Team:WLC-Milwaukee/Probiotics

From 2014.igem.org

Definition of probiotic

Bacteria are known to inhabit even the most extreme environments on earth. They are everywhere, and in large numbers. For every human cell of your body, there are approximately 10 microbial cells in or on you. Thankfully, relatively few are pathogenic. Many are merely opportunistic pathogens and some are harmless to us. Some are actually beneficial, participating in mutualistic relationships and composing what is known as the microflora. “The gut microflora is an essential component of the healthy human and is essential for optimal resistance to disease.”[1]

Coined in 1965, the word ‘probiotic’ literally means ‘for life.’ Thus it follows that anything that supports life, rather than inhibits it, could be considered a probiotic. This very broad definition is misleading if taken at face value. For instance, any pharmaceutical drug (that does not cause extreme side effects) could be a probiotic. What makes probiotics unique is the application of live microorganisms. While often applied to the digestive system, it is not limited to this application. Probiotics have been developed for the mouth, gastrointestinal tract, upper respiratory tract, and urogenital tract[2]. A more accurate definition of a probiotic is “a mono- or mixed culture of live microorganisms, which when applied to animal or man, affect beneficially the host by improving the properties of the indigenous microflora.”[2]

Regulations for probiotics

Probiotics must pass rigorous testing and documentation before becoming suitable for the market. It is necessary to clearly identify the genus, species, and strain of the bacteria used and a sample must be deposited in the international culture collection.[3] Safety tests are the first conducted; in vitro scenarios are performed followed by in vivo scenarios.[3]

Common in vitro tests include resistance to gastric acidity, bile acid resistance, adherence to mucus and/or human epithelial cells and cell lines, antimicrobial activity against potentially pathogenic bacteria, ability to reduce pathogen adhesion to surfaces, bile salt hydrolase activity, and resistance to spermicide depending on the purpose and location of implementation of the probiotic strain.[3] Strains selected for probiotic use should have a GRAS (Generally Regarded As Safe) status as well. Characterization for antibiotic resistance patterns, metabolic activities, side-effects, toxin production, and hemolytic activity should also be performed.[3] The mechanism of the probiotic should be determined.

In vivo tests on animals and humans are also necessary. Administration of the probiotic strain to immunocompromised animals to test against opportunistic pathogenicity is encouraged.[2] Probiotics must cause statistically significant biological improvements when compared to a placebo. These improvements may be evident in condition, symptoms, reduced risk of disease, longer remission times, or faster recovery from pathogens. Finally, repetition of trials is required for validation of effectiveness and potential side effects are monitored.

Selection of a probiotic strain is very important. Ideally the selected strain can be initially found in the target organism. For example, a probiotic strain for humans could originally be found in the gastrointestinal tract of a human. Furthermore the GRAS status is needed.[1] Lactic acid bacteria are frequently used for this reason. Probiotics intended for the gastrointestinal tract must be able to survive the acidic environment of the stomach. The presence of the probiotic must not elicit the host’s immune system to produce antibodies against it. However, some probiotics are used to increase the activity of the immune system.[1],[4] Other advantages characteristic of a probiotic strain may include adherence to the host’s cells, antibiogram profiles, production of antibacterial factors targeting pathogens, metabolic activity, technological suitability, anti-inflammatory properties, antimutagenic and anticarcinogenic properties, or site specific delivery of recombinant proteins.[1]

When the probiotic has passed sufficient testing and is ready for market, proper labeling must be observed. An appropriate label should contain the genus, species, strain, shelf-life, viable number of bacteria at the end of the shelf-life, effective dose, specific health claim(s), storage conditions, and corporate contact information.[3] The probiotic product can take many forms: freeze-dried powders, liquid suspensions, granules, paste, or gels.[2]

Difficulties arise for multiple reasons. It is common to have mixed cultures in probiotics. Unfortunately this makes it more difficult to isolate and test any one component or strain for probiotic effects and even more unlikely to determine the exact mechanism by which the probiotic functions. Additionally in vitro and in vivo tests are not always representative of the complex system of a living mammal.[4] It is very difficult to predict the effects of introducing a probiotic because there are variations between the microflora of any two individuals.[2] This is because both humans and livestock obtain their microflora after birth from exposure to the environment.[4],[5] It is a very complex dynamic composed of the mammal, bacteria, protozoa, fungi, archaea, and even viruses.[5] Finally, it may also be difficult to attain statistically significant results. If a probiotic vaguely claims to improve digestive health, it becomes very difficult to obtain quantitative data that verifies the claim.[3],[1] This in part has led to the public’s lack of confidence in probiotics. There must be scientific data to validate any probiotic claims.

