Sciencemadness Discussion Board

Microbial production of lactic acid

Polverone - 16-4-2006 at 15:34

From Experimental Organic Chemistry pages 110-111:
Quote:
In a 750-cc. flask dissolve 50 grams of cane-sugar or anhydrous glucose in 500 cc. of water; add 20 cc. of sour milk and 20 grams of precipitated calcium carbonate. Set the flask in a warm place (temperature about 40°) and allow the fermentation to proceed for one week. The flask should be taken at least twice a day. At the end of the time add a small amount of calcium carbonate, heat to boiling, filter, pour the solution into a flat dish, and let it evaporate spontaneously. Filter off the solid by suction, dissolve it in a small amount of boiling water, filter if necessary, and set the solution aside to crystallize. Separate the crystals as before and let them dry on a porous plate. The yield is about 35 grams.

NOTE. (a) The sour milk used in the preparation contains the lactic acid bacteria. The milk should be used before the curd has separated. The latter is needed for the development of the bacteria during fermentation. Calcium carbonate is added to neutralize the lactic acid as it is formed, since the fermentation stops if the acid accumulates. Toward the end of the fermentation butyric acid is usually formed. The odor produced is due to this cause. When the sugar has been fermented the calcium carbonate passes into solution. If there is a large residue at the end of a week the milk was not satisfactory.


It says "the flask should be taken at least twice a day" and this is in the original text; it's not an OCR error. I presume that it actually meant shaken twice a day.

I intended to follow this procedure using sucrose. However, since Norris's day, pasteurization of milk has become much more common. I was unsure if this would affect the ability of my milk to properly sour. Searching online, I found sites like this one claiming that "You may notice that raw milk left out will sour naturally but pasteurized milk will rot. This is because the beneficial bacteria in the raw milk helps to keep putrefactive bacteria under control. Pasteurized milk, however, does not have any of the beneficial bacteria left to keep it from rotting." I don't know if this is just fringe nonsense or if lactic acid producing bacteria really are less able/likely to grow and thrive in pasteurized milk that has been left to warm.

Wary of leaving milk to sour, I decided to try jumpstarting the process by using live culture yogurt. I dissolved the sucrose, added 20 grams of precipitated CaCO3, added 20 cc of fresh pasteurized milk, added a couple grams of live culture yogurt from a freshly opened container, mixed it all, and set the capped jar of material on a hot plate that kept it just a bit above 40 C.

Over the course of a week there has definitely been some activity, but a large portion of the CaCO3 apparently remains undissolved. It does smell rather offensively of butyric acid and maybe H2S also.

The procedure doesn't make any mention of sterilization. I had treated the jar I was using (and its lid) with diluted chlorine bleach for a day prior to starting the procedure, washing it out with hot water only immediately prior to adding the sugar water (the sugar water itself was briefly brought to a boil before cooling). Is contamination something to worry about, or will lactic acid bacteria triumph over their competitors if they start out in large enough numbers?

Can (should?) I let ordinary pasteurized milk sour, and use that in place of unsoured milk + yogurt?

Can the bacteria get all the nutrients they need to process all that sucrose from the relatively small quantity of milk added? Would the production go faster or more reliably if I added vitamins or other trace nutrients?

Would using glucose instead of sucrose, or making invert sugar from sucrose, be a better food than sucrose for lactic acid bacteria? Would lactose be a better food?

I am going to try this again, but on a smaller scale until I can get it right, because making pure precipitated CaCO3 starting from CaCl2 is rather tedious.

garage chemist - 16-4-2006 at 15:51

Sounds interesting, I hope that you get it to work.

I'm not sure if yoghurt contains the correct bacteria for this.

Can you get a hold of fresh milk? A local supermarket here carries it, I think that you should be able to get some too.
It is found among the cooled products in the supermarket.

...........some talk of nutrients for your project

solo - 16-4-2006 at 16:38

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate
Sunhoon Kwon, Pyung Cheon Lee, Eun Gyo Lee, Yong Keun Chang, Nam Chang*
Enzyme and Microbial Technology Volume 26, Issues 2-4 , February 2000, Pages 209-215

Abstract
' Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4¢/kg lactic acid, which was only

Attachment: Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate .pdf (220kB)
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denatured - 17-4-2006 at 13:43

"Over the course of a week there has definitely been some activity, but a large portion of the CaCO3 apparently remains undissolved. It does smell rather offensively of butyric acid and maybe H2S also."

