key: cord-0853888-gphit34s
authors: Norouzbeigi, Sahar; Yekta, Reza; Vahid‐Dastjerdi, Leily; Keyvani, Hossein; Ranjbar, Mohammad Mehdi; Shadnoush, Mahdi; Khorshidian, Nasim; Yousefi, Mojtaba; Sohrabvandi, Sara; Mortazavian, Amir M.
title: Stability of severe acute respiratory syndrome coronavirus 2 in dairy products
date: 2021-07-04
journal: J Food Saf
DOI: 10.1111/jfs.12917
sha: ad16149b5f83665a2c1853cf73a948739ce60689
doc_id: 853888
cord_uid: gphit34s

The present investigation was performed to determine the stability of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) under several industrial processing situations in dairies, including pasteurization, freezing, and storage in acidic conditions. Ten treatments were selected, including high‐temperature short‐time (HTST)‐pasteurized low‐fat milk, low‐temperature long‐time‐pasteurized low‐fat milk, extended shelf life (ESL)‐pasteurized low‐fat milk, HTST‐pasteurized full‐fat milk, LTLT‐pasteurized full‐fat milk, ESL‐pasteurized full‐fat milk, pasteurized cream, ice cream frozen and stored at −20 or −80°C, and Doogh (as a fermented milk drink with initial pH < 3.5) refrigerated for 28 days. The viral particles were quantified by RT‐PCR methodology. Besides, the virus infectivity was assessed through fifty‐percent tissue culture infective dose (TCID(50)) assay. These products were seeded with a viral load of 5.65 log TCID(50)/mL as a simulated cross‐contamination condition. Pasteurization techniques were sufficient for complete inactivation of the SARS‐CoV‐2 in the most dairy products, and 1.85 log TCID(50)/mL virus reduction in full‐fat milk (fat content = 3.22%). Freezing (either −20°C or −80°C) did not result in a virally safe product within 60 days of storage. Storage at high acidic conditions (initial pH < 3.5) completely hampered the viral load at the end of 28 days of refrigerated storage. This research represents an important practical achievement that the routine HTST pasteurization in dairies was inadequate to completely inactivate the viral load in full‐fat milk, probably due to the protective effect of fat content. Furthermore, freezing retain the virus infectivity in food products, and therefore, relevant contaminated foods may act as carriers for SARS‐CoV‐2.

shelf life (ESL)-pasteurized low-fat milk, HTST-pasteurized full-fat milk, LTLT-pasteurized full-fat milk, ESL-pasteurized full-fat milk, pasteurized cream, ice cream frozen and stored at À20 or À80 C, and Doogh (as a fermented milk drink with initial pH < 3.5) refrigerated for 28 days. The viral particles were quantified by RT-PCR methodology. Besides, the virus infectivity was assessed through fifty-percent tissue culture infective dose (TCID 50 ) assay. These products were seeded with a viral load of 5.65 log TCID 50 /mL as a simulated cross-contamination condition. Pasteurization techniques were sufficient for complete inactivation of the SARS-CoV-2 in the most dairy products, and 1.85 log TCID 50 /mL virus reduction in full-fat milk (fat content = 3.22%). Freezing (either À20 C or À80 C) did not result in a virally safe product within 60 days of storage. Storage at high acidic conditions (initial pH < 3.5) completely hampered the viral load at the end of 28 days of refrigerated storage. This research represents an important practical achievement that the routine HTST pasteurization in dairies was inadequate to completely inactivate the viral load in full-fat milk, probably due to the protective effect of fat content.

Furthermore, freezing retain the virus infectivity in food products, and therefore, relevant contaminated foods may act as carriers for SARS-CoV-2.

RNA in patients' feces indicates the feasibility of the fecal-oral transmission path (Dhama et al., 2020; Yekta, Vahid-Dastjerdi, Norouzbeigi, & Mortazavian, 2020; Zhang, Wang, & Xue, 2020) . As yet, there is no proof of COVID-19 transmission through breast milk (Centeno-Tablante et al., 2020). Owing to the possibility of cross-contamination by infected foodstuffs during the outbreak, the ingestion of unheated milk should be avoided. Beyond that, not only thermal processing at each temperature-time combination may not be adequate for the inactivation of viral particles, but also due to the stability of SARS-CoV-2 at À20 C for up to 2 years, some frozen milk products like ice cream may consider as a carrier for the novel coronavirus pandemic (Yekta et al., 2020) .

The probable fecal-oral transmission route should not be ruled out because of the probability of food contamination by either carryover or carry through paths. The recognition of the novel coronavirus on the frozen chicken wing from Brazil has propounded the first discovery of SARS-CoV-2 on real foods (Han, Zhang, He, & Jia, 2020) .

Recently, we published a review article focusing on the risk of disparate foods being vehicles of the novel coronavirus (Yekta et al., 2020) .

Accordingly, and considering that no report was present regarding the effects of food processing on the durability of SARS-CoV-2 in food matrices, this research was designed to evaluate the stability of SARS-CoV-2 under several industrial processing situations in dairies, including pasteurization, freezing, frozen storage, and acidic conditions (low pH and high titratable acidity).

