Methods of isolating yeast cultures are described, together with the scheme of

Transcription

1 Vol. 58, 1952] 347 PRACTICAL YEAST PROPAGATION By H. F. P. Webber, B.Sc, F.R.I.C., J. Comfton, B.Sc., A.R.I.C, and L. Taylor. B.Sc., F.R.I.C. {The Laboratory, Anchor Brewhouse, Horselydown, London, S.E.I) Received 30M April, 1952 Methods of isolating yeast cultures are described, together with the scheme of developing such yeasts in the laboratory, and a detailed account is given of the wort collection and yeast propagation plant. The importance of sterilizing both the worts and the plant before use is emphasized, and the methods adopted to this end are outlined. The technique of collecting the yeast crop is described; worked as a contin uous process, approximately 80 Ib. of yeast can be produced weekly. It is advisable to keep records at every stage of the propagation procedure. Introduction The principle underlying the system of pure yeast production now to be outlined is similar to that suggested by B. M. Brown (this Journal, 1934, 9). The scheme is applicable to the development of mixed colony, single colony or single cell cultures at will, and results in the skimming or collec tion of a weekly crop of yeast from the actual propagation plant. The work described below is related in particular to mixed colony cultures, as our present policy specifies that only these are to be employed. Experience shows, however, that this method of selection gives satisfactory results and maintains the characteristics of the brewery yeast. Technique Preparation of new cultures.there are various methods of maintaining a selected culture in active operation, i.e., in a condition from which it may be drawn upon when required for propagation, and the following are found to be convenient starting points for the propagation procedure: (a) A stock slope on unhopped wort-agar medium. (b) A stock culture in liquid wort. (c) An active brewery fermentation by previously cultured yeast. (d) A brewery crop from previously cultured yeast. In his paper Brown describes the first two methods, viz., (a) the production of a stock slope from colonies after plating a suitable dilution of the selected yeast, and (b) the maintenance of 200 ml. of liquid culture in active fermentation by addition of fresh wort twice weekly. In the third method (c), a sample of actively fermenting wort from a brewery vessel is plated, incu bated in unhopped wort-agar medium, and then either single or multiple colonies are selected. Method (d) is the present starting point, streak plates on 4-in. petri dishes being prepared, and well-isolated colonies chosen after incubation at 70 F. for 3 days. These colonies are inoculated into a 10-ml. quantity of sterile liquid wort, incubated at 70 F. for 1 day, and pitched into 200 ml. of sterile wort in a 500 ml. flask which is em ployed as described in the next section. The small amount left in the test tube is used to inoculate four streak plates, while the final residue is microscopically examined. The plates are incubated for 3 days at 80 F., and examined for infection or atypical colonies. Should any be found the culture is rejected, and an older liquid stock culture kept in reserve as in (b) is made use of. Laboratory propagation procedure.3 Gal. of first wort are collected at the wort refrig erators (Excise indulgence), broken down to S.G. 1,045 with distilled water and distributed as follows: 1x2 gal. in large stainless steel laboratory vessel (2 gal. working capacity). 1 X 1,800 ml. in small stainless steel laboratory vessel ( gal. working cap acity). 2 x 300 ml. in litre flasks. 2 x 200 ml. in 500 ml. flasks. The various portions are sterilized by boiling, and subsequently serve in the propagation procedure.

