The FDA is collecting samples of frozen berries from processors, distribution centers, warehouses, and retail locations throughout the year to test for hepatitis A virus and norovirus.

The sampling assignment began in November and is estimated to last approximately 18 months. FDA is collecting domestic samples of frozen berries. It is also collecting import samples from ports of entry, importer warehouses, or other storage facilities where foreign goods are cleared for entry into the country. The agency plans to collect and test 2,000 samples in all.

Some consumers use frozen berries as ingredients in foods without first cooking them, increasing their risk of exposure to harmful viruses, says FDA. The agency reported three hepatitis A virus outbreaks and one norovirus outbreak linked to frozen berries in the U.S. from 1997 to 2016.

Strawberries, raspberries, and blackberries are delicate and may become contaminated with bacteria or viruses if handled by an infected worker who does not use appropriate hand hygiene, or if exposed to contaminated agricultural water or a contaminated surface, like a harvesting tote. Freezing preserves berries but generally does not kill viruses, which can survive at low temperatures.

If FDA detects hepatitis A virus or norovirus in a sample, the agency will notify the firm of the finding(s) and work with the firm to take appropriate action. Upon detecting a positive test result, FDA may also take actions such as placing a firm on an import alert, overseeing a recall, or issuing public warnings.

The FDA will post the sampling results on its FY 19-20 Frozen Berries Assignment page on a quarterly basis and will publish an analytical report once the assignment is complete.

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The world is awash with new rapid testing technology that is enhancing food quality and safety knowledge for the global food industry and consumers alike.

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(Editor’s Note: This is an online-only article attributed to December/January 2019 issue.)

Image credit: molekuul.be/Shutterstock

I don’t think that any of us would work in the food safety industry if we didn’t believe improvement was possible. Through my own work, I hope for a future where foodborne illness outbreaks are virtually eliminated and food recalls are a thing of the past. It’s a big dream. According to the CDC’s July 27, 2018 Surveillance Summaries published in the Morbidity and Mortality Weekly Report, foodborne contaminants cause on average almost 9.4 million illnesses every year. And a recent CDC FoodNet report says that foodborne illness is on the rise, increasing 96 percent in 2017 compared with the 2014-2016 average.

Clearly, it’s a multi-faceted problem that needs an equally complex and complete solution. So how can a safer food ecosystem that takes into account both consumer and food industry needs be achieved? The answer may rest in tech that has been around for quite some time.

Complacent Methods

As the industry becomes complacent with current tools, safety systems are in need of new, intelligent innovation. Polymerase chain reaction (PCR), which has been a popular method for detecting pathogens and adulterations for decades, is thought to be no longer as useful in preventing foodborne illnesses.

In the over 30 years PCR has been on the market, it hasn’t done enough to improve its processes. Instead, the technology has stagnated, especially over the past 10 years. PCR hasn’t kept pace with automation, which in any other industry or tech implementation is now a given. In contrast, PCR still largely relies on hands-on labor. This is problematic for multiple reasons, as it increases the chance of errors in processing and generally creates inefficiencies in a food safety platform.

Unfortunately, this extra effort doesn’t mean extra insight. PCR still just gives binary yes/no answers, meaning that suppliers frequently know the symptom but not the root cause of their problems. Additionally, with high rates of false positives and negatives, even a PCR diagnosis is not the absolute a company would hope for. And yet, this is the current industry standard that has not changed in decades.

PCR as the Canary in the Coal Mine

While foodborne illness should be a preventable problem, it has become prevalent due to the limits of current technology. By the time the scope and specifics of an outbreak are known, it’s often too late for both consumer and company. In a report released in December 2017, the Inspector General of Department of Health and Human Services found it took 57 days on average to recall food.

The industry needs efficiency and knowledge. PCR struggles to meaningfully improve on either vector. Instead, as a tool, it remains a stealthy and serious deficiency in the pursuit of public health.

This problem is reminiscent of another famous public health safety problem, which was also dangerous in its silence: carbon monoxide. For centuries, miners went underground not knowing what they were breathing or how mine conditions would affect their physiology in the wake of their work. What has become a cliché was once peoples’ reality; miners really did take canaries down into coal mines to gauge the safety of the area. If the canary stopped singing, the miners knew they had to get out. While it was industry standard, this methodology was certainly not perfect.

Relying on miners’ observation of a canary is a crude and incredibly fallible mechanism for determining risk. It also frequently left miners with insufficient time to respond to the danger. Sound familiar?

PCR is similarly rudimentary. The living and dying of a canary may be a primitive detriment for safety, but at least it’s an absolute one. In contrast, the false positives and false negatives of PCR in the food safety industry commonly require reevaluations of results, as each uncertainty has huge ramifications from both a food safety standpoint and from a cost consideration standpoint. A false positive means high operational costs, as inventory is held and results are double checked. Meanwhile, when a false negative occurs, it can have ramifications of up to $10 million.

Eventually, the carbon monoxide detector was invented, and mine safety was dramatically improved. However, it took many lives and tragedies for people to force this change and push on inventors for a better solution. The food industry also wants improvement. Companies want to implement whatever protocols that enable them to deliver better and safer food products to customers. Every food safety official, from top to bottom, already recognizes the problem of pathogens and contaminants, as well as their impact.

The Next Safety Revolution

The problem of food safety is even more nuanced than the above example. While the analogy is useful for understanding the prevalence and arch of the problem, it is imperfect. Most notably, with food safety, there isn’t merely one contaminant—like carbon monoxide—to worry about. There are many possible adulterations or issues. That’s why it’s crucial to have technology that is comprehensive in scope and execution.

A promising technology that is thorough and fast enough to speak to these unique challenges of the food safety industry is next-generation sequencing (NGS). NGS looks at the very DNA of foods to discover their composition. It’s a methodology that has revolutionized both the study and application of genomics and molecular biology. Now it’s taking the food safety world by storm.

Unlike PCR, which requires different tests for each pathogen, NGS can ask almost infinite questions about a sample and get the answers all in one test. Additionally, it can sequence hundreds of samples at a time. This amount of data enables companies to identify pathogens at the strain and serotype level even in mixed-ingredient and packaged foods. In addition, all tests go from sample to answer within 24 hours.

The rapid access to in-depth information is something that companies are hungry for. For safety and for consumer satisfaction, having product accuracy that borders on the absolute is not just crucial; it’s a competitive advantage. NGS can provide this certainty with an accuracy of 99.9 percent. PCR’s accuracy is approximately 98 percent, according to a study published in Applied and Environmental Microbiology on accuracy and sensitivity of commercial PCR-based methods for detecting Salmonella enterica in feed.

“Tunable” pathogen profiling is also available from such products as Clear Safety’s Clear Labs NGS platform, which enables companies to set the level of molecular characterization based on the information they need.

This awareness is empowering rather than overwhelming thanks to the complete capabilities of NGS. For every discovery, it offers a solution.

Food safety is a complex, ever-evolving conversation. NGS fits into that conversation with its own complexity of analysis and delivery of results.

Endeavoring Towards a Safer Future

NGS provides an opportunity to enter a new era of safety where companies have unprecedented information about their food products—and consumers have unparalleled peace of mind.

Of course, the obvious benefit to NGS is helping the industry better prevent and react to contamination along the supply chain. However, the key here is not that we’re just better at knowing what happened, we can actually begin to prevent the contamination from making it beyond its first touchpoint along the supply chain. With the speed, accuracy, and affordability provided by NGS, food safety testing can be done early and often, helping brands avoid the consequences of an extensive recall.

The rise of NGS means that we are building a future of food safety that is preventive as well as proactive.

Ghorashi is co-founder and COO of Clear Labs, where he leads commercial activities, including product, sales, and marketing. Reach him at mahni.ghorashi@clearlabs.com.

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Global regulators continue to focus on food fraud, whether deliberate or accidental. The food industry has risen to the challenge by finding innovative new tools to monitor food and ingredients along the supply chain. Its next step should be to bring the same level of care to the laboratory, where food samples are tested for quality.

Supply Chain Traceability to Mitigate Adulteration

Before discussing the laboratory, let’s look at the example of the supply chain where food manufacturers have been tremendously successful in using traceability to improve the safety of their products.

The post Integrity of Laboratory Data Crucial in Food Testing appeared first on Food Quality & Safety.

(Editor’s Note: This is an online-only article attributed to the April/May 2018 issue.)

