Starter Cultures

"The connection between wine fermentation and the development of the sugar fungus is not to be underestimated; it is very probable that, by means of the development of the sugar fungus, fermentation is started."

From A Preliminary Communication Concerning Experiments on Fermentation of Wine and Putrefaction by Theodor Schwann, 1837 (as recounted by Barnett, 2003.)


The successful manufacture of all fermented products relies on the presence, growth, and metabolism of specific microorganisms. In reality, however, it is possible to produce non-fermented counterparts of some fermented products. For example, sour cream, cottage cheese, and summer sausage can be produced in the absence of microorganisms simply by adding food-grade acidulants to the raw material. Similarly, carbon dioxide can be produced in dough by adding chemical leavening agents. Even products as complicated biochemically and microbiologically as soy sauce can be made by chemical means. However, despite the technical feasability of producing these products without fermentation, these non-fermented versions generally lack the desired organoleptic qualities that are present, and that consumers expect, in the fermented products. This is because microorganisms are responsible for producing an array of metabolic end-products and textural modifications, and replicating those effects by other means is simply not possible.

If microorganisms are indeed necessary for converting raw materials into fermented foods with the desired characteristics and properties, then what is the best way to ensure that the relevant organisms are present in the starting material? In other words, how are fermentations started? There are essentially three ways to induce or initiate a food fermentation.The oldest method simply relies on the indigenous microorganisms present in the raw material. Raw milk and meat, for example, usually harbor the very bacteria necessary to convert these materials into cheese and sausage. Grapes and grape crushing equipment, likewise, contain the yeasts responsible for fermenting sugars into ethanol and for transformation of juice into wine.

For these natural fermentations to be successful, however, requires not only that the "correct" microorganisms be present, but also that suitable conditions for their growth are established. Even if these requirements are satisfied, however, there is no guarantee that the product will meet the quality expectations, be safe to consume, or even be successfully produced. Still, many foods are produced by natural fermentation, including some sausages (Chapter 6), wines (Chapter 10), and pickles and other fermented vegetables (Chapter 7).

Once a successful fermentation has been achieved, a portion of that product could be transferred to fresh raw material to initiate a fermentation. This method, called backslop-ping, is probably nearly as ancient as the natural fermentation practice. It works for almost any fermented food, and is still commonly used for beer, some cheeses and cultured dairy products, sour dough bread, and vinegar. In addition, these methods are still practiced today for small-scale production facilities, as well as in less developed countries and in home-made type products. The principle, regardless of product, is the same. Any successfully fermented product should contain the relevant number and type of microorganisms, and, given a fresh opportunity, they will perform much the same as they had the previous time. Despite the detractors of the backslopping technique (see below), it can be argued that this practice actually selects for hardy and well-acclimated organisms with many of the desired traits necessary for successful production.

The demonstration by Pasteur that fermentation (as well as spoilage) was caused by microorganisms led Lister, Orla-Jensen, and other early microbiologists, more than a century ago, to isolate and identify the responsible organisms. Koch's postulates regarding the germ-disease connection could then be applied to fermentation science. Thus, an organism isolated from soured or fermented milk could be purified, re-introduced into fresh milk causing the expected fermentation, and then re-isolated from the newly fermented product. The implication of this discovery—that pure cultures could be obtained and used to start fermentations, did not go unnoticed. Indeed, these observations resulted in a third way to produce fermented foods, namely via the use of a starter culture containing the relevant microorganisms for that particular product.

Role of Starter Cultures

It is often argued by advocates of traditional manufacturing methods that natural fermentations, whether initiated by the endogenous flora or by backslopping, yield products that have unique or singular quality attributes. Naturally-fermented wines, for example, are often claimed to be superior to wines made using a starter culture. Even if a slow or "stuck" fermentation occurs occasionally, it would cer tainly be worth it (the argument goes) to end up with a truly exceptional product. This attitude may be perfectly fine on a small scale basis, given the inherent flexibility in terms of time and quality expectations. In contrast, however, modern large-scale industrial production of fermented foods and beverages demands consistent product quality and predictable production schedules, as well as stringent quality control to ensure food safety.These differences between traditional and modern fermentations, in terms of both how fermented products are manufactured and their quality expectations, are summarized in Chapter 1,Table 1-1.

