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Quote from: linuxboy on September 17, 2009, 12:24:39 AM
If you want to use agar, I would use a broth if you want to maintain and replicate. A plate is better for isolation. Look at Ellikers lactic agar, or MRS or M17. You can agitate the broth at a slow RPM, and use a buffer if you want, and centrifuge after to concentrate. If you have a lyophizer, you can take the centrifuged mass and freeze dry it, which would form a DVI culture .

I can find some links for detailed culture prep instructions if you want something more exact. But the gist of it, sure, you can propagate on your own with agar, just watch the pH and food needs.

this sounds like what i want to do. i assume dvi culture is powder culture and freeze drying it would make it last how long? by buffer what kind and where can i get this buffer. how large of a broth should i be using to get to the adiqute amount of dvi. i'm going to school to be a micro biologist. the more info the better.

Wayne,
While every one gets all bent out of shape about phage and loves to blame phage for cheese making issues, I have rarely seen it in real life.  We run flora danica 7 days a week, 351 days a year and only have minor phage issues which have developed over years.  I don't think it's a realistic concern for a home or even farmstead producer.

Quote from: linuxboy on September 17, 2009, 12:24:39 AM
I can find some links for detailed culture prep instructions if you want something more exact. But the gist of it, sure, you can propagate on your own with agar, just watch the pH and food needs.

yes please link me up.

Quote from: Sailor Con Queso on September 17, 2009, 02:24:09 AM
Linuxboy - more is good.

What I need to know is how in the world to adjust ripening times. The Direct Set packets are "calibrated" based on activity level. Is there a way to test BASIC activity level to calculate either culture volume or ripening times?

Peter Dixon suggests a shortcut for kick starting the starter cultures while you are heating milk to its starting temp. He says you should quickly heat a much small container of milk (a glass full?) to 80-85 F and add the starter to that container. So while the main vat is heating up, the starter is already becoming very active. He says that this is a way to greatly reduce ripening time. However, he does NOT suggest how to adjust the timing. I  guess if you know what the target pH is at renneting, you could just monitor and add rennet at the right time. Without pH levels, I'm lost about how to adjust times.

I have no idea how to adjust without pH because bacteria strains have wide ranges of acidification, and it's that pH that is so crucial for draining and brining. In theory, if you knew the CFU/gram, and the theoretical listed acidification rate, you could formulate an estimated pH for the milk volume based on an acidification curve using time, starter amount, and milk volume as variables. You would be invariably off, though, especially with DVI cultures because the CFU/g is something like 100-150 x 10^9.

Alternatively, you could culture a starter and then determine the bacterial concentration, but again, without pH, all you have are theoretical numbers from the lab of the acidification curve. Not as accurate in the real world when there are so many other variables.

The easier way really is to use pH. If you use too much or too little starter, or if the acidification rate is different among your strains, the ripening time won't matter much so long as you hit pH targets. Well, that's almost true. A drastic too little or too much starter affects the time to hit flocculation targets and time to rennet and to an extent time to heal. And later, on it will affect affinage. Say you have too few bacteria in there but the pH is fine. Well, as the cheese ages, there's a smaller volume of enzymes from the cell walls and membranes that contribute to flavor and texture. It's a delicate balance.

If you get a pH meter, you can also start plotting pH curves from your own cheesemakes so you can compare time vs pH for different cheese styles and makes.

Quote from: krops13 on September 17, 2009, 03:32:20 AM

Quote from: linuxboy on September 17, 2009, 12:24:39 AM
If you want to use agar, I would use a broth if you want to maintain and replicate. A plate is better for isolation. Look at Ellikers lactic agar, or MRS or M17. You can agitate the broth at a slow RPM, and use a buffer if you want, and centrifuge after to concentrate. If you have a lyophizer, you can take the centrifuged mass and freeze dry it, which would form a DVI culture .

I can find some links for detailed culture prep instructions if you want something more exact. But the gist of it, sure, you can propagate on your own with agar, just watch the pH and food needs.

this sounds like what i want to do. i assume dvi culture is powder culture and freeze drying it would make it last how long? by buffer what kind and where can i get this buffer. how large of a broth should i be using to get to the adiqute amount of dvi. i'm going to school to be a micro biologist. the more info the better.

Yes, DVI is basically freeze dried culture. Longevity depends on storage temps. Typical loss of viability is 5-10%/year. But, this depends on how it was prepared, handled, and freeze dried. The big names (chr hansen, etc) do a good job, so their cultures can last many years.

By buffer I mean two distinct things. One, buffer ions that help maintain pH equilibirum, and two, ammonium hydroxide to raise pH. For broth volume follow manufacturer guidelines.

Have a look at this for a quick primer. http://www.dairyscience.info/cheese-starters/108-starter-concentrates.html?showall=1

Before i start doing all this crazy stuff. i figured I'd better ask if freezing culture in ice cube trays and storing in the freezer last for a while. i read in my book (which I'm finding out isn't all that great) that they will only last for a month in the freezer then i have to re-propagate.

