COVER STORY
HOT POT Technician samples a simmering vat of soup for quality control.
AN EYE ON FOOD Analytical instrumentation and assays serve as important tools to ensure quality and safety in the food and dairy industry STEPHEN K. RITTER, C&EN WASHINGTON
M
OST PEOPLE I N T H E DEVELOPED WORLD TAKE FOOD
for granted. Ifyou need groceries, you go to the supermarket. Don't feel like cooking? Tbu might order a pizza or Chinese takeout. In a hurry? You're likely to hit a fast-food joint. Want something different? ^bu might try a new restaurant. In many cases, the food you buy is grown, processed, and even prepared by someone else. Do the products you buy have a consistent quality and are they safe to eat? Are you actually getting what you pay for? 28
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The answers to these questions are nearly always yes, thanks to numerous analytical measurements made behind the scenes at food-processing facilities to ensure that food ingredients and finished products meet federal specifications for different food categories and are free from pathogenic microorganisms. These tests range from simple to complex. Stroll through any supermarket aisle, and it will be apparent that many of the items for sale are processed foods—dairy products, ready-to-eat meats, soups, condiments, and more. The ability of companies to produce large quantities of these foods requires highly automated producWWW.CEN-0NLINE.ORG
tion facilities. Companies known as system integrators help food processors implement automation equipment to monitor and control production. All this is done with an eye toward maximizing usage of raw materials and minimizing waste to help boost profit margins, as well as to ensure food quality and safety More than 1,000 companies serve as system integrators, notes VernonJ. Spaulding, vice president of sales and marketing at ESE Inc., a process engineering company based in Marshfield, Wis. These companies, known as automation solution providers, set up program logic controllers and human-machine interfaces to control all aspects of a production facility A COMPLETE engineering system in a manufacturing plant typically has sensing devices such as temperature and pressure probes, p H meters, and in-line near-infrared (NIR) instruments, Spaulding explains. Program logic controllers process data from the sensing devices and then control plant operations, such as automatically opening and closing valves, to optimize production according to specifications. Human-machine interfaces provide production line operators and plant managers with a view into the process so they can monitor progress. "Today, the components of the instruments are pretty standard, and the accuracy is pretty good," Spaulding observes. "But what makes the difference for in-line instrumentation is the software-user interface—that is, how easy it is for the operators to program and calibrate. It's all about having a complete turnkey, closed-loop automation system." N I R spectroscopy is the workhorse of the process industry It's a simple, inexpensive method that focuses on light absorption in the 650-1,050-nm region. This is a "sweet spot" for the food and beverage industry to monitor C - H bonds of fats and oils; O - H bonds for moisture, alcohol, and sugars; and N - H bonds of proteins. Spectra can be acquired using in-line flow-through sample cells, or grab samples can be measured off-line, Spaulding notes. Most analyses are based on the spectrum profile over a range ofwavelengths and are facilitated by process software that quickly compares the data to calibration files derived from standard spectra that are generated during more extensive lab tests. ESE makes two N I R systems that use a halogen lamp source with a photodiode array detector. The Process Quantifier, designed for in-line use, has fiber-optic probes inside a flow-through cell that mounts in-
to a product stream. The spectral signal from the cell is processed in the spectrometer placed adjacent to the process line and is part of the plant's automated control system. "This type of system gives the customer the ability to quickly acquire a spectrum and analyze the necessary parameters, usually within about 30 seconds," Spaulding says. "Using traditional sampling methods, it would take an hour or several hours to do the same analysis in a lab. This faster response gives the producers the ability to
optimize their process by adjusting a parameter, such as protein, fat, or moisture, generally to less than 0.5%, depending on the constituent."The software interface is bidirectional, so someone monitoring a system can change a recipe or load a whole new set of parameters on the fly if needed. The company's second N I R instrument, the Food Quantifier, is used for rapid lab testing. Samples are placed in a plastic bag and inserted into the instrument to obtain the spectrum. Both N I R instruments are used to analyze samples for fat, protein,