During the 1970s in the U.S., sprouts became popularized as a healing food by people such as Adele Davis (Let’s Eat Right to Keep Fit) Frances Moore Lappe (Diet for a Small Planet), and Ann Wigmore (The Sprouting Book: How to Group and Use Sprouts to Maximize Your Health and Vitality). Increasingly, consumers have been exploring the relationship between highly processed foods involving nutrient depletion and chemical additives, and many long-term degenerative diseases. There is a growing interest in eating a healthy diet rich in sprouts, wheatgrass juice, berries and other primary foods. By “primary food,” I mean a food carrying all the nutrients it needs to get started on life; the seed or berry receives from the mother plant the highest quality nutrients available to ensure the survival of its species.

When the seed is first planted and begins to sprout, the nutrients burst forth and nutrients, including phyto- (plant) chemicals, which are known to have various healing properties, are often present at much higher concentrations than in the full-grown plant. For example, broccoli, radish, kale, and other plants in the Brassica family have a phytochemical (sulforaphane) at many times the level found in the broccoli branches, that has been shown to protect against a range of maladies, including cancer.

Not only are sprouts high in phytochemicals, they are exceedingly high in naturally occurring microorganisms. In the past, bacteria have often been seen as inherently undesirable, to be minimized or eliminated entirely. However, there is a growing realization that the human body exists in a complex relationship with countless types of microorganisms that are crucial not only to good health, but to our very existence.

My husband, Bob, and I have been sprout growers since 1976. I first got involved in the Massachusetts Department of Food and Agriculture, Promotional Advisory Committee, in the early 1980s. The offer of a federal/state marketing grant led me to contact other Northeast sprout growers. We set up a sprout association to both promote sprouts and solve some of our quality problems in conjunction with the University of Massachusetts Amherst plant pathology department (pathogens in those days were the bacteria that spoiled sprouts). This association led to the formation of the International Sprout Growers Association (ISGA). Our regional, national, and international involvement in the sprout industry followed from there.

In the late 1980s, CDC’s epidemiology began to identify illnesses related to consumption of sprouts and other vegetables, and suddenly we were involved with a new kind of microorganism, that made people sick. The ISGA formed a Technical Review Board and, among other projects, began work on our Code of Practice for the Hygienic Production of Sprouted Seeds and Beans.

The FDA released sprout grower guidance for growing safe sprouts in 1999. The guide basically consisted of three recommendations: treat seed with an effective disinfection process, test all production batches for pathogens of concern with hold-and-release pending negative test results, and maintain a clean and sanitary operation.

Through the ISGA, sprout growers became involved in forming a Task Force with the Institute for Food Safety and Health. A group of growers, professors, and related industry members began work with government on the Sprout Safety Audit, which I co-chaired with Tong-Jen Fu, PhD, research chemical engineer at FDA.

Sprout Regulations

After a year of work on the audit, followed by beta testing with three sprout companies, the USDA suggested converting the audit to a format listing not just the requirements of an audit, but also the procedure, verification, and corrective action for each requirement. Permission was granted by the United Fresh Produce Association to model a Sprout Grower Packer Operations Safety Standard on the Harmonized GAP Standards. Interested parties, such as Whole Foods and Sysco Corp., joined in on conference calls for the next year of conversion from Audit to Standard.

In 2016, FDA codified the growing and handling of all produce (the FSMA Rule) with a special section addressing (some of—I’ll get back to this) the unique qualities of sprouts. Upon issuance of the final Rule, the Task Force went into its third revision of the “Standard” to incorporate the appropriate elements for the applicable FSMA regulations (Produce Safety Rule, Sanitary Transport, and Intentional Adulteration). The Task Force will evaluate need for a fourth revision when the 2017 Draft FDA Guidance for Sprout Operations is finalized.

Concurrent with the development of sprout safety standards by industry and regulatory, in 2003 the Food Marketing Institute developed a Safe Quality Food (SQF) audit, within the framework of the Global Food Safety Initiative, which has become the primary food safety standard for many of the larger retailers.

Recently, SQF created a draft Sprout Module, using the language of the Sprout Standard. At the same time, due to budget concerns, the USDA has tabled the next round of beta testing and auditor training for their Sprout Safety Audit until they see more interest from growers, or industry QA, for a less expensive, thorough audit, based on the Sprout Safety Standard. Contact Ken Petersen, branch chief of specialty crops inspection division, Audit Services Branch, USDA-AMS, at ken.petersen@ams.usda.gov if you are interested in pursuing this option.

During the summer of 2017, the FDA released its Draft Guidance for Sprout Growers, a 123-page document with details appropriate to specific kinds of sprouting equipment (more or less costly to growers depending on the type of equipment they use) and in some cases containing unclear or insufficient scientific or statistical rationale for the specific recommendations given. Although the sprout industry and the public were given the opportunity to comment on both the FSMA Rule and the Guidance, the process of deliberation and decision does not involve further sprout grower input, and will at some point be published as “final” Guidance.

Sprout Sampling

Back to FSMA, there are several areas where regulators have codified practices for the sprout industry that are based on “the way things have been defined” and not on a scientific questioning of the applicability to the unique qualities of sprouts. For example, FDA classified sprouts as “Time/Temperature Control” for safety. It is expensive to growers and retail stores and ignores one of the most promising characteristics of sprouts. From the earliest guidance in 1999, the FDA has suggested sampling and testing of sprout irrigation water (SIW) for Salmonella and E. coli O157:H7, at about 48 hours from planting of every batch of sprouts grown (see graph).

Microbial growth in two types of samples ~30 CFU/g: seed and sprout samples (black circles) or irrigation water samples (clear circles). Solid line represents the logistic regression line from total aerobic growth.(A) Growth of E. aerogenes at ~30 CFU/g during the sprouting process. (B) Growth of Salmonella Stanley at ~30 CFU/g during the sprouting process. (Republished with permissions of Journal of Food Protection, from Quantitative Analysis of the Growth of Salmonella Stanley during Alfalfa Sprouting and Evaluation of Enterobacter aerogenes as Its Surrogate, Bin Liu and Donald W. Schaffner, Vol 70, Issue 2, 2007; permission conveyed through Copyright Clearance Center, Inc.)

Notice that the population of bacteria on the sprout increases from about 3.6 log cfu/g at the start of sprouting (about 600 cfu) to about 9 log cfu/g (100 million cfu) in 24 to 48 hours, after which it shows no further growth, and possibly a slight decline in population. Every experiment we have seen tracking normal or pathogenic bacterial growth in sprouts has almost the identical growth curve.

This is interesting because there is a strong implication that, if the sprouts pass their SIW pathogen test at 48 hours, after any pathogens on the seed will have had the opportunity to multiply by 6 log cfu/g, the risk that the sprouts might be contaminated is greatly reduced. If undesirable organisms are present at significant levels in sprouts, these organisms would have been detected in the routine, every-batch SIW testing that has become standard good manufacturing practice for sprout production, and would not have reached the consumer.

(Note that these graphs are measuring sprouts that are continuing to sprout in ideal growing conditions, 20-30 degrees Celsius, for up to 84 hours). This research gives a high level of confidence that the sprouts, subject to this procedure, are safe to consume.

There is also an implication that any cross-contamination with pathogens onto the sprouts, after the sampling, will not have significant growth, even under the most ideal conditions for growth. This is where the Time/Temperature Control is questionable, and not based on sufficient research. There has been some initial, promising research in this area.

Another implication from the growth curve of bacteria on these graphs is the possibility that healthy bacteria inoculated on the seed before sprouting may out-compete pathogens that might be present on the seed from prior contamination. Some research into competitive exclusion with friendly bacteria as a preventive pre-sprout step has good indicators. Sprout grower associations, universities, and the FDA are continuing to pursue this line of investigation, which would save sprouts from harsh pre-sprout sanitation steps that kill pathogens but almost always show re-growth during the first 24 to 48 hours of sprout growth.

The maintenance of a healthy sprout industry depends on every grower having access to relevant, affordable and up to date good practices and third-party audits. An industry standard, backed by an audit (unlike the Rule) can be easily updated to newer or better procedures that can save costs, be simpler for growers to use, and produce better quality product.

Industry needs government to distribute best practices, research expensive and sound scientific improvements, and regulate industry with some threat of consequence for slacking. Although we have worked together quite well over the years, we feel that there are a number of areas in the FSMA Rule as it applies to sprouts, and in the Draft Guidance, that did not adequately involve the sprout industry. To name a few: developing a mandatory seed supplier seed testing protocol; reviewing the scientific basis of the value of “Time/Temperature Control” for safety; the statistical rationale for defining an entire seed lot as “contaminated” following a single positive SIW test; the evidence for determining the best time in a watering cycle to collect the SIW sample; and the ongoing uncertainties around estimating treatment efficacy, resulting from the lack of a standard protocol for treatment research studies.

