Direct Lyophilized Milks Cultures

2,000,000 - 50 = 1,999,950

1:20

2,500

3,999,900 - 2,500 = 3,997,400

2:00

1.25 X 105

7,994,800 - 1.25 X 105 = 7,869,800

2:40

6.25 X 106

15,739,600 - 6.25 X 106 = 9,489,600

3:20

3.13 X 108

18,979,200 - 3.13 X 108 = less than 1

xGiven the following assumptions: Initial phage level = 1 phage/ml of milk Initial cell level = 1,000,000 cells/ml Phage latent period = 40 minutes Cell generation time = 40 minutes Average burst size = 50

xGiven the following assumptions: Initial phage level = 1 phage/ml of milk Initial cell level = 1,000,000 cells/ml Phage latent period = 40 minutes Cell generation time = 40 minutes Average burst size = 50

In the past twenty years, however, the incidence of phage infections against thermophilic cultures (consisting of S. thermophilus and either L. bulgaricus or Lactobacillus helveticus) has increased significantly. It is no coincidence that there has been a huge increase in the production of Mozzarella cheese, yogurt, and other products that rely on these thermophilic starter cultures.The emergence of thermophilic bacte-riophage underscores the problem faced by the starter culture industry and its customers: wherever and whenever lactic acid bacteria are used on a large scale, phages that infect those organisms will undoubtedly appear as potential adversaries.

In response to the bacteriophage problem, the dairy starter culture industry, as well as the cheese and cultured dairy products industries, have adopted several strategies (Table 3-5; also reviewed in Chapter 5). First, and perhaps most importantly, high standards of hygiene must be applied. In fact, sanitation and asepsis, appropriate plant design, and phage exclusion programs

Table 3.5. Phage control strategies.

Method

Purpose or Function

Sanitation Plant design Phage inhibitory media Phage resistant cultures Culture rotation

Kill and remove phages in plant environment

Keep phages out of production area, prevent cross contamination

Prevent phages from attaching to and infectingculture cells

Design starter cultures cells that will grow and perform well even in presence of phages Prevent proliferation of phages by limiting access to suitable host

are the first lines of defense against phages. Sanitizing agents that are ordinarily used in dairy plants (e.g., chlorine and hypochlorite solutions) are also effective against bacteriophages.

All areas where starter cultures are handled and grown should be isolated from the rest of the processing facility. Because phages are frequently transmitted via air or airborne droplets, the contained areas should be maintained under positive pressure, and filtered air should be used within the starter culture room, as well as in the starter tanks. It is also important to locate the actual production facilities down stream from the starter preparation area to ensure that waste flow (i.e., whey) does not contaminate the culture area. The latter point is critical, because whey is considered to be the major reservoir of phages within a dairy plant, and the primary vehicle by which phage dissemination occurs. In fact, in other lactic acid fermentations where opportunities for phage transmission are limited, infection of the culture by phages does not occur. For example, in the manufacture of fermented meats (Chapter 6), fermentation takes place within the individually encased sausages, and spread of phages from one link to another is not possible.

Another way to control phage is to use phage inhibitory media during the culture propagation step. As part of the infection process, calcium ions are required for phages to attach to and subsequently invade their bacterial hosts. The development of phage inhibitory media nearly fifty years ago was based on the principle that by reducing ionic calcium or by incorporating calcium chelating agents into the culture media, calcium ions would then be unavailable for phage attachment.The main chelating agents are phosphate and citrate salts, which have the added advantage of providing buffering capacity (see above). On the flip side, however, some phages exist that can effectively attach to and infect host cells even in the absence of calcium.

A third approach to reduce infection by bacteriophages takes advantage of the innate ability of some starter culture bacteria to defend themselves against phage attack (discussed below and in Chapter 5). Several types of natural phage-resistance mechanisms exist, including inhibition of adsorption, restriction of phage DNA, and abortive infection. Thus, it is possible to isolate strains that possess one or more of these systems and to use them in industrial fermentations.This approach, as described previously, forms the basis of most dairy starter culture systems. In particular, the multiple defined strain starter cultures used in cheese manufacture often contain as many as five different phage unrelated strains.These cultures can be used either on a continuous basis (the same culture every day) or rotated such that strains with the same phage sensitivity pattern are not used for consecutive fermentations. Rotations programs can also be performed even with single or paired strain starter. In either case, it is important to monitor the phage levels in the whey or milk on a regular basis.

When phage titers reach a particular threshold, signifying a strain has become phage-sensitive, that strain is removed from the mix ture and replaced by a resistant strain. Since phage proliferation requires susceptible host strains, when those strains are removed from the production environment, the background level and accumulation of phages will be reduced such that normal fermentation rates can be achieved. This practice, however, is constrained by the limited availability of phage-unrelated strains (discussed previously). Also, frequent exchange of one strain with another within a given culture may result in undesirable variations in product quality. In fact, many of the defined, phage-resistant cultures now contain only two or three strains to maintain greater product consistency. In theory, it should be possible to isolate new strains from nature that are both phage-resistant and that have good cheese making properties, a strategy that is now standard industry practice (Box 3-5).

