COMMUNICATION THERMAL DEATH POINTS OF MICRO-ORGANISMS IN BEER

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1 Nov.-Dec., 1946] lund: thermal death points of micro-organisms in beer 307 He considered that the combine harvester was an asset and had come to stay. Given favourable weather conditions and providing the barley was not cut before it was properly ripe, the combine en sured an even sample and a regular run of grain. The somewhat old-fashioned method of kiln drying was certainly perfection, as the maltsters had confidence that barley dried in that way would eventually grow satisfactorily after it had been properly rested, but in these days of progress it appeared that the quicker drying of large quantities was now necessary, and in that connection he knew of a system adopted by one large malting where the grain was first subjected to humid atmosphere for about an hour, and then passed to the drying chamber at 120 F. for one hour, and finally to the cooling chamber for an hour, the whole treat ment taking three hours at least, and the drying operation was continuous. The farm drier, how ever, was only intended to dry surface moisture. and he did not think it could be considered in the light of conditioning barley for safe storage, the whole operation in the farm drier was so rapid that there was a risk of the germinative capacity of the grain being affected. Maltsters would do their best to keep malt on the floors as long as possible, but brewers should re member that the barleys contained so defects that it might be that a shorter flooring period would be more advisable than a considerable amount of mould. It was a matter that should be left to the maltster's discretion. There was every indication that the acreage under barley would be decreased, and that of wheat increased. Further than this, it was difficult to express any opinion about next year's crops, especially owing to the present anxiety concerning suitable supplies of sound seed for sowing, but given a fine spell the present outlook could still be considerably improved. COMMUNICATION THERMAL DEATH POINTS OF MICRO-ORGANISMS IN BEER By Aage Lund Of the numerous micro-organisms which can enter breweries by air or other sources only a few can grow in normal beer. First and foremost one can leave out of consider ation the great group of moulds. Of yeasts, there are some types which can render beer turbid and give it an unpleasant taste. This applies to spore-forming wild yeasts, of which the main species are Saccharomyces Pastorianus and S. dlipsoideus. Thanks to the pure cultivated beer yeast and the advanced hygienic methods used such wild yeasts seldom occur to-day in large quantities in Danish breweries. Filmforming yeasts, Pichia, Hansenula, and especially Mycoderma are rather common, but generally of no great importance owing to their demand for oxygen. Species of Torula are frequent in breweries, but normally do not develop at all, or only to a small degree, in Danish beer. Sometimes culture yeast forms a sediment in beer and may impart a yeasty taste to the beer. Bacteria are the most dangerous micro organisms in breweries. Most dreaded are lactic acid bacteria, both the rod-shaped Laetobacillus species and the Pediococci, which cause turbidity and unpleasant taste due to their formation of lactic acid or diacetyl. Acetic acid bacteria, species of Acelobacter, occasionally cause sourness in top-fermented beer, whereas their chances of developing are rather poor in bottom fermentation breweries. Sometimes it is maintained that beer may contain spore-forming bacteria, that is specially heatesistant bacteria. It should be emphasized, however, that development of spore-forming bacteria in normal beer has hardly ever been established. It is true that during the war the growth of hay-bacilli (Bacillus subtilis) was stated in German beer, according to Zeller and Nickl,1 but the beer in question was of low original gravity and high pb. If there were spore-forming bacteria which developed in normal beer the usual pasteurization temperature would hardly be sufficient to kill them. In the literature on the subject disagree ments about the thermal death points of micro-organisms are sometimes found. Epstein and SnelP report for instance that Lactobacillus and 5. Pastorianus are particu larly thermoesistant as in some cases they were not killed below the temperature of F. (58-60 C.) for ten minutes, whereas the thermal death point of Acetobacter was lower, viz., " F. (52-54 C). Laufer and Brenner3 on the other hand state that some strains of Lactobacillus and 5.

