Current Status of the Preharvest Application of Pro- and Prebiotics to Farm Animals to Enhance the Microbial Safety of Animal Products

ABSTRACT

The selection of microorganisms that act as probiotics and feed additives that act as prebiotics is an ongoing research effort, but a sizable range of commercial pro-, pre- and synbiotic (combining pro- and prebiotics) products are already available and being used on farms. A survey of the composition of commercial products available in the United States revealed that Lactobacillus acidophilus, Enterococcus faecium, and Bacillus subtilis were the three most common species in probiotic products. Of the nearly 130 probiotic products (also called direct-fed microbials) for which information was available, about 50 also contained yeasts or molds. The focus on these particular bacteria and eukaryotes is due to long-standing ideas about the benefits of such strains, research data on effectiveness primarily in laboratory or research farm settings, and regulations that dictate which microorganisms or feed additives can be administered to farm animals. Of the direct-fed microbials, only six made a claim relating to food safety or competitive exclusion of pathogens. None of the approximately 50 prebiotic products mentioned food safety in their descriptions. The remainder emphasized enhancement of animal performance such as weight gain or overall animal health. The reason why so few products carry food safety-related claims is the difficulties in establishing unambiguous cause and effect relationships between the application of such products in varied and constantly changing farm environments and improved food safety of the end product.

HISTORY AND DEFINITIONS

Elie Metchnikoff, who is “regarded as the grandfather of modern probiotics” (1) mentioned in his book The Prolongation of Life, published in 1907, that a researcher at the Pasteur Institute, Dr. Belonowsky, had shown that administration of the “Bulgarian bacillus cures a special intestinal disease known as mouse typhus” (2). Although likely impossible to prove, this passage in a book might have been one of the first to describe experimental probiotic action against an intestinal pathogen. Whatever one might think today about Metchnikoff’s ideas and his preoccupation with “putrefaction” in the digestive tract, he provided what could still be considered the basis of the modern definition of a probiotic when he wrote with reference to lactic bacilli, “The latter become acclimatized in the human digestive tube as they find there the sugary material required for their subsistence, and by producing disinfecting bodies benefit the organism which supports them” (2). With the term “disinfecting bodies,” Metchnikoff was referring primarily to lactic acid, but he was also aware, based on Belonowsky’s research, that more than lactic acid was involved in the probiotic action of the “Bulgarian bacillus.” Nowadays, most authors have settled on a broad definition of probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit to the host” (3); however, with respect to food safety, this definition might not be broad enough. Certainly, a food animal host that is healthier as a consequence of probiotic administration would be less likely to be a food safety concern, but would live microorganisms that reduce a human pathogen such as Campylobacter jejuni in the chicken’s intestinal tract without any noticeable health benefits to the host not also be a probiotic? Similarly, a product that would reduce the Escherichia coli 0157:H7 carrier state in cattle would also fall into that category.

Likely unintentionally, Metchnikoff also embedded a reference to prebiotics in his statement on lactic bacilli by mentioning “sugary materials.” He apparently recognized that a suitable substrate for beneficial bacteria was the basis of desired effects of these microorganisms. It was not until 1995 that dietary compounds that would be able to modulate the microbiota were given the name “prebiotics” (4). Today, prebiotics are defined as “a nonviable food component that confers a health benefit on the host associated with modulation of the microbiota” (5). Usually there are some limitations to which food components actually count as prebiotics. The compounds need to be resistant to hydrolysis and absorption by the upper gastrointestinal tract so that they can reach the target organisms in the lower gastrointestinal tract. It is desirable that these compounds be substrates more or less only for those microorganisms that one intends to support. It has been argued that only fructooligosaccharides and inulin meet the criteria (6); however, numerous other compounds have been included in lists of prebiotics such as galactooligosaccharides, soy-oligosaccharides, xylooligosaccharides, pyrodextrins, isomaltooligosaccharides, lactulose, pectinoligosaccharides, lactosucrose, sugar alcohols, glucooligosaccharides, levans, resistant starch, and xylosaccharides (5).