I hope that the FSMA Rule is not considered to be a “Final Rule,” and believe that a further cooperative effort with the sprout industry could be beneficial in developing more effective and practical regulations.

There are valuable opportunities here, which can lead to significant improvements in sprout safety, and help increase confidence in sprouts as a safe and uniquely nutritious food. For more information on sprouts, and on some of these research opportunities, please contact the ISGA office at office@isga-sprouts.com.

Sanderson, with her husband Robert—who assisted in this article, are the owners of Jonathan Sprouts, Inc. since 1976. Reach her at barbaraasanderson@gmail.com.

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As readers of Food Quality & Safety well know, there has been an ever-increasing interest in recent years regarding testing for common food pathogens such as E. coli, Listeria, and Salmonella in commercial foods. Publicity around food disease outbreaks has increased in the media, which in turn has sparked an increased interest in governmental regulation around food safety issues, including the passage of the Food Safety Modernization Act in 2011. More tests are now being required than in the past and testing for food pathogens has become a global concern.

Traditional Pathogen Testing

Figure 1. Typical Food Testing Lab Process Flow

Testing for pathogens in a food testing lab is a multi-step process and preparing the enrichment media constitutes much of the activity. Figure 1 illustrates the typical process flow in the test cycle. After a food sample is received for testing, it is documented for recordkeeping purposes, and weighed. Prior to use, enrichment media is prepared and autoclaved for sterilization purposes, and must pass QC checks, which can take several days. The test sample is added to the QC approved enrichment media, and incubated for a period of time, depending on the test method. Finally, the test sample is analyzed, and the test results are recorded and released to the customer. The test method used determines how the sample is analyzed. For example, testing for common food pathogens is typically not a quantitative (or numeric) test, but rather a simple qualitative (i.e., pass/fail) test.

Testing for food pathogens using this traditional method is highly manual and time-consuming, and fraught with problems in the testing process. First, there are numerous opportunities for human error that can affect test results. For instance, incorrect data may be recorded about the test sample, both pre- and post-test. There may also be inconsistencies in the volumes of media prepared for testing, which can have an impact on test results. Evaporation during the sterilization (autoclave) step is a very common problem and can cause measurement uncertainties in the test results. Next, there are a number of safety concerns in the testing process, particularly around enrichment media preparation as it is traditionally done. Autoclaves are used repeatedly, both to heat the enrichment media and to sterilize test containers, leaving various opportunities for contact burns from the autoclave itself or from glassware/containers. Large volumes of heated enrichment media, and the transport of same, also brings the potential for burns caused by contact with hot fluids. Finally, the post-enrichment incubation times may be long due to the time required to bring samples up to test temperature. With many standard testing methods, the enrichment media needs to be at the target test temperature, and using standard media preparation practices, each media container of approximately 3375 milliliter (mL) will need to be pre-warmed prior to use. These media containers are typically placed in an incubator or other heating source in order to do this. Heating large volumes of enrichment media takes time and failing to have the enrichment media at the proper test temperature will obviously influence the test results.

Increasing Efficiency of Media Preparation

Automating the media preparation process in foods pathogen testing can alleviate many of the problems described above. Most notably, the throughput of test volume may be dramatically increased if QC-approved concentrated sterile enrichment media is added to test containers holding pre-heated and sterilized water prepared by an automated media preparator, which brings the enrichment media up to the final test volume. Tables 1 and 2 show an example of how the use of concentrated enrichment media in this manner can allow for up to an 85 percent reduction in the amount of enrichment media that would need to be autoclaved, allowing for far greater throughputs. Dramatically decreased labor and energy costs result from processes that are more efficient, and which require significantly less autoclave time.

Automated media preparators may be valuable in both large and small food testing labs. In using a media preparator in a large lab, with an incubator room, the lab manager first determines the approximate daily sample volume and the amount of enrichment media that would be required in total using traditional testing methods. Sterilized water is then pre-dispensed into test containers to which the concentrated enrichment media will later be added. These sterilized water containers are placed into the incubator room to maintain the proper test temperature prior to testing. When using the system in a smaller lab, without an incubator room, the media preparator is adjusted to dispense directly into the test container, just above the target test temperature, and concentrated QC approved enrichment media is added to the sterile heated water containers. This allows the enrichment media and the sample to maintain the proper temperature prior to and during incubation.

The following example outlines the testing economies that can be realized by using an automated media preparator. Using traditional media preparation methods, a lab receiving 40 test samples per day at 375-gram sample size each would require 3375 mL of heated enrichment media per sample, or 135 liters of enrichment media per day. By using an automated media preparation system, only 20 liters of concentrated media would be required each day, nearly an 85 percent reduction in volume. This concentrated enrichment media is then added to the remaining volume of sterile water—dispensed at predetermined temperature by the media preparator.

The reduction in costs associated with autoclave use to heat enrichment media in this manner is dramatic, as outlined in Table 1. In the standard procedure, 14 hours of autoclave time is required each day to heat the 135 liters of enrichment media, at a cost of about $245 in labor (14 hours x $17.50-hour labor cost). Using concentrated media and a media preparator, only four hours of labor would be required each day: two hours to make the 20 liters of concentrated sterilized enrichment media, and two hours to dispense 115 liters of pre-heated and sterilized test water. The daily cost savings would be $175; 10 fewer hours of labor; and 12 fewer loads in the autoclave.

The savings add up. In the example described above, the weekly labor cost savings comes to $1,225, or over $63k a year. Obviously, the larger the volume of media required each day for testing, the greater the cost savings, and the faster the automated media preparator will pay for itself. In addition to the number of hours required to prepare 135 liters of enriched media per day, the autoclaves in themselves are huge limiting factors in terms of production throughput in the testing lab. Smaller autoclaves aren’t capable of keeping up with the large volumes of enrichment media that may be required, and large autoclaves can easily cost more that the media preparation system itself and can require additional staff to keep up with the sample volume.

Heateflex’s Demeter is an example of an automated media preparator. (Image credit: Heateflex)

As an example of a media preparatory, the Demeter, manufactured by Heateflex Corp. (see image), automatically heats and dispenses sterile water at a pre-determined temperature into a test container, to which sterile concentrated enrichment media and the test sample is then added. The dispense is highly precise and accurate for each test, eliminating human error. Onboard electronics provide traceability for test temperature and volume, and up to 16 pre-programmed test recipes/dispenses are available for various volumes (225 mL to 5000 mL) and test temperatures (0 to 50 degrees Celsius). An ultraviolet light filtration system ensures that the test water is sterilized prior to the dispense. For recordkeeping, the system includes a scanner to record sample and batch data, and a barcode label printer for affixing test information to the sample container.

Economic arguments aside, there are other reasons for considering the use of an automated media preparation system in the food lab testing process. First, they’re easy to use, and sample accuracy is ensured due to the precise dispense capabilities (both volume and temperature) afforded by these types of systems. Lab recordkeeping can also be automated to a certain extent, as the data collected by these products can often be uploaded to a lab information management system if one is available. And finally, lab operational safety can be significantly improved. There are fewer autoclaves involved in the testing process, and both the heating and transport of large volumes of heated enrichment media may be eliminated.

In closing, using automated media preparation systems in the food testing process flow may make a great deal of sense in the operation of many food testing labs, but these products aren’t for everyone. They’re not ideally suited for labs where testing for food pathogens is minimal; e.g., in labs that are primarily focused on quantitative testing. And, in smaller labs, the traditional use of autoclaves and sterilizers may be adequate for test volumes, and there may not be a strong economic argument justifying the productivity advantages afforded by these systems. In most other situations, though, they’re worth a look by lab managers seeking to improve operational efficiencies.

Garcia is the SQF system manager and microbiology lab manager at Diamond Pet Food. Reach her at HGarcia@diamondpet.com. Castaneda is vice president of engineering at Heateflex Corp. Reach him at HCastaneda@heateflex.com.

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Hungary—a country with a growing wine export market—is composed of 22 wine regions. Despite winemaking playing an important role in Hungarian culture for hundreds of years, the country has not been as well-renowned globally as countries like France, Italy, and Germany. Tokaji—a sweet wine from the northeast region of Tokaj—is perhaps the most well-known Hungarian wine, but other wines of great value are produced in the country. For this reason, protection of Hungarian wine from wine fraud and forgery through the generation of a wine map of origin is of great importance.

