Author Topic: Propionibacteria - Propagating  (Read 3752 times)

Offline linuxboy

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Propionibacteria - Propagating
« on: November 30, 2009, 07:58:38 PM »
I've seen some mention here, maybe by Dave (Likespace), about spending a small fortune on Propionibacteria. Francois and I have both posted some methods for propagating cultures, such as making bulk mother culture and duplicating cheese styles by using commercial rind flora. I wanted to follow that up with a discussion on how to propagate Propionibacteria. This process applies to the common strains such as shermanii, as well as unclassified strains. In most cases, you will use the process to take a small amount of commercial DVI starter and multiply it to make your dollars stretch.

A few more notes and then the process. One, this does take a moderate initial investment. Two, if you failed biology/chemistry, don't worry, you can still do this, but pay very close attention to having a sterile environment. Three, propagation works best if you can use the culture quickly, like you would for a bulk culture. You can freeze this, but it will decrease viability. If you want to increase viability, I can post a followup of how to centrifuge the broth and preserve in glycerin in the freezer.

OK, enough theory (almost). The basic idea of propagating any culture is to give the bacteria enough food and the right temperature so that it can thrive. With Propionibacteria, the right food is a sodium lactate broth, and the right temp is 30 degrees C (86 F). Meaning to propagate, you need to inoculate the broth and keep it at 30 degrees. The length of time to propagate depends on a few factors. One, it depends on how much you start with. With a 1% inoculum, you can achieve culture saturation in as little as 3-5 days. With less, it may take 7-9 days. If you are money conscious, it is better to err on the side of too long than too short, within reason. Don't go too far past a week.

To prepare sodium lactate broth (SLB):

Ingredients:
10 g tryptone, or similar pancreatic digest of casein (this is 1% wt/vol)
10 g yeast extract (this is 1% wt/vol)
10 g sodium lactate (again, this is 1% wt/vol)
(optionally) .5 g of tween 80, this is an emulsifier, aka polysorbate 80
(optionally) .25 g K2HPO4, this is a buffer
1 L distilled H2O

Process:
 
1) Mix everything together in water and stir to dissolve a bit.
2) If you have an autoclave, heat to 121 C for 15, or follow your lab's procedure for broth prep. If not, boil for 20 mins and transfer to aseptic container, or as aseptic as you can find.



To culture Propionibacteria:

1) Take the broth, heat to 30C, put in anaerobic container that lets off gas, such as a bottle with a stopper and wine bubbler lock filled with alcohol, and add 1% inoculum. Measure the powder out beforehand. If you're using an anerobic kit, you're fine, but if you're using a stopper with bubbler, purge with CO2 or argon to get out the oxygen.
2) Keep at 30 degrees C for at least 5 days.
3) Put in fridge and use up as soon as you can. At 1C, you should still have some viability even after 6-9 months. At least enough to reculture the next batch.


I'm anticipating some questions, so I will try to answer them beforehand.

Q: Do I have to use sodium lactate broth? That's a lot of stuff to buy.
A: No, you can use MRS broth. It's almost as good. Propionic is picky, so those are your two best choices. For anyone who is a Simpsons fan: "Troy: If you can't find metal stucco lath, Homer: Uh-huh? Troy: Use carbon-fiber stucco lath! Homer: Uh-hohh! Troy: Now parge the lath!"

Q: Where do I get this stuff?
A: Look online. The major biology supply houses like cole palmer and VWR should have culture media. If all else fails, just buy MRS broth. Make sure you get the broth and not agar.

Q: How much should I use?
A: This one is tougher to answer because your colony size will depend on how much inoculum you use, how many days you let it grow, the style of cheese, etc. As a starting point, try 10-20 grams per 2-4 gallons for an emmenthaler style. This is about a teaspoon per gallon.

Q: What is the least amount of equipment that I will need?
A: Gram weight scale for solids, graduated measuring device for liquids, bottle with stopper and bubble lock. Also storage containers for prepared broth and cultured broth.

Q: Can I reculture from an existing cheese to isolate a swiss-type wild or native strain?
A: Yes, but this is time consuming, requires more equipment, and for this one you really should take a few classes in biology that have lab sessions.

