Microbial Update International
December 2008
VOL 14, IS 4
Microbial Update International (ISSN 1082-9296) is published every other month. © 2008 by Food Technology Intelligence, Inc., 215 Godwin Ave., P.O. Box 322, Midland Park, NJ 07432-0322 USA; phone 201-445-4227; fax 201-447-5904; email ftiinfo@ftipub.com. Visit our Web site at www.ftipub.com. Fee: one year, $250; two years, $400. Add $20 per year for service outside North America.
TABLE OF CONTENTS (Click on title to go to story)
Directly assess toxic behavior of contaminating bacteria
Use cranberry concentrate to control E. coli O157:H7 in ground beef
Mammalian cells speed pathogen detection
Analyze storage stability, antibacterial activity of the plant antimicrobial carvacrol
Correlate C. jejuni to the amount of biofilm present
Prolong the shelf life of bratwurst sausages using high hydrostatic pressure
Validate processes for fermented food
Eureka status awarded for rapid multiplex pathogen test
In this issue… Consider a new technique for detecting bacteria, based on the color changes in pigment-bearing cells from Siamese fighting fish. It is more directly related to toxicity assessment than conventional approaches are. It may be possible to use cranberry concentrate in hamburgers to ward off bacteria. A cell-based sensing technique simultaneously screens thousands of samples of food or water for several foodborne pathogens in less than two hours.
Directly assess toxic behavior of contaminating bacteria
Part of the problem with conventional food safety testing done with DNA-based tests or antibody-based methods is that they only indicate the presence of specific bacteria, and do not necessarily describe toxicity and the potential to cause harm.
Microbiologists at Oregon State University have developed a new technique for detecting bacteria, made possible by fundamental research on the color changes in pigment-bearing cells from Siamese fighting fish. It should be easier to use, faster—from a few minutes to hours for detection—and more directly related to toxicity assessment than conventional approaches. Rapid methods are not readily available that directly assess the toxicity of bacterial contamination in a user-friendly fashion. The technology has been patented and is available for licensing, although further studies are needed.
Existing tests only work to detect bacteria that have already been characterized, based on a specific sequence of DNA or type of protein they produce. Such tests can’t tell whether the contaminating bacteria are alive or dead. They can’t directly assess their toxic potential and sometimes don’t detect newly emerging or genetically rearranged strains as bacteria mutate.
The new approach, by contrast, is built on the unusual characteristics of certain chromatophore or pigment-bearing cells, called erythrophores, from Siamese fighting fish. When Siamese fighting fish encounter certain stressful or threatening environmental conditions, such as exposure to toxic chemicals, the erythrophores change appearance. Their pigment moves in a characteristic pattern to an internal part of the cell. The change in pigment location can be numerically described.
One stressful situation which causes the location of pigment to change is the toxic threat posed by illness-causing bacteria. The pigment-bearing cells respond immediately to certain food-associated toxin-producing bacteria. There is potential to directly assess the toxic behavior of the contaminating bacteria, not just the simple presence of the DNA or protein of these bacteria. This technology can detect such important food-associated bacteria as Salmonella, C. perfringens, B. cereus and C. botulinum.
Further studies are needed to define the pigment-bearing cell response to other important bacteria of concern, such as E. coli O157:H7 and Listeria. Research is also needed to immortalize a pigment-bearing cell line for mass production and commercial use. Portable kits could be developed that would not require specialized training to use. Results would be available in minutes and would allow food processors, distributors, handlers or even consumers to quickly assess food for contaminating bacterial toxicity.
Patent. 6,913,877—Methods for detecting bioactive compounds. Issued July 5, 2005. Inventors: Frank Chaplen, et al. Assigned to the State of Oregon.
Further information. Janine Trempy, Department of Microbiology, Oregon State University, 250 Nash Hall, Corvallis, OR 97331; phone: 541-737-4441; fax: 541-737-0496; email: trempyj@orst.edu.
Use cranberry concentrate to control E. coli O157:H7 in ground beef
A major source of infection by E. coli O157:H7 is undercooked ground beef. Other sources include unpasteurized milk and juice, raw sprouts, lettuce and salami, as well as contact with infected live animals.
Scientists at the University of Maine investigated the antimicrobial effects of cranberry concentrate on E. coli O157:H7 in ground beef, and then examined the sensory properties of burgers to which the concentrate had been added. Considering the antimicrobial effects and other health benefits, burgers with cranberry concentrate may be a potentially safe, healthy and popular product preferred by consumers.
