High-Technology Approaches to Microbial Safety ... - ACS Publications

Chapter 23

High-Technology Approaches to Microbial Safety in Foods with Extended Shelf Life Myron Solberg

Downloaded by AUBURN UNIV on December 26, 2017 | http://pubs.acs.org Publication Date: February 14, 1992 | doi: 10.1021/bk-1992-0484.ch023

Center for Advanced Food Technology, New Jersey Agricultural Experiment Station, Rutgers, The State University, New Brunswick, NJ 08903

The target for food processors and consumers that remains off in the distance is the shelf stable "chef-like" and "fresh-like" food which is ready to heat and eat. The interim goal is the extended shelf-life refrigerated product which, after reheating, will have quality attributes equivalent to food freshly prepared by a master chef. The first steps toward the identified objectives are manifest in supermarket and food service operations today. The supermarkets of England and Finland have far more space dedicated to refrigerated modified atmosphere packaged precooked entrees than to frozen foods. In the U S A , the refrigerated shelf space is increasing as precooked items, in many cases prepared within the store but in some situations brought into the store, continue to fill the demand for quick to prepare and "fresh" which is important to the two income with or without children families. The food service sector is now introducing the sous-vide entree. This precooked vacuum packaged refrigerated entree has had a few years of success in France and has now entered into the U.S. menu. At least one manufacturer has elected to distribute the product frozen in the U.S. The prerequisite for any food item is safety. Chemical and microbial considerations cannot be subject to compromise. Safety has traditionally been assured by sacrificing aesthetic quality. Military commanders have traditionally preferred a battle ready soldier griping about the overcooked food to one who is incapacitated, albeit temporarily, due to microbially induced food-borne illness. A similar approach is the " 12D" thermal process concept which yields overcooked but safe shelf stable canned, jarred or pouched food. Freezing of foods may also be looked upon as overprocessing. Concern for safety takes precedence over quality, which is often seriously affected by the thawing process. Transfer of the " 12D" thermal process concept to sterilization of food by ionizing irradiation is another overprocessing treatment which limits applications due to quality and has perpetrated a host of health related concerns. Physical approaches 0097-6156/92/0484-0243$06.00/0 © 1992 American Chemical Society Finley et al.; Food Safety Assessment ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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have been used to improve heat transfer through conversion of the package from a round "tin" can to a flat tray or pouch. The increased surface area tends to even out the process and reduce the overprocessing. The product quality is improved but remains overprocessed in the name of microbial safety. There is a striving toward the aseptic processing of particulate containing food systems. This process permits efficient heat transfer in small diameter or thickness heat exchangers followed by aseptic transfer of the commercially sterile food into presterilized containers. This system has the potential to reduce the overprocessing to a greater extent than the reconfigured packages described previously, but the over riding microbial safety need again demands overprocessing. Still another approach is the seldom used direct steam injection under pressure aseptic systems which overcome the problems of particulate flow systems since the product is in a confined space, eliminating the variable flow rate and therefore heat transfer concerns. In these systems, vacuum cooling prior to sealing the container removed the steam condensate which was added to the container during the direct steam injection heating. This seemingly most effective treatment system still produces highly overprocessed food relative to the amount of cooking which would take place in home, restaurant or food service establishment preparation. The overprocessing is demanded in the name of microbial safety. Attempts to reach the interim goal of extended shelf-life refrigerated products have relied upon minimal processing approaches. Thermal treatment is almost always the basis underlying these products. Some use of the hurdle concept (1) approach has been implemented. Lowered pH, incorporation of water immobilizing ingredients, and antimicrobial spices and condiments contribute to the progress toward the interim goal. A l l of the approaches mentioned thus far, may be considered as "sledgehammer" in nature. They consist of processes or systems which are interactive with every molecule of the food system and therefore result in considerable change from the fresh unprocessed food. The degree of change is often far greater than that which would be effected by home or food service establishment food preparation. There is a need to define the problem before solutions may be proposed. The microbial problems are similar for both the ultimate goal products which are shelf stable, "chef-like" and "fresh-like" and the interim goal products which are refrigerated, extended shelf-life, "chef-like" and "fresh-like." The primary target organism in all case is Clostridium botulinum. The objective is to prevent outgrowth and toxin production. It is now clear that the non-proteolytic C. botulinum types Β and Ε may grow and produce toxin at temperatures just slightly above 3°C and that time in the growth supporting temperature range tends to be cumulative (2). It is also clear that the potential for growth in an organic substrate of this strictly anaerobic organism in the presence of oxygen is possible due to the existence of anaerobic microenvironments within the system (3). Among the secondary target organisms are two pathogens capable of growth at refrigerator temperatures. These are Yersinia enterocolitica and Listeria monocytogenes. Among the room temperature growing mesophilic pathogens are the familiar Salmonella species, Staphylococcus aureus, Clostridium perfringens, and the