MICROBIOTA MANIPULATIONS AND FOOD SAFETY

Current products designed to manipulate the microbiota of food animals with live microorganisms (or with products directly derived from the culture of these organisms) fall into two rather lopsided categories. The predominant type attempts to improve or maintain the animals’ health status under the conditions encountered in modern animal husbandry practices without making specific claims to target pathogens that are of concern to human health. The smaller group claims to establish or modify intestinal microbiota that have a direct measurable effect on pathogens of concern to humans, such as Salmonella enterica and C. jejuni. The two categories are not mutually exclusive because healthier animals are expected to be less susceptible to colonization with certain human pathogens or to carry fewer of these pathogens. Similarly, microbiota changes designed to inhibit foodborne pathogen colonization can also improve overall animal health and, for example, lead to increased weight gains. As will be shown later in this article, the number of probiotic products sold in the United States that claim to be directed against pathogens or are competitive exclusion products is exceedingly small compared to products that claim to improve feed conversion efficiency, growth, immune system function, or resistance to stressful events.

The strategy of increasing food safety by microbiota interventions was the first to be addressed experimentally, primarily in chickens. In 1952 Milner and Schaffer (7) observed that a mature microbiota can confer resistance to infection of chicks by Salmonella, but it was not until the 1970s that effective and commercially viable microbial preparations were developed that, when administered to newly hatched chicks, were capable of reducing Salmonella infection (8). This approach to food safety enhancement in the poultry industry is sometimes termed the “Nurmi concept,” named after the author who first developed the concept. It is assumed that this approach works by the bacteria preparation competitively excluding Salmonella from sites that this pathogen would occupy if no other bacteria were present. Generally it is assumed that a combination of factors work together to exclude unwanted organisms. These factors include effects on the immune system, interference with adhesion to intestinal surfaces, competition for nutrients and perhaps oxygen, as well as production of inhibitory molecules such as volatile fatty acids, lactic acid, bacteriocins, and perhaps extracellular enzymes. Years of research showed that the more complex the mixture of bacteria that was administered to chicks, the more successful was the exclusion of Salmonella. Thus, administering bacterial mixtures from fecal or cecal sources was more protective than administering single bacterial isolates or a combination of just a few isolates (9).

The competitive exclusion concept was later also tested on pigs with undefined cultures of porcine origin plus Bacillus thetaiotaomicron (10). Interestingly, it appears that complex or undefined cultures were not prepared for use in cattle. Here, the route chosen was primarily to find single isolates that would be inhibitory to E. coli 0157:H7, for which cattle have long been considered the primary reservoir (11). For example, Brashears et al. (12) isolated numerous lactic acid bacteria and tested them for inhibition of E. coli 0157:H7 inoculated into manure and rumen fluid. The authors also determined the effect of the lactic acid bacteria on E. coli 0157:H7 carriage in live cattle (12, 13). The reported effects were generally positive, indicating that single strains or combinations of a couple of strains were capable of reducing E. coli 0157:H7 shedding and carcass contamination.

REGULATION OF PRO- AND PREBIOTICS

The trend for using single strains or a combination of several, characterized strains to improve animal well-being and growth, and directly or indirectly to improve food safety, has continued. Rather than calling these products “probiotics,” the term “direct-fed microbial products” is used in the United States for products that are given to animals. According to the Bovine Alliance on Management and Nutrition, which is comprised of representatives from the American Association of Bovine Practitioners, the American Dairy Science Association, the American Feed Industry Association, and the U.S. Department of Agriculture, the terms “probiotic” and “direct-fed microbial” (DFM) can be used interchangeably (14).

