Evaluation of ultraviolet‐C and spray‐drying processes as two independent inactivation steps on enterotoxigenic Escherichia coli K88 and K99 strains inoculated in fresh unconcentrated porcine plasma

Abstract The objectives of this study were to assess the effectiveness of an ultraviolet (UV‐C, 254 nm) irradiation system and the spray‐drying method as two independent safety steps on inactivation of Escherichia coli K88 and K99 spiked in porcine plasma at 6·46 ± 0·04 log10 ml−1 and 6·78 ± 0·67 log10 ml−1 respectively for UV‐C method, and at 7·31 ± 0·39 log10 ml−1 and 7·66 ± 0·11 log10 ml−1, respectively for the spray‐drying method. The UV‐C method was performed at different UV light doses (from 750 to 9000 J l−1) using a pilot plant UV‐C device working under turbulent flow. Spray‐drying treatment was done at inlet temperature 220 ± 1°C and two different outlet temperatures, 80 ± 1°C or 70 ± 1°C. Results indicated that UV‐C treatment induced a 4 log10 viability reduction for both E. coli at 3000 J l−1. Full inactivation of both E. coli strains was achieved in all spray‐dried samples dehydrated at both outlet temperatures. The special UV‐C system design for turbid liquid porcine plasma is a novel treatment that can provide an additional redundant biosafety feature that can be incorporated into the manufacturing process for spray‐dried animal plasma. Significance and Impact of the Study The safety of raw materials from animal origin such as spray‐dried porcine plasma (SDPP) may be a concern for the swine industry. Ultraviolet treatment at 254 nm (UV‐C) of liquid plasma has been proposed as an additional biosafety feature in the manufacturing process of SDPP. We found that UV‐C exposure in the liquid plasma at 3000 J l−1 reduces about 4 log10 ml−1 for E. coli K88 and K99. Full inactivation of both E. coli strains was achieved in all spray‐dried samples. The incorporation of UV‐C treatment to liquid plasma improves the robustness of the SDPP manufacturing process.


Introduction
Spray-dried animal plasma (SDP) is a protein source extensively used in pig feed due to its functional components that contribute to improved post-weaning performance and survival (Torrallardona 2010). However, the safety of raw materials from animal origins is a concern for the swine industry. Ultraviolet (UV) treatment of liquid plasma has been proposed to introduce an additional redundant inactivation step in the manufacturing process of SDP to further enhance biosafety of the final spraydried product (Polo et al. 2015;Bl azquez et al. 2017).
Ultraviolet exposure at a wavelength of 254 nm (UV-C) is a nonthermal process that has a germicidal effect by causing thymine-thymine and thymine-cytosine dimers in DNA and thymine-uracil dimers in RNA, which disrupts microbial reproduction (Jagger 1967). During the spray-drying process, thermal inactivation, high pressure and rapid dehydration are the phenomena involved in microbial inactivation. Although the most important site of damage caused by dehydration is the cytoplasmic membrane (Crowe et al. 1987;Lievense & van 't Riet 1994;Perdana et al. 2013;Huang et al. 2017), dehydration also produces damage to DNA/RNA and protein (Lievense 1992). Thus, the sequential action of both methods for the plasma production process is promising to inactivate micro-organisms, since both damage different targets involved in microbial inactivation.
Enterotoxigenic Escherichia coli is one of the main causes of enteric disease and death in newborn and weaned pigs (David 2002) and is the major cause of neonatal diarrhoea in calves (Acres 1985). E. coli requires the expression of adhesion fimbriae (adhesins), which are encoded in plasmids, to be adhered to the intestinal epithelium. E. coli expressing K88 adhesin is mainly found in pigs (Gaastra and De Graaf 1982), while K99 is the main adhesion antigen found in bovine species (Tzipori 1981), although K99 can also be found in ovine and porcine species (Gaastra and De Graaf 1982).
The aim of this study was to assess the effectiveness of a UV-C treatment system on E. coli inactivation after inoculation in fresh unconcentrated liquid porcine plasma. In addition, a second objective was to test the effectiveness of the spray-drying process on the inactivation of E. coli at two different outlet temperatures, at the regular outlet temperature normally used by the industry (80°C) and at lower outlet temperature (70°C).