Q: How much money can I save?
A: You really do need to be a serious swiss cheesemaker for this to be worthwhile considering that you can make about 80-100 lbs of cheese for $10 in Propionic culture. But, to make the same amount in broth form would take something like 10-30 grams of broth base, which costs about 10 cents/gram. Meaning for the average person, not very much. And it's a lot of hassle and takes time. For the maker of huge wheels or for someone who already has the equipment and also cultures using bulk starter, it's more worthwhile.

Q: Can I use this process to boost viability of my propionic and to add broth instead of powder to the milk?
A: Of course. This may be a sweet spot for the home cheesemaker. Use 1/5-1/10 the amount of DVI and make up a small batch of broth to use for a batch of cheese. No need to maintain cultures that way and you can stretch the commercial stuff. For this method ferment the propionic for 2-4 days beforehand.

Q: Can I use this method to reculture other single-strain commercial starters or propogate lactic bacteria.
A: Yes, in theory. At home, it's easier to use skim milk or reconstituted skim milk enriched with some yeast extract, though. Cheaper, too.

Hope this helps. I know some here have asked about more details on reculturing and propagation, and that's the gist of it.
« Last Edit: March 15, 2011, 10:20:13 AM by linuxboy »
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Offline DeejayDebi

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Re: Propionibacteria - Propagating
« Reply #1 on: November 30, 2009, 09:31:44 PM »
Excelent Linuxboy! Very interesting.

Offline tina

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Re: Propionibacteria - Propagating
« Reply #2 on: January 01, 2010, 12:46:09 PM »
Thanks for helping me understand what a good deal the prepackaged cultures and flora are.... and I was starting to think they were expensive.

I used to save and culture beer yeasts and thought maybe cheese cultures would be similar, (a fairly simple process), but it sounds like it's not worth the trouble for a very part-time cheesemaker.


Offline linuxboy

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Re: Propionibacteria - Propagating
« Reply #3 on: January 01, 2010, 01:10:59 PM »
Hi Tina,

For the quality control checkpoints and convenience one gets, I agree, they are a good deal.

I think with regard to ease, it depends on the person and comfort level with scientific processes, and frequency of making cheese. Keeping a yeast bank and keeping a culture bank are very similar. Instead of wort or malt extract, you have agar broth, or more simply, a skim milk formulation, and instead of letting the yeast settle into a layer by cold crashing, then harvesting, you can centrifuge and collect the cultures that way. You can keep test tubes of your cultures for a predefined batch size, and freeze them in glycerin, same as you do for yeast. That will keep them viable for years.

Also, propagating regular lactic bacteria cultures (in comparison with propionic) is a bit more straightforward and faster. You can have colony saturation in 12-24 hours, which is about similar to yeast.

If there's interest, I can post some more details on how to test for colony viability/size, and how to standardize so your pitch rate results in reproducible acidification curves. In that regard, yeast is more forgiving. Overpitching yeast by 20-30% doesn't have the same consequence as overpitching starter culture.
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Offline Likesspace

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Re: Propionibacteria - Propagating
« Reply #4 on: January 20, 2010, 06:57:33 PM »
LB,
I just saw this post and was initially excited. I have spent quite a lot on propionic bacteria but I've also since found that I was using it wrong.
I figured that if a little produced small holes then a LOT should produce the large holes. I was up to a teaspoon and a half in a 4 gallon wheel.
I am now using the recommended amount (1/8 tsp. per 4 gallons) and a package of the bacteria goes a LONG way using this method.
I am beginning to think that a 1/4 tsp. per 4 gallons might be better since it's taking me about 9 days to see even the beginnings of swelling but still.....even at that rate, a package will last a long time for me.
I will admit that I never took chemistry or biology, (even in high school) and it all seems really foreign to me. Considering that fact I think I'm much better off simply sticking with the commercial DVI cultures.
My oldest daughter is involved in microbiology right now and loves every second of it. She's always been a bit of a geek but I love her anyway.  :D
Had I known that my oldest daughter would be working towards a nurse practitioner degree and my middle daughter would be pursuing a career in the same field I probably would have concentrated more on ANY profession that would have earned me the greatest amount of money. I simply cannot get over the fact that I'm renting certain books for upwards of $300.00 a semester. Sheesh.
The bright spot in all of this is that my youngest daughter seems to be headed toward a career that is located somewhere between a Mother Teresa and an axe murderer. Either way, it should cost MUCH less for her education. :)


