酵母的长期冻存技巧

jianguowu

Homebrewtalk看到的一篇短文，内容概要是说如何有效的长期的保存酵母，以便在下次发酵的时候使用。

转发的目的是想告诉亲们，这个方法是好使的，有条件真可以试试，特别是对于一些来的珍贵的液体酵母。

我的经验是准备50%(比下文的要高)的食用甘油（灭菌），1：1加到制备好的酵母悬液中（最后的密度约0.5*10^11），混匀后慢冻于-20度冰箱中，保存越低越好。我不建议用下文的异丙醇，购买和使用上很麻烦，如果有卧式冰箱，你可以象我一样，先把细胞管放在冰箱的靠上部分，再慢慢下移到底部。虽然甘油在接种时含量很少，同时它在微生物培养过程中常以慢速碳源来使用，但解冻使用的时候仍建议您再洗一次为好！

仅供参考。

原文如下：

Freezing Starter-Sized Samples of Yeast for Long-Term Storage

Written by Brewitt with contributions from HBT members

WHAT: The purpose of this article is to summarize what has been learned about freezing yeast by a number of contributors to the thread "Do you know how to make a yeast starter? Then why not farm yeast and freeze it?" (http://www.homebrewtalk.com/f163/do-you-know-how-make-yeast-starter-then-why-not-farm-yeast-freeze-269488/) started by, and with extensive contributions from, BBL_Brewer. Other major contributors include Brewitt (me), Forkhead, katy bug, ScoRas, and IsItBeerYet. Many others have made contributions that can be read in their original form in the thread. Together, through reading and experimentation, we have come up with an effective and relatively simple method for long term storage of pitchable quantities of yeast in either standard frost-free freezers or, better yet, non-frost-free freezers often used for food storage. Keep in mind that some of the recommended procedures have not been fully tested and will undoubtedly be undergoing some modifications but the method outlined below works well. Some modifications and additional considerations are listed at the end of the article.

Figure 1: Tubes of frozen yeast. These were prepared from a starter of yeast from a bottle of Sculpin IPA (Ballast Point Brewery) and the yeast from a fermentation started with a bottle of Pranqster Belgian Golden Ale (North Coast Brewery). These have been stored in a non-frost-free freezer for 7 and 9 months, respectively.

WHY: The method(s) described below is sufficient for storing cells in portions large enough to pitch directly (50-200 billion cells, similar to the amount in a smack pack from Wyeast or a tube from White Labs) (See Figure 1). Nevertheless, we recommend an overnight starter to ensure a large number of viable cells and rapid initiation of fermentation. The cells to be frozen can be from a large starter culture made from a tube or pack of commercial yeast. A single large starter can be broken up and saved for multiple future brewing days. A similar approach can be taken with a small starter made from yeast recovered from commercial bottle-conditioned beer or home brews that you would like to save for later. Finally, the yeast can be washed cells from a recently completed fermentation (a single 5 gallon fermentation can provide starter samples for as many as 10 future brews). Keep in mind that you will save $7-$10 per 5 gallon batch of beer each time you pull a tube out of the freezer.

GOALS: The goals of the method that we have devised are three-fold:

• To allow the preservation of large samples of cells in freezers in common use by homebrewers
• To use materials available to homebrewers at a reasonable cost
• To achieve high viability of stored cells for weeks, months, or perhaps years
  

Figure 2: Examples of tubes useful for freezing yeast.Sanitized used White Labs tubes and 50 ml screw cap conical tubes (see Resources).

jianguowu

SUPPLIES: Materials required for preparation of frozen cells:
A large quantity of yeast culture that you want to store for future use. Any "clean" yeast culture can be used (for example: one or more quarts/liters of aerated or stirred starter culture; washed cells from a 5 gallon fermentation (see "Sticky: Yeast Washing Illustrated") (http://www.homebrewtalk.com/f163/yeast-washing-illustrated-41768/)).
Sanitized tubes for freezing samples of yeast. This could be plastic White Labs tubes saved from prior purchases (these are apparently also called "baby soda bottles"); 50 ml plastic screw top tubes commonly used in laboratories (see Resource section at the end of this article), any other small freezable container that can be sanitized and tightly sealed. See Figure 2.
Some form of tube holder.
Glycerin, food grade (must be diluted in sanitized water before use; a rate of 1 part glycerin to 4 parts water gives a 20% final concentration). Glycerin, glycerine and glycerol are all the same thing. See Resource section at the end of this article. See Figure 3.
Isopropyl alcohol (also called "rubbing alcohol", available at any drug store), freezable gel packs, other good insulator for tubes of yeast.
Container large enough to hold multiple tubes of yeast that will hold alcohol or gel packs. An insulated container is best (small Styrofoam ice chest or discarded shipping box for frozen goods).
Sanitized water

