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Offlinemogur
regnartS

Registered: 11/15/05
Posts: 322
Loc: Puget Sound
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Re: Low temp fractional sterilization? [Re: blackout]
    #5221149 - 01/25/06 09:50 AM (18 years, 1 month ago)

The jars that contained measured amounts of grain/water were just processed entirely in the jars. But the rest were boiled or pasteurized in excess water in large covered pots, then left to sit at room temp until the next day. On the final day, the grains were thoroughly drained and jarred, then processed to sterilize the grain and, of course, the jars.

My current experiment is comparing the results of different grain/water ratios. There is such a wide range of recommended ratios on the boards, that I wanted to see for myself. My conclusion is that it doesn't much matter, any ratio by volume from 1 to 1.5 (grain to water) produces acceptable results. Less than 1 to 1 ratio leaves standing water, and anything more than 1.5 to 1 ratio is too dry. This only applies to my particular bag of rye grain seed, but I think most grains have a similar moisture content of about 15%. Millet is probably very different.

Edited by mogur (01/25/06 09:55 AM)

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Offlineblackout
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Registered: 07/16/00
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Re: Low temp fractional sterilization? [Re: mogur]
    #5221382 - 01/25/06 11:23 AM (18 years, 1 month ago)

Great. I prefer drier grains than wet. I would have thought the grains would turn to mush if heated and left in the hot water, but maybe the low temps are ok.

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InvisibleMagash
Da Bud Guru
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Re: Low temp fractional sterilization? [Re: blackout]
    #5221472 - 01/25/06 11:53 AM (18 years, 1 month ago)

You have to get into temprature ranges that a PC can't reach to kill endospores.

The definition of an endospore is an extremely heat-and chemical-resistant, dormant, thick-walled spore that develops within bacteria. Endospores are extraordinarily resistant to environmental stresses including heat, UV radiation, gamma radiation, chemical disinfectants, and desiccation. Endospores can remain viable for thousands of years or longer. An example of this are some endospores that were found on Dominican bees that had been encased in amber for 25 - 40 million years. These dormant structures can be found in several different types of bacteria: Bacillus, Clostridium, and Sporosarcina. I will be trying to isolate Bacillus in my experiment, and after isolation prove that it is indeed an endospore forming bacterium by getting it to transform into its dormant/ spore state.

Bacillus is a gram-positive aerobe that can be found in soil. It appears as straight rods 0.5 - 2.5 x 1.2 - 10 mm, and is chemoorganotrophic, which means that it gets its energy by oxidizing chemical compounds, and organic molecules are its electron source.

Here are some examples of spore forming bacteria out in the world: Clostridium perfringens and Clostridium botulinum (both anaerobes) are food poisoning microorganisms. C. perfringens commonly occurs in meats that have been heated (O2 depletion) and then slowly cooled. As these are ingested the cells sporulate in the intestine, producing an enterotoxin that causes symptoms of nausea, diarrhea, and abdominal cramps. C. botulinum is found on potatoes that are not heated sufficiently and cause the production of toxins. Bacillus cereus is another spore forming bacterium that also is found on starchy foods like rice. Its symptoms are similar to those of C. perfringens.

Microorganisms are also used in bioterrorism. One such spore forming bacterium is Bacillus anthracis which results in anthrax. This organism's endospores can survive for decades in animal products and soil. It can enter the body through cuts and abrasions (cutaneous anthrax), or by inhaling endospores (pulmonary anthrax). Symptoms resemble that of the flu, and if it invades the bloodstream the disease can be fatal.

Vancomycin is an antibiotic that is bactericidal for some forms of Bacillus and Clostridium, as well as other microorganisms. It works by binding to the D-alanine-D-alanine sequence so the peptide interbridge cannot be formed. This results in the formation of weak peptidoglycan which eventually leads to the cells lysing because of osmotic differences.

Sporulation is the process by which an active vegetative cell turns into its dormant spore state. This occurs because of a lack of nutrients, or because the cell was exposed to environmental stress. There are several steps to the process which are discussed below:
Formation of nuclear filament within the vegetative cell.
Cell membrane folds inward enclosing part of the DNA and forming the septum.
Continuation of the cell membrane to fold inward and engulf the immature spore.
Spore cortex is formed in the space between the two membranes.
A protein coat is developed around the cortex.
The spore continues to mature within the sporangium (mother cell).
Lysis of sporangium and release of spore.

For a spore to go back to its active vegetative cell state, it goes through a process called germination. Amino acids and sugars are two examples of the nutrients that can trigger germination. This involves the spore swelling, either the absorption or rupture of the spore coat, loss of heat/chemical resistance, the release of spore components, and increased metabolic activity. The third stage, outgrowth, follows germination. This involves the synthesis of new components, and emergence from the spore coat ready to develop into an active bacterium.


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Offlinemogur
regnartS

Registered: 11/15/05
Posts: 322
Loc: Puget Sound
Last seen: 11 years, 3 months
Re: Low temp fractional sterilization? [Re: Magash]
    #5222316 - 01/25/06 03:58 PM (18 years, 1 month ago)

Very nice presentation, Magash. Most literature that I have found mentions the heat resistance of endospores, but fails to quantify that tolerance. While it is accepted knowledge in mushroom cultivation that PC temps are required for complete sterilization, some of my research points in another direction.

A 1948 study by Davis and Williams indicates that bacillus endospores succumb to boiling temps in less than 12 minutes. When selected for heat tolerance, subsequent colonies acquire increased resistance, up to 18 minutes within 10 generations. Now, I don't think this study justifies a conclusion that a 20 minute boil will guarantee sterility; but, I am hard pressed to find any scientific studies that claim that endospores have to heated beyond PC temps.

In a microbiology textbook by Prescott, Harley, and Klein, a very thermophilic bacteria genera, Thermoactinomyces, commonly found in damp haystacks, compost piles, and other high-temperature habitats have very heat-resistant endospores. Yet their survival in 90C temps is only 30 minutes.

As a side note, I have experimented with oven sterilization of rye. One hour at 300F thoroughly dries the grain and removes all residual moisture. I would assume endospores could not survive this heat. Soaking in excess water required 48 hours or so to totally rehydrate the grain. I did not continue the experiment, but will probably repeat it soon and then jar, sterilize, and inoculate to see if it contams and/or colonizes.

PS edit: Blackout, the only jar that really mushed out was the one that was boiled thoroughly on three separate days. All the others were fine, with only a rare burst kernal or two.

Edited by mogur (01/25/06 04:08 PM)

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Offlineblackout
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Re: Low temp fractional sterilization? [Re: mogur]
    #5224785 - 01/26/06 02:58 AM (18 years, 1 month ago)

I have had success with roasted grains. In the microwave they got up to about 150C/300F. I dried them completely and rehydrated with LC. I have a jar at the moment that I added water to and microwaved until roasting for about just 20-30mins. I inoculated with LC which proved unviable. My new LCs are taking off and I will inject next week again. I have added LC to my 48hr barley jar.

I have also wondered about combination sterilization. In some texts I see some endospores are supposed to be killed in say 10mins moist heat but 200mins dry heat, then another maybe the exact opposite, 200mins moist 10 mins dry. So if you were to heat 10min dry then 10min moist it would kill them both. These are not real figures, I am just exagerrating the point.

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Offlinemogur
regnartS

Registered: 11/15/05
Posts: 322
Loc: Puget Sound
Last seen: 11 years, 3 months
Re: Low temp fractional sterilization? [Re: blackout]
    #5224989 - 01/26/06 05:37 AM (18 years, 1 month ago)

Check this study, by Nicholson et al.-

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