Of course, if some strains are naturally resistant to phage infections, then there must be a genetic basis for the phage-resistant pheno-types described above. Indeed, genes responsible for these phage-resistant phenotypes have been identified and characterized. Importantly, these genes can be introduced into phage-sensitive strains, making them phage-resistant. In addition, it is also possible to obtain spontaneous phage-resistant mutants by simply exposing sensitive, wild-type strains to lytic bac-teriophages. The phage-resistant derivatives that are then selected must be evaluated for cheese-making properties before they are reintroduced into a starter culture, because pleiotropic mutations frequently occur that render them defective as cheese cultures (discussed previously).

Engineered Phage Resistance

Genes in Lactococcus lactis subsp. lactis that conferred a phage-resistance phenotype were identified in the early 1980s by researchers at North Carolina State University. These genes were located on a 46 kb, self-transmissible plas-mid, named pTR2030. Initially, it was thought that pTR2030 contained a single phage-

Box 3-5. Looking Far and Wide for Lactococci

Over the thousands of years that lactic acid bacteria have been used in the manufacture of fermented foods, and the 100 years that lactococci, in particular, have been used as dairy starter cultures, these organisms have undoubtedly been exposed to considerable selective pressure. To grow and compete well in a milk environment, for example, an organism must transport and metabolize lactose rapidly and also be able to hydrolyze milk proteins and use the resulting peptides efficiently. Other traits, specific for cheese fermentations and for which strains are routinely screened, include the ability to produce good cheese flavor and texture, to grow within the relevant temperature range, to resist bacteriophage infection, and to be amenable to large-scale propagation, handling, and storage.

Because of these strict phenotypic requirements, it has been suggested that relatively few distinct strains of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris actually exist and that the overall genetic diversity of dairy lactococci is limited (Salama et al., 1995). Moreover, many of the strains that are marketed commercially, even those from disparate geographical locations, are derived from common stock cultures or dairy products (Ward et al., 2004). It is reasonable to assume that in many cases, these cultures, even if marketed by different culture companies, may not be very different from one another.Therefore, the discovery of new strains that satisfy culture requirements would be quite valuable, especially since these strains might provide a rich source of new genetic information, including genes encoding for novel phage resistance mechanisms.

In an effort to obtain new lactococcal strains that could potentially be used as starter cultures, researchers at Oregon State University obtained environmental and dairy samples from diverse geographical locations (Salama et al., 1993, 1995). Samples (more than 200) were obtained from local plant sources, including vegetables, wildflowers, weeds, and grasses, as well as milk and dairy products from China, Morocco, Ukraine, and the former Yugoslavia.Their screening method was modeled after procedures used for large, rare clone library screening, and involved an enrichment step followed by colony hybridization using lactococci- or L. lactis subsp. cremoris-specific 16rRNA probes.The selected isolates were then characterized based on various phenotypic characteristics (growth at 10°C, 40°C, and 45°C or in 10% salt or at pH 9.2, and arginine hydrolysis). In general, L. lactis subsp. lactis were isolated from both plant and dairy sources, whereas L. lactis subsp. cremoris could only be found in dairy samples.

A second level of screening was then performed, based on rapid acid production and flavor profiles (i.e., tasting milk fermented with each strain).These results indicated that 61 out of 120 strains grew well in milk and produced acceptable flavor characteristics. Additional investigations indicated that ten new strains were phage resistant and suitable for cheesemaking (Urbach et al.,1997).

Collectively, these studies suggest that new strains, with the necessary phenotypes for dairy fermentations, certainly do exist in nature. However, having a rational isolation and screening plan is also required if such "bio-prospecting" efforts are to be successful.

References

Salama, M.S.,W.E. Sandine, and S.G. Giovannoni. 1993. Isolation of Lactococcus lactis subsp. lactis from nature by colony hybridization with rRNA probes.Appl. Environ. Microbiol. 59:3941-3945. Salama, M.S.,T. Musafija-Jeknic,WE. Sandine, and S.G. Giovannoni. 1995.An ecological study of lactic acid bacteria: isolation of Lactococcus lactis subsp. cremoris. J. Dairy Sci. 78:1004-1017. Urbach, E., B. Daniels, M.S. Salama,W.E. Sandine, and S.G. Giovannoni. 1997.The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not phenotypes.Appl. Environ. Microbiol. 63:694-702. Ward, L.J.H., H.A. Heap, and WJ. Kelly. 2004. Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity. J.Appl. Microbiol. 96:144-148.

resistance determinant (responsible for a heat-sensitive abortive infection phenotype), but it was later discovered that other genes, encoding for a restriction and modification system, were also present. When the plasmid was transferred via a simple conjugal mating procedure into a phage-sensitive, industrial cheese-making strain, transconjugants with resistance to a broad range of lytic industrial phages were obtained. This was a noteworthy accomplishment, in part, because it represented the first application of biotechnology to improve dairy starter cultures, but also because the actual technique of gene transfer did not involve recombinant DNA technology. Thus, these modified strains could be used commercially.