Freezing Yeast - Brewitt - figure-3-240.jpg
Figure 3: Glycerine for freezing yeast.One example of glycerine suitable for freezing brewing yeast (see Resources).

HOW: A single method is outlined. Alternatives are discussed in below.

Freezing:

1. Collect yeast by chilling the culture or washed cells in a refrigerator without disturbing for 4 hours or more (preferably overnight). Pour off spent wort or washing water leaving just enough to allow cells to be suspended as a very dense slurry. The goal is to get sufficient cells for an overnight starter culture in a volume that is convenient to freeze. A good sample size is about 50-200 billion yeast cells (0.5-2 x 1011 cells). One quart of starter culture can usually be reduced to 1/40th the volume or less giving you about 100 billion cells in 25 milliliters (about 1 1/2 Tablespoons).

2. Split up the slurry of settled yeast into samples of about 100 billion cells (cells from about 1 quart of starter or a half gallon of fermented beer) in sanitized freezable screw top containers. That amount of slurry should fill the container less than half way.

3. Add an equal volume of chilled 20% glycerol to each of the samples of yeast and cap tube tightly. Swirl or stir until fully mixed without frothing. This will lead to a final concentration of glycerin of 10%. Concentrations of glycerin higher than 15% appear to be detrimental to cells when stored at home freezer temperature (20C, -3F). Make sure there is sufficient room for expansion during freezing.

4. Loosen the caps of the tubes and place into the large container, standing up to avoid spilling, and add enough isopropyl alcohol to the large container submerge the tubes to the level of the glycerin solution but not to the level of the cap. You don't want isopropyl alcohol mixing with your yeast. If using gel packs, make sure tubes are completely surrounded by the gel.

5. Place the container in the freezer. The isopropyl alcohol or the gel packs will slow the freezing of the cells, which is an advantage for maintenance of viability, and will avoid thawing during frost-free cycles.

6. After freezing is complete, tighten the caps of the tubes. If you are in a non-frost-free freezer, you can remove them from the alcohol and save it for a future use.

Thawing:

1. Remove tube of yeast from the large freezing/storage container.

2. Immediately submerge tube in water at approximately body temperature (37C, 98F). This can be running warm water or a large volume of water for submersion of the tube. Swirl continuously until completely thawed.

3. Remove tube of cells from the water, wipe dry with sanitizer and pitch into 1-4 liters of wort to prepare an overnight starter. In general that inoculation of the starter will grow to maximum density within 12-24 hours with appropriate shaking or aeration.

Other useful considerations and information:

Keep in mind that the cleanliness of your starter culture, washed yeast, and freezing supplies is key. Your subsequent beers will only be as good as your starting yeast strain and any contamination in your starter culture will be perpetuated in brews made with the frozen yeast prepared from it. If you have a beer with flaws due to contamination, don't freeze the yeast for later use. Also, yeast blends and blends of yeast and bugs will not necessarily be present at the same ratios after growing starters or using them for a fermentation. They can be frozen but results may vary.

Isopropyl alcohol for freezing and for storage can be cumbersome. The goal is to freeze slowly and avoid thawing during frost-free cycling in home freezers. Another approach to slow freezing is to chill in a refrigerator overnight and then to place in an insulated container like a Styrofoam ice chest or box and place in the freezer. It is useful to use gel packs or some similar frozen insulator when storing in a frost-free refrigerator.

Glycerin is a cryoprotectant. It helps maintain the viability of frozen cells. We have tested final concentrations from 7.5% to 50% and found 7.5%-15% to be optimal for maintaining viability (upwards of 75% viability after several months of freezing). Lower final concentrations may also work but higher concentrations are detrimental.

Glycerin can be added to the yeast slurry before or after splitting into smaller portions. If you want a more concentrated slurry, you can pour off more spent wort and/or resuspend your slurry in more concentrated glycerin to achieve a final concentration of 7.5%-15%.