Finley et al.; Food Safety Assessment ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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less well known Bacillus cereus and Campylobacter jejeuni. The discouraging truth is that there are pathogens of concern in both the refrigerated and the shelf stable products. There are also spoilage causing microorganisms which cause quality losses and need to be controlled if success is to be achieved. The quality losses of concern include texture, color, flavor and nutritional value. The quality losses described are attributable to chemical changes within the food system. The rates at which the chemical reactions occur can be increased by the presence of active enzymes. Intrinsic properties of the food such as acidity, moisture, salt and free radical moderators as well as extrinsic factors in the food environment, such as temperature, gaseous environment, light, and humidity; can regulate the chemical reactions. The problem may be reduced to preventing microbial growth, enzymatic activity and chemical reaction, including changes of physical state within a food system after it has reached an optimum state of quality which may be described as "chef-like" and "fresh-like." The key question relative to microbial safety is, "why do we need to overprocess the food?" The answer is that since we have no way of knowing how many of what type of microorganisms are present, we are unable to design a system with a precise effect. The treatments used have excessive safety factors built into them so as to err on the side of caution. The result is highly overprocessed food. There are two approaches which appear obvious and probably many more which are obscure. The first approach is to devise, design and develop a non-destructive, non-invasive system for on-line identification and enumeration of microorganisms. Such information could be fed to a computer which would determine the minimum safe treatmenttimeand choice of end products for which such treatment could be used with the expectation of quality required. The computer integrated manufacturing system would determine which of the suitable products was needed for inventory and would drive the production toward the product. The second approach is "magic bullet" based. The objective is to attack the microorganisms without affecting the food. Energy, specifically targeted to a relatively unique and critical site of an enzyme key to all microbial life, would be a possibility. A substance which could be added or produced from a precursor in the product which would specifically interact with microbial membranes to prevent transport of key components through the membranes thus terminating cell viability (3) is another possibility. Other similar cell mobility disrupting scenarios can be imagined. Let us examine the present state of the art with respect to detecting microorganisms so as to estimate the time frame for real time identification and enumeration. There is no need to discuss the traditional selective media approaches which require one or more days. The furthest advanced methods emanating from the new biotechnology are those which depend upon monoclonal or polyclonal antibodies or upon D N A hybridization. These identification devices are linked to detectors which may be enzymes, fluors, or radioisotopes. The challenge to which these systems have been directed is specific organism detection by genus or specie. The developed techniques do this task well but are lacking in sensitivity. Thus, there is a need for large concentrations of the specific microorganism to be present for detection to be effected.



