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Bivalve molluscan shellfish such as oysters are important vectors for the transmission of foodborne pathogens including both viruses and bacteria. Photoinactivation provides a cold-sterilization option against the contamination as excited photosensitizers could transfer electronic energy to oxygen molecules producing reactive oxygen species such as singlet oxygen, leading to oxidative damage and death of the patho- gens. However, the efficacy of photoinactivation is very often compromised by the presence of food matrix due to the nonselective reactions of short-lived singlet oxygen with organic matter other than the target pathogens. In order to address this issue, we encapsulated a food-grade photosensitizer rose bengal (RB) in alginate microbeads. An extra coating of chitosan effectively prevented the release of RB from the microbeads in seawater, and more importantly, enhanced the selectivity of the photoinactivation via the electrostatic interactions between cationic chitosan and anionic charge of the virus particles (bacteriophage MS2 and Tulane virus) and the Gram-negative bacteria Vibrio parahaemolyticus (V. parahaemolyticus). The treatment of oysters with microencapsulated RB resulted in significantly higher reductions of MS2 phage, Tulane virus and V. parahaemolyticus than free RB and non-RB carrying microbeads (P<0.05) tested with both in vitro and in vivo experimental set-ups. This study demonstrated a new strategy in delivering comprehensively formulated biochemical sanitizers in bivalve shellfish through their natural filter-feeding activity and thereby enhancing the mitigation efficiency of foodborne pathogen contamination. Key words: Bacteriophage MS2; Tulane virus; Vibrio parahaemolyticus; photosensitizer; encapsulation; oyster. Introduction environments and is the leading cause of seafood-associated gastroenteritis throughout the world. The bacteria can be Bivalve molluscan shellfish such as oysters are filter feeders concentrated in oysters and cause infection through the fecal– and could potentially bio-accumulate pathogenic micro- oral route, especially if the time–temperature abuse at post- organisms such as human noroviruses (hNoVs) and Vibrio harvest stage provides ample opportunity for these bacteria to parahaemolyticus (V. parahaemolyticus) from large quan- replicate and reach infective doses (Drake and DePaola, 2007; tities of water (Westrell et al., 2010; Schaeffer et al., 2013; Sudha et al., 2012). Although thorough cooking is absolutely Lowmoung et al., 2017; Ryu et al., 2019). Recent evidence effective in inactivating both viral and bacterial pathogens, suggests that oysters are not just passive filters, but can se - it alters the organoleptic qualities of shellfish. The consump - lectively and specifically accumulate virus particles based tion of raw or minimally cooked shellfish is still a common on the carbohydrate ligands in their intestine tissues shared practice worldwide. Therefore, alternative management and with humans as well as the diverse functionalities of their gut mitigation strategies are in need to lower the safety risks as- microbiota (Tian et al., 2007; Le Guyader et al., 2012; Su sociated with shellfish consumption. et al., 2018; Eshaghi Gorji et al., 2021). Shellfish are often Photoinactivation with the use of photosensitizers pro- cultivated in coastal waters where there is the potential of vides a cold-sterilization against a wide range of patho - human faecal contamination from multiple sources including gens including both bacteria and viruses (Luksiene and variable freshwater input from rivers and point source dis- Brovko, 2013; Randazzo et al., 2016; Majiya et al., 2018). charges. Heavy rainfall and flooding may increase with cli - Photosensitizers are organic molecules act as sensitizers mate change and result in greater discharge of untreated that become energetically excited by UV-Vis light and