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Microbial antagonists to food-borne pathogens and biocontrol
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Microbial antagonists to food-borne pathogens and biocontrol Antonio Ga´lvez, Hikmate Abriouel, Nabil Benomar and Rosario Lucas Application of natural antimicrobial substances (such as bacteriocins) combined with novel technologies provides new opportunities for the control of pathogenic bacteria, improving food safety and quality. Bacteriocin-activated films and/or in combination with food processing technologies (highhydrostatic pressure, high-pressure homogenization, inpackage pasteurization, food irradiation, pulsed electric fields, or pulsed light) may increase microbial inactivation and avoid food cross-contamination. Bacteriocin variants developed by genetic engineering and novel bacteriocins with broader inhibitory spectra offer new biotechnological opportunities. Infarm application of bacteriocins, bacterial protective cultures, or bacteriophages, can decrease the incidence of food-borne pathogens in livestock, animal products and fresh produce items, reducing the risks for transmission through the food chain. Biocontrol of fungi, parasitic protozoa and viruses is still a pending issue. Address A´rea de Microbiologı´a, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jae´n, 23071 Jae´n, Spain Corresponding author: Ga´lvez, Antonio ([email protected])
Current Opinion in Biotechnology 2010, 21:142–148 This review comes from a themed issue on Food biotechnology Edited by Dietrich Knorr and Carmen Wacher Available online 9th February 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.01.005
Introduction Microbes elicit a variety of mechanisms that facilitate colonization and prevalence in ecological niches. These include adherence, competition for available nutrients, production of toxic metabolites, and secretion of dedicated antimicrobial substances such as antibiotics and bacteriocins. The wise exploitation of these mechanisms of microbial interference can be beneficial to human and animal health, and economy. The transmission of foodborne pathogens through the food chain is still an unresolved issue. The globalization of the food market, and the new trends in food production and distribution, together with changes in consumer habits and population susceptibility (such as the elderly or immuno-compromised people) are always pointed as the main contributing factors. In addition, the substantial economic losses Current Opinion in Biotechnology 2010, 21:142–148
because of spoilage of raw materials or processed products and the costly recalls because of microbial contamination are matters of concern in a world that periodically faces economic crisis and increasingly suffers from population overgrowth, malnutrition and overexploitation of natural resources. In developing countries, the incidence of illnesses caused by food-borne pathogens in the younger people has also a clear influence in malnutrition, which in turn has a negative impact on health status and cognitive potential. Among the wide array of strategies being currently used or proposed for food preservation, control strategies based on living organisms and/or their antimicrobial products (biocontrol, or biopreservation) have been used since ancient times (such as in food fermentation) and are becoming increasingly popular for several reasons: firstly, natural preservation methods are regarded as healthfriendly by consumers, and are expected to have a lower impact on the food nutritional and sensory properties (as opposed to chemical or physico-chemical treatments); secondly, they may decrease the processing costs while at the same time extending the product shelf life period, do not require advanced technological equipment or skills and therefore can be exploited by smaller economies; thirdly, may offer new opportunities to solve emerging issues such as the increase of antibiotic resistance in the food chain, the need to improve animal productivity by natural means, or the control of emerging pathogens.
Microbial cell factories for biocontrol Microbes may produce a wide spectrum of antimicrobial substances. Most studies have focused on antimicrobials produced by lactic acid bacteria (LAB) and associated bacteria such as the propionic acid bacteria and the bifidobacteria. The decreased pH value and antibacterial activities of organic acids produced by LAB are the main mechanisms for biopreservation of fermented foods. Specific strains of LAB may also produce other inhibitory substances (such as diacetyl, reuterin, reutericyclin), antifungal compounds (such as propionate, phenyl-lactate, hydroxyphenyl-lactate, cyclic dipeptides, and 3-hydroxy fatty acids), bacteriocins and bacteriocin-like inhibitory substances (BLIS), which can be exploited against foodborne pathogens and spoilage bacteria (Figure 1). LAB bacteriocins can be grouped in different classes, such as the lantibiotics, the Class II bacteriocins and subclasses, or the circular bacteriocins [1]. Nisin is the paradigm of lantibiotic bacteriocins, and is licensed as a food preservative. The pediocin AcH/PA-1 is also available as a commercial preparation, while other bacteriocins such as lacticin 3147 or the cyclic peptide enterocin AS-48 www.sciencedirect.com
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Figure 1
Biocontrol of pathogenic bacteria through the food chain using microbial antagonistic bacteria and/or their antimicrobial products. Antagonistic strains can be applied: (1) as living cultures on livestock and fresh produce; (2) as protective cultures on ready-to-eat food products; (3) as starter or protective cultures in fermented foods. They are expected to grow and produce antimicrobial substances in situ, displacing unwanted bacteria. Alternatively, food-grade preparations containing antimicrobials produced at industrial scale by antagonistic strains can be applied as biopreservatives or as food additives to inhibit transmission of food-borne and/or spoilage bacteria through the food chain (1–4). Since the food microbiota may change considerably from farm to fork, biocontrol strategies must be designed specifically for each type or category of food product.
can be produced on cheap by-products from the dairy industry in the form of lyophilized powders amenable for commercial exploitation [2]. Bacteriocins offer a wide spectrum of potential applications against pathogenic and spoilage bacteria in foods [3]. One of the main limitations of many bacteriocins is their narrow antibacterial activity. Genetic engineering investigation led to the discovery of nisin derivatives with increased activity against Gram-positive pathogens including Listeria monocytogenes and/or Staphylococcus aureus [4]. This major step forward in the bioengineering of nisin may open new possibilities to modify the spectrum and specific activity of other lantibiotics. Although most bacteriocins are only active on Gram-positive bacteria, some LAB bacteriocins described recently are active on Gram-negative bacteria of concern in foods [5,6,7,8]. Although there are very scarce or no reports yet on their efficacy in food systems, they may provide novel tools to control food-borne pathogens. Bacteriocins from non-LAB bacteria, such as variacin (from Kocuria varians), cerein 8A (from Bacillus cereus) or the colicins and microcins are also being investigated for food biopreservation [3,9,10]. Microcins are interesting small peptides for the inhibition of Gram-negative bacteria. Microcin J25 (MccJ25) is active against Salmowww.sciencedirect.com
nella spp., Shigella spp., and Escherichia coli O157:H7. Since MccJ25 is highly resistant to digestive proteases and could affect the normal gastrointestinal microbiota when ingested with foods, a chymotrypsin-susceptible MccJ25 variant has been developed recently, which may be used as a food preservative against the Gram-negative pathogens [11].
