foods Article Drastic Microbial Count Reduction in Soy Milk Using Continuous Short-Wave Ultraviolet Treatments in a Tubular Annular Thin Film UV-C Reactor María Martínez-García 1 , Jezer N. Sauceda-Gálvez 1 , Idoia Codina-Torrella 2 , María Manuela Hernández-Herrero 1 , Ramón Gervilla 3 and Artur X. Roig-Sagués 1, * 1 Centre d’Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), TECNIO CERTA-UAB, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Travessera dels Turons S/N, 08193 Barcelona, Spain; martinez_maria91@hotmail.com (M.M.-G.); jezer_noe@hotmail.com (J.N.S.-G.); manuela.hernandez@uab.cat (M.M.H.-H.) 2 Departament d’Enginyeria Agroalimentària i Biotecnologia, Edifici D4C, Esteve Terradas, 8, 08860 Castelldefels, Spain; idoia.codina@upc.edu 3 SPTA-Servei Planta Tecnologia Aliments, Universitat Autònoma de Barcelona, c/de l’Hospital S/N, 08193 Barcelona, Spain; ramon.gervilla@uab.cat * Correspondence: arturxavier.roig@uab.cat Abstract: Vegetative cells of Listeria monocytogenes and Escherichia coli and spores of Bacillus subtilis and Aspergillus niger were inoculated in soy milk at an initial concentration of ≈5 log CFU/mL. Inoculated and control (non-inoculated) soy milk samples were submitted to three types of treatments using a tubular annular thin film short-wave ultraviolet (UV-C) reactor with 1 mm of layer thickness. Treatments applied depended on the flow rate and the number of entries to the reactor, with UV-C doses ranging from 20 to 160 J/mL. The number of entries into the reactor tube (NET) was established as the most determining parameter for the efficiency of the UV-C treatments. Conidiospores of A. niger were reported as the most resistant, followed by B. subtilis spores, while vegetative cells Citation: Martínez-García, M.; were the most sensible to UV-C, with Listeria monocytogenes being more sensible than Escherichia coli. Sauceda-Gálvez, J.N.; Treatments of just 80 J/mL were needed to achieve a 5 log CFU/mL reduction of L. monocytogenes Codina-Torrella, I.; while 160 J/mL was necessary to achieve a similar reduction for A. niger spores. Hernández-Herrero, M.M.; Gervilla, R.; Roig-Sagués, A.X. Drastic Keywords: Listeria monocytogenes; Escherichia coli; Bacillus subtilis; Aspergillus niger; soy milk; short- Microbial Count Reduction in Soy wave ultraviolet (UV-C) Milk Using Continuous Short-Wave Ultraviolet Treatments in a Tubular Annular Thin Film UV-C Reactor. Foods 2023, 12, 3813. https:// doi.org/10.3390/foods12203813 1. Introduction The consumption of soy products has increased in the last years due to the health Academic Editor: Joao Laurindo benefits of soybean compounds, such as reduced LDL cholesterol and protection against Received: 5 September 2023 osteoporosis [1]. Soy milk is the liquid extract obtained by mixing and grounding soybeans Revised: 9 October 2023 with water, resulting in a product with a similar appearance to cow’s milk, although with a Accepted: 13 October 2023 different composition [2]. Commercial soy milk is usually stabilized using thermal methods. Published: 17 October 2023 These processes may cause undesirable changes in nutritional value, such as the destruction of essential amino acids and vitamins, as well as modification of sensorial properties [3,4]. Nonthermal technologies, such as short-wave ultraviolet (UV-C) radiation, are cur- Copyright: © 2023 by the authors. rently being investigated as an alternative to heat pasteurization processes. UV-C showed Licensee MDPI, Basel, Switzerland. good capability to reduce or eliminate spoilage and pathogenic microorganisms in foods [5]. This article is an open access article The application using reactors that allows for processing fluid foods in a continuous process distributed under the terms and increased interest in this technology as an alternative to thermal treatments, as it is easy to conditions of the Creative Commons use, does not generate chemical residues, and can be effective at a lower cost [6,7]. More- Attribution (CC BY) license (https:// over, it preserves the nutritional and quality properties of foods better [8]. UV-C radiation