The primary reason for suppliers of such products to stay away from complex, partially, or completely undefined probiotics is based on regulations for the use of such products in animals that require spelling out the microbial composition of the products. For this reason, the commercial product originally developed by Nurmi and coworkers, is only available in countries that do not require product labeling that includes a list of the microorganisms included in the product. Furthermore, some countries have established lists of microorganisms that are allowed to be used in such products. For example, the European Union and the United States have published lists of acceptable organisms (Table 1). In the United States, Compliance Policy Guidelines (Sec. 689.100 Direct-Fed Microbial Products) (15) specify how probiotic products have to be labeled and what claims can be made. A particular influence on what kind of probiotics can be marketed is Policy 3 of the guidelines, which states that a product that “contains one or more microorganisms not listed in the AAFCO Official Publication is a food additive and is adulterated under Section 402(a)(2)(C) unless it is the subject of a food additive regulation.” Therefore, a substantial investment will have to be made by anyone who wants to bring to market a product that contains microorganisms not currently on the list. The previously approved poultry competitive exclusion product, Preempt (NADA 141-101 PREEMPTTM) (16), has been removed from the list of FDA-approved products as of 2013 (17).

TABLE 1.

List of microorganisms approved for use in DFBs in the United States and the European Union^a

Microorganisms approved by FDA and AAFCO (61)		Microorganisms with QPSb status by EFSAc
Bacillus coagulans	Lactobacillus curvatus	Bacillus amyloliquefaciens	Lactobacillus coryniformis	Pediococcus acidilactici
Bacillus lentus	Lactobacillus delbrueckii	Bacillus atrophaeus	Lactobacillus crispatus	Pediococcus dextrinicus
Bacillus licheniformis	Lactobacillus fermentum	Bacillus clausii	Lactobacillus curvatus	Pediococcus pentosaceus
Bacillus pumilus	Lactobacillus helveticus	Bacillus coagulans	Lactobacillus delbrueckii	Propionibacterium acidipropionici
Bacillus subtilis	Lactobacillus lactis	Bacillus fusiformis	Lactobacillus farciminis	Propionibacterium freudenreichii
Bacteroides amylophilus	Lactobacillus plantarum	Bacillus lentus	Lactobacillus fermentum	Streptococcus thermophiles
Bacteroides capillosus	Lactobacillus reuteri	Bacillus licheniformis	Lactobacillus gallinarum	Xanthomonas campestrisb
Bacteroides ruminicola	Leuconostoc mesenteroides	Bacillus megaterium	Lactobacillus gasseri	Yeasts:
Bacteroides suis	Megasphaera elsdenii (cattle only)	Bacillus mojavensis	Lactobacillus helveticus	Candida cylindraceab
Bifidobacterium adolescentis	Pediococcus acidilactici	Bacillus pumilus	Lactobacillus hilgardii	Debaryomyces hansenii
Bifidobacterium animalis	Pediococcus cerevisiae (damnosus)	Bacillus subtilis	Lactobacillus johnsonii	Hanseniaspora uvarum
Bifidobacterium bifidum	Pediococcus pentosaceus	Bacillus. vallismortis	Lactobacillus kefiranofaciens	Kluyveromyces lactis
Bifidobacterium infantis	Propionibacterium acidipropiconici (cattle only)	Bifidobacterium adolescentis	Lactobacillus kefiri	Kluyveromyces marxianus
Bifidobacterium longum	Propionibacterium freudenreichii	Bifidobacterium animalis	Lactobacillus mucosae	Komagataella pastoris b
Bifidobacterium thermophilum	Propionibacterium shermanii	Bifidobacterium bifidum	Lactobacillus panis	Lindnera jadinii b
Enterococcus cremoris	Rhodopseudomonas palustris (broiler chickens only)	Bifidobacterium breve	Lactobacillus paracasei	Ogataea angusta b
Enterococcus diacetylactis	Yeasts and molds:	Bifidobacterium longum	Lactobacillus paraplantarum	Saccharomyces bayanus
Enterococcus faecium	Aspergillus niger	Carnobacterium divergens	Lactobacillus pentosus	Saccharomyces cerevisiae
Enterococcus intermedius	Aspergillus oryzae	Corynebacterium glutamicum b	Lactobacillus plantarum	Saccharomyces pastorianus
Enterococcus lactis	Saccharomyces cerevisiae	Geobacillus stearothermophilus	Lactobacillus pontis	Schizosaccharomyces pombe
Enterococcus thermophilus		Gluconobacter oxydans b	Lactobacillus reuteri	Wickerhamomyces anomalus b
Lactobacillus acidophilus		Lactobacillus acidophilus	Lactobacillus rhamnosus	Xanthophyllomyces dendrorhous
Lactobacillus brevis		Lactobacillus amylolyticus	Lactobacillus sakei
Lactobacillus buchneri (cattle only)		Lactobacillus amylovorus	Lactobacillus salivarius
Lactobacillus bulgaricus		Lactobacillus alimentarius	Lactobacillus sanfranciscensis
Lactobacillus casei		Lactobacillus aviaries	Lactococcus lactis
Lactobacillus cellobiosus		Lactobacillus brevis	Leuconostoc citreum
Lactobacillus farciminis (swine only)		Lactobacillus buchneri	Leuconostoc lactis
		Lactobacillus casei	Leuconostoc mesenteroides
		Lactobacillus cellobiosus	Microbacterium imperiale b
		Lactobacillus collinoides	Oenococcus oeni

