Human Norovirus (HuNoV) belongs to the genus Norovirus of the family Caliciviridae. It is a small, non-enveloped, icosahedral symmetric virus with a positive-sense, single-stranded RNA genome organized in three open reading frames (ORFs) (Hall et al., 2013). HuNoV has a low estimated infectious dose of ~18 viral particles (Teunis et al., 2008) and is excreted in high concentrations (4–9 log genome copies/g) in feces for at least 2–3 weeks in both symptomatic and asymptomatic cases (Teunis et al., 2015; Sabria et al., 2016). HuNoV bioaccumulates in bivalve molluscs in contaminated waters through their filter-feeding mechanism and they retain the virus for long periods of time (Le Guyader et al., 2012).

Jeotgal is a salted fermented food consumed in Korea and some Asian countries, made from various types of seafood such as shrimps, oysters, shellfish, fish eggs and fish guts. Traditionally, the Jeotgal is processed with 20–30% salt as the main ingredient and is prized for its distinctive flavor and high nutritional content. In Korea, fresh jogaejeotgal (jogae, meaning clam; jeotgal, meaning salted seafood) is prepared by adding 10–20% salt and seasonings (such as sesame oil, minced garlic, ginger and finely chopped spring onions). Jeotgal is usually eaten raw after fermentation and is not sterilized during this process. Although many foods could be involved in the transmission of HuNoV, bivalve molluscs such as oysters, mussels and clams are considered relatively high-risk foods because contaminated water can lead to the accumulation of HuNoV in the digestive tissues of these organisms (Lees, 2000; Bellou et al., 2013).

Real-time quantitative polymerase chain reaction (RT-qPCR) is a methodology that accurately quantifies nucleic acid through monitoring the entire PCR process by utilizing a real-time fluorescence quantifier. However, although RT-qPCR is a very sensitive technique, it has certain limitations in the analysis of viruses in food, as it cannot clearly differentiate between infectious and non-infectious viruses and therefore does not allow efficient risk assessment (Hassard et al., 2017). As a rapid tool for the detection and quantification of viable cells of different microorganisms in various food matrices, qPCR tests combined with nucleic acid intercalating dyes based on propidium monoazide (PMA) have been developed (Liu et al., 2018; Laidlaw et al., 2019). PMA can be used as a pre-treatment prior to RT-qPCR. It helps to distinguish between live and dead cells (Nocker et al., 2006). Analytical applications based on the PMA treatment technique may benefit from its excellent and unique properties, such as high-water solubility, high foam stability and strong sorption capacity to proteins (Zi et al., 2018). Since the PMA dye only penetrates cells with damaged membranes and intercalates DNA/RNA, the PMA RT-qPCR technique is based on the integrity of the cell membrane (Fittipaldi et al., 2012; Elizaquível et al., 2013). Sodium lauroyl sarcosinate, also known as sarkosyl, is an anionic surfactant that is derived from sarcosine. The anionic detergent can help PMA penetrate damaged cells and improve the discrimination between dead and live cells (Lee et al., 2018).

High-pressure processing (HPP) has been identified as a promising form of nonthermal intervention to treat food with a high risk of viral contamination without significantly changing the nature of the food (López-Caballero et al., 2000). HPP technology entails hermetically sealing food in a flexible container and applying high-pressure of 100–600 MPa at room temperature. A liquid (typically water) is used as the pressure transfer medium to subject the interior and surface of the food to an even amount of pressure to achieve pasteurization (Balasubramaniam et al., 2015). In recent years, HPP has been used to treat high-risk foods, including fruit and vegetable products (e.g., salsa, apple sauce, and various fruit blends and purees) and shellfish (e.g., oysters and clams) for virus contamination. HPP disrupts living organisms by either destroying or deactivating microbial cells using a combination of physiological and biochemical effects on the microorganisms (San Martin et al., 2002). Treatment of oyster homogenate contaminated by inoculation with HuNoV genogroup II.4 at 300 MPa for 5 min at 6°C resulted in a 3.51 log reduction in the HuNoV genome (Ye et al., 2014). HPP at 600 MPa for 5 min at 6°C, but not at 400 MPa (at 6 or 25°C), completely inactivated HuNoV in seeded oysters, as reported by Leon et al. (2011).