Indeed, this approach was successful in actual cheese manufacturing environments. However, with prolonged use, bacteriophages eventually appeared in cheese plants that were able to circumvent the resistance of these strains. Nonetheless, these early efforts marked the beginning of a new era in starter culture technology and led to the development of other molecular strategies aimed at controlling bacteriophages.One approach, for example, involved introducing different phage-resistance genes into a single strain, on an individual basis, thereby generating several isogenic phage-resistant derivatives. When used in a rotation scheme, the properties of the parental strain remain the same, while the resistance pattern against different phage types is expanded. Other examples of engineered phage resistance are described in Chapter 5.

Starter Culture Technology in the Twenty-first Century

Phage is not the only concern of the dairy starter culture industry. How fast a given strain grows, what sugars it ferments, and what end-products are formed are also important issues that influence culture performance and that have been addressed by various research groups. In particular, much of the research on lactic acid bacteria in the past thirty-five years was based on the pioneering work of McKay and co-workers at the University of Minnesota (Box 3-6). It was the McKay lab that showed that most of the phenotypic traits necessary for growth and activity of lactic starter cultures, including lactose fermentation, casein hydrolysis, and diacetyl formation, were encoded by plasmid DNA. In addition, genes encoding for nisin production and immunity, for phage resistance, and for conjugal transfer factors were also identified by the McKay group.

These discoveries, and the development of gene transfer and gene exchange techniques, have made it possible to manipulate the physiological, biochemical, and genetic properties of lactic starter cultures in ways hardly imaginable a generation ago. Cultures are now customized to satisfy individual customer demands. Not only are improved strains of lactic acid bacteria now used in dairy, meat, vegetable, soy, and wine fermentations to satisfy modern manufacturing requirements and enhance product quality, they also are being used for novel applications. Lactic acid bacteria, by virtue of their ability to survive digestive processes and reach the intestinal tract, have been found to serve as excellent delivery agents for vaccines and antigens.The use of lactic acid bacteria as probiotics has advanced to the point where the medical community is now actively involved in evaluating these bacteria, in well-designed clinical trials, for their ability to treat important chronic diseases (Chapter 4). Certainly, the information mined from the "omic" fields of genomics, prote-omics, and metabolomics is bound to lead to many other new applications (Chapter 2).

Of course, research on starter culture microorganisms is not limited to lactic acid bacteria. The genome of Saccharomyces cere-visiae was sequenced in 1996, and efforts aimed at manipulating the physiological and biochemical properties of bread, beer, and wine yeasts have been ongoing for many years (Chapters 8, 9, and 10). Despite the apparent simplicity of these fermentations, there are many opportunities for improvement. Bakers' yeasts, for example, can be made more cryotol-erant so that leavening will still occur when

Box 3-6. Plasmid Biology, Gene Transfer Technology, Microbial Starter Cultures, and Softball: Life in the Larry McKay Laboratory

It can be fairly argued that research interest in lactic acid bacteria is at an all-time high. Nearly twenty genomes have been sequenced, sophisticated culture improvement programs are underway throughout the world, and pharmaceutical companies have developed strains of lactic acid bacteria that are now being used to deliver human and animal vaccines.

Of course, lactic acid bacteria were among the very first groups of bacteria studied by the very first microbiologists more than a century ago. Pasteur, Lister, Metchnikoff, and Koch all focused (literally) their microscopes and attention on these bacteria. As the applied significance of these bacteria became evident, researchers devoted their entire careers to understanding their physiological and biochemical properties and their particular performance characteristics in fermented dairy foods. Indeed, great discoveries were made by Orla-Jensen, Sherman, and Whitehead, and research groups led by Paul Elliker, Bob Sellars, Marvin Speck, Bill Sandine, Robert Lawrence, and many others.

Research on lactic acid starter cultures moved into an entirely new, uncharted, and ultimately revolutionary direction in the early 1970s. It was at that time when Larry McKay, a new assistant professor at the University of Minnesota, embarked on a research career that essentially created the field of starter culture genetics. The McKay lab, managed by his extraordinary assistant, Kathy Baldwin, also became the main teaching laboratory for the next thirty years, responsible for training a cadre of students and future scientists that has continued to make advances in starter culture research.

McKay had been a graduate student in the Sandine lab at Oregon State University when he first became interested in lactic acid bacteria and when he developed a keen sense of observation. It was his ability to understand what those observations meant that led him to discover the biochemical pathway by which lactic acid bacteria metabolized lactose, a key finding that was of fundamental as well as applied significance.

