Yeast Propagation and Scale-Up
Yeast serial repitching is commonly done in breweries in order to reduce the costs associated with yeast. This process includes pitching (inoculating the yeast in the fermentation vessel), harvesting or cropping (i.e., collecting yeast from the fermentation vessel), evaluating the yeast viability, and re-pitching (i.e., pitching the collected yeast in a subsequent fermentation vessel). In some breweries, particularly traditional ale top-cropping types, single yeast cultures have been in use for many years. More commonly, however, and without exception breweries periodically introduce new cultures of yeast to replace existing stocks.
Replacing Yeast Cultures
There are a number of reasons for replacing yeast cultures. Flavor may be impacted, but other qualities are often affected first. Slower fermentation rates, reduced flocculation, declines in viability, and delayed diacetyl reduction are all indicators that may indicate poor yeast performance as a result of age. While yeast most certainly can and does undergo genetic mutations, a common reason in breweries that is frequently overlooked is selection. Selection of yeast occurs, to some extent, every time that yeast is cropped from a fermenter. Continued serial fermentation and cropping carries with it the risk that variants may be selected for within the yeast population.
Yeast cultures suitable for use as brewing yeast can be obtained in several ways. The most common is to have a regular or “house” strain stored by a company. These cultures are typically stored in liquid nitrogen to maintain genetic integrity. When a new culture is requested, the company that maintains the culture collection will subculture the yeast and send it out as either a fresh agar slant or in freeze-dried form. Freeze-dried (lyophilized) yeast cultures are usually shipped in a sealed glass container in either flaked or powdered form.
Direct Propagation from Freeze-Dried Yeast Culture
Direct propagation from freeze-dried yeast culture consists of re-hydration, inoculation of a Carlsberg Flask, inoculation of a yeast propagation vessel, pitching to a fermenter, and harvest. Re-hydration can be accomplished by using just sterile deionized water. At room temperature, the yeast should rehydrate thoroughly in about 2 to 3 hours. Once hydrated the culture can be aseptically added directly to the Carlsberg Flask that contains aerated sterile wort (Figure 4.5).
Carlsberg Flask - How it Works. The Carlsberg Flask consists of a cylindrical container with a flat bottom and top cover equipped with breathing filters, a membrane sampling valve for aeration and product transfer, and a micro sample port for aseptic introduction of pure yeast culture. It is recommended the flask is filled with wort to the net capacity (approximately 80% of the total volume) sterilized using an autoclave. The flask is then placed in a refrigerator or a cold room to cool the wort to the desired working temperature. The cold wort is aerated through the membrane sampling valve connected to the aeration lance. The cold wort is then aerated through the membrane sampling valve connected to the aeration lance.
Single Cell Culture
The brewer’s ability to pitch the correct number of yeast cells to initiate fermentation is crucial to consistently producing a product of superior and constant quality (Section 12.1). Pitching rates are governed by a number of factors, including yeast strain, fermentation capacity of the yeast, yeast viability, flocculation characteristics, previous history of the yeast, and desired beer flavor characteristics. Other considerations when choosing pitching rates include wort gravity, wort constituents, fermentation temperature, and the degree of wort aeration.
Rehydration of Active Dried Yeast
Rather than using a starter culture, as discussed above, the brewer can opt to use active dried yeast (ADY), as the name states, is dried yeast which once rehydrated will be reactivated. Active dried yeast must be rehydrated in wort or water, between 20 and 30 degrees C (68 and 86°F) for up to one hour, prior to pitching. The rehydration can easily be performed in existing yeast storage or pitching vessels and once the dried yeast is reconstituted into cream in such vessels, it can be pumped into the fermentation vessel containing wort at whatever temperature is used in the brewery.
The objective of the laboratory phase of propagation is to grow sufficient biomass to enter the brewery propagation phase without issue. This is achieved by serial transfers of increasing culture volume for scale-up. The number of transfer steps in the laboratory varies according to the final weight of yeast required for the propagation plant. Of course, the more transfers, the greater the risk of infection.
Ideally, the propagation should be completed in a controlled and sterile environment. Preferably the room should be tiled or some other surface suitable for cleaning and sanitizing. Walls, floor, ceiling sprayed with suitable sanitizer at regular intervals. A dehumidifier keeps moisture levels low, helping prevent unwanted cultures from growing on surfaces.
The key to successful laboratory propagation includes:
A typical brewery yeast propagation schedule is described as follows, but details will vary greatly with the size of the brewery and the particular propagation equipment available. Laboratory propagation for yeast is listed below
Once the lab completes propagation in small stages, it transfers the culture to the brewery where it is added to a propagation vessel filled with sterile wort and aerated with sterile air. Wort may be sterilized, in situ, in the holding tank by application of steam to external jackets. Alternatively, the wort may be sterilized by passage through a heat exchanger during filling. In the absence of a holding tank the wort may be sterilized in the propagation vessels themselves.
Temperature and Oxygenation
The only means of regulating yeast growth and extent is by manipulation of temperature and oxygenation. There is a wide variety of recommendations in propagation temperatures. Some brewers prefer to propagate their yeast at temperatures identical to those employed during fermentation in order to prevent temperature shock to the yeast (Hough et al., 1982). Others will start out at slightly higher temperatures with intermittent aeration to stimulate growth, dropping the temperature at each step until normal fermenting temperatures are reached at plant scale (Maule, 1980).
Batch versus Semicontinuous Systems
If fresh yeast for use in the brewery is only required every 3 weeks or propagate different strains, the one- or two-tank batch system is fully sufficient and economically preferred. The disadvantage with this process, however, is that it involves much laboratory work, since propagation must be started each time in the laboratory.
Basic elements to consider when designing an optimal propagator system include the following: (1) a composition of stainless steel, with highly polished internal surfaces so that cleaning and sterilization are easily done; (2) there shouldn’t be any “dead legs,” and everything should be designed to the highest hygienic standards; (3) a means to efficiently introduce, measure, and control a sufficient supply of air; (4) a means for controlled homogenization of slurry/wort mixture; (5) an ability to control and adjust temperature via jacketed walls; (6) an ability to apply top pressure (0.5–1.0 bar) to control foaming and prevent contamination; (7) a built-in freeboard to contain the foam head; (8) an ability to measure and retain volume/mass for recharge; (9) a dedicated clean-in-place (CIP) system; (10) an inoculation point for the Carlsberg Flasks, should be included; and lastly, (11) the system should contain a sterile and coalescing filter for aeration with the capability to clean and sterilize the housings (Maca, 2015).
One-Tank System. Small breweries typically use single-vessel propagation plants. A single-vessel propagation plant utilizes one vessel to sterilize, aerate, and cool the wort, followed by inoculation with the laboratory culture in the same vessel.
Two-Tank System. A two-tank plant utilizes one vessel for wort sterilization and cooling and second vessel for the actual propagation (Figure 4.6). The two-tank process is a self-sufficient, self-contained system. The biological safety is ensured by possible wort sterilization. .
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