Novel Tools and Partnerships

New, innovative tools are being developed to advance wine authenticity and identification methods. Nuclear magnetic resonance (NMR) is a powerful technique capable of making precise measurements of thousands of different wines, and its recent adoption in Hungary is providing wine customers and producers with confidence and trust in the content and origin of their wine. NMR has been used in western Europe for a number of years, but Hungary is the first eastern European country to adopt it.

The Hungarian Ministry of Agriculture and Diagnosticum Zrt., a Hungarian diagnostics company, have been working together in recent years to achieve two goals: 1) to develop a wine map of origin and 2) to contribute data on Hungarian wines to the existing international database. The recognition of a need of alliance between the Ministry and Hungarian wine associations led to the signing of a contract in July 2017, propelling the Hungarian wine authentication and identification program through the use of innovative technology.

The Ministry has now acknowledged the importance of an inclusive Hungarian wine map—the first in Eastern Europe—to extend the existing European Union (EU) map consisting of French, Italian, Spanish, and German wines. The addition of Hungary to the map will positively impact international business, wine producers, and dealers.

Ensuring Wine Authenticity

Wine fraud encompasses intellectual property infringement, wine adulteration, and counterfeiting, which can be done by misrepresentation and mislabeling of grape variety, blend origin, or vintage. The Hungarian Ministry of Agriculture is looking to tackle this problem using NMR screening technology.

The Hungarian Ministry of Agriculture and the EU commissions and funds the Hungarian wine identification and authentication program. Diagnosticum carries out screening of wine samples sent to its laboratory by wine producers who are keen to reinforce their customers’ trust by authenticating their product. When wine producers from across Hungary send their samples in, Diagnosticum uses Bruker’s NMR FoodScreener to screen for 52 different measurement parameters and produce a report. Each individual wine sample is then compared to a broad authentic database of reference samples and a detailed certificate is produced. The parameters include tests for decomposition, markers of fermentation, amino acids, phenol derivatives, and stabilizing agents.

“At the moment, we can produce a measurement report from sample receipt to report delivery within one month,” says Sándor Fazekas, Minister of Agriculture, who signed the agreement between the Hungarian Ministry of Agriculture and Diagnosticum. “In the next year, we’re hoping to bring this time down further. The testing itself is very speedy—we can measure 120 samples per day across the two instruments we own, and only need a small sample to get an accurate measurement.”

Wine Screening with NMR

Using NMR allows Diagnosticum to acquire spectroscopic profiles, or fingerprints, from wine samples that are specific to individual samples, and compare these to a large database of authentic wine samples using a multivariate statistical approach. This high-throughput technique provides a wide range of targeted and non-targeted information, such as the detailed chemical composition of the wines, the geographical origin (including influence of soil), identification of wine variety, vintage year, any form of adulteration, and aging of the wine. The resulting test certificate provides foreign and Hungarian traders with a greater guarantee of the origin and quality of the wines than previously available.

“The importance of wine goes beyond its pure market value—it empowers the whole economy,” says Fazekas. “It is therefore imperative that the wine is of excellent and authentic origin for domestic and overseas customers. In order to implement the program, the Ministry will enter into a strategic agreement with Diagnosticum where they will provide the technical background needed to draw the map of origin of Hungarian wines, creating a database based on an internationally authentic mathematical model. In return for submitting their samples, Hungarian wineries will be given a year’s free access to provide their wines for analytical studies, which has not been available to them until now. We see great potential in the innovative work that Diagnosticum are undertaking, which will unquestionably make the self-identification of Hungarian wine possible.”

The wine analysis certificate gives both foreign and Hungarian traders a greater guarantee for the origin and quality of the wines, significantly improving the market position of Hungarian wines and strengthen consumer confidence. The “fingerprint” of the individual wines are visualized and verified in the database, and the technology used demonstrates the chemical characteristics of the wine, as well as information on the soil in which the grapes were grown. Consumers are increasingly wary of wine fraud, so validating authenticity will increase consumer trust on a global basis.

“NMR is the most reactive high-resolution spectrum technology, which is uniquely placed for generating unique wine identifiers (fingerprints),” says Ferenc Péterfy, PhD, chairman of Diagnosticum. “The NMR spectrum can be used to identify the wine’s region, vintage, and variety, using a database based on authentic patterns. This is incredibly valuable to us and is the driving force of the Hungarian wine authentication and identification program.”

International Support

Diagnosticum and the Ministry are in direct contact with Italian and French wine laboratories, which have been using NMR technologies for wine screening for some time. As part of the wine map of origin project, Diagnosticum has open access to these NMR facilities and the countries are able to discuss the latest advances in techniques.

“The same sample can be measured in different countries, but with NMR we should all get the same results,” says Péter Szaszák,who is project director of the program at Diagnosticum and is leading the partnership with the Ministry to develop the Hungarian wine map of origin and the international database. “We can directly ask other countries’ wine laboratories how they are using these new technologies and what their workflow is. We’re still learning, and we still have a lot of questions which, with the help of other countries, we will gain more answers to.”

Looking Forward

Wine fraud and forgery is an industry-wide global issue, where significant investments are being made to bring new sophisticated solutions to market to improve authentication and identification methods. Mathematical modeling of wine analyses to create the wine map of origin is a work-in-progress, where professionals must be trained to interpret the data output from NMR screens. It is thought that in the next two years, a robust mathematical model will be available to wine producers, and the turnaround time for analysis and reporting will be cut in half. The advances in NMR technology could mean that countries not using this technique will be left behind.

Dr. Mangelschots is president of Bruker Corp.’s BioSpin’s Applied, Industrial & Clinical division. Reach her at iris.mangelshots@bruker.com.

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As is often the case in business, it is always about the bottom line.

In the food industry, producers continually evaluate their processes to ensure the highest level of profitability, and a significant portion of the equation focuses on risk management. Risks like drought, flood, and pest infestation are considered acts of nature to which consumers are forgiving; however, other risks, such as releasing contaminated food items to consumers, is viewed by the public as preventable and less forgivable.

According to a study commissioned by the Grocery Manufacturers Association, 77 percent of respondents estimated the financial impact of a Class I recall to be up to $30 million dollars; 23 percent reported even higher costs. The prospect of these types of losses frighten large companies and can bankrupt small companies.

The Food Safety Modernization Act rules and regulations now require food producers to perform pathogen testing to minimize the probability of recalls. Although there is a willingness to perform pathogen testing, food producers don’t want to excessively pay for testing, as it bites into their bottom line. Hence, food safety testing programs are all about sufficiently managing risk, while preserving the bottom line.

Over the past few decades, the incidence of food recalls has not declined, which is troubling. However, there is a reason for optimism as new technologies are under development that may provide better tools for food safety officers to carry out their jobs, presuming that they are more sensitive than current methods and can detect problems in a timelier manner.

The Problems with Culture

The practice of growing pathogens (i.e. culture) has long been used in the industry since it is relatively affordable, simple to perform, and confirms viability, but it does have two major drawbacks. First, it is slow and takes several days to return a result. For perishable products, every day that’s lost waiting for results impacts the product’s value since there is less time for those products to be sold. The delay in getting test results is responsible for additional incurred expenses for transporting the food products to a storage facility and then paying for refrigerated storage, if needed. Although indirect, these costs need to be factored into the cost per sample tested.

The second drawback is that no single medium and growth condition works for all pathogens. This is problematic since splitting a sample across two or more growth strategies can double or triple the cost, which forces a decision as to whether or not to screen for certain pathogens. This is not a decision that’s taken lightly, considering that foodborne illnesses are not only caused by bacteria but can also be caused by viruses and fungi. The failure to screen for pathogens like norovirus, hepatitis A, and mycotoxin-producing fungi leaves many companies exposed to more risk than is desirable, but the added cost of screening for these pathogens is often prohibitive with the current methods.

Pros and Cons of Molecular Testing

Antibody/immunoassay methods are inexpensive, generally look for just one pathogen at a time, and are easy to perform. Although these tests take just a few minutes to perform, their overall time-to-result is relatively poor because culture is first required to overcome their poor sensitivity. In contrast, molecular DNA-based methods are so sensitive that skipping culture can be entertained in some cases. Some have even argued that polymerase chain reaction (PCR)-based testing is too sensitive and would cause a dilemma in deciding how to handle samples since many would come up as positive, where previously they were thought to be negative. This is where quantitative PCR (qPCR) may have utility since a threshold in quantity for a positive sample can be set. Another benefit of molecular testing is that it is more amendable to multiplex analysis, allowing for samples to be screened for multiple pathogens at a time.