then human-derived wastewater into the coastal zone (Hassard et transfer electronic energy to oxygen molecules producing re- al., 2017). Consequently, shellfish are recognized as important active oxygen species (ROS) such as singlet oxygen, leading vectors for foodborne virus transmission and cause outbreaks to oxidative damage and death of microorganisms ( Kessel continually worldwide (Nenonen et al., 2009; Thebault et and Luo, 1998; Kudinova and Berezov, 2009). Similar al., 2013; Meghnath et al., 2019). V. parahaemolyticus, on with many other (bio)chemical antimicrobial reactions the other hand, is ubiquitously present in marine and coastal Received 2 January 2022; Revised 6 February 2022; Editorial decision 1 March 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 M. Eshaghi Gorji and D. Li (Li et al., 2012; López-Gálvez et al., 2012), the efficacy of In order to coat the alginate microbeads, the precipitated photoinactivation is very often compromised by the pres- particles were added into 0.1% low molecular chitosan solu - ence of organic matter due to the nonselective reactions of tion (50–190 kDa, 75%–85% deacetylated, Sigma-Aldrich, singlet oxygen with food matrix other than the target patho- Arklow, Iceland) containing 1% acetic acid, with the pH ad - gens (de Oliveira et al., 2018; Liu et al., 2021). In accord- justed at 6.2, and stirred at 100 r/min for 15 min. After settling ance, this study encapsulated rose bengal (RB), a food-grade down of the microbeads, the aqueous phase was collected to photosensitizer, in alginate microbeads coated with chitosan. measure the RB released during the coating procedure, and Due to the lack of a robust cell culture system for hNoVs, the microbeads were washed once with the acetate buffer (pH we employed two commonly used surrogates including bac - 5.5) and stored in the same buffer afterwards. teriophage MS2 (MS2) and Tulane virus (TV). Encapsulated As a control, chitosan-coated alginate microbeads without RB with chitosan coating was tested over TV, MS2 and V. RB were prepared with the same procedures as above (to be parahaemolyticus in oyster-derived matrices with both in referred as non-RB carrying microbeads). vitro and in vivo assays. Characterization of RB encapsulated microbeads The morphology of the RB encapsulated microbeads was Materials and Methods observed with an optical microscope Olympus BH2-UMA Encapsulation of RB in alginate microbeads with equipped with a Cue-2 image analyzer (Olympus, Tokyo, chitosan coating Japan). The granulometric size distribution was determined Alginate beads were prepared by the emulsification/internal by a laser diffractometer (Fraunhofer model) using a Coulter gelation technique as described by Ribeiro et al. (2005) LS130 particle analyzer (Beckman Coulter Inc., Fullerton, with some modifications as shown in Figure 1. Briefly, so - CA, USA). Particle size is expressed as volume mean diam- dium alginate (2%, mass concentration, medium viscosity; eter (µm)±standard deviation (SD) values of the mean. Sigma-Aldrich, Oslo, Norway) was dispersed into deionized Measurements were made in triplicate for each batch. water and stirred overnight at 500 r/min with a magnetic The encapsulation efficiency of RB in the microbeads was stirrer (Corning , Mexico City, Mexico) to be fully dis- calculated by an indirect method using the following equation: solved. Thereafter, 10 mg RB (Sigma-Aldrich, Bangalore, India) was dissolved in 10 mL of the 2% alginate solution C1 − C2 Encapsulation efficiency (% ) = × 100 % , and stirred at 500 r/min for 10 min, followed by adding C1 1.25 mL of 5% (mass concentration) calcium carbonate. The mixture was then poured into 50 mL canola oil con- where C1 was the amount of RB used for encapsulation; taining 1% Span 80 (Sigma-Aldrich, Madrid, Spain). After C2 was the RB remaining in the aqueous solution after re- 15 min hard stirring at 700 r/min, 60 µL of glacial acetic moving the newly formed