Microbiology of Fermentation

Fermentation of foods has enjoyed a long history, nearly as long as the history of human civilization. In fact, it would be several thousand years before microbes were identified as the source of fermentation (Shurtleff & Aoyagi 2004). The earliest traces of fermentation are found in Iraq, 8000 years before present (BP), where it is thought cheese-making originated after the domestication of animals (Ross et al. 2002). Ancient settlers of the Fertile Crescent region may have inadvertently (or purposefully) added salt to food material. The salt may have then selected harmless microorganisms that fermented the product (Shurtleff & Aoyagi 2004).

Over the next eight millennium, many food types would be fermented. The Egyptians discovered how to use yeast to leaven bread and brew beer (~6000 BP). The Sumerians fermented barley to make beer. In East Asia (~2500-500 BP), yogurt, milk products, pickles, sauerkraut, and vinegar were used. The Chinese would be the first to inoculate basic foods with molds. Indeed, the usefulness of fermentation was immediately obvious, not only in food preservation, but in enhancements of flavor, odor, and texture, and making food more digestible (Ross et al. 2002).

While I will not be focusing this review on fermented beverages, it is worth noting their impact on early societies. The Egyptians praised Osiris for the brewing of beer and the Greeks invented Bacchus, god of wine. Early Japanese miso and shoyu breweries had shrines for patrons to venerate before (Shurtleff & Aoyagi 2004). The word fermentation is derived from the Latin ‘to boil’, due to the bubbling and foaming of the beverages. Fermentation was a considerable achievement and played (and continues to play) a significant role in human health and happiness.

Fermentation is the anaerobic catabolism of organic compounds, usually carbohydrates, in the absence of an external electron acceptor (Madigan & Martinko 2006). This chemical process was first described in the late 18^th century by Antoine-Laurent de Lavoisier, when he experimented by transforming sugar to alcohol and carbon dioxide (Donovan 1993). In 1810, J.L. Guy-Lussac summarized the process with the equation C6H12O6 ® 2 CO2 + 2 C2H6O. Louis Pasteur was the first to identify living organisms as the initiators of fermentation. In 1857, he demonstrated that lactic acid fermentation was caused by bacteria. Four years later he again showed that bacteria soured milk, leading to the ubiquitous practice of pasteurization (Shurtleff & Aoyagi 2004).

Modern fermentation practices have developed exponentially from those early times. With improved technology and molecular techniques, fermentation plays a crucial role in human society. Steinkraus (1995) has identified five major purposes of modern fermentation:

1. Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates.

2. Preservation of substantial amounts of food through lactic acid, alcoholic, acetic acid, and alkaline fermentations.

3. Enrichment of food substrates biologically with protein, essential amino acids, essential fatty acids, and vitamins.

4. Detoxification during food fermentation processing.

5. A decrease in cooking times and fuel requirements.

The microbes involved with fermentation come from a relatively small number of genera: eight genera of molds, five genera of yeasts, and six genera of bacteria (Ross et al. 2002). The bacteria are characterized by lactic acid bacteria (LAB), acetic acid bacteria, and propionic bacteria. The most common yeast is Saccharomyces cerevisiae (Madigan & Martinko 2006). LAB are the most important fermenting microbe and the primary focus will be on them.

Food fermentation can be broken into three groups: dairy (e.g. milk, cheese, yogurt, sour cream), meat (e.g. dry and semidry sausages), and vegetable (e.g. sauerkraut, pickles, olives, onions, peppers). LAB are the most significant microbe group and are often introduced as starters in a mixed culture. Secondary inoculation is not uncommon. For example, secondary inoculums of Lactobacillus and Penicillium roqueforti are responsible for the blue veins, taste, and aroma of blue cheese. Secondary fermentation also catabolizes lactic acid to propionic acid, acetic acid, and carbon dioxide (Madigan & Martinko). Enterococci are also introduced as a component of some mixed starters but this is still widely debated, as many species of this genus are known human pathogens and are thought to contribute to antimicrobial resistance (Ross et al. 2002).

Although the most common metabolic pathways in LAB use sugars (hexoses and pentoses) as the primary substrates for lactate and pyruvate formation, there are other uncommon pathways as well. These include polyols (e.g. pentitols, hexitols), organic acids (e.g. citrate, aspartate), and amino acids (e.g. alanine, serine) (Liu 2003). With respect to the primary metabolic pathways, LAB can be grouped into two separate classes based upon their carbohydrate metabolism. Group 1 is the homofermenters, which includes Lactococcus, Pediococcus, Enterococcus, and Streptococcus. This group converts one mol of glucose into two mol of lactate. Heterofermentive bacteria, group 2, produce equimolar amounts of lactate, ethanol, and carbon dioxide using the pentose pathway (Liu 2003; Ross et al. 2002). Only half as much energy is generated this way. Bacteria included in this group are the Leuconostoc, Wessella, and some lactobacilli.

