1 UV light-emitting diode (UV-LED) as a potential technology to reduce Campylobacter and Salmonella

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2 in chicken meat and its impact on the microbial community composition compared to a UV pilot-

3 plant scale device

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4 Arturo B. Soro1, 2, Daniel Ekhlas1, 2, Sajad Shokri3, Ming Ming Yem3, Rui Chao Li3, Soukaina Barroug3,

5 Shay Hannon1, Paul Whyte2, Declan J. Bolton1, Catherine M. Burgess1, Paula Bourke3, Brijesh K. Tiwari1

6 1Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.

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7 2UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.

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8 3UCD School of Biosystems and Food Engineering, University College Dublin, Belfield, D04 V1W8,

9 Dublin, Ireland. er
10 Contact information for corresponding author: Arturo B. Soro, Belgian Public Health Institute

11 (Sciensano), Juliette Wytsman 14, 1050 Ixelles, Brussels, Belgium, Arturo.BlazquezSoro@sciensano.be,

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12 tel.: +32 470972862

13 Email addresses: Daniel Ekhlas: daniel.ekhlas@teagasc.ie

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14 Sajad Shokri: ss4664@bath.ac.uk

15 Ming Ming Yem: ming_yem96@hotmail.com

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16 Rui Chao Li: ruichaoli7@gmail.com

17 Soukaina Barroug: soukaina.barroug@ucdconnect.ie

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18 Shay Hannon: shay.hannon@teagasc.ie

19 Paul Whyte: paul.whyte@ucd.ie

20 Declan J. Bolton: declan.bolton@teagasc.ie

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21 Catherine M. Burgess: kaye.burgess@teagasc.ie

22 Paula Bourke: paula.bourke@ucd.ie

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23 Brijesh K. Tiwari: brijesh.tiwari@teagasc.ie

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 24

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25 Abstract

26 This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology

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27 treatment with refrigerated storage of chicken breast meat over 7 days on Campylobacter jejuni,

28 Salmonella enterica serovar Typhimurium, total viable counts (TVC) and total Enterobacteriaceae

29 counts (TEC). An optimised UV-LED treatment at 280 nm for 6 min decreased inoculated S.

30 Typhimurium and C. jejuni populations by 0.6-0.64 log CFU/g, and TVC and TEC population by 1-1.2 log

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31 CFU/g in chicken samples. During a 7-day storage at 4 °C, a 2.62 log reduction in C. jejuni was achieved

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32 compared with non-treated samples. A reduction in Salmonella counts of 1.12 log CFU/g from non-

33 treated meat was observed. Moreover, the UV-LED effectiveness to reduce TVC and TEC during

34
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refrigerated storage was compared with a conventional UV lamp and a similar efficiency was observed.

35 The impact of UV-LED and UV lamp devices on the microbial community composition of chicken meat
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36 during storage was further examined using 16S rRNA gene amplicon sequencing. Although similar

37 bacterial reductions were observed for both technologies, the microbial communities were impacted

38 differently. Treatment with the UV conventional lamp increased the proportion of Brochothrix spp. in
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39 meat samples, whilst Photobacterium spp. levels were reduced.

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48 Keywords

49 Meat bacterial community; UV-LED; Campylobacter; Salmonella; food spoilage; chicken meat

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 50

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51 1. Introduction

52 Fresh chicken meat can be highly contaminated with pathogenic and spoilage bacteria. Some of the

53 key causes of contamination can include poor biosecurity measures on farms, leakage of faecal matter

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54 in abattoirs, and cross-contamination between batches during processing (Golden et al., 2021). As a

55 consequence, broiler meat can act as a vector for cross-contamination on kitchen surfaces and spread

56 of pathogens to foods, posing a health risk to consumers (Possas and Pérez-Rodríguez, 2023).

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57 Campylobacter and Salmonella have been identified as two of the most significant foodborne

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58 pathogens associated with poultry meat worldwide, with one study indicating that 30 and 25% of the

59 total global outbreak cases in humans associated with Campylobacter and Salmonella are linked to

60 poultry, respectively (Thames and Sukumaran, 2020). Although a range of different serovars of
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61 Campylobacter and Salmonella can be found in chicken meat, Campylobacter jejuni and Salmonella
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62 enterica serovars Typhimurium and S. Enteritidis are of particular concern given the number of human

63 infections they cause (Wessels et al., 2021). Bacterial contamination and cross-contamination of

64 equipment, surfaces, workers and air, among others, within the poultry chain, can impact the initial
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65 microbial burden and the microbial community composition of chicken meat, which can impact its

66 safety, quality, and consumer acceptance (Marmion et al., 2021).

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67 Novel decontamination strategies such as cold plasma, ultrasound, pulsed electric fields, and light

68 technologies have been investigated in recent decades to improve the safety and to extend the shelf
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69 life of food without impacting on its quality (Priyadarshini et al., 2019). Ultraviolet (UV) light has been

70 described as an emergent non-thermal technology for the preservation of liquid foods and food

71 surfaces. This technology is based on the emission of photons in a particular wavelength spectrum
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72 ranging from 200 to 400 nm, also known as UV light spectrum (Kebbi et al., 2020). The UVC spectrum

73 (200-280 nm) has been identified as the most germicidal range and therefore, it has been evaluated
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74 for decontamination of several food products. UVC has been found to be effective against bacteria,

75 fungi, yeasts, and viruses due to its ability to damage DNA, RNA, and other cellular structures, which

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 76 can result in cellular dysfunction and cell death (Singh et al., 2021). Conventional UV devices such as

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77 mercury lamps have been applied at an industrial level over the past number of years. However, UV

78 light-emitting diode (LED) devices are currently being evaluated with the purpose of replacing

79 conventional UV equipment due to their advantages, including the absence of toxic mercury, easy

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80 adaptability low power, heat emissions, and cost effectiveness (Soro et al., 2023).

