Effect of milk film thickness on the efficiency of UVC radiated sterilization of raw cow’s milk

ABSTRACT In this study, the effect of UV-C radiation at different milk thickness levels on the bacterial count of raw and pasteurized milk contaminated with Escherichia coli was investigated. A total of 27 samples were irradiated with different UV-C doses (1302.0, 33839.7, and 64,060.7 J/m2) and at different thicknesses (2, 4, and 6 mm). A quadratic model was developed to predict the bacterial count as a function of the UV-C dose and the thickness of the milk. The results showed that the bacterial count decreased exponentially with increasing UV-C dose and that the effect of the dose was more pronounced at lower thicknesses. The statistical analysis of the data revealed that the model was highly significant, with an adjusted R-squared of 0.981. Additionally, physical and chemical analyses were performed on the irradiated pasteurized and E. coli-contaminated milk and compared to the respective standard for pasteurized milk. The results showed that the quality and composition of the milk treated with UV-C were not affected. The results of this study indicate that UV-C radiation can be used as an effective method for reducing bacterial counts in milk while preserving the quality and composition of the milk.


Introduction
Raw cow's milk is an important source of food for many people worldwide, as it is rich in essential nutrients for human health [1 ] but also a potential carrier of foodborne pathogens.These pathogens predominantly include Escherichia coli, a central focus of our study, as well as others like Staphylococcus aureus, and Listeria monocytogenes. [2]Such bacteria can lead to severe health complications like food poisoning and infections, with a higher impact on vulnerable demographics such as children, pregnant women, and the elderly. [3]o ensure raw milk's safety, it undergoes various sterilization processes.While pasteurization, a conventional method, has been in wide use for years. [4,5]It can potentially alter the milk's nutritional and sensory properties, [6][7][8] Recognizing this, researchers and the dairy industry alike are constantly seeking novel techniques to simultaneously improve the safety and preserve the quality of raw milk.
[11][12] UVC radiation is a form of ultraviolet light with a wavelength of between 200 and 280 nm, [13] can effectively inactivate a broad spectrum of microorganisms, including bacteria and viruses, by damaging their DNA. [14,15]][19] UVC radiation also has several advantages over traditional pasteurization methods, including the elimination of heating and cooling, higher efficiency in pathogen inactivation, and less impact on the nutritional and organoleptic quality of milk. [20,21]evertheless, comprehensive knowledge on how the thickness of the milk film affects UVC radiation sterilization efficiency is lacking.Considering that milk is an opaque liquid and hence a poor conductor of UVC radiation, evaluating the impact of milk film thickness on sterilization efficiency is crucial.This could aid in the design of reactors that allow for the application of appropriate UVC radiation doses.With this in mind, the current study aims to investigate the impact of milk film thickness on the efficiency of UVC radiation sterilization in raw cow's milk

Materials
Raw cow's milk was sourced from a local dairy farm.The selected bacteria for contamination, Escherichia coli, was obtained from a trusted commercial supplier.The UVC radiation was achieved using two 12 watt mercury lamps, with an approximate wavelength of 254 nm to ensure sterilization. [22,23]he milk samples were exposed to UVC radiation in 3D printed containers having specific film thicknesses of 2 mm, 4 mm, and 6 mm.The irradiance was measured using the LS126C equipment, which has a range of UVC wavelengths from 200 nm to 280 nm.