microbeads. RB was measured acid was added to the emulsion to cause gelation. After using UV-visible spectrophotometers (Shimadzu UV-1800, another 15 min constant stirring at 700 r/min, 100 mL of Duisburg, Germany) at an absorbance of 540 nm. A calibra- acetate buffer (0.1 mol/L, pH 5.5) was added to the gelled tion curve was plotted with RB standards of 1–20 µg/mL. emulsion followed by gentle stirring at 100 r/min for 5 min. In order to follow up the release dynamics of RB from the The mixture was left still overnight for partition of the oily microbeads in an oyster-derived environment, freshly pre - and aqueous phases and settling down of the microbeads. pared microbeads carrying 5 mg of RB were suspended in After removing the top oily phase, the aqueous phase was 200 mL artificial seawater with a salinity of 2% (pH 8.2) collected to measure the residual RB which was not encap- under continuous stirring at 200 r/min. Five milliliters of the sulated, and the microbeads were washed once with the suspension was collected every 15 min and centrifuged at acetate buffer (pH 5.5). The release of RB from the alginate 12 000×g, followed by measuring the concentration of RB in microbeads was negligible when stored in the acetate buffer the supernatants as described above. The released rate of RB of pH 5.5. was calculated with the following equation: Figure 1. Schematic illustration for the encapsulation of rose Bengal (RB) in alginate microbeads with chitosan coating using an emulsification/internal gelation technique. Photoinactivation by microencapsulated rose bengal 3 In vitro photoinactivation of the microorganisms in t oyster-derived matrices RB release rate (%) = × 100 %, 0 Live pacific oysters ( Crassostrea gigas) were purchased from a local supermarket in Singapore. Two types of oyster-derived where M was the amount of RB in the microbeads at the matrices were prepared, including oyster chopped digestive starting time point and M was the amount of RB detected tissue (OCDT) and oyster digestive tissue extract (ODTE). from the supernatant at time t. All experiments were per- The oysters were shucked, the digestive tissue was excised formed in triplicate. using sterile scissors and tweezers, chopped into fine pieces with sterile blades and used as OCDT. As for ODTE, 2 g of the excised oyster digestive tissue was mixed with 4 mL of Virus stock and culture conditions sterile phosphate-buffered saline (PBS) followed by shaking MS2 coliphage (Escherichia coli (E. coli) bacteriophage with an orbital shaker at 150 r/min for 10 min and centrifu- 15597-B1TM) and E. coli 15597 were obtained from gation at 3000×g for 5 min. The resulting supernatant was the American Type Culture Collection (ATCC; Rockville, collected and used as ODTE. MD, USA). Freeze-dried MS2 was recovered and propa - MS2, TV, and V. parahaemolyticus were independently gated with E. coli 15597 according to the product sheet. examined over the photoinactivation treatment. Briefly, The titer was determined as plaque-forming units (PFU)/ OCDT or ODTE was individually spiked with MS2 phage mL by plating assays using the recommended double- lysate at 1:1000 to a concentration of 6.50 log PFU/mL, layer tryptone yeast glucose agar (TYGA) containing E. TV lysate at 1:5 to a concentration of 5.80 log TCID /mL, coli 15597 in the semisolid TYGA layer (Debartolomeis and a cocktail of V. parahaemolyticus suspension at 1:100 and Cabelli, 1991). Propagated MS2 stocks were stored at to a concentration of 6.00 log CFU/mL. Following the in- –80 °C in 20% glycerol until use. oculation, OCDT and ODTE were mixed with free RB, en- TV, kindly provided by Professor Xi Jiang, Cincinnati capsulated RB (final concentration of RB at 50 μmol/L and Children’s Hospital Medical Center (Cincinnati, OH, 250 µmol/L) or non-RB carrying microbeads (equal amount USA), was propagated and assayed in monkey kidney cell of beads with 50 μmol/L and 250 µmol/L RB) suspended in line, LLC-MK2 (ATCC