81 The application of UV-LED to inactivate Campylobacter spp. and Salmonella spp. on poultry meat has

82 been reprted (Calle et al., 2021; Haughton et al., 2012; Wang et al., 2021). However, studies evaluating

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83 the combined effect of UV-LED and storage on Campylobacter spp. and Salmonella spp. on chicken

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84 meat are scarce and are required to inform successful future implementation of this technology in

85 poultry processing. Additionally, the decontamination efficacy of UV-LED to reduce spoilage bacteria

86
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of poultry meat should also be considered when assessing this technology (Soro et al., 2021). Apart

87 from Soro et al. (2021) and Haughton et al. (2012) which evaluated UV-LED to reduce total viable
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88 counts (TVC) and total Enterobacteriaceae counts (TEC), no other study has investigated the

89 effectiveness of UV-LED combined with refrigerated storage to reduce the total microbial burden on

90 chicken meat. The present study examined the combined effects of UV-LED and aerobic storage of
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91 chicken breast meat for 7 days at 4 °C on C. jejuni, S. Typhimurium, TVC, and TEC. Moreover, the

92 effectiveness of UV-LED to reduce TVC and TEC during refrigerated storage was compared with a UV
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93 pilot-plant scale device. Lastly, the impact of UV-LED and UV lamp devices on the microbial community

94 composition of chicken meat was also examined using 16S rRNA gene amplicon sequencing in order to
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95 investigate the changes that may occur during storage and how this may be influenced by treatments.

96 Studies investigating the impact of this technology on processed chicken meat microbial community

97 composition are lacking and could provide an insight into its future implementation in the broiler
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98 production chain.

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100 2. Materials and methods

101 2.1. Bacterial culture and inoculum preparation

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 102 The NCTC 11168 C. jejuni strain was selected from the stock collection at Teagasc Food Research

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103 Centre, Ashtown (Dublin, Ireland), where it was preserved in defibrinated horse blood (Cruinn

104 Diagnostics, Dublin, Ireland) at -80 °C. S. enterica serovar Typhimurium ATCC 14028 was obtained from

105 the stock collection of the School of Biosystems and Food Engineering in University College Dublin,

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106 where it was stored using protective beads (Pro-Lab Microbank®, Birkenhead, UK) at - 80 °C.

107 Resuscitation of the C. jejuni stock was conducted on Mueller Hinton agar (MHA, Oxoid, Basingstoke,

108 UK) with micro-aerobic incubation for 48 h at 42 °C. A micro-aerobic atmosphere of 5% O2 and 15%

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109 CO2 was achieved using Campygen 2.5 and 3.5 L sachets (Oxoid, Basingstoke, UK). Isolated colonies

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110 were streaked onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA, Oxoid, Basingstoke,

111 UK) supplemented with Campylobacter growth supplement (Oxoid, Basingstoke, UK) and incubated

112 for 48 h at 42 °C under micro-aerobic conditions. A bead of the Salmonella stock was streaked onto
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113 Tryptic Soy agar (TSA, Oxoid Ltd., Basingstoke, UK) and incubated aerobically for 24 h at 37 °C. Fresh
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114 bacterial cultures were prepared every week to avoid cell damage. Isolated colonies of Campylobacter

115 and Salmonella were inoculated in Mueller Hinton broth (MHB, Oxoid, Basingstoke, UK) and Tryptic

116 Soy broth (TSB, Oxoid Ltd., Basingstoke, UK) and incubated micro-aerobically at 42 °C for 48 h and
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117 aerobically for 18 h at 37 °C, respectively. After incubation, bacterial suspensions were centrifuged at

118 4000 x g for 15 min and washed in sterile maximum recovery diluent (MRD, Oxoid Ltd., Basingstoke,
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119 UK). To achieve a bacterial concentration of approximately 8 log CFU/mL in the working inoculum, cell

120 density was measured using the 0.5 McFarland standard kit (BioMérieux, Marcy-l'Etoile, France).

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122 2.2. Sample preparation, inoculation of pathogenic bacteria and UV-LED light treatment

123 Skinless chicken breast fillets were obtained 1-day post-mortem at a local butcher shop. Samples were
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124 stored at 4 °C for less than 2 h and fillets were cut into cubes of approximately 10 ± 2 g with a thickness

125 of 1-1.5 cm. A 100 µL volume of Campylobacter or Salmonella suspensions were transferred onto the
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126 surface of the chicken pieces to achieve a bacterial concentration of approximately 4-5 log CFU/g,

127 respectively. Inocula were spread on the chicken surface using cell spreaders. Subsequently, samples

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 128 were left for 30 min at room temperature and under sterile conditions in order to facilitate bacterial

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129 attachment. UV treatment was carried out according to the parameters described in our previous

130 study (Soro et al., 2021). Briefly, UV treatments were conducted with an UV-LED device (PearlLab

131 Beam, Aquisense Technologies, NC, USA) at a wavelength of 280 nm for 3 and 6 min, which were

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132 previously selected as the most effective inactivation treatments in other studies (Soro et al., 2021).

