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Laboratory propegation and Brewery propegation of yeast (SOPs)

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  • Laboratory propegation and Brewery propegation of yeast (SOPs)

    This SOP is intended for people who have ample knowledge in micro, lab, and sterile techniques. You must also be comfortable and able to work with agar petri dishes and culture media without contamination issues.


    The Goals of this SOP is to.
    1. Describe the laboratory step-up procedure of yeast from a pure culture (from a slant of ordered culture) to a large enough volume to pitch the 3 barrel propagator

    2. Detail Yeast Propagation in the 100 Gallon Propagator

    3. Deliver 1st generation yeast to the brewery at pitchable quantities(40-60bbl fermentations) in a short period of time(about two weeks) without the cost associated with buying large pitches

    First of all you'll want to obtain a clean culture from your culture collection (Refrigerated petri dishes with colonies, Slants, or your LHBS vials)

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    streak out your yeast onto YPD(yeast peptone dextrose) agar plates. and incubate them at 22-24C for a couple days until you get nice distinct isolated colonies. It will take some practice to achieve good streak plates if you've never done this before

    The objective here is to get nice individual colonies to pick, these colonies will be the inoculant for 100mL flasks as the first stage of propagation.

    you want to avoid petite colonies and ones that otherwise look out of the ordinary.

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    picked colonies will get transferred directly into 250mL flasks filled with 100mL of sterilized wort with very low BUs or none.
    The wort you use for propagation should be about 8-12 plato

    as you can see in the above pictures 500mL media bottles were used to collect and autoclave the wort. the flasks were autoclaved separately with their stoppers on a dry cycle. then the sterilized wort is transferred into the flasks leave the trub behind, remember good sterile technique in transferring the sterile wort to the flasks, I like to transfer the wort at boiling temperatures into the flasks and then let it cool in the flasks. You can see the trub left behind in the media bottles, you don't want to let that stuff get into the flasks.

    Pick a bunch of colonies from your YPD plate with a sterilized inoculation loop and inoculate the wort in the flasks, flame the lip of the bottle for transfers and inoculation (good micro technique)

    target for inoculation is 15-20 million cells per mL
    Incubate the flasks with agitation on an orbital shaker at 150 RPM for 2-3 days at 22-24C

    you should end up with 100-200 million cells per mL
    I hope I encouraged you!

  • #2
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    if you go much above 12 plato with your propegation media(wort) you will get fermentation even if you supply air to the yeast(aerobic)
    though the effect happens at a half a plato you still really don't want to go above 12 or so with the prop media.


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    as said above it's a good idea to leave the trub behind after autoclaving the wort you will use for propegation.
    Last edited by Yeast; 04-01-2016, 07:56 PM.
    I hope I encouraged you!

    Comment


    • #3
      now that we have the 250mL flasks grown up they should look nice and "milky"

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      as you can see we will do the same practice again scaled up, so you'll need more wort sterilized, bigger flasks sterilized etc...

      the 250mL flasks get moved up to 1L flasks which then in turn are used to inoculate 4L flasks

      the 4L flasks are used to inoculate the propegator in the brewery

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      once the 4L flasks are grown up I use 4 of them to pitch the 3bbl propegator.
      the 4L flasks have 1L of yeast slurry in them at usually about 150-200 million cells per mL

      (200cells/ml) * (4000 mL) = (? concentration) * (354,000mL) (because 4L + 350L is the total volume)
      800 billion cells = (? concetration)(354,000mL)
      2259887
      cells per each mL

      that's 4L at 200 cells into 350 liters which is pitching at about 2 million cells and that's plenty appropriate for the propagator
      Last edited by Yeast; 02-25-2017, 01:25 PM.
      I hope I encouraged you!

      Comment


      • #4
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        here is the 3bbl propegator

        it's set up like this

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        as you can see the red hose is the air line, it's fed into a sterile filter and a regulator. Ideally you should filter the air before it goes to the regulator but I pasteruize/sanitize the regulator 180F or hotter water(not ideal but it works)

        the regulator goes to an airline that pumps through the bottom cone for agitation and air to keep the yeast aerobic. I find that 10PSI works well for this.

        I also keep the regulator above the level of the liquid in the propegator, even though there's an inline check valve, this is an additional measure to prevent any backflow to the filter or regulator.

        to sanitize the propegator I turn the hot liquor tank up to 180F or higher. Then I attach a TEE with a sight glass to the bottom of the propegator as seen in the first picture there. I fill the propegator til overflowing then keep going until the thermometer on the tank reads at least 170F and hold it with the water in there for at least 10 minutes. Then I close up the top ports and the manway. and dump out the hot water.

        To fill the propegator we use the beer being brewed that day. If it needs to be watered down to get to 12 plato for propegation I'll leave some of the hot liquor used to sanitize the propegator in the propegator. Ideally you would steam sterilize the propegator but that's not an option right now with this setup.