Therefore, the objective of our study is to assess the efficiency of HPP treatment against HuNoV GII.4 infectivity of jeotgal assessed by PMA + Sarkosyl pretreatment combined with RT-qPCR. We also aimed to determine whether higher pressure levels (100–600 MPa) of HPP treatment would adversely affect the pH value and Hunter color attributes of jeotgal.

In the last 3 years, Korean fermented shellfish jeotgal, including clam jeotgal, have been identified as a cause of microbial food poisoning in Korea. Choi et al. (2018) reported that coliforms and Bacillus cereus were qualitatively detected in 17 (48.6%) and 20 (57.1%) of 35 seasoned jeotgals, respectively, and the total number of aerobic bacteria detected quantitatively in clam jeotgal was 5.9 log CFU/g. In 2018, oyster jeotgal and raw crab soy sauce jeotgal were found to contain HuNoV (Ministry of Food and Drug Safety (MFDS), 2018). The following year, hepatitis A virus (HAV) outbreaks were reported at specific restaurants in Busan, Korea, where 64 individuals who consumed clam jeotgal became infected with HAV (Anonymous, 2019). Therefore, the development of food processing technologies to deactivate pathogenic microorganisms mainly including HuNoV in potential high-risk jeotgal is urgently needed, especially in Korea. Contaminated shellfish are accountable for HuNoV outbreaks, and it is important to have competent techniques to detect and retrieve infectious HuNoV in shellfish. This study is involved on gauging the efficacy of high-pressure processing (HPP) treatment against HuNoV GII.4 infectivity in jeotgal by using RT-qPCR. To evaluate HPP-induced damage to the viral capsid proteins, the samples were subjected to proteinase K and PMA + Sarkosyl pretreatment prior to RT-qPCR assessment. Proteinase K was utilized to break down the protein capsid, facilitating the release of genomic RNA, which could then be degraded by RNase. The Proteinase K digestion methodology has undergone comprehensive evaluation and was included in the ISO 15216 standard procedure for the quantitative detection of Norovirus and hepatitis virus in 2017 (Anonymous, 2017). Recently, several studies have assessed the use of PMA intercalating dye pretreatment before RT-qPCR to distinguish between infectious and non-infectious viruses. Jeon et al. (2020) demonstrated a significant reduction in norovirus load in PMA treated crustacean samples compared to untreated samples using an optimized PMA treatment. Specifically, Jeong et al. (2017) reported a decrease in log copies of <1.5 for non-dye treated HuNoV on spinach, while spinach associated HuNoV was not detected after PMA treatment at 95°C for 2 min. Mohammad et al. (2015) reported that PMA/RT-PCR was able to differentiate between infectious and non-infectious murine norovirus following inactivation by chlorine. Although both of these viruses belong to the Caliciviridae family, the efficacy of PMA treatment may vary depending on the virus type, such as feline calicivirus (FCV) and murine norovirus (MNV-1). The differences in the ability of PMA treatment to distinguish between infectious and non-infectious viral particles may be due to the degree of damage to RNA or capsids caused by the various inactivation methods, including ultraviolet light, plasma, heat treatment, and gamma radiation. However, it is commonly known that intercalating dyes have a where the inactivation method may cause damage to the nucleic acid resulting in the loss of infectivity due to damaged nucleic acids, but the capsid remains intact (Leifels et al., 2015). Mohammad et al. (2015) reported that the addition of chlorine and exposure to heat can damage the viral capsid, allowing the genome to access the dye. Our findings indicate that there was no significant difference in the reduction of log copies between PMA pretreated and non-PMA pretreated samples subjected to HPP treatment, despite statistical differences between the two groups. This implies that, in these HPP conditions, the viral capsid may have remained partially intact or that the denatured capsid protein can safeguard a portion of the genome, preventing PMA binding.

