Science Buddies: "Ask an Expert"

Hi,
I'm going to be participating in a science fair that is coming up in March and I was thinking of doing an experiment involving lactobacillus bulgaricus or L. acidophilus (I have an interest in fermented foods) and how they either enhance parts of the immune system or inhibit the growth of competing microorganisms.

I only have access to my high school for equipments, so I understand that that will be one of my limiting factors. However, I'd still like to do something with one of my favorite bacteria . Based on some of the research I have done, lactobacilli are shown to inhibit the growth of certain E. coli and also enhance the production of proteins in lymphocytes. I was considering of doing an experiment that identifies which end products from fermentation is responsible for the inhibition of bacteria. So far, I know that lactobacillus produce lactic acid, bacteriocins and (although debated) hydrogen peroxide. I am sure there are more, but I haven't found any articles about the components of what lactobacilli produce after fermentation. If any experts know of any research, could you post a link?

Do you think this would be a possible experiment for me to do?

A bit about myself: This is the first time I am participating in a science fair and I don't expect to win first place or anything. My purpose for participating in this fair is solely for learning more about the health benefits of lactobacillus and getting to experience how scientists communicate information to others. I am currently a junior (I know it is a bit late to start participating), so I have the fundamentals covered for biology. I do not have a mentor and am still looking for one (Hopefully my biology teacher would be willing to assist me). I cannot access certain websites (google, yahoo), but other sites are generally ok.

I have some ideas for science projects involving lactobacillus, and I posted the links to the project underneath each recommendation.

1. Demonstrate how lactobacillus acidophilus is used to acidify milk, creating yogurt. In dairy products and in the human gut, L. acidophilus digests coverts lactose into lactic acid. Some lactose-intolerant people take tablets containing this bacteria, so that they can consume dairy products. You can also observe L. acidophilus under the micrscope by staining yogurt with methylene blue solution. Maybe you can provide free yogurt samples at the fair.

http://www.eng.umd.edu/~nsw/ench485/lab8.htm
http://www2.mrc-lmb.cam.ac.uk/microscop ... oghurt.php

2. Demonstrate inhibition of bacteria by L acidophilus. An experiment innococulated MRS broth with E. Coli, S. Aureus, and L acidophilus. Some MRS broth was innoculated without L. acidophilus to serve as controls. Cultures were plated onto Violet Red Bile Agar (VRBA) and Mannitol Salt Agar (MSA) in order to count the numbers of E. Coli and S. Aureus, respectively. I do not know if MRS broth, VRBA, and MSA are available at your school. To distinguish E. Coli, you can use EMB agar, because E. Coli colonies appear dark with a metallic green sheen.

http://www.ansi.okstate.edu/research/re ... Report.pdf

3. Since L. acidophilus ferments lactose, you can grow this bacteria on differential media that distinguishes lactose-fermenting bacteria such as EMB and MackConkey (MCK) agar.

Hope this helps!

Hi!
Thank you for your response! I think I have an interest in the second proposed experiment on the inhibition of bacterial growth. Do you happen to know any good sources that describe how lactobacilli produce the by products? I would like to know the exact mechanism behind the process.

I have thought up of some question I am interested in:
1. How does anaerobic respiration and aerobic respiration of lactobacillus affect its ability to inhibit bacterial growth?
2. How does live lactobacilli and secreted products of lactobacilli differ in ability to inhibit bacterial growth?

I was wondering how do lactobacilli produce bacteriocins? As in, what is the exact mechanism that happens when the bacteria produces it? What reactants are involved? If any experts out there know the answer, I'd like to hear from you!

Hi Karin,

I think you have chosen a wonderful topic and have some great ideas to test. I’m a big fan of probiotics like yogurt and like to think that it is really good for me—now you can figure out how it is beneficial!

