A Rapid Method to Evaluate Bacterial Content in Food

1 Center for Advanced Sensor Technology, University of Maryland Baltimore County, Baltimore, MD, 21250, 5 USA; e-mail@e-mail.com 6 2 Department of Mechanical Engineering, University of Maryland Baltimore Count, Baltimore, MD, 21250, 7 USA; e-mail@e-mail.com 8 * Correspondence: kostov@umbc.edu; Tel.: + (410) 455-6569 9 10 Abstract: The importance of detecting bacteria in various food products is ever-increasing, due to 11 recent food trends that lend themselves to food contamination. Additionally, the detection of 12 probiotics in food products is of increasing importance to consumers, who realize the benefits of 13 probiotics on one’s diet. Existing technologies for detection of bacteria in food are accurate, but most 14 are slow, increasingly costly and unsuitable for applications outside of research laboratories. Optic 15 approaches have recently emerged as an alternative, allowing rapid detection of bacterial presence. 16 This study employs a portable kinetics fluorometer, fabricated in-house, in conjunction with NADH 17 sensitive fluorescence reporter for analysis of various food products. The presence of bacteria is 18 detected in 5 minutes. Both pathogenic and probiotic bacteria were detected in food products, such 19 as raw chicken and beef, spoiled lettuce and contaminated water, yogurt, and kombucha tea. The 20 cellular activity of two probiotic pills was also verified. All samples displayed varying levels of 21 bacterial activity. The study indicates the viability of biosensors being used as an alternate method to 22 detect bacteria in food products – and the viability of a fluorescence-based biosensor to detect viable 23 bacteria. The approach is suitable for both laboratory and field determinations. 24


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In 2018, an E. coli outbreak due to contaminated romaine lettuce sickened nearly 200 people 28 across the United States, and led to the deaths of five people [1]. Food contamination most often 29 occurs at various stages at the production level; this, coupled with the rising popularity of raw foods 30 and fresh produce [2], makes the detection of pathogens in food increasingly important.

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Ready-made meals also have the potential to host pathogenic bacteria [3,4], and should therefore be 32 a source of concern. As long as these food products remain popular with buyers, the detection of 33 potentially harmful bacteria should be a primary concern in order to protect consumers and ensure 34 food quality.

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On the other hand, there are types of beneficial bacteria, or probiotics, that are present in 36 many types of food, and consumers are increasingly aware of the benefits presented by these 37 probiotics. However, "there is no FDA regulatory definition for the term probiotic" and thus the 38 labeling of foods that may contain them is somewhat ambiguous [5]. It is of increasing interest to be 39 able to analyze these foods for the presence of probiotics. Despite improvements in bacterial 40 detection technology, there is still a need for universal bacterial detection processes [6]. results, and are generally more cost-effective than methods that require bacterial culture. However, 48 they are not always accurate, as false positive results are possible. Additionally, it is favorable to use 49 monoclonal antibodies as opposed to polyclonal antibodies for immunoassays because they are 50 more sensitive and return more precise results; however, they are also much more costly [9].

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The most recently adopted method of detection is biosensors, generally comprised of a 60 bioreceptor and a transducer, which translates the reactions within the sensor into electrical signals 61 [7]. The optic biosensor has proven to be a highly specific type of biosensor in terms of detecting 62 pathogens. Optic biosensors utilize transducers to detect the physical changes of an analyte after 63 bonding with a biological recognition element; this change is then converted into qualitative or 64 quantitative units in order for easy interpretation by the end user [11]. The use of biosensors in the 3 of 11 the detection of the change in fluorescence of resazurin dye in the presence of viable cells. The device 83 operation and sample preparation are briefly discussed below.

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The kinetics fluorometer is a single-excitation, single emission photometer that can detect

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The microfluidic cassette acts as a sample holder for the device. It is fabricated using three 96 poly (methyl methacrylate) (PMMA) sheets; one 1.5 mm in thickness, and two 0.2 mm thick. A laser 97 cutter is used to cut the 1.5 mm sheets, as well as to engrave the channels. The cassette channel is set 98 to hold 350 μL of a sample. The reading chamber was designed in a way that allows the most 99 efficient filling of the cassette when the sample is injected.

