Influence of Epicuticular Physicochemical Properties on Porcine Rotavirus Adsorption to 24 Leafy Green Vegetables and Tomatoes.

Foodborne diseases are a persistent problem in the United States and worldwide. Fresh produce, especially those used as raw foods like salad vegetables, can be contaminated, causing illness. In this study, we determined the number of rotaviruses adsorbed on produce surfaces using group A porcine rotaviruses and 24 cultivars of leafy vegetables and tomato fruits. We also characterized the physicochemical properties of each produce's outermost surface layer, known as the epicuticle. The number of rotaviruses found on produce surfaces varied among cultivars. Three-dimensional crystalline wax structures on the epicuticular surfaces were found to significantly contribute to the inhibition of viral adsorption to the produce surfaces (p = 0.01). We found significant negative correlations between the number of rotaviruses adsorbed on the epicuticular surfaces and the concentrations of alkanes, fatty acids, and total waxes on the epicuticular surfaces. Partial least square model fitting results suggest that alkanes, ketones, fatty acids, alcohols, contact angle and surface roughness together can explain 60% of the variation in viral adsorption. The results suggest that various fresh produce surface properties need to be collectively considered for efficient sanitation treatments. Up to 10.8% of the originally applied rotaviruses were found on the produce surfaces after three washing treatments, suggesting a potential public health concern regarding rotavirus contamination

Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.

<p>Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.</p

Physical properties of epicuticular layers of 24 vegetable leaves and tomato fruits.

<p>(Contact angle is presented in °, and roughness is in μm.). Ad and ab indicate adaxial and abaxial leaf, respectively. Stoma lengths were measured on adaxial leaf surfaces.</p><p>Physical properties of epicuticular layers of 24 vegetable leaves and tomato fruits.</p

Chemical composition of epicuticular waxes from 24 vegetable leaves and tomato fruits and the genome copies from adsorbed rotaviruses on these produce surfaces.

<p>^a The percentage was calculated using number of adsorbed rotaviruses divided by OSU rotavirus genome copies in the initial viral solution (7.17 ± 0.05 log₁₀ genome copies/ml). Wax components were quantified by GC-FID and the total amount of wax was calculated as the sum of single components. LSD value was calculated by Student’s T-test at <i>P</i> = 0.05.</p><p>Chemical composition of epicuticular waxes from 24 vegetable leaves and tomato fruits and the genome copies from adsorbed rotaviruses on these produce surfaces.</p

Comparison of physiochemical epicuticular properties between cultivars with 2-D or 3-D wax crystals on leaf surfaces.

<p>Absence or presence of 3-D wax crystals was determined by SEM. LSD test was used to indicate significant difference for the variables between cultivars with and without wax crystals on leaf surface. Tomato cultivars were excluded because of different tissue type.</p><p>Comparison of physiochemical epicuticular properties between cultivars with 2-D or 3-D wax crystals on leaf surfaces.</p

Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.

<p>Partial least squares prediction model for the number of adsorbed viral particles on produce surfaces using six epicuticular physicochemical properties, including concentrations of alkanes, fatty acids, alcohols, and ketones, contact angle, and surface roughness.</p

Correlation coefficients (r) between epicuticular physiochemical properties and the number of rotaviruses adsorbed on the produce surfaces.

<p>Contact angle, surface roughness, No. of stomata, stoma length, and wax extraction from the produce surface. N = 24 except for the correlations involving numbers and lengths of stomata (N = 21). Pearson’s correlation coefficients (r) were calculated by mean values of each variables from each cultivar, and the r values in bold are significantly correlated at P < 0.05.</p><p>Correlation coefficients (r) between epicuticular physiochemical properties and the number of rotaviruses adsorbed on the produce surfaces.</p

SEM images of epicuticular surfaces before and after 1 min chloroform extraction for ‘Top Bunch’ collards and ‘Starbor’ kale.

<p>A and B are intact ‘Top Bunch’ leafy collards before and after 1 min chloroform extraction, respectively. C and D are intact ‘Starbor’ leafy kale before and after 1 min chloroform extraction, respectively. Scale bar in the image is 10 μm.</p

Epicuticular images from various vegetable leaves and tomato fruits.

<p>All SEM images were generated at 500 × resolution. Scale bar in the image is 50 μm. Inset images were taken at higher resolutions. White arrows indicate stomata. Alphabetical order matches sample list from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132841#pone.0132841.t001" target="_blank">Table 1</a>. A: Tokyo bekana; B: ‘Perseo’ radicchio; C: ‘Rhodos’ endive; D: ‘Southern Giant Curled’ mustard; E: Mizuna; F: ‘Tyee’ spinach; G: ‘Racoon’ spinach; H: ‘Carmel’ spinach; I: Tatsoi; J: ‘Top Bunch’ collard; K: ‘Starbor’ kale; L: ‘Red Russian’ kale; M: Arugula; N: ‘Totem’ Belgian Endive; O: ‘Two Star’ lettuce; P: ‘Tropicana’ lettuce; Q: ‘Outredgeous’ romaine lettuce; R: ‘Super Red’ cabbage; S: ‘Gonzales’ cabbage; T: ‘Ruby Perfection’ cabbage; U: ‘Alcosa’ cabbage; V: ‘Sun Gold’ cherry tomato; W: ‘Indigo Rose’ tomato; X: ‘Rose’ tomato.</p

Experimental schema for the OSU rotavirus adsorption assay to leafy vegetables and tomato fruits.

<p>Each piece of vegetable leaf or tomato fruit skin was transferred carefully and gently onto the top of a droplet of 300 μl OSU rotavirus solution in PBS on a 35-mm-glass-bottom-dish. The edge effects (viruses might diffuse into the piece through its edge) were avoided by cutting the piece into a smaller disk for later RNA extraction.</p