Comparison of Enrichment Media for Routine Detection of Beer Spoiling Lactic Acid Bacteria and Development of Trouble-shooting Medium for Lactobacillus backi

Abstract

J. Inst. Brew. 116(2), 151-156, 2010The beer spoiling lactic acid bacteria (LAB) are known to have a substantial financial impact in the brewing industry and their rapid detection is essential. Thus more effective media for the cultivation of LAB in both routine quality control and special trouble-shooting situations are needed. In this study, different media were tested for the routine detection of LAB at a commercial brewery. The results showed that the use of an enzyme controlled glucose delivery system, in combination with beer-MRS medium, can significantly decrease the total analysis time. For more effective trouble-shooting in contamination incidents a trouble-shooting media, including the reducing agents L-cysteine-HCl and sodium bicarbonate, was developed. The presented medium was shown to improve the growth of beer spoiling L. backi and L. brevis, and is thereby suggested for faster detection of these strains at the breweries.

Brewing microbiology - Lactic acid bacteria and cultivation methods of detection - part II

“…ABD půda podle autorů (Suzuki et al, 2008) umožňuje průkaz pivu škodících bakterií ve vzorku, které jsou hůře detekovatelné běžnými půdami jako je MRS, BMB, Raka--ray a NBB-A. Oproti tomu Taskila et al (2010a) prezentují porovná-ní půdy ABD s B-MRS na 19 reálných vzorcích piva a nedoporučují ji k rutinní mikrobiologické kontrole piva a pivovarského provozu. Na K-MRS agaru, na jehož povrch byl aplikován enzym katalasa, rostly všechny kmeny mléčných bakterií a to výrazně rychleji v porovnání s ostatními kultivačními médii.…”

“…According to the authors (Suzuki et al, 2008) ABD medium allows determination of beer-spoiling bacteria which are harder to detect by conventional media such as MRS, BMB, Raka-Ray and NBB-A. In contrast, Taskila et al (2010a) compared ABD with B-MRS on 19 real samples of beer and recommended it for routine microbiological control of beer and brewing operation.…”

Lactic acid bacteria and cultivation methods of their detection - Part I.

“…2)� Někdy jsou souhrně označovány jako "mléčné koky" nebo nesprávně "sarciny"� Jedná se o grampozitivní, nesporulující, katalasa-negativní, chemoorganotrofní, anaerobní bakterie� Jsou homofermentativní, glukosu fermentují na kyselinu mléčnou bez produkce plynu� Optimální teplota růstu je uváděna v rozmezí 25-35 °C, ale např� P. damnosus roste v rozmezí 8-30 °C, u druhu P. acidilactici je undesirable sensory substances already during beer production, and secondary (post-pasteurization) contamination, which can damage the final product by causing turbidity and adversely affecting its taste and smell (Back, 2005 (Back, 1987;Back et al�, 1996;Bohak et al� 1998;2006;Bokulich and Bamforth, 2013;Dobson et al�, 2002;Ehrmann et al�, 2010;Funahashi et al�, 1998;Hollerová and Kubizniaková, 2002;Chaban et al�, 2002;Priest, 2003;Russell and Walker, 1953;Storgårds et al�, 1998;Suzuki et al�, 2004;Taskila et al�, 2010a)� Table 1 gives a survey of the species of Lactobacillus and Pediococcus which may come to the fore as beer contaminants� Not all isolates of these species, however, have the ability to spoil beer -lactic acid bacteria typically include both beer spoilers and nonspoilers� The species L. brevis, L. lindneri and P. damnosus are reported as the most risky and most common contaminants of finished beer� According to estimates, L. brevis is responsible for more than approximately 50 % of cases of microbial spoilage of beer (Sakamoto and Konings, 2003)� The species P. damnosus is found mostly in beer during final fermentation and in finished beer, less frequently in yeast� The species P. inopinatus and P. pentosaceus are mainly detected as yeast contaminants and seldom in beer (Priest, 2003)� Other representatives of lactic bacteria such as Leuconostoc, Oenococcus, Lactococcus, Streptococcus and Enterococcus do not show resistance to bitter substances of hops and are not commonly found in beer (Bokulich and Bamforth, 2013)�…”