Biocontrol of Gram-positive bacteria in foods L. monocytogenes
Among the Gram-positive bacteria, L. monocytogenes is considered the food-borne pathogen of greatest concern owing to its capacity to survive and grow in a wide variety of food substrates and environmental conditions, including refrigeration. Many studies have investigated the effects of bacteriocins and bacteriocin-producing strains on L. monocytogenes in different food systems and in combination with different physico-chemical hurdles [3], although the efficacy of lantibiotics and Class IIa bacteriocins against this food-borne pathogen is compromised by the emergence of bacteriocin resistant strains and the cross-resistance observed between bacteriocins [12,13]. Among the different approaches tested, surface application of bacteriocins (by dipping, washing, or film immobilization) alone or in combination with other hurdles or treatments is gaining attention to decrease the levels of listeria and avoid cross-contamination in raw as Current Opinion in Biotechnology 2010, 21:142–148
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well as processed foods such as sprouts, fruits, liquid foods, ready-to-eat meat products such as ham and sausages, cheeses, and fish products such as smoked salmon [3,14–21]. Novel food processing technologies such as pulsed electric fields (PEF) [22], high-hydrostatic pressure (HHP) [17], high-pressure homogenization [23], inpackage pasteurization [24], pulsed light [21], or ionizing radiation [20] could be used in combination with bacteriocins as effective antilisterial steps in the production of ready-to-eat foods and drinks. Selected LAB protective cultures can control L. monocytogenes in foods [3,25–28]. These can be applied either by surface inoculation or mixed with the food, and produce antilisterial substances during storage, fermentation or ripening. Understanding the microbial ecology of food-borne pathogens is crucial to control their transmission. L. monocytogenes can survive attached to biofilms in food processing plants. Protective culture bacteria with antilisteria activity could find application in the control of Listeria biofilms in places refractile to cleaning and disinfection, such as floor drains [29].
Other Gram-positive bacteria Biocontrol of staphylococci has focused considerably on the prevention and treatment of mastitis and improvement of animal health. In food systems (such as sauces, desserts, meat or milk) addition of bacteriocins alone or in combined treatments causes a variable degree of inactivation of S. aureus [30–32]. Microbial inactivation of staphylococci in milk increases remarkably with combined treatments by PEF and antimicrobial peptides (such as nisin, enterocin AS-48, lysozyme, or combinations of these) [33]. In fermented sausages, survival of S. aureus decreases after the addition of nisin or inoculation with bacteriocinogenic strains [34,35]. Although staphylococci are much more resistant than L. monocytogenes to bacteriocin treatments, different biocontrol approaches are now available to inhibit proliferation and staphylococcal toxin production in foods. Inactivation of spores from food poisoning bacteria (mostly B. cereus and Clostridium botulinum) is still a concern in the food industry. Many bacteriocins have shown antimicrobial activity against endospore-forming bacterial cells and germinating spores in food systems, as exemplified by nisin [3]. Promising results have been reported recently for combined treatments (such as HHP, nisin, or moderate heat) on the inactivation of C. botulinum and B. cereus spores [36,37]. Such combined treatments could improve food safety and decrease the impact of the intense heat treatments required for endospore inactivation. In addition, the residual bacteriocin in the finished product affords natural protection against bacterial growth and toxin production during the product shelf life. Current Opinion in Biotechnology 2010, 21:142–148
Biocontrol of Gram-negative bacteria in foods One of the main concerns in food safety is the transmission of pathogenic enterobacteriaceae, because of their high incidence in food-borne illness and the emergence of new virulent serotypes and transmission routes. Outbreaks related to the consumption of fresh produce have been increasingly reported. Pathogenic bacteria may contaminate fresh produce from dust, animal excreta, manure, irrigation water, cross-contamination from processing wash or postharvest handling. Adhesion of human pathogenic bacteria to leaf surfaces and invasion of the inner leaf tissue have been documented, decreasing the efficacy of decontamination treatments [38]. Tissue damage may enhance the growth of human pathogens on produce [39]. Control of food-borne pathogens in fresh produce is difficult as a result of the limited treatments that can be applied without compromising the food’s organoleptic properties and shelf life. LAB bacteriocins could be applied for the inactivation of Gram-negative pathogens in foods in combination with other hurdles or treatments to induce cell damage and partial disorganization of the outer cell membrane protective layer [3]. Application of bacteriocins in combination with other antimicrobials such as sanitation or washing treatments can reduce the microbial load on fresh produce and inactivate Gram-negative bacteria (including S. enterica, E. coli O157:H7, Shigella spp., Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila and Pseudomonas fluorescens) [40]. Such treatments can also decrease the risk for transmission of pathogenic bacteria from fruit surfaces to sliced fruits during processing [3]. Biocontrol based on bacteriophages or protective LAB cultures has also been proposed [41,42], although the efficacy of such treatments greatly depends on ecological factors such as phage specificity and inactivation, or the capacity to grow and produce antimicrobials in situ by the protective cultures. Microbial inactivation of E. coli and S. enterica in fruit juices can be enhanced with treatments based on various types of bacteriocin combinations (with organic acids, chelators, lysozyme, PEF, high-pressure homogenization, or HHP) [22,23,43,44,45]. These mild treatments allow a better preservation of nutrients and organoleptic properties of the juices compared to thermal treatments. In foods of animal origin such as meat and poultry products, bacteriocins have been tested for carcass decontamination by washing or spraying, with varying degrees of success [3]. Spray intervention with colicin E1 provides a strong reduction of E. coli O157:H7 on beef carcasses under refrigeration, although high bacteriocin concentrations are required [9]. In meat and poultry products, application of bacteriocins (like nisin, pediocin, or enterocins A, B and AS-48) in film coatings or in combination with HHP can reduce the growth and www.sciencedirect.com
