# Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of Bacillus subtilis in Whole and Skim Milk

^{1}

^{2}

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## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Preparation of the Spore Suspension of B. Subtilis

_{4}·H

_{2}O (Merck, Darmstadt, Germany) in 1 L of distilled water), which were incubated at 30 °C for up to 30 days. The formed spores were collected by adding 20 mL of sterile distilled water to the Roux bottles and scraping the surface with a Digralsky stick. Spore suspensions were pooled and washed four times in 15 mL cold sterile water by centrifugation at 10,000× g for 20 min at 4 °C using a Sigma 4K15 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The resulting sediment was then suspended in 30 mL of sterile distilled water and subjected to a heat treatment at 75 °C for 10 min to ensure the inactivation of vegetative cells. The resulting spore suspension was stored at 4 °C until use. One mL of the spore suspension was used to inoculate 1 L of milk to guarantee a minimum initial load close to 10

^{6}CFU/mL.

#### 2.2. Types of Milk Used

#### 2.2.1. Absorption Coefficient at 254 nm (α(254))

#### 2.2.2. Turbidity

#### 2.3. Application of UVC Radiation Treatments

#### 2.3.1. UVC Reactor Features

^{2}. Each of the lamps is protected by a 2 mm thick quartz tube (UV-Consulting Peschl, Geldo, Spain), leaving a radial space to circulate for the food of 1 mm. The internal section of the UVC reactor consists of different concentric cylinders (Figure 1): between sections A and B air circulates to cool the UVC lamp; between sections B and C the food matrix circulates in a layer of 1 mm; externally (between sections C and D) water flows to control the temperature of the treatments. In this survey, the temperature was adjusted to 20 °C.

#### 2.3.2. UVC Treatments

#### 2.3.3. Dosimetry Using a Potassium Iodide/Iodate Actinometer

_{MP}is the applied UVC dose of the UVC lamp in mJ/cm

^{2}; ΔOD

_{352}${\Delta \mathrm{OD}}_{352}$ is the difference of the absorbance of the irradiated sample and absorbance of the untreated (white) sample measured at 352 nm; V is the volume of the irradiated sample in L; I is the path length of light passing through the solution in cm (in the reactor used it was 0.1 cm); T is the temperature of the treatment, expressed in °C; A is the area of sample facing the light source (700 cm

^{2}) and 23,786.4 is the constant (mJ/cm

^{2}) specific for LMP-UVC lamps.

#### 2.3.4. Determination of the Type of Flow into the Reactor

^{3}), and η is dynamic viscosity of the fluid (Pa·s). The relative viscosity was obtained using an Ostwald 1293 model viscometer (CIVEQ, Mexico City, Mexico) and density with a hydrometer–aerometer densitometer HYDR-100-001 (Labbox, Vilassar de Dalt, Spain).

#### 2.3.5. Determination of the Effective Depth of UVC

_{254 nm}radiation is effective. This can be determined by applying the Lamber-Beer law by the equation:

_{0}correspond to the intensity of the UVC radiation expressed in mW/cm

^{2}; I corresponds to the intensity that the matrix receives at a given point, and I

_{0}corresponds to the intensity emitted by the reactor. The constant k corresponds to the α(254) of the matrix expressed in cm

^{−1}; c is the concentration of solutes capable of absorbing UVC of the sample expressed in mol/L, and finally, d is the depth or distance at that must UVC light pass through expressed in cm.

#### 2.4. UHPH Treatments

#### 2.5. Microbiological Analysis of the Samples and Calculation of the Lethality Achieved

_{2}PO

_{4}in 1 L of distilled water and pH adjusted to 7.4 (PBS, Panreac). Aliquots of each dilution were plated in Petri plates with trypticase soy agar medium enriched with 0.6% yeast extract (TSA-YE, Oxoid). The plates were incubated for 24 h at 37 °C.

_{0}is the initial amount of spores of B. subtilis present in the samples before the treatments and N is the number of remaining viable spores after the treatments, both expressed as in CFU/mL.

#### 2.6. Determination of the Inactivation Kinetics and Effect of Treatment Variables

#### 2.7. Determination of the UVC Four Decimal Reduction Value (4Duvc)

^{®}Excel [38]. The suitability of the adjustment was determined by determining the root mean square error (RMSE) value and the coefficient (R

^{2}). The 4D

_{uvc}value that is the UVC dose necessary to reduce 4 log CFU/mL of the initial load of the microorganisms tested was determined from the models that show the best adjustment.