creativecommons.org/licenses/by/ forms dimers of pyrimidine in the exposed microorganisms, blocking DNA duplication 4.0/). Foods 2023, 12, 3813. https://doi.org/10.3390/foods12203813 https://www.mdpi.com/journal/foods Foods 2023, 12, 3813 2 of 16 what kills microbial cells [9,10], including bacterial spores that cannot be eliminated using other nonthermal technologies [11,12]. The microbiocidal effect of UV-C has been evaluated in different types of food, such as fruit juices, vegetable smoothies, liquid egg, and milk, demonstrating effectiveness similar to thermal pasteurization [13–16]. Few studies have been conducted on soy milk, although the potential of this technology to eliminate both vegetative and sporulating bacteria has been demonstrated [17,18]. However, soy milk presents a high UV-C absorption coefficient, which is a limiting factor that reduces the possibility that UV-C radiation reaches all the microorganisms present in the foodstuff and, consequently, treatment effectiveness [19,20]. To increase the efficiency of UV-C treatments using reactors, both the UV-C radiation dose and the flow pattern (laminar flow or turbulent flow) must be correctly adjusted. The absorbed UV-C radiation dose is influenced by both the distance from the source of the UV-C radiation during exposure and the presence of food particles [8], but the presence of food particles may also interfere. Different designs of continuous flow UV-C reactors have been described in the literature. Recently, Hirt et al. [21] compared the overall efficacy of inactivation in Saccharomyces cerevisiae between different designs of UV-C reactors (annular thin film, serpentine, and coiled tube reactors), concluding that the inactivation was less efficient when a laminar flow was generated in the reactor since vortices are needed to interchange the portion of the volume directly facing the UV-C source. Another proposed strategy is to reduce the space between the UV-C source (lamp) and the farthest particle of the matrix, reducing the path length by creating a thin film around the UV-C source. However, this system makes liquid flow under laminar flow conditions, which is an inconvenience when treating high absorbance coefficient matrices. Reverter-Carrión et al. [22] reported that in annular thin film UV-C reactors, efficacy against spores of Bacillus subtilis, Geobacillus stearothermophilus, Alicyclobacillus acidoterrestris, and Aspergillus niger increased when UV-C treatments were applied through several cycles. This strategy allowed for overcoming the inconvenience of the laminar flow generated in this type of UV-C reactor. More recently, Martinez et al. [13] introduced the concept of the number of entries in the reactor tube (NET), consisting of applying several cycles, as a parameter to establish the efficiency of the UV-C treatments in reactors with laminar flow, reporting that increasing NET improves the efficacy of treatment. This study aimed to evaluate the effectiveness of UV-C treatments consisting of ap- plying a different number of passes (cycles) to different microorganisms (spores of Bacillus subtilis and Aspergillus niger, and vegetative cells of Escherichia coli and Listeria monocyto- genes) inoculated in soy milk. To determine the importance of the variables on the efficacy of the treatments, some mathematical models were proposed. 2. Materials and Methods 2.1. Soy Milk Physicochemical Characteristics An organic commercial UHT-treated soy milk containing 14% soybean was used to evaluate the lethal effect of the treatments tested. The coefficient of absorbance at 254 nm (AC254 ) was measured with a Nanophotometer Pearl spectrophotometer (IMPLEN, München, Germany), and the result was 500 ± 0.1 cm−1 . pH was measured directly using a pH meter (pH basic 20, Crison, Barcelona, Spain), giving a mean value of 6.34 ± 0.01; turbidity was measured with a portable EPA 2100Q turbidimeter (HACH, Hospitalet de Llobregat, Spain), and the value obtained was 35,983 ± 0.041 NTU (nephelometric turbidity units). The relative viscosity was obtained using an Ostwald 1293 model viscometer (CIVEQ, Mexico