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Offlineblackout
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Low temp fractional sterilization?
    #5206113 - 01/21/06 11:09 AM (18 years, 1 month ago)

Has anybody ever heard of fractionally sterilizing grains at lower temperatures for longer time. e.g 24hrs at 70C, 24hr incubation, on 3 occassions.

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Re: Low temp fractional sterilization? [Re: blackout]
    #5207225 - 01/21/06 04:53 PM (18 years, 1 month ago)

Nope. 70C is pasteurization temperature. It's only meant to kill most nasty contams, and leave beneficial bacteria alone. Problem with grain is bacterial endospores which can withstand 100C. Need to PC them at 121C, or fractionally sterile at 100C. Also called Tyndallization, the method is as follows- boil 15-30 mins first day, allow to incubate at 20C to 40C for 24 hours. Boil 15-30 mins second day, incubate for 24 hours. Boil 30-60 mins third day. Cool and inoculate. What happens according to theory is that endospores will germinate when triggered by boiling. Subsequent boiling will then be able to kill the germinated spores. In addition to a lot of time and hassle, the grain turns to mush after all that boiling, as I found out. Will still colonize, but rye at least, gets all slimy.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5207417 - 01/21/06 06:19 PM (18 years, 1 month ago)

i can atest to the sucess of this. however it will just promp you to go buy a damn PC. youll see how great gain is.


--------------------
im sorry about your mother. She was a terrible attrative woman.

Get back to nature; hunting burgers and gathering fries.

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Re: Low temp fractional sterilization? [Re: skeletor]
    #5207818 - 01/21/06 08:38 PM (18 years, 1 month ago)

When I first began, I would do "fractural sterilization". Method: Boil jars of pre-soaked grains for 1 hour, once a day, for 3 days. Never had a problem with it. Hope I helped!

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Re: Low temp fractional sterilization? [Re: FungusMan]
    #5207846 - 01/21/06 08:44 PM (18 years, 1 month ago)

I forgot to mention that I soaked the grains for 24-48 hours, then did a 5 minute flush before jarring. No simmering was performed.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5209517 - 01/22/06 09:16 AM (18 years, 1 month ago)

Quote:

mogur said:What happens according to theory is that endospores will germinate when triggered by boiling. Subsequent boiling will then be able to kill the germinated spores.



Exactly. I have been trying to find the thermal death times (TDT) for grains at various temperatures. This is the minimum time needed at a given temperature to kill various contaminants in the grain. Everybody knows that sterilizing at 10psi requires more time than at 15psi. I am convinced grains can be sterilized at 100C is given a long enough time. I have barley at 99C for 48hrs now. The germinated endospores are easily killed so I wonder if they could be killed if kept at 70C for long enough. Many people have water tanks in the house and some are kept at this temperature. People could just leave jars floating in them and fractionally sterilize.

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Re: Low temp fractional sterilization? [Re: blackout]
    #5210784 - 01/22/06 04:01 PM (18 years, 1 month ago)

To understand the contam, we need to become the contam. There are hundreds of molds, mildews, bacterium, and fungi that plague the mycologist. But Bacillus subtilis is one of the most common, and has an understood sporulation mechanism typical of the most heat resistant contams. Here?s an interesting little story that demonstrates that this relative of anthrax is not entirely our enemy-

[EDIT: the following story is complete horse-pucky, I discovered that it is NOT TRUE. Some greedy company manufactured this story as pseudoscience validation of their rip-off cure-all medications. DO NOT eat animal dung to cure ANYTHING. Sorry for posting it, should have recognized it as bullshit when the first sentence claimed that Germans discovered Bacillus subtilis in 1941, although I did note this discrepency. Will leave it posted only as an interesting myth-buster.]

Quote:

The bacillus subtilis was discovered by the Nazi German medical corps in 1941, toward the end of their African campaign. [ed. Note: In 1872, Ferdinand Cohn recognized and named the bacterium Bacillus subtilis.] At the time, the German military victory was at its height. But the German high command became genuinely alarmed when hundreds upon hundreds of soldiers in North Africa suddenly began dying every week. Oddly, the Nazi soldiers weren't dying because of British General Montgomery's retaliatory bombs and shrapnel, but instead, they were dying of uncontrollable dysentery.

Of course, the Germans were aware that dysentery was caused by pathogenic (i.e. disease-causing) bacteria from local food and water sources. But in those days, there were no antibiotics. Sulfur was already on the market, but only in a topical non-ingestible form. So with no medication available with which to stop the plague of dysentery, the Nazis quickly began looking for other means to help their dying soldiers.