“…pro pivu-škodící bakterie (Nishikawa a Kohgo, 1985)� Kindraka (1987) uvádí, že modifikované NBB médium je více specifické a má větší záchyt pivu škodících mikroorganismů než UBA� Crumplen et al� (1988) naopak vyhodnotili původní i modifikované NBB médium jako méně účinné než UBA� Médium LGM (Lactobacillus growth medium; Lactobacillus růs-tové médium) bylo navrženo a porovnáno s dalšími půdami na zá-kladě rychlosti růstu a velikosti kolonií bakterií rodu Lactobacillus ( Van Keer et al�, 1983)� LGM je komplexní médium s glukosou jako zdrojem uhlíku a bromkresolovou zelení jako indikátorem pH� Růst mléčných bakterií na této půdě je srovnatelný s Raka-Ray 3 (co se týče počtu kolonií z původního inokula), ale u všech testovaných kmenů (33) bylo na LGM agaru dosaženo zkrácení doby inkubace, po které lze vyhodnotit počet kolonií, z 48 na 24 hodin� Půda není selektivní, přídavkem β-fenylethanolu lze zabránit růstu gramnegativních bakterií� V pivovaru Kirin byla v roce 1981 navržena půda KL-2B obsahující maltosu jako zdroj uhlíku, aktidion pro potlačení růstu kvasinek a azid sodný (NaN 3 ) pro inhibici gramnegativních bakterií� Půda však neumožňovala detekci některých pediokoků a byla proto upravena přídavkem piva (Taguchi et al�, 1990)� Nové médium KOT (Kirin-Ohkochi-Taguchi) obsahovalo 800 ml piva v 1 litru půdy, kromě maltosy také glukosu, a další látky (např� kyselinu jablečnou, či cytidin)� Pro zvýšení selektivity pro pivu škodící mléčné bakterie byl do půdy při-dán cysteinhydrochlorid� Při srovnání KOT půdy s původní KL-2B na ní rostlo více kmenů Pediococcus a médium KOT bylo vyhodnoceno jako vhodnější pro kontrolu mikrobiologické kvality� V porovnání s půdami NBB a VLB-S7 vykazovala vyšší záchyt pediokokové kontaminace (Taguchi et al�, 1990)� Půda KL-2B ani KOT nejsou komerč-ně dostupné� Jejich kompletní složení je uvedeno v práci Taguchi et al� (1990)� Jednou z modifikací MRS půdy je B-MRS, kde při přípravě 1000 ml kultivační půdy je k základní MRS přidáno 250 ml piva (Holzapfel 1992, Taskila et al�, 2010a LGM medium (Lactobacillus growth medium) was devised and compared with other media on the basis of growth rate and colony sizes of test strains of Lactobacillus ( Van Keer et al�, 1983)� LGM is a complex medium with glucose as carbon source and bromocresol green as the pH indicator� The growth of lactic acid bacteria on the medium is comparable with that on Raka-Ray 3 in terms of the number of colonies from the original inoculum� However, with the LGM agar a shortening of the incubation time after which the number of colonies can be evaluated from 48 to 24 hours was achieved in all 33 tested strains� The medium is not selective; addition of β-phenylethanol can inhibit the growth of Gram-negative bacteria�…”

“…In 1981 the Kirin brewery designed a KL-2B medium containing maltose as a carbon source, actidione for suppressing the growth of yeasts and sodium azide (NaN 3 ) to inhibit Gram-negative bacteria� The medium did not allow the detection of some pediococci and was therefore modified by adding beer (Taguchi et al�, 1990)� The new KOT medium (Kirin-Ohkoch-Taguchi) contained 800 ml beer in 1 liter of medium, maltose and glucose, and other substances (e�g� malic acid or cytidine)� To increase the selectivity for beer-spoiling lactic acid bacteria the medium was supplemented with cysteine hydrochloride� When compared with the original KL-2B, KOT medium supported the growth of more strains of Pediococcus and it was assessed as more suitable for microbiological quality control� Compared with NBB and VLB-S7 media it showed a higher capture of pediococcal contamination (Taguchi et al�, 1990)� Media KL-2B or OTC are not commercially available� Their complete composition is given in the report by Taguchi et al� (1990)� One of the modifications of the MRS medium is B-MRS, in which 250 ml beer is added to 1000 ml of the basic MRS culture medium (Holzapfel, 1992;Taskila et al�, 2010a)� Modification MRS B + was described by Gillet et al� (2004)� Basic MRS medium was enriched with sugars, growth substances and 10% beer� Compared to the basic MRS medium the test strains of the genera Lactobacillus and Pediococcus produced larger colonies, and in liquid medium they had a higher growth rate� MRS-broth containing beer was further used for the determination of beer-spoiling lactic acid bacteria by using so-called EnBase system consisting of continuous addition of glucose into the medium during the cultivation (Taskila et al�, 2009)� The supply of glucose is done through a biphasic gel which releases starch into the culture media; this is then immediately enzymatically degraded to glucose� Beer--spoilage lactobacilli (L. brevis, L backii, L. lindneri and L. malefermentans) proliferated in this system faster than during the comparative cultivation on B-MRS medium� When testing the system on contaminated samples bacterial growth was spectrophotometrically detected in 68% of the samples after 18 hours with EnBase and in 50% of the samples without enzyme system� After 48 hours of cultivation the growth was detected in all samples, with or without EnBase� When checking real beer samples, lactic acid bacteria propagated in several samples only when using EnBase� The advantage of the EnBase system is a gradual supply of glucose to the medium during the incubation period without an initial high glucose concentration that could cause osmotic shock to the cells (Taskila et al�, 2009)� Haakensen et al (2009 used MRS agar modified by the addition of beer for testing of hop-gradient agar plate technique for detection of lactic acid bacteria adapted on the stringent conditions of beer� Another modification of the B-MRS broth for determination of beer spoilage lactic acid bacteria is the so-called S-MRS (supplemented MRS) broth containing L-cysteine-HCl and sodium carbonate to reduce the dissolved oxygen content (Taskila et al�, 2010a)� On comparing the medium with the B-MRS, beer-spoiling L. brevis and L. backii grew faster in MRS-S while with real beer samples a slightly higher growth of bacteria was observed in B-MRS medium� Only those samples which contained the species...…”