Light-activated antimicrobial activity of turmeric residue edible coatings against cross-contamination of Listeria innocua on sausages

Accepted Manuscript Light-Activated Antimicrobial Activity of Turmeric Residue Edible Coatings Against Cross-Contamination of Listeria innocua on Sausages

Juliano V. Tosati, Erick F. de Oliveira, J. Vladimir Oliveira, Nitin Nitin, Alcilene R. Monteiro PII:

S0956-7135(17)30372-9

DOI:

10.1016/j.foodcont.2017.07.026

Reference:

JFCO 5719

To appear in:

Food Control

Received Date:

24 May 2017

Revised Date:

21 July 2017

Accepted Date:

22 July 2017

Please cite this article as: Juliano V. Tosati, Erick F. de Oliveira, J. Vladimir Oliveira, Nitin Nitin, Alcilene R. Monteiro, Light-Activated Antimicrobial Activity of Turmeric Residue Edible Coatings Against Cross-Contamination of Listeria innocua on Sausages, Food Control (2017), doi: 10.1016/j. foodcont.2017.07.026

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Light-Activated Antimicrobial Activity of Turmeric Residue Edible Coatings Against Cross-

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Contamination of Listeria innocua on Sausages

3 4

Juliano V. Tosatia, Erick F. de Oliveirab,c, J. Vladimir Oliveiraa, Nitin Nitinb,d, Alcilene R.

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Monteiroa #

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a Departamento

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Santa Catarina, Florianópolis, SC, Brazil

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b Department

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c CAPES

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d Department

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USA.

de Engenharia Química e Engenharia de Alimentos, Universidade Federal de

of Food Science and Technology, University of California, Davis, CA, USA.

Foundation, Ministry of Education of Brazil, Brasilia, DF, Brazil. of Biological and Agricultural Engineering, University of California, Davis, CA,

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# Address correspondence to Alcilene R. Monteiro, [email protected] +554837212512

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ABSTRACT The high number of foodborne outbreaks in ready-to-eat (RTE) food such as cooked

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sausages illustrate the importance of controlling microbial safety during post-processing stages

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of food products. Edible antimicrobial coatings can be applied to food after processing and

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sanitation with the goal of preventing microbial cross-contamination, decreasing the risk of

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foodborne illness and increasing the product shelf-life. In this study, edible hydrogel coatings

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that can present strong antimicrobial activity when combined with UV-A light were prepared.

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The hydrogels coatings were prepared using either turmeric residue and gelatin hydrogels (TGH)

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or cassava starch and gelatin hydrogels with added purified curcumin (CGH). The coatings were

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characterized regarding their thickness, encapsulated curcumin concentration and water swelling,

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in addition to their light-activated antimicrobial activity against different initial loads of Listeria

27

innocua at different incubation temperature. Additionally, the coatings were applied to the

28

surface of cooked sausages and evaluated for their ability to prevent bacterial cross-

29

contamination. It was observed that UV-A light-exposed hydrogels coatings could inactivate

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more than 5 log CFU/mL of L. innocua after light treatments as short as 5 min. In addition, the

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light-activated antimicrobial activity of the hydrogel coatings were not affected by the incubation

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temperature. Hydrogel-coated sausages exposed to UV-A light experienced a reduction from 4

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log CFU/mL of incubated bacteria to levels below the detection limit of 1 log CFU/mL after 5

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and 15 min of light exposure, for CGH and TGH, respectively. Further mechanistic studies

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suggested that L. innocua inactivation was due to the photo-irradiation of low levels of curcumin

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released from the coatings to solution. Lastly, it was shown that the combination of curcumin-

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loaded hydrogels coatings and UV-A light have great potential as antimicrobial coatings to

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prevent cross-contamination of L. innocua in refrigerated sausages.

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1

INTRODUCTION Food contamination continues to be a big concern for public health, consumer, regulatory

42

agencies and food industries around the world (Giaouris et al., 2014). Read-to-eat (RTE) meat

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products are subjected a proper heat treatment for elimination of non-sporogenic pathogens

44

(Brasileiro et al., 2016). Nevertheless, in America and in Europe several outbreaks of foodborne

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diseases in RTE have been reported by Listeria monocytogenes contamination (Buchanan,

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Gorris, Hayman, Jackson, & Whiting, 2017). The main factor responsible for their occurrence is

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cross-contamination during post processing operations (slicing, chopping, comminuting) and the

48

potential of L. monocytogenes to grow under various conditions, such as the presence or absence

49

of oxygen, over a large pH range (4.7 - 9.2) and at low temperature (Magalhães et al., 2016).

50

Post-processing protection using antimicrobial edible films or coatings has been proposed

51

as a potential approach to prevent or minimize cross-contamination of sausages (Cagri, Ustunol,

52

Osburn, & Ryser, 2003; Cagri, Ustunol, & Ryser, 2002; Gadang, Hettiarachchy, Johnson, &

53

Owens, 2008; Marchiore et al., 2017; Nguyen, Gidley, & Dykes, 2008; Siripatrawan & Noipha,

54

2012). Natural biopolymers such as proteins, polysaccharides and lipids or mixture of them, have

55

been used to develop biodegradable coatings and films (Alemán et al., 2016). Furthermore, the

56

use of industrial residues to form edible coatings has become popular in the recent years

57

(Maniglia, Domingos, de Paula, & Tapia-Blácido, 2014). Turmeric (Curcuma longa L.) residue

58

is a by-product obtained after supercritical fluid extraction (SFE) and pressurized liquid

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extraction (PLE) of curcumin and curcuminoids from turmeric rhizomes. The turmeric residue

60

obtained is mostly composed of starch, fibers and traces of oleoresin and curcuminoids, which

61

confers the turmeric residue great potential to be used for the preparation of edible coatings

62

(Maniglia et al., 2014; Osorio-Tobón, Carvalho, Rostagno, Petenate, & Meireles, 2014). In

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Brazil, sausage are commonly wrapped using natural or artificial coatings and immersed in

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yellow colorant (MAPA, 2000). In that scenario, turmeric residue could be applied on sausage

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from an antimicrobial coating with the goal of preventing microbial cross-contamination during

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food handling (Maniglia et al., 2014).