The German high command immediately sent out a contingent of scientists, physicians, chemists, biochemists, bacteriologists and other experts to help solve the problem. With typical German circumspection, these top experts reasoned that there must be a natural way to counteract the deadly bacteria causing the dysentery because, if there wasn't, the millions of Arabs living in the region would have been dead long ago.

Therefore, the Germans' first step was to closely scrutinize the native Arabs, and see whether or not they were affected by dysentery. What they discovered was that the Arabs also caught dysentery, but at the first sign of diarrhea [the #1 symptom of dysentery --- Ed.] the Arabs would do something quite incredible: They would immediately begin following around a horse or camel until it would drop its dung. Then, the affected Arab would pick up the warm dung droppings, and quickly gulp them down! This strange procedure effectively eliminated the dysentery almost overnight.

Once the good hygienic Germans finally recovered from the shock of seeing the Arab natives gulping down warm camel dung, they quickly realized that there must be something in the dung that somehow counteracted the harmful bacteria that caused the dysentery. They questioned the Arabs, who told them that they had no idea why it worked, but that their fathers had always done so, as had their forefathers, and it had always worked. The only caveat was that the camel or horse dung had to be ingested while still warm and fresh, because it had no effect on the dysentery if ingested cold.

So the Nazis began carefully examining fresh camel and horse dung. What they discovered was that it was teeming with a powerful bacterial microorganism which later came to be called Bacillus subtilis. This bacteria, it turned out, is so strong that it practically cannibalizes all harmful microorganisms in the human body --- particularly pathogenic bacteria like the virulent strain which was causing dysentery in the German troops.

Within a very short time, the Nazis began producing hundreds and thousands of gallons of active Bacillus subtilis cultures for their troops to ingest. And bingo, no more dysentery! Soon afterwards, the Germans even discovered the process by which the Bacillus subtilis cultures could be dried and placed into easily ingestible capsules. From that time forward, the resourceful Germans had no more problems with losing troops from dysentery.




But, more to the point of sterilization techniques, here is what Bacillus subtilis looks like.

And, here are the hardened spores encased within the cells.

The sporulation process in outline form.


Endospores are formed by vegetative cells in response to environmental signals that indicate a limiting factor for vegetative growth, such as exhaustion of an essential nutrient, or dehydration. They germinate and become vegetative cells when the environmental stress is relieved. Hence, endospore-formation is a mechanism of survival rather than a mechanism of reproduction. Mature spores have no detectable metabolism, a state that is described as cryptobiotic. They are highly resistant to environmental stresses such as high temperature (some endospores can be boiled for several hours and retain their viability), irradiation, strong acids, disinfectants, etc. Although cryptobiotic, they retain viability indefinitely such that under appropriate environmental conditions, they germinate into vegetative cells. What triggers germination? Extremes of heat, and an ample supply of moisture. Now, I have yet to pin down the exact amount of heat required to ensure sufficient germination to nail them with subsequent sterilization/pasteurization, but I suspect that 70C won?t do it. I will keep looking. Meanwhile, wipe that shit-eating grin off your face, you Nazi!!!

Edited by mogur (01/23/06 09:13 AM)

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InvisibleJaeger
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Re: Low temp fractional sterilization? [Re: mogur]
    #5210793 - 01/22/06 04:06 PM (18 years, 1 month ago)

Well presented mogur!!
5 for you!!

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Re: Low temp fractional sterilization? [Re: Jaeger]
    #5213049 - 01/23/06 02:31 AM (18 years, 1 month ago)

mogur- thanks a lot for that!
"some endospores can be boiled for several hours and retain their viability". That is very encouraging, I thought it may say days. I have barley I kept at 99C for 48hrs. I have access to boilers constantly turned on. people could get a 50gallon drum with a cheap element and simmerstat/thermostat and dump 100's of jars in.

The formation of endospores is also intresting, some of my techniques may have actually made more new endospores than killed old ones!

I only hope the Nazi story wont cause a new trend of post questions.

"I have these mouldy shrooms and hear if I eat camel shit after them I will be ok, I have searched walmart and they wont have it in stock until summer, do sponsors sell it?"  :evil:

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Re: Low temp fractional sterilization? [Re: blackout]
    #5213096 - 01/23/06 03:05 AM (18 years, 1 month ago)

First off, GO SEAHAWKS ! ! !