Methods
The study adopted a 3^k factorial design, precisely 3^2, indicating the inclusion of 3 levels with 2 factors.This entails a total of 9 treatment combinations.Each of these treatment combinations was replicated three times, resulting in a sum of 27 experimental runs.The factors evaluated during the experimentation were Dose and Thickness.The Dose was calculated by multiplying the measured radiation by the exposure time to which the sample was subjected.The values of radiation obtained for each sample were averaged to obtain an average value for each treatment.The results were 21.70 W/ m^2, 70.499 W/m^2, and 71.179 W/m 2 , with an exposure time of 60s, 480s, and 900s, respectively.
For the contamination of pasteurized milk, meticulous cleaning, and hygiene standards were adhered to.We also employed the use of protective gear to prevent any unintended milk contamination and ensure the reliability of the microorganism count results.The milk film thicknesses of 2 mm, 4 mm, and 6 mm were examined.For this, we utilized 3D printed containers of varying dimensions, each with a capacity of 20 ml.To calculate the Dose, the following formula was used: D=[I*t], where D is the applied dose (J/m^2), I is the UVC irradiance (W/m^2), and t is the time the material or product is exposed (s).The dose levels obtained were 1302.016J/m^2, 33839.73J/m^2, and 64,060.7 J/ m^2.These doses were applied to samples as shown in Figure 1.
Bacterial growth and contamination protocol were implemented to ensure consistency in the samples used in the experiment following a similar procedure as. [24]The process began by cultivating a sample of Escherichia coli in a solution of 10 g of the contaminated sample in 90 ml of peptone water.The solution was then allowed to sit for 10 minutes before 1 ml of the sample was seeded on a plate with medium for E. coli.The plate was then incubated at 37°C for 24 hours to allow for bacterial growth.Next, the contaminated solution was used to contaminate pasteurized milk by taking 10 ml of the contaminated peptone water solution and adding it to 1 liter of milk.The contaminated milk was then allowed to sit at room temperature for 5 hours to allow for bacterial reproduction.
For the UVC irradiation, the volume of milk used per replicate was 20 ml, selected specifically to maintain a consistent film thickness of 2 mm, 4 mm, or 6 mm in the 3D printed containers.It is important to note that the key parameter in this study was the thickness of the milk film exposed to the UVC radiation, rather than the surface area.This made it possible to maintain the specified film thicknesses despite variations in the surface area of the milk due to the different dimensions of the 3D printed containers used.
Sample collection was performed following UV-C irradiation.After irradiation, the milk was thoroughly mixed to ensure that any bacteria affected by the radiation were evenly distributed throughout the sample.A sterile pipette was used to collect a 1 ml sample from the mixture, which was then placed onto agar plates for bacterial colony count using the method of mass seeding described in the International Organization for Standardization (ISO) ISO 7251:2005 standard. [25]nitially, the number of colonies present before incubation was 50 UFC.
To determine the properties of the milk after exposure to UVC radiation, tests and analyses were carried out to verify the efficiency of the sterilization procedure.The fat content of the milk was calculated using the Gerber method described in the International Organization for Standardization (ISO) method ISO 19,662:2018. [26]The total solids were measured using the method described in the ISO method ISO 6731:2010, [27] while the fat solids were obtained by subtracting the total percentage of fat from the percentage of total solids.The protein percentage was calculated using the Kjeldahl method described in the ISO method ISO 8968-1:2014. [28]he statistical analysis was performed using the Statgraphics software.The response variable was the number of colony-forming units (CFUs) of the bacteria.The statistical model included the effect of the factors (dose and thickness) and their interaction.The model adequacy was checked by analyzing the residuals using the normal probability plot.The results were considered significant at a 95% confidence level.

