Aitana SANTAMERAa, María Antonia BAÑUELOSb, Carlos ESCOTTa, Iris LOIRAa, Carmen GONZÁLEZa, Juan Manuel DEL FRESNOa, José Antonio SUÁREZ-LEPEa, Antonio MORATAa
a. EnotecUPM, Chemistry and Food Technology Department, ETSIAAB, Universidad Politécnica de Madrid, Avenida Complutense S/N, 28040 Madrid, Spain
b. Dept. Biotecnología-Biología Vegetal, ETSIAAB, Universidad Politécnica de Madrid, Avenida Complutense S/N, 28040 Madrid, Spain
Pulsed light can be considered a non-thermal emerging technology suitable for foodstuff sanitization. Sanitization may occur through the use of high energy pulses during extremely short periods of time (ns to μs). The light used in the process comprises UV-C irradiation that would produce irreversible damages at DNA level and on the microbial membranes and thus, the reduction of microbial populations. The use of few pulses (1 and 2) are able to reduce 1 log10 UFC/cm2 while 3 pulses reduce 2 log10 UFC/cm2 with little influence on the surface temperature. 5 pulses may increase the temperature up to 4º C with no lasting effect. The fermentative performance observed in treated and non-treated grapes showed the effect of PL on microbial populations. Two out of three fermentations with treated grapes (5 pulses) produced ethanol after the 4th day yielding 4.7% ethanol (v/v) while the third did not produced significant amount of ethanol. Lastly, no difference in colour was observed between treated and control grapes. In summary, pulsed light, as non-thermal sanitization technology may not modify the product sensorial properties nor its nutrimental benefits but would afford the reduction of native microbiota. This allows the use of emerging fermentation biotechnologies and the production of wines with less preservatives since the sanitization of vinification grapes would comprise a reduction of SO2 content in winemaking.
The use of pulsed light (PL) in food technology dates back to the 1970s when this technology was first used in Japan due to its antimicrobial activity to preserve food (Escott et al, 2017). One of the reasons this technology is able to reduce microbial populations on food substrates comes from the UV light component that inactivates microbial DNA and disrupts cell membranes.
PL can reach 105 folds the energy intensity produced by the sun at sea level in several discontinuous pulses which can last from 1 μs to 0.1 s. The energy produced by xenon lamps in each pulse can reach up to 35 MW released during 85 ns (Morata et al, 2017), or up to 100 W in continuous arrays (Fig 1). Even though the energy is high, and the temperature can rise locally, the average temperature increase observed in treated matrices is ca. 6º C; this feature would include this technology as non-thermal preservation technology in the food industry.
Fig 1. Continuous array proposed for pulsed light sanitization of vinification grapes.
PL can be considered an emerging technology that, similar to ultrasound (US), UV – radiation (UV), high hydrostatic pressure (HHP), pulsed electric fields (PEF), electron beam irradiation (e-beam) and, more recently, ultra-high pressure homogenization (UPHP), can contribute to reduce, or eliminate, microorganisms from a wide variety of foodstuff (Morata et al, 2017). This includes vinification grapes where the microbial population reduction is desired for the proper implantation of fermentative yeasts, either wine active dry yeasts or fresh inoculum, and avoid sensorial deviations from uncontrolled spontaneous fermentations. In this way, PL can reduce both, yeast and bacterial populations as a function of amount of pulses and the energy applied (Fig 2).
Fig 2. Bacterial population (CFU/mL) after 5 or 10 pulses at medium (me) or maximum (ME) energy released on vinification grapes Vitis vinifera L. cv. Tempranillo (From Escott et al, 2017).
Similar to what happens with the use of other emerging technologies, the use of PL on Vitis vinifera grapes may produce disruption in cellular structures enhancing the diffusion of anthocyanins from the skin of berries into the flesh (Fig 3a). This disruption has been documented by Fava et al (2011) after the use of C-type UV light on treated grape berries (Fig 3b). The damage is found to happen on the epicarp, one of the most outer layers of the grape skin. Nonetheless, the diffusion effect observed after the use of PL is not as intense as it was observed when other techniques were used as in the case of high hydrostatic pressure or the electron beam irradiation.
Fig 3. A) Effect of PL on the external appearance and on the pulp coloration (From Escott et al, 2017). B) Damage caused by UV-C light on the epicarp of Vitis labrusca grapes (From Fava et al, 2011).
The effectiveness, the cost associated and the potential to scale-up the pulsed light machinery would make this non-thermal emerging technology suitable for grape sanitization in the winemaking industry.