The following was paraphrased, pasted, or just plain stolen from too many sources to list:

Endospores can remain dormant for many years. Several scientists have been able to recover viable endospores from bees trapped in amber that is 25-40 million years old. The microbe isolated was found to be most closely related to Bacillus sphaericus. There are even claims of endospores that are over 250 million years old being able to germinate when placed in appropriate medium, but there is significant skepticism about this.
The change from endospore to vegetative state involves three stages:
1. Activation. Spores can be activated by heat (60 ? 65C for 10 or more minutes), oxidizing or reducing agents, low pH and some nutrients such as amino acids (L-alanine) or nucleotides (inosine or adenine). The process is reversible if there are no nutrients available for germination. Activated spores remain resistant to heat and are refractile.
Several researchers have studied heat activation of Bacillus spp. (Doyle, 1989). The experimental procedure involved three steps:
1) Heat shock at 60C, 70C, 80C, 90C and 100C for 12 minutes
2) Incubation at 35C for 30 minutes
3) Second heat shock (repetition of step 1).
The results indicate that the temperature of the second heat-shock variably affects the growth rate between the Bacilli. Overall, a net reduction occurred in the growth rate of all the strains studied. Two heat treatments at 100C have the greatest effect. The survival from the initial counts is reduced by more than 5 log units for B. amyloliquifaciens, B. subtilis 23059, B. pumilus, B. licheniformis 12759, B. megaterium; and by more than 3 log units for B. subtilis 6051, B. licheniformis 14580

2. Germination. In the presence of suitable nutrients, the activated spores germinate. This stage is associated with loss of refractility, SASPS and calcium dipicolinate. While germinated spores lack some of the metabolic activities of vegetative cells (such as synthesis of macromolecules), they are metabolically active. Germination of spores is considered a rapid process, which usually takes 60 to 90 min (Talaro and Talaro, 1996) and depends upon several parameters such as incubation temperature, pH, prior heat treatment, and reducing conditions (Doyle, 1989). One strain of C. perfringens germinated well at a pH of 5.5 and 7.0 but the germination rate was reduced by two-thirds when the pH was increased to 9.5. Lowering incubation temperature from 45C to 7C resulted in a decrease in the germination rate from 85% to 75% (at a pH of 6.0). Moreover, oxygen is known to inhibit germination. The addition of sodium bicarbonate and CaCl2 enhanced germination of C. perfringens spores. Finally, the presence of lysozyme, a protein found in hen egg white, mucus, tears, and blood, helps germination of endospores (Doyle, 2002). . In another study, of the ten temperatures tested, ranging from 4C through 50C, the optimum reduced temperature for spore germination was 30C.
3. Outgrowth. This is the transitional stage between germination and the vegetative cell. Water uptake results in swelling and the spore coat is broken. Synthesis of macromolecules such as DNA, RNA and proteins resumes and the cell enters the vegetative state.

Although I could not seem to find a Thermal Death Rate chart for typical grain inhabiting bacterial endospores, I still believe 70C to be way too low to achieve any significant population reduction, since the only references to effective sterilization seem to be at 100C and above. 70C, however, can to used to ?activate? the spores, but they will remain resistant to any heat less than 100C. If the endospores are incubated at 30C shortly after activation, however, then the reapplication of 70C will kill the resulting vegetative cells.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5213120 - 01/23/06 03:27 AM (18 years, 1 month ago)

Thanks again.
So you are saying 70C will not kill the endospores, but using 70C could possibly fractionally sterilize the grain, i.e. activate and germinate all the endospores, then treat again at 70C to kill the vegetative cells. (or do it several times as usually recommended)

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Re: Low temp fractional sterilization? [Re: blackout]
    #5213403 - 01/23/06 07:34 AM (18 years, 1 month ago)

That's exactly what I'm getting out of this research. Mind you, the sands seem to shift with each different study that I read. An example is that some researchers claim that oxidation is an endospore activation inhibiting factor, while the next paper lists hydrogen peroxide as an activation facilitator. Go figure, but the overall flavor seems to be that sub 100C temps will both activate endospores and kill vegetative cells, if interspersed with incubation temps near 30C. That sounds a lot like 'fractional pasteurization' to me, or as you say, "low temp fractional sterilization". I wouldn't recommend these methods to anyone here, except as an experiment. I plan to play around a little myself with them, and appreciate your endeavors. Keep us posted.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5213439 - 01/23/06 07:57 AM (18 years, 1 month ago)

Like I said, I did it many times, sometimes with/ sometimes without a water-peroxide soak. Hasnt failed me yet.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5213539 - 01/23/06 09:05 AM (18 years, 1 month ago)

Quote:

mogur said:I wouldn't recommend these methods to anyone here, except as an experiment.