When McKay arrived at Minnesota in 1971, he began to address another question that had long puzzled dairy microbiologists—specifically, why is it that some lactococci appear to lose their ability to ferment lactose (referred to as a lac- phenotype, in contrast to a lactose-fermenting or lac+ phenotype). Similarly,there were also strains that had spontaneously lost the ability to degrade milk proteins (aprt~ phenotype).This loss of function phenotype had been observed by Orla-Jensen as long ago as 1919, by Sherman in 1937, and Hirsch in 1951, but as McKay noted, "The question remains, however, as to how the loss of lactose metabolism is induced in cultures of lactic streptococci" (McKay et al., 1972).

The answer, while obvious today, was not so obvious in 1971, when McKay rightly hypothesized that these bacteria "could be carrying a genetic element which is responsible for the cells' ability to ferment lactose" and that "the loss of this element would cause the cell to become lac-" (McKay et al., 1972). Shortly thereafter, the McKay lab provided the first experimental proof that these genetic elements were indeed plasmids (Cords et al., 1974; the reader is reminded that plasmids had only been "discovered" in 1969).The McKay group then published an entire series of seminal papers demonstrating that other essential traits in lactic acid bacteria, including protein metabolism, citrate fermentation, nisin production and resistance, conjugation factors, and phage resistance, were also encoded by plasmid DNA.These (and subsequent) reports all had the McKay signature—they were written in a simple and concise style; the experimental approach was straightforward, precise, and well controlled; and the results were presented clearly and definitively.

As it was becoming clear that important plasmid-encoded functions were widespread in lactic acid bacteria and lactococci in particular, McKay quickly realized the implications.Already, in 1974, McKay and Baldwin had shown that strains which had lost the ability to ferment lactose or produce proteinase could be transduced by tranducing bacteriophages, generating "Lac+"

Box 3-6. Plasmid Biology, Gene Transfer Technology, Microbial Starter Cultures, and Softball: Life in the Larry McKay Laboratory (Continued)

and "Lac+, Prt+" transductants (McKay, Cords, and Baldwin, 1973; McKay and Baldwin, 1974). The development of a conjugation or cell-to-cell mating system (Kempler and McKay, 1979; McKay et al., 1980; and Walsh and McKay, 1981) provided a basis for transferring genes from one organism to another.

Although transformation, the direct uptake of DNA into the cells, proved to be more elusive, Kondo and McKay reported the first successful use of a transformation technique for lactococci in 1982 and later described optimized procedures in 1984. Over the next several years, studies on gene cloning, construction of food-grade cloning vectors, and mechanisms of gene integration and conjugation were reported (Harlander et al., 1984;Froseth et al., 1988;Mills et al., 1996; Petzel and McKay, 1992). Strain improvement strategies were also devised, including efforts aimed at isolating strains that could accelerate cheese ripening, over-produce bacteriocins, and resist bacteriophage infection (Feirtag and McKay, 1987; McKay et al., 1989; Scherwitz-Harmon and McKay, 1987).

During his career, McKay enjoyed nothing more than perusing the lab, examining plates, looking at gels, or asking questions of the lab personnel. He was just as passionate about teaching and mentoring. His course on microbial starter cultures,which was taught every other year, was so valuable that students would often take the class a second time, just to stay caught up in the field. Upon leaving the lab, McKay's students and postdoctorates were always well prepared, scientifically and professionally, and assumed prestigious positions in academia and industry. Of course, as productive as the McKay lab was for more than three decades, the lab did have other important interests.There are stories, perhaps apocryphal, that upon interviewing a potential new graduate student, McKay's final question would have something to do with the applicant's skills on the softball field and whether they could contribute to the department's chances for the next season.While there was no substitute for having a solid background in biochemistry, genetics, and microbiology, having a good arm at shortstop would certainly have helped your chances at joining one of the best research laboratories in the world.

References

Cords, B.R., L.L. McKay, and P. Guerry. 1974. Extrachromosomal elements in group N streptococci. J. Bacte-riol. 117:1149-1152.

Harlander, S.K., L.L. McKay, and C.F. Schachtele. 1984. Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis.Appl. Environ. Microbiol. 48:347-351. Feirtag, J.M., and L.L. McKay. 1987. Isolation of Streptococcus lactis C2 mutants selected for temperature sensitivity and potential in cheese manufacture. J. Dairy Sci. 70:1773-1778. Froseth, B.R., R.E. Herman, and L.L. McKay. 1988. Cloning of nisin resistance determinant and replication origin on 7.6-kilobase i"coRI fragment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3. Appl. Environ. Microbiol. 54:2136-2139. Kempler, G.M., and L.L. McKay. 1979. Genetic evidence for plasmid-linked lactose metabolism in Streptococcus lactis subsp. diacetylactis.Appl. Environ. Microbiol. 37:1041-1043. Kondo, J.K., and L.L. McKay. 1982. Transformation of Streptococcus lactis protoplasts by plasmid DNA.