Here is what happened: at the beginning the streptococcus started to grow producing acidity .. then lactobacillus is activated(it likes acidity) and grows until all the lactose in the medium is consumed and finished.

At last Oidium lactis fungus and yeast is activated in which they consume all the acid produced turning the medium's pH into neutral. which suits very well the growth of fungi which uses all the carbs in the medium also the esters , alcohols until the medium contains proteins only. here starts the bacteria which decomposes the protein or Proteolytic bacteria ... and H2S evolves and the milk or the medium will be or look like a turbid whey....

Yoghurt contains two bacterium Lactobacillus bulgaricus and Streptococcus thermophilus, the latter ferments Lactose into lactic acid. which is the wanted one .. right?

Lactic Acid Bacteria belongs to :
Lactobacillus
Streptococcus
Leuconostoc
Pediococcus

And all of those are Microaerophilic which means they became active in very very low oxygen.

The pH in which LAB is very active is around 3 to 4... and you should avoid pH of 7 because butyric acid bacteria loves this number and grows at excellent rate, like Cl. butyricum.

So i think to make it work you should try to
1-reduce the air in the container you are using.
2-if you have lactose sugar ... use it.
3-adjust the pH by lactic acid or any other acid to 4.
4-lower the temperature to 37C.
5-add more yoghurt.

"Can the bacteria get all the nutrients they need to process all that sucrose from the relatively small quantity of milk added?"

Yes , Milk is natural medium by itself.

Hope that helps ... and let us know...

ordenblitz - 17-4-2006 at 15:20

Lactobacillus delbruckii and Lactobacillus amylolyticus produce essentially only lactic acid are easily obtained in a home brew supply store.

If they don't have the prepared cultures on hand, whole malted barley is loaded with lactobacillus delbruckii. One would only need buy a fraction of a pound, preferably coarsely ground but not necessary, soak in warm water.. filter out the solids and you have a liquor rife with bacillus.

Polverone - 17-4-2006 at 20:11

Thanks for all the tips. Solo, it looks like the industrial procedure uses higher sugar concentrations, higher nutrient concentrations, and much less time. Even if it had worked as intended, the Norris procedure is much less efficient.

Alnokta, I wouldn't think that much change in pH occurred over the course of the fermentation. That was the purpose of the CaCO3. I don't think I can start the pH out any lower, but I can try using lactose instead of sucrose and leaving very little headspace in the jar. I think the initial liquid is fairly well degassed by the brief boiling before I let it cool.

Oh, one possible problem: the initial pH of the liquid before fermentation started was around 8, or so it seemed (hydrion paper isn't that accurate). I think the problem might have been from hard to remove sodium carbonate that was initially trapped inside small clumps in my precipitated CaCO3. I gather that, if nothing else, I might want to ensure that the pH is no higher than 7 before I add the bacterial source.

Ordenblitz, I don't think I have seen a homebrew place around here, but I will check out the yellow pages. I'm sure I can order online if there isn't one. Some of the challenge is in working with household items, but I'm not above using a superior article if I can't avoid it ;)

denatured - 18-4-2006 at 09:20

After checking , you cannot let the milk sour, if you do let it sour .. what microorganisms would be the responsible for the sour/acidity?

"live culture yogurt"

What is the bacteria types it contains? if you mean by that the starter you add to make yogurt then it won't last for too long cause they engineer it so you cannot use the produced yogurt in making new patchs.

Try to find a natural yogurt or homemade or something like that i don't know what do you call it there ... and then you should try to purify the bacterium lactobacillus bulgaricus.

ordenblitz - 18-4-2006 at 15:58

Polverone:
There is probably more home brew supply shops in "The Sunny Pacific Northwest" then anywhere else in the USA.

I think you may want to stay away from yogurt cultures because they are not going to produce only lactic acid but a mix of things because they are typically a mix three different bacteria . You really should try to get good cultures of Lactobacillus delbrueckii.