Molecular analysis also has drawbacks. Namely, it requires a skilled molecular biologist, is more expensive, and it cannot confirm viability. As such, it is not expected to entirely replace culture. However, PCR, if properly implemented, should allow food safety officers to rapidly assess the risk of some food items, thereby allowing them to quickly decide how to handle food lots of varying risk levels.

For example, samples that are found to not have DNA from pathogenic organisms would be deemed as low-risk items that could be shipped directly to customers, whereas samples that are found to contain DNA from pathogenic organisms would be deemed higher risk and slated to be either processed differently (i.e. heated to kill the pathogens) or tested by culture to confirm whether the positive genetic test could be attributed to residual dead pathogens or if the signal was due to viable pathogens that could cause disease.

Another drawback to PCR is that major sample types cannot easily be processed for PCR because some matrices are just too challenging. For example, it is hard to envision genetic analysis being performed directly on a 25-gram beef sample, as the technology is just not designed to handle this volume or type of matrix. Likewise, it is very difficult to process viscous food items like peanut butter. For these types of matrices, upfront culture will be required to achieve the desired sensitivity, which eliminates the speed advantage of molecular analysis.

In contrast, it is easy to envision genetic analysis being performed on liquid samples that don’t have too much particulate matter and are not too viscous (i.e. the media from swabs, fruit and vegetable wash, and the water that’s used to rinse grains). So, companies that are interested in exploring the advantages of genomics must first realize that the initial scope of use for genetic analysis within the food safety sector is limited. Nonetheless, sufficient testing happens on these types of matrices to warrant serious attention.

The incredible sensitivity of PCR makes it the most attractive molecular technology for detecting pathogenic organisms in food processing plants. However, although it is sensitive, it doesn’t currently fully address the desire for a shortened time-to-result since the work must be done by trained molecular biologists who typically do not work night shifts, which is problematic for companies that operate 24/7. The better solution is to take the “skilled” human entirely out of the equation and have a fully automated instrument perform PCR analysis on the samples. This way, sample testing can happen around the clock.

Multiple companies are working hard to simplify the complexity of PCR into an automated solution. Successes have already been realized in other industries, namely human clinical diagnostics. However, these same successes have not yet filtered down to the food safety industry, where the acceptable price point for each sample that’s tested is substantially lower. Nonetheless, advances are being made on reducing the cost per sample down to a price point that potentially will spur widespread adoption in the food industry. This advancement will likely become commercially available in the next year or two.

The Ideal Solution

For many in the food safety industry, the ideal solution would be to have an instrument that is easy enough to be used by factory workers who have no training in microbiology or molecular biology and, as such, could be placed inside of the factory close to where the final products are packaged. The same factory workers who now package up samples to be sent to a food contract lab for testing, would instead load samples directly onto an instrument in their facility for automated onsite testing.

Ideally, the instrument will be able to process large volumes of fluid to minimize the chances of a false negative result. The automated instrument will need to have the capability to concentrate the particulates in liquid samples, purify the genetic material from these particulates, and then assemble, perform, and analyze the results of multiplex qPCR tests that are designed to detect the most common pathogens that cause foodborne illness.

An added benefit would be to simultaneously quantify the level of indicator species so that the cleanliness of the product and cleaning processes in the facility could be monitored. To not hold up the packaging and shipping processes, the instrument will need to be able to return results in about an hour. This will allow food safety officers to quickly make decisions as to whether or not food items should be loaded onto trucks that are destined for the consumer or onto trucks that are destined for a test-and-hold warehouse (while they await results from samples that are pulled for traditional culture analysis).

The bottom line is that the food safety industry is in need of better tools to prevent foodborne illnesses. The industry has a willingness to pay for more expensive methods if the new methods translate into operational efficiencies and lower risk. Advancements in the industry are moving quickly, and prices are coming down. Expectations are that the wait for new technologies isn’t far away.

These new technologies are expected to empower food safety officers to change business practices where most food lots can be shipped directly to the customer, reserving only those that are found to be at a higher risk to be tested via culture. The hope is that new technologies will allow food producers to deliver fresher and safer foods to consumers, while also allowing them to maintain economic efficiencies.

Dr. Regan is the CEO and founder of LexaGene, a biotechnology company that develops automated and sensitive instrumentation for rapid pathogen detection. Reach him at info@lexagene.com.

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Figure 1. Sodium in milk powder calibration curve for benchtop EDXRF.

Image Credit: Bruker

Energy dispersive X-ray fluorescence (EDXRF) spectroscopy is a rapid and non-destructive elemental analysis technique. It helps food labs optimize production processes and minimize downtime. EDXRF is used to measure nutrients and fortificants, screen for contaminants and incidental adulterants, and identify foreign body contaminants found during production or packaging.

EDXRF performs measurements on all kinds of samples including liquids, solids, or loose powders. It combines high accuracy and precision with minimal sample preparation. It provides simultaneous analysis of elements from carbon to americium and for elemental concentrations from ultra-trace levels up to 100 percent, depending on the specific instrument configuration.

EDXRF is a powerful, green alternative to traditional atomic spectroscopy methods. Sample preparation is rapid and non-destructive with no hazardous waste disposal regulations to be concerned with. Additionally, EDXRF has comparatively low operation or maintenance requirements and costs.

Options of this type of spectroscopy include the following.

• Benchtop EDXRF is the food lab method of choice for dedicated applications in quality and process control with its ease of use and compact size. It delivers speed and analytical flexibility for a multitude of research and monitoring tasks.
• Micro-XRF is the food lab method of choice for high-speed, two-dimensional elemental analysis of non-homogeneous or irregularly shaped samples as well as small samples or inclusions.
• Total reflection XRF (TXRF) spectrometry is the food lab method of choice for rapid ultra-trace elemental analysis, and low parts-per-million (ppm) and parts-per-billion (ppb) of multiple sample types.
  

  Figure 2. Elemental nutrient distribution analysis of banana slice with laboratory micro-XRF.

  Image Credit: Bruker

• Handheld XRF (HHXRF) is the food lab method of choice when an analyzer needs to be brought to the sample for immediate analysis rather than transporting the sample to the lab.

Analysis of Elemental Nutrients and Fortificants

Benchtop EDXRF analyzers quickly measure elemental nutrient and fortificant content in food products at any stage of production, from incoming raw materials to end products. This includes elemental additives such as sodium and potassium or fortificants such as iron and calcium in milk products. EDXRF also measures elemental nutrient content such as selenium and molybdenum in dietary supplements or magnesium and iron in animal feed.

Micro-XRF goes one step further by providing visual images of the nutrient or fortificant distribution on or within the food product. A slice of produce is measured to determine elemental nutrient rich locations, such as in bananas and apples. Micro-XRF also provides elemental fortification distribution maps of crackers, chips, or cereal to help optimize food processing. Mapping images for the distribution of phosphorus, sulfur, and iron on cereal as well as salt distribution on snacks help determine effective fortification process steps.

Analysis of Incidental Adulterants and Contaminants

Figure 3. Elemental nutrient distribution analysis of apple slice with laboratory micro-XRF.

Image Credit: Bruker

EDXRF is ideal for routine analysis of incidental adulterants and contaminants in foods at any stage of the product. These efficient analyzers quickly identify and quantify incidental adulterants such as lead or chromium from colorants, mercury or copper from fungicides, lead from water, or arsenic and bromine from pesticides. Minimal sample preparation is required to achieve high precision and accuracy of results.

TXRF is best suited for ultra-trace elemental analysis. While it is a powerful tool for food fraud prevention in globalized supply chains, it’s particularly relevant for food safety as outlined by the Food & Agriculture Organization/World Health Organization (FAO/WHO) standards, stating it can directly analyze low levels of arsenic in rice or lead in tea drinks. Its versatility for the analysis of multiple sample types as well as minimal sample preparation requirements for even complex samples makes it much faster than inductively coupled plasma emission spectroscopy, which requires fully dissolved liquid samples for analysis.

Identification of Foreign Body Contaminants Found

Contaminants are the last thing anyone wants in their final products, but with virtually non-stop use of production line equipment such as food augers, roller mills, air locks, and drying conveyors, it happens. When contaminants are found, the use of handheld XRF can help food labs quickly identify the foreign body and find its source to fix the problem before any more product is contaminated.

Figure 4. Elemental fortificant distribution analysis of cereal with laboratory micro-XRF.