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The time to detection is short, generally a few hours at most; the time to reach a detectable level is as long as several days i f there are only a few organisms present initially. If the antibody or hybridization techniques are to be useful in defining process requirements, they will need to be made very sensitive, quantitative and capable of broad as well as specific interaction with microorganisms. One scenario might be a cocktail of antibodies with differing detectors which would simultaneously indicate the presence of a variety of microorganisms. There is a potential biotechnology approach using both the antibody and hybridization techniques. Antibodies could be used to "fish" out specific cells from a production slip stream. The D N A of the cells could be amplified quickly using a polymerase chain reaction (PCR) (4). An enzyme linked D N A probe could then be used for identification. Such a system would identify a microorganism in 3 or 4 hours from the capture of a single cell. Problems that remain include quantification and the inability to determine whether the cell originally recovered was dead or alive at the time of capture. An approach to quantification may be possible by fixing the number of available capture sites, controlling the D N A amplification rigorously and measuring relative enzyme catalyzed response quantitatively. Every living cell and only living cells contain adenosine triphosphate (ATP) which when released from cells can be reacted with the enzyme luciferase to produce luminescence. The relatively new technique of photon counting imaging in combination with an optical microscope is able to directly visualize bioluminescence in a single cell (5). If such a system could be integrated with the antibody capturerPCR D N A amplification system previously described it would indicate whether the captured cell was dead or alive at the time that its D N A was released for initiation of the PCR process. Another biotechnology based microorganism identification and enumeration scheme utilizes bacteriophages which are bacteria specific virus like systems. The lux gene which causes bioluminescence can be inserted into the genome of a bacteriophage (6). When this phage infects a bacteria it becomes amplified and utilizes the bacterial ATP to make the bacteria luminescent. This event occurs within 30-50 minutes and the light emission intensity is directly related to the number of bacterial cells available for infection. Standard bioluminescence measuring technology is capable of detecting emissions representing a few hundred cells (5) and improved reagent systems can detect ATP from as few as 10 cells (7). The possibility of combining this phage system to the previously described photon counting imaging microscope system could increase the sensitivity dramatically. Although all of the described systems are existent, there is considerable research and development needed before any one of them will be ready for commercial application within a broad range of food products. It does seem reasonable to believe that the 1990's decade will see some of these advanced methods in use. Predictive mathematical models of microbial growth and survival in foods could permit quality, shelf life and stability judgments to be made (8). Such predictions are critical as a processor moves toward perceived freshness via minimal processing or to reduced salt or preservative formulations to make foods which will be perceived as more healthful. This approach could be applied on an interim basis through two



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systems. The first would be dependent upon real time detection and enumeration of microorganisms followed by predictive modelling of product life. A second approach, one which is closest to application, would be to assure conditions within the product through ingredient and environment control which would insure, beyond any doubt, the prevention of growth or toxin production by the more difficult to destroy organisms. These would include the sporeforming bacilli and Clostridia. Processing could then be done on a traditional probability basis to reduce the more susceptible organisms to an essentially zero level without significant loss in "chef-like" quality. Predictive mathematical modelling is dependent upon experimental definition of combined effects of various factors affecting microbial growth and survival. Traditionally such data collection has been very difficult and time consuming involving standard microbial methods of incubation and plate counting or turbidity measurements when clear broths are used. Recent development of automated systems such as the Lab Systems Bioscreen machine, which can examine up to 200 cultures simultaneously using optical density for measurement, has simplified the model medium part of the data collection. The establishment of a more useful data base appears feasible. The combination of the data base with high speed computerized curve fitting (9) will permit some application of predictive modelling in the near future. Next, let us consider the state of know-how relative to "magic bullet" approaches to selective inhibition of microorganisms and chemical reactions. Availability of high-power pulsed lasers with output frequencies in the ultraviolet region as well as broadly tunable infrared color center lasers provide a range of high intensity stable energy sources which may interact uniquely with proteins and thus control enzyme activity and microbial viability (10). Although still far from economically feasible, even tunable monowavelength X-ray lasers are available and offer opportunities to exert closer control over irradiation processes by conferring some specificity of interaction (77). These outgrowths of the "Star Wars" program yield an opportunity for specific targeting of finely tuned energy which may result in excitation of molecular regions which could inactivate enzymes and microorganisms. The end result is not completely dependent upon the total energy input but may be in part dependent upon the vibrational mode established by the laser. The approach is to utilize ultra short pulses of high energy so that molecules are made to vibrate with ensuing bond disruptions prior to the energy being dissipated as heat (72). Successes to date are limited but the concept is intriguing. The availability of high pressure hydrostatic based process equipment with working volumes as large as 1 liter, for use in the cold isostatic pressing of ceramic powders, opens possibilities in food processing (13). High pressure unfolds proteins and disrupts membrane structure. These two events, occurring simultaneously result in inactivation of enzymes and loss of viability in microorganisms. The question remaining is in specificity. Is the pressure required for enzyme inactivation one at which structural protein will be left intact? Is the pressure which disrupts the microbial membrane one which will also disrupt plant or animal cells? The Japanese formed a Research and Development Association for High Pressure Technology in the Food Industry within the past year (13). Answers to many questions should emanate from this four year program which is funded with $1.0 million in its first year.

