SIGNIFICANCE OF BACTERIOPHAGES IN FOOD PRESERVATION
Food preservation is being practised from ancient era to increase its shelf-life and make it available at all the places. It can done using simple techniques like freezing, drying and fermentation etc. which are able to preserve the food but all of them suffer from one or the other limitation(s). Exploring the novel methods and technologies to ensure food safety are still under research considerations and one such method is the use of bacteriophages for the preservation of food items. Virus which is capable of infecting and killing bacteria is known as bacteriophage or can be simply termed as “phage”. They can be used as means to destroy foodborne bacteria and thus preventing food spoilage. Due to their host-specific nature, there is an increase in their usage. Bacteriophages were first used in food preparation for human consumption on August 18, 2006 with the approval of Food and Drug Administration (FDA).
All phages are composed of nucleic acid which is either DNA or RNA (genome) in nature, encapsulated by a protein coat referred to as capsid. Phage replicates in the cytoplasm of bacteria after injection of their genome. Bacteriophages may undergo lytic or lysogenic cycle. In lytic phage, bacterial cells are lysed and destroyed after immediate replication of the virion and new phage progenies are released, to infect new hosts. Whereas in lysogenic phage, viral genome get integrated with host DNA and replicate along with it. After many reproductive cycles it gets dissociated from the host genome and may undergo a lytic cycle. Bacteriophages which are to be used for food preservation should be lytic in nature not lysogenic.
Ecology of bacteriophages in food
Bacteriophages may be present on the surface of meat, fish and other foods, wherever the bacterial host is present. In addition, many fermented foods are also contaminated with bacteriophages, either from the environment or from the host bacteria themselves if these are lysogenic. Bacteriophages can be isolated from foods using their ability to lyse specific bacteria. Poultry and pig meat get contaminated extensively if conditions prior to and immediately after slaughter are not hygienic or if skin is retained on the carcass. There are various evidences that support the fact that bacteriophages are active against E. coli and Campylobacter, they are frequently isolated from poultry. The presence of bacteriophages in foods can be an indicative of a number of reasons, including,
(i) indicators of contamination with intestinal/faecal bacteria,
(ii) presence of spoilage bacteria and as a means to prevent them,
(iii) fermentative deterioration of certain foods, or
(iv) the recent interest reawakening in bacteriophages for control of bacterial food-borne pathogens
Bacteriophages application to control foodborne pathogens
Bacteriophages are ubiquitous in nature. They are unable to infect the human gastro-intestinal tract, consequently have a great potential for use as biocontrol agents to control unwanted bacteria in food. However, an oral treatment by phages cannot be employed due to various limitations such as:
- inactivation of phages due to the acidic conditions of stomach
- suitable number of phages are unable to reach to the gastrointestinal tract
- nonspecific binding of phages to food particles and other debris in the gastrointestinal tract.
Phages can be exploited to improve the quality in both pre-harvest and post-harvest foods of animal and plant origin. The evidences regarding the control of bacterial blotch (Pseudomonas tolaasii) in cultivated mushrooms, control of Salmonella in experimentally infected broccoli and mustard seeds are well documented. In a number of diverse species like fish, cattle, pigs, lambs and chicken etc. under experimental conditions phages have been evaluated to control pathogens. Phage efficacy is enhanced when mixture of phages with complementary activities is administered. Post-harvest control of pathogens has been successful with the use of phages under a variety of environmental conditions. A mixture of four different phages of Salmonella Enteriditis can reduce the bacterial numbers during storage under refrigeration. Further phages can control spoilage of raw skim milk and major foodborne pathogens of animal origin i.e. E. coli, Campylobacter, Salmonella and Listeria. These bacteria are common contaminants of ruminants, poultry, and swine and are usually carried asymptomatically in their gastrointestinal tract.
Qualities of bacteriophages which favour their usage for control of foodborne bacteria include their self-perpetuating nature, selective modification of bacterial flora, stability in foods, ability to survive processing, ubiquitous presence, easy isolation, cost-effectiveness, ease of preparation and application, non toxic nature to eukaryotic cells and no effect on food quality. Disadvantages for developing a bacteriophage approach to the control of foodborne bacteria comprises of limited host range, phage-resistant bacterial mutants, food environments barriers, transduction of undesirable characteristics, antigenicity (immune response, allergenicity), consumer perception of adding viruses to foods.