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COVER STORY carbohydrates, pH, salt, moisture, percent low speed—only one to three scans per trol, and report material mass balance insolids, and more. second—reducing the usefulness of realformation from incoming raw milk to endESE works with some of the largest milk time, in-line measurements, he says. product shipments and to convey the inprocessors and natural formation into a financial and process cheese mansystem that can track NEW TO THE marketplace are hybrid IR ufacturers in the world, as trends and evaluate perinstruments that get around some of these well as manufacturers of formance, he notes. For limitations by taking advantage of the best butter and cultured milk now, Spaulding believes features of process-grating instruments products such as yogurt ESE is the only company and FTIR. One example is Aspectrics' Enand sour cream. Other that can offer this type of coded Photometric IR (EP-IR) instrutypes ofproducts the comcomplete package. ment, which the company introduced at pany handles include pudthe Pittsburgh Conference in Orlando, ESE is a midsized comdings, pasta sauces, soups, Fla., in March. pany with about a 2 % salad dressings, condimarket share in the diffuse EP-IR is similar to FTIR in that indiments such as ketchup $430 million automation vidual wavelength segments are modulatand mustard, and nonferand in-line instrumentaed and the spectral intensity is computed mented beverages. tion market in the U.S. via a Fourier transform, Hawker notes. There are about 100 But with time, the comLight from a broadband source passes dominant system intepany would like to see its through a sample and a diffraction grating, grators in the food and market share expand to and then onto a rotating disk that is made by a photolithographic process. The disk beverage sector, Spauldmore than 10%, he says. ing notes, but only a few The advantage of procontains up to 256 concentric encoding dozen companies focus cess N I R instruments is tracks, which effectively replace the dethat they are rugged yet simple in design tector elements of a conventional array deon dairy products. "Of those companies, there's only one—and that's ESE—that and generally cost-effective, according to tector, he explains. manufactures N I R instrumentation and Donald A. Hawker, vice president of marIndividual grating-dispersed wavedoes the system integration," he says. keting at Aspectrics, a process instrument lengths of the sample signal are reflected manufacturer based in Pleasanton, Calif. off the disk to a single lead sulfide or lead ESE has started a "Lean Enterprise" iniThe disadvantage is that analysis is limited selenide detector. The detector signal can tiative, akin to Lean Manufacturing and be combined at a rate of 100 scans related business management per second, giving the instrument strategies such as Six Sigma, to the ability to accurately measure promote maximizing efficiency low levels of compounds in the and ingredient utilization in the visible through mid-IR range. food processing industry. "It's a The cost of the EP-IR spectromvery natural fit to go out into the eter is slightly more than single marketplace and educate our cusN I R units, but much less than tomers on how we can apply these FTIR, he says. methodologies to their benefit," Spaulding says. "The EP-IR spectrometer's inherent sensitivity, ruggedness, and One example, from the dairy reasonable cost should prove to industry, involves the price of milk be an excellent tool for food and and other products, 60% of which beverage manufacturers to assist is due to the cost of the raw milk, in quality control in the process Spaulding notes. The situation is environment, where real-time reparallel to that of natural gas in the sults are important," Hawker chemical industry Manufacturer notes. In February, the company margins are sometimes squeezed SAY CHEESE Technician at an Associated Milk began shipping evaluation instrubetween the price of the raw milk Producers' plant checks cheese for moisture using ments to companies that manuand the price at which a product ESE's benchtop Food Quantifier near-IR spectrometer facture analytical and process incan be sold. For a product like (unit below computer monitor). strumentation. Aspectrics is cheddar cheese, the whey byworking with system integrators and their product can sometimes be as valuable as to only a few compounds of interest, he suppliers to use EP-IR for food analysis of the cheese itself, he says. says. If multiple components are to be meafats, proteins, and sugars, but the details are sured, then multiple NIRunits are required, "That makes it important for the manthus reducing the cost advantage and confidential for now, he says. ufacturer to be able to manage the incomadding complexity to plant engineering. Polychromix, based in Wilmington, ing raw milk and accurately know paramMass., introduced another new type of eters such as percent butterfat and proteins Fourier transform infrared (FTIR) prodcompact process NIR instrument at Pittcon ucts have better resolution and work well as the milk is being processed," Spaulding in the lab environment, Hawker adds. But 2005: the Digital Transform Spectrometer says. "The key really comes down to inbecause the moving mirror of the inter(DTS), which is based on diffractive microstrumentation. Ifyou can't measure it, you ferometer is sensitive to vibrations, it can electromechanical system (MEMS) chip can't control it. For us, we do it with NIR." be costly to employ in a production facilitechnology It joins the MEMS-based chemThe goal for ESE with Lean Enterprise ty Also, FTIR instruments scan at a fairly ical process N I R instrument made by Axis to be able to automatically measure, con-
"Components of instruments are pretty standard, and the accuracy is pretty good. But what makes the difference for in-line instrumentation is the softwareuser interface."