Probiotics in use

An array of organisms has already been used as probiotics. As of 1992 probiotics have frequently utilized bacteria from the following genera: Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, Propionibacterium, Pediococcus, Saccarharomyces, Escheriscia, and more.[1] Lactic acid bacteria are often favored for their GRAS status and pre-existing presence in the gut. One of the most visible uses of probiotics for consumers is the yogurt section of the grocery store. Some of the brands that claim probiotic status in some of their products are Dannon, Yoplait, Stonyfield Farms, and Chobani. These products typically claim to aid in digestive health with Lactobacillus bulgaricus, Streptococcus thermophiles, Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus casei being the most common bacteria used.[6] Probiotics have also been used successfully in the treatment of upper respiratory infections and otitis by outcompeting pathogens.[7] The probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 suspended in skim milk as an oral intake has been clinically shown to treat bacterial vaginosis in women in as little as one week.[8] Work has also been done with some success on probiotics for oral health itself. “Probiotics may control dental caries in children due to their inhibitory action against cariogenic streptococci.[9]” The future seems bright for oral probiotics.

[9]

Probiotic use is not limited to humans but is common for livestock too. Most of these probiotics target the gastrointestinal tract and aim to increase feed efficiency and weight gain, limit ruminal acidosis, reduce the risks associated with weaning, reduce pathogen colonization, increase milk production and quality, improve meat quality, limit diarrhea, and to promote optimal microbiota for general health.[5] Ruminants, horses, pigs, poultry, and even fish and shrimp have benefited from probiotics.[5] Probiotics are an alternative to antibiotics, the use of which risk increasing bacterial resistance to the limited number of known antibiotics.

Probiotic Mechanisms

There are various mechanisms by which a given probiotic affects a benefit to its host. Livestock probiotics may encourage additional growth due to a better feed conversion, balance a disrupted microflora with healthy colonization, or pre-digest anti-nutritional factors for the host.[2] In humans probiotics may also be used to re-establish a healthy microflora, outcompete pathogenic bacteria for adherence to the intestinal epithelium, lower serum cholesterol, reduce mutagenicity and tumor formation, metabolize lactose, improve the absorption of calcium, or synthesize vitamins to name a few.[2] Some probiotics produce acid thereby lowering the gut pH which promotes the growth of the native microflora and successively inhibits pathogen colonization[5]. Pathogen colonization may also be inhibited by the secretion of bacteriocins or other toxins.[5] A given probiotic may also function by producing enzymes which degrade harmful bacterial toxins. Others have been shown to increase metabolism of the mammal’s intestinal cells, increase mucosa development, or stimulate positive immune responses.[5] “Probiotics regulate immune responses by enhancing the innate immunity and modulating pathogen-induced inflammation via toll-like receptor-regulated signaling pathways.[10]” Probiotics have already been shown to have an array of mechanisms; many more application are yet to be seen.[11]

Ruminant digestion

The probiotic created by this project is designed to be active in the foregut of ruminants. Ruminants like cattle, sheep, goats, and deer are foregut fermenters as opposed to monogastric hindgut fermenters like humans, horses, and dogs. While monogastric digestion is characterized by a single stomach chamber, ruminants have a four chambered stomach: rumen, reticulum, omasum, and the abomasum. At the beginning of ruminant digestion food is ingested and chewed between the bottom teeth and a thick upper pad on the roof of the mouth. This chewing phase of digestion is known as mastication. The new bolus of partially chewed plant matter and saliva travels down the esophagus to the rumen. The rumen is colonized by bacteria that aid in the breakdown and digestion of plant matter by secreting cellulases. The rumen and reticulum are only separated by a reticular fold, which allows mixing between the two compartments. Food that is not completely chewed is regurgitated via antiperistalsis to be re-chewed and re-swallowed. This is known as chewing the cud or rumination. This process is repeated multiple times until the food matter is sufficiently broken down. The next step of digestion occurs in the omasum. The omasum lining is highly folded for maximum surface area to absorb water and salts from the food. The omasum then pumps the food to the abomasum, the true stomach. The abomasum secretes enzymes and hydrochloric acid to denature and cleave proteins into peptides.