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survival of Salmonella [19,30,32,46,47] and E. coli O157:H7 [16,19]. Application of bacteriophage mixtures dramatically reduces the levels of enterobacteria (such as Salmonella Typhimurium and Campylobacter jejuni) on meats [48]. Reducing the concentrations of enterobacteria in foods decreases the risks of human infection through food consumption. Nevertheless, incomplete inactivation of microbial populations may increase the risks for the selection of resistant or adapted strains. This phenomenon has not yet been studied in depth on Gram-negatives. Cronobacter (Enterobacter) sakazakii is an emerging pathogen of concern in infant formula. C. sakazakii can be inhibited under laboratory conditions by antimicrobial peptides (lactoferrin, nisin, and nisin combination with diacetyl) [49] but not in reconstituted infant formula [50]. Therefore, it is necessary to investigate other antimicrobial treatments against this bacterium in infant foods. In ethnic cereal foods, survival of the surrogate E. aerogenes as well as other Gram-negative bacteria decreases after fermentation with LAB strains producing BLIS [51]. Since much ethnic fermented gruel is consumed as infant weaning foods as well as by elderly people, the application of specific protective cultures to produce antimicrobial substances could improve the sanitary conditions of such fermented foods for these higher risk populations. Bioactive extracts from such BLIS-producing strains could also find applications as hurdles against transmission of pathogenic bacteria in several other kinds of infant formula.
Biocontrol of enteric pathogens in farm animals Livestock (such as pigs, cattle and poultry) are the main reservoirs of important food-borne pathogens such as E. coli, Salmonella, Shigella or Campylobacter strains. The application of antibiotics in animal breeding and in animal health results in training of pathogenic bacteria against antibiotics of clinical use, and infections caused by the resulting multi-resistant strains are increasingly difficult to treat. Several microbial antagonists (mostly colicins and microcins) have been proposed or marketed for application in livestock [52]. Administration of bacteriocins in feed before poultry slaughter appears to provide the control of C. jejuni to effectively reduce human exposure. Microencapsulated bacteriocin preparations from Paenibacillus polymyxa and Lactobacillus salivarius in chicken feed dramatically reduced both intestinal levels and frequency of colonization by campylobacters (C. jejuni, C. coli) in broiler chickens and in turkey poults [5,53,54]. Bacteriocin administration also had an impact on the gut morphology of turkey poults, reducing crypt depth and goblet cell density [54], although it is not clear whether this was a direct or indirect effect of treatment. The influence of www.sciencedirect.com
such alterations on colonization by Campylobacter as well as by other enteropathogens remains to be deciphered. Some enterocins produced by enterococci isolated from poultry also show promising results. Beneficial effects reported for the administration of enterocins E50-52 and E-760 in feed or water to broilers included a dramatic reduction of cecal levels of C. jejuni and Salmonella enteritidis, and reduction of the systemic dissemination of salmonellae in liver and spleen [7,8]. Although the total LAB content in the ceca of birds apparently was not affected by the administration of enterocins, the impact of bacteriocins on the global composition of the intestinal microbiota (including the LAB composition) needs to be further investigated. Since these enterocins also display strong antibacterial activity against other food-borne pathogens (Salmonella spp., Shigella spp., E. coli O157:H7 and Y. enterocolitica) they could probably be used to control pathogen colonization of poultry as well as other farm animals. Nevertheless, bacteriocin inactivation by proteases of the gastrointestinal tract, differences in strain sensitivity to bacteriocins, and the emergence of bacteriocin resistant or adapted strains need to be evaluated. For these reasons, administration of cocktails containing several bacteriocins should be recommended rather than single bacteriocins. It is also likely that bacteriocin treatments eliminate pathogenic bacteria from most but not all enteric locations. In order to prevent recolonization of the gut by the remaining survivors, bacteriocin challenges could be applied just before marketing.
Conclusions and perspectives Research on biocontrol of pathogenic bacteria through the food chain has provided a wide array of treatment options based on natural antimicrobials. In spite of the large number of laboratory studies carried out with different bacteriocins, there is still a long way to industrial applications, with little innovation with respect to classical commercial preparations such as nisin and pediocin PA-1/ AcH. Antimicrobial activity of bacteriocins can be modified by genetic engineering, and hybrid bacteriocin molecules are to be expected in the future. However, heterologous bacteriocin production and development of large-scale production processes are challenging. Ethnic foods are valuable sources for new bacteriocin-producing strains better adapted to their food substrates and maybe also with novel and interesting biotechnological properties. Several novel bacteriocins capable of inhibiting Gram-negative bacteria of concern in foods have been described recently, and conceivably this will open new possibilities for their application in food preservation. Some of them have been already tested with satisfactory results in the control of pathogenic bacteria in farm animals. However, the impact of bacteriocin-producing strains on the microbial ecology of the gastrointestinal tract and on animal health is far from being understood. Current Opinion in Biotechnology 2010, 21:142–148
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One major pending issue is extending the spectrum of bacteriocins to food-borne parasitic protozoa and viruses. These are of major concern in foods that are eaten without cooking or not cooked sufficiently. Remarkably, enteric viruses always rank in outstanding positions in statistics of food-borne illnesses. The antifungal activity of LAB has not been exploited satisfactorily, in spite of the great interest to control food spoilage caused by yeasts and filamentous fungi as well as mycotoxin production. Screening for novel antifungal LAB strains from less explored environments such as ethnic foods, together with genetic engineering approaches, may contribute to fill the existing gap in this field. Nevertheless, antifungal compounds may also have toxic effects on other eukaryotic cells, and may not meet the qualified presumption of safety (QPS) status attributed to bacteriocins and other antimicrobials produced by LAB.