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During the last decade, food industries have explored the use of non-thermal processes

68

such as UV-C light irradiation, ionizing radiation or hydrostatic high pressure to inactivate

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microorganisms or to reduce microbial cross-contamination on food, with minimal impacts on

70

the sensorial properties of food (Bayındırlı, Alpas, Bozoğlu, & Hızal, 2006; Janisiewicz, Takeda,

71

Glenn, Camp, & Jurick, 2015; Mukhopadhyay, Ukuku, & Juneja, 2015; Shahbaz et al., 2014;

72

Sommers, Sheen, Scullen, & Mackay, 2017; Tawema, Han, Vu, Salmieri, & Lacroix, 2016).

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However, many of these non-thermal processes have disadvantages such as significant

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investment in specialized equipment that may limit their use on food. Thus, there is an unmet

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need for technologies which are safe, efficient, practical and preferably inexpensive for

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inactivating pathogenic microorganism at a local food processing, handling and service facilities

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(Liu et al., 2016).

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Among various technologies, light-mediated inactivation of microbes has emerged as a

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potential approach that could be implemented in various settings with low investment costs. One

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of the challenges with UV-C light-mediated approaches to inactivate microbes is the potential

81

loss in organoleptic processes induced by oxidation processes triggered by UV-C radiation

82

(Guerrero-Beltran & Barbosa-Canovas, 2004). To address this challenge few studies have

83

evaluated the use of food grade compounds with longer wavelength light frequencies to

84

inactivate a diversity of microbes (Cossu et al., 2016; Dovigo et al., 2011; Shirai, Kajiura, &

85

Omasa, 2015; Yin et al., 2013). In this process, which is similar to the conventional photo-

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dynamic therapy (PDT) approach used in biomedical applications, food grade compounds such

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as organic acids, food grade dyes and polyphenolic compounds can generate oxidative stress

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species such as free radicals and singlet oxygen species that can lead to microbial inactivation

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(Haukvik, Bruzell, Kristensen, & Tønnesen, 2009; Qian et al., 2016; Sarkar & Hussain, 2016).

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The use of longer wavelengths such as blue light or UV-A can reduce the photonic energy

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required for the light-mediated process and thus reduce any significant impact on the

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organoleptic properties of food (Liu et al., 2016). Many of the current studies using light-

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activated compounds have been conducted without the presence of food materials and are often

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conducted only in aqueous solutions. To the best of our knowledge, only purified food grade

95

compounds have been used as photo-activated compounds in food-related applications

96

(Buchovec, Lukseviciute, Marsalka, Reklaitis, & Luksiene, 2016; López-Carballo, Hernández-

97

Muñoz, Gavara, & Ocio, 2008; Oliveira, Cossu, Tikekar, & Nitin, 2017; Penha et al., 2016;

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Temba, Fletcher, Fox, Harvey, & Sultanbawa, 2016; Tiwari et al., 2009; Wu et al., 2016). This

99

could represent a potential limitation as purified compounds can add a significant cost to the

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process. In this study, turmeric residue obtained after the combination of supercritical fluid

102

extraction and pressurized liquid extraction processes was used to prepare antimicrobial

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hydrogels to inhibit bacterial cross-contamination in meat products (Maniglia, de Paula,

104

Domingos, & Tapia-Blácido, 2015; Osorio-Tobón et al., 2014). The selection of curcumin as a

105

photo-activated antimicrobial agent was inspired by photodynamic therapy (PDT) treatments

106

used in oral or dental applications (Araújo, Fontana, Bagnato, & Gerbi, 2012; Dovigo et al.,

107

2011; Paschoal et al., 2013). Furthermore, applications of the PDT approach based on the

108

combination of light and curcumin in biomedical applications suggest that the generation of toxic

109

products was not of significant concern (Araújo, Fontana, Bagnato, & Gerbi, 2012; Dovigo et al.,

110

2011; Paschoal et al., 2013). In contrast to biomedical applications, this study is focused on using

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a food-grade by-product of the curcumin extraction process for the inactivation of

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microorganisms on a meat surface (Sandikci Altunatmaz et al., 2016). Unlike previous studies

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with food-grade compounds in aqueous solution, this study is focused on application of a photo-

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activated antimicrobial agent on a solid food product (López-Carballo et al., 2008). To deliver

115

curcumin from turmeric residue to a meat surface, the hydrogel was selected as a model delivery

116

system. One of the potential advantages of the hydrogel approach are that this proposed

117

antimicrobial treatment could be combined with edible coatings on food surfaces. These edible

118

coatings can provide controlled release of encapsulated residual curcumin from the turmeric

119

residue maintaining a significant concentration of the photo-activated antimicrobial compound

120

on the surface of a meat product (Liu et al., 2016).

121

In this way, edible hydrogels of turmeric residue and gelatin that could be activated in

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combination with UV-A light showing enhanced antimicrobial activity were prepared and

123

characterized. In addition, hydrogels of cassava starch and gelatin supplemented with purified

124

curcumin were prepared and characterized in order to compare with turmeric residue hydrogels

125

which have residual curcumin naturally present. The hydrogels were used to form coatings and

126

the UV-A light-activated antimicrobial activity of these coatings was assessed against Listeria

127

innocua, a non-pathogenic surrogate for L. monocytogenes, at both 23ºC and 4ºC. The hydrogels

128

were also applied on sausages and the ability of sausage-coated hydrogels to prevent L. innocua

129

cross-contamination were evaluated. Furthermore, the possible light-mediated antimicrobial

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mechanisms of the hydrogel coatings were investigated by monitoring curcumin release from the

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hydrogels and UV-A light-mediated curcumin degradation within the hydrogels.