2 348 WEBBER, COMPTON AND TAYLOR: PRACTICAL YEAST PROPAGATION []. Inst. Brew. After pitching the 10 ml. of fermenting wort mentioned in the previous section into 200 ml. contained in a 500 ml. flask, the fermentation is allowed to proceed for 3 days at 70 F., when the following transfers are made: (i) 25 ml. of the fermenting wort into 300 ml. of sterile wort in a litre flask for the propagation procedure, (ii) 25 ml. to 200 ml. of sterile wort in a 500 ml. flask for stock (e.g., against possible failure in the next propaga tion). Both flasks are kept at 70 F. for 24 hr., after which the 300 ml. portion of fermenting wort in the litre flask is used to inoculate 1,800 ml. of sterile wort in the small S.S. vessel, and fermentation proceeds at 70 F. for 3 days. Transfer is then effected to the large stainless steel laboratory vessel con taining 2 gal. of sterile wort and fermentation continues at 70 F. for another 4 days. This represents the final stage in the laboratory propagation procedure, the total volume of fermented wort being approximately 2\ gal. which experiment has shown contains up wards of \ lb. of yeast. This is now ready for transfer, as described in the section on propagation room procedure, but some description of plant and Excise requirements is necessary before continuing. Stainless steel laboratory vessels.these are hemispherical in shape, being provided with thick flush-fitting Perspex lids. Each has a spout rising from near the bottom and terminating level with the rim, and these spouts joined by rubber tubing may be used for transferring from one vessel to another or to the first vessel in the yeast propagation room (see Fig. 1). Materials of construction.the plant in contact with the process is fabricated in stainless steel, this applying to wort collection vessels, heat exchanger, wort mains, and fermenting (or propagation) vessels. Wort collection.outside the propagation room are situated two covered cylindrical collecting vessels. The smaller (C.V.21) has a total capacity of 4 brl. and commands the first two vessels in the propagation room, while the larger (C.V.20) holds 18 brl. gross and is used to supply the large culture vessel. The collecting vessels are supplied by a special wort charge main used only for this purpose. Both are entered and gauged vessels. A note of the following week's wort requirements is handed to the duty brewer (conveniently on a Friday morning) in order that the necessary arrangements may be made in the brewery and the appropriate entries written in the Excise book. 120 gal. of P.A. first wort are collected on Monday and 14 brl. of P.A. wort at current O.G. on Thursday. Excise requirements.worts cannot be moved from the collecting vessels until the Excise Officer has passed them, or, in his absence, until 12 hr. after the dips and gravities have been entered. In making the declaration the brewer may give notice for the worts to be removed at 8 a.m. the follow ing morning. The 3 gal. of hopped P.A. first wort referred to under laboratory procedure are collected at the wort refrigerators, and the Excise require a record to be kept, showing the amount of each collection, how much is used, and how much destroyed. Wort sterilization and transfer.usually it is necessary for the worts to remain covered in the collecting vessels until the following day, but this will depend on the time of brewing. A certain amount of sludge settles out if there is a waiting period, and allowance is made at wort collection so that this sludge may remain behind on transfer to the propa gation room. A plate heat-exchanger (used as a sterilizer) and fitted with a holding tube is included in the flow line to the propagation room. The holding tube temperature is maintained at 212 F., following which the worts are cooled, and leave the sterilizer at F. The transfer is effected by means of an impellor pump through mains specially assembled on each occasion. Compressed air.a small air-compressor is included in the assembly, the air being filtered and fed to a wort aeration point on the input main of the sterilizer. It is also distributed to the propagation room vessels, where it is used for transferring from one vessel to another and for aeration or rousing as neces sary. Description of propagation plant.this consists of three totally enclosed vessels marked Y.P.V.l, Y.P.V.2, and Y.P.V.6, housed in a special room, supplied with air from the brewery conditioned-air system and temperature controlled to 60 F. The vessels are connected to each other and to the wort

3 l-'ic. I.Laboratory propagation ^vessels. I'u;. :.'. -Propagation vessels of, respectively, 3J l»rl. and 22.1 gal. working capacity.

4 fcife';^'.1 ^ ^ l-'io. 3.Propagation vessel of 17J brl. working capacity.