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34. Sparo M, Nun˜ez GG, Castro M, Calcagno ML, Garcı´a Allende MA, Ceci M, Najle R, Manghi M: Characteristics of an environmental strain. Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages. Food Microbiol 2008, 25:607-615.
46. Jofre´ A, Aymerich T, Garriga M: Assessment of the effectiveness of antimicrobial packaging combined with high pressure to control Salmonella sp. in cooked ham. Food Control 2008, 19:634-638.
35. Hampikyan H: Efficacy of nisin against Staphylococcus aureus in experimentally contaminated sucuk, a Turkish-type fermented sausage. J Food Prot 2009, 72:1739-1743. 36. Black EP, Linton M, McCall RD, Curran W, Fitzgerald GF, Kelly AL, Patterson MF: The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk. J Appl Microbiol 2008, 105:78-87. www.sciencedirect.com
47. Santiago-Silva P, Soares NFF, No´brega JE, Ju´nior MAW, Barbosa KBF, Volp ACP, Zerdas ERMA, Wu¨rlitzer NJ: Antimicrobial efficiency of film incorporated with pediocin W (ALTA 2351) on preservation of sliced ham. Food Control 2009, 20:85-89. 48. Bigwood T, Hudson JA, Billington C, Carey-Smith GV, Heinemann JA: Phage inactivation of foodborne pathogens on cooked and raw meat. Food Microbiol 2008, 25:400-406. Current Opinion in Biotechnology 2010, 21:142–148
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This work exemplifies the use of bacteriophages in biocontrol strategies to reduce the microbial load of relevant food-borne pathogens (Salmonella Typhimurium and Campylobacter jejuni) on meat.
An excellent overview on application of bacteriocins and bacteriocinproducing strains in livestock for preharvest control of food-borne pathogens or as growth promoters.
49. Lee SY, Jin HH: Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii. Lett Appl Microbiol 2008, 47:315-321.
53. Stern NJ, Svetoch EA, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, Mitsevich EV, Mitsevich IP, Levchuk VP: Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. J Food Prot 2005, 68:1450-1453. The authors investigated paenibacilli as a novel source of antimicrobial substances for application in broiler chickens, and demonstrated the protective effects of microencapsulated bacteriocin against chicken colonization by campylobacters. Feeding bacteriocin before slaughter appears to effectively control campylobacters to reduce human exposure.
50. Al-Nabulsi AA, Osaili TM, Al-Holy MA, Shaker RR, Ayyash MM, Olaimat AN, Holley RA: Influence of desiccation on the sensitivity of Cronobacter spp. to lactoferrin or nisin in broth and powdered infant formula. Int J Food Microbiol 2009, 136:221-226. 51. Sa´nchez Valenzuela A, Dı´az Ruiz G, Ben Omar N, Abriouel H, Lucas Lo´pez R, Martı´nez Can˜amero M, Ortega E, Ga´lvez A: Inhibition of food poisoning and pathogenic bacteria by Lactobacillus plantarum strain 2.9 isolated from ben saalga, both in a culture medium and in food. Food Control 2008, 19:842-848. 52. Diez-Gonzalez F: Applications of bacteriocins in livestock. Curr Issues Intest Microbiol 2007, 8:15-23.
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54. Cole K, Farnell MB, Donoghue AM, Stern NJ, Svetoch EA, Eruslanov BN, Volodina LI, Kovalev YN, Perelygin VV, Mitsevich EV et al.: Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults. Poult Sci 2006, 85:1570-1575. Dietary administration of bacteriocins (B602 from P. polymyxa and OR7 from L. salivarius) reduced the levels of C. coli (the most prevalent Campylobacter species in turkey) in turkey poults. This work also demonstrates for the first time the effect of bacteriocin ingestion on gut morphology.
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Microbial antagonists to food-borne pathogens and biocontrol Antonio Ga´lvez, Hikmate Abriouel, Nabil Benomar and Rosario Lucas Application of natural antimicrobial substances (such as bacteriocins) combined with novel technologies provides new opportunities for the control of pathogenic bacteria, improving food safety and quality. Bacteriocin-activated films and/or in combination with food processing technologies (highhydrostatic pressure, high-pressure homogenization, inpackage pasteurization, food irradiation, pulsed electric fields, or pulsed light) may increase microbial inactivation and avoid food cross-contamination. Bacteriocin variants developed by genetic engineering and novel bacteriocins with broader inhibitory spectra offer new biotechnological opportunities. Infarm application of bacteriocins, bacterial protective cultures, or bacteriophages, can decrease the incidence of food-borne pathogens in livestock, animal products and fresh produce items, reducing the risks for transmission through the food chain. Biocontrol of fungi, parasitic protozoa and viruses is still a pending issue. Address A´rea de Microbiologı´a, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jae´n, 23071 Jae´n, Spain Corresponding author: Ga´lvez, Antonio ([email protected])
Current Opinion in Biotechnology 2010, 21:142–148 This review comes from a themed issue on Food biotechnology Edited by Dietrich Knorr and Carmen Wacher Available online 9th February 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.01.005
Introduction Microbes elicit a variety of mechanisms that facilitate colonization and prevalence in ecological niches. These include adherence, competition for available nutrients, production of toxic metabolites, and secretion of dedicated antimicrobial substances such as antibiotics and bacteriocins. The wise exploitation of these mechanisms of microbial interference can be beneficial to human and animal health, and economy. The transmission of foodborne pathogens through the food chain is still an unresolved issue. The globalization of the food market, and the new trends in food production and distribution, together with changes in consumer habits and population susceptibility (such as the elderly or immuno-compromised people) are always pointed as the main contributing factors. In addition, the substantial economic losses Current Opinion in Biotechnology 2010, 21:142–148
because of spoilage of raw materials or processed products and the costly recalls because of microbial contamination are matters of concern in a world that periodically faces economic crisis and increasingly suffers from population overgrowth, malnutrition and overexploitation of natural resources. In developing countries, the incidence of illnesses caused by food-borne pathogens in the younger people has also a clear influence in malnutrition, which in turn has a negative impact on health status and cognitive potential. Among the wide array of strategies being currently used or proposed for food preservation, control strategies based on living organisms and/or their antimicrobial products (biocontrol, or biopreservation) have been used since ancient times (such as in food fermentation) and are becoming increasingly popular for several reasons: firstly, natural preservation methods are regarded as healthfriendly by consumers, and are expected to have a lower impact on the food nutritional and sensory properties (as opposed to chemical or physico-chemical treatments); secondly, they may decrease the processing costs while at the same time extending the product shelf life period, do not require advanced technological equipment or skills and therefore can be exploited by smaller economies; thirdly, may offer new opportunities to solve emerging issues such as the increase of antibiotic resistance in the food chain, the need to improve animal productivity by natural means, or the control of emerging pathogens.