133 Chicken pieces were placed into sterile plastic petri dishes and placed centrally in the UV-LED chamber

134 at a distance of 5 cm from the light source. Untreated samples were considered as controls. To

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135 determine the UV light dose applied, the UV light fluence rate (W/cm2) at a wavelength of 280 nm was

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136 measured with a radiometer (Opticalmeter, model ILT2400, International Light Technologies, MA,

137 USA). The measured values were multiplied by the time of exposure (min). The UV light doses

138 calculated for UV light exposure at 280 nm for 3 and 6 min were 0.204 and 0.408 W x min x cm-2,
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139 respectively, with a fluence rate of 0.068 W x cm-2.
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140

141 2.3. Evaluation of the effect of UV-LED light and storage on artificially-inoculated chicken meat

142 The effect of UV light at 280 nm was assessed using two different UV treatment lengths (3 and 6 min)
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143 in chicken meat inoculated with C. jejuni or S. Typhimurium. Moreover, the influence of storage of the

144 inoculated treated samples at 4 °C was determined by sampling on 0, 1, 3, and 7 days’ post-treatment.
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145 Treated and control samples were packed in polyethylene terephthalate film laminated with

146 polyethylene film (PET/PE, 400 µm, Multivac, Dublin, Ireland) and sealed in air.

147
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148 2.4. Evaluation of the effect of UV-LED light and storage period on the background bacteria of chicken

149 meat
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150 To study the effect of UV light on the culturable bacteria of chicken meat, TVC and TEC were

151 determined in chicken pieces exposed to UV-LED for 3 and 6 min at 280 nm. Chicken samples were
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152 prepared and treated as described in Section 2.2. UV exposure for 6 min was found to be the most

153 effective treatment and these samples were packed in polyethylene terephthalate film laminated with

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 154 polyethylene film (PET/PE, 400 µm, Multivac, Dublin, Ireland) and heat sealed to amorphous PET/PE

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155 trays (Multivac, Dublin, Ireland) in a compressed air atmosphere using a tray sealer (TPS XL, Henkovac

156 international, Hertogenbosch, Netherlands). Subsequently, samples were stored at 4 °C and TVC and

157 TEC were determined on days 0, 1, 3, and 7 post-treatment.

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159 2.5. Comparison of the effectiveness of UV-LED technology with UV pilot-plant scale device

160 Chicken pieces were exposed to UV at 254 nm for 3 and 6 min using a UV conveyor lamp (UV Torpedo®

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161 Conveyor, 101098-T501-U001, Jenton Group, Hampshire, UK) in the Prepared Consumer Food Centre

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162 (PCFC), a pilot-plant facility located in Teagasc Ashtown Food Research Centre (Dublin, Ireland). A

163 previous publication described the structure of this machine, which has two arrays of UV lamps at the

164 top and bottom (Soro et al., 2022). The UV light dose applied at 254 nm for 3 and 6 min was 0.603 and
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165 1.206 for the top lamps (UV fluence of 0.201); and 0.873 and 1.748 for the bottom lamps (UV fluence
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166 of 1.748), respectively, and was calculated as described in Section 2.2. To determine the most effective

167 UV exposure, TVC and TEC were determined in chicken pieces treated with the UV lamp conveyor at

168 254 nm for 3 and 6 min. The treatment of 6 min was selected and the procedure as described in Section
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169 2.4 was conducted in order to compare the effectiveness of both devices for the decontamination of

170 chicken samples.

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172 2.6. Microbiological analysis

173 UV-treated and control chicken samples inoculated with C. jejuni and S. Typhimurium were blended
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174 (Star Blender, LB 400, VWR, USA) for 90 s in maximum recovery diluent (MRD, Oxoid, UK) in a 1/10

175 (w/v) proportion. From these suspensions, ten-fold serial dilutions were prepared and 0.1-mL volumes
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176 were spread plated in duplicate onto Xylose Lysine deoxycholate agar (XLD, Oxoid, Basingstoke, UK)

177 and incubated for 24 h at 37 °C to enumerate Salmonella; and onto mCCDA and incubated for 48 h at
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178 42 °C under micro-aerobic conditions to determine Campylobacter concentrations. Enumeration of

179 bacterial colonies was conducted after incubation and calculated mean values were expressed as log

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 180 CFU per g of chicken meat. To determine TVC and TEC in chicken meat, a volume of 1 mL was inoculated

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181 onto aerobic counts and Enterobacteriaceae counts 3MTM PetrifilmTM (Analab Analytical Lab. Supplies,

182 Dublin, Ireland) and incubated for 48 h at 30 °C and 24 h at 37 °C to determine TVC and TEC,

183 respectively.

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185 2.7. DNA extraction and 16S rRNA gene amplicon sequence analysis

186 DNA extraction was conducted from bacterial suspensions obtained after blending of treated chicken

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187 as well as control samples of non-inoculated chicken meat, stored for 1, 3, and 7 days to investigate

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188 treatment and storage effects on the microbial community profile of the meat during storage. The

189 DNeasy PowerFood microbial kit (Qiagen, Manchester, UK) was employed according to manufacturer’s

190 instructions. Purification and quantification of extracted DNA was carried out using with a
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191 NanodropTM 1000 Spectrophotometer (ThermoFisher Scientific, Eaton Socon, UK) and Qubit 4.0
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192 Fluorometer (Invitrogen, ThermoFisher Scientific, Eaton Socon, UK), correspondingly. From the

193 extracted DNA, PCR amplification, DNA purification, DNA library preparation, and sequencing steps for

194 16S rRNA amplicon generation were conducted by Novogene Bioinformatics Technology Co., Ltd.
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195 (Beijing, China). DNA amplification of 16S rRNA genes of the V3-V4 region was performed using the

196 primer set of 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) with barcodes
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197 included in the PCR product (Yu, Lee, Kim, & Hwang, 2005). PCR reactions were conducted using the

198 Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). Subsequently,

199 quality and quantification of PCR products were checked and these products were electrophoresed on
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200 a 2% agarose gel. Bands of 400-450 bp were excised using the Qiagen Gel Extraction Kit (Qiagen,

201 Manchester, UK). Preparation of DNA libraries was performed using the NEBNext® Ultra TM DNA
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202 Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and quantified using Qubit and

203 qPCR. Libraries were analysed using the Illumina NovaSeq 6000 platform to generate 250 bp paired-
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204 end reads (100 K tags per sample).