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2

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2.1

MATERIALS AND METHODS Materials

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Glycerol, Curcumin and Gelatin were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Turmeric was supplied by Office Herbal Pharmacy Handling Ltda (Ribeirão Preto, SP,

137

Brazil). Phosphate-buffered saline (PBS), Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA)

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were obtained from Fisher Scientific (Pittsburgh, PA, USA). Commercial cassava starch (Bob’s

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Red Mill®, Milwaukie, OR, USA) and commercial sausages (Chicken Franks Foster Farms®,

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Livingston, CA, USA) were purchased from local markets. The commercial sausages used in this

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study were 57% water, 28% total fat, 12.5% protein and 1.78% carbohydrate. Ultrapure water

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was obtained using a Milli-Q filtration system (EDM Millipore; Billerica, MA, USA).

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2.2

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2.2.1

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Hydrogel preparation Turmeric-gelatin solution Turmeric residue was obtained after curcuminoids extraction from turmeric by

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Supercritical Fluid Extraction followed by Pressurized Liquid Extraction as previously described

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(Osorio-Tobón et al., 2014). The resulting starch-rich turmeric residue was then ground, sieved

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(mesh 0.075 mm) and stored until further use. Turmeric-gelatin solution was prepared following

149

the methodology described before (Maniglia et al., 2014). First, aqueous starch-rich turmeric

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solution (5 w/w) was prepared by dispersing turmeric residue in hot water at 85 °C under

151

magnetic agitation for 4 h (IKA Works, Wilmington, NC). During this time, the turmeric-starch

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solution was further homogenized every hour using a high-shear probe mixer (2 min at 12000

153

rpm; Ultra Turrax T25, IKA Werke, Germany). Gelatin and glycerol were then incorporated into

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the starch-rich turmeric solution and kept under magnetic agitation for 30 min at 70 °C. The

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resulting turmeric-gelatin solution was cool down to 50 ºC and further used to prepare turmeric-

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gelatin hydrogel coatings.

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2.2.2

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Cassava-gelatin hydrogel solution The cassava-gelatin solution was prepared as follows. First, cassava starch aqueous

159

solution (8.4 w/w) and gelatin aqueous solution (12.0 w/w) were separately prepared at 71 ºC

160

under magnetic agitation until completely solubilized. Then, the cassava starch and the gelatin

161

solutions were combined, glycerol was added and the mixture was homogenized at 70 ºC under

162

magnetic agitation for 30 min. The cassava-gelatin solution was cool down to 50 ºC, curcumin

163

was added to a final curcumin concentration of approximately 150 µg/mL and the resulting

164

cassava-gelatin solution was further used to prepare cassava-gelatin hydrogel coatings.

165

2.2.3

166

Turmeric-gelatin hydrogel and Cassava-gelatin hydrogel coatings In order to prepare the hydrogel coatings, 0.5 mL of turmeric-gelatin solution or 0.5 mL

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of cassava-gelatin solution were placed in an individual well of a sterile 24-wells flat bottom

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polystyrene plate (1.56 cm of diameter) and stored at 4 ºC for 30 min. The resulting turmeric-

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gelatin hydrogel coating and cassava-gelatin hydrogel coating were labeled TGH and CGH,

170

respectively. Control hydrogels (K) were prepared using cassava-gelatin solutions without the

171

addition of curcumin. The composition of the final hydrogels coatings was approximately 88 %

172

(w/w) water, 6 % (w/w) gelatin, 1.8 % (w/w) glycerol and either 4.2 % (w/w) turmeric residue,

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for TGH, or 4.2 % (w/w) cassava starch, for CGH and K.

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Turmeric-gelatin solution and cassava-gelatin solution were also applied on the surface of

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commercial sausages to produce TGH-coated sausages and CGH-coated sausages, respectively.

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Commercial sausages were cut to perfectly fit into an individual well from a 24-wells plate (1.85

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cm of diameter and 0.5 cm of thickness). Then, the 24-wells plate containing sausages were used

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to prepare hydrogel-coated sausages following the same methodology as described above.

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2.3

Hydrogels coatings characterization

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The hydrogels coatings were characterized regarding the coating thickness, the

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concentration of curcumin in the final coatings and the swelling behavior of the hydrogels in

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water. Hydrogel thickness were measured using a manual digital micrometer (Mitutoyo Co,

183

Japan) to the nearest 0.01 mm. Thickness measurements were performed at different locations of

184

the hydrogels and the average calculated . The concentration of curcumin in the hydrogels was

185

evaluated through UV-Vis spectroscopy (Spectramax 340, CA, USA). Briefly, curcumin was

186

extracted from TGH and CGH using ethanol (50 % v/v), the absorbance of the extract was

187

determined at 425 nm and the curcumin concentration was inferred based on a standard

188

calibration curve. The hydrogels swelling behavior in water was evaluated by monitoring the

189

increase in hydrogel mass before and after incubation in water. Hydrogels coatings prepared

190

inside individual wells from a 24-wells polystyrene plate were incubated with 4 mL of water for

191

30 min and the swelling behavior was expressed in percentage of mass increase due to water

192

uptake.

193

2.4

Microbial culture

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Listeria innocua (ATCC 33090, Manassas, VA, USA) modified with a Rifampicin

195

resistant plasmid were supplied by Dr. Trevor Suslow from the Department of Food Science and

196

Technology at University of California, Davis. Fresh L. innocua suspensions were prepared

197

before each experiment as follows. First, bacteria stored in liquid nitrogen were streaked onto

198

TSA plates and grown overnight at 37ºC. Then, a single colony of L. innocua was picked from

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the plate and cultured overnight in TSB broth containing Rifampicin 50 μg/mL at 37°C and 150

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rpm until stationary phase was reached (around 9.8 log CFU/mL). Lastly, working bacterial

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suspensions were prepared by serially diluting the stationary phase bacteria in sterile water to

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final L. innocua concentrations of 4.3 and 6.3 log CFU/mL.