#### 2.8. Statistical Analysis of the Results

## 3. Results

#### 3.1. Lethal effect of UVC Treatments on Bacillus Subtilis Spores

^{2}. Whole milk had the highest α(254), and in consequence, the distance that UVC radiation can penetrate without interference was shorter. Due to this reduced effective distance, increasing the number of passes (determined by the NET), increased the probability for a B. subtilis spore to get into this effective zone, increasing the real UVC received dose and consequently the effectiveness of the treatment.

#### 3.2. Lethal Effect of UHPH-UVC Combined Treatments on Bacillus Subtilis Spores

#### 3.3. Mathematical Modeling of Treatments

^{2}that should be as close as possible to 1; (2) the square root of the mean square error (RSME), that should be as low as possible, and finally, (3) the model where the number of variables is the most reduced. According to that, the best model was the Model 3 in both whole and skim milk. This model was a quadratic approach that takes into consideration NET and log C as variables. The most significant weight in this model was given by the NET value, if the log C variable is not taken into account. Therefore, the applied dose does not play a determining role in terms of the lethality achieved in the different UVC treatments. This fact confirms that increasing the NET increases the chances for one spore to get into the effective area of the reactor, increasing the time that this spore is exposed to a lethal dose.

#### 3.4. Kinetics of Inactivation and Estimation of the D_{uvc} Value for the Different UVC Treatments

^{2}value were used to determine the fitting accuracy.

## 4. Discussion

^{2}to reduce one log the initial load of B. subtilis in water while Sommer et al. [40] achieved this goal with a dose of 20–22 mJ/cm

^{2}in a liquid food model with an α(254) of 0.42 cm

^{−1}. Zhang et al. [41] obtained an inactivation of 0.81 log when treated water with a dose of 78 J/mL. Reverter-Carrión et al. [33] obtained a reduction of 5 log CFU/mL of spores of B. subtilis in PBS adjusted to an α(254) of 26 cm

^{−1}with caramel after applying a dose of 7.5 J/mL using the same reactor and the same strain of B. subtilis that the present study, where the maximum lethality achieved with a single pass was slightly higher than 1 log CFU/mL after a dose of 160 J/mL, due to the greater α(254) of milk.

^{2}in a reactor with an inner diameter of 1.6 mm and a flow with a Re number of 713, obtaining a reduction of 2.65 log CFU/mL in whole milk and 1.78 log in skim milk. These lethalities were similar to those obtained in the present study with a T3 treatment at a dose of 40 J/mL (4165.6 mJ/cm

^{2}) and 20 J/mL (2082.8 mJ/cm

^{2}) in whole and skim milk, respectively. As in the study of Choudhary et al. [18], in the present survey the efficiency of UVC treatments was higher in skim milk than in whole milk at small doses (20 J/mL and 40 J/mL), probably due to the α(254) of both types of milk and its turbidity value. However, these α(254) (220 cm

^{−1}and 170 cm

^{−1}for whole and skim milk, respectively) were lower than the observed ones in the milk samples used in this study (801 cm

^{−1}and 264 cm

^{−1}for whole and skim milk, respectively). This difference may be because, in this study, commercial milk was used, which in addition to the UHT treatment had suffered a homogenization treatment. The greater effectiveness observed in skim milk is also explained because of the estimated distance at which B. subtilis spores receive the most effective UVC radiation is higher than in whole milk, as can be seen in Table 2, and then when NET increases so do the probability of the presence of the spores within this zone.

^{2}on Bacillus cereus spores in a soybean drink with an α(254) of 163 cm

^{−1}, lower than that reported in the milks used in this study. The reactor used was a spiral tube with two different internal diameters (1.6 mm and 3.2 mm), generating two values of Re number that were higher than those achieved in this study (Table 1). Due to that, they reported reductions of 3.22 and 1.66 log CFU/mL with the smallest and largest diameter, respectively. In this study, reductions of 1.47 log were reported for a dose of 20 J/mL equivalent to 2,082.8 mJ/cm

^{2}in the T3 treatment and 4 log CFU/mL for a dose of 80 J/mL (equivalent to 8,323 mJ/cm

^{2}).