UV-C test
Plasma inoculated with E.coli K88 strain had an initial count of 6Á46 AE 0Á04 log 10 ml À1 . After UV-C treatment at 3000 J l À1 , bacterial counts showed a significant reduction of 4Á34 log, describing a curve adjusted to the log linear plus tail model (Fig. 1) with a regression coefficient of R 2 = 0Á95 (Table 1). At doses of 6000 and 9000 J l À1 , residual E. coli populations of 1Á18 AE 0Á30 and 1Á12 AE 0Á30 log 10 ml À1 were counted, respectively. The UV-C doses required to have 4 log 10 reduction (log10R) was predicted to be 3105 J l À1 .
Plasma inoculated with the strain E. coli K99 had an initial count of 6Á78 AE 0Á67 log 10 ml À1 . After UV-C treatment, bacterial counts decreased significantly, showing a curve adjusted with the Weibull plus tail model, with a regression coefficient of R 2 = 0Á923 (Table 1). There was a 3Á97 log 10 ml À1 reduction of the initial count between 0 and 3000 J l À1 (Fig. 1). Residual populations of 2Á30 AE 0Á08 and 2Á11 AE 0Á15 log 10 ml À1 were counted after irradiation at doses of 6000 and 9000 J l À1 , respectively. The 4 log10R was predicted to be achieved at 3427 J l À1 .

Spray-drying test
Full inactivation of strains E. coli K88 and K99 was achieved in all spray-dried samples dehydrated at an inlet temperature of 220 AE 1°C and both outlet temperatures of 80 AE 1°C or 70 AE 1°C (Table 2).
Numerous studies have demonstrated the effectiveness of the spray-drying process used during the manufacturing of SDP, providing evidence that SDP is a biologically safe product relative to multiple pathogens of concern for the swine industry (Polo et al. 2005;Pujols et al. 2007Pujols et al. , 2008Gerber et al. 2014). However, it is prudent to evaluate additional biosafety features that may further enhance the robustness of the SDP production process. Exposure to UV-C is extensively used for the disinfection of liquid media and surfaces due to its germicidal activity (Guerrero-Beltran 2004;Lin et al. 2012). Previous research has demonstrated that UV-C treatment of liquid plasma was effective to inactivate Porcine parvovirus (Polo et al. 2015) and Salmonella spp. (Bl azquez et al. 2017) inoculated in liquid plasma. During the spray-drying process, temperature and dehydration are the mechanisms that contribute to microbial mortality (Perdana et al. 2013;Huang et al. 2017), whereas, UV-C treatment causes damage to nucleic acids (Jagger 1967) and protein-DNA cross links (Smith 1962).
In this study, UV-C inactivation kinetics of two strains of E. coli from porcine (K88) and bovine (K99) origins were very similar, although such kinetics fit better to different models, as indicated by the lower RMSE in each case. For both strains of E. coli, a rapid decrease in bacterial count was observed between 0 and 3000 J l À1 of UV-C, with the appearance of a residual population (Nres) afterwards. These results agree with other UV-C inactivation studies performed with E. coli (Hijnen 2006).
The reduction of the inactivation rate at high UV fluencies (tailing) could be caused by micro-organism aggregation, appearance of a resistant subpopulation, hydraulic design (Hijnen 2006), matrix effect or particle size effect (Winward 2008). Porcine plasma is a dense, coloured, liquid matrix with 8-10% solids, and contains a complex blend of different proteins with some of the proteins having binding properties (Burnouf 2007). Therefore, the matrix and particle size effects of porcine plasma may have had a special impact on the tailing effects of UV-C treatment in the present study.
The residual population of E. coli after UV-C treatment should apparently be eliminated in the subsequent spraydrying process based on the total inactivation results by the spray-drying methods at the two outlet temperatures tested ( Table 2). The outlet spray-drying temperature is 80°C for commercial manufacturing of SDP (P erez-Bosque et al. 2016) and results of the present study suggest that both E. coli strains are very susceptible to spray-drying even at a lower outlet temperature (70°C). These results provide confidence that current commercial spray-drying conditions are highly effective for inactivation of E. coli.
Processing steps should be able to remove or inactivate a wide range of pathogens, according to the World Health Organization (WHO, 2004) guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. These guidelines recommend that two or more robust, effective and reliable processes will be able to remove or inactivate 4 logs or more of viruses. Although the inactivation of viruses has to be considered separately, these guidelines used for virus safety in human plasma transfusion products can be applied to pathogens in general that affect animal blood then UV-C light treatment at 3000 J l À1 and spray-drying can be considered two robust safety procedures in the production of SDP since both methods individually inactivated at least 4 log 10 E. coli.
In addition, the manufacturing process of SDP has other safety features, such as blood collection from ‡4D reduction: UV-C dose irradiation in J l À1 at which a 4 Log reduction was achieved. healthy animals declared fit at slaughter for human consumption, pooling of inherent neutralizing antibodies (NA) against potential pathogens, and post-packaging storage in a dry environment at room temperature for at least 14 days (P erez-Bosque et al. 2016). Plasma pooling is also a recognized safety step in the production of certain human plasma products (Solheim et al. 2000(Solheim et al. , 2006(Solheim et al. , 2008, since there is successful neutralization of antigens in the presence of NA. Some micro-organisms in dehydrated form and stored under appropriate constant conditions can remain viable in a unique vitrified state for very long times, even years (Perdana et al. 2013 However, it is unknown if these storage conditions affect E. coli or other bacteria survival in spray-dried plasma. In the present study, the SDP storage temperature effect (20°C for 14 days) on E. coli survival was not tested because both E. coli strains did not survive the spray-drying process. All the above-mentioned safety features involved in the manufacturing process of SDP use different inactivation mechanisms, and collectively ensure the biosafety of SDP.
In conclusion, this study provides evidence that affordable levels of UV-C treatment (3000 J l À1 ) of liquid porcine plasma can significantly decrease E. coli bacterial counts (4 log 10 ml À1 at 3000 J l À1 ). Furthermore, the study indicated that both UV-C treatment and spray-drying as independent safety procedures are very effective for inactivating E. coli K88 and K99. This novel UV-C technology can be adapted to further enhance the robustness of the manufacturing process for assuring the biosafety of spray-dried plasma.

