Effect of Far-UVC and Violet Irradiation on the Microbial Contamination of Spinach Leaves and Their Vitamin C and Chlorophyll Contents ^†

Abstract

Microbial contamination of food can lead to faster spoilage and infections. Therefore, disinfection processes are required that have a low detrimental effect on the nutritional content. Concerning radiation disinfection, two spectral ranges have recently become important. The Far-UVC spectral range, with a wavelength below 230 nm and visible violet light. In this study, leaf spinach was used to investigate the extent to which these radiations inactivate Escherichia coli, but also to determine if the vitamin C or chlorophyll content was reduced. Frozen spinach leaves (Spinacia oleracea) were contaminated with E. coli × pGLO and irradiated with either a 222 nm krypton chloride lamp or 405 nm LEDs. The achieved bacterial reduction was determined by plating the irradiated samples on agar plates and subsequent colony counting. The vitamin C concentration was determined by means of redox titration, and the concentrations of chlorophyll a and chlorophyll b were determined using spectrometry. Both irradiations exhibited a strong antimicrobial impact on E. coli. The average log reduction doses were about 19 mJ/cm² (222 nm) and 87 J/cm² (405 nm), respectively. The vitamin C concentration decreased by 30% (222 nm) or 20% (405 nm), and the chlorophyll concentrations decreased by about 25%. Both irradiation approaches are able to substantially reduce microorganisms on spinach leaves by two orders of magnitude, but this is associated with a reduction in the nutrient content.

1. Introduction

The production of food is not a completely sterile process, which means that food can be microbially contaminated. This is especially true for plant-based foods, as plants are usually grown in soil that is rich in microorganisms and may have been fertilized with manure, which also contains many bacteria. This microbial contamination of (plant-based) foods by bacteria and fungi can have two undesirable consequences: (1) They lead to premature spoilage of the food before it is consumed. Every year, fungi alone spoil food that could have fed 600 million people [1,2]. (2) Microbial contamination causes millions of foodborne infections every year, hundreds of thousands of which are fatal [3,4].

Washing plant-based foods alone is not enough to get rid of the microorganisms. More successful is the heat sterilization proposed by Louis Pasteur in 1866 [5], which was named pasteurization after him. Unfortunately, however, this pasteurization not only leads to a longer shelf life but also undesirably changes other properties, such as texture and vitamin content. Therefore, a gentler disinfection technique would be desirable.

A completely different microbial reduction approach is radiation disinfection. For example, the 254 nm UVC radiation of a low-pressure mercury vapor lamp can, in principle, reduce microorganisms by several orders of magnitude within minutes or even seconds [6,7], and in recent years, two other spectral ranges have gained in importance with regard to disinfection applications. One of these is the spectral range of approx. 200–230 nm, known as “Far-UVC”, which has a similarly strong antimicrobial effect as conventional UVC radiation. However, Far-UVC has the special property that the strong absorption of the radiation by proteins results in human DNA and human cells hardly being damaged by this radiation [8,9,10,11,12]. This leads to the question of whether plant foods can also be irradiated without damage. The other spectral range mentioned concerns blue and violet light. This short-wavelength visible light also exhibits an antimicrobial effect if the irradiation dose is high enough [13,14], and here too, human cells cope better with the radiation than microorganisms [15,16], which gives rise to the second question of whether this also applies to plants.

To answer the question of whether the above-mentioned radiation is capable of reducing bacteria without damaging the plant, irradiation experiments with the wavelengths 222 nm (Far-UVC) and 405 nm (visible violet light) will be carried out in this study. Spinach leaves that have been artificially contaminated with E. coli are irradiated to test the antimicrobial effect of these radiations. The possible negative influence of the irradiation on the spinach leaves is examined on the basis of their vitamin C and chlorophyll content before and after irradiation.