CCL-7™). LLC-MK2 cell were PBS at a volume ratio of 2:1. The mixtures were proceeded maintained in complete M199 medium (Life Technologies, to light treatment in 6-well plates (1 mL mixture per well) in Grand Island, NY, USA) supplemented with 10% fetal a 30 cm×25 cm×18 cm treatment box. The light treatment bovine serum (HyClone, Logan, UT, USA), 100 µg/mL at 20 mJ/s (measured by an optical power meter, Thorlabs, penicillin, and grown at 37 °C under 5% CO atmos- Dachau, Germany) for 1 h was delivered by an illuminator (il- phere. Virus stocks were obtained by infecting confluent lumination of 340–800 nm, Thorlabs, Shanghai, China) from monolayer of LLC-MK2 cells for 3 d followed by three 10 cm distance. The temperature in the treatment box was freeze–thaw cycles, pelleting of cellular debris by cen- monitored constantly and did not exceed 30 °C. After light trifugation at 1000×g (gravitational acceleration) for treatment, the mixtures were serially diluted and the MS2, 10 min. Then, viral lysate in complete M199 was filtered TV, and V. parahaemolyticus were determined as described through a 0.22-µm pore size sterile filter and stored in above. All samples were tested in triplicate. aliquots at –80 °C. The titer of TV stocks was measured using 50% tissue In vivo photoinactivation of the microorganisms in culture infectious dose (TCID ). Briefly, each sample was −1 −8 oysters serially diluted (10 to 10 ) in medium and inoculated onto LLC-MK2 cells in 96-well culture plates (200 μL Live pacific oysters ( Crassostrea gigas) were purchased from per well, four repetitive wells per dilution), and incubated a local supermarket in Singapore and placed in tanks con- for 7 d at 37 °C. Observation of visible cytopathic ef- taining artificial seawater (three oysters in each tank with 5 L fects (CPE) such as cell balling was converted into TCID seawater). The salinity of the seawater was 2% and the pH units using the Reed and Muench calculation method was 8.2. The tanks were kept in an environment chamber (Lindenbach, 2009). A negative control was included on (BMT Technologies, Mannheim, Germany) with temperature each plate. set at 15 °C. Aquarium air pumps were used to supply suffi - cient amounts of oxygen for the shellfish. After 6 h of acclimation, 10 mL of MS2 phage lysate Bacterial strains and growing conditions (9.50 log PFU/mL), 15 mL of TV lysate (6.50 log TCID /mL), Two V. parahaemolyticus strains (ATCC 17802 and 17803) or 100 mL of V. parahaemolyticus cocktail (8.00 log were individually cultivated in tryptic soy broth (TSB; CFU/mL) was added to each tank with four oysters. Twenty- Oxoid, Hampshire, UK) supplemented with 2% NaCl and four hours afterwards, each tank received 15 mg RB either incubated for 24 h at 37 °C, to prepare a cell suspension in free format or the encapsulated microbeads, or the same of approximately 10 colony-forming units (CFU)/mL. The amount of non-RB carrying microbeads. The control tanks freshly cultured bacteria suspensions were centrifuged at were not treated with RB or microbeads. Following 3 h ex- 8000×g for 5 min, resuspended with deionized water, and posure, the oysters were harvested, rinsed with deionized mixed at a volume ratio of 1:1 to form a bacterial cocktail water, and shucked with a sterile knife. The shucked oysters as the inoculum for both in vitro and in vivo tests. were placed in sterile Petri dishes individually and received V. parahaemolyticus was enumerated on thiosulfate–cit- the light treatment as described above. rate–bile salts sucrose (TCBS) agar (Oxoid, Hampshire, UK) After the light treatment, each oyster was analyzed individu - plates with 24 h incubation at 37 °C. ally for the microorganisms’ viability. In order to titer MS2 and 4 M. Eshaghi Gorji and D. Li TV, the digestive tissues of oysters were excised using sterile scis- A paired t-test was used for data with two groups; one- sors and tweezers, cut into fine pieces with sterile scissors, and way