_{uvc}values for B. subtilis in whole and skim milk once the inactivation curve was fit to a Weibull with tail model. The most effective treatment in whole milk showed to be the T3, being necessary a dose of 91.2 J/mL to inactivate 4 log CFU/mL of spores, but the difference with the T2 treatments was small. On the contrary, T3 treatments showed to be much more efficient in skim milk, where a dose of 41.6 J/mL would be enough to achieve this goal. No previous references were found of D

_{uvc}values for B. subtilis or any other sporulated bacteria in milk. Crook et al. [2] reported a D

_{uvc}value of 0.73 J/mL for L. monocytogenes in milk, and of 0.556 J/mL for E. coli, but these bacteria are much more sensitive to UVC than B. subtilis [9]. Concerning other matrices, Nicholson et al. [45] calculated the D

_{uvc}value for spores of B. subtilis in phosphate-buffered saline solution, estimating it in 120 J/m

^{2}, what implies a dose of 480 J/m

^{2}to achieve a 4D reduction. In the present survey, for the most effective treatment (T3), the estimated 4D

_{uvc}would be above 43,300 J/m

^{2}. In that case, the least interfering matrix used there would justify the differences.

## 5. Conclusions

_{uvc}values of the most effective treatments are too high.

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

- Gouma, M.; Gayán, E.; Raso, J.; Condón, S.; Álvarez, I. UV-Heat Treatments for the Control of Foodborne Microbial Pathogens in Chicken Broth. Biomed. Res. Int.
**2015**, 2015, 1–12. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Crook, J.A.; Rossitto, P.V.; Parko, J.; Koutchma, T.; Cullor, J.S. Efficacy of ultraviolet (uv-c) light in a thin-film turbulent flow for the reduction of milkborne pathogens. Foodborne Pathog. Dis.
**2015**, 12, 506–513. [Google Scholar] [CrossRef] [PubMed] - Guerrero-Beltrán, J.A.; Barbosa-Cánovas, G.V. Advantages and Limitations on Processing Foods by UV Light. Food Sci. Technol. Int.
**2004**, 10, 137–147. [Google Scholar] [CrossRef] - Koutchma, T. Ultraviolet Light for Decontamination and Preservation of Beverages, Liquid Foods, and Ingredients. In Reference Module in Food Science; Elsevier: Amsterdam, The Netherlands, 2016. [Google Scholar]
- Tran, M.T.T.; Farid, M. Ultraviolet treatment of orange juice. Innov. Food Sci. Emerg. Technol.
**2004**, 5, 495–502. [Google Scholar] [CrossRef] - Coohill, T.P.; Sagripanti, J.-L. Overview of the Inactivation by 254 nm Ultraviolet Radiation of Bacteria with Particular Relevance to Biodefense. Photochem. Photobiol.
**2008**, 84, 1084–1090. [Google Scholar] - Choudhary, R.; Bandla, S. Ultraviolet Pasteurization for Food Industry. Int. J. Food Sci. Nutr. Eng.
**2012**, 2, 12–15. [Google Scholar] [CrossRef] - Hijnen, W.A.M.; Beerendonk, E.F.; Medema, G.J. Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: A review. Water Res.
**2006**, 40, 3–22. [Google Scholar] [CrossRef] - Gayán, E.; Álvarez, I.; Condón, S. Inactivation of bacterial spores by UV-C light. Innov. Food Sci. Emerg. Technol.
**2013**, 19, 140–145. [Google Scholar] [CrossRef] - Cilliers, F.P.; Gouws, P.A.; Koutchma, T.; Engelbrecht, Y.; Adriaanse, C.; Swart, P. A microbiological, biochemical and sensory characterisation of bovine milk treated by heat and ultraviolet (UV) light for manufacturing Cheddar cheese. Innov. Food Sci. Emerg. Technol.
**2014**, 23, 94–106. [Google Scholar] [CrossRef] - Matak, K.E.; Churey, J.J.; Worobo, R.W.; Sumner, S.S.; Hovingh, E.; Hackney, C.R.; Pierson, M.D. Efficacy of UV light for the reduction of Listeria monocytogenes in goat’s milk. J. Food Prot.
**2005**, 68, 2212–2216. [Google Scholar] [CrossRef] - Matak, K.E. Effects of UV Irradiation on the Reduction of Bacterial Pathogens and Chemical Indicators of Milk. Ph.D. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA, 2004. [Google Scholar]
- Yin, F.; Zhu, Y.; Koutchma, T.; Gong, J. Inactivation and potential reactivation of pathogenic Escherichia coli O157: H7 in bovine milk exposed to three monochromatic ultraviolet UVC lights. Food Microbiol.
