 832 US-A- 5 164 301 US-A- 5 601 998 (43) Date of publication of application: US-A- 5 601 998 US-A- 5 786 167 25.04.2012 Bulletin 2012/17 US-B2- 6 756 225 US-B2- 7 150 977 (73) Proprietor: 3M Innovative Properties Company • DARUKARADHYA J ET AL: "Selective Saint Paul, MN 55133-3427 (US) enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria (72) Inventors: and non-starter lactic acid bacteria from Cheddar • YOUNG, Robert, F. cheese", INTERNATIONAL DAIRY JOURNAL, Saint Paul, Minnesota 55133-3427 (US) ELSEVIER APPLIED SCIENCE, BARKING, GB, • MACH, Patrick, A. vol. 16, no. 5, 1 May 2006 (2006-05-01), pages Saint Paul, Minnesota 55133-3427 (US) 439-445, XP024963231, ISSN: 0958-6946, DOI: • HUGHES, Michael, E. 10.1016/J.IDAIRYJ.2005.06.009 [retrieved on Saint Paul, Minnesota 55133-3427 (US) 2006-05-01] • BINSFELD, Christine, A. • BUJALANCE C ET AL: "A selective differential Saint Paul, Minnesota 55133-3427 (US) medium for Lactobacillus plantarum", JOURNAL • BJORK, Jason, W. OF MICROBIOLOGICAL METHODS, ELSEVIER, Saint Paul, Minnesota 55133-3427 (US) AMSTERDAM, NL, vol. 66, no. 3, 1 September •REIF-WENNER, Mara, S. 2006 (2006-09-01), pages 572-575, XP027926939, Saint Paul, Minnesota 55133-3427 (US) ISSN: 0167-7012 [retrieved on 2006-09-01] • LUBRANT, Henry, J. • MINNESOTA MINING AND MANUFACTURING Saint Paul, Minnesota 55133-3427 (US) COMPANY: ’3M Petrifilm Coliform Count Plate’, [Online] 1999, Retrieved from the Internet: <URL: http://www.3m.com/intl/kr/microbiology /p_ coliform/use3.pdf> Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 443 249 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 443 249 B1 Description BACKGROUND 5 [0001] Acid-producing bacteria comprise a relatively diverse group of Gram positive microorganisms that share com- mon metabolic and physiological characteristics. This group of bacteria produces acid as the major end product of the fermentation of carbohydrates. The group is divided into two metabolic subgroups - homolactic fermenters, which convert carbohydrates essentially into acid; and heterolactic fermenters which, in addition to producing acid, also convert car- bohydrates into other metabolites including ethanol and carbon dioxide, for example. 10 [0002] Although some acid-producing bacteria, such as lactic acid-producing bacteria (LAB) have a beneficial role in the production of fermented foods, they also are known as a principal agent of food and beverage spoilage, particularly in vacuum-packaged meats, meat products, and beer. The metabolic activity of acid-producing bacteria in certain food products can lead to significant deterioration of the organoleptic properties (e.g., smell, taste) of the food or beverage. [0003] Traditional microbiological techniques are typically used to identify and/or enumerate acid-producing bacteria, 15 such as LAB. Agar and broth culture media (e.g., MRS and APT media) are used to promote growth and/or identify acid- producing bacteria. The cultures are incubated in microaerophilic environments to improve the growth of the acid- producing bacteria, such as LAB. Typical methods include the growth and isolation of presumptive acid-producing bacteria colonies on a selective agar plate, followed by subculture in a broth medium containing a fermentation tube to detect the production of gas by heterolactic fermenting species. These methods may take up to four days to detect or 20 identify the acid-producing bacteria. In some cases, the methods may take from 7 -10 days to identify the acid-producing bacteria. [0004] Darukaradhya et al., International Dairy Journal 16(5), 2006, 439-445 discloses a medium for the selective enumeration of lactic acid-producing bacteria, however it is not described to differentiate between different sub-groups of the acid-producing bacteria. 25 [0005] There exists a need for simple articles and methods for the detection of lactic acid-producing bacteria. SUMMARY [0006] In view of the current general methods to detect and/or identify acid-producing bacteria, which typically require 30 specialized culture media, lengthy incubation periods, specialized incubation conditions, and/or subculture procedures, the present disclosure includes simple articles and methods to detect and/or identify lactic acid-producing bacteria. In some embodiments, the inventive methods provide for the differentiation of lactic acid-producing bacteria. Additionally, or alternatively, some embodiments provide for the enumeration of lactic acid-producing bacteria. In some embodiments, the inventive methods provide for the automated detection and/or enumeration of lactic acid-producing bacteria. 