analysis of variance was used for data with more than mixed with sterile PBS at a volume ratio of 1:2. The mixture was two groups. Significant differences were considered when P vortexed vigorously and centrifuged at 3000× g for 5 min. The was <0.05. supernatant was collected and used for the viral titrations as de - scribed above. Regarding V. parahaemolyticus, each oyster was Results placed into a filtered stomacher bag with 25 mL sterile PBS and then squeezed with hand thoroughly. Thereafter, the supernatant Characterization of RB encapsulated microbeads was serially diluted before plating out on TCBS agar plates. RB encapsulated alginate microbeads with and without chitosan coating were both spherical in shape without sig- In vivo imaging of free and encapsulated nificant aggregation ( Figure 2A). The mean diameters of fluorescence dye in oysters uncoated and coated microbeads were (98.7±33.0) µm and Cyanine5.5 NHS ester (Lumiprobe, Hunt Valley, MD, USA) (151.2±91.6) µm, respectively, without significant difference was encapsulated in alginate microbeads and coated with (P>0.05; Figure 2B). chitosan with the same procedures as described above. The An encapsulation efficiency of 88.10%±3.10% was characterizations of Cyanine5.5 NHS ester microbeads (size, achieved for RB in the alginate microbeads (Figure 2C). The encapsulation efficiency, and release rates) were comparable release of RB from the alginate microbeads was negligible to the RB microbeads. when stored in the acetate buffer of pH 5.5. A marginal loss Live Pacific oysters kept in artificial seawater as described of RB was observed during the coating procedure afterwards above (three oysters in each tank with 5 L seawater) received due to the pH adjusted at 6.0 but eventually resulted in no 100 mg Cyanine5.5 NHS ester either in free format or in the significant difference in the encapsulation efficiency ( P>0.05; encapsulated microbeads. After 3 h, the oysters were shucked Figure 2C). and observed with an in vivo imaging system (IVIS, Perkin- RB was released rapidly from the alginate microbeads Elmer, Waltham, MA, USA). The excitation/emission combin- when suspended in artificial seawater with a pH of 8.2 (up ation of 675 nm/720 nm was employed using epi-fluorescence to 80.00%±3.10% within 120 min; Figure 2D). In contrast, for visualization. the chitosan coating supplied evident protection of RB re- lease from the microbeads at higher pH by resulting in a less Statistical analysis than 10% release rate within 120 min (Figure 2D) and thus Statistical analyses were performed using the software SPSS showed potential for use in the following photoinactivation for windows, version 22.0 (SPSS Inc., Chicago, IL, USA). studies. Alginate beads Alginate beads with chitosan coang Alginate beads Alginate beads with chitosan coang 015 30 45 60 75 90 105 120 Alginate beads Alginate beads with Time (min) chitosan coang Figure 2. Characterizations of the rose bengal (RB) encapsulated microbeads. (A) Observation of the coated and uncoated microbeads under optical microscope; (B) The diameters of the coated and uncoated microbeads as measured by a laser diffractometer using a Coulter LS130 particle analyzer; (C) The RB encapsulation efficiencies of the coated and uncoated microbeads; (D) The release of RB from the coated (triangles) and uncoated (squares) microbeads to the artificial seawater. Each data point represents the average of triplicates, and each error bar indicates the standard deviations. Encapsulation efficiency (%) Release rate of RB (%) Diameter of beads (μm) Photoinactivation by microencapsulated rose bengal 5 In vitro photoinactivation of MS2, TV, and V. In vivo photoinactivation of MS2, TV, and V. parahaemolyticus in oyster-derived matrices parahaemolyticus in oysters RB encapsulated in the chitosan coated alginate microbeads The in vivo photoinactivation of MS2, TV, and V. at 250 µmol/L resulted in significantly higher reductions parahaemolyticus