**2015**, 49, 74–81. [Google Scholar] [CrossRef] [PubMed] - Lu, G.; Li, C.; Liu, P. UV inactivation of milk-related microorganisms with a novel electrodeless lamp apparatus. Eur. Food Res. Technol.
**2011**, 233, 79–87. [Google Scholar] [CrossRef] - Donaghy, J.; Keyser, M.; Johnston, J.; Cilliers, F.P.; Gouws, P.A.; Rowe, M.T. Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment. Lett. Appl. Microbiol.
**2009**, 49, 217–221. [Google Scholar] [CrossRef] [PubMed] - Engin, B.; Karagul Yuceer, Y. Effects of ultraviolet light and ultrasound on microbial quality and aroma-active components of milk. J. Sci. Food Agric.
**2012**, 92, 1245–1252. [Google Scholar] [CrossRef] - Krishnamurthy, K.; Demirci, A.; Irudayaraj, J.M. Inactivation of Staphylococcus aureus in milk using flow-through pulsed UV-light treatment system. J. Food Sci.
**2007**, 72, M233–M239. [Google Scholar] [CrossRef] - Choudhary, R.; Bandla, S.; Watson, D.G.; Haddock, J.; Abughazaleh, A.; Bhattacharya, B. Performance of coiled tube ultraviolet reactors to inactivate Escherichia coli W1485 and Bacillus cereus endospores in raw cow milk and commercially processed skimmed cow milk. J. Food Eng.
**2011**, 107, 14–20. [Google Scholar] [CrossRef] - Pereira, R.V.; Bicalho, M.L.; Machado, V.S.; Lima, S.; Teixeira, A.G.; Warnick, L.D.; Bicalho, R.C. Evaluation of the effects of ultraviolet light on bacterial contaminants inoculated into whole milk and colostrum, and on colostrum immunoglobulin G. J. Dairy Sci.
**2014**, 97, 2866–2875. [Google Scholar] [CrossRef] [Green Version] - Reinemann, D.J.; Gouws, P.; Cilliers, T.; Houck, K.; Bishop, J.R. New Methods for UV Treatment of Milk for Improved Food Safety And Product Quality. In Proceedings of the American Society of Agricultural and Biological Engineers, Portland, OR, USA, 9–12 July 2006; p. 1. [Google Scholar]
- Altic, L.C.; Rowe, M.T.; Grant, I.R. UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture. Appl. Environ. Microbiol.
**2007**, 73, 3728–3733. [Google Scholar] [CrossRef] - Rossitto, P.V.; Cullor, J.S.; Crook, J.; Parko, J.; Sechi, P.; Cenci-Goga, B.T. Effects of uv irradiation in a continuous turbulent flow uv reactor on microbiological and sensory characteristics of cow’s milk. J. Food Prot.
**2012**, 75, 2197–2207. [Google Scholar] [CrossRef] - Bintsis, T.; Litopoulou-Tzanetaki, E.; Robinson, R.K. Existing and potential applications of ultraviolet light in the food industry—A critical review. J. Sci. Food Agric.
**2000**, 80, 637–645. [Google Scholar] [CrossRef] - Koutchma, T.; Parisi, B.; Patazca, E. Validation of UV coiled tube reactor for fresh juices. J. Environ. Eng. Sci.
**2007**, 6, 319–328. [Google Scholar] [CrossRef] - Koutchma, T.; Forney, L.J.; Moraru, C.I. Ultraviolet Light in Food Technology: Principles and Applications; CRC Press: Boca Raton, FL, USA, 2009. [Google Scholar]
- López-Malo, A.; Palou, E. Ultraviolet Light and Food Preservation. In Novel Food Processing Technologies; CRC Press: Boca Raton, FL, USA, 2004; pp. 405–421. [Google Scholar]
- Gayán, E.; Monfort, S.; Álvarez, I.; Condón, S. UV-C inactivation of Escherichia coli at different temperatures. Innov. Food Sci. Emerg. Technol.
**2011**, 12, 531–541. [Google Scholar] [CrossRef] - Pereda, J.; Ferragut, V.; Quevedo, J.M.; Guamis, B.; Trujillo, A.J. Effects of ultra-high-pressure homogenization treatment on the lipolysis and lipid oxidation of milk during refrigerated storage. J. Agric. Food Chem.
**2008**, 56, 7125–7130. [Google Scholar] [CrossRef] [PubMed] - Picart, L.; Thiebaud, M.; René, M.; Guiraud, J.P.; Cheftel, J.C.; Dumay, E. Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments. J. Dairy Res.
**2006**, 73, 454–463. [Google Scholar] [CrossRef] - Hayes, M.G.; Fox, P.F.; Kelly, A.L. Potential applications of high pressure homogenisation in processing of liquid milk. J. Dairy Res.