Microbial cell factories for biocontrol Microbes may produce a wide spectrum of antimicrobial substances. Most studies have focused on antimicrobials produced by lactic acid bacteria (LAB) and associated bacteria such as the propionic acid bacteria and the bifidobacteria. The decreased pH value and antibacterial activities of organic acids produced by LAB are the main mechanisms for biopreservation of fermented foods. Specific strains of LAB may also produce other inhibitory substances (such as diacetyl, reuterin, reutericyclin), antifungal compounds (such as propionate, phenyl-lactate, hydroxyphenyl-lactate, cyclic dipeptides, and 3-hydroxy fatty acids), bacteriocins and bacteriocin-like inhibitory substances (BLIS), which can be exploited against foodborne pathogens and spoilage bacteria (Figure 1). LAB bacteriocins can be grouped in different classes, such as the lantibiotics, the Class II bacteriocins and subclasses, or the circular bacteriocins [1]. Nisin is the paradigm of lantibiotic bacteriocins, and is licensed as a food preservative. The pediocin AcH/PA-1 is also available as a commercial preparation, while other bacteriocins such as lacticin 3147 or the cyclic peptide enterocin AS-48 www.sciencedirect.com
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Figure 1
Biocontrol of pathogenic bacteria through the food chain using microbial antagonistic bacteria and/or their antimicrobial products. Antagonistic strains can be applied: (1) as living cultures on livestock and fresh produce; (2) as protective cultures on ready-to-eat food products; (3) as starter or protective cultures in fermented foods. They are expected to grow and produce antimicrobial substances in situ, displacing unwanted bacteria. Alternatively, food-grade preparations containing antimicrobials produced at industrial scale by antagonistic strains can be applied as biopreservatives or as food additives to inhibit transmission of food-borne and/or spoilage bacteria through the food chain (1–4). Since the food microbiota may change considerably from farm to fork, biocontrol strategies must be designed specifically for each type or category of food product.
can be produced on cheap by-products from the dairy industry in the form of lyophilized powders amenable for commercial exploitation [2]. Bacteriocins offer a wide spectrum of potential applications against pathogenic and spoilage bacteria in foods [3]. One of the main limitations of many bacteriocins is their narrow antibacterial activity. Genetic engineering investigation led to the discovery of nisin derivatives with increased activity against Gram-positive pathogens including Listeria monocytogenes and/or Staphylococcus aureus [4]. This major step forward in the bioengineering of nisin may open new possibilities to modify the spectrum and specific activity of other lantibiotics. Although most bacteriocins are only active on Gram-positive bacteria, some LAB bacteriocins described recently are active on Gram-negative bacteria of concern in foods [5,6,7,8]. Although there are very scarce or no reports yet on their efficacy in food systems, they may provide novel tools to control food-borne pathogens. Bacteriocins from non-LAB bacteria, such as variacin (from Kocuria varians), cerein 8A (from Bacillus cereus) or the colicins and microcins are also being investigated for food biopreservation [3,9,10]. Microcins are interesting small peptides for the inhibition of Gram-negative bacteria. Microcin J25 (MccJ25) is active against Salmowww.sciencedirect.com
nella spp., Shigella spp., and Escherichia coli O157:H7. Since MccJ25 is highly resistant to digestive proteases and could affect the normal gastrointestinal microbiota when ingested with foods, a chymotrypsin-susceptible MccJ25 variant has been developed recently, which may be used as a food preservative against the Gram-negative pathogens [11].
Biocontrol of Gram-positive bacteria in foods L. monocytogenes
Among the Gram-positive bacteria, L. monocytogenes is considered the food-borne pathogen of greatest concern owing to its capacity to survive and grow in a wide variety of food substrates and environmental conditions, including refrigeration. Many studies have investigated the effects of bacteriocins and bacteriocin-producing strains on L. monocytogenes in different food systems and in combination with different physico-chemical hurdles [3], although the efficacy of lantibiotics and Class IIa bacteriocins against this food-borne pathogen is compromised by the emergence of bacteriocin resistant strains and the cross-resistance observed between bacteriocins [12,13]. Among the different approaches tested, surface application of bacteriocins (by dipping, washing, or film immobilization) alone or in combination with other hurdles or treatments is gaining attention to decrease the levels of listeria and avoid cross-contamination in raw as Current Opinion in Biotechnology 2010, 21:142–148
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well as processed foods such as sprouts, fruits, liquid foods, ready-to-eat meat products such as ham and sausages, cheeses, and fish products such as smoked salmon [3,14–21]. Novel food processing technologies such as pulsed electric fields (PEF) [22], high-hydrostatic pressure (HHP) [17], high-pressure homogenization [23], inpackage pasteurization [24], pulsed light [21], or ionizing radiation [20] could be used in combination with bacteriocins as effective antilisterial steps in the production of ready-to-eat foods and drinks. Selected LAB protective cultures can control L. monocytogenes in foods [3,25–28]. These can be applied either by surface inoculation or mixed with the food, and produce antilisterial substances during storage, fermentation or ripening. Understanding the microbial ecology of food-borne pathogens is crucial to control their transmission. L. monocytogenes can survive attached to biofilms in food processing plants. Protective culture bacteria with antilisteria activity could find application in the control of Listeria biofilms in places refractile to cleaning and disinfection, such as floor drains [29].