5 Vol. 58, 1952] WEBBER, COMPTON AND TAYLOR: PRACTICAL YEAST PROPAGATION 340 collection plant by a suitable mains system. Each vessel is jacketed for attemperation purposes, being supplied with brewery liquor (temperature 54 F.). Thermometers, safety valves, sample cocks, sight glasses, and in ternal illumination are provided. Vessels 1 and 2 are cylindrical with steamjacketed hemispherical bottoms and clamped loose lids. Their depth in each case is approximately twice their diameter. Y.P.V.l has a working capacity of 22J gal. and Y.P.V.2 one of 3 brl. (see Fig. 2). Y.P.V.6 is a totally enclosed vessel approx imately 8 ft. high by 5 ft. diameter with a working capacity of 17J brl. measured to the top hatchway which is lipped for yeast collection. It is fitted with an impellor rouser, a lower manway for cleaning opera tions, and an anti-vortex device at the beer outlet (see Fig. 3). Plant sterilization.this falls naturally into three main sections: (a) Process line (collecting vessels, pump, heat exchanger and wort mains). (b) Fermenting vessels (yeast propagation vessels 1, 2 and 6). (c) Compressed air mains. (a): The process line is dealt with by means of a sterilizing solution of a commercial quaternary ammonium compound, the whole line being flushed with and left full of the solution for as long as possible before use. The heat exchanger is dismantled for cleaning after every run. When the plant is required the solution is run to waste and rinsed away with cold liquor. Finally the system is scalded with hot liquor for 10 min. by pump ing through the sterilizer with the holding tube maintained at 212 F.; this is followed by cold liquor in preparation for the wort transfer. (b): The collecting and fermenting vessels receive the usual scouring and cleaning, which is assisted by hosing down with hot liquor as soon as possible after use. All moveable fittings are stored in a germicide bath until required further. On the morning of wort running, vessels Y.P.V.l and Y.P.V.2 are fitted up, and a quantity of softened liquor is introduced into each, to be boiled for } hr. by means of the steam jackets. The vessels are allowed to cool, and the water is run to waste immediately before the wort transfer. The large vessel Y.P.V.6 cannot be sterilized in this manner, and, after scouring and thor oughly hosing down, the various fittings are placed inside. The germicide solution is pumped along the process line, the day before wort running, in such quantity that sufficient remains in the dish of the vessel to provide a sterilizing bath for fittings and vessel inlet alike. On the following day the vessel is drained and hosed down, the process line flushed with cold liquor, scalding liquor, and finally with cold liquor again. It is advisable to give Y.P.V.6 a thorough sterilization every other week by completely filling with a solu tion of the germicide. The air main serving this vessel often becomes yeasty, and requires dismantling and cleaning after every fermen tation. (c): Air mains are sterilized in stages from a steam point on the compressor, each vessel being taken in turn as required in order to minimize condensation in the propagation room. A new-filter pad is placed in the air cylinder, the head is screwed down, and the compressor started in order to blow conden sate out of the air line. The sterilization of all sections of the plant is highly important. Experience demon strates that, without such sterilization, the resulting yeast crops may carry infection which will defeat the object of the whole process of yeast selection and propagation. In fact it is clear from the records of many propagations that the only failure experienced was due to inadequate plant preparation and sterilization. Obviously, different modes of sterilizing procedure will suggest themselves to different operators. Propagation room procedure.the 120 gal. of P.A. 1st wort, collected in C.V. 21 as previously described, are transferred to the propagation room on the following day, 20 gal. being run to Y.P.V.l, and 100 gal. to Y.P.V.2, the latter being left overnight with a trickle of liquor through the attemperating jacket. The wort in Y.P.V.l is boiled for 10 min., and then cooled back to pitching temperature, the whole process taking about 3 hr. After aeration for 5 min. the wort is ready for seeding with the laboratory culture, this operation being performed at F. Care is taken to effect the transfer from the laboratory culture vessel to Y.P.V.l under completely aseptic conditions, and the worts are thoroughly mixed after the operation by air rousing for 2 min.

6 350 WEBBER, COMPTON AND TAYLOR: PRACTICAL YEAST PROPAGATION [J. Inst. Brew. The fermentation is allowed to proceed for two days, and usually no attemperation is required. S.G. and ph values are recorded before, during and after the fermentation in this vessel and, in fact, throughout the whole process of yeast production. In the meantime, during the day before use, the 100 gal. of wort in Y.P.V.2 are boiled for 15 minutes by means of the steam jacket, thus receiving a second sterilization, and then cooled, a small trickle of attemperation liquor being allowed to flow again throughout the night. On the following day the transfer main is set up between Y.P.V.l and Y.P.V.2 Fig. 4.Collection of bottom yeast. and the wort in the latter adjusted to pitching temperature (64-65 F.) and air roused for 5 min. The fermenting wort is then blown from the smaller to the larger vessel, care being taken to transfer all yeast. The contents of the vessel are air roused for 2 min., and the fermentation is allowed to proceed for 24 hr., attemperation being rarely necessary. The 14 brl. of P.A. wort collected in C.V. 20 are pumped to Y.P.V.6 in the propagation room on the following day, and sterilized in so doing by passing through the heat ex changer at 212 F., the outgoing temperature being 65 F. During this particular steriliza tion the worts are aerated. The flow of wort is halted when Y.P.V.6 is about one-third full, top pressure is applied to Y.P.V.2 and its contents blown to Y.P.V.6, back washing if necessary to transfer all bottom yeast. Wort running is then resumed until the vessel is full to the lip, the hatchway is closed, and the contents are impellor-roused for 1 min. The temperature of pitching is usually either 64 or 65 F., according to circumstances, and in this vessel temperature is not allowed to rise above 67-6 F. though care is taken not to check back the fermentation at any stage. Skimming.The "dirty" head is removed through the skimming hatch by means of a rake on the morning following pitching. The first crop of top yeast is ready for skimming two days after pitching, and is removed by the same method into a yeast wagon. A further crop is taken off on the following day into the same wagon and placed in the yeast cold room for draining. The bottom yeast is now ready for removal, and preparation is made to collect from the bottom of the vessel by means of an assembly as shown in Fig. 4. On opening the valve the thick yeast siphons over into the tub, and is collected until it becomes diluted with beer. The operation is repeated on the following day, and any further top yeast is skimmed. After 24 hr. the vessel is tapped for the third time, all bottom crops being placed together in the cold room for draining. By this time the top yeast has drained, and is transferred to a yeast tub and weighed. Seven days after pitching, the beer in Y.P.V.O is blown to another vessel for racking and blending with the main volume of the gyle. A thick deposit of yeast remains behind, which is very clean, requires no draining, and is added to the bulk of the bottom yeast. The top and bottom crops together compose the yeast made available by the process for the first brewery fermentation with the new strain. As a rule 80 lb. of yeast are produced when P.A. wort (O.G. 1,041) is fermented in Y.P.V.6, although the crop has been as low as 50 lb. and as high as 92 lb. weighed as wet or liquid yeast, in which form the brewery pitchings are made. Such a crop may be produced each week and provides sufficient yeast for pitching a 100-brl. vessel. If it is preferred to use only top yeast from the propagation room crop there is as a rule sufficient (e.g., up to 40 lb.) to pitch a 50-brl. brewery vessel. Experience shows, however, that the mixed top and bottom crops result in a normal brewery fermentation. The whole process from laboratory culture to final collection takes 20 days, the programme being arranged to fit in with brewery requirements. At any one moment there are 3 propagations in process, and when working continuously in this way a weekly crop results. Storage.The yeast crops examined micro scopically present a healthy and vigorous appearance, and there is an absence of bacterial infection. After draining and until required for use, the yeast is stored in the