In experiments, researchers inoculated samples of ground beef with E. coli O157:H7 at approximately 6 logs CFU per g. These were combined with cranberry concentrate at 0%, 2.5%, 5% and 7.5% w/w, then stored at 7 C for five days.
E. coli O157:H7 and total aerobic bacteria were detected on days 0, 1, 3 and 5. A nine-point hedonic test was performed. Fifty panelists evaluated the appearance, flavor, texture and overall acceptability of burgers to which cranberry concentrate had been added at various levels. Compared to the control beef samples, cranberry concentrate significantly reduced both total aerobic bacteria and E. coli O157:H7 after three days.
On the fifth day of testing, cranberry concentrate at 2.5%, 5% and 7.5% levels reduced total aerobic bacteria by 1.5 logs, 2.1 logs and 2.7 logs CFU per g, respectively, when compared to the control. During the same period, levels of E. coli O157:H7 were reduced by 0.4 log, 0.7 log and 2.4 logs CFU per g, respectively.
It appears that storage time and the amount of cranberry concentrate in the beef had a synergistic effect on inhibiting both total aerobic bacteria and E. coli O157:H7. Sensory evaluation found no differences among burgers with 0%, 2.5% and 5% levels of cranberry concentrate in terms of appearance, flavor and taste. Burgers with both 2.5% and 5% of concentrate did well in terms of overall acceptability.
Further information. Vivian Chi-Hua Wu, Department of Food Science and Human Nutrition, 5735 Hitchner Hall, Room 106, University of Maine, Orono, ME 04469; phone: 207-581-3101; fax: 207-581-1636; email: vivian.wu@umit.maine.edu.
Mammalian cells speed pathogen detection
Researchers at Purdue University have developed a cell-based sensing technique that can simultaneously screen thousands of samples of food or water for several foodborne pathogens in one to two hours. The technique also can estimate the amount of microbes present and determine whether they pose an active health risk.
The technology utilizes live mammalian cells that release a measurable amount of a signaling chemical when harmed. Optics and software analyze this quantity to estimate the amount of harmful microbes present. The technology can recognize very small amounts of L. monocytogenes—about 105 cells. It also recognizes several species of Bacillus. Investigators found a way to immobilize cells and were able to simultaneously perform multiple tests on a large number of samples. The technology is available for licensing and is the subject of a patent application.
By using live cells, or biosensors, this approach can identify actively harmful pathogens but ignore those that are inactive or harmless.
Cells are suspended in collagen gel and placed into small wells within multi-well plates. Each well can test one sample, so tests can be expanded to quickly analyze as many samples as desired. Researchers indicate that the sensor also could help optimize processes to kill harmful microbes or deactivate toxins.
The multi-well plates and their contents of gel-suspended mammalian cells could be efficiently prepared in a central location. When desired, the plates could then be shipped to the test location, a processing plant, so that analysis can take place on-site.
This technology tests for bacteria and toxins that attack cell membranes. For this reason, researchers employed cells with high amounts of alkaline phosphatase, the signaling chemical released upon damage to the cell membrane. Researchers could conceivably employ other types of cells to detect additional pathogens. Samples of food and water are added to biosensor wells before being incubated for one to two hours. To each well a chemical is added that reacts with the biosensor’s alkaline phosphatase, yielding a yellow product quantified by a special camera and software. A precise calculation may not always be necessary.
Researchers are trying to get these cells to live within the gel beyond four to six days. But this time span could be expanded to two weeks, the shelf life necessary for the technique to have commercial value.
Further information. Arun Bhunia, Department of Food Science, Purdue University, 1160 Food Science Building, West Lafayette, IN 47907; phone: 765-494-5443; fax: 765-494-7953; email: bhunia@purdue.edu.
Analyze storage stability, antibacterial activity of the plant antimicrobial carvacrol
We all know of the problems and concerns caused by E. coli O157:H7. So it appears there’s a need to discover new, food-compatible ways to protect foods against this bacteria and other pathogens.
Edible films made from fruits or vegetables containing plant essential oils have the potential to protect food against contamination by bacteria and fungi. USDA-ARS scientists wanted to evaluate the antimicrobial activity as well as the storage stability and physical properties of apple-based films containing carvacrol, the main component of oregano oil.
Carvacrol’s antimicrobial activity against E. coli O157:H7 and stability were evaluated during the preparation and storage of apple-based films made by two different casting methods: continuous casting and batch casting. After storing the films at 5 C and 25 C, the researchers performed physico-chemical and antimicrobial assays on the films. They also used high-performance liquid chromatography to analyze the film extracts.