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sun Technologies, Billerica, Mass., which was introduced at Pittcon 2004. Polychromix' original focus was on us ing MEMS technology to develop electrooptical devices that can manage multiple wavelengths on a single optical fiber for telecommunications, notes Yariv Geller, the company's director of marketing and business development. "The work in the telecommunications field really lends it self nicely to spectroscopy because the wavelength ranges are nearly the same," Geller says. The hand-sized DTS is designed to re ceive a spectrum from an external sample cell via an optical fiber. The incoming sig nal is dispersed across the MEMS chip so that each part of the chip interacts with a different wavelength. "Essentially we have a programmable filter with which we can control the MEMS chip to block or reflect any combination of wavelengths to a sin gle InGaAs detector, with spectra acquired in less than a second," Geller notes. The detector signals are then processed using a digital transform method. Like Aspectrics' EP-IR, the DTS sys tem is essentially a cross between an FTIR
instrument and a grating instrument with an array detector, except it has no mov ing parts. One instrument covers the 900-1,700-nm range, while a second in strument covers the 1,700-2,500-nm range. A third high-resolution version is designed for specific narrow-band appli cations in the 1,100-1,300-nm range. For now, the DTS instruments depend on using a separate light source and sam ple cell or probe, but Polychromix is de veloping accessories, such as a portable light source. "Ultimately, by developing ac cessories, we will be able to have a com plete portable solution," Geller adds. In general, the devices can be used for any application in which IR is currently used, Geller says. In May, Polychromix an nounced a partnership with Ocean Optics, Dunedin, Fla., to distribute the DTS in struments. Polychromix also is looking to work directly with process solution providers to incorporate the instruments into their offerings. Geller doesn't believe the new devices will necessarily replace existing N I R or mid-IR systems, but he expects the new instruments to sell more for new applica-
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COVER STORY tions. One example is measuring trans-fat content of foods, an important parameter now that the Food & Drug Administration has issued rules for listing of trans fat on nu tritional labels beginning next January. "When a new regulation like this comes along, a company like Polychromix with a low-cost solution could have a tremendous advantage in the food and beverage in dustry," Geller says. A promising technique for the direct and more complete analysis of the thou sands of compounds naturally found in foods is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), according to chemistry professor Alan G. Marshall of Florida State Univer sity, Tallahassee. Marshall is a coinventor of the technique and founding director of the ICR program at the National High Magnetic Field Laboratory in Tallahassee. FT-ICR MS offers much higher resolu tion than a standard mass spectrometer,
MASSMATICIAN Florida State's Marshall poses among the components of an ion cyclotron resonance mass spectrometer, which has a resolving power to 0.0005 dalton and can be used to analyze natural products such as vegetable oils that contain thousands of compounds. allowing Marshall and his coworkers to dis tinguish between ions that differ in mass by as little as 0.0005 dalton—less than the mass of an electron. 32