205

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 206 2.8. Statistical analysis

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207 2.8.1 Microbiological analysis

208 Three independent experiments were performed together with treatments in triplicate (N = 9) to

209 evaluate the effect of UV light and storage on Campylobacter, Salmonella, TVC and TEC levels in

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210 chicken meat. Differences in TVC, TEC, Campylobacter and Salmonella concentrations of treated and

211 control samples were explored using factorial analysis of variance (ANOVA) with treatment and days

212 as the grouping variables. Tukey’s multiple comparison post-hoc test (α < 0.05 level) was performed to

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213 detect any statistical differences. Statistical analysis and graph creation were conducted using

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214 GraphPad Prism 7.0 for Windows (GraphPad Software Inc, San Diego, CA, USA).

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216 2.8.2 16S rRNA gene amplicon sequence analysis

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217 Assignment of paired-end reads to samples was performed using unique barcodes which were
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218 removed together with the primer sequences. Truncated paired-end reads were merged with FLASH

219 (v1.2.7) and were called raw tags (Magoč and Salzberg, 2011). High-quality clean tags were obtained

220 after quality assessment of these raw tags under specific filtering conditions following the quantitative
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221 insights into microbial ecology (Qiime, v1.7.0) process for quality control (Caporaso et al., 2010). The

222 reference in the SILVA database (release 138) was compared with the raw tags through the UCHIME
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223 algorithm in order to detect chimera sequences that were later removed to obtain the effective tags

224 (Edgar et al., 2011; Haas et al., 2011). All the effective tags were analysed by the Uparse software

225 (v7.0.1090) and sequences with >97% similarity were assigned to the same operational taxonomic
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226 units (OTUs) (Edgar, 2013). A representative sequence for each OTU was screened using Qiime (v1.7.0)

227 and Mothur pipelines and conducted against the SSU rRNA database of SILVA (release 138) database
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228 to provide species annotation at each taxonomic level (Altschul et al., 1990; Quast et al., 2012; Wang

229 et al., 2007). The MUSCLE (v3.8.31) tool was used to obtain the phylogenetic relationships of all the
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230 OTUs (Edgar, 2004). Then, OTU abundance findings were normalised with a standard which was

231 considered as the sequence number of the sample with the least sequences. Relative abundance

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 232 visualisation of the chicken meat microbial community composition was performed using R (v4.2.1;

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233 2022-06-23) and RStudio (v2021.09.2). The quality of the 16S rRNA gene amplicon sequence findings

234 was improved using Affinity Designer (v1.8.5.703, Serif). Lastly, the indices of Observed species,

235 Shannon, Inverse Simpson and Chao1 were calculated in the normalised data to analyse the

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236 biodiversity of each sample, also known as alpha diversity analysis. Beta diversity analysis was not

237 conducted due to the small number of collected samples.

238

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239 3. Results and discussion

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240 3.1. Evaluation of the effect of UV-LED light and storage period on artificially-inoculated chicken meat.

241 A UV-LED device was employed to investigate the effects of UV light on C. jejuni and S. Typhimurium

242 inoculated on chicken meat. In Figure 1, the concentration of Salmonella and Campylobacter (log
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243 CFU/g) in treated (at 280 nm for 3 and 6 min) as well as untreated chicken fillet samples are presented.
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244 Initial mean concentrations of Campylobacter and Salmonella after Inoculation in the chicken pieces

245 were 4.86 and 4.67 log CFU/g, respectively. Application of UV-LED at 280 nm for 3 min achieved

246 significant reductions of 0.6 log CFU/g in S. Typhimurium and 0.51 log CFU/g in C. jejuni (p < 0.05)
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247 (Figure 1A and 1B). Extending the UV treatment to 6 min did not increase the inactivation of Salmonella

248 on chicken meat (reduction of 0.6 log CFU/g). However, Campylobacter levels were reduced by 0.64
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249 log CFU/g in chicken meat (as compared to non-treated samples) after UV exposure for 6 min (p <

250 0.05). Thus, a UV treatment at 280 nm for 6 min was selected as the most efficient to reduce both

251 species in chicken meat using an UV-LED device. Studies evaluating the ability of UV-LED technology
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252 to inactivate Campylobacter spp. are scarce. Haughton et al. (2012) used a UV-LED device at a

253 wavelength of 395 nm to reduce C. jejuni inoculated on skinless chicken meat and a reduction between
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254 1.43 and 2.62 log CFU/g was achieved. In the case of Salmonella spp., some authors have studied the

255 potential application of UV-LED technology to inactivate this pathogen in chicken meat (Calle et al.,
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256 2021; Wang et al., 2021). In both studies, Salmonella spp. was reduced by 1.90 log CFU/g in chicken

257 breast fillets after exposure with UVC-LED at 250-280 nm. Despite of the high inactivation rates

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 258 observed for Salmonella spp. and Campylobacter spp. with this technology, the combined effect of

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259 storage at 4 °C and UV-LED was not considered in any of these studies.