7 Vol. 68, 1952] WEBBER, COMPTON AND TAYLOR: PRACTICAL YEAST PROPAGATION 361 liquid or wet condition in the yeast cold store which is maintained at 44 F. Yeast transport.the plant initiates yeast for two breweries, the second and distant brewery being supplied from later fermenta tions in the first brewery. When required, this yeast is transported in the liquid condi tion in covered stainless steel tubs, a small insulated motor van being used for the pur pose. An ice-box is provided to maintain cool conditions during the 50-mile journey in very hot weather, but normally no cooling is necessary. Records.Each yeast is given a detailed code number by which it can be completely identified. Details of the laboratory pro cedure are entered in the yeast culture book, while in the yeast propagation room a record sheet is kept for each of the three vessels. These show all relevant details for identifica tion, together with dates, times, volumes, temperatures, gravities, ph values, etc. After the yeast has been passed into the brewery, entries on a record sheet follow each yeast through every brewery generation until the strain is rejected or its identity becomes lost. Without such records there is no insurance that the products of the propagation room will be fully utilized. TABLE I Propagation Room Data Wort at collection Wort after pitching After 1 day in vessel After 2 days in vessel After 3 days in vessel After 0 days in vessel Y.P.V.l F. S.G. ph (Then to Y.P.V.2) Y.P.V.2 F. S.G. ph (Then to Y.P.V.6) Y.P.V.6 F. S.G. ph * J (Racked) TABLE Typical Brewery Fermentations II Brew No. Code No. Yeast Pitchin ; data Weight (Ib.) Gyle Wort i Brl. Yeast crop Weight (Ib.) Bacteria per 10 fields Beer at racking S.G. ph 1 26/ P.A / P.A / P.A / P.A

8 352 WEBBER, COMPTON AND TAYLOR: PRACTICAL YEAST PROPAGATION [J. Inst. Brew. Typical propagation room and brewery data.the observations made in the course of a propagation are given in Table I, 80 lb. of yeast having been collected from Y.P.V.6 on this occasion, with a bacterial count of zero per 10 microscopical fields, each field including approximately 50 yeast cells. The Hyde (Trans. N.E. Eng. Inst. Tech. Brew., 1894, 3, 39). The scheme described above has now been in operation for over a year, and it is evident that the measure of pitching yeast control provided, and trade experience with the resulting beers, both give a high degree of uniform satisfaction. Tut tube Fksk fy 3 YEAST PROCESS LINE 3HBH. Y.RV I 22<iGal. WORT LINE Heat exchanger Wort pump Air compressor Fig. 5.Flow sheet, yeast propagation plant. yeast is followed through the brewery for 4 generations with results such as are shown in Table II. Conclusion.The scope of this paper does not include a discussion as to the advantages of pure yeast application in the brewery. This subject has been dealt with by Brown (he. cit.) and more recently by B. V. S. Seed (this Journal, 1952, 124), while early pioneer work was described by A. K. Miller & C. F. Synopsis.The sequence of events des cribed in the foregoing is illustrated diagrammatically in the flow sheet shown in Fig. 5. Acknowledgements.The Directors of Cour age and Co., Ltd., have given permission for this work to be published, and we desire to thank Mr. J. Grant for his enthusiastic interest and encouragement, and Mr. S. W. T. Paine for his expert advice on matters of plant erection and layout.