Appl. Environ. Microbiol. 43:1213-1215. Kondo,J.K., and L.L. McKay. 1984. Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.Appl. Environ. Microbiol. 48:252-259. McKay, L.L., and K.A. Baldwin. 1974. Simultaneous loss of proteinase- and lactose-utilizing enzyme activities in and reversal of loss by transduction.Appl. Microbiol. 28:342-346. McKay, L.L., K.A. Baldwin, and P.M.Walsh. 1980. Conjugal transfer of genetic information in Group N strep-tococci.Appl. Environ. Microbiol. 40:84-91.

Box 3-6. Plasmid Biology, Gene Transfer Technology, Microbial Starter Cultures, and Softball: Life in the Larry McKay Laboratory (Continued)

McKay, L.L., M.J. Bohanon, K.M. Polzin, P.L. Rule, and K.A. Baldwin. 1989. Localization of separate genetic loci for reduced sensitivity towards small isometric-headed bacteriophage ski and prolate-headed bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2. Appl. Environ. Microbiol. 55: 2702-2709.

McKay, L.L., K.A. Baldwin, and E.A. Zottola. 1972. Loss of lactose metabolism in lactic streptococci.Appl. Microbiol. 23:1090-1096.

McKay, L.L., B.R. Cords, and K.A. Baldwin. 1973.Transduction of lactose metabolism in Streptococcus lactis C2.J. Bacteriol. 115:810-815.

Mills, D.A., L.L. McKay, and G.M. Dunny. 1996. Splicing of a group II intron involved in the conjugative transfer of pRSO1 in lactococci. J. Bacteriol. 178:3531-3538.

Petzel,J.P, and L.L. McKay. 1992. Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of Lactococcus lactis subsp. lactis .Appl. Environ. Microbiol. 58:125-131.

Scherwitz-Harmon, K.M., and L.L. McKay. 1987. Restriction enzyme analysis of lactose and bacteriocin plas-mids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of Bcll fragments coding for bacteriocin production.Appl. Environ. Microbiol. 53:1171-1174.

Walsh, P.M., and L.L. McKay. 1981. Recombinant plasmid associated with cell aggregation and high-frequency conjugation in Streptococcus lactis ML3. J. Bacteriol. 146:937-944.

frozen dough is thawed. Likewise, increasing the metabolic capacity of these yeasts to ferment maltose and other sugars, whose use is ordinarily repressed by glucose, would accelerate the fermentation rate and shorten the leavening step. Modern brewers' yeast strains can be modified such that they produce beers with specific compositional characteristics (e.g., low carbohydrate, low ethanol), that have specific performance traits (e.g., attenuation, flocculating properties), or that simply produce better-tasting beer (e.g., no diacetyl or hydrogen sulfite). Several desirable traits in wine yeasts have also been targeted for improvement. Development of osmophilic wine yeast strains would be desirable due to their ability to grow in high sugar-containing musts. The malolactic fermentation, ordinarily performed by malolactic acid bacteria, could be carried out by S. cerevisiae harboring genes encoding for the malic acid transporter and malolactic enzyme.

Despite the opportunities that now exist to modify and improve starter culture organisms, strain improvement programs have been limited by regulatory and public perception considerations. While tools for introducing DNA into food grade starter microorganisms or for altering the existing genetic makeup of these organisms are now widely available, commercialization of genetically modified organisms (GMOs)on a world-wide basis has not occurred. Although GMOs are common in the United States, they are generally not marketed in Europe or the Far East. The debate over GMOs is not likely to abate anytime soon, even as researchers continue to demonstrate the potential benefits GMO technologies may offer (Box 3-7).

Encapsulated and Immobilized Cells

In the fermentation industry, there has been considerable interest in the development and application of encapsulated and immobilized cell technologies. In general, encapsulation refers to a process whereby cells are embedded or enrobed within a gel-containing shell. Encapsulated cells are metabolically active and fully capable of performing fermentations. In addition, encapsulated cells may be either freely suspended in the medium or immobilized to an inert support material. Encapsulated and immobilized cell systems offer several advantages. First, they provide a means for conducting fermentations on a continuous rather

Box 3-7. Genetically Modified Organisms: Current Status and Future Prospects

As noted elsewhere in this chapter, as well as throughout this text, one of the most important developments in the starter culture industry has been the application of molecular biology to improve starter culture microorganisms. It is now rather easy to modify or manipulate the phe-notype of starter bacteria or yeasts, either by the introduction of heterologous (i.e., foreign) DNA into selected organisms or by increasing expression or inactivating particular genes.These genetically modified (GM) or genetically engineered organisms may then have traits, that when used to make fermented foods, result in products having better flavor, texture, nutritional value, or shelf-life. In addition, GM organisms may possess other processing advantages, such as immunity to bacteriophages, resistance to biological or chemical agents, and tolerance to low temperature and osmotic and other inhibitory conditions.

