DISINFECTION OF BREWING YEAST WITH ACIDIFIED AMMONIUM PERSULPHATE By C, W. Brucx, A. Horrman, R. M, Gosine ann M. W. Brenner (Schwarz Laboratories, Inc., Mount Vernon, New York) Received 8th January, 1904 Acidified (pH 2:8) ammonium persulphate (0-759%) was more effective than an acid wash at pH 28 or straight 0-75%, ammonium persulphate in the disinfection of contaminated yeasts obtained from breweries. The contaminating bacteria in the samples of brewery yeast were isolated and grown in mixed fermentations with yeast. The data from disinfection trials with the contaminated yeasts showed that acidified ammonium persulphate provided the most effective treatment, The following patterns of bacterial resistance to the various treatments were noted: (a) lactobacilli tend to be resistant to disinfection with 0-75%, ammonium persulphi (b) acetic acid hacteria resist acid treatments, unless the pH Is 2-4 or lower; and (©) flavobacteria are sensitive to acid washes and to treatments with ammonium persulphate, but the acidified ammonium persulphate killed this bacterial type more rapidly. Intropuction destruction of bacteria in yeast cultures took Tue problem of infection by undesirable ;nisms is common to all microbiological processes in which pure cultures are used. The practical brewer, trying to free his itching yeast of bacterial contamination, Taces essentially the same problem as the virologist who attempts to secure microbe- free tissue cultures. ‘One of the earliest methods for the advantage of the differential sensitivity of these organisms to low pH. Pasteur noted that yeasts tolerate exposure to low pH better than do bacteria, and he suggested the disinfection of yeast by dilute solutions of tartaric acid. “Since then, other investi- gators have reported the use of phosphoric acid? and sulphuric aci As Uhlig* has pointed out, the chief disadvantage in theVol. 70, 1964] BRUCK ef al.: YEAST DISINFECTION WITH ACIDIFIED PERSULPHATE use of acid washes is the possible preferential selection of wild yeasts, if the acid wash is not carefully controlled. The application of other materials, such as sodium or calcium bisulphite and ammonium persulphate, for disinfection of yeast suspensions has been advocated since the turn of the century. Although there are few data in the literature on the dis- infecting efficiency of ammonium persulphate, it is generally recognized in the brewing industry that washes with dilute ammonium persulphate solutions (0-5-1%) are usually as effective as acid washes. Gray & Kazin* were the first to report disinfection of brewery yeast cultures with antibiotics. Strandskov & Bockelmann* have described the effectiveness of polymyxin against gram-negative bacteria that con- taminate yeast and beer, and the usefulness of penicillin against gram-positive cocci and in yeast or beer, The present investigation of yeast disinfee- tion is primarily a by-product of a larger programme initiated by this laboratory to determine the biological factors involved in the formation of diacetyl in beer. Because it has been shown that some beer lactobacilli can produce diacetyl, and that lager yeasts free of infection will yield significant amounts of this flavour compound in some fermenta~ tions, we have been carrying out ecological studies with mixed fermentations of beer bacteria and lager yeast to see what inter- relationships exist in the biological forma- tion of diacetyl. After the beers were removed from these fermentations, a suppl of contaminated yeast was available whic could be used in yeast disinfection studies, A second motive for this investigation was found in the repeated comments by brewers that disinfection by acid washes or ammonium, persulphate was not effective against the bacteria contaminating their yeasts. Since the bacteria used in’ these studies were isolated from either pitching yeast or beer, the comparative resistance of a spectrum of lager yeast contaminants to several disinfec- ting procedures could be evaluated, MATERIALS AND MetHoDs Microbial cullures.—All of the bacteria used in this study were isolated from con- taminated brewing yeasts or from cellar beers high in diacetyl. They were maintained 243, as stock cultures in hopped wort-yeast broth and were transferred every three months. ‘The lager yeast used in the mixed fermenta- tions was obtained in the pressed state from abrewery. The level of bacterial contamina- tion is very low, being usually less than one bacterium per million yeast cells. Of the three naturally contaminated brewing yeasts that were treated duri these studies, two were obtained as liquic slurries. The remaining lager yeast (yeast B) was received in the dried state on blotting paper and was grown and harvested after a normal fermentation in a 3-litre vessel, The mixed fermentations were obtained in the following manner. Tubes of ho} wort-yeast, broth, were inoculated with 8% inoculum from the broth stocks of each bacterium. After growth had proceeded at 28°C. for 24-48 hr. 4ml. of this culture were added to 100 mi. of hopped wort-yeast broth, which was incubated at 28°C. for 48 hr. The 100 ml, of bacterial culture was inoculated into 3 litres of hopped wort in an