Image Credit: Bruker

HHXRFs configured with internal libraries of standard alloy and metal grades and compositions identify the contaminants. However, to determine the source of foreign bodies, an XRF audit of all equipment on the production floor is performed first. Simple 30 second test results of all metal surfaces that come in contact with food, or have a potential for breaking, provide a production floor matching catalog. This contains the metal or alloy grade and elemental composition of each piece of equipment, component, piping, or part tested. When more than one source of an identified contaminant is possible from the matching catalog, spectral fingerprint matching is used to take a closer look. Advanced qualitative PC software for HHXRF is used to match the spectral fingerprint of the contaminant to that of its source.

How EDXRF Measures Elements Quickly

Atomic spectroscopy is the most commonly recommended technique for evaluating the elemental composition of samples. It analyzes the interaction between light (energy) and matter (samples). EDXRF is a non-destructive, versatile, and fast spectroscopy technique with minimal sample preparation requirements; and, it can be designed as a laboratory or portable analyzer.

In a way, EDXRF is like a high-powered flashlight that sees beyond what humans can. When the light source is turned on to illuminate a sample, it “sees” the energy of any elements present. It also “senses” how much of those elements are present by their energy’s magnitude. For example, when EDXRF illuminates a sterling silver coin, it detects silver at 22.163 keV and copper at 8.046 keV; and, it determines the coin’s composition to be 92.5 percent silver and 7.5 percent copper.

Figure 5. Ultra-trace analysis capability of arsenic in rice with mobile TXRF.

Image Credit: Bruker

The process of EDXRF elemental analysis of a sample is as follows:

• Energy from an EDXRF source aimed at a sample can eject the sample’s atoms’ inner orbital electrons;
• Outer electrons move into those voids to regain stability;
• While moving in, the outer electrons generate energy characteristic of elements in the sample;
• Resultant energy is detected and processed to determine which elements are present in the sample;
• EDXRF spectrometry results are represented as graphs or spectra showing intensity as a function of energy; and
• The intensity (number of photons) measured at a given element’s energy determines its relative abundance or concentration.

Benchtop EDXRF. These analyzers have the widest range of elemental detection, from light elements such as carbon to heavy elements such as americium with short analysis times, high precision, and excellent detection limits. They are the most versatile in

Figure 6. Internal camera view of metal fragment contaminant in analysis window of handheld XRF.

Image Credit: Bruker

terms of setting up user specific calibrations for virtually any analysis scenario. And, they typically have the most advanced and comprehensive qualitative and quantitative data analysis software capabilities available.

Benchtop EDXRF analyzers are closed-beam systems that can be configured with air, helium, nitrogen, or vacuum atmospheres. Closely coupled thin window X-ray tubes with power up to 50 watts and 50 kV excitation voltage for direct excitation, automatic filter changer selection and high energy resolution silicon drift detectors (SDD) enable the wide elemental analysis and low detection limit range. They are self-contained with a touch screen for user-friendly routine analysis and a variety of connectivity ports. Options typically include internal cameras, automatic sample changers and spinners.

Micro-XRF. This elemental analysis technique with a spatial resolution significantly smaller than conventional EDXRF enables micron size sample analysis. It is especially helpful for analyzing small particle wear debris found during production or particle inclusions in plastic film found during packaging. When micro-XRF is combined with sophisticated elemental mapping software, it is ideal for studying the distribution of nutrients in foods, such as produce, and of fortificants on foods, such as cereal and snacks.

Micro-EDXRF is configured as a closed-beam benchtop two-dimensional micro-XRF spectrometer, typically with a 30W powered rhodium X-ray tube, SDD detector, programmable X-Y-Z stage, fisheye camera, optical video microscopes, polycapillary X-ray optics for spot sizes of 25 micrometers, and software designed for collecting large elemental data sets and mapping distribution via “stitching.”

Figure 7. Fast alloy ID results screen on handheld XRF.
Image Credit: Bruker

TXRF. These analyzers provide ultra-trace (PPB and PPM) quantitative and semi-quantitative multi-elemental microanalysis. This capability is especially critical for ultra-low, but dangerous levels of heavy metals like arsenic and lead. TXRF spectrometers provide fast quantitative and semi-quantitative multi-element analysis of liquids, suspensions, and contaminants. TXRF is optimally suited for trace elemental analysis reaching ppb and ppm detection limit ranges.

TXRF analyzers are configured with a 50W, 50 kV X-ray tube, multilayer monochromator optics and an SDD detector to provide fast and accurate measurement of ultra-trace elements as low as 0.1 ppb in liquids. They have a variety of sample chamber tray configurations; and, in contrast to most analytical methods, sample amounts in nanograms to micrograms are sufficient.

HHXRF. When you can’t take samples to the analyzer, you can bring a portable XRF to them. HHXRF analyzers are the most agile XRF analyzers for the simultaneous measurement of elements anywhere they’re needed. Although they are primarily used for in-situ

Figure 8. Cataloged spectral fingerprints of relevant process equipment in production line folder for contaminant spectral fingerprint matching with handheld XRF analyzers.

Image Credit: Bruker

measurements, such as alloy or metal identification of in-use equipment or incoming materials, they can also be set up in benchtop stands for use with prepared or small samples. They are ideal when immediate results are needed on the production floor.

HHXRF is an open-beam technology, typically with a 2-4W powered X-ray tube, silicon PiN or SDD detector, internal camera, variable spot sizes up to 8 mm, application-specific filters, and software capable of qualitative and quantitative analysis. Some HHXRF analyzers provide the ability to use customized filters and even vacuum or helium flush for light element analysis.

Russell works in business and market development for the Bruker Nano Analytics Division. Reach her at kimberley.russell@bruker.com.

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If you’re a laboratory professional who thinks a programmable coffee maker is the greatest thing since sliced bread, you’re in for a pleasant surprise. Not only can you wake up to fresh brewed java at your prescribed time, you can now arrive at your workplace and find freshly made culture media ready and waiting to be used, thanks to the recent development of a new programmable media preparator.

Say hello to the Masterclave 20 Automated Media Preparator, the brainchild of bioMérieux, Inc., Hazelwood, Mo., introduced in November 2016.

“With its automatic water-filling and autostart features, the Masterclave 20 has the ability to prepare fresh agar or broth that is ready when lab operations begin,” says J. Stan Bailey, PhD, director of scientific affairs for bioMérieux Industry.

With this preparator, one medium can be made at a time, during a cycle that takes less than 90 minutes.

“The Masterclave 20 is compact, mobile, and adaptable to any workspace,” Dr. Bailey relates. “A built-in cleaning gun provides fast and efficient cleaning. Our proprietary ‘I Connect’ technology offers fully automated traceability with built-in RFID (radio frequency identification), email alerts, and laboratory information management system (LIMS) connectivity capability. And the instrument is ISO 11133, ISO 7218, and GMP (good manufacturing practices) compliant.”

bioMérieux’s GENE-UP real-time PCR solution for pathogen detection allows for a high level of specificity.

Photo credit: Christian GANET

Another recent bioMérieux offering is GENE-UP, a proprietary real-time three-step polymerase chain reaction (PCR) pathogen detection system, which the company touts is fast, simple, accurate, and requires minimal training.

Dr. Bailey explains that GENE-UP’s first step, sample preparation and enrichment, includes a standardized protocol and workflow with single enrichment and incubation time between 8 and 24 hours. Step two consists of a simplified, generic 5-minute mechanical lysis. (What bioMérieux calls its Magic Cap eliminates the need to cap/decap tubes, so there is less hands-on time required, plus there is a decreased risk of cross-contamination, Dr. Bailey notes.)

The third step, amplification and detection, features the same PCR run for all parameters. “This allows for accurate results within one hour, a higher level of specificity than other molecular methods, and real-time PCR analysis coupled with end-point melt peak analysis,” Dr. Bailey says. “GENE-UP uses a different kind of FRET (fluorescence resonance energy transfer) technology with three levels of specificity, namely primers, FRET probes, and melt peak analysis. This offers an additional level of sensitivity valuable for detecting low level samples.”

Since GENE-UP was introduced in 2016, it has really taken off, Dr. Bailey boasts. “Laboratories are encountering workflow improvements in a molecular platform, equivalent or better data performance than their current method, and with the internal control and melt peak analysis, an immediate value and confidence in the results they are reporting,” he elaborates.

In July 2016, bioMérieux also introduced a new EHEC GENE-UP PCR Kit that combines stx and eae virulence genes, and the top six serogroups in one solution.