        The wort comes from the brewery heat exchanger at ~68-72F. if you had to dilute the wort to get to 8-12 plato you'll have to turn the glycol or city water to the jackets on to get it back down to propegation temperature 68-72F for ale yeast


        the ports on the propegator are not very important but having two ports on the top is ideal. The CIP port with the spray ball removed acts as the inoculation port

        the other top port acts as the gas arm and has a ball valve placed on it for inoculation(i'll explain)

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        I hope I encouraged you!

        Comment


        • #5
          To inoculate the propagator I put 70% IPA in a wash bottle.
          I grab a torch and a lighter
          and get my 4 4L flasks of yeast

          you can see in the last post that you can fill the ridge on the TC fitting with IPA and flame sterilize the "inoculation port" on the propegator

          I also use that ball valve I put on the gas arm and shut it off at this time. I've already attached the air to pump through the bottom of the propegator, when you close the gas arm valve the air creates a positive pressure and your inoculation port is always going to have a net positive pressure then blowing air out of the vessel during inoculation greatly reducing the chances for infection.

          I flame sterilize the lips of the 4L flasks one at a time. and flame the inoculation port on the top of the propegator and then slowly and with great dexterity pour them in one at a time.


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          Ready for inoculation

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          inoculated

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          and now you're propagating
          I hope I encouraged you!

          Comment


          • #6
            now after 2-3 days you should reach a cell count of 150-250 million cells.

            where american ale yeast usually grows to 100-200 at 70F
            I find that Wyeast 3711 (french saison) grows to a potential of about 250million at 75F (saccharomyces delbrueckii)
            and lager yeast does well too but propegate at lower temperatures like 60F

            you can crash the propegator after it reaches peak cell count to retain the high glycogen content, I'll crash it to 45F and you can get a slurry off the cone of about 600-900 million cells per mL with most yeasts if you don't want to push the whole 3bbl volume forward (cone to cone)

            I suggest pitching immediately after peak cell count is reached(during the same day it hits not the same hour) the whole 3bbl can go in to the new fermentation or like I said crash it and harvest

            since you have 3bbl in the propagator and your cell count is say 150million that means you have enough yeast to pitch these examples
            assuming you want 750,000 cells per mL per plato for your ale fermentation

            30bbl at 20 plato
            60bbl at 10 plato

            you could also pitch bigger batches say you have a brew length of 40bbl and have a 120bbl fermentor
            you could split that up over 2 days do a AM brew then the next day do AM PM. the first day brew will grow the yeast enough to deal with the next days fill it's not the most ideal but it can work in a pinch

            I suggest doing 30-60bbl batches and then re-using the yeast for larger batches if you need.

            if there are any questions, comments, or concerns feel free to contact me here in this thread or in private message
            I know I left out a lot of details but if you're able to perform propegation you should already have the skills you need, this is more of a proof that it can be very effectively done in a modest sized craft brewery 10K-100K bbl per year, with multiple yeast strains.

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            remember to re-streak and re-slant your cultures regularly and since you just made 1st generation yeast it's a good idea to rotate the new stuff into your culture collection every time you prop. since you'll be taking a sample for micro anyway
            Last edited by Yeast; 03-20-2016, 05:11 PM.
            I hope I encouraged you!

            Comment


            • #7
              Yeast Propagation

              Interesting yeast propagation procedure, however I have done it with some differences, instead of the YPD agar, we used wort agar to propagate and keep the yeast and place it in the incubator at a higher temperature.
              The wort used is exactly the same that is brewed, 2000 ml are sterilized. Place 1000 ml in a bottle with a 2000 ml capacity, pitch the selected yeast, stir and keep at a higher temperature, later after a day add the other 1000 ml. This can be divided.
              Later, this propagated yeast in the wort is taken to the yeast propagator (Assimilator) to continue with the propagation and produce enough yeast for a 320 HL open fermenter. Besides to determine the yeast cell count, it was done with the use of a spectrophotometer.

              Likewise, at the brewery I am working, the yeast is bought, grown in 10BBl or 20 BBL yeast brinks and enough yeast is produced to pitch 120 BBL or 240 BBL fermenters. For the 960 BBL fermentation vessels the 20 BBL brink is used and then grown in a 120 BBL fermenter.

              Comment


              • #8
                wort agar

                wort agar does work great but at our brewery we make so many different brands that the wort agar would be inconsistent at the primordial stage of growth. YPD has predictable morphology for yeast colonies. our wort could come from anything from a light ale to a imperial stout and would need to be watered down etc...

                Wort agar does work great though. YPD is very cheap and is almost the same price as just buying agar powder (unless you go to the asian grocery store and buy the telephone brand stuff)

                there's many ways to go about propagating but the company already had the 3bbl propagator at my disposal so I used what I had. as far as the lab side of propegation goes it's mostly taken from MBAA and ASBC guidelines and adopted to the equipment available.

                I use wort for the propagation flasks and for the 3bbl propagator. I could make YPD broth without the agar-agar but since wort is such a easy resource to come by at a brewery it's used. YPD is just good for streaking out colonies since it's always going to look a predictable way.
                I hope I encouraged you!

                Comment


                • #9
                  Wort Agar (W├╝rzeagar) description

                  Attached is the wort agar description, sorry it is in German.