Factors affecting bacteriophages survival in foods
During storage there may occur degradation of some bacteriophages which makes it difficult for them to survive intact independently of their host bacterium. So, the need for the study of all individual bacteriophages in all aspects of their properties arises. The survival of Listeria bacteriophages appeared to be very stable in most of the foods of animal origin even during storage but not in plant origin foods. Sometimes, even though bacteriophages are not inactivated, but can’t show their activity due to their immobilization in solid foods and therefore became inactive by limited diffusion. To determine the bacteriophage survival in foods so that they would remain protective during processing and prevent re-contamination, some of the below mentioned factors need consideration:
- pH – Mostly bacteriophages are stable at pH 5-8
- Temperature – Thermotolerance of bacteriophages is in relation with the environment or host system from which they are originated. Bacteriophages found in cheese and yoghurt tends to be highly thermotolerant, whereas those from psychrotrophic bacteria are less thermotolerant. Bacteriophages can survive the pasteurisation process, this being bacteriophage strain dependent. Bacteriophages are found to be more thermotolerant than the host bacteria which indicates their survivability even after the destruction of the host bacterium. Bacteriophage activity is only evident when the environmental and nutritional conditions are favourable for growth of the host. At refrigeration temperatures growth rates of enteric pathogens is lowered and the length of the bacteriophage infection cycle, is increased. However, psychrotrophic bacteriophages can multiply on their hosts at 1o
- Light – Bacteriophages are inactivated exponentially by ultra violet light at variable rates which is probably the reason for inactivation by sunlight in water. Such DNA damage can be repaired after infection by bacterial DNA repair mechanisms.
- Osmotic shock and pressure – Bacteriophages under osmotic shock produce ghost particles, in which the DNA has been lost.
- Disinfectants and other chemicals – A wide number of antiseptic chemicals can inactivate bacteriophage particles rapidly, some of them include periacetic acid, ethanol and sodium hypochlorite. Bacteriophages are more resistant than bacteria to inactivation by chemical and physical stresses. A large number of coliphages are resistant to chlorine. As bacteriophages are more resistant than coli to waste water treatment they become persistent in processing plants. So, the disinfection regimens may need to be developed to monitor efficacy of their application in the food industry.
- Miscellaneous factors – Information on the effects of fermentation, freeze-drying or irradiation on bacteriophage stability is scarce. A proportion of bacteriophages survive in fermented sausage. Freeze-drying is known to reduce titres initially but lower titres persistence has been reported for many weeks. The food matrix can have an important protective effect on bacteriophages.
Effective roles played by bacteriophages in treatment of some of the commonly encountered bacteria –
- Bacteriophage treatment of Campylobacter
Campylobacter is the most common cause of foodborne disease. Two species of major concern of this genus are Campylobacter jejuni and Campylobacter coli. The widespread infection of campylobacteriosisis can occur because of ingestion of low doses (400–500 cells). The number of reports available on Campylobacter phages is relatively few probably due to the fastidious growth conditions of their host and also due to unique features that phages exhibit. Appreciable numbers of reports regarding the phage biocontrol against Campylobacter are present for poultry but only few reports are available on infected livestock. Overall, studies suggest that high multiplicity of infection value is more effective in the control of Campylobacter in foods. When reach to the human intestine, bacteria increases its metabolic activity and phages eventually get attach and lyse the target bacteria, leading to its control.
- Bacteriophage treatment of Salmonella
After Campylobacter, Salmonella is the second most common cause of foodborne illnesses distributed ubiquitously. It has a broad host range, high zoonotic value and high physiological adaptability. The major serovars involved are S. Typhimurium and S. Enteritidis. Majority of Salmonella outbreaks are reported due to consumption of contaminated eggs, and poultry and pig meats. A number of phages infecting Salmonella have been reported, till now the best known Salmonella phages are the lytic phage Felix 01 and the temperate virus P22. Felix 01 is known for its broad lytic spectrum among Salmonella and has been used in its identification. Recently, one new phage with a broader host range known as Salmonella phage PVP-SE1 has been described which presents great potential not only as a therapeutic agent but also as a diagnostic tool. Several studies on the use of phages to control Salmonella have been elicited. Among all the models studied so far poultry represents the most common model. Two phage products which are available:
(1) BacWash from OmniLytics Inc. which received, USDA Food Safety and Inspection Services approval in 2007 for commercialization and is applied as a mist, spray, or wash on live animals prior to slaughter.