203

2.5

204

UV-A light chamber The UV-A light source consisted of four UV-A lamps (320 – 400 nm; 18 W; Actinic BL,

205

Philips, Holland) located on the inside-top of a plastic chamber. Samples were positioned at the

206

center of the chamber, 8 cm away from the lamps. The average UV-A light intensity at the center

207

of the chamber was 32 ± 0.2 W/m2 as measured by an UV-A light meter (Model UV-340A,

208

Lutron Electronics, Taiwan).

209

2.6

UV-A light activated antimicrobial activity of hydrogels coatings

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The antimicrobial activity of TGH and CGH in combination with UV-A light were

211

evaluated against L. innocua. TGH and CGH coatings were prepared on the surface of individual

212

wells from a 24-wells polystyrene plate as described above. Then, half milliliter of a fresh L.

213

innocua suspension was added to the wells containing each hydrogel coatings leading to a final

214

bacteria concentration of 6 log CFU/mL. The plate was then placed at the center of the UV-A

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light chamber and exposed to UV-A light for up to 30 min. After each time point, the plate was

216

removed from the chamber and aliquots were thoroughly homogenized using a pipette tip. After

217

homogenization, a fixed volume of 0.1 mL was aspirated from top of the hydrogel coating,

218

serially diluted in sterile PBS and spread onto TSA plates containing Rifampicin (50 μg/mL).

219

The plates were incubated at 37 ºC for 48 h, before L. innocua populations were determined by

220

the standard plating count method and expressed as colony forming unit CFU/mL. Control

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hydrogel coatings without curcumin (K) were used to evaluate the effect of UV-A light alone on

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bacterial inactivation. In addition, L. innocua incubated with TGH and CGH and not exposed to

223

UV-A light were performed as dark controls.

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The UV-A light activated antimicrobial activity of the hydrogels coatings was evaluated

225

at both 23 ºC and 4 ºC. When the experiment was performed at 4 ºC, the hydrogels and the

226

bacterial suspension were pre-incubated at 4 ºC for one hour before bacteria was added to the

227

hydrogel coatings. The UV-A light chamber was placed inside a refrigerated room (T = 4 ºC) and

228

UV-A light exposure and further bacteria quantification was performed as described above.

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2.7

UV-A light activated antimicrobial activity of hydrogel-coated sausages The TGH and CGH coatings were also applied on the surface of commercial sausages

232

and the UV-A light activated antimicrobial activity of TGH and CGH coated sausages were

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evaluated at refrigerated temperature (4 ºC). Since UV-A light exposure of L. innocua

234

suspensions on the surface of hydrogel-coated sausages were performed at refrigerated

235

temperature, both hydrogel coatings and working bacterial suspensions were pre-incubated at 4

236

ºC for one hour before the experiment. After pre-incubation, half milliliter of fresh L. innocua

237

suspensions was added to individual wells from a 24-wells polystyrene plate containing the

238

hydrogel-coated sausages. Two different initial bacterial loads of L. innocua were evaluated: 4

239

and 6 log CFU/mL. The samples were then placed inside the UV-A light chamber at 4 ºC and

240

exposed to UV-A light for up to 30 min. After UV-A light exposure, bacterial populations were

241

determined as described previously and expressed as CFU/mL. Sausage coated with hydrogel

242

without curcumin (K) were used as control.

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The ability of TGH- and CGH-coated sausages to inactivate bacteria after multiple cycles

244

of bacterial contamination and UV-A light exposure was evaluated at 4 ºC. L. innocua

245

contamination of TGH and CGH coated sausages was performed as described above achieving

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an initial bacterial load of 4 log CFU/mL. Next, the samples were exposed to UV-A light for 5

247

and 20 min, for CGH and TGH coatings respectively. The sample-coated sausages were then re-

248

contaminated with 4 log CFU/mL of L. innocua and exposed again to UV-A light for the same

249

period of time. A third cycle of bacterial contamination and UV-A light exposure were also

250

performed. Sample-coated sausages were either exposed to one, two or three cycles of

251

contamination-decontamination and bacterial count after each cycle was determined and

252

expressed as previously described.

253

2.8

254

UV-A light activated antimicrobial activity of curcumin released from hydrogel coatings The UV-A light activated antimicrobial activity of curcumin released from hydrogels

255

were performed in order to evaluate the effect of curcumin release on the UV-A light activated

256

antimicrobial activity of the hydrogels. Curcumin release was achieved by incubating 0.5 mL of

257

water with either TGH or CGH for 5 and 30 min. After the incubation time, the water solution

258

was removed from the top of the hydrogel coating and transferred to an individual well of a 24-

259

well polystyrene plate. Half milliliter of a fresh bacterial solution was added to the solution

260

containing curcumin released from the hydrogels giving an initial bacterial concentration of 1 x

261

106 CFU/mL. The samples were exposed to UV-A light at 23 ºC for 5 min and the bacterial

262

count was determined and expressed as previously described. In addition, in order to evaluated

263

the impact of the concentration of curcumin released from the hydrogels, curcumin solutions

264

with different concentrations were prepared, combined with L. innocua (final concentration of 6

265

log CFU/mL) and exposed to UV-A light at 23 ºC for 5 min.

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2.9

Degradation of curcumin in hydrogel coatings by UV-A light irradiation

267

Degradation of curcumin was performed by exposing the TGH and CGH coatings to UV-

268

A light for 5, 15 and 30 min and measuring the concentration of curcumin before and after UV-A

269

light exposure. The concentration of curcumin was determined by extracting curcumin from the

270

hydrogels with ethanol 50% (v/v), reading the absorbance at 425 nm with an UV-Vis

271

spectrophotometer (Spectramax 340, CA, USA) and calculating the curcumin concentration

272

based on a standard calibration curve. Degradation of curcumin was expressed as percentage loss

273

of initial curcumin.

274

2.10 Statistical analysis

275

All experiments described were performed at least in triplicate and the results were

276

evaluated using analysis of variance (ANOVA) by the software Statistic 6.0 (StatSoft Inc., USA)

277

and the factors showing significant difference (p ≤ 0.05) were submitted to Tukey’s test.