**2005**, 72, 25–33. [Google Scholar] [CrossRef] - Poliseli-Scopel, F.H.; Hernández-Herrero, M.; Guamis, B.; Ferragut, V. Sterilization and aseptic packaging of soymilk treated by ultra high pressure homogenization. Innov. Food Sci. Emerg. Technol.
**2014**, 22, 81–88. [Google Scholar] [CrossRef] - Amador Espejo, G.G.; Hernández-Herrero, M.M.; Juan, B.; Trujillo, A.J. Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization. Food Microbiol.
**2014**, 44, 204–210. [Google Scholar] [CrossRef] - Reverter-Carrión, L.; Sauceda-Gálvez, J.N.; Codina-Torrella, I.; Hernández-Herrero, M.M.; Gervilla, R.; Roig-Sagués, A.X. Inactivation study of Bacillus subtilis, Geobacillus stearothermophilus, Alicyclobacillus acidoterrestris and Aspergillus niger spores under Ultra-High Pressure Homogenization, UV-C light and their combination. Innov. Food Sci. Emerg. Technol.
**2018**, 48, 258–264. [Google Scholar] [CrossRef] - Asociación Española de Normalización y Certificación. Quantitative Suspension Test for the Evaluation of Sporicidal Activity of Chemical Disinfectants Used in Food, Industrial, Domestic and Institutional Areas-Test Method and Requirements; AENOR: Madrid, Spain, 2002. [Google Scholar]
- Rahn, R.O. Potassium Iodide as a Chemical Actinometer for 254 nm Radiation: Use of lodate as an Electron Scavenger. Photochem. Photobiol.
**1997**, 66, 450–455. [Google Scholar] [CrossRef] - Linden, K.G.; Mofidi, A.A. Disinfection Efficiency and Dose Measurement of Polychromatic Uv Light; IWA Publishing: London, UK, 2004. [Google Scholar]
- Müller, A.; Günthner, K.A.; Stahl, M.R.; Greiner, R.; Franz, C.M.A.P.; Posten, C. Effect of physical properties of the liquid on the efficiency of a UV-C treatment in a coiled tube reactor. Innov. Food Sci. Emerg. Technol.
**2015**, 29, 240–246. [Google Scholar] [CrossRef] - Geeraerd, A.H.; Valdramidis, V.P.; Van Impe, J.F. GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves. Int. J. Food Microbiol.
**2005**, 102, 95–105. [Google Scholar] [CrossRef] [PubMed] - Chang, J.C.H.; Ossoff, S.F.; Lobe, D.C.; Dorfman, M.H.; Dumais, C.M.; Qualls, R.G.; Johnson, J.D. UV inactivation of pathogenic and indicator microorganisms. Appl. Environ. Microbiol.
**1985**, 49, 1361–1365. [Google Scholar] [PubMed] - Sommer, R.; Cabaj, A.; Sandu, T.; Lhotsky, M. Measurement of UV radiation using suspensions of microorganisms. J. Photochem. Photobiol. B Biol.
**1999**, 53, 1–6. [Google Scholar] [CrossRef] - Zhang, Y.; Zhou, L.; Zhang, Y. Investigation of UV-TiO
_{2}photocatalysis and its mechanism in Bacillus subtilis spore inactivation. J. Environ. Sci.**2014**, 26, 1943–1948. [Google Scholar] [CrossRef] [PubMed] - Bandla, S.; Choudhary, R.; Watson, D.G.; Haddock, J. UV-C treatment of soymilk in coiled tube UV reactors for inactivation of Escherichia coli W1485 and Bacillus cereus endospores. LWT-Food Sci. Technol.
**2012**, 46, 71–76. [Google Scholar] [CrossRef] - Lagrée, P.-Y. Boundary layer separation and asymptotics from 1904 to 1969. Comptes Rendus Mécanique
**2017**, 345, 613–619. [Google Scholar] [CrossRef] - Pereda, J.; Ferragut, V.; Quevedo, J.M.; Guamis, B.; Trujillo, A.J. Effects of ultra-high pressure homogenization on microbial and physicochemical shelf life of milk. J. Dairy Sci.
**2007**, 90, 1081–1093. [Google Scholar] [CrossRef] - Nicholson, W.L.; Galeano, B. UV resistance of Bacillus anthracis spores revisited: Validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne. Appl. Environ. Microbiol.
**2003**, 69, 1327–1330. [Google Scholar] [CrossRef] - Sauceda-Gálvez, J.N.; Roca-Couso, R.; Martinez-Garcia, M.; Hernández-Herrero, M.M.; Gervilla, R.; Roig-Sagués, A.X. Inactivation of ascospores of Talaromyces macrosporus and Neosartorya spinosa by UV-C, UHPH and their combination in clarified apple juice. Food Control
**2019**, 98, 120–125. [Google Scholar] [CrossRef]