^a Abbreviations: AAFCO, Association of American Feed Control Officials; QPS, qualified presumption of safety; EFSA, European Food Safety Authority.

^b QPS status subject to qualifications.

^c Adapted from Table 1 in ref. 62.

DFMs AND THEIR COMPOSITION

Despite the regulatory constraints imposed on probiotic products in the United States and the European Union, numerous products are currently on the market. Based on listings provided in the Microbial Compendium (18), nearly 130 products were being offered in the United States that contained either live bacteria or bacterial culture-derived components that may or may not include live bacteria. Of all these products, only two make the claim that the product has competitive exclusion functions. Two additional products mention activity against pathogens. One product consisting of Lactobacillus acidophilus live cells mentions “food safety.” The remainder of the products either do not make specific claims about the effects of the product or claim benefits to the target animal only. Some producers or distributors describe the bacteria in the product as “beneficial,” as promoting or enhancing a “balanced” microbiota, as improving feed digestion and feed conversion, and as being helpful during or following stressful events. The obvious absence of specific claims, especially food safety claims, stems from the requirements that would have to be met to substantiate such claims. Challenge trials with pathogens under controlled conditions are frequently carried out to test the efficacy of microbial interventions, because such trials are easier and cheaper to conduct than actual field trials; however, challenge trials have drawbacks, and it has been argued that “the public health benefits of preharvest interventions to reduce zoonotic pathogens in livestock might be best served by field trial results alone” (19).

The scope of bacteria and products from these bacteria is limited as a consequence of regulations and agreements between the U.S. government and the organization representing the DFM industry (Fig. 1). L. acidophilus leads the list of bacteria included in DFMs, closely followed by Enterococcus faecium. L. acidophilus, in particular, has a long history of use in food and feed and has been evaluated for its probiotic properties such as survival under gastrointestinal conditions, adhesion to tissue, immunomodulatory properties, and production of bacteriocins and other antimicrobial activities in numerous trials (reviewed in 20, 21). The bacterium features prominently in studies aimed at reducing the prevalence of fecal shedding of E. coli 0157 in beef cattle that showed significant reductions in shedding (22).

FIGURE 1.

Number of products entered in the Microbial Compendium (18) that contain particular bacteria or groups of bacteria. Abbreviations: L., Lactobacillus; B., Bacillus; Ped., Pediococcus; Bif., Bifidobacterium; Prop., Propionibacterium; E., Enterococcus.