Quote from: krops13 on September 17, 2009, 06:23:19 PM
Before i start doing all this crazy stuff. i figured I'd better ask if freezing culture in ice cube trays and storing in the freezer last for a while. i read in my book (which I'm finding out isn't all that great) that they will only last for a month in the freezer then i have to re-propagate.

Yes, that's a good way of doing it at home. It's tough to say how long they will last, but there's no sound reasoning in the arbitrary number of one month. It depends on the concentration of live cells at time of freezing, and the freezing prep. For example, if you flash freeze, the ice crystals are much smaller, and when you defrost, the viability will be better. Also, if you defrost quickly, such as tossing ice cubes in the milk, the cells don't reconstitute as well. It's better to defrost slowly in the fridge to give the cells a chance to become acclimated.

Also, the longevity depends on how you freeze. You could add glycerin or other adjunct, like you would if keeping a yeast bank in the freezer. If you do that, cell viability greatly increases and your ice cubes or test tubes will last for years. The storage temp also matters. The colder, the better.

There are other storage methods. If you can up the CFU/g count and centrifuge, you could take a buffered water solution and dump the culture in it, and just keep in the fridge like you do with rennet. That would last for years, too, with 10-20% loss of viability/year.

If you want to go the easy route and maintain cultures in ice cubes using just skim milk as the medium, the cubes should be viable for 6-12 months. A good practice with the cubes is to prep a starter culture the night before using 1-2 cubes and a few cups of milk. That way you can tell if the bacteria are alive, and cut down on ripening time.

What would be a good PH to hit before freezing. I figured by the previous post this is the best way to tell how much the culture has grown/ how much there is?

Quote from: krops13 on September 17, 2009, 07:07:38 PM
What would be a good PH to hit before freezing. I figured by the previous post this is the best way to tell how much the culture has grown/ how much there is?

Eh, it's not that straightforward, so in some ways you have to guess if you want to determine colony size based on pH. You can have bacteria that metabolize faster than they reproduce, so your colony size will be smaller. In that case, you'd need to raise pH to get colony size up if you want an apples to apples comparison to another strain.

But, there are clear guidelines for when you know that bacteria cannot grow any more because they cannot tolerate the pH. In general, it is 4.0 for S thermophilus, and about 4.5 for Lactobacillus species. You can confirm visually or with a meter. The isoelectric point of milk if 4.6; this is when casein precipitates and milk coagulates. So not much beyond that is the optimal min. I would stop a little after coagulation for both meso and thermo.

If you want to try a quick experiment, leave a culture and check pH at 6, 12, 18, 24, 48, and 72 hours. You should notice it leveling off at around 4 for S thermophilus and 4.5 for Lactobacilli after 24 hours, with just marginal changes until you get to 72 hours.

Ok sooooooooo when it gets to the ph it likes (im using just meso right now) it will start to reproduce. There's really no way to know how much they reproduce so I should let set say 24 hours after the targeted ph is reached to grow I guess the longer its let to grow the faster it will acidify the milk later when making cheese so its just experamentation right  I kinda lost myself

Quote from: krops13 on September 17, 2009, 09:03:40 PM
Ok sooooooooo when it gets to the ph it likes (im using just meso right now) it will start to reproduce. There's really no way to know how much they reproduce so I should let set say 24 hours after the targeted ph is reached to grow I guess the longer its let to grow the faster it will acidify the milk later when making cheese so its just experamentation right  I kinda lost myself

You almost got it. Meso pretty much always reproduces at room temp so long as it has food. It doesn't exactly have a trigger for starting, only stopping. With a healthy starter, your culture shouldn't need more than 24 hours to reach peak density. Target 24 hours or a pH of ~4.5 for meso. You don't wait 24 hours after target pH; they should coincide, with the time varying based on temp and initial starter amount.

It's not the case that the more you wait, the faster it will acidify the milk later during the cheesemake. Without manipulation of pH, you can reach a colony level of only 2-5 x 10^9 in skim milk culture. This is reached at the pH levels I already specified. If you wait more, the bacteria start to die. That's why I said in the other thread to freeze only fresh culture, and not old culture. You don't want to freeze dead bacteria.

You're getting it. Think about what's happening at the biological level in terms of metabolization, mitochondria, Krebbs, active/passive transport, DNA/RNA replication, etc. Krebbs happens all the time. Heck it even happens after cell death for a little while.

Ok soooo long story short follow the directions on making a meso starter from the front of the page chill before freezing use dry ice to freeze quickly smaller ice crystals less damage to cell walls and when propagating wait till a ph of 4.5 before adding to milk

linuxboy - what in the world do you do for a living??? 

Quote from: Sailor Con Queso on September 18, 2009, 02:14:36 AM
linuxboy - what in the world do you do for a living??? 

I make people feel good about their decision to pay me money, and then try to convince them that it's a good idea to pay a little more .

  LMAO 
,,,and I'm sure you deserve every penny.