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bodhisatta 
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Endospores in Cerial Grains Discussion 13
#21604725 - 04/27/15 04:37 PM (9 years, 2 months ago) |
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This thread/post examines whether or not you end up with more endospores after a cereal grain soak for the explicit purpose of germinating endospores to aid in sterilization cycles.
A sufficient and correctly done Autoclave/PC cycle WILL kill endospores
endospores DO NOT need to be "softened" "soaked" "hatched" "germianted" etc... to be killed
it is my belief, that endospores are likely more numerous after a typical "endospore germinating soak for cereal grain" than when you started if you were to simply hydrate and then sterilize without a prolonged soak.
if you wish to debate please back up relevant facts or evidence. Please don't cite quotes from other users than yourself, and if you cite yourself please include the relevant facts sources and or evidence to support the claim.
There's a few species of bacteria that produce a spore like state to survive harsh environmental conditions.
The model organism in science is Bacillus Subtilis it's the most well studied species of these spore forming bacullus spp.
these spores are called endospores and the process they're made is called sporulation.
Sporulation is driven by stress response, most namely nutrient deprivation.
Quote:
A qualitative simulation method is used to model the sporulation network and simulate the response of the cell to nutrient deprivation. Using this method, we have been able to reproduce essential features of the choice between vegetative growth and sporulation, in particular the role played by competing positive and negative feedback loops.
article behind a paywall edit 10/16/16 http://sci-hub.io/10.1016/j.bulm.2003.08.009 (check the top of page 4 for more info)
I'll work on sourcing a graph of this trend. Bacillus will not simply be all or nothing. endospores are formed in an increased trend when nutrient deprivation signals increase; however this does not mean some small percentage of cells find it appropriate to form an endospore in a non-critical environment. this trend modulates back and forth depending on the substrate quality and other methods of survival that have been triggered. some subset of a large population over millions in a quart jar will form endospores despite conditions an inappropriate sporulation response as a human may call it. as conditions actually become more intolerable a large subset of the population will initiate sporulation.
it is actually favorable for a small subset of the population of spore forming bacteria to produce spores in "adequate" environments. this is the "head start" idea.
Quote:
We revisited the previously postulated cell cycle dependence of the initiation of spore formation. We show that the sensitive stage within the cell cycle during which spore development can commence is due to oscillations in the concentration of the master regulator of sporulation, Spo0A∼P. These oscillations are, in turn, partly driven by periodic expression of the sda gene in response to normal cell cycle cues dependent on the activity of DnaA protein. The results provide a molecular explanation for the coupling of sporulation initiation to cell cycle progression and underscore the need to coordinate DNA replication with cellular development to achieve maximal spore frequency and fitness.
http://genesdev.cshlp.org/content/23/16/1959.full
Spo0A and SDA initiate and modulate spore forming. it's a modulatory process in which a part of the population will be
Quote:
In particular, the first cells to initiate sporulation, ∼9 h into microcolony development, showed significantly lower levels of sda transcription than other cells (0.24 ± 0.01 vs. 0.37 ± 0.01 AU; t-test, P < 0.05; >900 cells measured from three independent movies). Early spore formers represented ∼10% of cells at that time. Importantly, it was also clear that sda expression was highly heterogeneous under these microcolony conditions
in this study they find 10% are early spore formers in their heterogeneous colonizes. 10% being an appropriate frequency for some subset of the population to be early spore formers to give a whole colony more options for survival long term.
Quote:
The time-lapse movies described above, using microcolony growth, revealed that there is a great deal of cell-to-cell variation in sda expression, even during the exponential growth phase, when all cells are engaged in DNA replication
cell to cell variation is diversity and diversity ensures survival.
I find it highly likely that in the "endospore germinating soak" that people are inadvertently creating more endospores than what was started with.
as for endospors needing to be treated in some way to be killed by sterilization, that point is definitively wrong.