Several studies have indicated the potential efficacy of surfactants, including triton-x-100, sodium deoxycholate, and sodium lauroyl sarcosinate (Sarkosyl), in improving the penetration of monoazide into slightly damaged capsids of HuNoV and HAV (Moreno et al., 2015; Fuster et al., 2016). In this study, the average difference between PMA/RT-qPCR and PMA + Sarkosyl/RT-qPCR was 0.14 log copies/μL (data not shown). Lee et al. (2018) reported that a combination of PMA and sarkosyl treatment of chlorine disinfected HuNoV GII.4 in water improved the discrimination between infectious and chlorine inactivated viruses by RT-qPCR. These observations were consistent with our findings, which revealed that HuNoV viability in the shellfish samples was more effectively assessed by PMA + Sarkosyl/RT-qPCT compared with the typical RT-qPCR sarkosyl enhanced permeability of dead or non-infectious particles to PMA. Taken together, the PMA and Sarkosyl pretreated method may indicate the infectivity of HuNoV in jeotgal following HPP treatment. As a result, it was utilized in this investigation.

High-pressure processing (HPP) has demonstrated potential as a non-thermal technology to enhance the safety and extend the shelf life of a wide variety of foods. This approach can maintain the organoleptic properties of food items whilst making them microbiologically safe with a prolonged shelf life (Patterson, 2005). The level of virus inactivation resulting from HPP treatment increases as the pressure and treatment duration increase. Nonetheless, the effectiveness of the treatment can significantly differ among various viruses. The potential of HPP treatment to inactivate HuNoV was investigated in preliminary studies Buckow et al. (2008) through the use of FCV and MNV-1 surrogate viruses. Tang et al. (2010) reported that application of pressure of 400 MPa for 5 min inactivated 8.22 log PFU of MNV-1. In other similar studies, FCV was reduced to undetectable levels in shellfish (4.21 and 3.83 log reductions in mussels and oysters, respectively) by applying pressure in excess of 300 MPa (Murchie et al., 2007). Kingsley et al. (2007) indicated that a temperature of 5°C, when 350 MPa pressure was applied, resulted in the greatest degree of inactivation, reducing the MNV-1 titer by 5.56 log, whereas warmer temperatures of 20 and 30°C yielded appreciably less inactivation of 1.79 and 1.15 log, respectively. In contrast, we found that treatment of HuNoV GII.4 at 600 MPa and 18°C resulted in a 1.62 log reduction in viral RNA using the PMA + Sarksoyl/RT-qPCR assay compared to a 1.38 log reduction at 20°C. In the study of Ye et al. (2014), HPP treatment of 300 and 600 MPa at 25°C resulted in 1.1–1.8 log reduction of HuNoV GII.4 and GI.1 in oysters. Li et al. (2013) reported that a reduction of 3.7 log RNA was achieved when oysters treated at 350 MPa for 2 min at 4°C were inoculated with HuNoV GII.4 strain, whereas a reduction of only 1.2 log RNA was observed when the virus inoculated oyster was treated by using 350 MPa for 2 min at 20°C. Our data are consistent with those of the above two studies indicating that HuNoV was more pressure resistant than FCV and MNV-1, a surrogate for the norovirus. This phenomenon may be attributed to various factors such as the innate properties of the virus, the size and structure of its particles, its high thermodynamic stability, discrepancies in the binding properties of the viral receptor, or differences in the protein structure and amino acid composition. Additionally, they concluded that a low initial sample temperature substantially increased HPP treatment of HuNoV compared with high temperature. Although the HPP-treated jeotgal sample was not subjected to cold temperatures (0–10°C) (a constant temperature of 18°C was used) in the current study, HuNoV inactivation could be speculated to occur at HPP of more than 600 MPa at cold temperatures. The mechanism underlying the HPP anti-HuNoV effects in foods including jeotgal has not been fully elucidated. However, typically, HPP treatment inactivates the virus through denaturation of the viral capsid proteins. This leads to the incapacitation of infection virions, preventing their attachment and penetration into host cells. Furthermore, variations in pH, salt concentration, fat composition, and protein composition have a significant impact on the sample matrix, resulting in pathogen inactivation. This has also been demonstrated to be true for viral inactivation (Buckow et al., 2008). In HPP treatment, salt in the food environment can provide protective benefits because it can stabilize viral capsid proteins. However, NaCl concentrations of up to 1% do not seem to offer significant protection to the virus. In contrast, higher concentrations of salt exhibit baro-protective characteristics (Grove et al., 2006; Kingsley et al., 2007; Kingsley and Chen, 2009). Comparing data from studies with varying treatment times, starting temperatures, and NaCl concentrations requires caution, as these factors can impact the extent of microbial inactivation.