If you have yeast that have been settled at room temperature, you can add glycerin solution that is at room temperature and then chill.

Although yeast can be used directly, this is likely to lead to under-pitching and a slow start to fermentation.

Resources: (The following are meant as examples, not necessarily recommended vendors or products)

Tubes for freezing yeast: Some examples from Amazon
http://www.amazon.com/Centrifuge ... plastic+tubes+50+ml

http://www.amazon.com/Karter-Sci ... trifuge+tubes+50+ml

http://www.amazon.com/150MM-5-75 ... s=baby+soda+bottles

Glycerin:
http://www.dudadiesel.com/search.php?query=glycerin

http://www.starwest-botanicals.com/category/vegetable-glycerin/

http://www.bulkapothecary.com/glycerin.htm

Gel Packs:
http://www.amazon.com/Hot-Cold-P ... freezable+gel+packs

http://www.amazon.com/Vesture-Pa ... s=hot+cold+gel+pack

chiongwong

好帖～～要能翻译一下就更好了

gnomonshadows

求翻译啊！

可乐加冰

零下20度！ 估计光指着冰箱够呛

wujianguo

谢谢chiongwong，这两天家里的小宝贝生病了，得照顾她。我译一下作者的操作部分吧，希望大家能看得懂，要是你们在没有翻译前还有些明白，看过我翻译后就糊涂了，这就哈哈了。
希望大家指正！

Freezing:
酵母冻存：
1。静置酵母于冰箱冷藏柜4小时以上（最好是过夜）来收集扩培或一发后洗涤回收的酵母。静置结束后，倒出了大部分麦汁或洗涤的水，留下少部分液体，足够让细胞重悬成酵母悬液就可以了。这步的目的是用使用少量液体获得高密度的细胞，小体积便于冻存和下次发酵前的过夜培养。一个比较好的样本大小约500-2000亿的酵母细胞（0.5-2×1011细胞）（注：基本上是20L体积投放的量，前面也说了，相当是一包11.5g商用酵母）。950ml过夜扩增的发酵体积通常可以减少到1/40体积或更少。约1×10^11的细胞在25毫升的液体中（约1.5汤匙）。
2。将酵母悬液分装到可冷冻的螺口的无菌离心管中，每管的酵母数量在1×10^11左右。（这个数量约1夸脱-950ml过夜扩培的酵母或半加仑-1.9L的一发后的啤酒产的酵母*注：这个我觉得可能会不同）。酵母悬液的装量不应超过管最大容量的一半。
（管子大家可在在淘宝搜索：离心管 无菌 50ml，这是这个样子）
3。在每个管中添加与酵母悬液相同体积的预冻的20%甘油，盖紧帽管。涡流（这个混合方式需要设备）或搅拌直到充分混匀，混匀过程中尽量不产生泡沫。这样冻存的最终甘油浓度为10%。存放在家里冰箱的温度（20C，- 3F）条件下，甘油浓度在15%以上时，可能对有害细胞。在液体冷冻过程会膨胀，确保管子上部有足够的空间。
4。旋松管盖，直立的放进大的容器内，此时应避免液体溢出，然后在加入足够的异丙醇大容器里，注意异丙醇的液面，防止漏入管中。如果使用凝胶包，使酵母管完全被凝胶包围。
5。把容器放在冰箱里。异丙醇或凝胶包会减缓细胞的冻结的速度，这个对维持酵母的活力是有利的，同时在冰箱在除箱周期可避免酵母被冻溶。
6。冷冻完毕后，拧紧管子的盖。如果是在一个无霜的冰箱，你可以把他们移出异丙醇，备用。

Thawing:
细胞复苏：
1。从冷冻贮存盒中取出酵母管。
2。立即淹没在37度水浴中（37度，98f）。这可以流动的温水或者一定体积的水浴锅中。摇动管子直到完全融化。
3。从水浴中取出管子，用消毒液擦干管壁周围的水并投放到1-4L的麦汁中进行种子扩培。一般来说，在适当摇动或充气下，接种的酵母将在12-24小时增长到最大密度。

gnomonshadows

马克一下，多谢！

悠闲的狮子

大家好，一起学习交流啊
来自: 微社区

liouyingjiei

看得非懂似懂的，超出我的理解。就是好贴