in oysters was consistent with the in of MS2 ((1.26±0.24) log PFU/mL), TV ((0.86±0.27) log vitro results. Live oysters treated with chitosan-coated TCID /mL), and V. parahaemolyticus ((1.32±0.21) log RB microbeads resulted in significant reductions of MS2 CFU/mL) in OCDT compared to control groups of non-RB ((0.60±0.22) log PFU/mL), TV ((0.47±0.29) log TCID /mL) carrying microbeads and free RB (P<0.01; Figures 3A, 3C, and V. parahaemolyticus ((1.01±0.28) log CFU/mL) in com- and 3E). Except for TV, the RB encapsulated microbeads parison with their respective control groups of non-RB generated higher reductions than free RB but without sig- carrying microbeads and free RB (P<0.05; Figure 4). nificance ( P=0.10; Figure 3C). The photoinactivation effect In order to simulate the fate of encapsulated RB in oys- was obviously matrix-dependent, because in ODTE with ters along with their filter-feeding activities, a far-red (and fewer organic components than OCDT comparable reduc- near-infrared) emitting dye, Cyanine5.5 NHS ester, was en- tions were achieved with 50 µmol/L of encapsulated RB in capsulated in alginate microbeads and coated with chitosan. the chitosan-coated microbeads for MS2 ((1.35±0.18) log Three hours after adding free and encapsulated Cyanine5.5 PFU/mL reduction), TV ((0.86±0.29) log TCID /mL reduc- NHS ester to the seawater, comparable fluorescence signal tion), and V. parahaemolyticus (1.07±0.12) log CFU/mL; strengths and distributions were observed from the oysters Figures 3B, 3D, and 3F). with free and encapsulated dye, which covered almost the A A C E 2.5 2.5 2.3 2.0 2.0 1.8 *b *b 1.5 1.5 1.3 *a *a 1.0 1.0 *b *a 0.8 a a a a a a a 0.5 0.5 a 0.3 0.0 0.0 -0.2 1 1 250 1 250 50 50 250 50 RB concentraon (μmol/L) RB concentraon (μmol/L) RB concentraon (μmol/L) D F B 2.5 *b 2.5 2.5 *b 2.0 2.0 *b 2.0 *b *a 1.5 1.5 *b 1.5 *b *a *a *c 1.0 1.0 ab 1.0 *a a 0.5 0.5 0.5 *a a 0.0 0.0 0.0 50 250 50 1 250 50 250 RB concentraon (μmol/L) RB concentraon (μmol/L) RB concentraon (μmol/L) Figure 3. The in vitro photoinactivation of MS2 (A and B), TV (B and C), and V. parahaemolyticus (E and F) in oyster-derived matrices OCDT and ODTE, respectively. Left-hand bars represent the reductions by free RB, central bars represent the reductions by RB encapsulated in chitosan-coated alginate microbeads, and right-hand bars represent the reductions by non-RB carrying microbeads. Each data point represents the average of triplicates, a–c and each error bar indicates the standard deviations. * indicates a significant reduction ( P<0.05) compared to the control group; different letters indicate a significant ( P<0.05) difference detected between the treatments. MS2, bacteriophage MS2; TV, Tulane virus; V. parahaemolyticus, Vibrio parahaemolyticus; OCDT, oyster chopped digestive tissue; ODTE, oyster digestive tissue extract; RB, rose bengal; TCID , 50% tissue culture infectious dose. A B C 1.8 1.8 1.8 *b 1.4 1.4 1.4 1.0 *b 1.0 1.0 *b *a 0.6 0.6 0.6 *b 0.2 0.2 0.2 1 1 -0.2 -0.2 -0.2 Treatment Treatment Treatment Figure 4. The in vivo photoinactivation of MS2 (A), TV (B), and V. parahaemolyticus (C) in oysters. Left-hand bars represent the reductions by free RB, central bars represent the reductions by RB encapsulated in chitosan-coated alginate microbeads, and right-hand bars represent the reductions by non-RB carrying microbeads. Each data point represents the average of triplicates, and each error bar indicates the standard deviations. * indicates a significant reduction ( P<0.05) compared to the control group; a–b different letters indicate a significant ( P<0.05) difference detected between the treatments. MS2, bacteriophage MS2; TV, Tulane virus; V. parahaemolyticus, Vibrio parahaemolyticus; OCDT, oyster chopped digestive tissue; ODTE, oyster digestive tissue extract; RB, rose bengal; TCID , 50% tissue culture infectious dose. Reduction of MS2 Reduction of MS2 (Log PFU/oyster) Reduction of MS2 (Log PFU/mL) (Log PFU/mL) Reducon