                  Wurze Agar 356e.pdf



                  In the USA you can obtain the wort agar from the Brewing Science Institute.

                  http://www.brewingscience.com/
                  Last edited by Fausto Yu-Shan; 03-20-2016, 09:19 PM.

                  Comment


                  • #10
                    Originally posted by Fausto Yu-Shan View Post
                    Attached is the wort agar description, sorry it is in German.

                    [ATTACH]31720[/ATTACH]



                    In the USA you can obtain the wort agar from the Brewing Science Institute.

                    http://www.brewingscience.com/
                    Mein Deutsch ist sehr scheiss zu sprechen.. Aber wenn es lesen ist, kann ich ein bisschen besser.

                    Maybe that made no sense. It's been years...

                    I've seen BSI's wort agar before. I guess it didn't cross my mind you were talking about the product rather than the home made stuff

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                    this GW kent 3bbl set up as a propegator can achieve some pretty thick slurries when crashed to the mid 40F range. 1272 recently yielded 80L of 8.85x10^8th slurry with the propegator filled to 3.5bbl (with anti foam) of ~10 plato wort
                    Last edited by Yeast; 03-20-2016, 09:56 PM.
                    I hope I encouraged you!

                    Comment


                    • #11
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                      here's all 3 sizes of flasks 250ml 1L and 4L

                      the 250ml are ready to be transferred to the 1L

                      and I'm going to combine the four 4L into just two 4L flasks since I don't have the room in the incubator shaker.

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                      the 250mL flasks have lager yeast in them right now it's extremely flocculant and as soon as you stop agitation it starts to floc out.

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                      all 3 250mL flasks transferred into the 1L flasks


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                      the four 4L flasks had 1L each in them. There's no room on the shaker for all these flasks so the big ones were consolidates so there is now two 4L flasks with 2L each in them of Wyeast 3711 which grows to 250 million cells in propegation easily. the lager yeast usually hits 175-200.
                      I hope I encouraged you!

                      Comment


                      • #12
                        Thank you so much for taking the time out to put this together! We're looking to start propagating in house for cost/availability reasons and I'm tasked with it. We're only looking to do a 10 HL pitch so even a smaller vessel would suffice. I am fairly comfortable doing propagation with flasks so one way we can achieve this right now is to ferment 'half' a batch and then use the harvested yeast from that for successive batches. However from a production stand point it's a little inefficient and it might make more sense for us to get a smaller propagation vessel instead of locking up a large conical. I would like to get a smaller ~2 HL dedicated vessel for yeast prop but I am still trying to wrap my head around the logistics of it, aeration being one of my biggest concerns. Does simply bubbling pure, filtered O2 through the dump port work well or will a sintered diffusion stone be necessary? I was thinking of using one of these and modifying them.

                        Comment


                        • #13
                          Originally posted by chinsi View Post
                          Thank you so much for taking the time out to put this together! We're looking to start propagating in house for cost/availability reasons and I'm tasked with it. We're only looking to do a 10 HL pitch so even a smaller vessel would suffice. I am fairly comfortable doing propagation with flasks so one way we can achieve this right now is to ferment 'half' a batch and then use the harvested yeast from that for successive batches. However from a production stand point it's a little inefficient and it might make more sense for us to get a smaller propagation vessel instead of locking up a large conical. I would like to get a smaller ~2 HL dedicated vessel for yeast prop but I am still trying to wrap my head around the logistics of it, aeration being one of my biggest concerns. Does simply bubbling pure, filtered O2 through the dump port work well or will a sintered diffusion stone be necessary? I was thinking of using one of these and modifying them.
                          I had simply bubbled filtered AIR through the bottom. I wouldn't use O2 unless you can measure it very accurately
                          "The regulator goes to an airline that pumps through the bottom cone for agitation and air to keep the yeast aerobic. I find that 10PSI works well for this.

                          I also keep the regulator above the level of the liquid in the propegator, even though there's an inline check valve, this is an additional measure to prevent any backflow to the filter or regulator"
                          I hope I encouraged you!

                          Comment


                          • #14
                            Thanks for Sharing it !!!!!

                            Very Well Written, Good Information, Thanks for Sharing it !!!!!
                            Gregg
                            Stockton

                            Comment


                            • #15
                              A few questions

                              I am looking at bringing yeast propagation in house, and have been doing quite a bit of research over the last 2 weeks, so i appreciate the post, but have a few questions:

                              At what point(s) do you collect prop samples for micro analysis before pitching?

                              What Media do you use to look for spoilage organisms during propogation phase and what are typical incubation times?

                              I am considering supplementing olive oil (in addition to shaker tables) into wort to help with yeast building up cell mass during early stage of prop (10ml-10L), but was wondering if that would cause the yeast to loose some of the genes early on for aerobic respiration (not familiar with yeast genetics enough to know unfortunately) and was wondering if you or anyone else on this forum had experimented around with it. Link to article explaining this here:

                              https://byo.com/malt/item/1206-olive-oil-aeration

                              How many generations are you able to get out of your yeast propogating it this way and how often do you reculture your slants/yeast sources?

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