The color, flavor, and texture are important quality characteristics of seafood and vegetables and are major factors that affect the sensory perception and consumer acceptance of foods. Many studies do not include a sensory evaluation or do not report whether HPP induced textural changes would have a detrimental effect on consumer acceptance of a product. In this experiment in which jeotgal was treated with HPP, the Hunter color values were darker, with more yellow and redness. This fact was possibly connected with the observation that the midgut gland of the shellfish was forced out as a result of the pressure. The pH value is widely regarded as a standard chemical indicator of alterations in the quality of foods. In our work, the pH value of the control (0 MPa) sample did not differ from that of all the HPP (100–600 MPa) treated jeotgal samples. These pH values were in accordance with those of a study by Jo (2019) who reported that the pH of jeotgal was 6.25. A few panelists mentioned that the jeotgal treated with HPP >500 MPa darkened in appearance whereas the non-treated jeotgal did not exhibit such color differences (data not shown). Few studies that investigated changes in the quality of seafood following HPP treatment have been reported, although both textural and visual changes to other bivalve shellfish following HPP treatment were reported. Arcangeli et al. (2012) found that subjecting clams to a pressure of 500 MPa for 1 minute at a temperature of 20°C is a viable method for MNV-1 activation, while leaving the appearance and texture of the clam unaffected. Ye et al. (2014) also reported that HPP (300–500 MPa) treatment for 2 min at 25 and 0°C did not significantly change the color or texture of oyster tissue. Similarly, López-Caballero et al. (2000) reported that HPP (400 MPa) treatment for 10 min at 7°C did not significantly change the appearance of oyster tissue. They concluded that HPP treatment has excellent antibacterial efficacy and that the sensual quality is preserved. HPP is therefore considered to be a potential and promising alternative to seafood conservation technology. However, additional HPP inactivation data for enteric viruses is necessary, across various treatment times and pressures, in both buffered systems and food matrices.

The research presented here establishes the efficacy of employing PMA + Sarkosyl pretreatment alongside RT-qPCR to assess the efficacy of HHP at 100–600 MPa for 5 min at ambient temperature against HuNoV GII.4 infection in jeotgal. The average viral RNA of HuNoV was reduced by around 43% (=0.24 log) in samples treated with PMA + Sarkosyl when compared to those not treated with PMA + Sarkosyl. To achieve a reduction in excess of one log in jeotgal treated with PMA + Sarkosyl, exposure to 400 MPa of HPP. The Hunter “L” color values decreased significantly (p < 0.05) with incremental increases in experimental pressure (0–600 MPa). Meanwhile, the Hunter “a” and “b” color values significantly increased (p < 0.05) as experimental pressure increased stepwise (0–600 MPa). The pH value ranged from 6.14 to 6.19. A statistically significant preference (p < 0.05) was found between untreated controls and all pressure-treated samples for the parameter “Color.” This outcome could potentially be explained by the midgut gland of the shellfish being extruded due to the applied pressure. These results suggest that RT-qPCR/PMA + Sarkosyl could be regarded as a tool to determine the viability of using HPP to treat Korean fermented shellfish product for HuNoV. Furthermore, our experiments have indicated that utilizing ≥400 MPa of HPP at 18°C for 5 min can be an effective means of reducing HuNoV by ≥90%. This process had no significant influence on the pH nor most sensorial qualities of jeotgal, with the exception of sensorial and mechanical colors.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

EBJ: Data curation, Validation, Writing – original draft. HSJ: Methodology, Writing – review & editing. SYP: Conceptualization, Writing – review & editing.