Taking question 2 first, it’s my opinion that the greatest health benefits accrue from live lactobacilli. Isolating separate bacterial products and testing them is ok in the lab to understand the mechanism of the probiotic’s health-giving properties, but for actual therapy I would opt for the holistic approach. Here’s a good paper that discusses the ‘ecology’ of lactobacilli in the gastro-intestinal tract:

Probiotic lactobacilli in the gut.
Published ahead of print 6 June 2008, doi: 10.1128/AEM.00753-08 Appl. Environ. Microbiol. August 2008 vol. 74 no. 16 4985-4996 [http://aem.asm.org/content/74/16/4985.full]

As to question 1 about aerobic vs anaerobic metabolism, this is complicated because the lactobacilli are facultative anaerobes, which means they can metabolize either way—with or without oxygen. In the gut their position would be essentially anaerobic (http://www.ncbi.nlm.nih.gov/books/NBK7670/). The picture is further clouded because some species of lactobacillus produce only lactose from fermentation while others can produce both lactose and ethanol (http://en.wikipedia.org/wiki/Lactobacillus). Which species of lactobacillus did you have in mind to experiment with? They all increase the local acidity of their environment which inhibits growth of many human pathogens.

Your last question about bacteriocins, the proteins produced by some bacteria to kill other bacteria, is especially interesting. They are a sort of natural, narrow-spectrum (usually) antibiotic specific for certain types of bacteria usually including the producing species itself (http://www.researchgate.net/post/What_i ... d_antibody).

I did a search for ‘lactobacillus bacteriocin’ on PubMed and came up with 53 pages of hits! http://www.ncbi.nlm.nih.gov/pubmed/?ter ... acteriocin This is an extremely complex subject because there are so many different bacteriocins. They are, I think, all proteins that are synthesized by the producing bacteria and probably secreted into the intestine or wherever the bacteria are. I did not see a paper explaining how bacteriocins kill their target bacteria, but the regulation of their synthesis may be by a recently discovered activity called quorum sensing: http://en.wikipedia.org/wiki/Quorum_sensing Quorum sensing is just a scientific term for the observation that bacteria regulate their protein production by ‘sensing’ the population of bacteria around them.

I have seen the bacterium Lactobacillus plantarum mentioned frequently as a promising probiotic, so you might consider doing some tests with that species to see how well it kills other bacteria and what conditions promote its bacteriocin production http://www.ncbi.nlm.nih.gov/pubmed/24242246

I don’t want to give you TOO much information so I’ll stop here. Let us know what hypothesis you decide to test and we can help you design good experiments to test it.

Good luck!

Sybee

Hello there,

I also agree with the previous experts that you have chosen a wonderful topic and I think that you've already come up with some excellent questions! Just to chime in about how bacteriocins are synthesized, the different types of bacteriocins known (not limited to just the ones produced by Lactobacilli), and how they kill their target bacteria, here are some other review articles that you can check out:

http://www.ncbi.nlm.nih.gov/pmc/article ... -S1-S3.pdf
http://journal.frontiersin.org/Journal/ ... 00241/full

As you do more reading and come up with additional ideas for questions/experiments, feel free to post back here and we'd be happy to help you brainstorm!

Connie

http://www.sigmaaldrich.com/technical-d ... cilli.html

I posted the link to a site on sigmaaldrich that gives a general overview of lactobacilli. If you want basic info about lactobacilli metabolism and byproducts, you can search this sight. Wish you the bet on your experiment.

Katherine

Thank you so much for your help!! I'm extremely grateful to have multiple experts reply to my post!
As Sybee mentioned, I have also come across the topic of quorum sensing while I was researching about the production of bacteriocins. Apparently, nisin is the most commonly used bacteriocin on the market because of its ability to kill various kinds of bacteria. I am probably going to be working with lactobacillus bulgaricus for my experiment. I am still seeing if I can get a hold of lactobacillus acidophilus or streptococcus thermophilus.

About lactobacillus bulgaricus, the bacteriocin they produce is called "bulgarican". I was considering of doing an experiment on how to enhance the production of bulgarican. Before this, maybe I should see how effective bulgarican is on inhibiting bacterial growth? The bacteria that I am planning to use to test for inhibition is E. Coli-K12. I hope this would work out.

Since bacteriocins are peptides, I am assuming that they require amino acids. Maybe I could have the amount of nitrogen available when growing lactobacilli as my independent variable? I have read that for lactobacilli to synthesize amino acids, they have a system outside of their membrane that regulates the transportation of components needed for building bacteriocin. Since it is a proteolytic system, perhaps coenzymes would have some sort of impact on the efficiency?

There are still too many things that I am unsure of to create a clear idea on what I want to do with my project. I will look at all the resources that the experts provided! Thank you so much for your help! I will write again when planning my methodology.