City, Mexico) 1.047 ± 0.003 mPa·s. Density was determined with a hydrometer–aerometer–densitometer HYDR-100-001 (Labbox, Vilassar de Dalt, Spain), and the value obtained was 1.014 ± 0.000 g/mL. 2.2. UV-C Equipment In this survey, a UV-C reactor was used (European Patent EP 2965766-A1, UV-Therm, Ypsicon S.L., Barcelona, Spain). The reactor consists of two low-pressure mercury UV- Foods 2023, 12, x FOR PEER REVIEW 3 of 17 Foods 2023, 12, 3813 2.2. UV-C Equipment 3 of 16 In this survey, a UV-C reactor was used (European Patent EP 2965766-A1, UV-Therm, Ypsicon S.L., Barcelona, Spain). The reactor consists of two low-pressure mercury UV-C lamps C lamps(UV-Consulting (UV-Consulting Peschl Spain, Peschl Castellón, Spain, Spain), Castellón, with 55 Spain), W of with 55total W ofelectrical power total electrical and 41 and power mW/cm 2 41 mW/cm 2 of irradiance, that arethat of irradiance, protected by a 2 mm are protected by a thick 2 mmquartz tube (UV-Con- thick quartz tube (UV- sulting Peschl, Consulting Geldo, Peschl, Spain). Geldo, TheThe Spain). reactor is fed reactor is fedusing usinga aperistaltic peristalticpump pump(Flowmaster (Flowmaster FMT300, FMT300, ISMATEC ISMATEC Lab. Lab. GmbH, GmbH, Wertheim-Mondfeld, Wertheim-Mondfeld,Germany) Germany)connected connected through through aa pipe pipe of of silicone, silicone, having having aa total total volume volume capacity capacityof of 673 673 mL. mL. Figure Figure 11 shows shows the the different different concentric cylinders that forms concentric forms the the UV-C UV-Creactor: reactor:airaircirculates circulatesbetween betweensections sectionsAAandandB to B cool to coolthe UV-C the UV-C lamp; lamp;food foodmatrix matrixcirculates circulatesbetween betweensections sections BB and and C in a layer layer of of11mm; mm; water flows water flows between between sections sections CCand andDDtotocontrol controlthethetemperature temperature of of the thetreatments. treatments. InInthis this survey, the the temperature temperature was adjusted ◦ C. adjusted to 20 °C. survey, Figure1. Figure UV-C reactor 1. UV-C reactor structure. structure. A: A:UV-C UV-C lamp; lamp; B: B: quartz quartzprotection protectionglass; glass;CCand andD:D:stainless stainlesssteel steel tubes. Section A–B: air circulation; section B–C: food matrix circulation area; section C–D: water tubes. Section A–B: air circulation; section B–C: food matrix circulation area; section C–D: water coolingsystem cooling system[13]. [13]. 2.3. UV-C Treatments 2.3. UV-C Treatments Table 1 shows the features of the treatments applied (UV-C dose, flow rate, retention Table 1 shows the features of the treatments applied (UV-C dose, flow rate, retention (exposure) time, Reynolds number, and number of entries in the reactor tube (NET)). All (exposure) time, Reynolds number, and number of entries in the reactor tube (NET)). All treatments were applied at 20 ◦ C. NET was calculated by the following formula: treatments were applied at 20 °C. NET was calculated by the following formula: Table 1. Characteristics of the treatments along with their ∙ ∙ variables. 𝑁𝐸𝑇 . (1) Flow Rate 1 2 Retention Dose Treatments where t corresponds NET to the treatment time,RPM expressed inTime seconds; Reynolds (mL/s) (s) L to(J/mL) the number of lamps in the reactor (2); CA the flow rate at which the matrix circulates, expressed as mL/s; and 43 12 10 105 VC to the total volume of food circulating through 21 the circuit, 24 expressed 20 as mL (70 52 mL). Reynolds number (Re) was determined 12 according to 48 the formula described 40 by Müller 26 et al. [23]: T1 <2.9 2 6 96 80 13 5 ∙ ∙ 120 100 10 𝑅𝑒 3 144 120 9 (2) 1.5 192 160 7 where d is the diameter of the tube 15 (m), v is the velocity (m/s), 180 ρ is the fluid 10 density (kg/m3), and η is the dynamic viscosity 30 of the fluid (Pa·s). Flow 360is considered20laminar when the value of Re is 2100 or lower. 60 720 40 T2 42 121 300 1440 80 658 151 1800 100 Table 1. Characteristics of the treatments along with their variables. 