Other Gram-positive bacteria Biocontrol of staphylococci has focused considerably on the prevention and treatment of mastitis and improvement of animal health. In food systems (such as sauces, desserts, meat or milk) addition of bacteriocins alone or in combined treatments causes a variable degree of inactivation of S. aureus [30–32]. Microbial inactivation of staphylococci in milk increases remarkably with combined treatments by PEF and antimicrobial peptides (such as nisin, enterocin AS-48, lysozyme, or combinations of these) [33]. In fermented sausages, survival of S. aureus decreases after the addition of nisin or inoculation with bacteriocinogenic strains [34,35]. Although staphylococci are much more resistant than L. monocytogenes to bacteriocin treatments, different biocontrol approaches are now available to inhibit proliferation and staphylococcal toxin production in foods. Inactivation of spores from food poisoning bacteria (mostly B. cereus and Clostridium botulinum) is still a concern in the food industry. Many bacteriocins have shown antimicrobial activity against endospore-forming bacterial cells and germinating spores in food systems, as exemplified by nisin [3]. Promising results have been reported recently for combined treatments (such as HHP, nisin, or moderate heat) on the inactivation of C. botulinum and B. cereus spores [36,37]. Such combined treatments could improve food safety and decrease the impact of the intense heat treatments required for endospore inactivation. In addition, the residual bacteriocin in the finished product affords natural protection against bacterial growth and toxin production during the product shelf life. Current Opinion in Biotechnology 2010, 21:142–148
Biocontrol of Gram-negative bacteria in foods One of the main concerns in food safety is the transmission of pathogenic enterobacteriaceae, because of their high incidence in food-borne illness and the emergence of new virulent serotypes and transmission routes. Outbreaks related to the consumption of fresh produce have been increasingly reported. Pathogenic bacteria may contaminate fresh produce from dust, animal excreta, manure, irrigation water, cross-contamination from processing wash or postharvest handling. Adhesion of human pathogenic bacteria to leaf surfaces and invasion of the inner leaf tissue have been documented, decreasing the efficacy of decontamination treatments [38]. Tissue damage may enhance the growth of human pathogens on produce [39]. Control of food-borne pathogens in fresh produce is difficult as a result of the limited treatments that can be applied without compromising the food’s organoleptic properties and shelf life. LAB bacteriocins could be applied for the inactivation of Gram-negative pathogens in foods in combination with other hurdles or treatments to induce cell damage and partial disorganization of the outer cell membrane protective layer [3]. Application of bacteriocins in combination with other antimicrobials such as sanitation or washing treatments can reduce the microbial load on fresh produce and inactivate Gram-negative bacteria (including S. enterica, E. coli O157:H7, Shigella spp., Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila and Pseudomonas fluorescens) [40]. Such treatments can also decrease the risk for transmission of pathogenic bacteria from fruit surfaces to sliced fruits during processing [3]. Biocontrol based on bacteriophages or protective LAB cultures has also been proposed [41,42], although the efficacy of such treatments greatly depends on ecological factors such as phage specificity and inactivation, or the capacity to grow and produce antimicrobials in situ by the protective cultures. Microbial inactivation of E. coli and S. enterica in fruit juices can be enhanced with treatments based on various types of bacteriocin combinations (with organic acids, chelators, lysozyme, PEF, high-pressure homogenization, or HHP) [22,23,43,44,45]. These mild treatments allow a better preservation of nutrients and organoleptic properties of the juices compared to thermal treatments. In foods of animal origin such as meat and poultry products, bacteriocins have been tested for carcass decontamination by washing or spraying, with varying degrees of success [3]. Spray intervention with colicin E1 provides a strong reduction of E. coli O157:H7 on beef carcasses under refrigeration, although high bacteriocin concentrations are required [9]. In meat and poultry products, application of bacteriocins (like nisin, pediocin, or enterocins A, B and AS-48) in film coatings or in combination with HHP can reduce the growth and www.sciencedirect.com
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survival of Salmonella [19,30,32,46,47] and E. coli O157:H7 [16,19]. Application of bacteriophage mixtures dramatically reduces the levels of enterobacteria (such as Salmonella Typhimurium and Campylobacter jejuni) on meats [48]. Reducing the concentrations of enterobacteria in foods decreases the risks of human infection through food consumption. Nevertheless, incomplete inactivation of microbial populations may increase the risks for the selection of resistant or adapted strains. This phenomenon has not yet been studied in depth on Gram-negatives. Cronobacter (Enterobacter) sakazakii is an emerging pathogen of concern in infant formula. C. sakazakii can be inhibited under laboratory conditions by antimicrobial peptides (lactoferrin, nisin, and nisin combination with diacetyl) [49] but not in reconstituted infant formula [50]. Therefore, it is necessary to investigate other antimicrobial treatments against this bacterium in infant foods. In ethnic cereal foods, survival of the surrogate E. aerogenes as well as other Gram-negative bacteria decreases after fermentation with LAB strains producing BLIS [51]. Since much ethnic fermented gruel is consumed as infant weaning foods as well as by elderly people, the application of specific protective cultures to produce antimicrobial substances could improve the sanitary conditions of such fermented foods for these higher risk populations. Bioactive extracts from such BLIS-producing strains could also find applications as hurdles against transmission of pathogenic bacteria in several other kinds of infant formula.