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"In effect, we are about 200 times bet ter at separating peaks than the best sin gle-stage gas chromatograph, high-per formance liquid chromatograph, or gel technique. Plus, we can identify the ele mental formulas of the peaks," Marshall points out. The lab's flagship instrument uses an electrospray ion source operating in either positive- or negative-ion mode. Ions are initially collected in a trap just outside the magnet and then injected into an 11-cm-diameter cyclotron that sits inside the 22cm bore of a 9.4-tesla su perconducting magnet. That's the same mag net strength as that in 4 0 0 - M H z proton nu clear magnetic reso nance spectrometers. Each ion circulates in side the cyclotron at a frequency that depends on its mass. Like NMR, ICR MS uses a radiofrequency signal for ex citation and detection of molecules. Over the years, Marshall's group has fur ther developed the technique and used it to analyze drugs, oligosaccharides, phos pholipids, and peptides and proteins. More recently, he has turned to the composi tional analysis of complex chemical mix tures, such as coal extracts, crude oil, gaso line and diesel fuels, and even wine. The high mass resolution of FT-ICR MS allows the "fingerprints" of different classes of compounds in these samples to be used to identify their origin and properties.
sis of crude oil and its distillates "petroleomics" (Ace. Chem. Res. 2 0 0 4 , 37, 53). In a similar vein, Marshall, Rodgers, and grad uate student Zhigang Wu have taken on the analysis of the complex composition of vegetable oils, an area they are calling "oleomics." "There is considerable health-related interest in the identity and levels of satu rated and unsaturated fatty acids and their glycerides in food, especially vegetable oils used for cooking," Marshall notes. Addi tionally, the authenticity of oils can be im portant to consumers because of cost. For ex ample, relatively expen sive olive oil can be di luted with cheap olive oil or another oil. With regard to safety one need is the ability to quickly identify the presence of a toxic adul terant. A famous case a few years ago involved an olive oil diluted with canola oil that had been denatured with aniline for industrial use, Marshall relates. The oil was later linked to several hundred deaths in Europe. Although all vegetable oils have similar components, such as di- and triglycerides, free fatty acids, and sterols, the mix of com ponents and their relative abundances vary quite a bit, according to Marshall. Ά key point about vegetable oils is that one can't simply rely on single-mass-unit resolution for analysis because there can be a dozen or more chemically different species all at the same nominal mass, and only one of them is the compound you think it might be. % u really need the high resolution available with FT-ICRMS to figure it all out." In one set of experiments, Marshall, Rodgers, and Wu dissolved canola, olive, and soybean oils purchased at a local su permarket in a chloroform-methanol so lution for analysis (J. Agric. Food Chem. 2 0 0 4 , 52, 5322). In negative-ion mode, the instrument detected about 3,000 dis tinct components in the oils, and in posi tive-ion mode, about 2,000 components. The differences between the two modes reflect the acid and base properties of the compounds, Marshall notes. Carboxylic acids and tocopherols tend to deprotonate to become negatively charged; di- and triglycerides tend to be protonated and be come positively charged, he says. The team identified several markers that could be used to routinely characterize veg etable oils. For example, canola and olive oils have a higher proportion of C18 fatty
Chemists take it for granted that it's necessary to separate and purify a chemical in order to identify it uniquely."