From the abomasum food travels to the small intestine where enzymes break carbohydrates into monosaccharides, lipids into monoglycerides, nucleic acids into nucleotides, and proteins into amino acids which can be absorbed and utilized by the body. The small intestine has three regions: the duodenum, jejunum, and ileum. In the duodenum bile from the liver along with enzymes and bicarbonate from the pancreas digest of lipids into monoglycerides and the neutralization of stomach acid. The lining of the jejunum in covered in villi and microvilli to increase surface area for enzymes to contact their respective substrates. Nutrients are either passively or actively transported across the intestinal wall. The sugar xylose diffuses passively into the bloodstream while amino acids, vitamins, and glucose require active transport into the capillaries. The ileum absorbs the remaining nutrients, notably vitamin B12. From the ileum, food moves through the ileocaecal valve to the caecum, the beginning of the large intestine. In some animals the caecum functions to store food material while microorganisms further degrade cellulose. The large intestine is composed of the ascending colon, traverse colon, sigmoid colon, rectum, and anus regions. The colon is necessary for osmosis and the active transport of sodium. Bacteria also inhabit this region, synthesizing vitamin K, thiamine, and riboflavin. Unused food and bodily wastes are excreted via the rectum and anus.

The ruminant digestive system is dramatically more efficient at extracting nutrients from high-cellulose plant material than the monogastric systems. Some nonruminant herbivores have other systems for extracting nutrients from high cellulose plant matter. Horses compensate by consuming more food and having a faster digestive system to process it. Lagomorphs, like rabbits, have a very fast metabolism and cannot wait for food to sit in the gut for any extended time. Thus they eat small portions often. Bacteria in the caecum digest the plant material and release volatile fatty acids which are used by the mammal. The mammal passes caecal pellets which are re-eaten to further extract the remaining nutrients before passing normal feces. Humans are unique in that they have an appendix, but they still cannot survive on high-cellulose diets.


Written by: Christa Winslow

References

[1] Collins, J. K., Thornton, G., and Sullivan, G.O. (1998) Selection of Probiotic Strains for Human Applications. Int. Dairy Journal 8, 487-490.
[2] Havenaar, R., and Huis in’t Veld, J. H. J., (1992) Probiotics: A general view. In: The Lactic Acid Bacteria Volume 1, ed. Wood, B. J. B. pp 151-170.
[3] Joint FAO/WHO Working Group Report. (2002) Guidelines for the evaluation of probiotics in food. http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf.
[4] Fuller, R. (1992) The effect of probiotics on the gut micro-ecology of farm animals. In: The Lactic Acid Bacteria Volume 1, ed. Wood, B. J. B. pp.171-192.
[5] Chaucheyras-Durand, F. and Durand, H. (2010) Probiotics in animal nutrition and health. Beneficial Microbes 1(1), 3-9.
[6] "Probiotic Yogurts: Gems Amongst the Supermarket Brands?" The Beneficial Bacteria Site. N.p., n.d. Web. http://beneficialbacteria.net/probiotic-yogurt/
[7] Popova, M., Molimard, P., Courau, S., Crociani, J., Dufour, C., Le Vacon, F., and Carton, T. (2012) Beneficial effects of probiotics in upper respiratory tract infections and their mechanical actions to antagonize pathogens. J Appl Microbiol 113(6), 1305-18.
[8] Reid, G., Bruce, A. W., Fraser, N., Heinemann, C., Owen, J. and Henning, B. (2001), Oral probiotics can resolve urogenital infections. FEMS Immunology & Medical Microbiology, 30: 49–52.
[9] Stamatova, I. and Meurman, J. K. (2009) Probiotics: Health benefits in the mouth. American Journal of Dentistry, 22(6), 329-338.
[10] Vanderpool, C., Yan, F., and Polk, D. B. (2008) Mechanisms of probiotic action: Implications for therapeutic applications in inflammatory bowel diseases. Inflamm Bowel Dis. 14(11), 1585-96.
[11] University of Waikato. “Adaptations to High Fibre Diets." Animal Structure & Function., n.d. Web. http://sci.waikato.ac.nz/farm/content/animalstructure.html.
Picture credit:
www.forbes.com
https://www.mun.ca/biology/scarr/Ruminant_Digestion.html
Ronnie Tackett