278 279

3

280

3.1

RESULTS Hydrogels characterization

281

In this study, the two model hydrogels were formed using turmeric residue-gelatin

282

hydrogel (TGH) and cassava-gelatin hydrogel supplemented with purified curcumin (CGH). The

283

physical properties of the TGH and CGH hydrogels are summarized in Figure 1. Curcumin

284

concentration within the hydrogels was estimated based on the UV-VIS absorbance of the

285

extraction solution at 425 nm and the estimated concentration varied between 129.37 ± 3.43 and

286

140.51 ± 3.21 µg/mL for CGH and TGH, respectively. The hydrogels presented similar

287

thickness, around 1.31 ± 0.14 and 1.33 ± 0.15 mm for TGH and CGH, and similar diameters,

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around 1.56 cm, reflecting the geometry of the mold used during preparation of both hydrogels.

289

In addition, the hydrogels presented similar swelling in solution, where the uptake of water

290

increased the hydrogels mass by 3.63 % and 1.08 % (w/w) for CGH and TGH, respectively.

291 292

Figure 1 3.2

UV-A light activated antimicrobial activity of hydrogel coatings

293

With the overall goal of reducing the risk of cross-contamination, this study is aimed at

294

evaluating the UV-A light activated antimicrobial properties of CGH and TGH after coating on

295

the surface of a sausage selected as a model system in this study. The first step was to evaluate

296

the antimicrobial activity of the CGH and TGH without the sausage and then evaluate these

297

materials in combination with the sausage. The antimicrobial activity of TGH and CGH against

298

L. innocua under UV-A light exposure was evaluated at room temperature (23 ºC) and

299

refrigerated temperature (4 ºC), temperatures at which sausages are commonly stored, and the

300

results were represented in Figure 2. Bacterial suspension incubated with CGH and exposed to

301

UV-A light (L+) at 23 ºC showed significant antimicrobial activity, where more than 5 log

302

CFU/mL reduction in the initial bacterial suspension count was achieved after 5 min of UV-A

303

light exposure (Fig. 2a). In contrast, bacteria incubated with TGH and exposed to UV-A light

304

(L+) at the same temperature presented a significantly (p < 0.05) lower bacteria inactivation rate,

305

where around 3 log CFU/mL reduction in the initial bacterial suspension count was observed

306

after a considerably higher exposure time of 30 min (Fig. 2c). As shown in Figure 2b and 2d, the

307

UV-A light activated antimicrobial activity of both CGH and TGH was not influenced by the

308

temperature change from 23 ºC to 4 ºC, illustrating that this treatment process can be conducted

309

both at room temperature and refrigerated conditions. When L. innocua were incubated with

310

CGH and TGH without UV-A light exposure (L-), no bacterial inactivation was observed. In

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addition, there was no reduction in bacterial count for L. innocua cells incubated with curcumin-

312

free hydrogels (K) and exposed to UV-A light (L+). These results suggest that both the presence

313

of curcumin and UV-A light exposure are necessary to achieve rapid inactivation of L. innocua.

314 315

Figure 2 3.3

316

UV-A light activated antimicrobial activity of sausage-coated hydrogels After validating antimicrobial activity of CGH and TGH, the hydrogels were coated on

317

sausage samples and incubated with a bacterial suspension. Inactivation of incubated bacteria on

318

coated sausage was evaluated using the experimental approaches described in the Materials and

319

Methods (Section 2.6). UV-A light exposure reduced the initial load of bacteria on CGH coated

320

sausage from 6 log CFU/mL and 4 log CFU/mL to below the detection limit (less than 1

321

CFU/mL) after 15 min and 5 min of treatment. In the case of TGH coated sausages, 30 min and

322

20 min of UV-A light reduced the initial load of bacteria on TGH coated sausage from 6 log

323

CFU/mL and 4 log CFU/mL, respectively, to below the detection limit (less than 1 CFU/mL).

324

Sausages coated with curcumin-free hydrogel (K) and exposed to UV-A light presented no

325

reduction in the initial bacterial load of 6 log CFU/mL and 4 log CFU/mL.

326 327 328

Figure 3 3.4

Antimicrobial activity of sausage-coated hydrogels exposed to multiple cycles of UV-A light

329 330

In the next step, our goal was to evaluate the potential of CGH- and TGH-coated

331

sausages to inactivate L. innocua with multiple cycles of bacterial contamination in the presence

332

of UV-A light (Fig. 4). CGH-coated sausages was able to inactivate more than 3 log CFU/mL of

ACCEPTED MANUSCRIPT 16 333

bacteria after each 5 min UV-A light exposure cycle, presenting excellent antimicrobial activity

334

even after 3 cycles of bacterial suspension incubation with CGH coated sausage. In contrast,

335

TGH-coated sausages presented a progressive decrease in antimicrobial activity after each 20

336

min UV-A light exposure cycle, achieving bacterial inactivation of more than 3 log CFU/mL

337

after the first incubation cycle, 2.5 ± 0.3 log CFU/mL after the second incubation cycle and 1.4 ±

338

0.3 log CFU/mL after the third incubation cycle.