Yes, try it on small batches of grain. Probably best not to waste/risk spores on it, it would also rule out contamed syringes/LC as a cause of the contams, if they do. If after a few weeks there is no contams then try it. Or if you make your own syringes or LC there is no real loss.

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Re: Low temp fractional sterilization? [Re: blackout]
    #5213566 - 01/23/06 09:24 AM (18 years, 1 month ago)

Quote:

blackout said:

I only hope the Nazi story wont cause a new trend of post questions.

"I have these mouldy shrooms and hear if I eat camel shit after them I will be ok, I have searched walmart and they wont have it in stock until summer, do sponsors sell it?"  :evil:




Lol. Your joke, however, prompted my curiosity, and to my embarrassment, I found the Nazi story is just clap-trap pseudo-science, fabricated to promote some sleazy marketing effort. Oops, can't believe I bought into that 'shit'. I disclaimed my above post, and sincerely hope that no one here is as gullible as me. Sorrrrrrrry. Can't believe everything you read on the WWW, unfortunately.

FungusMan, thanks for the input. My experience is the same as yours.

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Re: Low temp fractional sterilization? [Re: blackout]
    #5213595 - 01/23/06 09:42 AM (18 years, 1 month ago)

Quote:

blackout said:
Yes, try it on small batches of grain. Probably best not to waste/risk spores on it, it would also rule out contamed syringes/LC as a cause of the contams, if they do. If after a few weeks there is no contams then try it. Or if you make your own syringes or LC there is no real loss.




In fact, about a month ago, I did exactly that. I took 12 quart jars of rye grain and varied the sterilization process for each. From single pasteurization to triple fractional sterilization. Only one contamed, the single pasteurization jar. I didn't have a decent thermometer at that time, but I was trying for about 180-190F for a pasteurization temp, and about a half hour in length. All other jars are still contam free, including the jars that were only subjected to two pasteurizations and a 24 hour incubation at room temp of 65F. At the other extreme, boiling for an hour on three separate days, produced rye grain mush with standing water in the jars, but contam free, nonetheless.

Edited by mogur (01/23/06 09:44 AM)

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Re: Low temp fractional sterilization? [Re: mogur]
    #5213673 - 01/23/06 10:34 AM (18 years, 1 month ago)

Quote:

mogur said: I found the Nazi story is just clap-trap pseudo-science, fabricated to promote some sleazy marketing effort.



ahh  :frown: why ruin a good story with the truth!

Those jar experiments are VERY interesting. Some people got by with minor microwaving of grains too. My 48hr 99C barley did pop a few grains and is a bit sticky. Any more info on your technique? I think most people fail with fractional sterilization due to using large jars which never get a chance to reach 100C in the centre. Are you shaking the jars to get even heating?

Were the grain soaked prior to boiling/pastuerizing? I would think 2 30min sessions at 180-190F would be barely enough to hydrate the grain!

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Re: Low temp fractional sterilization? [Re: blackout]
    #5214404 - 01/23/06 03:46 PM (18 years, 1 month ago)

Im glad you mentioned the shaking. Yes, I did shake between boils. After each 1 hr boil,jars were so hot, they had to be shaken with potholders, lol. Glad you mentioned that, cuz I forgot, lol. Very important.

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Re: Low temp fractional sterilization? [Re: FungusMan]
    #5216497 - 01/24/06 01:20 AM (18 years, 1 month ago)

It's easier to just post an image of the spreadsheet, but basically, four jars were measured grain and water, then sterilized. The rest were boiled/pasteurized in a covered pot, and then drained on the last day for final treatment in the jar. The water in the jar was observed to boil at least 15 mins for the measured jars, and soaked grain was jarred already hot. All were gently shaken/redistributed after heating, while still hot.

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Re: Low temp fractional sterilization? [Re: mogur]
    #5220787 - 01/25/06 06:20 AM (18 years, 1 month ago)

Thanks for that. I am still not certain though. You were pastuerizing at 180-190F in excess water? then it was left to cool in this excess water? and drained only on the last day?
My LC is taking off so I will inoculate my 48hr barely hopefully next week.

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