Bacterial strains and test products
Two strains of E. coli were used in the present study: an isolate from swine expressing the K88 adhesin, and a second isolate from bovine expressing the K99 adhesin (both isolates were kindly provided by Dr. Antonio Ju arez. University of Barcelona, Spain). A 0Á3 ml volume of E. coli isolates was cultured in 100 ml of LB media (Sigma-Aldrich) at 37°C and 150 rev min À1 for 18 h. The cells were subsequently concentrated by centrifuging (1000g for 20 min at 4°C) using sterilized 40 ml tubes containing 20 ml of culture media. The remaining culture media was removed by resuspending the cell precipitate in 20 ml of sterile 0Á01 g mol À1 phosphate buffer saline (PBS). After resuspension, it was centrifuged again as described above and the resulting cell precipitate that was resuspended again in 500 ml of PBS reaching a final titre of 8Á98 log 10 CFU per ml for K88 and 8Á91 log 10 CFU per ml for K99 .
Fresh liquid porcine plasma from the production plant of APC Europe S.A., (Granollers, Spain) was used for these trials. This plasma was obtained by centrifugation of blood from pigs processed at a local officially inspected abattoir.

Settings of pilot scale UV-C system
The UV-C reactor system (SP1) was designed and manufactured by Sure Pure Operation AG (Zug, Switzerland) that has already been described by Bl azquez et al. (2017). The configuration of the pilot scale UV-C reactor consisted of a closed system with one low pressure mercury UV lamp (30 UV-C Watts, 254 nm) surrounded by a quartz crystal. The plasma flowed through a steel tube containing a vortex (internal grooved spiral tube that generated a turbulent flow) between the spiral tube and the quartz sleeve. The tangential inlet of the reactor created high velocity and turbulence in the inlet chamber improving liquid contact with the UV-C light. The liquid was pumped from the inlet chamber into the reactor at a constant flow rate of 4000 l h À1 to achieve a Reynolds value greater than 2800 which is indicative of a turbulent flow (Simmons et al. 2012). Plasma was pumped from the tank to the UV-C lamp and recirculated many times through this circuit to achieve the required UV-C dose vs time. The time spent by the liquid to pass through the system once was 7Á2 s, delivering 22Á95 J l À1 or 23Á40 mJ cm À2 per cycle.