upright Povitsky bottle. Pressed. brewer's lager yeast (6 g.) was added to the flasks, and the contents were fermented at 15°C. for 5-7 days, After fermentation, the beer was decanted from the flask and held for diacetyl deter- minations, About 200 ml. of beer was left on the contaminated yeast deposit at the bottom of each flask. “The yeast was mixed thoroughly with the beer by vigorous shaking of the bottles. Yeast slurry (60 ml.) was placed in each of three 200-ml. beakers, After the yeast slurries were chilled, 60 ml, of cold double-strength disinfecting solution was added to each beaker; the contents were thoroughly mixed with a stirrer, and the HH was adjusted with 1-0-n sulphuric acid, Bisinfection then proceeded at the selected temperature for the desired length of time, Any rise in pH during disinfection was determined after the viability assays were performed, The final yeast slurries had a packed cell volume of 8-10%. The dry yeast solids of these slurries was between 3 and 4%, Recovery media and assays—The viability of bacteria was determined by plating on WL Differential Agar medium (Difco) and the agar medium of Kissel & Dakin! The growth of each organism was measured both 2erobically on agar plates and anaerobically in Prickett tubes at 28°C, for 4-7 days.BRUCH ¢t al,: YEAST DISINFECTION WITH ACIDIFIED PERSULPHATE [J. Inst. Brew. TABLE 1 SURVIVAL OF BACTERIAL CONTAMINANTS IN THREE Brewing YEASTS AFTER DISINFECTION BY THREE PxoczDURES* Lager yeast culture A c c (diluted) Survival of bacteria (%): O-76% ammonium persulphate + slphuric acid to * Cultures A and B were treated for 20 hr. at 1-2°C.; culture C was treated for 20 hr. at 7-8°C, ‘The assays for yeast numbers were per- formed on aerobic plates of hopped wort-agar. Changes in the respiratory character of the yeast were examined by colonial growth on cies plates of WL nutrient agar (Difco). e interpretation of the respiratory defici- ency test has been described by Czarnecki & van Engel Aiter some trials, the treated yeast along with the disinfecting solution was pitched into 3 litres of wort." The activities of these fermentations were visually compared with untreated yeast fermentations. Final gravity and pH readings were taken after @ or 7 days at 16°C. REsuLTs At the request of several breweries, pilot- geale, disinfection ‘trials were made with infected lager yeasts furnished . Thear breweries'had encountered ifiultas in the purification of their yeast with a cold 0-75% ammonium persulphate wash or with an overnight acid wash at pH 2°8 in the cold. These two treatments were compared with a combination of the two, #.c., acidified ammonium persulphate. The results of these trials are presented in Table I. ‘These data show that each infected lager yeast had a different level of bacterial resis- tance to these treatments. Only the acidified ammonium persulphate treatment was con- sistently effective in all of these trials. The Presence of different bacterial types in each of the cultures was indicated; accordingly, the cultures were plated to isolate both the aerobic and the anaerobic contaminants. From lager yeast A, two different types of bacteria were isolated. Culture A-2P was a gram-positive rod form, anaerobic, slow growing, and produced ‘acid to a pH of about 45. Culture A-2U was a gram- negative rod form, aerobic, and a strong acid producer. Some diacetyl-favoured beer TABLE It Rusistance oF Bactenia Contaminants Isotatep prom Lacer Yeast SAMPLE A To Distnrection IN PRESENCE or ViABLE Yeast ‘Survival (%) pH42 | ps2 Bacterial ‘ahr | /20 hr, isolates 12°C, | 78°C. A-2P 00 40 A-2U 1 ‘O04 Ae 6 00 AT ca 60 '* The pH was adjusted with sulphuric acid.Vol. 70, 1964] BRUCH et al.: YEAST DISINFECTION WITH ACIDIFIED PERSULPHATE that had been fermented with this yeast in the was examined; two more bacterial types similar to, but more vigorous than, the culture A-2P were isolated. “These cultures were grown in mixed fermentations with lager yeast, and results with several disinfecting procedures are given in Table IT. 245 fermentations, harvested, and subjected to several disinfectis lures, ‘The resulting data are presented in Table IIT. ‘Alt of these cultures showed some resis- tance to a short-time, low-temperature cycle in 075% ammonium persulphate. Only culture B-5, which is an Acetobacter type, ‘TABLE IIT Resistance oF BACTERIAL ConTAMINANTS ISOLATED Rom LAGER YEAST SAMPLE B To ‘Disturnction 1 PRESENCE OF VianLE Yuast 075% ammonium Survival (9% (0-75% ammonium pereulphate Sulphuric acid ersulphatet pH42 | pee | pH22 pH2-8 Bacterial ‘the | Bone | "ane. 20 br. isolates rere. | arc, | are. 78°C, 16 0 05 o 5 ° 2 ° © The pH was adjusted with sulphuric acid. The gram-positive cultures, A-2P, A-24, and A-27, ate resistant to the ammonium persulphate treatment. This finding con- ed the difficulties that this particular brewery had experienced in trying to disinfect its yeast with ammonium per- sulphate. The aerobic acid-producin, culture, A-2U, tended to resist the aci treatment at pH 28, but it was susceptible to a pH of 2:2. Additional tests indicated that a pH of 2-4 has to be reached before an acid wash will be fully effective in killing this organism. jame