Despite the relative ease with which genetically modified organisms (GMOs) or microorganisms can be constructed in the laboratory, there are few actual examples of genetically modified starter culture organisms that are currently used in the fermented foods industry.This is in contrast to the GMO crops that are now fully integrated within the U.S. food supply. For example, most of the soybeans, corn, and canola produced in the United States come from genetically modified seeds. Moreover, even those commercially-available starter cultures that have been "modified" are not really genetically modified(i.e., made via recombinant DNA technology), but rather are derived via classical genetic techniques.They do not contain foreign DNA, and only natural gene exchange systems (i.e., conjugation or hybridization) are used.The notion of "natural" gene transfer is worth emphasizing, since the European Union (EU) defines GMOs as being altered genetically via processes that do not occur naturally (Kondo and Johansen, 2002).

If GM starter culture organisms, possessing highly desirable properties, can be developed in the laboratory, then why aren't these cultures available in the marketplace? This simple question has many answers. First, GMO products must satisfy the regulatory requirements of the country or countries in which the products will be marketed. Because GMO regulations differ throughout the world (and even what constitutes a GMO varies from country to country), gaining approval from one jurisdiction in no way guarantees acceptance elsewhere. Moreover, the ease with which approval for GMOs is obtained varies considerably. For example, in the United States, GM lactic acid bacteria can qualify as GRAS (generally recognized as safe), provided the "bioengineered" organism is not different, in any significant manner, from the original organism (i.e., it satisfies the substantial equivalence concept).

In the EU, however, the GMO application process is much more elaborate, requiring risk assessment and monitoring and detection plans. Labeling also is required in the European Union, than batch mode. This increases throughput and generally reduces overall production costs. In addition, the cells could be recovered after a fermentation and then used repeatedly (within limits) in subsequent fermentations. Because encapsulated cells are surrounded by a protective, inert material, they may be more stable during the fermentation, as well as during storage. In addition, cells that are encapsulated within alginate beads or other matrices may be less sensitive to phage infections due to the limited access phage would have to the cells' surface.

Despite these advantages and extensive research, the number of actual applications of these technologies are relatively few in number. For products such as cheese or yogurt, the non-fluid nature of the food medium places obvious restrictions on the ability to pass substrate through immobilized cell bioreactors or to recover encapsulated cells from fermented products. Still, food fermentation processes are among the applications receiving much of the attention.

One potential application of immobilized cell technology is production of culture bio-

Box 3-7. Genetically Modified Organisms: Current Status and Future Prospects (Continued)

whereas no such requirement exists in the United States for most GMO foods.The labeling issue is not a minor point—in fact, it may be a deal-breaker, because many consumers are either opposed to GMOs or confused enough by the GMO declaration on the label that it influences their purchasing decisions.

Although it would appear that the U.S. market (and perhaps others) would be more receptive to GM cultures than the EU market, it must be recognized that most foods or food ingredients, including starter cultures, are marketed globally. Thus, it seems likely that a culture supplier would commit resources to development of GM microorganisms only if approval on a near worldwide basis is expected. Nonetheless, there are some cultures, derived using molecular techniques, that are sold in the United States but are not marketed in Europe.

For example, a high diacetyl-producing derivative strain of Lactococcus lactis (MC010), obtained via a recombinant plasmid-mediated mutagenesis procedure, is now widely used in the United States while its approval in the EU is still being considered (Curic et al., 1999; Pedersen et al., 2005). Importantly, this strain contains no foreign DNA and is identical to the parent strain except for a mutation in a single gene. Moreover, there are now a variety of molecular approaches that can be adopted to construct food-grade GM starter culture organisms. Plasmid vectors, for example, that are comprised of only lactococcal DNA with natural selection markers (and devoid of antibiotic-resistance genes), are now available. It remains to be seen, however, whether these so-called "self-cloning" strategies will result in greater acceptance by both regulatory agencies and consumers.

References

Curic, M., B. Stuer-Lauridsen, P. Renault, and D. Nilsson. 1999.A general method for selection of a-acetolac-tate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation. Appl. Environ. Microbiol. 65:1202-1206.