Several essential variables exist that determine fermentation productivity. The type and amount of carbohydrates is important. If it is a milk product, the degree of hydrolysis of milk proteins is taken into consideration, as this determines the availability of essential amino acids. Also, the composition and degree of hydrolysis of milk lipids is taken into account. Lastly, temperature of the raw product, pH (under a pH of 4, LAB won’t ferment), moisture content, and presence of any microbes obstructive of the fermentation process, must all be adjusted appropriately (Heller 2001; Steinkraus 1995).

Due to their small genomes and considerable importance in industrial fermentation, LAB have been the subject of intensive genetic research (de Vos 2005; Liu 2003). Genetic modification has been used to address a number of pertinent issues, including: reduction in food spoilage, avoidance of food borne diseases, preservation/development of attractive flavors, tastes, and appearance (Hansen 2002); improving their use in food technology/manufacturing, improving product safety, production of therapeutic molecules, and creating bacteriophage resistant strains (Renault 2002; Konings et al. 2000). Most modification is done by introducing new genes or by altering their metabolic functions. Any introduced genetic elements are screened so as to be as safe as the host organism, and have a long history in food technology. The majority of genetic elements are derived from plasmids and genes from bacteria of the same species. Increased research in LAB has led to the development of cloning systems, chromosome modification systems, and gene expression systems (Renault 2002). LAB continue to be the subject of many studies in genetic and metabolic engineering.

In recent years there has been an increasing recognition of the health benefits associated with consuming fermented products. These microbes are referred to as probiotics, or microorganisms that when consumed in adequate numbers confer a health benefic to the host (Stanton et al. 2005; Soomro et al. 2002). Preliminary research has shown probiotics to provide relief from lactose intolerance, diarrhea, Crohn’s disease, depressed immune function, and even cancer (Saxelin et al. 2005; Stanton et al. 2005; Wollowski et al. 2001). In addition, some LAB produce as secondary metabolites beneficial vitamins (like B vitamins) and bioactive peptides (Stanton et al. 2005).

Fermenting microbes have had a significant role in human history, from food preservation to modern day medicine. Continued research is sure to yield even more interesting insights and developments.

References

de Vos, W.M. 2005. Frontiers in food biotechnology-fermentations and functionality. Current Opinion in Biotechnology 16: 187-189.

Donovan, A. 1993. Antoine Lavoisier: Science, Administration, and Revolution. Cambridge University Press, Cambridge, UK.

Hansen, E.B. 2002. Commercial bacterial starter cultures for fermented foods of the future. International Journal of Food Microbiology 78: 119-131.

Heller, K.J. 2001. Probiotic bacteria in fermented foods: product characteristics and starter organisms. American Journal of Clinical Nutrition 73 (suppl): 374S-379S.

Koning, W.N., Kok, J., Kuipers, O.P. and B. Poolman. 2000. Lactic acid bacteria: the bugs of the new millennium. Current Opinion in Microbiology 3: 276-282.

Liu, S.Q. 2003. Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations. International Journal of Food Microbiology 83: 115-131.

Madigan, M.T. and J.M. Martinko. 2006. Brock Biology of Microorganisms, 11^th Ed. Pearson Prentice Hall, Upper Saddle River, NJ.

Renault, P. 2002. Genetically modified lactic acid bacteria: applications to food or health and risk assessment. Biochimie 84: 1073-1087.

Ross, R.P., Morgan, S. and C. Hill. 2002. Preservation and fermentation: past, present, and future. International Journal of Food Microbiology 79: 3-16.

Saxelin, M., Tynkkynen, S., Mattila-Sandholm, T. and W.M. de Vos. 2005. Probiotic and functional microbes: from markets to mechanisms. Current Opinion in Biotechnology 16: 204-211.

Shurtleff, W. and A. Aoyagi. 2004. A Brief History of Fermentation, East and West.
http://www.thesoydailyclub.com/SFC/Fsoyfoods39.asp. Retrieved on: 19 June 2005.

Soomro, A.H., Masud, T. and K. Anwaar. 2002. Role of Lactic Acid Bacteria (LAB) in Food Preservation and Human Health – A Review. Pakistan Journal of Nutrition 1(1): 20-24.

Stanton, C., Ross, R.P., Fitzgerald, G.F. and D. Van Sinderen. 2005. Fermented functional foods based on probiotics and their biogenic metabolites. Current Opinion in Biotechnology 16: 198-203.

Steinkraus, K.H. 1995. Handbook of Indigenous Fermented Foods. Marcel Dekker, Inc. New York, NY.