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Offline linuxboy

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Re: Propionibacteria - Propagating
« Reply #5 on: January 20, 2010, 07:23:41 PM »
Woah, 1.5 tsp is quite a bit. Dave, I had some more ideas for your swiss if you ever want to give it another go. Propionic is very sensitive to both salt and temp. So whereas pressing under whey helps to compact the curd together and eliminate mechanical pockets, aging in a very warm environment and salting to about 1-1.2% should give the bacteria a happy environment to grow and produce enough gas to make large holes.

Also, to follow up on this thread, I have a good storage idea for long-term bacteria and mold storage in buffered sterile water. No need to freeze it and little loss of viability for 1-2 years. Also, I thought up of some good recipes for making media broth at home without the need to buy expensive stuff from science supply companies. So if anyone is feeling adventurous, let me know. It's not much harder than making starter from yogurt or buttermilk.
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Offline DeejayDebi

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Re: Propionibacteria - Propagating
« Reply #6 on: January 20, 2010, 08:31:42 PM »
Soumds like an interesting adventure to me. What's next?

Offline linuxboy

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Re: Propionibacteria - Propagating
« Reply #7 on: January 21, 2010, 03:00:01 PM »
Hi Debi,

Next steps are for me to write up and post technical snippets about how to go about making a very small lab and supplies using store-bought materials, and then procedures for making and storing cultures, and keeping a cheese culture bank.

It's interesting, similar works have been done for mushrooms (mostly for hallucinogenic ones), and beer (for yeast), but presumably when it doesn't get one high, there's less published home-geared procedural documentation. Maybe those guys have never stepped into a cave full of ripening blue or camembert. j/k :O
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Offline Likesspace

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Re: Propionibacteria - Propagating
« Reply #8 on: January 21, 2010, 03:50:32 PM »
similar works have been done for mushrooms (mostly for hallucinogenic ones)

HA!
I KNEW you were Owsley Stanley!!
 ;D


Dave

Offline Likesspace

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Re: Propionibacteria - Propagating
« Reply #9 on: January 21, 2010, 03:53:33 PM »
Oh, and I have been getting the "itch" to give Emmantal another try.
As soon as I can get things to slow down a little I will give it a go with your suggestions. I really do love making swiss styles of cheese and they do taste good, even without the nice holes.
Thanks for the information, LB.
I really appreciate it.

Dave


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Offline DeejayDebi

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Re: Propionibacteria - Propagating
« Reply #10 on: January 21, 2010, 08:14:58 PM »
My curiosity is definitely piqued!

Offline linuxboy

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Re: Propionibacteria - Propagating
« Reply #11 on: January 21, 2010, 08:28:27 PM »
This project, like my other 2,000+ projects will take some time. But I'll work at it a little at a time. I might put together a Web site for it, because the volume of content by the time I finish will be about 60,000 words.
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Offline DeejayDebi

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Re: Propionibacteria - Propagating
« Reply #12 on: January 21, 2010, 09:22:25 PM »
Works for me!

Offline Minamyna

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Re: Propionibacteria - Propagating
« Reply #13 on: May 14, 2010, 12:21:22 AM »
This project, like my other 2,000+ projects will take some time. But I'll work at it a little at a time. I might put together a Web site for it, because the volume of content by the time I finish will be about 60,000 words.

Did you end up putting together a website?

I am really interested in a home lab.

Offline linuxboy

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Re: Propionibacteria - Propagating
« Reply #14 on: May 14, 2010, 12:23:51 AM »
No, all my other projects are keeping me busy. There are not many people who have the interest, time, and money to put a home lab together, especially because most of the ones interested work in a lab, anyway.

I'd like to, though. I'll keep working on it when I have some time.

In the meantime, here's a good site with links to protocols http://www.protocol-online.org/prot/
« Last Edit: May 14, 2010, 12:36:53 AM by linuxboy »
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