Specific protocols (which are simply enriched in prewarmed buffered peptone water) are available for raw milk and raw milk cheese (25 grams), raw beef and veal (25 grams and 50 to 375 grams), and produce (200 grams).

This kit’s EHEC/STEC (enterohemorrhagic Escherichia coli/Shiga toxin-producing E. coli) solution marries GENE-UP in combination with bioMérieux’s long-established VIDAS automated food pathogen detection system to provide what Dr. Bailey describes as “unrivaled specificity.”

“As a result, false positives are dramatically reduced,” Dr. Bailey emphasizes. “That makes this assay a real game-changer.”

BCN Research Laboratories, Inc., Rockford, Tenn., a commercial laboratory that tests food, ingredients, and environmental sponge samples for food manufacturers, has been using GENE-UP since July 2016.

“We typically run about 100 GENE-UP samples every day,” says Amy Pass, BCN’s senior lab technician. “However, one of our clients operates 20 manufacturing plants throughout the U.S. Twice a year, in March and September, that company conducts biannual heavy environmental testing at all of its facilities. So then we are evaluating an additional 150 Salmonella swabs and 150 Listeria swabs per month for each of those 20 plants, which means we are running an average of 400 tests, but up to about 900 to 1,000 tests, per day, during those two months.

“We have a better work flow with GENE-UP compared to other lateral flow methods we have used in the past,” Pass adds. “Since we started using this technology, we have experienced a 25 percent increase in our sample load, but the amount of time our employees spend running the tests has stayed the same.”

Another benefit of GENE-UP, Pass mentions, is that it provides a definite positive or negative result. “So there is no subjective decision looking at the lateral flow strip to see one line or two,” she says.

Colony Tests and Counter

Charm Sciences’ Peel Plate Colony Counter features results in five seconds or less.
Photo credit: Charm Sciences

Results in five seconds or less are one of the charms of Charm Sciences, Inc.’s, Lawrence, Mass., new Peel Plate Colony Counter, which became commercially available in February 2017.

The instrument is designed to analyze a variety of Charm’s Peel Plate microbial tests, which were introduced in August 2015, according to Robert Salter, MS, the firm’s vice president of regulatory affairs.

The tests are prepared media in a shallow dish with an adhesive top. “They are aseptic ready-to-use tests that are simply rehydrated with the food or food dilution, and incubated at times and temperatures appropriate to the microbes being detected,” Salter explains. “Colonies appear as colored spots that are visually counted or automatically counted by the Peel Plate Counter. An air gap between the plate and cover allows colony quantitation, picking and determination of microbial morphology.”

Currently there are Peel Plate tests for aerobic bacteria (Peel Plate AC, introduced in August 2015), coliform bacteria (Peel Plate CC, introduced in 2016), Enterobacteriaceae (Peel Plate EB, introduced in 2017), yeast and mold (Peel Plate YM, introduced in 2016), heterotrophic bacteria in water (Peel Plate HET, introduced in January 2016), and coliforms/E. coli (Peel Plate EC, introduced in August 2015) for use in dairy products, ground meats, other foods, contact surfaces, and water.

“The Peel Plate AC uses standard plate count formulation with 48-hour incubation,” Salter relates. “Peel Plate EC and CC use coliform selective media with enzyme color substrates with 24-hour incubation. The Peel Plate YM (yeast and mold) tests use conventional potato dextrose formulation with three to five-day incubation. The Peel Plate EB (Enterobacteriaceae) tests use selective EB formulation with 24 to 48-hour incubation. And the Peel Plate HET (heterotrophic plate count) test uses R2A formulation for quantifying bacteria in water with three to five days incubation.”

The new Colony Counter reads all of the aforementioned Peel Plates, Salter says.
Additionally, Charm offers a high volume version of Peel Plates CC-HVS, YM-HVS, EB-HVS, and EC-HVS, designed for a 5 milliliter (ml) sample, typically used for food plant sponge sampling of the production environment, for greater sensitivity in ready to eat foods, and to test water. “These are viewed visually and not yet supported by the Peel Plate Counter,” Salter says.

Salter believes the Peel Plate offerings are an improvement on many existing simple-to-use microbial test products. “The Peel Plate provides a ready-to-use platform that is self-wicking, stackable, and resistant to sample pH/disinfectant effects,” he relates. “Charm Peel Plate tests are simple to interpret color spots that are specific without the need to confirm, but will allow traditional picking of colonies for additional microbial isolation, testing, and identification.

“Peel Plate CC and EC tests use traditional gram negative selective media with bacterial species specific color producing enzyme substrates and a 24-hour incubation,” Salter elaborates. “Coliforms produce easy to interpret red colonies, and E. coli blue colonies, that do not need additional confirmatory steps or difficult to interpret and time dependent gas production.”

The Peel Plate EC test holds Performance Test Method (PTM) Status 061501 with the AOAC Research Institute for total coliform in dairy products tested at 32 degrees Celsius and for E. coli and coliform detection in water, surface rinses, environmental sponges, and foods such as ground meats, eggs, chocolate, and dry dog food tested at 35 degrees Celsius.

Peel Plate AC uses a red color indicator for aerobic bacteria growth in a 48-hour incubation. It holds PTM certificate 071501 for dairy products at 32 degrees Celsius and ground meats, eggs, chocolate, environmental sponges, and dry dog food tested at 35 degrees Celsius.

Peel Plate YM uses a blue/green indicator for yeast and mold growth in a 3- to 5-day incubation. It holds PTM certificate 061601 for fruit, juices, dairy, flour, tortillas, hummus, and environmental sponges.

Based on additional multi-laboratory reference method comparative data, the Peel Plate EC test and the Peel Plate AC test were voted in the 2015 National Conference on Interstate Milk Shipments for inclusion into the Pasteurized Milk Ordinance governing U.S. milk testing requirements.

“Many of our customers are using Peel Plate tests to verify their sanitation and hygiene practices and to monitor and improve food product shelf life,” Salter notes. “They are competitively priced with other simple and ready to use microbial methods, saving time, skill, and labor required by the traditional microbial methods.”

The Colony Counter is a plug and play computer/camera/software that provides complete data management with a real-time print option, Salter points out. Date, time, operator, sample ID, test type, test matrix, count, sample dilution, calculated colonies/ml or gram product, raw plate image, and processed plate image are stored in folders and hyperlinked in a SQL database spreadsheet. Barcode scanning capability provides for a simple one button analysis.

“Onboard Ethernet enables real-time downloading to network SQL databases,” Salter says. “The images are saved as .jpeg files and are viewable at a later date. All data is reviewable by day, week, and month with the touch of a button. It provides for a simple integration with LIMS.

(Without the Peel Plate Counter, Peel Plate tests are scored based on visual counts of colonies, Salter notes. “Visual count is how the methods were compared to the reference methods—also visual—for the approvals,” he says.)

“Inasmuch as food companies are updating their microbial sanitation verification and their end product microbial monitoring programs to address the new Food Safety Modernization Act regulations, we believe the Peel Plate Counter is a valuable tool to assist in automated documentation and record keeping that will enable food production stakeholders to more easily meet audit and inspection requirements,” Salter adds.

Molecular Detection Chemistries

Roka Bioscience’s fully automated Atlas System enhances the accuracy, speed, and efficiency of testing through detection of molecular pathogens.

Photo credit: Roka Bioscience

Roka Bioscience, Inc., Warren, N.J., stands out in many state-of-the-art food laboratories by offering differentiated molecular chemistries for pathogen detection. One such cutting-edge chemistry is target capture, Roka’s proprietary sample preparation method that is integrated into the company’s fully automated testing instrument called the Atlas System.

Simply stated, target capture uses highly specific nucleic acid hybridization to purify and concentrate only the target RNA of interest, according to W. Evan Chaney, PhD, Roka’s director of customer applications and microbiology.

“Roka’s target capture technology is the only fully integrated nucleic acid based sample preparation technology in the industry,” Dr. Chaney says. “This technology is the initial step in all of Roka’s automated assays and serves to not only add specificity, but to also clean up the sample prior to detection.”

The diversity of sample matrices in food related analyses results in very unique diagnostic application challenges, Dr. Chaney points out. “Our target capture technology helps to address these challenges by providing an ideal sample for downstream amplification and detection by molecular chemistries called transcription-mediated amplification (TMA) and hybridization protection assay (HPA),” he relates.

An RNA based amplification system, Roka’s TMA has been used in clinical diagnostics for many years and was first commercially introduced to the food industry in 2012.

“TMA is still novel within the food industry and many food safety professionals are not aware of the differences between it and incumbent testing methods, like PCR,” Dr. Chaney says.