**Figure 1.**Structure of the UVC reactor used in this survey. A: UVC lamp; B: quartz protection glass; C and D stainless steel tubes. Section A–B: air circulation; section B–C: food matrix circulation area; section C–D: water cooling system.

**Figure 2.**Lethality of B. subtilis spores inoculated in whole milk caused by different UVC treatments consisting of different doses (J/mL) and number of entries to the reactor (T1, T2 and T3). Results are expressed as log CFU/mL ± standard deviation. Different lowercase letters in the columns indicate significant differences (p < 0.05) between processes with the same flow rate (T1, T2 and T3, see Table 1), but different doses of UVC. Different capital letters indicate significant differences (p < 0.05) between processes with different flowrate, but the same dose of UVC.

**Figure 3.**Lethality of B. subtilis spores inoculated in skim milk caused by different UVC treatments consisting of different doses (J/mL) and number of entries to the reactor (T1, T2 and T3 Results are expressed as log CFU/mL ± standard deviation. Different lowercase letters in the columns indicate significant differences (p < 0.05) between processes with the same flow rate (T1, T2 and T3, see Table 1), but different doses of UVC. Different capital letters indicate significant differences (p < 0.05) between processes with different flowrate, but the same dose of UVC.

**Figure 4.**Lethality of B. subtilis spores inoculated in whole milk from UHPH treatments at 200 MPa and UVC treatments at different doses (J/mL) and their combination with different number of passes (T1 and T3). Results are expressed as log CFU/mL ± standard deviation. Different lowercase letters in the columns indicate significant differences (p < 0.05) between processes with the same flow rate (T1 and T3, see Table 1), but different doses of UVC. Different capital letters indicate significant differences (p < 0.05) between processes with different flowrate, but the same UVC dose.

**Figure 5.**Lethality of B. subtilis spores inoculated in skim milk caused by UHPH treatments at 200 MPa and UVC treatments at different doses (J/mL) and their combination with different number of passes (T1 and T3). Results are expressed as log CFU/mL ± standard deviation. Different lowercase letters in the columns indicate significant differences (p < 0.05) between processes with the same flow rate (T1 and T3, see Table 1), but different doses of UVC. Different capital letters indicate significant differences (p < 0.05) between processes with different flowrate, but the same dose of UVC.

**Figure 6.**Surviving spores of B. subtilis inoculated in whole milk after T2 (

**A**) and T3 (

**B**) treatments at different UVC doses and adjustment of the data to the Weibull with tail inactivation model using the tool GInaFIT. (◊) indicates the experimental data and the black line the estimated curve once adjusted to the model.