Quote:
Because the destruction of endospores has been the benchmark of sterilization since the autoclave was invented, the only test of sterilization effectiveness accepted by regulatory agencies such as O.S.H.A. and state boards is endospore testing
autoclaves are tested with heat resistant bacillus endospores to verify they work http://www.gru.edu/dentalmedicine/oralbio/sterilization.php
11/22/16 update
Quote:
Depletion of carbon, nitrogen, or phosphorous causes the process of sporulation to begin, however, the process needs to start before the entire exhaustion of nutrients (Perez 2000). Otherwise, the spore formation cannot be completed due to the fact that the nutrients are too low for the energy-requiring sporulation process. This allows the cells to avoid being stuck in a vulnerable position.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC94387/?tool=pmcentrez
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blindingleaf
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21604909 - 04/27/15 05:12 PM (9 years, 2 months ago) |
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nice! i have to read that second link over its pretty long.
u mentioned in the other thread that there is living bacteria right now on the dried grains we have stored? do u know what kind of bacteria is reproducing in that kind of dry environment? and that when we soak grains, its that living bacteria that is multiplying in the soak water, not the endospores germinating cause the smell? i think you had mentioned endospores cannot germinate in 24 hours, do u have the link to that?
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21604955 - 04/27/15 05:19 PM (9 years, 2 months ago) |
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Quote:
bodhisatta said: the debate is about whether or not you end up with more endospores after a cereal grain soak for the explicit purpose of germinating endospores to aid in sterilization cycles.
How long a soak are you talking?
The most commonly stated guidelines for "fractional sterilization"/tyndallization is 24hr intervals before heat cycles. I always found it odd that 24hrs would be the optimal time, seeing as it is coincidentally 1 day cycle, I did not believe it was optimal and rather just easy to quote/instruct. When I found John Tyndall's actual texts from the 1800's he specifically said 24hrs is too long, and will lead to too much growth. He advised 10-12hr cycles which would "destroy all germs then approaching their point of final development"
I have had "intermittently heated" wheat & rye grain jars which showed no contamination after 5+ years. -not I am wary about how I word what I do, so as not to give any pedantic/semantic cunts anything to cry & bitch about.
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bodhisatta 
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21604999 - 04/27/15 05:27 PM (9 years, 2 months ago) |
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Quote:
blindingleaf said: nice! i have to read that second link over its pretty long.
u mentioned in the other thread that there is living bacteria right now on the dried grains we have stored? do u know what kind of bacteria is reproducing in that kind of dry environment? and that when we soak grains, its that living bacteria that is multiplying in the soak water, not the endospores germinating cause the smell? i think you had mentioned endospores cannot germinate in 24 hours, do u have the link to that?
when you soak your grain all the ambient bacteria on your hand on the dust in the air the stuff that's been on the grains as you get the soak going etc.. those are all going to start to go to work, endospores can germinate in minutes if conditions are perfect but I don't know the average time to germination in the environment of a grain jar or a grain soak. I would assume some percentage of the population of endospores will germinate in the soak duration, some wont, and I don't know of any study done on grain in a soak environment to determine those percentages. but during normal vegatative growth lots of endospores are being formed daily. 10% of a few million bacteria forming an endospore in a 12 hour soak and then that endospore is not able to germinate before the soak is done you are probably ending up with more than you started with. but as we have seen from how well no-soak teks work the point is moot as the sterilization kills the endospores anyway. I would still like to know though if the soak is actually inadvertently worse than no soak at all.
for example about that bacteria making the soak smelly quickly. you could take water and sugar and put it pot and warm it up to your soak temp and then swipe your hand over your counter top then shake your hands above the sugar water and it would get smelly by tomorrow morning. no grain involved.
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bodhisatta 
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21605048 - 04/27/15 05:36 PM (9 years, 2 months ago) |
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my understanding of tyndallization was that yes they are short intervals of 12 hours between treatments but the overall time of all three treatments is greater than 48 hours to ensure that all endospores have germinated.
day 1 kills vegitative to prevent them from forming more endospores.
day two kills any endospores that have germinated and formed vegitatives so that the vegitatives won't make new endospores
day three is the time in which you can be reasonably certain all endospores have germinated and you kill the final vegatative bacteria from those germinated bacteria.
so yes 12 hours is the tyndal thing but overall it's greater than 48 hours because some population of the endospores (extra vegatiave in the literature) take more than a day to germinate, the sole purpose of the multiple treatments is to prevent more endospores from forming while you wait for the extra vegitative ones to germinate more than 48 hours later
it's the entirety of the temporal part of tyndalization that matters it's not the individual treatments. so yes tyndall was right 24 is too long because you don't want new vegitative bacteria to form endospores which would require you to restart the three day tyndallization procedure
tyndallization also helps prove you can create more than you started with, hence those intermitent treatments every 12 hours to keep new ones at bay.
tydallization also helps prove that a soak is actually ineffective for germinating the damn things anyway since tyndallization requires greater than 48 hours to ensure that extra vegitative endospores are killed.