TMA uses two enzymes to drive the reaction, RNA polymerase and reverse transcriptase, he explains. According to Dr. Chaney, TMA is very rapid, resulting in a billion-fold amplification of the target RNA within 15 to 30 minutes.

“One component of this efficiency is the greater abundance of RNA target in cells as compared to DNA,” he says. “TMA is different from older DNA based chemistries such as PCR in that it is isothermal and autocatalytic. The higher RNA copy number per cell, combined with TMA, results in a very robust amplification that may occur in a shorter timeframe as compared to PCR, which can translate into quicker turnaround times for results.”

All rapid methods have various analytical limits of detection in the enriched sample, Dr. Chaney points out.

“For example, most PCR methods require 10⁴ or more cells per ml, whereas, TMA only requires 10² to 10³,” he says. “This becomes quite important to prevent false negatives when considering industry’s move to larger sample sizes and reduced incubation times across an increasingly diverse and complex range of matrices.”

Post TMA, all Roka assays detect any amplified product utilizing HPA, which Dr. Chaney describes as a highly specific chemiluminescent reaction from which the intensity is measured by the Atlas instrument.

“In addition to the detection of any pathogen, each individual sample processed by Roka’s technology includes an internal amplification control, which ensures all reactions occurred,” he says, adding that all of these technologies are automated on the Atlas instrument.

“Our chemistries and controls, coupled with integrated sample preparation on a fully automated platform, translate into faster result times, laboratory efficiency, full traceability, and more accurate foodborne pathogen screening results, particularly for challenging sample matrices,” Dr. Chaney elaborates. “Roka’s technology is routinely utilized in many industry segments, including commercial laboratories, poultry, ready-to-eat meats, produce, dairy, confectionary, ingredients, cereals, multi-component foods, snack foods, and as a tool in pre-harvest food safety.”

TMA is routinely used by Marshfield Food Safety, LLC (MFS), Marshfield, Wis., a firm that specializes in providing customized, onsite process control laboratory services for U.S. food processing operations.

Holding accreditation to ISO 17025:2005 standards with the American Association for Laboratory Accreditation at all nine of its U.S. food testing laboratories, the MFS portfolio includes an extensive list of microbiology and chemistry laboratory test offerings.

“We have been using TMA for qualitative, semi-quantitative, and limits testing for Salmonella for four years,” says Roy Radcliff, PhD, chief executive officer, MFS. “We started using TMA for identifying Listeria species in early 2016, and we have been using it for L. monocytogenes since August 2016.”

Dr. Radcliff says that the ease of use and decreased hands on time are benefits of TMA enjoyed in MFS labs.

“Roka’s Atlas System integrates well with our LIMS,” Dr. Radcliff relates. “The automatic importation of results into the LIMS and tracking of TMA kit lot numbers simplifies the workflow and traceability, which in turn makes them easily auditable.”

Dr. Chaney mentions that, along with continually expanding its footprint by developing unique applications for its current food testing products, Roka endeavors to provide support to its customers, and also works with strategic partners. “We strive to partner with the industry we serve in collectively advancing food safety,” he emphasizes.

Roka’s menu of automated pathogen detection kits includes Listeria spp., L. monocytogenes, Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli, as well as applications including semi-quantitative Salmonella. All of these kits utilize target capture, TMA, and HPA molecular chemistries, and these kits are utilized in Roka’s new applications, Dr. Chaney says.

“More recently, we have introduced a new kit for detection of Listeria spp. specifically in environmental samples, in addition to a novel workflow for use in mitigating the diagnostic challenges that may arise with use of industrial phage based processing aids,” Dr. Chaney adds. “We are currently validating some new and exciting options, such as media alternatives and new assay application parameters for our customers that we anticipate will confer a number of benefits and efficiencies to their operations.”

MALDI-TOF

As general manager of Mérieux NutriSciences, Wendy McMahon, MS, CFS, oversees the company’s Silliker Food Science Center (SFSC) contract research laboratory, Crete, Ill.

McMahon believes matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectroscopy (MS) is an important tool for bacterial and fungal identification in food laboratories today. “It’s really used for determining unknown organisms, mostly spoilage and contaminations, with mold being a good example,” she points out.

Available commercially for less than 10 years, MALDI is a three-step soft ionization technique that allows the analysis of biopolymers such as DNA, proteins, peptides, and sugars, and also large organic molecules. The TOF is the type of mass spectrometer most widely used with MALDI, primarily because of its large mass range.

McMahon says it’s interesting that the microbiology world is using MS for bacterial identification, since MS is a tool used for chemical analysis. “Chemists get a kick out of this,” she quips.

Under McMahon’s leadership, the SFSC is launching the use of MALDI-TOF in the lab during the spring of 2017. “We expect hundreds of ID requests per month due to its quick time to result,” she predicts.

The SFSC is using bioMérieux’s VITEK MS to run its MALDI-TOF tests. “We made that decision based on the database,” McMahon relates. “Specifically, bioMérieux’s database has been established with an average of greater than 14 isolates per species and an average of 26 spectra per species, making it very specific. If an organism is not a part of the database (unidentifiable), then 16S ribosomal RNA (rRNA) gene sequencing can be used for identification.”

The time to result was also a deciding factor in selecting VITEK MS, McMahon adds, noting that it allows for faster investigations and decisions than getting identifications with gene sequencing affords.

“Microbiologists appreciate the quick turnaround time MALDI-TOF offers, less than 30 minutes once the isolate is ready, while requiring very little hands on time from a technician,” McMahon elaborates. “In contrast, the gold standard of 16S rRNA gene sequencing for bacterial identification takes a day of operations and a significant amount of hands on time.”

The SFSC has been using the 16S rRNA method for more than 10 years.

“MALDI-TOF is becoming more widely used throughout the food industry due to the quick results and ease of use,” McMahon says. “The clinical and pharmaceutical industries took to it first and the food industry is quickly catching on. MALDI-TOF’s use in food will increasingly provide companies with faster results when investigating spoiled product, mold contaminations or out of specification raw ingredient or finished product.”

Details to Work Out

There are details to work out in the increasingly more sophisticated world of food laboratory technology, especially with regard to the pathogen testing and detection end of things, says Lee-Ann Jaykus, PhD, the William Neal Reynolds Distinguished Professor in the Department of Food, Bioprocessing, and Nutrition Sciences at North Carolina State University, Raleigh, and also the scientific director of the USDA-NIFA Food Virology Collaborative.

“In recent years, several assays have been designed to meet the need of providing testing results in near real-time (same day), but by and large, they still require some cultural enrichment for pathogen detection, even though enrichment may be abbreviated,” Dr. Jaykus relates.

To get true real-time (in a matter of minutes) pathogen detection will require methods that are completely culture-independent, she says.

“Such pathogen detection will also require pre-analytical sample processing methods, also called ‘sample prep,’ to concentrate the organisms from the sample matrix, and remove matrix-associated inhibitory compounds,” she elaborates. “While some novel sample prep technologies have been launched in the past several years, no silver bullet has been found yet.”

Many groups, be they academic, industry, or government, are actively developing biosensor technologies, Dr. Jaykus points out. “Many of these technologies are novel and ‘sexy’ but still do not have the low detection limits necessary for pathogen detection in foods,” she says. “In addition, the sample matrix can be a significant impediment to analytical sensitivity. Another reason for sample prep, and a personal caution, is that without one (sample prep) we cannot have success in the other (biosensors).”

Dr. Jaykus believes that as detection become less dependent upon culture and more dependent upon nucleic acid sequence, the issue of bacterial cell or virus viability becomes more important.  “Just because we can detect DNA does not mean that the organism is alive,” she notes. “This issue is of importance in making decisions about prevention and control in food safety, as well as management of recalls and outbreaks. It has not yet been resolved.”

Metagenomics

Metagenomics, a term that reportedly first appeared in peer reviewed literature in 1998 (Handelsman et al), basically the study of genetic material recovered directly from environmental samples, promises to impact laboratory analysis with ever increasing significance. In 2005 Chen and Pactor defined metagenomics as “the application of modern genomics technique without the need for isolation and lab cultivation of individual species.”

What some scientists call the metagenomic revolution has resulted in a lot of DNA sequence data for various foodborne pathogens, Dr. Jaykus says, while emphasizing that, “relative to the volume of data available, we currently do not have the critical mass of scientists necessary to interpret it. We are also not entirely certain as to the practical use of those data in food safety management. This will become clearer in years to come, but the field is currently in its infancy stage.”

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