339 340 341

Figure 4 3.5

UV-A light activated antimicrobial activity of curcumin released from hydrogel coatings One of the potential mechanisms for the antimicrobial activity of the hydrogel coatings

342

could be related to the release of curcumin from the hydrogel upon contact with solutions

343

containing bacteria. CGH and TGH hydrogels were incubated with aqueous solution for fixed

344

periods of time. The aqueous solution were then incubated with bacteria (initial concentration of

345

6 log CFU/mL) and exposed to UV-A light as described in the Materials and Methods (Section

346

2.8) and the results are presented in Figure 5. In the case of CGH, the fraction of curcumin

347

released after 5 min of incubation of the CGH with an aqueous solution was sufficient to

348

inactivate more than 5 log CFU/mL of L. innocua after UV-A light exposure. In the case of

349

TGH, fraction of curcumin released from TGH to solution after 30 min of incubation was able to

350

inactivate only 2.1 ± 0.2 log CFU/mL of bacteria after UV-A light exposure for the same time

351

interval as in the case of CGH. The concentration of curcumin released from the hydrogels was

352

also evaluated using UV-Vis spectroscopy. The concentration levels of curcumin released from

353

CGH and TGH were lower than the detection limit of a UV-Vis spectrometer (detection limit

354

was around 50 µg/mL; data not shown). In order to overcome the detection limit issue and to

355

assess the concentration of curcumin released from both hydrogels, UV-A light-exposed

ACCEPTED MANUSCRIPT 17 356

curcumin solutions (0.05 to 10 µg/mL) were evaluated against 6 log CFU/mL of L. innocua and

357

the results were expressed in Figure 5b. It was observed that curcumin concentrations as low as

358

0.05 µg/mL was enough to inactivate 2.4 ± 0.5 log CFU/mL of bacteria while more than 5 log

359

CFU/mL reduction in bacterial count could be achieved by only 0.1 µg/mL of curcumin. It can

360

be infered that CGH releases at least 0.1 µg/mL of curcumin to solution after 5 min of release

361

time, while curcumin released from TGH is not more than 0.05 µg/mL even after 30 min of

362

release time. Together, these results suggest that release of curcumin from the hydrogels can

363

result in inactivation of bacteria in the presence of UV-A light and also that the CGH releases

364

curcumin faster than TGH, resulting in the enhanced antimicrobial activity of CGH in the

365

presence UV-A light as compared to TGH.

366 367

Figure 5 3.6

Degradation of curcumin after hydrogel exposure to UV-A light

368

In order to evaluate the UV-A light oxidative effect on the curcumin encapsulated within

369

the hydrogels, the degradation of curcumin initially presented on CGH and TGH was monitored

370

after UV-A light exposure and the results were shown in Figure 6. Exposure of the hydrogels to

371

30 min of UV-A light led to the degradation of 22 and 47 % of the curcumin initially presented

372

in TGH and CGH, respectively, showing that curcumin degradation in TGH was lower than in

373

CGH. The lower degree of curcumin degradation observed for the TGH suggests that TGH

374

offers a protective effect against light mediated degradation of encapsulated curcumin as

375

compared to CGH.

ACCEPTED MANUSCRIPT 18 376

4

377

4.1

DISCUSSION CGH and TGH coatings act as a carrier for the delivery of curcumin

378

Light exposure of curcumin has been reported to present strong antimicrobial properties

379

against a variety of microorganism, including bacteria, viruses, fungi and spores (Dovigo et al.,

380

2011; Haukvik et al., 2009; Randazzo, Aznar, & Sánchez, 2016; Temba et al., 2016; Tortik,

381

Spaeth, & Plaetzer, 2014). It has been suggested that curcumin acts a photosensitizer, where,

382

upon excitation by a light source, curcumin molecules can generate reactive oxygen species

383

(ROS) in solution that can lead to microbial inactivation (Bernd, 2014; Haukvik et al., 2009).

384

The results show that TGH and CGH coatings exposed to UV-A light could lead to rapid

385

inactivation of L. innocua (Fig. 2). Furthermore, the results illustrate that exposure of bacterial

386

cells to a combination of UV-A and curcumin released from the TGH or CGH (Fig. 6a) resulted

387

in reduction of bacterial count similar to the UV-A light exposure of bacterial cells incubated

388

directly on the surface of the hydrogels (Fig. 2a and 2c). This observation suggests that the

389

observed antimicrobial activity of these hydrogels is due to the release of curcumin to solution

390

from the hydrogels and subsequent UV-A light excitation of these molecules.

391

TGH and CGH coatings, despite having similar initial concentration of curcumin,

392

presented very different light mediated antimicrobial activity against L. innocua. The higher

393

antimicrobial efficacy of CGH over TGH could be attributed to differences in the release of

394

curcumin from CGH and TGH over the same period of time. The observed differences in

395

curcumin release between both matrices could be related to the structure of the hydrogels. While

396

CGH was mainly consisted of cassava starch and gelatin with added purified curcumin

397

encapsulated into the polymeric matrix, the turmeric residue used in the preparation of TGH

398

consisted of a starch and cellulose-based fibrous array with residual curcumin naturally

ACCEPTED MANUSCRIPT 19 399

entrapped in the matrix (Kuttigounder, Lingamallu, & Bhattacharya, 2011; Leonel, Sarmento, &

400

Cereda, 2003). The starch turmeric residue have been reported to present a rough and porous

401

structure with a higher degree of crystallinity after gelatinization than cassava starch (Hmar,

402

Kalita, & Srivastava, 2017; Kuttigounder et al., 2011). These fibrous and crystalline starch

403

domains present in the composition of TGH could strongly bind to curcumin and hinder the

404

release of curcumin to solution. In addition, the compact structure of TGH could also be

405

responsible for the lower swelling of TGH in water and for the lower light mediated degradation

406

of curcumin as compared to CGH, highlighting the protective effect of the turmeric matrix on the

407

entrapped curcumin molecules.

408

4.2

409 410

UV-A light exposure of coated sausages with CGH and TGH reduced cross-contamination load of bacteria Edible antimicrobial coatings developed using natural sources and activated by physical

411

stresses such as light and pressure could present a new and promising strategy to reduce

412

microbial contamination in ready-to-eat foods (Acevedo-Fani, Salvia-Trujillo, Rojas-Graü, &

413

Martín-Belloso, 2015; Alkan & Yemenicioğlu, 2016; Guo, Jin, Wang, Scullen, & Sommers,

414

2014). Chitosan coatings infused with mandarin essential oil and applied on green beans have

415

been reported to present strong antimicrobial activity against L. innocua when combined with

416

high pressure treatments, highlighting the potential of combining natural antimicrobial agents

417

and physical stresses (Donsì et al., 2015). In addition to high pressure, some light-activated

418

antimicrobial coatings have also been reported. Strawberries dip-coated with chitosan and

419

chlorophyllin have been shown to inactivate up to 2.2 log CFU/g of gram-negative bacteria when

420

exposed to LED light (Buchovec et al., 2016). In addition, chlorophyllin-based gelatin coatings

ACCEPTED MANUSCRIPT 20 421

on sausages were reported to inactivate between 1 and 2 log CFU/mL of S. aureus and L.