THE NUMBER OF nitrogen, sulfur, and oxygen atoms; aromatic rings plus double bonds; and degree of alleviation of molec ular ions in a complex spectrum can be de termined using a computer search algo rithm, he explains, and the elemental formulas for each peak can be calculated. "It's actually pretty easy—all you have to do is count," Marshall quips. In one example, Marshall and Ryan P. Rodgers used the technique to resolve some 20,000 chemical species in a single spectrum of crude oil without having to first use standard chemical pretreatments or chromatographic separation. "Chemists take it for granted that it's necessary to sep arate and purify a chemical in order to iden tify it uniquely," Marshall says. Because petroleum has about as many components as there are human genes, Marshall and Rodgers have used a spin-off of the word "genomics" to dub the analy
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acids with a single double bond, while soybean oil has a higher proportion of C18 fatty acids with two double bonds. In a proof-of-concept experiment, the researchers diluted olive oil with various amounts of soybean oil and analyzed the samples. Soybean oil can be readily distinguished even at low concentration by the presence of a fatty acid that doesn't occur in olive oil and by overall differences in glyceride abundances, they report. "The tremendous resolving power of F T ICRMS shows us that these oils are more complex than previously believed," notes Gary R. Takeoka, a research chemist at the Department of Agriculture's Western Regional Research Center in Albany, Calif. Takeoka co-organized a symposium on the authentication of food and wine last March at the American Chemical Society's national meeting in San Diego, in which Marshall presented the vegetable oil research. "This technique gives us the ability to drastically reduce the overlap and interferences that characterize other chemical and spectroscopic analyses," Takeoka adds. "I believe FT-ICR MS has great potential as a new tool in detecting food adulteration."
One drawback is the cost, which currently is between $500,000 and $1.5 million per instrument, Marshall says. By his count, there are about 600 FT-ICR MS instruments worldwide. They have been commercially available from Bruker Daltonics and IonSpec Corp. for several years and more recently from Thermo Electron Corp., he notes. These instruments are targeted mostly for the study of peptides and proteins in the proteomics arena. Mass spectrometry is the fastest growing segment of commercial spectrometers, Marshall points out, and FT-ICR MS is growing twice as fast as mass spectrometry overall. MARSHALL BELIEVES the technique could be very useful in food science and other areas to identify the critical markers needed to analyze a material, as his group has shown with wines and vegetable oils. "Then you would only need to test for those markers using a more routine technique," he says. Quality is only part of the story in food science. Much research is aimed at improving methods for killing microorgan-
isms that cause illnesses and ensuring that foods treated by these methods are safe before they enter the supply chain. The key disease-causing culprits in foods are Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella enteritidis, Campylobacterjejuni, and Norwalk virus (Norovirus), notes microbiologist Richard H. Linton, director of the Center for Food Safety Engineering (CFSE) at Purdue University CFSE uses teams ofbiochemists, microbiologists, physicists, and engineers to develop better approaches for detecting and preventing food-borne hazards, Linton says. "Our focus is to be able to bring in a food sample, run it through a detection platform, and find out if it contains a pathogen and how much is there," he notes. One of the current hot topics in food safety is finding a quicker method to detect L. monocytogenes. This bacterium can contaminate almost any food, from vegetables to ready-to-eat meats. It's responsible for about 2,500 cases of listeriosis in the U.S. each year. Although that number may not seem high, listeriosis has a 25% mortality rate, the highest rate among food pathogens, Linton says.