181 2160 120 241 Treatments Flow Rate (mL/s) NET RPM Retention Time (s) Dose 1 2 2880 160(J/mL) Reynolds 23 180 10 46 43 12360 10 20 105 93 21 24720 20 40 52 T3 62 186 12 500 481440 40 80 26 1015 T1 <2.9 2332 6 961800 100 80 13 279 2160 120 5 120 100 10 372 2880 160 1 3 144 120 9 NET: number of entries in the reactor tube. 2 RPM: revolutions per minute. Foods 2023, 12, 3813 4 of 16 t· L·C A NET = . (1) VC where t corresponds to the treatment time, expressed in seconds; L to the number of lamps in the reactor (2); CA the flow rate at which the matrix circulates, expressed as mL/s; and VC to the total volume of food circulating through the circuit, expressed as mL (70 mL). Reynolds number (Re) was determined according to the formula described by Müller et al. [23]: d·v·ρ Re = (2) η where d is the diameter of the tube (m), v is the velocity (m/s), ρ is the fluid density (kg/m3 ), and η is the dynamic viscosity of the fluid (Pa·s). Flow is considered laminar when the value of Re is 2100 or lower. 2.4. Dosimetry The UV-C energy emitted by the lamp per volume unit at 254 nm was measured using the chemical iodide/iodate actinometer developed and described by Rahn [24] using a potassium iodide solution passed through the UV-C reactor. Its absorbance was then measured at 352 nm using a Nanophotometer Pearl spectrophotometer (IMPLEN) after diluting the sample with distilled water ten times. The formula proposed by Linden and Mofidi [25] was used to calculate the power of the UV-C reactor lamps: (∆OD352 )(V )(23, 786.4) H MP = (3) ( I )(1 + 0.02( T − 20.7)) A where HMP is the applied UV-C dose of the UV-C lamp in mJ/cm2 ; ∆OD352 is the difference between 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 cm2 ); and 23,786.4 is the constant (mJ/cm2 ) specific for LMP-UV-C lamps. 2.5. Effective Depth of UV-C To determine the depth at which the action of the UV-C254 nm radiation was most effective (lethal zone), the Lambert–Beer equation was applied: I = I0 ·e−k·c·d (4) where I and I0 correspond to the intensity of the UV-C radiation, expressed in mW/cm2 ; I corresponds to the intensity that the matrix receives at a given point and I0 corresponds to the intensity emitted by the reactor. The constant k corresponds to the AC254 of the matrix, expressed in cm−1 ; c is the concentration of solutes capable of absorbing UV-C of the sample, expressed in mol/L; finally, d is the depth or distance at the must UV-C light passes through expressed in cm. According to Koutchma [19], to inactivate microorganisms, they must be exposed to a dose of at least 1 mJ/cm2 to cause irreversible genetic damage, and consequently, the limit of the effective distance is where the dose achieved is 1 mJ/cm2 . The width of the lethal zone varies depending on the matrix and its absorbance. For determining the exposed dose, a particle size of 1 µm and a matrix pass of 1000 µm were considered to determine this zone. Moreover, the number of times that a particle passes through the lethal zone (NEL value) was calculated from the probability that any particle of the matrix passes through the lethal zone using the following formula: d NEL = · NET (5) AT Foods 2023, 12, 3813 5 of 16 where NET is the number of entries in the reactor tube, d is the width of the lethal zone, expressed in µm, and AT is the width of the space through which the matrix passes, expressed in µm (in that case, 1000 µm). The ratio d/AT is the probability that a particle can enter the lethal zone. 2.6. Preparation of Microbial Strains and Inoculation in Soy Milk Samples Strains of Escherichia coli (CECT423), Listeria monocytogenes (CECT4031), Bacillus subtilis (CECT 4002), and Aspergillus niger (CECT 2574) were obtained from the Spanish Type Culture Collection (CECT, Universitat de València, Valencia, Spain) as a freeze-dried culture. E. coli and L. monocytogenes were first recovered in tryptone soy broth (Oxoid, Basingstoke, UK) incubated overnight (18–20 h) at 37 ◦ C and later streaked on a Petri plate with tryptone soy agar (Oxoid, Basingstoke, UK) and incubated overnight (18–20 h) at 37 ◦ C. After testing the purity of cultures, some colonies were transferred to 0.9% (w/v) saline solution, and density was adjusted to a number 