The investigators obtained optimum antimicrobial benefits when they added carvacrol at about 1% content to the purees before film preparation. The carvacrol in the films and the weight of the films remained unchanged over a storage period of up to seven weeks. The researchers found that the casting methods used affected the carvacrol concentration, bactericidal activity and the physical properties of the films. Also, adding carvacrol to the purees used to prepare the films reduced the water vapor and oxygen permeability of the films.
It appears that adding carvacrol has a dual benefit: it can be used to both impart antimicrobial activities and to enhance the barrier properties of edible films. This research facilitates relating compositional and physico-chemical properties of apple films containing volatile plant antimicrobials to their use in foods.
Further information. Wen-Xian Du, USDA-ARS Western Regional Research Center, Processed Foods Research, Room 1105, 800 Buchanan St., Albany, CA 94710; phone: 510-559-6148; fax: 510-559-5818; email:
wen-xian.du@ars.usda.gov.
Correlate C. jejuni to the amount of biofilm present
C. jejuni is found in chickens and is the leading cause of foodborne bacterial diarrhea in the United States. Poultry producers must look for ways to control this pathogen before their birds are processed. The good news is that the bacterium is susceptible to stress and is vulnerable.
But how does it continue to function? Apparently, the bacterium latches onto other colonies of bacteria—biofilms—and uses them as places to thrive in ways the C. jejuni would be less likely to do on their own. These biofilms are complex structures that adhere to surfaces and consist of colonies of bacteria.
Scientists at the University of Arkansas indicate that the capture of C. jejuni can be correlated to the amount of biofilm present. C. jejuni can be captured and harbored by a biofilm regardless of its bacterial constituents. So, most biofilms should be considered as having the potential to promote and harbor C. jejuni. This makes controlling biofilms critical.
Being in a biofilm is an advantage to bacteria. The biofilm provides protection from antibiotics and other threats to the bacteria’s existence. C. jejuni has had a major disadvantage in that unlike many other bacteria, it doesn’t do well at making its own biofilm. So it moves into biofilms that are already protecting other bacteria. C. jejuni becomes a secondary colonizer.
The host colonizers can be any of several bacteria, but C. jejuni’s most prevalent host turns out to be Pseudomonas, which also serves as the main spoilage bacteria on chicken carcasses and is an excellent biofilm former, according to the researchers. They examined the ability of C. jejuni to survive in biofilm populations isolated from four locations: a drinking unit in a chicken growout house, a drain under a plucker in a processing plant, a retail chicken carcass and a crate used to haul live chickens. No C. jejuni was found on the growth surfaces outside of biofilms that had already been established. The biofilms were cultured and showed varying levels of ability to harbor C. jejuni.
Further information. Irene Hanning, Department of Food Science, University of Arkansas, 2650 N. Young Ave., Fayetteville, AR 72704; phone: 479-575-4206; fax: 479- 575-6936; email: ihanning@uark.edu.
Prolong the shelf life of bratwurst sausages using high hydrostatic pressure
High hydrostatic pressure (HHP) processing can significantly extend the shelf life of foods as well as improve their organoleptic quality. For example, prepacked bratwurst sausages are limited to a few days of shelf life under chilled storage conditions. Nitrates are used as preservatives, although they are not needed to fix the product’s color.
The shelf life of bratwurst sausages can significantly benefit if they are cold-processed using high hydrostatic pressure.
The objective of Greek scientists was to evaluate the effectiveness of HHP processing on the production of extended shelf life, preservative-free, packed bratwurst sausages. For HHP cold pasteurized samples, shelf life just about tripled compared to untreated preservative-free samples, and it doubled compared to those samples that contained nitrates and which were stored at 0 C.
The scientists vacuum-packed bratwurst sausages in laminate polymer films of ethylene vinyl alcohol and low-density polyethylene. These were processed under HHP conditions for five minutes at 600 MPa and 25 C. Then they were stored at different temperatures ranging from 0 C to 15 C. Changes in selected quality indices were kinetically studied. Color, texture, sensory and microbiological measurements were conducted on preservative-free HHP-treated samples and on non-treated samples made with and without nitrates.
The HHP process did not alter the color or the texture of the treated samples, compared to the untreated ones that didn’t contain nitrates. The nitrates led to an uncharacteristic pink color. The growth of lactic acid bacteria, the main spoilage mechanism, was modeled by the Baranyi equation and correlated to sensory hedonic scores that exhibited an apparent zero order behavior.