**Figure 7.**Surviving spores of B. subtilis inoculated in skim milk after T2 (

**A**) and T3 (

**B**) treatments at different UVC doses and adjustment of the data to the Weibull with tail inactivation model using the tool GInaFIT. (◊) indicates the experimental data and the black line the estimated curve once adjusted to the model.

Treatment | Flow Rate (mL/s) | NET | RPM | Retention Time (s) | UVC Dose (J/mL) | Reynolds Number |
---|---|---|---|---|---|---|

T1 | ≤2.9 | 2 | 21 | 24 | 20 | 52 |

12 | 48 | 40 | 26 | |||

6 | 96 | 80 | 13 | |||

5 | 120 | 100 | 10 | |||

3 | 144 | 120 | 9 | |||

1.5 | 192 | 160 | 7 | |||

T2 | 41.9 | 30 | 300 | 360 | 20 | 658 |

60 | 720 | 40 | ||||

121 | 1440 | 80 | ||||

151 | 1800 | 100 | ||||

181 | 2160 | 120 | ||||

241 | 2880 | 160 | ||||

T3 | 64.6 | 47 | 500 | 360 | 20 | 1015 |

93 | 720 | 40 | ||||

186 | 1440 | 80 | ||||

233 | 1800 | 100 | ||||

279 | 2160 | 120 | ||||

372 | 2880 | 160 |

**Table 2.**Calculation of the effective distance, expressed in mm where the dose received is close to 1 mJ/cm

^{2}.

Matrix | Doses (mJ/cm^{2}) | Effective Distance (mm) |
---|---|---|

Whole milk | 1.034 | 0.02 |

0.861 | 0.02 | |

Skim milk | 1.005 | 0.06 |

0.946 | 0.06 |

**Table 3.**Mathematical models depending on the lethality UVC treatments on the B. subtilis spores obtained in whole and skim milk.

Models | R^{2} | RMSE | |
---|---|---|---|

Whole milk | |||

1 | $\mathrm{Lethality}=3.56-0.71\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.015\times \mathrm{UVC}+0.05\text{}\times \text{}\mathrm{R}$ | 0.83 | 0.70 |

2 | $\mathrm{Lethality}=19.17-3.02\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.012\times \mathrm{NET}$ | 0.83 | 0.69 |

3 | $\mathrm{Lethality}=6.557-1.002\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.029\text{}\times \text{}\mathrm{NET}-0.00005\text{}\times {\text{}\mathrm{NET}}^{2}$ | 0.94 | 0.40 |

4 | $\mathrm{Lethality}=4.86-0.77\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.0048\text{}\times \text{}\mathrm{UVC}+0.029\times \mathrm{FR}-0.0078\text{}\times \text{}\mathrm{NET}$ | 0.86 | 0.63 |

5 | $\mathrm{Lethality}=2.696-0.39\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.0012\text{}\times \text{}\mathrm{UVC}+0.009\text{}\times \text{}\mathrm{FR}+0.027\text{}\times \text{}\mathrm{NET}-0.00005\text{}\times {\text{}\mathrm{NET}}^{2}$ | 0.94 | 0.40 |

Skim milk | |||

1 | $\mathrm{Lethality}=-14.74+2.79\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.014\text{}\times \text{}\mathrm{UVC}+0.017\text{}\times \text{}\mathrm{FR}$ | 0.91 | 0.52 |

2 | $\mathrm{Letalidad}=-10.1+2.12\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.0086\text{}\times \text{}\mathrm{NET}$ | 0.90 | 0.53 |

3 | $\mathrm{Lethality}=-4.2+0.97\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.023\text{}\times \text{}\mathrm{NET}-0.00004\text{}\times {\text{}\mathrm{NET}}^{2}$ | 0.95 | 0.36 |

4 | $\mathrm{Lethality}=-12.53+2.47\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.0077\text{}\times \text{}\mathrm{UVC}+0.0072\text{}\times \text{}\mathrm{FR}+0.00472\text{}\times \text{}\mathrm{NET}$ | 0.92 | 0.48 |

5 | $\mathrm{Lethality}=-4.367-0.92\text{}\times {\text{}\mathrm{Log}}_{\mathrm{C}}+0.0049\text{}\times \text{}\mathrm{UVC}+0.01\text{}\times \text{}\mathrm{FR}+0.02\text{}\times \text{}\mathrm{NET}-0.00004\text{}\times {\text{}\mathrm{NET}}^{2}$ | 0.96 | 0.34 |

**Table 4.**Adjustment to kinetic model and estimation of the corresponding 4D

_{uvc}value (Four decimal reduction value: UVC dose necessary to reduce 4 log CFU/mL) of B. subtilis spores in whole and skim milk according to the UVC treatments applied using the GInaFIT tool.