A similar wealth of studies has attempted to elucidate the probiotic properties of E. faecium, and such studies have shown properties of this bacterium similar to those of L. acidophilus. Recently, the impact of the administration of E. faecium NCIMB 11181 on the fecal microbiota of swine has been studied using high-throughput 16S rRNA gene pyrosequencing. The bacterium, when added to feed over a 2-week period, produced various changes in the proportions of the phyla and genera of the fecal bacteria (23). The authors pointed out that the level of E. coli decreased, perhaps indicating a beneficial effect on food safety. The application of a commercial product containing E. faecium, Lactobacillus salivarius, and Bifidobacter animalis to broilers housed on floor pens and challenged with three Eimeria spp. resulted in lower Eimeria counts in the probiotic-treated birds than in the untreated controls (24). Since associations between Eimeria infection and infection with other pathogens such as Salmonella have been established (25, 26), decreased carriage of Eimeria could be interpreted as a positive influence of the probiotic on food safety. A similar commercial preparation consisting of E. faecium, Lactobacillus spp., and Bifidobacterium thermophilum showed evidence for causing reduced Salmonella levels in poultry litter from flocks given the preparation (27). Such a reduction potentially correlates with improved microbial safety of birds after processing, although such a connection was not established.

The third most common bacterial component of DFMs, Bacillus subtilis, has a shorter history of use than lactic acid bacteria, but because of its obvious advantages over other bacteria with respect to storage and survival, it is now frequently included in probiotic products. Several studies have shown that the spores of various Bacillus spp. are able to germinate in the intestinal tract (for example, reviewed in 28–30); thus, they can be expected to produce metabolites such as acids, extracellular enzymes, bacteriocins, or antibiotics able to influence intestinal microbiota. The vast majority of studies examining the antipathogenic effect of Bacillus spp. so far have been done in vitro, but a number of challenge trials have been carried out, primarily with poultry (reviewed in reference 29), and immunomodulation by Bacillus spp.-containing preparations has also been observed in pigs and cattle (for example, references 31, 32). A commercial preparation containing a B. subtilis strain reduced Clostridium perfringens counts when administered to C. perfringens-challenged chicks (33). Since some level of association between C. perfringens and Salmonella infection may exist (25), the probiotic might be able to contribute to food safety of chickens.

Various species of Bifidobacterium and Pediococcus and Propionibacterium freudenreichii are also represented among the bacteria in DFMs. The probiotic effects of Bifidobacterium spp. have been studied extensively (reviewed, for example, in reference 34), including their protective effects against Salmonella and E. coli 0157:H7. Studies that showed protective effects against these pathogens were primarily carried out with animals under laboratory conditions such as those with streptomycin-treated (35) or germ-free mice (36), and it is obviously not possible to make direct connections to animals in farm settings. The same is true for studies using bioreactor models simulating the proximal colon of pigs (37). Animal trials involving Bifidobacterium longum in conjunction with fructo- and galactooligosaccharides revealed that this combination reduced Campylobacter spp. (38).

A commercial probiotic preparation containing Pediococcus parvulus and L. salivarius was administered to broiler chicks to study its effect on Salmonella colonization under conditions simulating commercial practices. Significant differences were found in the Salmonella counts in the ceca of birds receiving the probiotic supplement (39). The same product had earlier shown similar results with respect to S. enterica serovar Heidelberg colonization of broiler chickens and poults (40). Propionibacteria have a history of use as probiotics (41), and their probiotic properties such as adhesion to cells, survival at low pH, and survival in the presence of bile, as well as their antimicrobial activities are the subject of ongoing research (42). Antimicrobial activity has primarily been studied in vitro, but P. freudenreichii in conjunction with L. acidophilus was successfully utilized in trials with cattle carrying E. coli 0157:H7 (22).