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bodhisatta 
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta] 1
#21605083 - 04/27/15 05:44 PM (9 years, 2 months ago) |
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Quote:
1 COURSE: MCB 4403L (MICROBIOLOGY LABORATORY ñ FALL 2005) Supplemental Reading Material for Chapter 12: STERILIZATION PRINCIPLES AND METHODS
TYNDALLIZATION
Tyndallization is used for media which can't withstand autoclaving and relies upon the germination of spores to form cells that can be killed at 100C
A. Principles
The principle of Tyndallization depends upon spores germinating within the 49 hour period. On day 1, most of the vegetative bacteria, molds and mold spores are killed, but some bacterial spores will survive. The surviving spores may germinate overnight and be killed during the 2nd steaming. The third steaming is an added precaution.
B. Drawbacks
Tyndallization is not 100% dependable. While it was once considered a means of sterilization, it is not used much today. The two problems with this method are
1) If there are many spores in the broth, then there can be significant growth of bacteria. While these cells are killed in the next boiling step, the dead cells remain as debris in the medium/broth.
2) This procedure works only for broth media that support the growth of spore-forming organisms. It is not useful for sterilizing water or buffers.
C. Timetable for Tyndallization
1) Steam heating to 100C for 15-30 min Vegetative cells are destroyed but endospores survive 2) Incubate at 30C-37C overnight Most bacterial endospores germinate 3) Second heat treatment, 100C, 15-30 min Germinated endospores are killed. 4) Second incubation at 30C-37C overnight Remaining endospores germinate 5) Third heat treatment, 100C, 60 min Last remaining germinated endospores are killed
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blindingleaf
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21605120 - 04/27/15 05:51 PM (9 years, 2 months ago) |
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so u think the smelly bucket of soaking grains is not from the endospores themselves, but from ur hands that are mixing the grains, or just swiping ur hand over the grains?
in ur opinion, a qt of rye that was soaked, simmered, dried, then loaded, and cooked for 1 hour will succumb to bacteria is same amount of time as rye + water (no soak, just combine)>PC 1 hour, if the jars are kept next to one another in the same conditions?
in that example above, Quote:
The surviving spores may germinate overnight and be killed during the 2nd steaming. The third steaming is an added
wouldn't there be more endospores as well that would need another treatment, if replication and sporulation are happening?
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bodhisatta 
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605149 - 04/27/15 05:57 PM (9 years, 2 months ago) |
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Quote:
blindingleaf said: so u think the smelly bucket of soaking grains is not from the endospores themselves, but from ur hands that are mixing the grains, or just swiping ur hand over the grains?
in ur opinion, a qt of rye that was soaked, simmered, dried, then loaded, and cooked for 1 hour will succumb to bacteria is same amount of time as rye + water (no soak, just combine)>PC 1 hour, if the jars are kept next to one another in the same conditions?
in that example above, Quote:
The surviving spores may germinate overnight and be killed during the 2nd steaming. The third steaming is an added
wouldn't there be more endospores as well that would need another treatment, if replication and sporulation are happening?
probably a bit of both some endospores will germinate quickly probablu but a large part of the things making a soak go funky are natural contaminants. it's how spotaneous fermentations happen yeast and bacteria on the walls and dust in the air, and the air being blown from outside and making it through your furnace filter etc...
adding a few grains into that water will surely make it succumb to smell faster though
I would think yes that if you did a
soak simmer dry load sterilize for an hour and a simmer boil dry load sterilize for an hour they would both contaminate in the same time left side by side on average
if you just take water and grain and PC though you're trying to sterilize dry grains and it won't work well the grain has to be hydrated in both cases but one without the soak and one with the soak both are hydrated both are sterilized for one hour both will contaminate in the same time I would expect. though if you took water and grain and tried to sterilize that it would contaminate sooner as you're really not sterilizing dry grains effectively.
as for this part
Quote:
in that example above, Quote:
The surviving spores may germinate overnight and be killed during the 2nd steaming. The third steaming is an added
wouldn't there be more endospores as well that would need another treatment, if replication and sporulation are happening?
probably that's why tyndallization sucks.
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blindingleaf
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605161 - 04/27/15 06:00 PM (9 years, 2 months ago) |
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im gonna mix some sugar into water with my hands tonight, and hope that tomorrow night, its a smelly mess.