Kondo, J.K., and E. Johansen. 2002. Product development strategies for foods in the era of molecular biotechnology.Antonie van Leeuwenhoek 82:291-302. Pedersen, M.B., S.L. Iversen, K.I. S0rensen, and E. Johansen. 2005.The long and winding road from the research laboratory to industrial applications of lactic acid bacteria. FEMS Microbiol. Rev. 29:611-624.

mass. Starter culture bacteria are mass produced in a batch mode. Even when pH, atmosphere and oxygen, nutrient feed, and other environmental factors are properly controlled, high cell densities may be difficult to achieve. Continuous processes also do not fare well, mainly because plasmid-borne functions may be lost and because such configurations cannot be used for co-culture or mixed strain situations due to the difficulty in maintaining strain balance. It is possible, however, to produce continuous cell biomass from encapsulated cells. This approach takes advantage of the fact that cell-containing gel beads tend to release free cells at a regular rate. Those free cells can then be collected on a continuous ba sis while the encapsulated cells are maintained and contained either in an immobilized form or within in a fixed compartment.

Another application for encapsulated or immobilized cells is in liquid fermentations. For example, immobilized lactic acid bacteria can be used in yogurt fermentations to "preferment" the milk, on a continuous basis, prior to the batch incubation. In this approach, the milk pH is reduced to 5.7 during the pre-fermentation step, allowing an overall 50% reduction in the total fermentation time. Likewise, immobilized lactic acid bacteria can be used to produce industrial lactic acid and other organic end-products from whey, whey permeates, or corn-based feedstocks. Other value-added fermentation products that could be produced on a continuous basis by immobilized cells include exopolysaccharides (Chapter 4), bacteriocins, and diacetyl.

Perhaps the encapsulated cell applications with the greatest potential are those involving alcoholic fermentations, especially for wine production. Indeed, several such products, including both encapsulated yeasts and bacteria, are available commercially for use in specific product applications. Encapsulated strains of S. cerevisiae, for example, can be used in the manufacture of sweet wines (whose manufacture is described in Chapter 10).These wines, in contrast to dry wines, contain residual or unfer-mented sugar, due to cessation of the fermentation via a decrease in temperature to near freezing, addition of sulfites, or removal of yeast by filtration. In contrast, bead-encapsulated yeasts can be placed in nylon bags which are then added to the must, such that when the desired sugar concentration is reached, the bags (and yeasts) can simply be removed. Encapsulated yeast cells can also be used to re-start stuck or sluggish wine fermentations and to metabolize malic acid to ethanol. Similarly, encapsulation and immobilization of the malolac-tic bacterium Oenococcus oeni has been proposed to be an effective way to reduce malic acid and deacidify wine on a continuous basis.

Finally, it is relevant to mention that encapsulated starter cultures, in an entirely natural form, have long been used in the manufacture of kefir, a fermented dairy product widely consumed in Russia and Eastern Europe (Chapter 4).The key feature of this product is that it is traditionally made by inoculating milk with a culture contained within inert particles called kefir grains.These irregularly-shaped particles, which can be as large as 15 mm in diameter and consist of polysaccharide and milk protein, harbor a complex microflora. Included are homo- and heterofermentative lactic acid bacteria, as well as several yeasts.The grains can be separated from the fermented kefir by filtration or sieving, and then washed and re-used for subsequent fermentations. Although kefir cultures, which contain pure strains of Lacto-

bacillus spp. and Lactococcus spp., are now available commercially and are often used in the United States for kefir manufacture, traditional kefir grains still remain popular in many parts of the world.

Probiotics and Cultures Adjuncts

In addition to producing starter cultures for specific food fermentations, the starter culture industry also manufactures microbial products for many other applications. For example, there is now a large market for probiotic microorganisms in both fermented and non-fermented foods. As described in detail in Chapter 4, probiotics are defined as "live microorganisms which when administered in adequate amount confer a health benefit on the host." The main probiotic organisms include species of Bifidobacterium and Lactobacillus, although Saccharomyces and Bacillus sp., and even E. coli, are also commercially available as probiotics. In general, these organisms are produced industrially in the same manner as starter cultures (i.e., under conditions that maximize cell density). However, because they are not used to carry out a subsequent fermentation, these culture products are of the direct-to-vat type, rather than in bulk culture form.

Another group of culture products used by the cheese industry consists of lactic acid and related bacteria whose function is to accelerate and enhance cheese ripening and maturation. Specifically, these organisms, usually strains of Lactobacillus helveticus and Lacto-bacillus casei, produce peptidases and other protein-hydrolyzing enzymes that are necessary for proper flavor and texture development. Citrate-fermenting LAB that produce the flavor compound diacetyl as well as heterofer-mentative LAB that produce carbon dioxide may also be added as adjunct cultures for specific cheeses.

Finally, adjunct cultures and culture preparations are now available that serve a food safety and preservation function.That is, the microorganisms contained in these products do not necessarily make fermentation acids or modify texture or flavor, but rather they are included in the culture because they inhibit pathogenic or spoilage organisms. The inhibitory activity of these organisms may be due to one of several substances, including hydrogen peroxide, organic acids, diacetyl, and a class of inhibitory compounds called bacteriocins. The latter are proteinaceous, heat-stable materials produced by a given organism that inhibits other closely related organisms (Chapter 6).