A sizable number of DFM products include not just bacteria and/or their culture products, but also yeasts and molds. Of the approximately 130 products currently listed in the Microbial Compendium (18), more than 70 contain these eukaryotes as well as bacteria. Some DFMs are composed entirely of yeasts, although none of those products make any claim about food safety or antipathogen activity. The number of eukaryotic species allowed is limited, as can be seen in Table 1. The reason why yeasts and mold species and their culture products are included in DFMs is likely 2-fold. Based on studies with biomass from yeast fermentations fed to animals, it can be concluded that such preparations have performance-enhancing properties that might be related to the provision of additional nutrients such as vitamins, but also substrates that can be utilized by intestinal bacteria or enzymes that aid in the breakdown of plant polymers. The nutritional benefits of DFMs can be enhanced by including yeasts grown on inorganic selenium sources. The resulting organic selenium fraction is considered more nutritionally available to consuming animals than inorganic sources fed directly to the animals (43, 44).

In addition to the nutritional support provided by yeasts and molds, these organisms can potentially also act as antagonists of certain bacteria or as supporters of bacteria considered beneficial. Antagonism can be exerted as envisioned for bacteria simply through competition for nutrients and production of inhibitory metabolites. Obviously, the production of ethanol by Saccharomyces and other organisms has to be high on the list. Blocking of attachment sites on intestinal surfaces has also been proposed to be a factor that could prevent the establishment of pathogens (45). Due to their relatively large size and ability to aggregate, yeasts and molds can also be attachment sites for bacteria and provide environments that are conducive for the bacteria or that prevent them from interacting with the animal host. The presence of mannan oligosaccharides in yeast cell walls and therefore also in many preparations derived from yeasts has been proposed as providing structures to which enterobacteria can adhere and be prevented from adherence to intestinal surfaces; however, mechanisms and extent of attachment have not been fully elucidated (46).

PREBIOTICS

For virtually all DFM products, the more than 50 prebiotic products listed in the Microbial Compendium (18) also do not make any claims about food safety enhancement. Usually, the products emphasize contributions to animal health. Many of the products are not solely prebiotic preparations, but also contain live bacteria and/or yeasts. These products would fall into the category of synbiotics. Yeasts and molds are added not just in live form, but also in the form of autolysates, hydrolysates, or extracts. In this form, the components of these organisms are not only nutritional supplements, but are also a potential source of prebiotics in the form of mannan oligosaccharides or other oligosaccharides originating from the cell walls. Almost half of the prebiotic products listed in the compendium contain these types of oligosaccharides. An only slightly lower number lists fructo-oligosaccharides (FOS) and inulin as part of their content, and about 10 mention beta glucans. Rarely is the source of these oligosaccharides provided. Beta glucans might originate from yeasts, but also from plant material. FOS including inulin are likely exclusively of plant origin, and some products list yucca or chicory as sources. A review of the literature regarding the use of FOS-type prebiotics made it clear that studies of the effect of these probiotics have not yielded definitive proof of effectiveness (46–48). In part, this outcome can be attributed to the potential for differences in the FOS products utilized due to differences in origin and processing.

By definition, prebiotics are intended to be unavailable to the host but be a substrate for beneficial bacteria resulting in the exertion of inhibitory effects on pathogens. Many, but not all, lactic acid bacteria (LAB) and bifidobacteria have the ability to utilize prebiotic oligosaccharides (48), but it is difficult to document in animal trials that reductions in pathogens such as Salmonella and Campylobacter are indeed correlated with effects on these bacteria. Since not all LABs or bifidobacteria are able to utilize prebiotic oligosaccharides (49, 50), and not all of them can be expected to be equally effective as antipathogen agents, the presence of these bacteria and changes in their abundance do not automatically result in increased pathogen reduction. These circumstances might explain the observed heterogeneity in results with prebiotics, and they might also give impetus to combine prebiotic with live bacteria known to be inhibitory to pathogens. A recent review of prebiotic-related studies involving poultry (51) again highlighted the paucity of experiences with prebiotics as efficient contributors to food safety; however, the authors expressed their conviction that high-throughput approaches to intestinal microbiology will advance knowledge of the effects of prebiotics on intestinal microbiota and reveal suitable applications of prebiotics for the promotion of food safety. Perhaps microarray-based instrumentation such as the FDA’s GutProb (52), which allows rapid detection and analyses of probiotic strains, will also accelerate development of pro- and prebiotic products. Without approaches that allow a comprehensive analysis of the microbial composition of intestinal sites, it is difficult to detect changes in the microbiota in response to the application of prebiotics. For example, a recent study on the effects of a mannanoligosaccharide-based commercial prebiotic on the cecal microbiota of broiler chickens that utilized denaturing gradient gel electrophoresis was not able to reveal obvious and consistent differences in the cecal microbiota of birds fed the prebiotic and control birds (53). The study also highlighted the difficulty in demonstrating potential food safety benefits of prebiotic application in large-scale settings because the frequency of Salmonella isolations from the ceca of the treated and control birds was low regardless of the treatment.