Biocontrol of enteric pathogens in farm animals Livestock (such as pigs, cattle and poultry) are the main reservoirs of important food-borne pathogens such as E. coli, Salmonella, Shigella or Campylobacter strains. The application of antibiotics in animal breeding and in animal health results in training of pathogenic bacteria against antibiotics of clinical use, and infections caused by the resulting multi-resistant strains are increasingly difficult to treat. Several microbial antagonists (mostly colicins and microcins) have been proposed or marketed for application in livestock [52]. Administration of bacteriocins in feed before poultry slaughter appears to provide the control of C. jejuni to effectively reduce human exposure. Microencapsulated bacteriocin preparations from Paenibacillus polymyxa and Lactobacillus salivarius in chicken feed dramatically reduced both intestinal levels and frequency of colonization by campylobacters (C. jejuni, C. coli) in broiler chickens and in turkey poults [5,53,54]. Bacteriocin administration also had an impact on the gut morphology of turkey poults, reducing crypt depth and goblet cell density [54], although it is not clear whether this was a direct or indirect effect of treatment. The influence of www.sciencedirect.com
such alterations on colonization by Campylobacter as well as by other enteropathogens remains to be deciphered. Some enterocins produced by enterococci isolated from poultry also show promising results. Beneficial effects reported for the administration of enterocins E50-52 and E-760 in feed or water to broilers included a dramatic reduction of cecal levels of C. jejuni and Salmonella enteritidis, and reduction of the systemic dissemination of salmonellae in liver and spleen [7,8]. Although the total LAB content in the ceca of birds apparently was not affected by the administration of enterocins, the impact of bacteriocins on the global composition of the intestinal microbiota (including the LAB composition) needs to be further investigated. Since these enterocins also display strong antibacterial activity against other food-borne pathogens (Salmonella spp., Shigella spp., E. coli O157:H7 and Y. enterocolitica) they could probably be used to control pathogen colonization of poultry as well as other farm animals. Nevertheless, bacteriocin inactivation by proteases of the gastrointestinal tract, differences in strain sensitivity to bacteriocins, and the emergence of bacteriocin resistant or adapted strains need to be evaluated. For these reasons, administration of cocktails containing several bacteriocins should be recommended rather than single bacteriocins. It is also likely that bacteriocin treatments eliminate pathogenic bacteria from most but not all enteric locations. In order to prevent recolonization of the gut by the remaining survivors, bacteriocin challenges could be applied just before marketing.
Conclusions and perspectives Research on biocontrol of pathogenic bacteria through the food chain has provided a wide array of treatment options based on natural antimicrobials. In spite of the large number of laboratory studies carried out with different bacteriocins, there is still a long way to industrial applications, with little innovation with respect to classical commercial preparations such as nisin and pediocin PA-1/ AcH. Antimicrobial activity of bacteriocins can be modified by genetic engineering, and hybrid bacteriocin molecules are to be expected in the future. However, heterologous bacteriocin production and development of large-scale production processes are challenging. Ethnic foods are valuable sources for new bacteriocin-producing strains better adapted to their food substrates and maybe also with novel and interesting biotechnological properties. Several novel bacteriocins capable of inhibiting Gram-negative bacteria of concern in foods have been described recently, and conceivably this will open new possibilities for their application in food preservation. Some of them have been already tested with satisfactory results in the control of pathogenic bacteria in farm animals. However, the impact of bacteriocin-producing strains on the microbial ecology of the gastrointestinal tract and on animal health is far from being understood. Current Opinion in Biotechnology 2010, 21:142–148
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One major pending issue is extending the spectrum of bacteriocins to food-borne parasitic protozoa and viruses. These are of major concern in foods that are eaten without cooking or not cooked sufficiently. Remarkably, enteric viruses always rank in outstanding positions in statistics of food-borne illnesses. The antifungal activity of LAB has not been exploited satisfactorily, in spite of the great interest to control food spoilage caused by yeasts and filamentous fungi as well as mycotoxin production. Screening for novel antifungal LAB strains from less explored environments such as ethnic foods, together with genetic engineering approaches, may contribute to fill the existing gap in this field. Nevertheless, antifungal compounds may also have toxic effects on other eukaryotic cells, and may not meet the qualified presumption of safety (QPS) status attributed to bacteriocins and other antimicrobials produced by LAB.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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37. Gao YL, Ju XR: Exploiting the combined effects of high pressure and moderate heat with nisin on inactivation of Clostridium botulinum spores. J Microbiol Methods 2008, 72:20-28. Bacterial endospores are refractile to high-hydrostatic pressure (HHP) treatments. This work describes an interesting approach to inactivate endospores in foods processed by HHP. 38. Kroupitski Y, Golberg D, Belausov E, Pinto R, Swartzberg D, Granot D, Sela S: Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Appl Environ Microbiol 2009, 75:6076-6086. This work is an illustrative example on the microbial ecology of enteric pathogens outside their natural hosts and how they can interact with vegetables in order to survive and multiply. 39. McEvoy JL, Luo Y, Conway W, Zhou B, Feng H: Potential of Escherichia coli O157:H7 to grow on field-cored lettuce as impacted by postharvest storage time and temperature. Int J Food Microbiol 2009, 128:506-509. This interesting work illustrates how the changes in harvesting practices may have an impact on transmission of food-borne pathogens such as E. coli through fresh produce. 40. Cobo Molinos A, Abriouel H, Lucas Lo´pez R, Valdivia E, Bem Omar N, Ga´lvez A: Combined physico-chemical treatments based on enterocin AS-48 for inactivation of Gram-negative bacteria in soybean sprouts. Food Chemical Toxicol 2008, 46:2912-2921. The proposed decontamination treatments based on the cyclic bacteriocin in combination with other antimicrobials reduce the microbial load of Gram-negative bacteria on sprouts and afford protection against regrowth during refrigeration storage. 41. Abuladze T, Li M, Menetrez MY, Dean T, Senecal A, Sulakvelidze A: Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl Environ Microbiol 2008, 74:6230-6238. This work strengthens the potential of bacteriophages as biocontrol agents for decontamination against E. coli O157:H7, decreasing the risks for transmission of this pathogenic bacterium through different routes such as beef meat, fresh produce, and contact surfaces. 42. Trias R, Ban˜eras L, Badosa E, Montesinos E: Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria. Int J Food Microbiol 2008, 123:50-60. 43. Martı´nez-Viedma P, Sobrino A, Ben Omar N, Abriouel H, Lucas Lo´pez R, Valdivia E, Martı´n Belloso O, Ga´lvez A: Enhanced bactericidal effect of high-intensity pulsed-electric field treatment in combination with enterocin AS-48 against Salmonella enterica in apple juice. Int J Food Microbiol 2008, 128:244-249. The combined treatment of HIPEF and enterocin AS-48 increased the microbial inactivation of salmonellae in fruit juice, decreasing the risks for transmission of this pathogen through fresh fruit juices. 44. Buzrul S, Alpas H, Largeteau A, Demazeau G: Inactivation of Escherichia coli and Listeria innocua in kiwifruit and pineapple juices by high hydrostatic pressure. Int J Food Microbiol 2008, 124:275-278.