Five industrial dairy products were obtained from local commercial sources, including raw low-fat milk (fat content of 1.14 ± 0.21%), raw full-fat milk (fat content of 3.22 ± 0.16%), cream (fat content of 33.76 ± 0.15%), ice cream (fat content of 10.19 ± 0.07%), and Doogh (a typical Iranian fermented milk drink with initial pH 3.48 ± 0.02 and titratable acidity of 141.56 ± 1.03 D). These products were seeded with SARS-CoV-2 at a virus titer of~6 log TCID 50 /mL and 3.5 Â 10 6 RNA copy numbers to simulate the cross-contamination condition.

Then, each product was subjected to special processing as following:

1. The low-fat milk was pasteurized by HTST (high-temperature short time) method (72 C, 15 s). 7. The cream was pasteurized (90 C, 1 min).

8. The ice cream was stored at two frozen temperatures, that is, À20 C or À80 C. 9. The Doogh was stored for 28 days at a refrigerated temperature of 5 C.

The samples were assessed for total protein content by the macro-Kjeldahl procedure (AOAC, 2000) . The chemical analyses on samples were performed in parallel with virus seeding. Fat contents were measured by the Mojonnier method (Patel, Baer, & Acharya, 2006) . The pH was determined at ambient temperature using a pH meter (Metrohm, Switzerland). Titratable acidity (TA), reported as Dornic degrees, was calculated by adding 0.1N NaOH to the pink endpoint using phenolphthalein as an indicator (Mortazavian, Khosrokhavar, Rastegar, & Mortazaei, 2010) . Table 1 shows the pH, acidity, protein, and fat contents of dairy products.

A T-175 flask of Vero cell line culture prepared from Reference Keyvan Laboratory and trypsinize, centrifuged, and resuspended in 10% fetal bovine serum (Gibco) and 90% media composed of DMEM (Dulbecco's minimum essential medium). Also, cells subcultured in T25 flasks and incubated at 37 C and 5% CO 2 until 80% confluency. These cells were used for virus culture and micro-titration tests.

Previously, samples isolated from the nasopharyngeal cavity by swabs were placed in viral transportation medium (VTM), which supplied by Pasteur Institute (Tehran, Iran). COVID-19 positive diagnosed patients according to their real-time PCR analysis (cycle thresholds under [CT] values 15) used for the present study. We called the isolated virus stain K1 isolate. All investigations on the virus were performed in a biosafety level 3 laboratory. Virus quantification was performed after K1 isolate SARS-CoV-2 propagations and real-time PCR and fifty-percent tissue culture infective dose (TCID 50 ) assay for food seeding performed in 96-well plate and measured by the Reed-Muench method. K1 isolate with log TCID 50 /mL (virus titer)~7 and CT value 10 selected for seeding into food.

The treatments were contaminated under conditions presented in Table 2. 2.3.4 | Virus concentration and evaluation its infectivity (virus titer) by TCID 50 assay In this study, polyethylene glycol (PEG) precipitation was used to concentrate the virus and remove cytotoxic agents and/or PCR inhibitors from food samples. 10 mL of each sample (9 mL sample + 1 mL virus) was mixed with 1.5 mL of the PEG 6000 stock solutions. The suspensions were agitated on a shaking incubator at 150 rpm for 8 hr at 4 C, and the supernatant was transferred into the centrifuge tube. Then, they were centrifuged at 3,635g for 50 min. The PEG-containing supernatants were removed, and the resulting pellet was dissolved in 1 mL phosphate-buffered saline (PBS) and recentrifuged at 4,173g for 40 min. The resulting supernatant was passage through a 0.2 μm sterile membrane filter and added to the both 96-well plates and cell culture flasks for each test group. The virus was titrated in serial 1 log dilutions (from 1 log to 10 log) to obtain a 50% tissue culture infective dose (TCID50/mL) on 96-well culture plates of Vero cell line. The plates were observed every 24 hr for a total of 6 days for the presence of CPEs (cytopathic effects) employing an inverted optical microscope. The end-point titers were calculated according to the Reed-Muench method (Reed & Muench, 1938 ) based on four replicates for each food titration. In parallel in the cultured flasks, after seeding selected dilution of virus (base on TCID50 in micro-titration) they were slowly agitated on a shaking incubator at 37 C for adsorbtion. Finally, the upper phase was discarded, and the medium culture with a little FBS (%1) was added. All the cultured flasks similar to micro-titration were monitored every 24 hr for CPE (cytopathic effect) until 6 days. 

The experiments as well as measurements accomplished in triplicate.