260 The combined effect of UV-LED light with the storage of chicken meat at 4 °C was evaluated in chicken

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261 fillet samples inoculated with C. jejuni or S. Typhimurium and exposed for 6 min to UV at 280 nm

262 employing the UV-LED device. Salmonella and Campylobacter concentrations in untreated chicken

263 samples and those treated with UV-LED were compared at different time points during storage (Day

264 0, 1, 3, and 7) and are presented in Figure 1. Maximum reductions were for S. Typhimurium (0.59 log

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265 CFU/g) and C. jejuni (0.66 log CFU/g) populations in chicken samples treated with UV-LED on day 0.

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266 Nevertheless, the Salmonella concentrations for treated samples were higher on days 3 and 7 than day

267 0 (p ≥ 0.05), although were still significantly lower than the untreated controls on each day of sampling

268
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(Figure 1C). A systematic review and meta-analysis conducted by Silva et al. (2022) showed how

269 Salmonella spp. concentrations may vary, increasing or decreasing in meat subjected to cold storage.
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270 This may explain the variation in Salmonella levels observed in our study as a consequence of the

271 resilience of this food-borne pathogen for cold temperatures. In samples inoculated with C. jejuni, a

272 reduction of 1.72-1.52 log CFU/g was observed in non-treated and UV-LED treated chicken samples by
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273 day 3 of storage (p < 0.05) (Figure 1D). This was the greatest level of reduction for C. jejuni levels

274 observed in samples treated for 6 min with UV-LED at 280 nm. Thus, the concentration of
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275 Campylobacter in treated samples was significantly reduced by 0.94 log CFU/g at Day 1, 1.14 log CFU/g

276 at Day 3, and 0.73 log CFU/g at Day 7, compared with non-treated chicken meat at equivalent time
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277 points (p < 0.05). Consequently, reduction of C. jejuni populations of 1.52 log CFU/g at Day 3 and 1.44

278 log CFU/g at Day 7 in chicken meat treated with the UV-LED technology were observed. In some

279 studies, Campylobacter spp. persisted for up to 18 days in refrigerated chicken meat without changes
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280 in bacterial counts (Thames et al., 2020). According to Meredith et al. (2014), the high concentration

281 of O2 in chicken packaging can reduce Campylobacter levels due to the micro-aerophilic nature of this
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282 bacterium. In the present study, the combined effect of UV-LED light at 280 nm and air packaging of

283 chicken samples reduced C. jejuni concentrations by 2.62 log CFU/g after 3-days of storage at 4 °C

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 284 compared with untreated samples at Day 0. Reducing Campylobacter by 2 log CFU/g in chicken meat

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285 could lead to a significant food safety benefit, as concluded by previous risk assessments (Soro et al.,

286 2020).

287

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288 3.2. Evaluation of the effect of UV-LED light and storage on the background microflora of chicken meat

289 To investigate the antibacterial effect of the UV-LED technology on TVC and TEC of chicken samples,

290 two different UV treatment times (3 and 6 min) at 280 nm were compared with non-treated samples.

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291 Figure 2 shows the concentration of TVC and TEC in chicken samples before and after UV exposure for

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292 3 and 6 min using the UV-LED device. The initial concentration of TVC and TEC in chicken meat was

293 5.13 log CFU/g and 2.53 log CFU/g, respectively. Application of UV treatment at 280 nm for 6 min

294 caused a significant reduction of 1.04 and 1.23 log CFU/g in TVC and TEC, respectively (p < 0.05).
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295 Moreover, although TVC were significantly reduced by 0.53 log CFU/g (p < 0.05) in chicken meat
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296 treated with UV for 3 min, this treatment was not sufficient to significantly reduce TEC in chicken

297 samples (p ≥ 0.05) (Figure 2). Therefore, a UV treatment of 6 min was selected to maximise the

298 inactivation of TVC and TEC in chicken meat. A limited number of studies to date have investigated the
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299 effect of the UV-LED technology on TVC and TEC in chicken meat (Haughton et al., 2012; Soro et al.,

300 2021). Haughton et al. (2012) and Soro et al. (2021) achieved similar reductions in TVC and TEC (~1-1.2
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301 log CFU/g) to the those observed in the presented study.

302 The combined effect of UV-LED and refrigerated storage for 7 days on TVC and TEC in chicken meat
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303 was also studied (Figure 2). Treating chicken pieces with UV at 280 nm for 6 min resulted in significant

304 reductions of TVC of 0.71 log CFU/g on Day 1, 0.86 log CFU/g on Day 3, and 0.52 log CFU/g on Day 7;

305 and reductions of TEC of 0.82 log CFU/g on Day 1, 0.81 log CFU/g on Day 3, and 0.68 log CFU/g on Day
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306 7 (p < 0.05). The application of the UV-LED resulted in counts of 6.15 log CFU/g for TVC and 3.60 log

307 CFU/g for TEC at Day 3 of storage, compared to TVC and TEC of 7.01 log CFU/g and 4.41 log CFU/g in
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308 untreated control samples, respectively. To the best of the authors’ knowledge, this is the first study

309 evaluating the combined effect of the UV-LED technology and storage of chicken meat on TVC and TEC.