2 308 lund: thermal death points of micro-organisms in beer [Nov.-Dec., 1946 Pastorianus did not survive a temperature of 122 F. (50 C.) for minutes, whereas an Aceiobacter species was not killed below F. (62 C.) for the same period. Discrepancies between the results of various researches may be due to several different causes. Not only may species of the same genus, but also various strains of the same species react differently to tempera ture. Organisms which have been cul tivated for a long time under laboratory conditions are often extremely sensitive to heat, and on the other hand it is sometimes seen that the heatesistance of organisms which have survived pasteurization (inad equate pasteurization) is particularly high. The medium in which the organisms used for the experiments are grown is probably an important factor; beer should be the medium used, or, if the organisms grow slowly in this, hopped wort. The pasteurization medium is of great importance; to obtain results of practical value beer must be used. Shimwell4 found that Lactobacillus Pastorianus did not develop in beer kept at a temperature of 120 F. (49 C.) for five minutes, but that it was still growing in unhopped wort kept at a temperature of 125 F. (52 C.) for five minutes. Attention should also be paid to the type of the beer to be heated, to the rate at which the temperature is reached, and to the duration of the heating process. Experimental The pasteurization medium used was bottom fermented beer (Danish Pilsner, original gravity 40, alcohol 3-2 per cetu. by weight, pb 4*4) pasteurized in beer bottles at 140 F. (60 C). The inoculum consisted of pure cultures, which had been grown in pasteurized beer, or sometimes culture yeast, S. ellipsoideus, Torula sp. in sterile hopped wort for 6- days at 77 F. (25 C). Yeasts with spores, S. Pastorianus and S. turbidans originated from four days' old gypsum block cultures. /. Experiments in c.c. beer. Pasteurized beer was transferred under sterile conditions to sterile 16 c.c. bottles, c.c. beer per bottle. 0-1 c.c. suspension of the inoculum containing from 20,000 to 1,000,000 organisms, determined by counting of colonies on unhopped wort-agar plates, was sown on each bottle. The bottles were corked with sterile corks and then immersed in a waterbath with cold water at F. (-15 C). The water was then heated over gas burners and kept in motion by constant stirring. The desired temperature measured in control bottles with beer, was reached in the course of -15 minutes. The bottles were held at this temperature for 15 minutes; then they were cooled at room temperature. Inoculated, but unheated, control bottles were also used in each experiment. The effect of the heating was tested by the counting of colonies developed on unhopped wort-agar plates, by inoculation in unhopped wort, and by allowing the beer to stand. Plates and bottles were kept at a temperature of 77 F. (25 C.) and the final results were assessed by microscopic examina tion after one month. If there was no growth in the above media during this period, the organisms were considered killed. The results of the experiments are stated in Table I in order of increasing heatresistance. It appears that culture yeast (bottom fermenting yeast), S. Pastorianus with spores, Hansenula anomala, Mycoderma cerevisits, Torula sp. and Pediococcus perniciosus were killed at a temperature of about 122 F. (50 C). The counting of colonies on agar plates proved that the majority of the cells of several of these organisms already were killed at a temperature of F. (48 C); this temperature prevented the growth in beer of culture yeast, Mycoderma and Pediococcus, but these organisms developed in unhopped wort. After heating at F. (48 C.) they did not grow in any of the media. The thermal death points of S. ellipsoideus and 5. turbidans (with spores) and of Acetobacler viscosum and LactobaciUus Pastor ianus were somewhat higher, viz., F. (54-56 C). The most heatesistant organism was LactobaciUus Lindneri (the common lactic acid rod in bottom-fermented beer), which survived heating at F. (58 C), but did not develop in beer heated at 140 F. (60 C). //. Experiments in 340 c.c. beer. To control the results of the experiments with c.c. beer, heating experiments were

3 Micro-organisms. S. cereritbc vbottomfermenting y«wt) Jftmdrrma nreritut Pedhcoccut pemmonu.. Haruenula anomala S. Pattoriamu (with spores) S. turbidatui (with spores).. Aatobacur tistonun 8. tuiptoidrtim.... Lactobadllvt Pcutoriama lactotadtttu lindneri.. Unhcnted control. Agar Wort lieer 33,000 30,000 2, ,000 80,000 0,000 80,000 0,000 TABLE I Heat-treatment of c.c. Infected for 16 minutes Agar = unhopped wort agar, number of organism? per cc. ip ca growth Wort = unhopjied wort (bottom fermented beer) -^ = no growth my f. (44 C) * F. (40 C) 'F. (48'C). 122 F. (50'C). 125-e F. (82 C) F. Agar Wort Agar Wort Agar Wort Itecr Agar Wort Agor Wort Agar Wort manr 203 1G BO s- - t- I s- 4- T* Beci F. (SO' C). Agar Wort A F. (58'C). 140* F. (60' C). s z o $ PI S3 i 2 2 i 2 w