2. Materials and Methods

Frozen spinach leaves (Spinacia oleracea) of Greenyard Fresh (Trevenzuoulo, Italy), which are available in supermarkets all year round, were chosen as the plant-based test food. For all the irradiation experiments below, new spinach packs were thawed for approx. one hour, and yellowed or wilted leaves were removed beforehand.

2.1. Microbial Irradiation Experiments

E. coli × pGLO was selected as the test organism for the determination of microbial reduction by radiation. This is E. coli K12 with a pGLO plasmid—both from Biorad (Hercules, CA, USA)—which gives the E. coli ampicillin resistance and produces green fluorescent protein (GFP) in the presence of arabinose [17]. This fluorescence and the ampicillin resistance helped to limit the inactivation experiments, and its analysis was carried out below on E. coli, without the interfering influence of other microorganisms. E. colis also represent contaminants actually found on spinach [18,19,20].

E. colis were propagated at 37 °C in Luria–Bertani (LB) medium, to which ampicillin and arabinose were added after autoclaving, to an optical density (OD) of approx. 0.3 at 600 nm. The LB medium was then centrifuged off for 5 min at 7000 rpm (rotations per minute), and a bacterial suspension with a concentration of approximately 10⁸ E. colis/mL in phosphate-buffered saline (PBS) was prepared.

The 222 nm Far-UVC irradiation was carried out with a krypton chloride excimer lamp type UV222 from uvmedico (Abyhoj, Denmark) at an irradiance of 0.4 mW/cm² for 100 s up to an irradiation dose of 40 mJ/cm².

For experiments with visible violet light, a strong 405 nm LED with the designation XEGavt-HV20-K60-0000-000-0001 from Cree LED (Shenzhen, China) was applied. This generated an average irradiance of 60.5 mW/cm² on the spinach leaf. After 55 min, the maximum applied 405 nm irradiation dose of 200 J/cm² was reached. During irradiation, the temperature of the irradiated sample was measured several times using an infrared thermometer from Raytek (Berlin, Germany).

The individual spinach leaves were contaminated twice with five drops of 10 µL of the above-mentioned E. coli suspension. After a drying time of 30 min, two areas with five dried drops each were punched out of each individual leaf. In both cases, one of the contaminated, punched-out leaf pieces was irradiated, and the remaining one was left unirradiated as a reference. At the end of the irradiation experiments, irradiated and non-irradiated leaf pieces were each submerged separately in 1 mL PBS and placed in a Sonorex RK100 ultrasonic bath from Bandelin (Berlin, Germany) for 5 min.

Then 33 µL of each of the samples thus obtained were plated at different dilution levels on LB agar plates (containing ampicillin and arabinose) in at least three replicates, and, after one day of incubation at 37 °C, the resulting colonies were counted to determine the inactivations for the different irradiation doses and wavelengths and compared with the unirradiated samples. These irradiation experiments were carried out three times independently of each other, and the results were averaged.

2.2. Determination of Vitamin C Concentration

From the spinach leaves, which were irradiated with different doses at the two different wavelengths, 1 g each was taken and crushed with a mortar. Vitamin C (ascorbic acid) was then extracted in 2 mL of 70% ethanol according to the instructions by LEIFI [21] with an additional 100 μL of 8% acetic acid and 15 μL of ethylenediaminetetraacetic acid—both from Merck (Darmstadt, Germany)—for vitamin C stabilizing [22].

The vitamin C concentration was determined via redox titration according to Staatsinstitut für Schulqualität und Bildungsforschung [23]. Water, starch, sulphuric acid, and potassium iodide—all from Merck (Darmstadt, Germany)—were added to the sample to be tested with the above extracted vitamin C. Potassium iodate was then added drop by drop until the entire solution turned blue–violet. The vitamin C concentration could then be calculated from the spent potassium iodate solution [23]. This procedure was carried out at least three times for each wavelength and each irradiation dose applied and then averaged.