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 310

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311 3.3. Comparison of the effectiveness of the UV-LED technology and a UV pilot-plant scale device

312 The application of a 6-min treatment of chicken samples with UV at 254 nm resulted in significant

313 reductions of 0.74 and 1.08 log CFU/g in TVC and TEC compared with non-treated meat (p < 0.05)

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314 (Figure 2). Interestingly, no statically significant reductions were observed for both bacterial groups

315 when chicken meat was exposed for 3 min using the UV lamp conveyor compared with non-treated

316 samples (p ≥ 0.05). To compare the effectiveness of both technologies to reduce TVC and TEC in

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317 chicken meat, the same experimental procedures in packaging and storage were followed in both

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318 untreated and treated samples. No significant difference in reductions for both TVC and TEC were

319 observed for any of the sampling points (Day 0, 1, 3, and 7) when the UV lamp system and the UV-LED

320 technology were compared (Figure 2). Germicidal effectiveness of the conventional UV lamp and UV-
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321 LED devices was previously compared by numerous studies, despite of differing wavelengths. In the
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322 majority of these, UV-LED devices showed a high inactivation efficiency, similar to the germicidal effect

323 of the conventional lamps (Kebbi et al., 2020). In our study, the effectiveness to reduce TVC and TEC

324 levels in chicken meat was similar for both technologies.

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325

326 3.4. Analysis of the microbial community composition of chicken meat

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327 The variation of the chicken microbial community composition in samples treated with both the UV

328 lamp and UV-LED devices was determined during subsequent storage using the 16s rRNA gene

329 amplicon sequencing. The relative abundance of bacteria observed at family and species level in
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330 selected chicken samples for the storage periods of Day 1, 3, and 7 is displayed in Figure 3.

331 Vibrionaceae (39%), Listeriaceae (10%), Streptococcaceae (8%) and Lactobacillaceae (7%) were the five
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332 predominant families in chicken meat stored for 1 day (Figure 3A). A clear decrease in the variability

333 of the taxonomical family and species of the microbiota present in the meat was observed by Days 3
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334 and 7 of storage at 4 °C. In Table 1, values of four calculated indices: observed species, Shannon-

335 Wiener, Inverse Simpson, Chao1, measuring the alpha diversity of the samples are presented. The

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 336 reduction in the species richness in the stored samples was also observed in these alpha diversity

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337 indices, with the observed species index of non-treated samples stored in Day 1 (866) reduced on Day

338 3 (556) and Day 7 (382). This index is not corrected for taxonomical groups that were not observed and

339 therefore, high values correspond to high richness of unique OTUs in a sample (Fisher et al., 1943). To

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340 address the potential bias in the alpha diversity, other indices such as Shannon, Inverse Simpson,

341 Chao1 were considered (Willis, 2019). Similarly, Chao1 and Shannon-Wiener values were higher in

342 samples stored for 1 day than those stored for 3 and 7 days. A study conducted by Zhang et al. (2021)

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343 also applied 16S rRNA gene amplicon sequencing in chicken meat. These authors reported a similar

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344 decreasing trend in the number of OTUs and the alpha diversity indices in chicken meat samples after

345 7 days storage (Zhang et al., 2021). The observed abundances of species belonging to these bacterial

346 families can be seen in Figure 3B. By Day 3, an increase of 33% and 4% resulted in a relative abundance
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347 of Photobacterium phosphoreum of 72% and Brochothrix thermosphacta of 14%, respectively, was
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348 detected in chicken samples. This increment in the relative abundance of these species lead to a

349 reduction of the “Others” group (from 29 to 9%), Lactococcus lacti (from 3 to 1%), Lactobacillus

350 murinus, Streptococcus salivarius and Yersinia ruckeri (from 2 to 0%), Pseudomonas fragi, Escherichia
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351 coli, Lactobacillus johnsonii, Romboutsia ilealis, Lactobacillus plantarum, Lactobacillus reuteri and

352 Sphingomonas leidyi (from 1 to 0%). Storing the chicken meat for 7 days increased the P. phosphoreum
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353 from 72 to 81% and P. fragi from 0 to 1%, and decreased B. thermosphacta from 14 to 10%, the

354 “Others” group from 9 to 7%, Lactococcus piscium from 3 to 0%, and L. lactis from 1 to 0%. Other

355 authors have also detected Photobacterium, Brochothrix, and Carnobacterium as potential dominant
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356 genera in the spoilage of chicken meat causing a decrease in microbial diversity (Liang et al., 2012;

357 Zhang et al., 2021). Moreover, Campylobacter jejuni and Salmonella spp. were not detected in any of
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358 the analysed commercial chicken meat samples stored for 7 days at 4 °C.

359 The impact of the UV-LED and UV lamp technologies on the microbial community composition of
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360 chicken meat was also studied. DNA from chicken meat samples stored for 0 and 1 days after UV-LED

361 and UV lamp treatment, and from samples treated with UV lamp and stored for 3 days failed in the

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 362 amplification step. Causes of this issue are unclear and were the reason that beta diversity analysis

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363 was not possible to be performed. Slight changes in the relative abundance (less than 1%) of P.

364 phosphoreum, B. thermosphacta, L. lactis, L. piscium were observed in the profile of samples treated

365 with UV-LED at 280 nm for 6 min compared to non-treated samples after 3-days storage. By Day 7,

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366 some differences were observed in samples treated with UV-LED and UV lamp devices in comparison

367 with untreated samples. P. phosphoreum and B. thermosphacta were the most affected species.

368 Although relative abundance of these species varied only by 8% (P. phosphoreum 73% and B.

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369 thermosphacta 18%) in chicken meat treated with UV-LED compared with untreated samples (P.