Simply defined, starter cultures consist of microorganisms that are inoculated directly into food materials to overwhelm the existing flora and bring about desired changes in the finished product. These changes may include novel functionality, enhanced preservation, reduced food safety risks, improved nutritional or health value, enhanced sensory qualities, and increased economic value.Although some fermented foods can be made without a starter culture, as noted above, the addition of concentrated microorganisms, in the form of a starter culture, ensures (usually) that products are manufactured on a timely and repeatable schedule, with consistent and predictable product qualities. In the case of large volume fermentations, specifically, cheese fermentations, there is also a volume factor that must be considered. In other words, there is no easy way to produce the amount of culture necessary for large scale cheese production without the use of concentrated starter cultures (see below). For all practical purposes (but with a few notable exceptions), starter cultures are now considered an essential component of nearly all commercially-produced fermented foods.


As noted above, microbiology was not established as a scientific discipline until the 1860s and '70s. And although Pasteur, Lister, Koch, Ehrlich, and other early microbiologists were concerned about the role of microorganisms as a cause of infectious disease, many of the issues addressed by these scientists dealt with foodstuffs, including fermented foods and beverages. In fact, many of the members of the microbiology community at the turn of the twentieth century were essentially food micro-biologists, working on very applied sorts of problems.The discovery that bacteria and yeast were responsible for initiating (as well as spoiling) food fermentations led to the realization that it was possible to control and improve fermentation processes. Although the brewing, baking, and fermented dairy industries were quick to apply this new knowledge and to adopt starter culture technologies, other fermented food industries did not adopt starter cultures until relatively recently. And some still rely on natural fermentations.

One of the first such efforts to purify a starter culture was initiated in 1883 at the Carlsberg Brewery in Copenhagen, Denmark. There, Emil Christian Hansen used a dilution method to isolate pure cultures of brewing yeast, derived from a mixed culture that occasionally produced poor quality beer. Subsequently, he was able to identify which strain produced the best (or worst) beers. Hansen also was the first to isolate the two types of brewing yeast, the top (or ale) yeast and the bottom (or lager yeast). Eventually, all of the beer produced by the Carlsberg Brewery was made using the Hansen strain, Saccharomyces carlsbergensis (later reclassified as Saccharomycer pasto-nanus),which also became the lager type strain.

Although Pasteur had also proven that wine fermentations, like those of beer, were performed by yeast, there was little interest at the time in using pure yeast cultures for wine-making. That satisfactory wine could be made using selected strains was demonstrated by the German scientist Hermann Muller-Thurgau in 1890; however, it wasn't until the 1960s that wine yeasts became available (and acceptable) as starter cultures for wine fermentations.

At around the same time and place, another Hansen, Christian Ditlev Ammentorp Hansen, was working on the extraction of enzymes from bovine stomach tissue. This work led to the isolation of the enzyme chymosin, an essential ingredient in cheese manufacture. Prior to this time, chymosin had been prepared and used as a crude paste, essentially ground-up calf stomachs. Using the Hansen process, chy-mosin could be partially purified in a stable, liquid form, and the activity standardized. Factories dedicated to chymosin production were built in Copenhagen in 1874 and in New York in 1878. Later, Hansen developed procedures to produce natural coloring agents for cheese and other dairy products. By the end of the century, the Chr. Hansen's company began producing dairy starter cultures, thus establishing a full-service business that continues even today to be a world-wide supplier of starters cultures and other products for the dairy, meat, brewing, baking, and wine industries.

In the United States, a culture industry devoted to bakers' yeast arose in the 1860s and '70s. Two immigrant brothers from Europe, Charles and Max Fleischmann, began a yeast factory in Cincinnati, Ohio, producing a compressed yeast cake for use by commercial as well as home baking markets. Bread produced using this yeast culture was far superior to breads made using brewing yeasts, which was the common form at the time. More than forty years later, in 1923, another bakers' yeast production facility was built in Montreal, Canada, by Fred Lallemand, another European immigrant.

With the exception of brewers' and bakers' yeasts, all of the initial cultures developed and marketed prior to the 1900s consisted of bacteria that were used by the dairy industry. There was particular interest in flavor-producing cultures that could be used for butter manufacture. Most of the butter produced in the United States and Europe was cultured butter, made from soured cream (in contrast to sweet cream butter which now dominates the U.S. market). The cream was soured either via a natural or spontaneous fermentation or by addition of previously soured cream or buttermilk. Both methods, however, often resulted in inconsistent product quality. By the 1880s, researchers in

Europe (Storch in Denmark and Weigman in Germany) and the United States (Conn) showed that strains of lactic acid bacteria could be grown in pure culture, and then used to ripen cream.