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bodhisatta 
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605170 - 04/27/15 06:02 PM (9 years, 2 months ago) |
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Quote:
blindingleaf said: im gonna mix some sugar into water with my hands tonight, and hope that tomorrow night, its a smelly mess.
aim for a concentration around 5-10% sugar by weight. like 5-10g sugar to 100ml(100g at room temp) of water.
tomorrow night it will be fucked if it had more essential nutrition like nitrogen sources (FAN) in it. but I suspect it will be noticeably funky
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605180 - 04/27/15 06:03 PM (9 years, 2 months ago) |
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Quote:
if you just take water and grain and PC though you're trying to sterilize dry grains and it won't work well the grain has to be hydrated in both cases but one without the soak and one with the soak both are hydrated both are sterilized for one hour both will contaminate in the same time I would expect. though if you took water and grain and tried to sterilize that it would contaminate sooner as you're really not sterilizing dry grains effectively.
but, u said it doesn't matter because the autoclave is meant to destroy endospores? if the comparison is with simmered grains vs soak>simmer, its a different comparison, because that initial simmer is going to reduce population and/or help to germinate existing endospores
and will do on the sugar. i'd think many iced teas i've made (with even more % sugar) in the past would have gone bad sitting in my sink waiting to be washed, but ill try again.
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605202 - 04/27/15 06:06 PM (9 years, 2 months ago) |
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sterilization doesn't work with dry heat though on those endospores. the autoclave kills them yes but they're protected by dry starch so they're not actually being sterilized
if you do your sugar water experiment with a little bit of BRF it will get funky way faster since the BRF has nutrition the bacteria will need but BRF has no endospores according to most people but I'm not entirely positive of that though BRF can be steam sterilized in the case of PF cakes which leads people to believe there's no endospores in it.
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21605212 - 04/27/15 06:08 PM (9 years, 2 months ago) |
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and yes maybe the simmer does kill some endospores but people soak for the explicit purpose to germinate endospores to make them easier to kill so the comparison should be good enough for those people if you compared hydrated rye without soak to hydrated rye with a soak since their belief is that the soak is germinating them and my simmer wouldn't cut it. (and it wouldn't but the PC would)
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta] 1
#21605234 - 04/27/15 06:12 PM (9 years, 2 months ago) |
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well...i still think that the soak does germinate endospores, and thats what makes the smell, not the random yeasts, etc that fall on my hand. ill add some peptone instead.
so, to sum it up, you think that the soak people do to hydrate their grains has nothing to do with endospore germination? and that a soak would not affect sterilization (i.e. leaving two jars side by side to see which contaminates first)?
by ur theory then, wouldn't the soaked grains actually contaminate first?
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605242 - 04/27/15 06:14 PM (9 years, 2 months ago) |
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Quote:
by ur theory then, wouldn't the soaked grains actually contaminate first?
I would think so yes.
if you make 10% sugar water and add some peptone it will be funk tomorrow if you make it open air never heat it past 140F and use your hands.
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Re: Endospores in Cerial Grains Discussion [Re: bodhisatta]
#21605298 - 04/27/15 06:22 PM (9 years, 2 months ago) |
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Quote:
B. Drawbacks
Tyndallization is not 100% dependable.
Indeed, especially if you do what John Tyndall specifically warned against. That is just one of many texts I have seen giving incorrect advise, not just some minor semantics, they are instructing you to do exactly what he warned against, most annoying that the ignorant fuckers put his name to it.
I will usually boil grains a little and let them sit to soak up water. The advise about soaking is not just about germinating endospores. The even soaking allows less chance of burst grains, due to over soaked grains at the bottom of jars and drier ones on top. It is also to allow better heat transfer, just like people soak chickpeas or indeed wheat or rye before pressure cooking to eat themselves.
If doing intermittent heat treatments I usually do more than 3 heat cycles.
If the newly developed endospores are weaker then the supposedly better heat transfer properties might offset the negatives. i.e. if you had a soaked and unsoaked jar, and pressure cooked for a short time, say 30mins then the soaked may fare better, even if it technically had a higher endospore count.
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605423 - 04/27/15 06:41 PM (9 years, 2 months ago) |
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I see this repeated a lot on sites talking about sugar as a preservative:
"High concentrations of sugar also exert osmotic pressure that will draw water out of bacteria, preventing them from growing."