4 3 lund: thermal death points of micro-organisms in beer [Nov.-Dec., 1946 made with the micro-organisms most detri mental to beer, viz., spore-forming wild yeast, beer pediococci, and lactic acid rods in normally bottled beer, 340 c.c. per bottle, pasteurized at 140 F. (60 C). Pure cultures of the organisms were grown in pasteurized beer for ten days, and 1 c.c. infected beer, with a number of germs from 0,000 to 800,000 counted on unhopped wort-agar plates, was transferred to each on the whole are in conformity with the results of the experiments with c.c. beer. Five minutes' heating at F. (48 C.) was sufficient to prevent the development of Pediococcus perniciosus. Lactobacillus Pastorianus did not grow when the beer had been kept at a temperature of F. (52 C.) for minutes, or at a temperature of F. (64 C.) for five minutes, and the growth of S. ellipsoideus was prevented by five TABLE Heat-treatment of 340 c.c. Infected Bottom-fermented for 5 20 minutes II f- e growth t = no growth 114»«P. (4(5 0.). iie-4» p. (48»O.). 186-e"F. (8a«o.). ls^s p. (84'C). 13S-8* P. (86=0.) P. (WO.). 140» P. (60 0.). min. mlo. rain. mln. rain. noin. mint Micro-orRanlMns. control. I SO SO 6 SO 20 5 so PaHoaeetti jwrnfcimiu -=- f Lactotadttut PaMloriamu i (Uiptoidms LaaobaaUai UndntrU Strain 1 S,. S s*» 4 bottle of beer. The bottles were stopped with crown corks sterilized with alcohol and heated as described in the experiments with c.c. beer, with the difference, however, that the temperatures wanted were only reached after 30 to 45 minutes. At each temperature bottles were removed after 5, and 20 minutes' heating respectively. These bottles and also inoculated, but unheated, control bottles^ were kept at room temperature (68 F., 20 C.) for one month, and the growth of the organisms was then examined microscopically. The results of these experiments are set out in Table II, and it will be seen that these minutes' heating at F. (54 C). Ex periments with Lactobacillus Lindneri proved that some strains did not develop when the beer was kept at a temperature of F. (56 C.) for 20 minutes, whereas other strains demanded a higher temperature; one strain did not grow when heated at F. (58 C.) for minutes, others survived 20 minutes heating at F. (58 C), but none developped after'20 minutes' heating at 140 F. (60 C). Among recently published papers these results seem to be in greatest conformity with those obtained by Epstein and Snella; most important of our disagreements are,1

5 Nov.-Dec, 1946] lund: thermal death points of micro-organisms in beer 311 the thermal death points of S. Pastorianus and 5. ellipsoideus, which these authors found higher than I up to 140 F. (60 C.) for -15 minutes. The possibility cannot be left out of account that my cultures of these yeasts have been impaired by being grown in the laboratory. We are in full agreement, on the essential point that pasteurization of beer at a temperature of 140 F. (60 C.) for a period of 20 minutes ensures that the development of micro organisms is prevented. Summary By experiments on the thermal death points of micro-organisms in beer it was found that most of the organisms used for the experiments, species of Saccharomyces, Hansenula, Mycoderma, Tortda, Acetobacter and Pediococcus, and also Lactobacillus Pastor ianus were killed within fifteen minutes when heated in bottom fermentation beer at F. (56 C.) or at even lower temper atures several of them were killed already at about 122 F. (50 C.). The most heatesistant organism was Lactobacillus Lindneri (the common lactic acid rod in bottom fermentation beer), certain strains of which survived twenty minutes' heating at F. (58 C), but not at 140 F. (60 C). References 1. F. Zcller and D. Nickl, Allgcm. Brauer-u. Hopfen-Zeitg., 42, S. S. Epstein and F. D. SneU, this Journ., 1940, S. Laufer and M. W. Brenner, Proe. Am. Soe. Brew. Chem., 19; this Journ., 1941, J. L. Shimwell, this Journ., 1936, 481. Tuborg Breweries, Copenhagen. July, 1946.

Washed agar gave such satisfactory results in the milk-powder. briefly the results of this work and to show the effect of washing

Washed agar gave such satisfactory results in the milk-powder. briefly the results of this work and to show the effect of washing THE USE OF WASHED AGAR IN CULTURE MEDIA S. HENRY AYERS, COURTLAND S. MUDGE, AND PHILIP RUPP From the Research Laboratories of the Dairy Division, United States Department of Agriculture Received for publication