FUTURE PROSPECTS

Prebiotic research in the past was clearly focused on how these compounds can support well-known intestinal populations such as LABs and bifidobacteria, but recently, evidence was provided, albeit in a zebra fish model, that even the minority component of the gut microbiota can have a strong effect on intestinal immune responses (54). Provided these findings are true not just in a model system, but apply to complex intestinal microbiota, it will be necessary to evaluate means by which even minor components of the microbiota might have to be supported in the future.

It is difficult to predict at what pace applications of pro- and prebiotics for the purpose of improved preharvest food safety will move forward. As this chapter has attempted to outline, a relatively conservative regulatory environment has been established in the United States and Europe that makes it necessary to invest in considerable research and other efforts prior to the introduction of novel types of bacteria as DFMs. Considerable gaps in the understanding of the interactions of intestinal microorganisms with each other and with the host organism still exist, but these gaps are expected to disappear due to high-throughput sequencing and other molecular and genetic techniques. In the food safety field, a strong impetus toward improved applications of pro- and prebiotics is not only the need for improved animal health and performance, but also the movement away from the application of antibiotics in animal feed (55, 56). Pro- and prebiotics are viewed as components of strategies to reduce or even eliminate routine antibiotic use in animal agriculture. Obviously, the concerns about the spread of antibiotic resistance also pose one more prerequisite for the selection of suitable probiotic strains because it is absolutely essential that such strains do not carry antibiotic resistance genes. Other genes that should, of course, be absent in probiotic strains are those that code for the production of toxins or compounds that in any way will interfere with an animal’s well-being and productivity.

In contrast, genes that should be present are those that enable the organism to grow in inexpensive media for cost-effective production, to survive processing and storage, to pass through the stomach or other inhospitable parts of an animal’s digestive tract without being overly decimated, to persist and perhaps grow under the conditions of the lower intestinal tract, and to produce compounds that inhibit unwanted bacteria and perhaps also those that stimulate the animal’s natural defenses and performance. Finding organisms that possess all these traits is not easy, and not surprisingly, the genetic modification of probiotic strains has been considered and even tested. Lactic acid bacteria, in particular, have been the target of genetic modifications, with an eye on ultimately utilizing them for human applications such as combatting or preventing colitis (57). A few genetically modified strains have been tested with the aim of improving animal performance. For example, Lactococcus lactis was engineered to express the epidermal growth factor EGF-LL in an effort to boost the performance of early-weaned piglets (58), and the yeast Pichia pastoris was modified by the introduction of the C. perfringens alpha toxin gene in an attempt to induce immunity against C. perfringens in broiler chickens (59).

If and when such genetically modified organisms will ever be utilized commercially depends on scientific and economic factors but most importantly on whether or not the farming community and the public will be willing to accept the widespread release of genetically modified microorganisms in the environment and the food supply. As a way to avoid the direct introduction of genetically engineered microorganisms but still be able to utilize the benefits of genetic engineering, the use of culture supernatants is an option. This method is now being pursued, for example, in the case of the L. lactis expressing EGF-LL (60).