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33
COVER STORY Commercial sterilization technologies to about 1 million per mL. An enrichment and graduate student Tom Τ Huang came use heat, high pressure, irradiation, and up with a quick way to make test chips in culture is used to increase the number of other methods to destroy pathogens dur the lab without needing time-consuming bacteria in the sample, but this causes a ing food preparation, Linton explains. But photolithography or stamping fabrication delay food can later be contaminated during methods. In their "press-fit" method, glass "If you had a concentrated population packaging. Cooking the food will kill the fibers are placed on a glass slide or silicon of microorganisms that can be probed at bacteria, but ready-to-eat products like a small volume, less than 1 μ ι on a chip, the wafer in a desired pattern, and then a small sandwich meats often aren't recooked by square of flexible poly(dimethylsiloxane) test could be much faster," Ladisch says. consumers. is "pressed" over the top of the fiber (Anal. The researchers achieve this by con Chem. 2005, 77, 3671). The polymer ad Manufacturers pull some samples for centrating the usual 250-mL extract down heres to the glass, creating a tight seal testing and ship out the remaining product to about 500 μL, then loading it onto an around the fibers but leaving a small chan while test results are pending, Linton says. antibody-based fiber-optic probe or mi nel on either side for fluid flow. The hyThe volume of dairy products and meats is crofluidic chip. The antibody specifically so large, though, that these products can't binds L. monocytogenes, trapping it inside drophilic glass fibers sandwiched between the hydrophobic top and bottom layers di be sequestered for up to a week while test the probe or chip. With the probe, the bac rect the flow of liquids through the device, results are awaited. An additional factor is teria are detected in just a few hours by but flow can also be controlled by a syringe that the number ofviable bacterial cells that measuring a change in the optical signal. pump. can contaminate food following heat treat On the chip, the bacteria also are quickly ment and before packaging may be low and detected by the electrical conductivity The fibers can be positioned to create difficult to detect. That's a problem linear channels, Tshaped junctions, withL. monocytogenes, because as few and right angles, Ladisch explains, as 100 lingering cells are thought to and two fibers laid across one an be enough to cause illness. other can create well-like junctions. In addition, the fiber surfaces can be "We would like to develop a de modified ahead of time by dip-coat tection system that will give good ing them into solutions containing information to the industry very commercially available ion-exchange quickly on the relative safety of a microbeads. Antibodies that selec product," Linton says. "We want to tively bind L. monocytogenes or other be able to do this in a matter of min bacteria can be subsequently at utes, rather than in days, which it tached to the microbeads. now takes." One approach being pursued by Press-fit devices effectively func Purdue scientists and engineers tion as microscale liquid chro working on CFSE projects is to de matography columns, and detection velop microfluidic devices to con of bacteria can be carried out using centrate bacterial cells for detection. fluorescent labels. It takes only Agricultural and biological engineer about 15 minutes to make one of the Michael R. Ladisch, microbiologist devices. Arun K. Bhunia, electrical and com "The idea with these chips is that puter engineer Rashid Bashir, and you wouldn't use them for produc their coworkers have focused on us tion, but only for research purpos ing antibodies that specifically bind es," Ladisch says. "They enable you L. monocytogenes. Bashir's group has to make microfluidic channels with developed silicon-based microflu different chemistries for quick tests. idic devices that allow for rapid de Once you have a system you want, tection of live bacterial cells by culyou can go back to design and fab turing them on a chip. This work, ricate a complete microchip and de combined with development oflowtection system."This is a general lab conductivity growth media from tool, he adds, and his group has not Bhunia's lab and sample concentra pursued a patent on the process. SPLITTING HAIRS FT-ICR MS can resolve tion and recovery techniques from This type of research being car dozens of peaks at the same nominal mass, as Ladisch's group, has led to forma ried out in food science helps to mo shown in this expanded view of a negative-ion tion of a start-up company to com tivate and excite young people to spectrum of an olive oil (top). Complete analysis mercialize the technologies. enter chemistry and technical fields, of vegetable oils has yielded markers, such as the Current food testing, which is Ladisch believes. "Basically, it's a relative abundance of C18 fatty acids with zero, very effective, involves taking a combination of molecular biology, one, two, or three double bonds (bottom), that can product and preparing about 250 nanotechnology, and microflube used to check the purity of different oils. mL of extract solution. If contami idics —bionanotechnology. But nated, the solution might contain chemistry is what makes it work," fewer than 10 L. monocytogenes cells per mL, caused by the metabolic activity of the bac he says. "If we can package this technolo terial cells, a technique developed and Ladisch says. Standard bioassay test kits gy and communicate the excitement we patented by Bashir's group. used by the industry to test the solution have for it, then it will encourage students As the work moved forward, Ladisch require a high concentration of cells—up to pursue science and engineering." • 34
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