35 [0007] Thus, a method of detecting a acid bacterium is described. A method of detecting acid-producing bacteria can comprise providing a thin film culture device, a culture medium to support the growth of acid bacteria, a pH indicator with a transition range that extends below pH 7.0, a carbohydrate that can be fermented by acid-producing bacteria, and a sample. The thin film culture device can comprise a cold water-soluble gelling agent. The method further can comprise combining, in the culture device, a predetermined volume of the sample, the culture medium, the pH indicator, 40 and the fermentable carbohydrate. The method further can comprise incubating the culture device for a period of time at a pH below 7 and detecting the presence or absence of an acid-producing microorganism. [0008] In one aspect of the present invention, a method of detecting lactic acid-producing bacteria is provided, com- prising providing a thin film culture device comprising a cold water-soluble gelling agent, a culture medium to support the growth of acid-producing bacteria, a pH indicator with a transition range that extends below pH 7.0, a carbohydrate 45 that can be fermented by lactic acid-producing bacteria, and a sample suspected of containing lactic acid-producing bacteria. The method further comprises combining a predetermined volume of sample and the culture medium to form a first mixture; combining, in the culture device, the first mixture, the pH indicator, and the fermentable carbohydrate; incubating the culture device for a period of time; and detecting the presence or absence of a lactic acid-producing bacterium. 50 [0009] In any of the above embodiments, the culture device can comprise the culture medium and/or the pH indicator. In some embodiments of the method, the culture device further can comprise a selective agent. In some embodiments of the method, incubating the culture device can comprise incubating the device aerobically and/or anaerobically. [0010] In any of the above embodiments, detecting the presence of a microorganism comprises differentiating a microorganism, wherein differentiating a microorganism comprises detecting a pH indicator reaction and detecting a 55 gas bubble associated with the microorganism. In any of the above embodiments, the method further can comprise combining the sample with a diluent capable of neutralizing a chemical sanitizer. In any of the above embodiments, the pH of the culture medium can be adjusted to a pH below 6.5, wherein detecting the presence or absence of a microor- ganism comprises detecting the presence or absence of an lactic acid-producing microorganism. 2 EP 2 443 249 B1 [0011] In any of the above embodiments, the method can further comprise providing an antifungal agent. In any of the above embodiments, the pH indicator can be selected from the group consisting of, for example, chlorophenol red, bromcresol purple, bromphenol blue and bromcresol green. In any of the above embodiments, the method can further comprise the step of enumerating microorganisms. 5 [0012] In any of the above embodiments, the method further can comprise providing an imaging system and obtaining an image of the culture device, wherein detecting the presence or absence of a microorganism comprises displaying, printing, or analyzing the image of the culture device. In some embodiments, the method further can comprise providing an image analysis system, wherein analyzing the image comprises analyzing the image with the image analysis system. [0013] In another aspect, the present disclosure provides a thin film culture device. The culture device can comprise 10 a body member comprising a self-supporting, water-proof substrate having upper and lower surfaces. The culture device can further comprise a dry coating on the upper surface of the substrate. The dry coating can comprise a culture medium to support the growth of acid-producing bacteria; a cold water-soluble gelling agent; a pH indicator with a transition range that extends below pH 7.0; and, optionally, an antifungal agent.
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