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Finley et al.; Food D.C. Safety 2 Assessment Washington, 0036 ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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An approach of high interest is through molecular biology and biotechnology. The ability to transfer genes into plants and animals and have them express anti-microbial substances which become part of a food system is an exciting prospect. The elements of such a scenario could include a substance like chitosan, a deacetylation product of chitin. Chitosan is a polycation which probably affects the permeability of cell membranes. Other substances which would fit the scenario are the bacteriocins. These are substances produced by microorganisms which specifically inhibit other micro organisms. Some bacteriocins are active against C. botulinum, the primary target microorganism previously mentioned. Other bacteriocins are active against L. monocytogenes, a low temperature growing pathogen which was previously cited among the secondary target microorganisms. It is possible that a unique bacteriocin exists which would be active against both of these bacteria and possibly others as well. The present day approach is to add the antimicrobial substance or to have the producing bacteria grow in the food system to produce it. The goal is to identify the genetic components responsible for production of the antimicrobial, whether it be bacteriocin or chitosan or some other substance. Insert those components into the animal or plant so that the substance is produced at the proper time and in the proper amount. The result would be the elimination of the primary and secondary threats thus permitting minimal processing and extended storage without microbiological concerns for safety. The potential for utilizing some of the presented approaches in combination could yield the desired results. The combined effects of "magic bullet" control and on-line real time detection would provide a potentially ideal system leading to a fulfillment of our desires. These desires include not only wholesomeness, healthfulness and freshness but also life-extension or adding years to our existence, satisfaction and pleasure or enjoyment in our eating. The additional factors of timeliness representing quick to prepare, availability all through the year and variety to satisfy our whims at any moment round out the list of desires represented by the terms "chef-like" and "fresh-like." Acknowledgment This is publication No. D10535-1-91 of the New Jersey Agricultural Experiment Station supported by State Funds and the Center for Advanced Food Technology (CAFT). The Center for Advanced Food Technology is a New Jersey Commission on Science and Technology Center. Literature Cited 1. 2. 3. 4. 5.

Scott, V . N . J. Food Protec. 1989, 52, 431-5. Post, L.S.; Lee, D. Α.; Solberg,M.;Furgang, D.; Specchio,J.;Graham, C.J. Food Sci. 1985, 50, 990-6. Knorr, D.; Popper, L.; Kuhne, K.; Boguslawki, S. Institute of Food Technolo gists Annual Meeting Abstracts. 1990, p. 241. Van Brunt, J. Biotechnol. 1990, 8, 291-4. Stewart, G.S.A.B. Lett. Appl. Microbiol. 1990, 10, 1-8.


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Ulitzur, S.; Kuhn, J. In Bioluminescence and chemiluminescence New Perspec tives; Schlomerich, J.; Ed.; Wiley: New York, 1987, pp. 463-73. Simpson, W.J.; Fernandez, J.L.; Hammond, J.R.M.; Senior, P.S.; McCarthy, B.J.; Jago, P.H.; Sidorowicz, S.; Jassim, S.A.A.; Donyers, S.P. Lett. Appl. Microbiol. 1990, 11, 208-10. Gould, G. Food Sci. Technol. Today. 1989, 3(2), 89-92. Buchanan, R.L.; Phillips, J.G. J. Food Protec. 1990, 53, 370-6. Grygon, C.Α.; Perno, J.R. ; Fodor, S.P.A.; Spiro, T.G. Biotechniques. 1988, 6(1), 50-5. Kim, K-J.; Sessler, A . Science. 1990, 250, 88-93. Rotman, D. Industrial Chemist. 1987, Aug., 32-5. Farr, D . Trends in Food Sci. and Technol. 1990, 1, 14-6.


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High-Technology Approaches to Microbial Safety ... - ACS Publications

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