Lactic acid bacteria are prolific producers of bacteriocins, with all of the food-related genera capable of producing bacteriocins. Therefore, they can be used either as part of a lactic acid-producing starter culture or as an adjunct in dairy, meat, and other foods to inhibit pathogens and spoilage organisms and to enhance shelf-life. Furthermore, some of these organisms used as adjuncts are capable of producing bacteriocin in the food, but with only minor production of acids or other fermentation end-products.Thus,the sensory characteristics of the product are not affected by the producer organism, a property that would be especially important in non-fermented foods such as ready-to-eat meats.

Another means by which bacteriocins can be introduced into a food without adding live organisms has also been developed. In this process, the producer organism is grown in a dairy- or non-dairy-based medium, and then the spent fermentation medium is harvested, pasteurized, and concentrated. This material would contain the bacteriocin (as well as organic acids), which could then be added to foods as a natural preservative. Although these so-called bioprotective products are mainly effective against Gram positive bacteria, some commercially-available products also inhibit yeasts, mold, and Gram negative spoilage bacteria, including psychrotrophs that spoil refrigerated foods.

The Starter Culture Industry

Although starter cultures are certainly the main product line, the starter culture industry, as noted previously, produces and markets a variety of other products. Dairy culture suppli ers, in particular, offer culture media, coagulants, colors, and other ancillary products used in cheese manufacture. Starter culture companies also provide technical services and support, perhaps more so than any other ingredient suppliers. This is because the manufacture of cheese, sausage, wine, and all fermented products depends on the activity of an inherently unstable biological material, i.e., the starter culture.Although there may certainly be quality issues that arise during the manufacture of corn flakes, canned peas, granola bars, or other non-fermented foods, the failure of a biologically-dependent process is not one of them. A cheese plant operations manager who is responsible for converting 2 million Kg of milk into 200,000 kg of cheese (worth at least $250,000) each day will not wait long before calling the culture supplier the minute he or she suspects a culture problem. Thus, a well-trained support staff is a necessary component of the modern culture industry.

Culture suppliers are also often asked to provide customized cultures for specific applications. This means that the phenotype (or in some cases, even the genotype) of each strain in an industrial culture collection must be cataloged. Fermentation rates, product formation and enzyme profiles, sugar fermentation patterns, osmotic- or halo-tolerance behavior, and flocculation properties (for yeasts) should be included in the strain bank data. In the case of lactic acid bacteria and beer and wine yeasts, sensitivities to phages and killer yeasts, respectively, must be determined for each strain. When multiple strains are used in culture blends, production of or sensitivity to antagonism factors must be known.

Finally, the starter culture industry has always been a major contributor to the research on lactic acid bacteria, yeast, and fungi. Several of the large culture companies have maintained active research and development programs that conduct basic and applied research that has led directly to major findings on the genetics and physiology of starter culture organisms. In other cases, their contribution has been indirect, namely by providing research funding, strains, and other materials to investigators at universities and research institutes. Industry sponsorship of conferences and scientific societies have also served an important function in the advancement of starter culture research.

Bibliography

Buckenhuskes, H.J. 1993. Selection criteria for lactic acid bacteria to be used as starter cultures for various food commodities. FEMS Microbiol. Rev. 12:253-272.

Caplice, E., and G.F. Fitzgerald. 1999. Food fermentations: role of microorganisms in food production and preservation. Int. J. Food Microbiol. 50: 131-149.

Cogan, T.M. 1996. History and taxonomy of starter cultures, p. 1—23. In T.M. Cogan and J.-P.Accolas (ed.). Dairy Starter Cultures. VCH Publishers, Inc., New York, New York. De Vuyst, L. 2000.Technology aspects related to the application of functional starter cultures. Food Technol. Biotechnol. 38:105-112.

Hammes W.P, and C. Hertel. 1998. New developments in meat starter cultures. Meat Sci. 49 (sup-plement):S125-S138.

Mäyrä-Mäkinen A, and M. Bigret. 1999. Industrial use and production of lactic acid bacteria, p. 175— 198. In S. Salminen, A. von Wright, and A. Ouwe-hand (ed.) Lactic Acid Bacteria. Microbiological and functional aspects. 3rd Ed., Marcel Dekker Inc., New York, New York.

Pedersen, M.B., S.L. Iversen, K.I. S0rensen, and E. Johansen. 2005.The long and winding road from the research laboratory to industrial applications of lactic acid bacteria. FEMS Microbiol. Rev. 29:611-624.

Ross, R.P., C. Stanton, C. Hill, G.F. Fitzgerald, and A. Coffey. 2000. Novel cultures for cheese improve-ment.Trends Food Sci.Technol.11:96-104.

Sandine,W.E. 1996. Commercial production of dairy starter cultures, p. 191—206. In T.M. Cogan and J.-P. Accolas (ed.) Dairy Starter Cultures. VCH Publishers, Inc., New York, New York.

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