While this forum is littered with all sorts of information regarding ergot isolation, cultivation, mutation, etc as well as info directly on what causes increases and decreases for alkaloids. It is still not clear what the very best mixture for ergot culture is when it comes to producing the highest amount of alkaloids.

The reason for this is simple, it probably isn't known (or shared) what the very best mixture is -- ignoring other things such as alginate, methods to increase oxygen, etc

I think working towards a theoretically high yielding alkaloid culture medium is  something that is important to find out, so everyone can know and continue to add to it later on down the road. It will be useful no doubt and we can at least get a good idea what the mixture might be with some group-brainstorming,

All right so this is what we know in brief:

What increased alkaloids:

*Low phosphate concentration
*Asparagine
*Other amino acids along side asparagine that increase alkaloid content, luicine, glycine?
*Arsenate salts
*Biotin
*High osmotic pressure - often 100g of sucrose or 10g or so grams of salt would be added to solutions for the ergot
*Citric acid, succinic acid, and other supplements of the citric acid acid apparently improve yields.
*Actively growing fungi doesn't produce much alkaloid.

I'll continue to add to this, as it is mostly my project, but please feel free to bring up things I have forgotten or what I might over look...

So far I can't think of what else increases it ,but I will review a good amount of my notes tomorrow and I'm sure i will find some, or just wake up tomorrow remembering a few more - its time for me to sleep now. goodnight.

Great info, I didn't realize it was a ratio nor about the need for an anti-foam agent. Why is it that reduced surface tension leads to better oxygen? Allows for formation of smaller oxygen bubbles and thus increased surface area?


Additionally it is important to use sucrose instead of glucose or fructose, correct? The disaccharides yield a medium that produces more alkaloids than one does with polysaccharides.

What about the use of other di or trisaccharides?

Common  available disaccharides include....
Sucrose monomers being glucose    and fructose    ?(1?2)
Lactulose  monomers being galactose and fructose    ?(1?4)
Lactose monomers being galactose and glucose    ?(1?4)
Maltose monomers being glucose and   glucose    ?(1?4)
Trehalose  monomers being glucose and glucose    ?(1?1)?
Cellobiose  monomers being glucose and glucose    ?(1?4)

related:
 
A P-D-Fructofuranosidase from Claviceps purpurea

By A. G. DICKERSON
Department of Biochemistry, Imperial College of Science and Technology, London S. W.7, U.K.
(Received 1 May 1972)

The link above talks about what ergot does with the sucrose, which is thought to be the main carbohydrate source under parasitic conditions. Considering the goal of wild growing ergot (to survive and reproduce) is different than our goal (to produce alkaloids) I think it can give some insight into what is good and bad about sucrose. It claims that most of the fructose is turned into an oligosaccharide and used for storage - yet at the same time it points out that fructose decreased growth. This could possibly mean that we want a disaccharide made out of fructose-fructose -- as actively growing ergot doesn't produce alkaloids as much -- or that we want a disaccharide with little to no fructose. I am not sure, I haven't fully read it and it does not mention much about this stuff anyways..

Here in the UK, gaviscon is alginate (I wonder....)

Depends on what the fungus likes to be bathed in, some synthetic antifoams decrease yield, ergot sclerotia oil is often used.

Tweens increase yield.

Tweens are polysorbate polymers and are added to almost everything. If I remember correctly warami posted a link back on the hive showing that they were a mutagenic addative added to prevent pseudoephedrine extraction from pills. The multiple ether bonds act to chew up HI in needless side reactions from the LWR's.

http://en.wikipedia.org/wiki/Tween_20

http://en.wikipedia.org/wiki/Maltose

Malt extract, which I assume is mostly matlose is one of the preferred carbohydrate sources for growing mycelium in liquid cultures and on agar plates when it comes to mushrooms, likely other fungi as well -- at least to my understanding.

The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.

-- hxxp://www.ncbi.nlm.nih.gov/pubmed/11852564

Favourable results were obtained by using 25 per cent maltose solution as a protective medium.

hxxp://www.ncbi.nlm.nih.gov/pubmed/2025122

So maltose might be good - I really don't know since it turns into glucose and that induces growth while fructose, if present, slows down growth - which might be good for alkaloid production. Any ideas?

Or other things to add to the mix before I start to try and compile everything into one super-ideal perfect ergot culture medium for alkaloid production?

Seed stocks are often made from a mixture containing corn steep solids, this causes rapid growth with lowered alkaloid production capability.  Once the seed cultures are done they are filtered and added to the production stock.  The corn steep will lower yields if not filtered out beforehand. 

think the corn steep solids ref is in one of otto snows books.

Here is the optimized overunity33 production stock, taking into account many patents and papers I posted.  If using a good strain you should be able to pump out 4g/L no problem.  First nute base option is from the patent where they worked with the standard paspali and also a UV mutant, the second is from a patent which states slightly higher yields using a standard paspali culture.

150 g/L mannitol + 50 g/L peptone “Torlak” (Sabouraud dextrose agar)    OR   50g/L mannitol + 20g/L nacl + .3g/L epsom salts
Tween 40/60/80 - .5% by volume
Simethicone (antifoam) - .5% by volume
Citric acid - 1% by volume
KH2PO4/Na2HAsO4 - 1% by weight/5.51mM (50-20:1 ratio optimal)
Boiled tapwater is used for trace mineral content
adjust pH to 5.2 with ammonia, maintain pH with NaOH throughout fermentation.

you can say that again it gets very confusing poring over all these patents trying to make heads or tails of it.

amino acids like luecine would'nt be necessary for c. paspali as that gets incorporated into the complex ergopeptides produced by c. purpurea trytophan and alanine are the main building blocks of the simpler lysergamides produced by c. paspali.
steeped corn is pretty much fructose by the way

Seed stage medium:
40 g/L mannitol, 10 g/L glucose, 2 g/L chicken peameal, 3g/L KH2PO4, 3g/L epsom salts, 30 g/L succinic acid OR 1% citric acid

I honestly have no idea, I just read this: http://en.wikipedia.org/wiki/Corn_syrup and it makes me thing it is going to be mostly glucose - but I could easily be wrong.

he glucose can then be transformed into fructose  by passing the glucose through a column that is loaded with the enzyme D-xylose isomerase, an enzyme that is isolated from the growth medium of any of several bacteria.[5][6]

The viscosity and sweetness of the syrup depends on the extent to which the hydrolysis reaction has been carried out. To distinguish different grades of syrup, they are rated according to their dextrose equivalent (DE).

Where is it you heard the fructose thing?

Anyways, we should get back on topic...

any benefit to using succinic acid over citric acid or vice versa?
I guess they are just two commonly bio-chem reagents in the laboratory employed for the same purpose and it probably doesn't make much of a difference.


So it seems like two different culture mediums will be needed - one for C. Purpurea and C. Papsali in order to yield the highest alkaloid production for each species.

overunity33, the more I look at your first mixture - the more I like it, because i did not realize the significant difference  needed between the Papsali vs Purpurea medium. I just wish it had more detail on the micro-nutrients needed. :/


That is good to know.