of TV Reducon of TV Reducon of TV (Log TCID /oyster) (Log TCID /mL) 50 (Log TCID /mL) Reducon of Reducon of Reducon of V. parahaemolycus V. parahaemolycus V. parahaemolycus (Log CFU/oyster) (Log CFU/mL) (Log CFU/mL) 6 M. Eshaghi Gorji and D. Li Figure 5. Representative imaging of fluorophore Cyanine5.5 NHS ester in oysters. complete flesh of oysters with the signal peaking at the loca - TV effectively, it negligibly inactivates the virus. In this study, tions of the digestive tissues (Figure 5). the dose-dependence of reductions induced by the non-RB carrying microbeads with chitosan coating was only observed for MS2 but not TV or V. parahaemolyticus. More import- Discussion antly, the reductions induced by non-RB carrying microbeads RB is a food-grade photosensitizer that can be induced by were significantly lower than reductions induced by encap - light within the visible spectrum and has demonstrated prom - sulated RB under all of the tested scenarios, indicating that ising microbicidal effects over foodborne viruses (Kingsley et the enhanced photoinactivation efficacy as observed in this al., 2018) and bacteria (Dahl et al., 1988). Our purpose of study is mainly due to microbial binding capability but not this study was to enhance the efficacy of photodynamic in - the direct microbicidal effect of the chitosan coating. activation against pathogenic viruses and bacteria in oyster The emulsification/internal gelation technique employed matrix. This is because one major drawback of applying in this study revealed negligible aggregation of alginate photoinactivation in foods is its lack of selectivity toward microbeads, particularly compared to emulsification/ex - the target. Localization of photosensitizers is a key factor ternal gelation in which calcium chloride could disrupt as the generated ROS and particularly singlet oxygen are the equilibrium of a water/oil emulsion (Wan et al., 1993, extremely short-lived and can only exert their antimicro- Chan et al., 2002). The use of Span 80 as a nonionic sur- bial effects on very close targets (Klaper et al., 2016). For factant with high hydrophilic degree (hydrophilic–lipophilic instance, as reported previously by Sun et al. (2019), conju- balance=4.3) further promoted the stability of the emulsion gation of cholesterol and chlorin e6 (Ce6) could enhance the system by decreasing the interfacial tension between the al- antibacterial efficiency of Ce6 effectively. Chitosan is a linear ginate droplets and the oil. The size of particles and encap - polysaccharide composed of repeated β-(1–4) linked units of sulation efficiency was affected by several factors, including either 2-amino-2-deoxy-β-d-glucopyranose (glucosamine) or the speed and duration of stirring, acid/calcium ratio, etc. and 2-acetamido-2-deoxy-β-d-glucopyranose (glucosacetamide; has been optimized in this study. Although a higher speed of Dadou et al., 2017). The primary amine groups of glucosa- mixing was able to lower the size of particles, it caused more mine result in the cationic nature and net positive charge of heterogenicity of particle size distribution. Theoretically, each chitosan under acidic and neutral conditions, and thus are mole of calcium carbonate reacts with two moles of acetic expected to ‘attract’ the negatively charged virus particles and acid. In this study, we used a higher ratio of acid/CaCO (2.5 bacteria in oyster-derived matrices (pH between 6.0 and 7.0 instead of 2.0) than the stoichiometric amount to ensure suf- as measured in this study) via electrostatic forces, enhancing ficient acid is available to dissolve the calcium carbonate, the photoinactivation efficacy from ROS generated by the en - which resulted in an improvement of the RB encapsulation capsulated RB. efficiency. It has been reported that the cationic charge of chitosan it- The release of RB from alginate microbeads is highly de- self can interact electrostatically with the lipopolysaccharides pendent