UV-C test
A total of 60 kg of plasma were used for the present study, 30 kg for each of the tested E. coli strains. For each isolate, the 30-kg batch was divided into three 10-kg subbatches to conduct tests in triplicate. Because liquid fresh plasma from the abattoir may contain different microorganisms, the initial 60 kg of plasma product was treated by UV-C at 10 000 J l À1 for 1 h to inactivate any potential bacteria prior to artificial inoculation with E. coli.
After each UV-C irradiation dose, 1 mL samples were 10-fold diluted in peptone water and 0Á1 ml inoculated by duplicates onto TBX agar plates (Sigma-Aldrich) and incubated for 24 h at 37°C. Plates with more than 20 and <300 colonies were counted and results expressed as log 10 ml À1 .

Spray-drying test
A total of 7 kg of fresh plasma from a commercial manufacturing plant was previously UV-C treated at 10 000 J l À1 prior to inoculation with the E. coli strains to eliminate any other bacteria present in the initial raw material. Half amount (3Á5 kg) of this UV treated plasma was spiked with the swine E. coli K88 isolate at a ratio of 1/47 reaching a final titer of 7Á31 AE 0Á39 log10 ml À1 and the other half with the bovine E. coli K99 isolate, at a ratio 1/ 18 reaching a final titre of 7Á66 AE 0Á11 log10 ml À1 . From each of the 3Á5 kg inoculated plasma aliquots, two bottles of 750 ml were obtained and spray-dried in a lab drier (B€ uchi Mini Spray Dryer B-290, B€ uchi Labortechnik, Switzerland) at two different conditions: inlet temperature at 220 AE 1°C and outlet temperature at 80 AE 1°C or 70 AE 1°C, after stabilization of the spray-drier with water and non-inoculated control plasma. All studies were performed in triplicate. Air flow through the column was set at 20-27 m 3 h À1 at 20°C. Estimated dwell time was <1 s.
Once SDP was obtained at the two designated outlet temperatures, three tubes containing 0Á5 g of dried plasma for each condition were obtained and the dry samples were resuspended in water at a ratio of 1 : 9. From this resuspension, 0Á1 ml was seeded in TBX agar for 24 h at 37°C. Colony counting was performed as indicated in the previous section. Results were expressed as a log 10 g À1 of solids according to the equation: log 10 g À1 = log 10 (CFU per ml)/ [(% solid content of resuspended sample)/100].

Modelling of inactivation
The GInaFiT software was used to calculate and plot nonlinear E. coli survival curves. The log linear plus tail (Geeraerd et al. 2000) and Weibull plus tail (Albert and Mafart 2005) models were tested. The log linear plus tail model (Geeraerd et al. 2005) follows the equation (1): log N ¼ logðð10 log N0 À 10 log Nres ÞÞ À e ðkmaxtÞ þ 10 log Nres ð1Þ where k max is the inactivation rate of the log linear part of the curve; N0 is the initial bacterial concentration; t is time and N res is the number of resistant bacteria subpopulation.
The Weibull model plus tail (Albert and Mafart 2005) uses the equation (2): log 10ðNÞ ¼ log 10ðð10 log N0 À 10 log N res ÞÞ Â 10 À t d ð Þ p þ 10 log N res ð2Þ where N0 is the initial bacterial concentration; t is time; d parameter represents the time of the first decimal reduction concentration for the part of the population not belonging to N res ; p parameter allows to determine concavity or convexity of the curve; and N res is the number of resistant bacteria sub-population.

Statistical analysis
Data were expressed by means of Log 10 values and standard deviations of three independent experimental batches. Mean, standard deviations, ANOVA and F-test for comparisons were calculated with Excel 2007 (Microsoft Office). The LSD (Least Significant Difference) test was calculated with Statgraphics Centurion XV ver. 15.2.14 (©StatPoint Technologies Inc, Warrenton, Virginia) to determine significant differences between treatments. Differences at P < 0Á05 were considered significant.
Mean square error (MSE), goodness of fit in terms of root mean square error (RMSE), correlation coefficient (R 2 ) and adjusted correlation coefficient (adj-R 2 ) values were calculated with the GInaFiT software (Geeraerd et al. 2005). The smallest RMSE determined the inactivation model with the best fit (Geeraerd et al. 2005).