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After the slides have been cut, each side is sanded for 5 seconds using wet, fine sandpaper.

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Two holes are drilled into the side of the slide using a 1/32 carbide drill bit. Then they are coated 102 with 70% ethanol and bonded in a conventional oven. This process requires a vice clamp, two metal 103 plates, and two rubber sheets. One side of the cassette is bonded at a time, using the setup described

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The metabolic activity of the various samples was evaluated in duplicates. Negative controls 163 were used for each individual sample. The contaminated water was compared against water filtered 164 through 0.2 micron filter, the yogurts against milk, the kombucha against filtered kombucha (black 165 tea), and the media containing probiotic pills against plain media. Both meats were tested multiple 166 times over a several-day time period, with fresh raw beef/chicken used as a negative control. Lettuce 167 was tested after being left in a sealed refrigerated bag for 2 weeks, and was compared to fresh 168 lettuce. All tests were performed at room temperature for 5 minutes and the activity was compared 169 to a 1000 cfu/mL concentration of E.coli in media. Figure 6 demonstrates the dynamics of the test.

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The chicken was tested twice: once fresh, to act as the negative control, and again two days 183 later, after having been left at room temperature. The fluorescence intensity increased when the 184 chicken was measured on the second day; the difference in the change in fluorescence intensity 185 between these two samples is indicated in Figure 8. 188 Contaminated lettuce was also tested; lettuce was left in a sealed plastic bag in a refrigerator 189 for 2 week, then the bacteria was cultured and tested. This lettuce was compared to a fresh piece of 7 of 11 lettuce. The spoiled lettuce displayed much more bacterial activity than the fresh lettuce. Figure 9 191 displays the changes in fluorescence of both samples.
192 193 Figure 9. The fluorescence intensity (a.u.) over time of spoiled lettuce was compared to fresh lettuce.

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Contaminated creek water was also tested, against filtered water. As expected, the 195 contaminated water displayed much more bacterial activity than the filtered water, which displayed 196 almost no activity. Figure

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Kombucha tea, homemade yogurt, and commercial yogurt were tested and compared to 202 each other; the resulting slopes of the change in fluorescence for each test are shown in Table 1.  209

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In addition to yogurt and kombucha tea, probiotic pills of different strains were tested 219 against each other in different concentrations. In order to demonstrate the device sensitivity, the 220 probiotic pills samples were diluted. Table 2    The lactobacillus probiotic pills were also tested after being pre-treated in a pH 2 228 environment. This was done to simulate the activity of the probiotic after passing through 229 stomach-like conditions. Figure 11 shows the results of this trial; the pH pre-treated probiotic 230 exhibited much less cell activity than its non-treated counterpart.
231 232 Figure 11. The fluorescence intensity (a.u.) of probiotic pills over time. One trial was performed after the 233 pill was exposed to an acidic environment, to simulate stomach-like conditions.

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principle also holds true for chicken: when tested two days later, the fluorescence of the chicken had 243 increased much more than that of the fresh, raw chicken.

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The results of the probiotic testing give insight into the cell activity in probiotic-heavy foods 245 such as yogurt and kombucha. Some commercial yogurts are subjected to heat treatment, a process 246 meant to prolong shelf life that also kills most of the live cultures that were used to produce the 247 yogurt. Yogurts that bear the "Live and Active Culture" seal, such as the yogurt that was tested in

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When the probiotic pills were tested, both pills displayed high levels of bacterial viability.

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Additionally, testing the pill after exposure to a strongly acidic environment resulted in a large 266 decrease in activity. The pH range for optimal lactic acid bacteria activity, including lactobacillus 267 activity, is 6.3-6.9 [19], and very acidic environments have been shown to greatly impact the viability 268 of these bacteria [20]. This indicates that the passing of probiotic bacteria, unprotected, through the 269 stomach would significantly reduce the activity of the bacteria.

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In this study, we showed that our approach allows for field detection of the presence of