on the pH. Under acidic conditions in the acetate in the outer membrane of Gram-negative bacteria and conse- buffer (pH 5.5), the cross-linked alginates with calcium quently kill the bacteria by the formation of pores (Alfei and maintained stable RB with barely any release. Along with Schito 2020). This has been reported with certain antiviral ef- the pH increase, the alginic acid started to be converted to fects over multiple cultivable enteric viral surrogates including a soluble salt of sodium alginate, leading to swelling and feline calicivirus (FCV-F9), murine norovirus (MNV-1), and disintegration of the particles. Therefore, a burst release of bacteriophages (MS2 and phiX174; Su et al., 2009; Davis RB from alginate microbeads occurred in the artificial sea - et al., 2012, 2015). Controversially, in a very recent study, water. The positively charged chitosan molecules could de - Barnes et al. (2021) reported that although chitosan binds to posit on the interface and provide significant electrostatic Photoinactivation by microencapsulated rose bengal 7 interactions with negatively charged alginate. As a result, Author Contributions chitosan formed an outer membrane around the microbeads Mohamad Eshaghi Gorji: Methodology, validation, and writ - and maintained their structure by protecting the alginate ing original draft preparation; Dan Li: Conceptualization, from dissolution and RB from releasing in the seawater at methodology, validation, writing, review and editing, super - higher pH. vision, and funding acquisition. Both authors have read and As viral and bacterial contaminants in bivalve shell- agreed to the published version of the manuscript. fish are mainly introduced by their filter-feeding activity, the sanitizing agents must ideally be introduced via the same route to achieve maximal reach toward the targets. Funding Therefore, before applying the encapsulated RB microbeads This study was supported by the Ministry of Education to test the photoinactivation efficacy in oysters in vivo, we (MOE) academic research fund (AcRF) TIER 1 Project and prepared microbeads with Cyanine5.5 NHS ester, which the ‘Study of important foodborne viruses from relevant is ideal for fluorescence measurements where background foods in Singapore’ (R-160-000-A79-114), Singapore. fluorescence is a concern, to simulate and image the fate and distribution of the microbeads in oyster flesh. Three hours after being added to the seawater, the fluorescence Conflict of Interest signal from the Cyanine5.5 NHS ester almost completely The authors declare no conflict of interest. covered the flesh of oysters and the highest signals appeared at locations with digestive tissues. This result is in good cor- respondence with the bio-accumulated hNoV and Vibrio References distribution in oysters as reported previously (Wang et al., Alfei, S., Schito, A. M. (2020). Positively charged polymers as promis- 2008, 2010; Maalouf et al., 2010). ing devices against multidrug resistant Gram-negative bacteria: a MS2 has commonly been used to study the sensitivity review. Polymers, 12(5): 1195. of human viral pathogens to disinfectants (Dawson et al., Arthur, S. E., Gibson, K. E. (2015). Physicochemical stability profile 2005; Kohn and Nelson, 2007; Mamane et al., 2007) and of Tulane virus: a human norovirus surrogate. Journal of Applied Microbiology, 119(3): 868–875. it is also a good indicator for human fecal pollution as fre- Chan, L. W., Lee, H. Y., Heng, P. W. S. (2002). Production of alginate quently co-occurs with its bacterial host E. coli in sewage microspheres by internal gelation using an emulsification method. and animal feces (Leduc et al., 2020). 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Food Quality and Safety – Oxford University Press
Published: Mar 11, 2022
Keywords: Bacteriophage MS2; Tulane virus; Vibrio parahaemolyticus; photosensitizer; encapsulation; oyster
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