Treatment | Type of Milk | Inactivation Model | RMSE | R^{2} | 4D_{uvc} (J/mL) |
---|---|---|---|---|---|

T1 | Skim | Log-Linear Regression | 0.2433 | 0.592 | - |

Log-Linear with tail | 0.2269 | 0.654 | - | ||

Weibull | 0.2087 | 0.707 | - | ||

Biphasic | 0.2101 | 0.710 | - | ||

Whole | Log-Linear Regression | 0.1143 | 0.242 | - | |

Log-Linear with tail | 0.2331 | 0.202 | - | ||

Weibull | 0.2318 | 0.210 | - | ||

Biphasic | 0.2330 | 0.224 | - | ||

T2 | Skim | Log-Linear Regression | 0.7050 | 0.769 | - |

Log-Linear with tail | 0.5092 | 0.883 | - | ||

Weibull with tail | 0.4491 | 0.911 | 89.6 | ||

Biphasic | 0.4749 | 0.901 | 116.8 | ||

Whole | Log-Linear Regression | 0.5441 | 0.839 | - | |

Log-Linear with tail | 0.2731 | 0.960 | - | ||

Weibull with tail | 0.2375 | 0.970 | 94.4 | ||

Biphasic | 0.0646 | 0.966 | 126.4 | ||

T3 | Skim | Log-Linear Regression | 0.6673 | 0.811 | - |

Log-Linear with tail | 0.3124 | 0.952 | 44.8 | ||

Weibull with tail | 0.2294 | 0.975 | 43.2 | ||

Biphasic | 0.2059 | 0.980 | 41.6 | ||

Whole | Log-Linear Regression | 0.7465 | 0.705 | - | |

Log-Linear with tail | 0.3263 | 0.571 | - | ||

Weibull with tail | 0.4637 | 0.892 | 91.2 | ||

Biphasic | 0.2157 | 0.464 | 97.6 |

**Table 5.**Effect of ultra-high pressure homogenization (UHPH) treatments on the absorption coefficient and the turbidity of whole and skim milk.

Optical Parameter | Skim Milk | Whole Milk | ||
---|---|---|---|---|

Before UHPH | After UHPH | Before UHPH | After UHPH | |

α(254) (cm^{−1}) | 264 ± 0.03 | 412 ± 0.02 | 801 ± 0.02 | 1012 ± 0.02 |

Turbidity (NTU) | 18,416 ± 0.04 | 21,630 ± 0.03 | 77,967 ± 0.01 | 102,458 ± 0.01 |

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Martinez-Garcia, M.; Sauceda-Gálvez, J.N.; Codina-Torrella, I.; Hernández-Herrero, M.M.; Gervilla, R.; Roig-Sagués, A.X.
Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of *Bacillus subtilis* in Whole and Skim Milk. *Foods* **2019**, *8*, 539.
https://doi.org/10.3390/foods8110539

**AMA Style**

Martinez-Garcia M, Sauceda-Gálvez JN, Codina-Torrella I, Hernández-Herrero MM, Gervilla R, Roig-Sagués AX.
Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of *Bacillus subtilis* in Whole and Skim Milk. *Foods*. 2019; 8(11):539.
https://doi.org/10.3390/foods8110539

**Chicago/Turabian Style**

Martinez-Garcia, María, Jezer N. Sauceda-Gálvez, Idoia Codina-Torrella, Mª Manuela Hernández-Herrero, Ramón Gervilla, and Artur X. Roig-Sagués.
2019. "Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of *Bacillus subtilis* in Whole and Skim Milk" *Foods* 8, no. 11: 539.
https://doi.org/10.3390/foods8110539