422

monocytogenes after exposure to light (López-Carballo et al., 2008).

423

In this manuscript, we developed coatings entirely from natural materials (starch,

424

turmeric residue and gelatin) that were successfully coated on the surface of ready-to-eat

425

sausages. These coatings demonstrated significant light-activated antimicrobial activity against

426

L. innocua, a surrogate of L. monocytogenes. CGH-coated sausages presented rapid inactivation

427

of high loads of L. innocua where more than 5 log CFU/mL of bacterial inactivation were

428

achieved after 15 min of UV-A light exposure at 4 ºC. Such strong antimicrobial action at low

429

temperature is very important due to the ability of Listeria to grow under refrigerated conditions

430

(Buchanan et al., 2017). Furthermore, CGH-coated sausages were able to inactivate bacteria even

431

after multiple cycles of bacterial contamination and UV-A light mediated decontamination,

432

indicating that CGH-coated sausages presented sustainable release of curcumin to the bacterial

433

suspension that could be activated via light exposure. Therefore, light-activated curcumin-loaded

434

coatings may be an excellent alternative to rapid inactivate cross-contaminated bacteria at

435

different stages of post processing handling of ready-to-eat sausage products.

436

4.3

437 438

Coated substrate influences the release of curcumin and the light-activated antimicrobial activity of CGH and TGH coatings Interestingly, the light-mediated antimicrobial activity of the hydrogel coatings reported

439

in this study varied depending on the coated surface. TGH-coated sausages presented higher

440

light-activated antimicrobial activity than plastic-coated TGH, where the time required to

441

inactivate 3 log CFU/mL of bacteria decreased from 30 to 15 min (Fig. 2d and Fig 3a). On the

442

other hand, CGH light-activated antimicrobial activity was reduced when CGH was coated onto

443

sausage surfaces, where 15 min of light treatment were required to inactivate more than 5 log

ACCEPTED MANUSCRIPT 21 444

CFU/mL of bacteria in contrast to 5 min of light treatment for sausage-free CGH (Fig. 3a and Fig

445

2b). Those differences in light-activated antimicrobial activity are probably related on how the

446

presence of sausage interferes with the release of curcumin to solution. From one viewpoint, the

447

fat-rich sausage composition may present a more attractive environment for the diffusion of

448

hydrophobic curcumin from the hydrogels, creating a balance between the release of curcumin to

449

the aqueous solution and the release of curcumin to the sausage substrate. Although that

450

hypothesis would help explain the decrease in light-activated antimicrobial activity of CGH-

451

coated sausages, it does not explain the increase of bacterial inactivation by TGH-coated

452

sausages. From another perspective, hydrophobic molecules (i.e. fatty acids) could be diffusing

453

from the sausage surface into the TGH coating, permeating through the hydrophobic and

454

crystalline domains of the turmeric-starch matrix and leading to the solubilization of curcumin

455

molecules that were strongly bond to the compact turmeric residue structure. These freed

456

curcumin molecules now present in an oil phase could be more easily released into the aqueous

457

phase. This phenomena would not provide a sustainable release of curcumin to the aqueous

458

phase since diffusion of curcumin to the sausage substrate would still be more favorable, but

459

instead would lead to an initial burst release of curcumin that would explain the increase in light-

460

activated antimicrobial activity of TGH-coated sausages. It is important to highlight that the

461

concentration of curcumin in solution required to inactivate high loads of L. innocua is very

462

small (less than 0.1 µg/mL; Fig. 5b) and therefore very small changes in released curcumin could

463

present a strong effect on the light-activated antimicrobial activity of the hydrogels. In fact, the

464

decrease in light-activated antimicrobial activity after the first cycle of bacterial inactivation

465

(Fig. 4b) suggests that the increased burst release of curcumin from TGH-coated sausages was

ACCEPTED MANUSCRIPT 22 466

very small and not constant over time. Overall, these results illustrate the importance of

467

understating the surface properties of food substrates in order to design effective coatings.

468

4.4

469

Potential of turmeric residue-based antimicrobial coatings Despite the inferior performance of TGH as compared to CGH regarding the rapid

470

inactivation of high initial loads of bacteria, it is important to highlight the potential of using

471

turmeric residue for the preparation of antimicrobial edible hydrogel coatings. Due to its

472

complex and fibrous composition, turmeric residue may be used to develop coatings presenting

473

desirable mechanical properties, lower water affinity and increased protective effects against

474

light mediated degradation of curcumin. Furthermore, the compact structure of turmeric coatings

475

could lead to a slow sustained release of curcumin and curcuminoids to solution. The described

476

characteristics of turmeric residue suggest that it could be an excellent wall material alternative

477

not only for the encapsulation of curcumin and curcuminoids but also for the encapsulation of

478

other bioactive compounds, especially hydrophobic compounds (Malacrida, Ferreira, Zuanon, &

479

Nicoletti Telis, 2015).

480

Another major advantage of turmeric residue-based coatings is the residual curcumin

481

naturally present in turmeric, which could avoid encapsulation of additional curcumin into the

482

coating. Turmeric residue-based coatings could be also further optimized by using turmeric

483

residues with slightly higher concentrations of curcumin, since the residue used in this study was

484

obtained after two consecutive extractions of curcuminoids and presented very low concentration

485

of curcumin (Osorio-Tobón et al., 2014). In addition, different experimental strategies could be

486

used during the preparation of turmeric-based coating in order to tweak the properties of the

487

coatings for a desired application, such as the addition of surfactants and hydrophilic

ACCEPTED MANUSCRIPT 23 488

biopolymers to improve the release of curcumin (Paula, Oliveira, Carneiro, & de Paula, 2016;

489

Ratanajiajaroen, Watthanaphanit, Tamura, Tokura, & Rujiravanit, 2012).