Hi Karin,

Looks like you have a lot to think about in deciding how to approach your project. Maybe we can help in narrowing down your hypothesis.
You suggested the possibility of altering bacteriocin production by changing the concentration of a nutrient such as nitrate in the culture medium. This is probably not the best way to regulate bacteriocin production since nutrient levels affect all protein synthesis.

I think you are on the right track with quorum sensing because bacteriocin production has to be keyed to the presence of bacteria that are similar to L bulgaricus but not the same. But what is it that the Lactobacilli ‘sense’ and how do they do it? Why does the bacteriocin from L acidophilus kill L bulgaricus but does not inhibit itself? I don’t know if these questions have been answered but they certainly would make a great project.

Some strains of E coli including K12 make their own bacteriocins, called ‘colicins’, but as far as I can tell they are only active against other strains of E coli or enterobacteria and not Lactobacilli. The main question would be does lacticin (from L acidophilus) or bulgarican (from L bulgaricus) kill E coli K12. I found a 2013 paper that cited a reference that lacticin does not kill E coli. The authors found, however, that if they used lacticin plus an antibiotic that it DID kill E coli. http://www.biomedcentral.com/1471-2180/13/212

Read the papers that the experts have suggested; learn more about this important area of antibacterial research and then come up with a hypothesis. We’ll be here to help.

Good luck!

Sybee

I read through all the resources provided by the experts and gained a lot of insight about bacteriocin. I have tried to research about bulgarican, but not much information popped out on the internet. Since bacteriocins are specific to the kind of bacteria they inhibit, I am not sure if bulgarican actually inhibits the growth of E. Coli-K12 because L. bulgaricus is a gram-positive bacteria and E. Coli is gram-negative.
A study that interested me was the lacticin 3147 experiment (http://www.biomedcentral.com/1471-2180/13/212) where they added polymyxin to see if that would inhibit the growth of gram negative bacteria. Will this also work with bulgarican? This experiment also proposed an explanation for why bacteriocin produced by gram positive may be ineffective for gram negative bacteria. I am still unclear with how pore forming bacteriocins actually form pores on the bacteria's membrane. For lacticin Q, it interacts with the negatively charged membrane, which leads to the creation of pores and "lipid flip-flop" (http://www.ncbi.nlm.nih.gov/pmc/article ... -S1-S3.pdf). Lacticin Q is also nontoxic for gram negative bacteria. Based on this information, I reasoned that there must be something about gram negative bacteria's outer membrane that is preventing the electrostatic interaction of lacticin Q with the membrane.
Then, like lacticin 3147, if some other chemical was combined with lacticin Q, or any other bacteriocin that is only effective for gram-positive bacteria, would it not be effective for gram-negative bacteria as well?

I also looked into quorum sensing, and it said that bacteria respond to autoinducers and produce bacteriocins. Is it possible to have isotopes of these autoinducers to enhance the production of bacteriocin?

Much of the material I read is extremely fascinating, it is just that I'm not sure how I would do anything in a high school lab. First, I'd have to some how separate just the bacteriocin, or eliminate the other antimicrobial substances secreted by lactobacilli to be sure that it is the bacteriocin that is inhibiting bacterial growth, not the other components.

This is what I have done so far today, I will consult my partner on monday when school starts.

My ideas for the science fair is due on Thursday. I think I will be selecting one of these topics:
1. which strand of lactobacillus inhibit growth of E. Coli most effectively?
2. if lactobacilli produce bacteriocins in response to quorum sensing, will accumulation of dead competitors still induce production of bacteriocins?
3. will increasing concentration of lactose increase production of bacteriocins?

Hi experts,

I have come across a really big problem; I can't purify bacteriocin from LAB with the equipment available. I have been reading journals regarding the purification of bacteriocin (Such as: http://aem.asm.org/content/58/10/3355.full.pdf+html), but many of them require material that is not inside my high school lab. I was thinking that maybe I could purchase purified bacteriocins, but they are not available on online stores in my country. How should I purify it?

Hi Karin,

I don't understand--why do you have to purify bacteriocin? Lactobacilli are known to produce it and you can see its effect as it kills other bacteria. Your list of project possibilities did not include using pure bacteriocins. Can you explain more?

Sybee