But that's just my opinion. I did a bit more research on the web.
Quote:
Starter Culture
The starter culture for most yogurt production is blend of Streptococcus salivarius subsp. thermophilus (ST) and Lactobacillus delbrueckii subspecies bulgaricus (LB). Although they can grow independantly, the rate of acid production is much higher when used together than either of the two organisms grown individually. ST grows faster and produces both acid and carbon dioxide. The formate and carbon dioxide produced stimulates LB growth. On the other hand, the proteolytic activity of LB produces stimulatory peptides and amino acids for use by ST. These microorganisms are responsible for the formation of typical yogurt flavour and texture. The yogurt mixture coagulates during fermentation due to the drop in pH. The streptococci are responsible for the initial pH drop of the yogurt mix to approximately 5.0. The lactobacilli are responsible for a further decrease to pH 4.0. The following fermentation products contibute to flavour:
lactic acid
acetaldehyde
acetic acid
diacetyl

Polverone - 18-4-2006 at 18:15

Quote:
Originally posted by ordenblitz
There is probably more home brew supply shops in "The Sunny Pacific Northwest" then anywhere else in the USA.

I think you may want to stay away from yogurt cultures because they are not going to produce only lactic acid but a mix of things because they are typically a mix three different bacteria . You really should try to get good cultures of Lactobacillus delbrueckii.

I've looked in the yellow pages. I can find breweries and brewpubs, but no brewing supplies or homebrew supplies. I will visit the health food store -- I think they probably have malted barley.

Many sites describe yogurt as being produced by bacterial conversion of lactose to lactic acid, so I thought (especially in light of the Norris preparation) that I might be able to get away with producing mostly lactic acid this way. I won't be able to visit the store until the weekend so I'll probably try again with yogurt on a reduced scale.

ordenblitz - 18-4-2006 at 19:04

Alternative sources of lactobacillus........

It is also present in flour and some wheat products depending on the processing. Raw cabbage has it as well. Yeasts are also present, which cause soft sauerkraut of poor flavor when the fermentation temperature is too high. Lactic acid fermentation gives the sour taste to sauerkraut and pickles. The sugars in the cabbage are converted into lactic acid and serve as a preservative.

Chucklz - 1-6-2006 at 12:54

Tomato Juice Agar can be used to isolate Lactobacilli

To make 1L

400mL tomato Juice
10.0g Peptone
10.0g Peptonized Milk
11.0g Agar

Autoclave at 121C for 20 minutes.

Lactobacilli can be isolated from malted barley, sour milk, yogurt, the mouth, and vagina.

Polverone - 1-6-2006 at 18:08

Thanks for reminding me of this topic. I bought some refrigerated L. acidophilus from the health food store shortly after my last post. I also continued experimenting with yogurt as a starter, despite misgivings about mixed acid production. The acidophilus seems to grow okay but it doesn't take off with as much vigor as the yogurt-started batches, perhaps because the yogurt contains additional nutrients, or just because there are more live bacteria in the dollop of yogurt than in one acidophilus capsule. I did see a rather disturbing report examining L. acidophilus probiotics shortly before I made the purchase. It turns out that many commercial preparations contain additional bacteria not listed on the bottle or are altogether non-viable. Fortunately, non-viability was found to be rare among refrigerated samples and most of the contaminations spotted would not terribly interfere with my lactic acid production.

I have yet to see a batch completely consume all the precipitated chalk that is initially added. Some sugar is also left which leaves me with hard-to-purify liquid. It definitely contains soluble calcium salts, meaning acid was produced, but I don't have a good way to isolate the acid from the thick brown syrup of sugar and salts that remains after I cook it down.

I think I need to use a better nutrient system. I had tried supplementing the sugar solutions with a crushed multivitamin pill. The one solution I most thoroughly crushed the pill into, though, completely failed to grow. My off-the-cuff rationalization was that maybe all that iron isn't good for organisms that can't easily repair oxidative free radical damage. And maybe the multivitamin really isn't that good even for the batches that it doesn't completely kill off.

I may have to try completely un-supplemented solution again; according to Norris at least the bacteria should eventually if not rapidly dissolve all the chalk with their acidic leavings, even if they are rather nutrient-deprived.

On the bright side, I now have my growth chamber ready: a styrofoam cooler with a heating pad inside. It is much more energy efficient than an uninsulated hotplate.

[Edited on 6-2-2006 by Polverone]

ordenblitz - 1-6-2006 at 18:19

Polverone,

Too bad you don't have that homebrew store in town because they sell yeast nutrients as well. Check out whats in it: http://www.wyeastlab.com/nutrient/nunutrie.htm

Also I forgot to mention in previous post that cultures are also available in a liquid form that is highly viable, fresh and can be had for a few bucks. http://www.wyeastlab.com/beprlist.htm#bre

Homebrew store... should be called home culture store!