mnick12 - 30-10-2013 at 13:46
Hello everyone,
It has been a while since I have done any home chemistry, but I have a few questions I hope you all could help me with. In the past year or so I have
really gotten into home-brewing, it is a lot of fun and can be very scientific. Anyway I am at the point where I culture and grow my own yeast.
Normally I use dried malt extract as the nutrient media, but it is quite expensive especially if you are growing a large quantity of yeast. While
looking for alternative nutrient sources I ran across brown rice syrup which has about a 1:1 ratio of maltose and maltotriose; good enough for me!
While significantly cheaper than malt extract I thought I could do better. I chose to make my own rice syrup from grocery store rice, since dried rice
is just about the cheapest thing in the world. The rice I am dealing with is about 83% percent Carbohydrate, 9% Protein, 3.4% fat, and the rest misc.
Anyway as a preliminary trial I cooked a pot of rice per the instructions, but with extra water. The rice soup was then blended to a goopy mix.
Following this the mix was cooled to 135F, and 1/4 teaspoon of amylase enzyme mix ( available at brew shops) was added. Quickly the goop began to thin
and over the course 26hrs the mix began to taste distinctly sweet, possibly indicating the presence of smaller sugars. However the solution never
cleared and something remained in suspension. My guess is this is mostly protein and insoluble fiber.
Now ideally I would like do digest the rice protein into smaller soluble peptides, since Saccharomyces likes free amino nitrogen when it is growing.
My question is what sort of protease enzyme could accomplish this? Both papain and Bromelain are freely available and very cheap, would they
effectively break down the rice protein?
Any ideas are welcome.
Thanks!
mnick12 - 1-11-2013 at 18:36
A brief update:
Two cups ( 170g) of brown rice was cooked on the stove top per the instructions on the bag. Following this an additional 1500ml of tap water was added
to the rice, followed by an additional hour of boiling. The rice began to break apart forming a thick syrup with rice particulate suspended in it.
Next a hand blender was used to puree the mix. This produced a very gelatinous mix, and it was decided full starch gelatinization had occurred. In
order to more easily process the starch, the goop was placed on a cookie sheet and allowed to dry in an oven set to 215F for >12hrs. This produced
a crumbly brown translucent material that was further processed into a coarse powder for storage. From 170g of rice I was able to recover 127g of
gelatinized product.
The following day the rice powder was added to 500ml of tap water plus 250ml of distilled H20. The solution quickly gelled in a mater of 2 or 3
minutes. The resulting goo was extremely thick and difficult to stir. Next the mix was allowed to cool to 150F, and an arbitrary amount (1/16th of a
tsp) of amylase formula was added. Rapidly the gelatinous goop thinned to the consistency of thin pancake batter. The mix was held at 150-155F for 4
hours to ensure starch breakdown.
That where I am at right now, I will dry the stuff overnight.
Additionally I purchased some bromelain tablets sold as a nutrional supplement, not sure when that will be here but it will be used to digest the rice
protein. Also the amylase formula I am using will not produce simple sugars, it will only make smaller dextrins from the large polysaccharides. So in
order to make brewers yeast friendly sugars I also purchased a different amylase enzyme capable of hydrolyzing the 1,4 and 1,6 -glycoside bonds. This
should chew up those dextrins and produce the fermentable sugars I am looking for.
Stay tuned...not that anyone reads the biochemistry forum anyway...
crazyboy - 1-11-2013 at 21:01
A little confused as to your goal, if it is turning rice into alcohol why bother with all this hassle? Aspergillus oryzae has been used for hundreds
of years for its ability to produce amylases. I would imagine yeast should have the requisite enzymes to digest the proteins in rice.
mnick12 - 2-11-2013 at 10:35
My goal is to turn rice into a completely fermentable media, so that I can grow large amounts of yeast to make beer. Normally people use malt extract,
but it is very expensive. My idea was to use rice as a starting material since it is dirt cheap and has all of the necessary nutrients. As for the
enzyme, one of the amylase enzymes I will be using is an isolate from Aspergillus Niger. It is very potent stuff .36ml of enzyme/lb of starch.
Also as far as I know brewers yeast can digest smaller peptides, but really prefers free amino-nitrogen. In this case it is able to make almost all
of the proteins it needs. Thus the main issue I am concerned with is making sure the protein is in solution, and bromelain should do a good chewing up
the larger peptides.
phlogiston - 2-11-2013 at 15:36
I suspect it is much cheaper and certainly easier to just add the nitrogen in the form of an ammonium salt.
I used to culture Saccharomyces cerevisiae for biochemical research, not brewing, and I used ammonium sulfate as a nitrogen source. It is also
approved as a food additive (E517 in the EU), so it is probably safe to use for your purposes too (but do check this first!). Especially if you wash
the yeast before using it for brewing. I don't know if it has any taste. (Many free amino acids, however, have a very disagreeable, bitter taste).
Usually, I do not add ammonium sulfate itself, but 'yeast nitrogen base', which contains several other essential nutrients in addition to ammonium
sulfate. I only very rarely prepare the whole mixture myself from the individual chemicals (only when I need a special compositon for experiments).