1.0 of McFarland scale for L. monocytogenes, and 1.5 for E. coli (around 108 CFU/mL) with a Densimat (Biomérieux, Crappone, France). A modification of the UNE EN 13704:2019 procedure [26] was used to obtain the B. subtilis spores suspension. Briefly, the lyophilized culture was rehydrated in 10 mL of glucose and tryptone broth (TGB: 2.5 g of yeast extract (Oxoid), 5 g of tryptone (Oxoid), 1 g of glucose (Sigma-Aldrich, St. Louis, MI, USA), in 1 L of distilled water, pH adjusted to 7.2), and incubated for 24 h at 30 ◦ C. Two milliliters of this culture was transferred to Roux bottles containing yeast extract agar (MYA: 10 g of meat extract (Oxoid), 2 g of yeast extract (Oxoid), 15 g of agar (Oxoid), and 0.04 g of MnSO4 ·H2 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 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. A modification of the UNE-EN 1650:2020 standard procedure [27] was used to prepare the A. niger spore suspensions. The freeze-dried culture was rehydrated in 9 mL of tryptic soy broth (TSB) (Oxoid) and incubated at 30 ◦ C for 24 h. This cultured broth was used to inoculate Petri dishes with potato dextrose agar (PDA) (Oxoid) and was incubated at 30 ◦ C for 7 days. After that, 3 mL of nutrient broth (NB) (Oxoid) were added to the Petri dishes, and the surface was scraped out with a Digralsky stick. One milliliter of the resulting suspension was transferred into 9 mL of NB and incubated at 30 ◦ C for 24 h. Subsequently, 2 mL of this culture was inoculated into Roux flasks containing PDA and incubated at 30 ◦ C until the culture color changed to black (maximum sporulation rate). Conidiospores were recovered with 10 mL of sterile distilled water with 0.05% (v/v) Tween 80 using a Digralsky stick. The obtained suspension was filtered in a sterile Büchner funnel with a pore size of 45 µm (LABBOX, SL, Mataró, Barcelona, Spain). Soy milk samples were inoculated by transferring 10 mL of the pure culture sus- pensions of E. coli and L. monocytogenes and 1 mL of pure spore suspensions of B. cereus and A. niger to 1 L of sterile commercial soy milk to achieve a final concentration above 105 CFU/mL of vegetative cells/spores. 2.7. Microbiological Analysis of the Samples and Lethality Determination Treated samples were collected aseptically in sterile containers in a biosafety cabinet (Telstar PCR Mini-V, Terrassa, Spain) and kept until analysis at 4 ◦ C. Phosphate-buffered saline solution (PBS) (0.24 g of KH2 PO4 in 1 L of distilled water, pH adjusted to 7.4; Panreac) was used to prepare tenfold dilutions from each sample. Dilutions were plated in Petri plates with trypticase soy agar enriched with 0.6% yeast extract (TSA-YE, Oxoid). Plates were incubated at 37 ◦ C for 24 h. Foods 2023, 12, 3813 6 of 16 The lethality caused by the UV-C treatments on the inoculated microorganism was estimated with the formula:   N Lethality =−Log10 (6) N0 where N0 is the initial number of vegetative cells or spores present in the samples before the treatments and N is the number of remaining viable cells or spores after the treatments, both expressed as CFU/mL. 2.8. Determination of the Inactivation Kinetics and Effect of Treatment Variables Survival data of vegetative cells or spores after the different UV-C treatments (T1, T2, and T3) were adjusted to different linear and nonlinear models of survival curves using the GInaFiT 1.6 software, a freeware add-in for Microsoft® Excel 365 version 2309 [28]. The suitability of the adjustment was evaluated by determining the root mean square error (RMSE) value, the adjusted R squared (adjusted R2 ), and the Akaike information criterion (AIC) value. The 4DUV-C value, that is, the UV-C dose necessary to reduce 4 log CFU/mL of the initial load of the microorganisms tested, was determined from data that showed the best adjustment to the models. From the results obtained with the different matrices after the UV-C treatments (T1, T2, and T3), different statistical models were elaborated based on the lethality as a response variable and different independent variables: (1) Log C: the amount of microorganisms inoculated, expressed