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370 phosphoreum 81% and B. thermosphacta 10%), the UV lamp treatment reduced the relative

371 abundance of the P. phosphoreum by 45% resulting in 36% and increased the B. thermosphacta in 42%

372 reaching 52%. An increase in P. fragi from 1 to 2 and 4% was also detected in samples treated with UV-
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373 LED and UV lamp, respectively. Moreover, treatment with both technologies increased by 1% the
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374 relative abundance of L. piscium in chicken meat stored at Day 7. L. lactis, L. plantarum and S. salivarius

375 shown an increase of 1% in relative abundance in samples treated with the conventional UV lamp and

376 stored by 7 days, unlike non-treated and UV-LED treated chicken meat samples. All these
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377 psychrotrophic bacteria have been associated with spoilage of meat stored aerobically and therefore,

378 they are dominant in the microbial community composition profile of chicken meat (Saenz-García et
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379 al., 2020). Rouger et al. (2020) observed that the environment in the abattoir and the mixture of gas

380 used in the packaging step may influence the microbial community composition of the chicken meat.
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381 Additionally, alpha diversity was investigated in chicken meat treated with UV-LED and the UV lamp

382 device and compared to untreated samples after a 7-days storage (Table 1). The observed species

383 index in samples subjected to UV-LED at 280 nm for 6 min and stored for 3 and 7 days resulted in 424
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384 and 77, respectively, which were lower than untreated meat stored for 3 (556) and 7 (382) days. Similar

385 reductions in OTU richness was observed in samples treated with the UV lamp for 6 min at 254 nm and
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386 stored for 7 days (73). Calculation of the Chao1, Shannon-Wiener and Inverse Simpson indices also

387 showed a reduction in richness in chicken meat exposed to UV-LED and UV lamp when comparing the

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 388 indices of untreated meat stored for 3 and 7 days. The low Inverse Simpson index may be due to a low

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389 microbial evenness in the sample (Zhang et al., 2021). This fact may be explained by the high increase

390 in relative abundance of B. thermosphacta found in chicken meat treated with the UV lamp and stored

391 for 7 days (Figure 3). Studies evaluating the impact of UV light on the microbial community composition

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392 of chicken meat are lacking and therefore, further investigation is required.

393 Differences in the bacterial profile of samples exposed to UV light detected with the 16S rRNA gene

394 amplicon sequencing may be linked with the observed bacterial reductions in TVC and TEC in chicken

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395 meat (Figure 2). However, the Enterobacteriaceae family was detected in low abundant in the

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396 sequence based analysis and therefore, it is difficult to make assumptions about this link. In the case

397 of samples exposed to UV light using the UV lamp, a change in the microbial community composition

398
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of meat was detected after 7-days storage at 4 °C, which reduced the prominence of P. phosphoreum

399 and may have facilitated an increase in the relative abundance of B. thermosphacta. Moreover,
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400 according with the current literature, it is unclear if species richness and diversity play a role in spoilage

401 (Cauchie et al., 2019). The 16S rRNA gene amplicon sequencing analysis conducted in this study is

402 preliminary and can help giving insights in meat microbial community composition. Nevertheless,
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403 limitations of this analysis method related to the determination of the taxa at species level and the

404 representation of viable bacteria in the sample remain. Furthermore, further investigation in order to
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405 measure the beta diversity or diversity between samples (Dourou et al., 2021).

406
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407 5. Conclusions

408 The present study has evaluated the effect of the UV-LED technology on S. Typhimurium and C. jejuni

409 on chicken breast fillets. The selected treatment of UV at 280 nm for 6 min was found to reduce the
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410 levels of S. Typhimurium and C. jejuni by 0.6-0.64 log CFU/g in chicken fillet samples. During

411 refrigerated storage following UV-LED treatment a reduction of 1.12 log CFU/g in Salmonella levels by
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412 day 7 was observed compared with untreated samples. In the case of C. jejuni, a decrease of 2.62 log

413 CFU/g was achieved by day 3 storage at 4 °C compared with untreated samples at Day 0 which may

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 414 mitigate the risk of Campylobacter in chicken meat. Similarly, the ability of both UV technologies to

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415 reduce TVC and TEC (1-1.2 log CFU/g) was observed, which was maintained during the 7-day storage

416 period. The 16S rRNA gene amplicon sequencing analysis showed dominance of Vibrionaceae and

417 Listeriaceae families in the microbial community composition profile of chicken meat, which were

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418 mainly represented by P. phosphoreum and B. thermosphacta, respectively. UV lamp was found to

419 have the greatest impact on the bacterial profile of chicken meat compared to the UV-LED device,

420 allowing a greater dominance of B. thermosphacta over P. phosphoreum. While TVC and TEC

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421 reductions may be similar in chicken meat for both UV technologies, the community shifts differ, which

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422 may impact on the spoilage profile of the product following treatment. UV-LED technology may be an

423 effective strategy to decontaminate chicken meat with low impact in the microbial community

424 composition.
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425
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426 Funding

427 This study was funded by the Teagasc Walsh Scholarship program, Department of Agriculture, Food,

428 and Marine (DAFM) under the Food Institutional Research Measure (FIRM) program [Grant number:
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429 DAFM/17/F/275].