2.3. Determination of Chlorophyll Concentration

The determination of the chlorophyll concentration started similarly to the vitamin C determination. For each wavelength and irradiation dose, 1 g of spinach was crushed in a mortar after irradiation, placed in 10 mL of methanol from VWR (Bruchsal, Germany), and then exposed to the ultrasonic bath for 5 min. After subsequent centrifugation, only the chlorophyll dissolved in methanol remained in the supernatant, the absorbance of which could be measured spectrally using a spectrophotometer type Specord+ from Analytik Jena (Jena, Germany). Based on the absorbance values at 652 and 665 nm, the individual concentrations of chlorophyll a and chlorophyll b could be calculated [24,25]. This procedure was carried out at least three times for each wavelength and each applied radiation dose and then averaged.

3. Results

3.1. Microbial Irradiation Experiments

Figure 1 and Figure 2 show the reduction in E. colis due to irradiation with 222 nm (Far-UVC) and 405 nm (visible violet light), respectively. Each log level corresponds to a reduction by a factor of 10. The numerical values are available in Tables S1 and S2 in the Supplementary Materials. The individual values are scattered, but the reduction is clearly recognizable, and the straight lines drawn in this semi-logarithmic representation correspond to an exponential decrease. The maximum reduction is about 2 log levels or 99% for the applied Far-UVC dose and approximately 2.5 log levels or 99.7% for the maximum violet irradiation. The mean log reduction dose or D90 dose is 18.7 mJ/cm² for Far-UVC radiation and 87 J/cm² for violet light. It should be mentioned that the determined leaf temperatures during the irradiation were between 23 and 29 °C.

3.2. Determination of Vitamin C Concentration

The course of the vitamin C concentration in the frozen spinach leaves as a function of the irradiation dose is given in Figure 3 and Figure 4 for Far-UVC and visible violet light, respectively. The maximum irradiation doses correspond to the maximum doses of the microbial inactivations presented above. The determined vitamin C concentration decreases under both irradiation conditions, whereby the reduction with Far-UVC irradiation is slightly higher, with a maximum of approx. 30% under these irradiation conditions, than the 20% observed with irradiation with visible violet light. The numerical values are available in Tables S1 and S2 in the Supplementary Materials.

3.3. Determination of Chlorophyll Concentration

The courses of chlorophyll a and chlorophyll b concentrations in the frozen spinach leaves as a function of the irradiation dose are illustrated in Figure 5 and Figure 6 for Far-UVC and visible violet light, respectively. The maximum irradiation doses again correspond to the maximum doses of the microbial inactivations described above. The total chlorophyll concentrations determined decrease by an average of 25% under both irradiation conditions. The numerical values are available in Tables S1 and S2 in the Supplementary Materials.

4. Discussion and Conclusions

These irradiation experiments have revealed that the radiation of both spectral ranges can, in principle, reduce microbial contaminations on spinach leaves. Reductions of ≥99% were achieved with the doses applied here.

However, although Far-UVC radiation and violet light have almost no effects on humans for low irradiation doses, the nutrient content of the spinach leaves has changed. For the investigated vitamin C and chlorophyll concentrations, maximum reductions of 20–30% were observed. Surprisingly, there was not much difference between the two spectral ranges when irradiation doses were chosen, so that the bacterial reductions were similar. This nutrient reduction was smaller than the bacterial reduction but higher than expected at the beginning of the study. The research hypothesis that these irradiation wavelengths might be capable of reducing bacteria without damaging the plant could not be confirmed for either wavelength.

To our knowledge, these are the first published results concerning antimicrobial effects and vitamin C or chlorophyll concentration when irradiated with Far-UVC or visible violet light. There are several published studies dealing with different consequences of conventional 254 nm UVC radiation from mercury vapor lamps in particular [26,27,28,29], but the experimental conditions are very different from those in this study. Partly, it is about fresh spinach or pre-harvest spinach, but never about frozen spinach. These discrepancies and the different spectral ranges prevent a meaningful comparison of the results.