as log CFU/mL; (2) UV-C: dose of UV-C radiation, expressed in J/mL; (3) treatment flow rate, expressed in mL/s; and (4) NET, expressed as its absolute value. Five different models were elaborated: Model 1 presents the effect of the combination of variables of dose and flow rate; Model 2 presents the influence of the NET value with a linear adjustment; Model 3 presents the influence of NET from a quadratic approach; Model 4 included the variables of UV-C dose, flow rate, and NET with a linear adjustment; and Model 5, a quadratic adjustment using the variables dose, flow rate, and NET. The suitability of the adjustment was evaluated by determining the root mean square error (RMSE) and R squared (R2 ) values. 2.9. Cleaning and Disinfection of the UV Equipment Prior to each treatment, the equipment was disinfected by recirculating a 0.1% sodium hypochlorite solution for 10 min at 20 ◦ C. Then, 10 L of sterile softened water was circulated to remove any residual disinfectant. After the treatments, the circuit was cleaned by recirculating a nonfoaming alkaline soap solution called Capture-VC16 (Diversey España, Viladecans, Spain) at 60 ◦ C for 15 min to eliminate any remaining debris. Subsequently, 10 L of water was circulated to rinse off the soap residue, and the circuit was then disinfected by circulating a 0.1% sodium hypochlorite solution for 10 min at 20 ◦ C. Finally, any remaining traces of disinfectant were eliminated by pumping 10 L of sterile softened water. Before each treatment, a sample of the rinsing water was microbiologically analyzed to ensure that the equipment used during the treatments did not contribute to contamination. 2.10. Statistical Analysis of the Results For each experiment, three independent trials were performed, with two different samples from each trial (n = 6). To compare results between treatments and/or matrices, analysis of variance (ANOVA) and Tukey test were used, considering differences significant when p < 0.05. This analysis was performed with the R system for statistical computation (R Foundation for Statistical Computing, Vienna, Austria 2014, http://www.R-project.org. 3. Results and Discussion 3.1. Effect of UV-C Treatments on the Inoculated Microorganism Figure 2 shows the lethality obtained in the spores of Bacillus subtilis, depending on the treatment conditions tested. After T1 treatments, lethality increased significantly with http://www.R-project.org. 3. Results and Discussion 3.1. Effect of UV-C Treatments on the Inoculated Microorganism Foods 2023, 12, 3813 7 of 16 Figure 2 shows the lethality obtained in the spores of Bacillus subtilis, depending on the treatment conditions tested. After T1 treatments, lethality increased significantly with the UV-C dose (p < 0.05), but maximum lethality was always less than 1 log. The lethal the UV-C effect dose for the (p 1 indicates a convex shape, indicating the rapid elimination of weaker members and the progressive survival of more UV-C-resistant Foods 2023, 12, 3813 13 of 16 individuals [44]. The 4DUV-C value, estimated using the GinaFiT tool, represents the UV-C dose required to achieve a 4 log reduction. This value is used for comparing nonlinear survival curves. Different resistance behaviors to UV-C treatments were observed among microorganisms. A. niger and B. subtilis strains showed higher δ, p, and 4DUV-C values after applying T2 and T3 treatments, indicating their higher resistance than vegetative Foods 2023, 12, x FOR PEER REVIEW 14 of 17 bacteria. A decrease in these values was observed for all microorganisms when T3 treat- ments were applied (Table 4). The resistance sequence from least to most resistant was L. monocytogenes < E. coli < B. subtilis < A. niger. Figure 6. Cont. Foods 2023, 12, 3813 14 of 16 Figure 6. Adjustment Adjustment of of the the data of surviving spores of Aspergillus niger and Bacillus subtilis, and vegetative vegetative cells of Escherichia coli cells of Escherichia coli and and Listeria Listeria monocytogenes monocytogenes to to the the Weibull inactivation model Weibull inactivation model (with (with and and without tail) using