490

Lastly, since turmeric and curcumin are natural food-grade compounds, curcumin-loaded

491

turmeric residue-based antimicrobial coatings could meet the current and growing demand from

492

consumers and food industries to replace synthetic-based preservatives for food-grade natural

493

antimicrobials in food systems (Oms-Oliu et al., 2010; Tiwari et al., 2009).

494 495 496

5

CONCLUSIONS The present study demonstrated that the combination of UV-A light and curcumin-loaded

497

edible coatings can be used as an antimicrobial approach to inactivate L. innocua preventing

498

cross-contamination on coated sausages. Although hydrogel coatings containing added purified

499

curcumin (CGH) presented higher antimicrobial efficacy, turmeric residue-based coatings were

500

also effective in reducing L. innocua load on the surface coated-sausages. Furthermore, the light-

501

mediated antimicrobial activity of the hydrogels coatings were not affected at refrigerated

502

temperature (4 ºC), suggesting that the proposed antimicrobial coatings approach could be

503

applied to refrigerated foods. Moreover, it was shown that light-activation of curcumin

504

molecules released from the coatings to solution was the main mechanism behind bacteria

505

inactivation and that the release of low concentrations of curcumin are sufficient to show

506

antimicrobial activity against L. innocua. In conclusion, turmeric residue obtained after two

507

extraction processes (SFE and PLE) can still be used to prepare hydrogels that can present high

508

antimicrobial activity when combined with UV-A light, suggesting that these hydrogels can be

509

applied as edible coatings to inhibit bacterial cross-contamination in sausages.

ACCEPTED MANUSCRIPT 24 510 511 512

FUNDING INFORMATION This project was supported by Agriculture and Food Research Initiative grant no. 2014-

513

67017-21642 from the USDA National Institute of Food and Agriculture (USDA-NIFA)

514

Program in Improving Food Quality (A1361) and grant no. 2015-68003-23411 from the USDA-

515

NIFA Program Enhancing Food Safety through Improved Processing Technologies (A4131).

516

CAPES/BR supported this work through the “Science without Borders” scholarship program

517

(recipient nº 11897-13-9) for Erick F. de Oliveira. Scholarship for Juliano V. Tosati was

518

supported by CAPES Foundation, Ministry of Education of Brazil, Brasilia, DF, Brazil (process

519

number 99999.006818/2015-03).

520 521 522 523

ACKNOWLEDGEMENTS The authors are grateful to the Brazilian Coordination for the Improvement of Higher Education Personnel (Capes) for the financial support.

524 525

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Figure 1: Physical properties (a) and optical images (b) of the Turmeric-Gelatin hydrogel (TGH) and the Cassava-Gelatin hydrogel (CGH). Control hydrogel (K) consists of Cassava-Gelatin hydrogel without the addition of curcumin. Figure 2: Inactivation of Listeria innocua incubated on Turmeric-Gelatin hydrogel (TGH) or on Cassava-Gelatin hydrogel (CGH) and exposed (L+) or not exposed (L-) to UV-A light at 23 ºC and 4 ºC. (a) and (b) represent CGH at 23 ºC and at 4ºC, respectively; (c) and (d) represent TGH at 23 ºC and at 4ºC, respectively. Control hydrogel (K) consists of Cassava-Gelatin hydrogel without the addition of curcumin. Initial bacterial load was 6 log CFU/mL. The limit of detection was 5 log CFU/mL of bacterial inactivation. Figure 3: Inactivation of Listeria innocua incubated on Turmeric-Gelatin Hydrogel (TGH)-coated sausages or on Cassava-Gelatin Hydrogel (CGH)-coated sausages and exposed to UV-A light at 4 ºC. Initial bacterial load was (a) 6 log CFU/mL and (b) 4 log CFU/mL. Control hydrogel (K) consists of CassavaGelatin hydrogel without the addition of curcumin. The limit of detection was (a) 5 log CFU/mL of bacterial inactivation and (b) 3 log CFU/mL of bacterial inactivation. Figure 4: Multiple inactivation cycles of Listeria innocua incubated on (a) Cassava-Gelatin Hydrogel (CGH)-coated sausages or (b) Turmeric-Gelatin Hydrogel (TGH)-coated sausages and exposed to UV-A light at 4 ºC. Initial bacterial load was 4 log CFU/mL. The limit of detection was 3 log CFU/mL of bacterial inactivation. Figure 5: (a) Inactivation of Listeria innocua incubated with curcumin released from CassavaGelatin Hydrogel (CGH) and Turmeric-Gelatin Hydrogel (TGH) and exposed to 5 min of UV-A light. Release of curcumin from hydrogels to water were conducted for 5 (white) and 30 (gray) min before L. innocua incubation and UV-A light exposure. (b) Inactivation of Listeria innocua incubated with different curcumin concentrations and exposed to 5 min of UV-A light. For (a) and (b) the initial bacterial load was 6 log CFU/mL and the limit of detection was 5 log CFU/mL of bacterial inactivation. Figure 6: Degradation of curcumin present in the Cassava-Gelatin Hydrogel (CGH) and the Turmeric-Gelatin Hydrogel (TGH) after exposure to UV-A light for 5, 15 and 30 min.

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HIGHLIGHTS 1) Turmeric residue was used to prepare light-activated antimicrobial hydrogel coatings 2) Hydrogel coatings exposed to UV-A light can inactivate 5 log CFU/mL of L. innocua 3) The antimicrobial activity of the hydrogel coatings were independent of temperature 4) Light-exposed hydrogel coatings can prevent bacteria cross-contamination on sausages 5) Curcumin release from hydrogels is the main inactivation mechanism of L. innocua