[Edited on 2-11-2013 by phlogiston]
mnick12 - 2-11-2013 at 22:00
I have product called "yeast nutrient" it is a mix of urea and ammonium phosphate, it is recommended for beers with low nutrient levels ( adjunct
laden), and things like cider.
I have in the past tried to prepare yeast media from sucrose and this yeast nutrient. The yeast will eat this stuff but fermentation is sluggish when
compared to malt extract. My hypothesis is the slightly more complicated peptides and sugars give the yeast more to work with. That is why I would
like to breakdown the rice protein into soluble peptides, so that the yeast might grow in conditions similar to those found in beer wort.
Hopefully the bromelain will work, otherwise I may just have to settle for the yeast nutrient.
Also an update on the rice:
The amylase treated rice syrup was dried on a cookie sheet over night in the oven producing a translucent brown crystalline "cracker" that is
extremely crumbly. I have not taken a weight yet, but I will do that next. When the other amylase and Bromelain arrive I will be doing a second
sacharification and protein digestion. I will be doing this in phosphate buffer to ensure maximum enzyme activity.
Also I think it would prudent to start from brown rice flour rather than the grain, this would save a lot of hassle.
Thanks for the input btw
jwpa17 - 14-12-2013 at 11:19
You cook simply cook the rice in, e.g., 0.1 M HCl, for an extended period, then neutralize the acid. Although I frankly suspect that the yeasts will
grow quite happily on your home-made rice syrup without worrying about the proteins.
mnick12 - 14-12-2013 at 12:39
Sorry this got put on hold for finals.
The bromelain arrived and I tried out the enzymes on a boiled mashed potato, buffered with phosphate. The resulting solution still had some white
insoluble material in it. This was filtered down and boiled to produce a dark sweet and umami flavored syrup. I am still working with the rice, but it
is harder to process.
@jwpa17:
Acid digestion is something I never considered, how well does it work?
acid digestion of proteins
jwpa17 - 4-1-2014 at 13:54
It's the preferred method for converting proteins to free amino acids for amino acid analysis. Used to be, 24, 48, and 72 hours in 6 M HCl were used
to convert (3 samples of the same protein) into amino acids, and each sample was chromatographed. Some AA are destroyed with time, so you'd
extrapolate to zero time. Some peptides are harder to digest than others, like Leu-Leu, so those you'd extrapolate to infinity.
Nowadays, the digestion is usually a single sample, at 121 C, with HCl in the vapor phase. Last I paid attention, a lot of work was being done on
microwave/HCl digestion, too.
Acids are a large part of how your stomach does it, too.
In your situation, I don't think you really need to reduce the proteins to amino acids. The yeast needs some nitrogen - they'll break down peptides
to get it. I'm still not sure that your crude extract, proteins and all, wouldn't be quite satisfactory to the yeasts.
mnick12 - 10-1-2014 at 13:53
Interesting, I don't have the equipment to autoclave a protein sample. Perhaps and extended reflux would accomplish similar results. Additionally I am
not to concerned with free amino acids, but rather making sure the peptides are in solution. Anyway I tried something similar to the potato experiment
but with rice flour. I ended up with a sweet liquid with quite I bit of material in suspension. I assumed this was some protein, but iodine test
suggested is was large starch particles that were unconverted. I ended up filter the solution (which was a pain) and reducing the volume to ~400ml of
solution with a specific gravity of 1.050. Just for kicks I pitched 4ml of mixed culture, containing various brettanomyces and sacharomyces strains as
well as pediococcus and lactobacillus. Fermentation ensued and there was a nice pellicle a few weeks later. So it looks like one of those species
enjoyed the crude extract.
What I am now trying to work on is reducing the particle size of the rice flour, so it can be converted quicker and more fully. A coffee grinder
doesn't work, I may have to get a ball mill.
jwpa17 - 24-1-2014 at 19:44
You can "fake" a ball mill pretty easily. Just keep in mind that the milling media should be harder than the stuff you're milling AND that the
container will be subject to abrasion, too. I once tried using aluminum spacers in a steel can with large steel nuts to mill paper. It worked quite
well at milling the paper (took a couple of weeks), but it also milled the aluminum.
I used an empty paint can, strips of aluminum angle bolted to the interior of the can, and steel nuts as media. It was rolled on an old
tissue-culture roller.
I had more success (i.e., no aluminum powder/pieces) by mounting a can on a rotary platform and simply allowing the nuts to shake along the bottom of
the can. Kind of a planetary mill. Both were quite noisy, though.
mnick12 - 26-1-2014 at 10:44
I was thinking about one of those rubber drum rock tumblers, I had one years ago when I was into making bp and the like. They are quite reliable and
~35$ from amazon. Anyway I bought some pepsin too, as I am thinking bromelain may not be the best protease for this job, and the pepsin is super
potent 1:3000.
The other enzymes work well, I made some oligosaccharides from a solution of gelatinized potato starch and the alpha-amylase. I am hoping to use this
for a more selective media for fastidious yeasts like Brettanomyces. Now I just need to find a source of cycloheximide to inhibit Saccharomyces.