the tool GInaFIT after T2 (left column) and T3 (right column) treatments at without tail) using the tool GInaFIT after T2 (left column) and T3 (right column) treatments at different UV-C doses. Black circles correspond to the data points, the solid line shows the estimated curve according to the model and the dotted lines the prediction bands. Some microbial models have been used to model the survival curves of microorgan- isms in milk, tiger nut, and soy milk products with UV-C light. Martínez-Garcóa et al. [13] reported similar results in whole cow’s milk for B. subtilis, in which the inactivation model was the Weibull tail with values at T2 and T3 of 94 and 91 J/mL. Croock et al. [6] also found a Weibull-type fitting model for E. coli and L. monocytogenes in milk, with p values greater than 1.6 and 4D values of 1.7 and 1.3 J/mL, respectively, which are lower than the results obtained in this study. In the case of tiger nut, the best fit for E. coli was also Weibull, with p values below 1 and a 4D value of approximately 20 mJ/cm2 [45]. However, in our study, the T2 and T3 values were significantly higher, corresponding to 13,121.64 and 5831.84 mJ/cm2 . However, Orlowska et al. [33] found that the inactivation kinetics for this microorganism in soymilk were linear with a D-value of 6.71 mJ/cm2 (4DUV-C of 26.72 mJ/cm2 ). The intrinsic factors of the liquid foods, the type of equipment, and its flow rate and mixing characteristics, as well as the applied dose, are very important factors to obtain a well-processed product. Although the first-order approach is the most commonly used approach to understand the effect of UV-C light on microorganisms, it seems that this approach is only suitable for water and clarified products. Therefore, it is necessary to use nonlinear models to fully describe the effect of UV-C light on liquid food products [43]. 4. Conclusions Recirculation appears to be a good strategy for applying UV-C treatments using an annular radial UV-C reactor in matrices with high interference, such as soy milk. The mixing effect would ensure that microorganisms will be in the maximum efficacy area (where the action of UV-C radiation is the highest) for enough time to be inactivated. This hypothesis seems to be demonstrated by NET being the most determining variable for the lethality prediction of the tested microorganisms. Although pasteurization of soy milk using UV-C seems an achievable goal, commercial sterilization is complicated since the estimated 4DUV-C values needed to inactivate microbial spores are too high. However, implementing correct hygiene practices in the preparation of raw materials and during treatments, or the combined application of other technologies, seeking a hurdle effect, could allow for a lethality of less than 4 Log to be sufficient to guarantee the quality of the product [13,22]. Author Contributions: Conceived and designed the experiments: M.M.-G., J.N.S.-G., M.M.H.-H., R.G. and A.X.R.-S.; performed the experiments: M.M.-G., J.N.S.-G. and I.C.-T.; analyzed the data: M.M.-G., M.M.H.-H., R.G. and A.X.R.-S.; wrote the paper: M.M.-G., A.X.R.-S. and M.M.H.-H. All authors have read and agreed to the published version of the manuscript. Foods 2023, 12, 3813 15 of 16 Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-60005-R), co-funded by the European Regional Development Fund (FEDER). Data Availability Statement: Data is contained within the article. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ramdath, D.; Padhi, E.; Sarfaraz, S.; Renwick, S.; Duncan, A. Beyond the Cholesterol-Lowering Effect of Soy Protein: A Review of the Effects of Dietary Soy and Its Constituents on Risk Factors for Cardiovascular Disease. Nutrients 2017, 9, 324. [CrossRef] 2. Poliseli-Scopel, F.H.; Hernández-Herrero, M.; Guamis, B.; Ferragut, V. Comparison of Ultra High Pressure Homogenization and Conventional Thermal Treatments on the Microbiological, Physical and Chemical Quality of Soy milk. LWT-Food Sci. Technol. 2012, 46, 42–48. [CrossRef] 3. Achouri, A.; Boye, J.; Zamani, Y. 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