(2) BIOTECTOR S1 phage product from CheilJedang Corporation that is to be applied on animal feed to control Salmonella in poultry.
- Bacteriophage treatment of E. coli.
Most of the Escherichia coli strains are harmless, but some serotypes can cause serious food poisoning. Fecal–oral transmission is the prime route through which pathogenic E. coli strains can cause disease. E. coli phage treatment aims to decrease contamination prior to slaughtering via applying or mixing directly onto or into the food product. The phage’s host range should also be ascertained in certain cases, for example with coliphage IMM-001, specifically infects enterotoxigenic E. coli strains, thereby eliminating chances of any negative impact on the intestinal microflora of the consumer if it were to be applied. Different approaches have been used but the best results were obtained by aerosol spraying and intramuscular (i.m.) injection and significant reduction in the mortality of broiler chicken was observed.
- Bacteriophage treatment of Listeria
Genus Listeria of facultative intracellular bacterium consists of 10 species, of which the most important pathogen of human importance is Listeria monocytogenes. It can grow in temperature from 4 °C i.e. refrigeration temperature to 37 °C i.e. body temperature. Listeria monocytogenes is often found in soil, stream water, sewage, which may result in vegetable contamination. Listeria phage studies were carried out employing a combination of phage and nisin (a broad spectrum antibacterial peptide). This strategy had a synergistic effect once added to melon and apple leading to reduction of Listeria compared to phage or nisin alone but not in case of ground beef. Hence, phage biocontrol should be optimized separately for every food matrices under study. The U.S. Food and Drug Administration (USFDA) in 2006 approved ListShieldTM, the LMP-102 phage preparation from Intralytix for the control of L. monocytogenes on ready-to-eat (RTE) foods. Besides this, anti-listerial agent, bacteriophage ListexP100 (phage P100), has been also approved by the USFDA for L. monocytogenes control in meat and cheese products.
- Bacteriophage treatment of Staphylococcus
Staphylococcus is a major etiologic agent in opportunistic and nosocomial infections. Toxins produced by Staphylococcus in the food are the main reason of food poisoning. Staphylococcus aureus bacteriophage K was evaluated to eliminate Staphylococcus infection in bovine mammary gland during lactation, but there was significant degradation or inactivation of the infused phage within the gland. This led to the conclusion that phage does not cause an irritation to the animal. In pasteurized milk, a combination of phage treatments with nisin and high hydrostatic pressure could synergistically be used to reduce staphylococcus when compared to each treatment alone. Care should be taken during this treatment, as nisin adapted strains can seriously compromise phage activity. S. aureus inactivation in cheese manufacturing has been accomplished in both fresh and hard-type cheese utilising a phage cocktail.
CONCLUSIONS
Bacteriophages are bacterial viruses that are omnipresent in nature. Nearly for every bacterial species, there exists one bacteriophage that can specifically infect and eventually destroy that particular bacterial group. They do not harm human or animal cells. With the above mentioned characteristics, bacteriophages can prove to be valuable tools in fight against human diseases and can be an alternative to traditional antimicrobials use in the control of foodborne pathogens. However, due care should be taken if this new technology is to be effectively and safely employed. Complete phage genome analysis is required to ensure safety and effectiveness of use of phages. Lastly, food industry acceptance and consumer preference are critical hurdles needed to be overcome for their commercial application. As such, bacteriophages are considered ideal antibacterial agents for use in foods to enhance the food safety.
Mamta Pandey*, Research Associate, Molecular Reproduction Lab, Animal Biotechnology Center, ICAR-NDRI, Karnal, Haryana, India drmamtavet@gmail.com
Neeraj, M.V.Sc., Scholar, Animal Reproduction Gynaecology and Obstetrics (LPM Section), ICAR-NDRI, Karnal, Haryana, India