The first dairy starter cultures were liquid cultures, prepared by growing pure strains in heat-sterilized milk. The main problem with these cultures was that they would become over-acidified and lose viability (discussed in more detail later in this chapter). To maintain a more neutral pH, calcium carbonate was often added as a buffer. Still, liquid cultures had a relatively short shelf life,which eventually led to the development of air-dried cultures. The latter were rather crude preparations and were produced simply by passing liquid cultures through cheese cloth, followed by dehydration at 15°C to 18°C.These cultures were more stable,yet required several transfers in milk to revive the culture and return it to an active state. Freeze dried dairy cultures had also become available by the 1920s, but cell viability remained a problem, and even these products required growth in intermediate or mother cultures to activate the cells. Frozen cultures, which are now among the most popular form for dairy cultures, were introduced in the 1960s. In the last twenty years, significant improvements in both freezing and freeze-drying technologies have made these types of cultures the dominant forms in the starter culture market.

The technologies described above were largely developed by starter culture companies and by researchers at universities and research institutes. Most of the culture houses started out as family-owned enterprises and then grew to rather large companies with significant research and production capabilities. By the 1980s, large pharmaceutical-based corporations acquired many of these companies, although a few smaller ones remain, selling mostly specialized products.

As noted above, cultures are not the only product the industry sells.There is a rather large amount of culture media that is used to propagate starter cultures and this represents a substantial market, as do the enzymes and coloring agents used for cheese production. Finally, due to the demanding nature of the cheese and cultured dairy products industries—successful product manufacture requires consistent culture performance—starter culture companies often provide nearly round-the-clock technical service and support to their customers. If a fermentation is delayed, slow, or is otherwise defective, the culture (and the culture supplier) will often be blamed. Cultures, after all, are about the only food ingredient supplied in a biologically active form. Enzymes also fall into the category, but are generally more easily standardized and stabilized.

A discussion of the history of starter culture science and technology would be incomplete without noting what has been perhaps the main driving force for much of the basic and applied research on lactic starter cultures.The observation that bacteriophages, viruses whose hosts are bacteria, could infect and then kill starter culture bacteria was first made in the 1930s.Since that time, but especially in the past thirty years (coinciding with the advent of molecular biology), several research groups in the United States and from around the world have devoted significant attention to understanding and controlling bacteriophages (Box 3-1).This research has led to countless other important and fundamental discoveries about lactic acid bacteria.

Starter Culture Microorganisms

Chapter 2 described many of the microorganisms that are important in fermented foods. Not all of these organisms, however, are produced or marketed as starter cultures. Some of the products do not lend themselves to starter culture applications, and for others, the market is simply too small or too specialized. For example, sauerkraut and pickle fermentations are mediated by the natural microflora; therefore, even commercial manufacturers can produce these products without a starter culture. Although cultures and manufacturing procedures now exist and can be used for controlled starter culture-mediated pickle fermentations,

Box 3-1. Bacteriophages of Lactic Acid Bacteria

The physical structure, biology, taxonomy, and life cycle of bacteriophages are quite unlike those of the bacteria, yeasts, and molds. In fact, bacteriophages, defined simply as viruses that attack bacteria, look and behave like nothing else in the biological world. In essence, bacteriophages are just packages of DNA (or RNA) contained within a protective "head" which is attached to structures that enable them to adhere to and ultimately parasitize host cells.

Despite their simplicity, their importance in food microbiology cannot be overstated. Bacteriophages that infect Escherichia coli O157:H7, Vibrio cholera, and other pathogens, for example, may carry genes encoding for toxins and other virulence factors, which are then transferred to and expressed by the host cell. In fermented foods made using lactic starter cultures, bacte-riophages are often responsible for causing manufacturing delays, quality defects, and other detrimental effects.The economic consequences of bacteriophage infection can be enormous, especially in large scale operations. Thus, understanding the ecology, mode of replication, and transmission of bacteriophages are essential for minimizing the incidence of bacteriophage problems and for developing strategies for their control.

Bacteriophage classification

Because bacteriophages are parasites and cannot grow outside of a host,they have no real physiological activities that can be used as a basis for systematic classification. That is, bacterio-phages are neither aerobic or anaerobic, they have no temperature or moisture optima, per se (aside from attachment kinetics or host-dependent functions), and biochemical or metabolic pathways do not exist. Thus, classification is based on other criteria, including morphology, structural composition, serology, host range, and DNA and genome analyses (McGrath et al., 2004). Morphological characteristics of bacteriophages that infect lactic acid bacteria are summarized in Table 1.

Table 1. Morphotypes of lactic acid bacteriophage.




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