The investigators estimated the rates of microbiological and organoleptic deterioration. The temperature dependence was modeled by the Arrhenius equation, a formula used to determine the rate of a chemical reaction. Activation energies were calculated at 90 kJ per mol and 102 kJ per mol for the untreated samples without and with nitrates respectively, and at 101 kJ per mol for the HHP-treated samples.
Further information. Petros S. Taoukis, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechneiou 9, Zografou 157 80, Athens, Greece; phone:+30 210772 3171; fax: +30 210772 3163; email: taoukis@chemeng.ntua.gr.
Validate processes for fermented food
Acidified and fermented foods have had an excellent safety record, with few or no cases of foodborne disease reported from consuming these products. However, there have been reports of disease outbreaks caused by E. coli O157:H7 and Salmonella in juice products that have pH values of less than 4.0.
These outbreaks have raised questions about the safety of acidified foods. As defined in the U.S. Code of Federal Regulations (21 CFR part 114), acidified foods contain acid or acid food ingredients. They have a water activity of 0.85 or greater and must have a final equilibrium pH of 4.6 or less.
These regulations were promulgated by the FDA in 1979 with the intent of preventing spore germination and growth of C. botulinum. At that time, vegetative cells of bacteria were not considered to be a significant health risk for acidified or fermented food products. Acid foods, which include fermented products, and refrigerated foods are exempt from these regulations.
In 2001, the FDA began holding commercial process filings for acidified foods that are processed using a heat treatment step. Exceptions included products with an equilibrium pH value at or less than 3.3 that are acidified with acetic acid. For these products, only a holding time of 48 hours is needed to ensure their safety before they can be commercially distributed.
For acidified foods with a pH greater than 3.3, the minimum heat treatments needed to achieve a 5-log reduction in the numbers of pathogenic bacteria have been determined. Research has been underway in several laboratories to determine how organic acids present in fermented and acidified vegetable products can be used to destroy acid-resistant pathogens. Scientists also want to determine the molecular mechanisms by which these pathogens resist destruction by acids.
Further information. Fred Breidt, USDA-ARS Food Science Research, Department of Food Science, North Carolina State University, 322 Schaub Hall, Box 7624, Raleigh, NC 27695; phone: 919-513-0186; fax: 919-513-0180; email: breidt@ncsu.edu.
Eureka status awarded for rapid multiplex pathogen test
The U.S. Centers for Disease Control and Prevention reports that 76 million illnesses, 325,000 hospitalizations and 5,000 deaths are caused annually by foodborne pathogens. In the United Kingdom, more than £350 million was spent in 2007 treating cases of food poisoning.
Now, an innovative pathogen detection test developed by RnA Assays bv, Utrecht, The Netherlands, and Alaska Food Diagnostics Ltd., Wiltshire, U.K., has been awarded intergovernmental Eureka status. The test is intended for use by meat producers and processors. The firms have been working together to provide meat producers and importers with improved tools, rapid and reliable food safety and quality control testing.
For use in diagnostic laboratories, the new RnA multi-analyte Plex assay detects microbial and non-microbial contamination simultaneously in a single animal-derived sample, providing multiple results in one sample run. The assay can be applied to groups of animals at time of slaughter to find contaminated product. It enables producers and resellers to predict the quality and assure the safety of final processed products. RnA is working with Alaska Food to develop a rapid high-throughput version of the Plex assay system.
Based at the U.K. government’s Defense Science and Technology Laboratory, Alaska Food has exploited government-patented adenylate kinase (AK) phage technology to develop a highly sensitive and specific fastrAK rapid microbiology system. The company claims this is the only technology available to offer same-day testing for Salmonella, E. coli O157, Listeria and other food pathogens.
RnA launched its first MultiPlex RnAssay, Sal Plex, in June 2007. With this test, serological analyses can be performed on thousands of samples within 24 hours. The Sal Plex results can differentiate simultaneously between five different Salmonella serogroups: B, C1, C2, D and E. The RnAssay technology enables users to make a fingerprint of the quality and safety of products.
Further information. Laurence Callow, Chief Executive Officer, Alaska Food Diagnostics Ltd., Building 227, Defense Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, U.K.; phone: +44 1980 590030; fax: +44 1980 590031; email: lc@alaskafooddiagnostics.com. Ron Wolbert, Chief Executive Officer, RnA Assays bv, Nieuw-Gildestein Building, Room 308, De Uithof, 3584 CM Utrecht, Netherlands; phone: +31 (0) 30 2535362; email: r.wolbert@rnassays.com.