My very uneducated understanding:
Not all species of bacteria produce endospores. Different species produce and germinate endospores at different rates and under different conditions.
The stink of grain soak water is from a combination of endospore producing and non-producing species of bacteria. (other stuff causes stink too, but we're talking endospores here)
Without a link to back my claim, I'll still go out on a limb and say that some endospores will germinate and produce more spores and endospores while others will not. But, I don't think it matters if more endospores are being produced because they are hydrated and are easy to kill in the PC. My understanding, from The Shroomery, is the dry endospores are the ones that are hard to destroy in the PC.
At some point, the exponential growth of the different species reproducing together in the same pot, would trigger a endospore producing response to the sudden decline in food and increase in competition. I think it's more at the end of the soak cycle is when the vast majority of endospores would produce, but they are soft because they are in water. And, the water still stinks because of the number of non-endospore producing bacteria still expanding.
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Re: Endospores in Cerial Grains Discussion [Re: SpitballJedi] 1
#21605527 - 04/27/15 07:04 PM (9 years, 2 months ago) |
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good point about the hydration of new endospores. but i don't know if a grain soak would run out of nutrients in 24 hours.
i know that not all species produce endospores, but i still think, if one were to say sterilize a 5 gallon bucket, and gloves, and dump dry grains into the bucket and stir in water in front of a hood/in lab, there would be spore germination from just the endospores on the grains. if there were non endospore forming bacteria growing, where would they come from? if they were on the grains, there would be signs of bacterial growth, and some moisture to allow survival thru storage.
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Re: Endospores in Cerial Grains Discussion [Re: blindingleaf]
#21605619 - 04/27/15 07:23 PM (9 years, 2 months ago) |
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I don't think they are running out of nutrients, but there is a lot stuff growing in there and the mere population and exponential growth of multiple species may be a stress trigger for various species.
I think if you were to sterilize your gloves, bucket, water, and every thing else except the grain, then you'd still get stink from some endospore germination. Because of the presents of so many molds, mildew, and fungi, I don't think it's possible for grains grown and harvested outside in the open, then packed and shipped, to be without them by the time they go in your sterile water bucket. I base this on zero amount of research, but I'm pretty sure "dry" doesn't kill everything. I could be wrong though, but I think grains are covered in more crap than just endospores that wake up once wet.
I think we don't see signs of bacteria on dry grains because there just isn't enough moisture for favorable conditions to germinate. They won't just germinate just because they are in the presents of moisture.
It may also go back to that thing about sugar as a preservative. Theoretically, the sugars from the grain may also be preventing a "favorable conditions to germinate" response even in the presents of minute moisture.
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Re: Endospores in Cerial Grains Discussion [Re: SpitballJedi] 1
#21605721 - 04/27/15 07:40 PM (9 years, 2 months ago) |
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Quote:
I think we don't see signs of bacteria on dry grains because there just isn't enough moisture for favorable conditions to germinate. They won't just germinate just because they are in the presents of moisture.
but, if they need to germinate, then isn't that either an endospore, or something like it? the grains we get are often stored for half a year before we receive them. if there is bacteria on there that does not form endospores for long term survival in less than ideal conditions, i'd be curious to learn what kind of bacteria that is
i would agree there is more on grains than endospores though, just not sure to what degree a soak would be ideal for them. i'd imagine, like how mold grows on top of a cup of coffee after a while instead of at the bottom of the cup, its the anaerobic nature of the soak that makes it ideal for bacteria. add to that hot water (i start with hot tap water, and add a gallon of near boiling before letting it soak overnight), and i think the conditions are ripe for bacteria, specifically those forming endospores that end up on the dry grain, which are then hydrated and softened by the hot water, inducing germination. similar to how jars/bags in too high a temp for incubation get bacteria (heat), or LC's/jars/bags with insufficient GE get bacteria (anaerobic), a soak would do a similar thing i'd imagine. i think a simmer/no soak would also be effective for similar reasons. whether a soak>simmer>PC is more effective than a no soak>simmer>PC, I'm not sure. i'd think the best case would be long soak (18-24 hours) with warm-hot water would be best if the idea was to germinate as many endospores as possible. i still don't have the link about average time for endospore germination, but i would bet its based on temp, all else being equal. whether the "new ones created" are as resistant to heat, or not as resistant, I'm not sure. i would guess less resistant to heat (PC) than ones that sat for a few months in dry storage, like u mentioned before
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