430
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431 Author Contributions

432 Arturo B. Soro: Conceptualization, performance of UV light experiments, methodology, investigation,

433 majority of formal analysis and visualization, writing—original draft preparation and writing—review
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434 and editing. Daniel Ekhlas: conceptualization, writing—review and editing, DNA extraction

435 methodology, and visualization of relative abundance graphs. Sajad Shokri: microbiological analysis
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436 methodology, writing—review and editing. Ming Ming Yem: microbiological analysis methodology. Rui

437 Chao Li: microbiological analysis methodology. Soukaina Barrou: sample preparation and bacterial
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438 inoculation methodology microbiological. Shay Hannon: writing—review and editing, supervision.

439 Paul Whyte: writing—review and editing, supervision. Declan J. Bolton: writing—review and editing,

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 440 supervision. Catherine M. Burgess: conceptualization, writing—review and editing, supervision. Paula

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441 Bourke: writing—review and editing, supervision, funding acquisition. Brijesh K. Tiwari: writing—

442 review and editing, supervision, project administration, funding acquisition.

443

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444 References

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 563 Table 1. Alpha diversity indices (Observed species, Shannon, inverse Simpson and Chao1) calculated in

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564 selected treated and untreated chicken samples stored at 4 °C for 7 days.

Index
Observed Inverse
Treatment Shannon Chao1
species Simpson

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Non-treated
866.00 4.60 1.21 920.89
Day 1
Non-treated
556.00 1.69 2.16 681.71
Day 3
UV-LED Day 3 424.00 1.67 2.23 468.59
Non-treated
382.00 1.47 2.95 408.66

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Day 7
UV-LED Day 7 77.00 1.40 2.30 112.10

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UV lamp Day 7 73.00 1.83 1.66 86.57
565

566

567
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568
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569

570

571
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572

573
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574

575
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576

577

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579

580
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581

582

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 583 Figure captions

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584 Figure 1. Concentration (log CFU/g) of S. enterica Typhimurium (A) and C. jejuni (B) in untreated and

585 chicken samples exposed to UV-LED at 280 nm for 3 and 6 min. The concentration of Salmonella (C)

586 and Campylobacter (D) in untreated and UV-treated samples for 6 min stored aerobically at 4 °C for 7

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587 days and sampled at days 0, 1, 3, and 7. Results are expressed as mean and standard deviation.

588 Significant differences between untreated and treated samples at each time point are indicated with

589 * (p < 0.05).

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590

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591 Figure 2. Concentrations (log CFU/g) of TVC (A) and TEC (B) in untreated and UV-LED treated chicken

592 samples with a UV-LED device at 280 nm and a UV lamp conveyor at 254 nm for 3 and 6 min.

593 Concentration of TVC (C) and TEC (D) of untreated samples and treated samples with UV lamp and UV-
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594 LED for 6 min stored aerobically for 7 days at 4 °C and sampled at days 0, 1, 3, and 7. Results expressed
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595 in mean and standard deviation. Significant differences between untreated and treated samples are

596 indicated with * (p < 0.05).

597
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598 Figure 3. Relative abundance (%) of the taxonomy at family (A) and species (B) level of the bacteria

599 detected in chicken meat stored for 1, 3, and 7 days treated with UV light at 280 nm for 6 min with an
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600 UV-LED or UV lamp device and untreated controls. Unassigned and low abundant species (< 1 %) were

601 summarized in the group “Others”.

602
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 A
*
B
*
e d
6

Salmonella concentration
* 6 *
w

Campylobacter concentration
e
4.67
5

i
4.86
4.07 4.07 5 4.35 4.22

(Log CFU/g)

v
4

(Log CFU/g)
4

e
3
3
2

r
2

1 r
0
Non-treated 3 min 6 min

ee 0
Non-treated 3 min 6 min

t p D

Campylobacter concentration
8 Non-treated * 6
Salmonella concentration

Non-treated
* *

o
7 * UV-LED/280/6 6.51
UV-LED/280/6
5.32 5.39
5
*

n
6 5.66 5.41 5.22 4.18
(Log CFU/g)

4.14

(Log CFU/g)
5.07 4.95
5 4 3.48 3.24 *

t
2.66
4 3 2.17

ir n
3 1.52 1.44
2
2
1 1

e p 0
Day 0 Day 1 Day 3 Day 7
0
Day 0 Day 1 Day 3 Day 7

r
603

604 Figure 1.

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 *
A
*
B
e d
w
Non-treated 6
6 * Non-treated

Bacterial concentration
5.13

Bacterial concentration
5.13 3 min 4.67
4.39 3 min

e
5
4.60 5
6 min

i
4.09
6 min

(Log CFU/g)
(Log CFU/g)
4
*

v
4
*
2.53 3 2.53
3

e
2.11
2.06 1.45

r
1.30 2
2

r
1 1

e
0 0
TVC TEC TVC TEC

p e D

t
Control * Control *
10 * 8
UV-LED/280/6 *

Total Enterobacteriaceae
UV-LED/280/6

o
* 8.75
8.23 7.97
UV lamp
6.33
Total bacteria counts

UV lamp *
8 * 5.65
*

n
*
7.01 6 * 5.36

(Log CFU/g)
* 6.33
(log CFU/g)

*
6.15
* 4.41

counts
t
5.46
6 5.13
4.39
4.75 4.56
4
* * 3.60 3.57
4.09
* 3.01

ir n
4 2.53
2.19
2.17

1.30 1.45
2
2

605

e p 0
Day 0 Day 1 Day 3 Day 7
0
Day 0 Day 1 Day 3 Day 7

606
r
Figure 2.

P 26

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4382846
 ed
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607

608 Figure 3.
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610
ep

611
Pr

612

27

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4382846