Material and Methods
Table grapes from a local greengrocer (Madrid) were used as samples. The preparation of the samples consisted on cutting the grapes crosswise in halves and placing them on glass Petri dishes faced down so that the skin, and therefore the contaminated part of the grape, was exposed to the PL treatment.
All the tests were carried out using the same pulsed light sterilization laboratory unit (Claranor, Avignon Cedex, France) with two xenon flash lamps of 254 mm, which produces maximum energy of 6 kVA. This equipment only allows to set the number of pulses and the exposure distance. In this case the exposure distance is fixed constant at 6 cm.
In order to assess how the number of pulses affect the microbial population of grapes, principally yeast and bacteria, the samples were exposed to a PL treatment of 1, 2 or 3 light pulses. Once the treatment was applied, the treated skin of the grape was put into contact with Yeast Peptone Dextrose (YPD) medium, leaving a mark, and incubated at 28° C for 48 h.
On the other hand, a 5-pulse treatment was performed to assess how PL affects the fermentation. The must was obtained manually by pressing all the grapes, and afterwards 30 mL of must were distributed in glass vials (50 mL capacity), with a needle to release CO2. The fermentation process was kept at constant 28° C.
Fermentation monitoring was followed up by measuring the percentage of ethanol produced, that is, the fermentative power of the treated and control samples.
Colour intensity and hue was determined using a UV – visible (UV – Vis) spectrophotometer 8453 from Agilent Technologies ™ (Palo Alto, CA, USA) with a photodiode array detector.
Thermal images were acquired using a FLIR E5 thermal imager (FLIR Systems France, Croissy-Beaubourg, France). The images were taken immediately after applying the PL treatment, that is, when the samples were still inside the chamber, and when the samples were outside the chamber.
Results and discussion
The microbial counts show that the prevalent microorganisms on grape’s skins are bacteria. The control (not treated) microbial population accounted for an estimated 3 log10 CFU per cm2 of skin mark. The use of 1 and 2 pulses reduces the population 1 log10 CFU/cm2 while the treatment with 3 pulses produces a reduction of 2 log10 CFU/cm2. This determination does not discriminate between yeast and bacteria populations (Fig 4a).
The temperature of the samples, measured right after the PL treatment, increases as a function of the number of pulses used. A sequence of 5 pulses entails an increase of 3 to 4º C on the surface of the grapes and the effect seems more intense towards the middle of the chamber. The temperature increase does not last after the samples are removed from the PL chamber where there is no thermal difference between treated and non-treated grapes (Fig 4b).
Fermentative wise, as expected, non-treated grapes have produced more ethanol than treated ones; up to 6% (v/v) ethanol was recorded after spontaneous fermentation whilst samples with 5-pulses treatment have produced less. The differences were more obvious during the first 4 days of fermentation. After this time, two out of three samples behaved differently with a final production of 4.7% ethanol (v/v) with lower production rate than the control and, only one did not produce ethanol during the span of the evaluation (<0.5% v/v) (Fig 4c)
Lastly, the colorimetric parameters measured did not allow to differentiate between treated and non-treated grapes at the beginning of the fermentation.
Fig 4. Effect of PL treatment on a) Ethanol yield (% v/v) after 5 pulses, b) thermal effect on grapes surface (right image after PL) and c) microbial population reduction after 2 pulses.
Pulsed light has proven to work as effective emerging technology for the sanitization of grapes since it is capable of reducing the microbial populations, both yeasts and bacteria, 1 to 2 log10 CFU/cm2. Some other advantages this technology has are: it can be considered non-thermal treatment since the increase of temperature observed was ca. 3 to 4º C; the time needed for the treatment is short and the cost is low. Further implementation of PL would include continuous treatment of destemmed grapes and a thorough evaluation of the reduction of SO2 addition.
Escott, C., Vaquero, C., del Fresno, J. M., Bañuelos, Loira, I., Han, S., Bi, Y., Morata, A., Suárez-Lepe, J. A. (2017). Pulsed Light Effect in Red Grape Quality and Fermentation. Food and Bioprocess Technology 10(8), 1540-1547.
Fava, J., Hodara, K., Nieto, A., Guerrero, S., Alzamora, S. M., Castro, M. A. (2011). Structure (micro, ultra, nano), color and mechanical properties of Vitis labrusca L. (grape berry) fruits treated by hydrogen peroxide, UV C irradiation and ultrasound. Food Research International, 44, 2938-2948. Morata, A., Loira, I., Vejarano, R., González, C., Callejo, M. J., Suárez-Lepe, J. A. (2017). Emerging preservation technologies in grapes for winemaking. Trends in Food Science and Technology 67, 36-43.