33. Sobrino-Lopez A, Viedma-Martı´nez P, Abriouel H, Valdivia E, Ga´lvez A, Martin-Belloso O: The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field. J Dairy Sci 2009, 92:2514-2523.
45. Ukuku DO, Zhang H, Huang L: Growth parameters of Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and aerobic mesophilic bacteria of apple cider amended with nisin-EDTA. Foodborne Pathog Dis 2009, 6:487-494.
34. Sparo M, Nun˜ez GG, Castro M, Calcagno ML, Garcı´a Allende MA, Ceci M, Najle R, Manghi M: Characteristics of an environmental strain. Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages. Food Microbiol 2008, 25:607-615.
46. Jofre´ A, Aymerich T, Garriga M: Assessment of the effectiveness of antimicrobial packaging combined with high pressure to control Salmonella sp. in cooked ham. Food Control 2008, 19:634-638.
35. Hampikyan H: Efficacy of nisin against Staphylococcus aureus in experimentally contaminated sucuk, a Turkish-type fermented sausage. J Food Prot 2009, 72:1739-1743. 36. Black EP, Linton M, McCall RD, Curran W, Fitzgerald GF, Kelly AL, Patterson MF: The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk. J Appl Microbiol 2008, 105:78-87. www.sciencedirect.com
47. Santiago-Silva P, Soares NFF, No´brega JE, Ju´nior MAW, Barbosa KBF, Volp ACP, Zerdas ERMA, Wu¨rlitzer NJ: Antimicrobial efficiency of film incorporated with pediocin W (ALTA 2351) on preservation of sliced ham. Food Control 2009, 20:85-89. 48. Bigwood T, Hudson JA, Billington C, Carey-Smith GV, Heinemann JA: Phage inactivation of foodborne pathogens on cooked and raw meat. Food Microbiol 2008, 25:400-406. Current Opinion in Biotechnology 2010, 21:142–148
148 Food biotechnology
This work exemplifies the use of bacteriophages in biocontrol strategies to reduce the microbial load of relevant food-borne pathogens (Salmonella Typhimurium and Campylobacter jejuni) on meat.
An excellent overview on application of bacteriocins and bacteriocinproducing strains in livestock for preharvest control of food-borne pathogens or as growth promoters.
49. Lee SY, Jin HH: Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii. Lett Appl Microbiol 2008, 47:315-321.
53. Stern NJ, Svetoch EA, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, Mitsevich EV, Mitsevich IP, Levchuk VP: Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. J Food Prot 2005, 68:1450-1453. The authors investigated paenibacilli as a novel source of antimicrobial substances for application in broiler chickens, and demonstrated the protective effects of microencapsulated bacteriocin against chicken colonization by campylobacters. Feeding bacteriocin before slaughter appears to effectively control campylobacters to reduce human exposure.
50. Al-Nabulsi AA, Osaili TM, Al-Holy MA, Shaker RR, Ayyash MM, Olaimat AN, Holley RA: Influence of desiccation on the sensitivity of Cronobacter spp. to lactoferrin or nisin in broth and powdered infant formula. Int J Food Microbiol 2009, 136:221-226. 51. Sa´nchez Valenzuela A, Dı´az Ruiz G, Ben Omar N, Abriouel H, Lucas Lo´pez R, Martı´nez Can˜amero M, Ortega E, Ga´lvez A: Inhibition of food poisoning and pathogenic bacteria by Lactobacillus plantarum strain 2.9 isolated from ben saalga, both in a culture medium and in food. Food Control 2008, 19:842-848. 52. Diez-Gonzalez F: Applications of bacteriocins in livestock. Curr Issues Intest Microbiol 2007, 8:15-23.
Current Opinion in Biotechnology 2010, 21:142–148
54. Cole K, Farnell MB, Donoghue AM, Stern NJ, Svetoch EA, Eruslanov BN, Volodina LI, Kovalev YN, Perelygin VV, Mitsevich EV et al.: Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults. Poult Sci 2006, 85:1570-1575. Dietary administration of bacteriocins (B602 from P. polymyxa and OR7 from L. salivarius) reduced the levels of C. coli (the most prevalent Campylobacter species in turkey) in turkey poults. This work also demonstrates for the first time the effect of bacteriocin ingestion on gut morphology.
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