Results
The results of the study are presented in Table 1, which shows the average number of colonies forming units (CFUs) of E. coli present in the milk samples after UVC radiation treatment.The table also  displays the dose of UVC radiation (J/m^2) and the thickness of the 3D printed container (mm) used in each treatment.The results were obtained by averaging the results of three replicates.
The assumptions of the model were thoroughly examined, and the results indicate that the model is suitable for the data.The normality of residuals was confirmed using the normal probability plot and a Shapiro-Wilk test, which returned a p-value of 0.6197, indicating that the residuals were normally distributed.The homogeneity of variances was verified by analyzing the scatter plot of residuals and no evidence of heteroscedasticity was found.Additionally, the independence of the data was assessed through the scatter plot of residuals against run order and no relationship was observed, further supporting the validity of the model.These findings provide strong evidence that the model can be used to accurately predict bacterial counts of E. coli in milk treated with UVC radiation.
As shown in Table 1, the highest average number of CFUs (150) was found in the samples treated with the lowest dose of UVC radiation (1302.016J/m^2) and with a thickness of 2 mm, 4 mm and 6 mm in the 3D printed container.However, as the dose of UVC radiation increased to 33,839.73J/m^2 and 64,060.7 J/m^2, the number of CFUs decreased significantly, with an average of 12 CFUs and 0 CFUs, respectively, at a thickness of 2 mm.Similarly, at a thickness of 4 mm, the number of CFUs decreased to an average of 24 CFUs and 8 CFUs, respectively, at the same radiation doses.Finally, at a thickness of 6 mm, the number of CFUs decreased to an average of 30 CFUs and 20 CFUs, respectively, at the same radiation doses.
These results suggest that increasing the dose of UVC radiation could reduce the number of E. coli present in milk.However, as the thickness of the milk increases, a higher dose of UVC radiation is required for effective sterilization.The results also show that the highest dose of UVC radiation (64060.7 J/m^2) was able to eliminate E. coli in milk samples at a thickness of 2 mm and 4 mm, but only reduced the number of CFUs at a thickness of 6 mm, further suggesting that thicker samples require higher doses for effective bacterial reduction.
The statistical analysis was performed using the Statgraphics software.The response variable was the number of colony-forming units (CFUs) of the bacteria.The statistical model included the effect of the factors (dose and thickness) and their interaction.The results of the ANOVA are shown in Table 2.
The ANOVA table shows that there is a significant effect of both Dose (J/m^2) and Thickness (mm) on the number of colony-forming units (CFUs) of bacteria present in the milk samples.The F-ratio for Dose is 1086 with a P-value of 0, indicating a strong effect of the dose level on the CFU count.Similarly, the F-ratio for Thickness is 16.18 with a P-value of 0.0006, indicating a significant effect of the thickness of the 3D printed container on the CFU count.
The interaction between Dose and Thickness also has a significant effect on the CFU count, with an F-ratio of 224.22 and a P-value of 0. The results of the ANOVA indicate that increasing the dose and the thickness of the container leads to a reduction in the number of CFUs present in the milk samples.
Additionally, the results show that there is a significant effect of the interaction between Dose and Repetition (blocks) with an F-ratio of 3.7 and a P-value of 0.0421.This indicates that the effect of dose level on the CFU count may vary depending on the repetition of the experiment.
Overall, the ANOVA table suggests that the UVC radiation treatment was effective in reducing the number of CFUs present in the milk samples, and that the effect of the treatment is dependent on both the dose and the thickness of the container used.The results were considered significant at a 95% confidence level.Figure 2 illustrates the main effects plot of the dose and the thickness on the mean CFUs of E. coli.From the main effects plot, as the dose increases, the bacterial count decreases exponentially.Additionally, as the thickness increases, the bacterial count also increases linearly.
Figure 3 shows the estimated response surface of the fitted model, which confirms these observations.Overall, these results suggest that the effectiveness of UVC radiation on the sterilization of raw cow's milk is dependent on both the dose and the thickness of the film.
The regression model obtained is presented in Equation ( 1), with an adjusted R-squared value of 98.11.This indicates that the model fits the data well and that the predictor variables (dose and thickness) explain a large proportion of the variance in the response variable (bacterial counts).
By setting the bacterial counts equal to zero and solving for the variable dose, we can determine the necessary quantity of the dose to eliminate bacteria at different milk thicknesses using Equation ( 2) It is worth noting that the equation for Dose yields two solutions.However, one of these solutions does not have any physical significance and should be disregarded.By plotting the remaining solution as showing in Figure 4, we can determine the required doses for a specific thickness.From this, we can see that the minimum dose required to eliminate Escherichia coli is 35,756.35J/m^2 at a thickness of 0 mm.
Based on these results, an additional experiment was conducted to validate the model.Three samples of 2 mm thickness were irradiated with 41,151.10J/m^2 resulting in 0 colony forming units (CFUs) of bacteria after counting.Furthermore, to evaluate the effects of UV-C treatment on the quality and composition of the milk, physical and chemical analyses were conducted on pasteurized and E. coli-contaminated milk and raw milk, and the results were compared to the standard for pasteurized milk.The findings indicated that the UV-C treatment did not negatively impact the quality and composition of the milk, as demonstrated in Table 3:

Discussion
The results of the statistical analysis showed that both dose and thickness have a significant effect on bacterial count.According to research by Singh et al. [12] UVC light (200-280 nm) is an effective alternative to inactivate a large set of microbial pathogens such as: fungi, viruses and bacteria.The adjusted R-squared value of 0.981 indicates that the model fits the data well.The ANOVA table also showed that the interaction between dose and thickness was not significant, which suggests that the effect of dose and thickness on bacterial count is independent.
Additionally, the analysis of the physical and chemical properties of the milk showed that the treatment with UV-C did not affect the quality or composition of the milk.UV-C technology helps in the effective prevention of foodborne diseases by simultaneously increasing the shelf life of food without compromising its quality by reducing the microbial load. [12]This agrees with previous studies that have shown that UV-C treatment is effective in eliminating bacteria without affecting the quality of the milk. [4,21]Define optimal process parameters to inhibit microbial growth without causing changes in physicochemical properties and sensory characteristics of milk is a great challenge, because  drastic UV-C conditions can have oxidative potential, while optimal conditions have no impact on the physicochemical, nutritional and sensory aspects. [4]ne crucial aspect to consider in future studies is the sensory effect of UV-C treatment on the milk, as well as consumer perception. [29]Despite our study's focus being primarily on bacterial count and physical-chemical characteristics, it is widely recognized that sensory aspects significantly influence consumer acceptance and preference.This is indeed an interesting perspective that needs to be explored further, and we are planning on including sensory methods and consumer perception in our future studies.In addition to this, we also aim to evaluate the effectiveness of UV-C treatment on other bacteria.
Furthermore, the results of the model validation experiment confirmed the validity of the model.It was observed that a dose of 41,151.10J/m^2 resulted in a bacterial count of zero for a thickness of 2 mm.This result is in line with the findings of the statistical analysis, which suggests that a higher dose is required to eliminate bacteria in thicker samples.It is important to note that the results obtained in this study assume that the bacteria are uniformly distributed in the milk sample.Additionally, it is assumed that the physical and chemical properties of the milk remain constant during the UV-C treatment.Further research is needed to verify these assumptions and to investigate the effect of other factors such as the type of bacteria and the initial bacterial concentration on the UV-C treatment.The effectiveness of microbial inactivation by UV-C varies according to the initial load of microorganisms, the dose of radiation applied and the optical density of the product. [16]e have obtained a regression model that describes the relationship between bacterial counts, dose and thickness.However, it is important to note that this equation gives two solutions for Dose, one should be discarded because it does not have any physical meaning.By plotting the second solution, we can observe the required doses for a specific thickness.We can see that even for the smallest thickness of 0 mm, the minimum dose required to eliminate E. coli was 35,756.35(J/m^2).
This result can be explained by the fact that the thickness of the milk does not only affect the number of bacteria present, but also the amount of UVC radiation that reaches the bacteria.The thicker the milk, the more UVC radiation is absorbed and scattered, making it harder to reach the bacteria and eliminate them.Additionally, the presence of fat and protein in the milk can also affect the effectiveness of the UVC radiation.Studies have shown that these components can act as natural sunscreens, reducing the penetration of UVC radiation. [30,31]The efficiency of UV-C disinfection is reduced by the presence of macromolecules, suspended matter such as casein micelles and organic compounds, which increase the resistance to penetration of UV-C energy. [4]To evaluate the effectiveness of UV-C treatment, not only the power to drastically reduce the number of pathogenic microorganisms in milk should be considered, but also other important aspects such as the ability not to affect the composition of fatty acids and other physicochemical properties that ensure a good quality of the treated milk. [16]t is also important to consider that the results of this study were obtained under specific conditions, such as the type of milk used, the specific strain of E. coli used as a contaminant, and the specific equipment and methods used to measure the UVC radiation and bacterial counts.Therefore, the results may not be directly applicable to other types of milk or other types of bacteria.

Conclusion
The developed model for predicting bacterial counts in milk exposed to UV-C radiation is highly accurate and reliable.The model has an adjusted R-squared value of 0.981, indicating that 98.11% of the variation in bacterial counts can be explained by the variation in dose and thickness.The ANOVA Analysis showed that there is a significant effect of both dose and thickness on the bacterial counts.The thickness of milk plays a significant role in the effectiveness of UVC radiation for sterilization.Thicker milk requires higher doses of UVC radiation to eliminate bacteria, highlighting the importance of considering the physical characteristics of the product when designing sterilization processes.However, it should be noted that the effectiveness of the UVC radiation could be influenced by other factors such as the type of bacteria present in the milk, and the initial bacterial concentration.Our study did not measure these variables, and further research is needed to better understand their influence.In addition, while our study found that UVC radiation did not significantly affect the quality or composition of the milk, we focused specifically on fat content, total solids, fat solids, and protein percentage.It's possible that other quality and composition indicators, such as flavor and color, could be affected by UVC exposure.Future studies should incorporate sensory analysis methods to assess any changes in these attributes.The results of this study support the potential use of UVC radiation as a safe and effective method for sterilizing raw cow's milk and to prevent foodborne illnesses.The obtained regression model can be used to predict the required dose for different thicknesses of milk.While the results are promising, we recognize that a more comprehensive approach, incorporating other microorganisms commonly found in raw milk, and a broader range of milk quality and composition indicators, would be needed to fully validate the use of UVC radiation for milk sterilization.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Samples irradiated under 12w lamps and measured with LS126C equipment.

Table 1 .
Bacterial counts (CFUs) for different doses and film thicknesses.

Table 2 .
Results of the ANOVA.

Table 3 .
Results of physical and chemical analysis (3 samples per type of milk).