Imagine an edible optical sensor that could be placed in produce bags to detect levels of bacteria and consumed right along with the veggies.
Scientists at Tufts University’s School of Engineering have demonstrated for the first time that it is possible to design such living optical elements that could make possible an entirely new class of sensors. These sensors would combine nanoscale optics with biological readout functions, be biocompatible and biodegradable, and be manufactured and stored at room temperatures.
The Tufts team used fibers from silkworms to develop early-stage platform devices. Tufts has filed a number of patent applications on silk-based optics and is exploring commercial opportunities, although a working device is a few years away. Eventually, there could potentially be a bioactive silk film in every bag of spinach, indicating whether E. coli were in the bag before the food was consumed.
Silk proteins are a natural for integrating optical and biological functions. They can be processed in water at ordinary temperatures and patterned on the nanoscale to generate a wide range of optical elements, including ultrathin films, thick films, and nanoscale and large-diameter fibers.
To form the devices, Tufts scientists boiled cocoons of the Bombyx mori silkworm in a water solution and extracted the glue-like sericin proteins. The purified silk protein solution was ultimately poured onto negative molds of ruled and holographic diffraction gratings with spacing as fine as 3600 grooves per mm. The cast silk solution was air-dried to create solid fibroin silk films that were cured in water, dried and optically evaluated. The elements were prepared, processed and optimized in all-aqueous environments and at ambient temperature, making it possible to include sensitive active biological receptors in the solution.
The Tufts team embedded three very different biological agents in the silk solution: a protein (hemoglobin), an enzyme (horseradish peroxidase) and an organic pH indicator (phenol red), which maintained their activity for long periods when simply stored on a shelf.
Further information. Fiorenzo Omenetto, Biomedical Engineering Department, Science and Technology Center, Tufts University, 4 Colby St., Medford, MA 02155; phone: 617-627-2580; fax: 617-627-3231; email: fiorenzo.omenetto@tufts.edu.
Hops contain substances that control pathogens in the intestines of chickens. Certain bacteria in the intestines not only can contaminate meat during processing, but also may lead to major production losses by causing disease in broiler chickens. Currently, poultry producers use sub-therapeutic amounts of antibiotics in poultry feed as growth promoters and to control pathogens or parasites. However, bacteria can become resistant to antibiotics, so scientists have been looking for alternatives.
The hop plant—Humulus lupulus—contains bitter acids known to be potent antimicrobials. One of these compounds, lupulone, may control levels of C. perfringens in chickens. Investigators delivered different concentrations of lupulone in water to chickens inoculated with C. perfringens. After 22 days, which is the timeframe associated with the onset of clostridial disease in broiler chickens, C. perfringens counts were significantly reduced—from 30% to 50%—in the lupulone-treated group compared to another group of chickens that did not receive lupulone. Based on these results, lupulone has potential for use as an antibiotic alternative in poultry rearing.
Contact: Gerhard Haas, School of Natural Sciences, Fairleigh Dickinson University, 1000 River Rd., Teaneck, NJ 07666. Phone: 201-692-2257. Fax: 201-692-7349. Email: haas@fdu.edu.
Researchers are using rapid genetic characterization to test the DNA of E. coli by examining single nucleotide polymorphisms (SNPs)—variations at single sites in DNA. Rapid genetic characterization opens up the possibility of identifying the bacterial culprits in disease outbreaks and finding out where they originated.
Using SNPs, scientists analyzed 96 markers, making the genetic analysis of pathogenic bacteria possible at an extremely fast rate. It used to take three months to score one gene individually. Now scientists are developing a more rapid system that can analyze thousands of genes per day.
In a study released in March 2008 in the Proceedings of the National Academy of Sciences, “Variation in Virulence Among Clades of Escherichia coli O157:H7 Associated With Disease Outbreaks,” researchers examined the DNA of more than 500 strains of E. coli O157:H7. The investigators discovered that individual bacteria could be separated into nine major groups, called clades. E. coli sickens us by producing Shiga toxins. These toxins block protein synthesis, an essential cellular function, particularly in the kidneys. The scientists found that different clades produced different kinds of Shiga toxins in varying amounts based on their DNA. They now know why some outbreaks cause serious infections and diseases, and others don’t. The different E. coli groups produce different toxins.
Contact: Thomas Whittam, Microbial Evolution Laboratory, National Food Safety and Toxicology Center, 165 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824. Phone: 517-884-2033. Fax: 517-432-2310. Email: whittam@msu.edu.