preliminary taxonomic studies were conducted with these and some of the later isolates that will be described. Culture A-2U is an Acetobacter type, while the other three cultures are lactobacilli variants. Four different types of bacteria were isolated from lager yeast B. Culture B-3 is a gram-positive aerobic rod that grows vigorously in wort but does poorly in a beer fermentation. Culture B-5 is a - negative rod, aerobic, and a strong producer of acid. Culture B-6 is a gram-negative aerobe, of extremely short rod form, that produces some acid. Culture B~7 is a gram- negative, facultatively anaerobic, thin, long rod form that produces little acid; it appears to belong to the flavobacteria. These isolates were grown with lager yeast in mixed showed much resistance to the acid treat- ments. As noted in Table II also, acidified ammonium persulphate was the most effec- tive treatment. From lager yeast C, three related gram: negative, facultatively anaerobic, thin rod forms were isolated. A fourth isolate, C-9 which is similar to the three isolates from t C, was found in almost pure culture in the yeast from another brewery. Mixed fermentations were carried out, and_ the contaminated yeasts harvested and disin- fected. The results are presented in Table IV. ‘These cultures, which are considered to be of flavobacteria, were treated only in low- temperature, short-time trials and were sensitive to all of the treatments, The highest survivals were obtained with the straight ammonium persulphate treatment, but a higher temperature ot a longer period. of time would have increased the effect of this treatment. The levels of yeast survival were good (over 70%) in nearly all of these trials. me sluggishness was noted in the fermenta- tions with most of the treated yeasts. This difficulty can be overcome by a slightly higher rate of pitching (or a higher pitching temperature). No tendency to produce or select for respiratory-deficient variants was noted in these experiments.248 Discussion The standards of microbiological control in breweries have changed over the years. A generation ago a pitching yeast was accepted as usable when it contained 1% of Dacteria; more recently careful brewers were still passing yeasts with 025%, and some looked pained when our laboratories said that 0-1 18 ‘was too high. ‘A contaminated yeast may harbour a massive reservoir of troubles. Brewers who might shudder at the thought of their cooled wort containing 10,000 bacteria per ml., seem untroubled by adding 10,000 bacteria per ml. when they add 10,000,000, yeast cells per ml. along with 10,000 bacteria as they BRUGH ef al.: YEAST DISINFECTION WITH ACIDIFIED PERSULPHATE (J. Inst. Brew. ammonium persulphate treatment may be expected to reduce the viable bacteria count to 0-0002% (2 bacteria per million yeast cells. Taking the same example, if the yeast {02% viable bacteria) is added to wort to provide a normal pitching yeast count of 10,000,000 yeast cells per ml. the bacterial count would be 20,000 per mi. "After washing the yeast with acidified ammonium per- sulphate, the bacterial count in the pitched wort would very probably be Jess than 20 per ml. ‘The highest survival rate in the recorded data was 3 per 1000; in 90% of the tests no viable bacteria remained after exposure to acidified ammonium persulphate. TABLE IV Rusistance oF BACTERIAL ConTAMINANTS ISOLATED FROM LAGER YEAST SAMPLE C TO DISINFECTION I PRESENCE OF VIALE YEAST Survival (%): ‘Ammonium 075% ammonium persulphate Sulphuric acid etaulphatet mae 20 | eee | ose | pres Bacterial Pee Pore | Pane |e Pon. isolates 12°C. Eve | ete. | cre te. 25 5 +3 ° ° ° ° cas 5 1 ° ° ° o e0 1 +3 ° ° ° ° cot 1 +6 ° o1 ° 0 * The pH was adjusted with sulphuric acid. t This bacterial culture was isolated from another brewing yeast; it is similar in character to the isolates from yeast C. do when they use yeast containing 0-1% of bacteria (1 bacterium per 1000 yeast cells), Arithmetic demonstrates that the contamina- tions are equal. Experience teaches that the bacteria accompanying yeast into the wort may be more hazardous, because the wort-contaminating organisms are usually sty by yeast fermentation. ith the availability of the simple yeast- purifying technique described in this paper, it is possible to achieve, quickly and easily, new levels of ‘microbiological purity in pitching yeast. The results of dozens of experiments indicate high probability of efiecting a 99-:0% kill of Bacteria with acidified ammonium persulphate. Assuming yeast crop contains 0-2% viable bacteria (2 bacteria per 1000 yeast cells), the acidified Acknowledgement.—The data of this paper, and a modified version of the text, were presented orally by Dr. Bruch at the 63rd Annual Meeting of the American Society for Microbiology, at Cleveland, Ohio, 5th-9th May, 1963. ‘REFERENCES |. Compton, J., Cronyn, J.B, & Read, W. F., ‘allerstéin Lab. Commun., 1954, 17, 19. . Czarnecki, H. T., & van Engel, E, L., Brewers, Digest, Mar. i969, 62. . Gray, & Kazin, D., Wallerstein Lab, ‘Master Brewers Assoc. Amer., 1950, 44. . Strandskov, F. B., & Bockelmann, J. B., ‘J. Agric. & Food Chem., 1953, 1, 1219. Uilig, K., ‘Amer. Beewer, Nov., i962, 41. ee keen ee 3 3