Data were analyzed using SPSS software version 26 with a confidence level of 95% to recognize any significance between treatments. Statistics paired t tests were used to evaluate differences in viral titer between the initial count and after the pasteurization process. A repeated measure ANOVA was used to analyze the data obtained from shelf life study. Although heating to 72 C for 15 s (HTST method) was adequate for the inactivation of SARS-CoV-2 in the low-fat milk, exposure to the latter treatment was not effective in eradicating the virus in the fullfat one. More to the point, there was only~34.5% (from 5.8 to 3.8 log TCID 50 /mL) reduction in the initial viral load of the full-fat milk after heat treatment. This could be attributed to the higher fat content of full-fat milk that presumably protected SARSCoV-2 towards heat, similar to the results from the heat inactivation of hepatitis A (HAV) virus in different dairy products (Bidawid, Farber, Sattar, & Hayward, 2000) .

Consequently, the routine pasteurization process (HTST) was insufficient to inactivate the viral load in the full-fat milk fully. In contrast, in the cream sample, the complete inactivation of SARS-CoV-2 (initially 5.6 log TCID 50 /mL present) was achieved at 90 C for 1 min, and the virus could no longer be detected. (Hirneisen et al., 2010) . Not only the membrane protein (M protein) of SARS-CoV is aggregated by heat processing, but also at 35 C, the nucleocapsid protein of SARS-CoV begins to unfold and is denatured entirely at 55 C (Kampf, Voss, & Scheithauer, 2020; Wang et al., 2004) . In a study performed to determine the durability of SARS-CoV-2 at various temperatures, the T A B L E 2 Contamination conditions of various kinds of dairy products Treatments Sample size SARS-CoV-2 addition dose Storage condition ( C)

Raw milk 9 mL $6 TCID 50 /mL 4 Cream 9 mL $6 TCID 50 /mL 4 Doogh 9 mL $6 TCID 50 /mL 5 Ice cream 9 g $6 TCID 50 /g À20 or À80

Abbreviation: TCID 50 , fifty-percent tissue culture infected dose.

T A B L E 3 The effect of different types of pasteurization on the stability of SARS-CoV-2 in dairy products (Chin et al., 2020) . Bidawid et al. (2000) investigated the thermal inactivation of HAV in cream, homogenized milk, and low-fat milk, which contained 18, 3.5, and 1% fat, respectively. They demonstrated that the higher the fat content, the more increased heat resistance of HAV, possibly due to the protective functions of fat. In other words, at any particular temperature, higher exposure times were required for dairies with more fat contents to obtain a specific decrement in HAV load. In a study performed to determine the inactivation of SARS-CoV-2 in breast milk through pasteurization, the results indicated that the exposure to whether 63 or 56 C for 30 min caused complete inactivation of SARS-CoV-2 in both human milk and the control medium (Walker et al., 2020) . In an experiment that was performed by Weismiller, Sturman, Buchmeier, Fleming, and Holmes (1990) , their data suggested that mildly alkaline conditions (pH 8) induce a conformational alteration in the spike (S) protein of the CoV, mouse hepatitis virus (MHV), that may be associated with the initiation of fusion and infectivity of the viral particle with the host cell. In other research, Xiao, Chakraborti, Dimitrov, Gramatikoff, and Dimitrov (2003) reported that the spike glycoprotein of SARS-CoV fused with the host cell at a neutral pH. However, Darnell, Subbarao, Feinstone, and Taylor (2004) determined the impact of various pH exposures on SARS-CoV infectivity.

The virus was fully inactivated either by alkaline (pH > 12) conditions at 4, 25, and 37 C after 1 hr, or acidic (pH < 3) conditions at 25 and 37 C after 1 hr exposure time. At 4 C, pH = 3 did not completely inactivate the virus. While the virus retained its infectivity at moderate variations of pH conditions. Their results illustrate that SARS-CoV infectivity is susceptible to extreme pH conditions. In another work, Wang et al. (2004) demonstrated that the nucleocapsid protein (N protein) of SARS-CoV at a pH near 5 starts to unfold and is completely denatured at a pH near 2.7 acid unfolding process is reversible. Compared to other coronaviruses, SARS-CoV-2 could maintain its durability in a broad spectrum of pH values (pH 3-10)

after 1 hr exposure time at room temperature (Chin et al., 2020) . It could concisely elucidate the rapid prevalence of SARS-CoV-2 compared to SARS and MERS (Aboubakr, Sharafeldin, & Goyal, 2020) . In one research, the high titer of SARS-CoV-2 remained stable under an acidic situation (pH 2.2), which imitating the gastric condition at room temperature for 30s, 30 min, or 60 min (Sun et al., 2020) .

The results of the log TCID 50 /g of ice cream treatments were demonstrated in Figure 2 . Under freezing temperatures at either À20 or À80 C, SARS-CoV-2 remained infectious in ice cream samples during 60 days study period. During the first 2 weeks of frozen storage, the virus infectivity did not considerably decline for both treatments.

However, the infectivity of ice cream maintained at À20 C was significantly (p ≤ .05) higher than that of À80 C (~5.6 vs. 5 log TCID 50 /g for samples stored at À20 and À80 C in the second week after virus F I G U R E 1 Alterations in viral titer during refrigerated storage of Doogh (5 C)

F I G U R E 2 Alterations in viral titer of ice cream in frozen storage at À20 or À80 C seeding, respectively). After Week 2, the infectivity loss was more

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