3.5. Viruses
Viruses remain a vexing problem for the shellfish industry. There are a large number of pathogenic viruses that can be shed from the human gastro-intestinal tract [
52]. These viruses can enter shellfish growing areas as a result of sewage overflows and floods, defective septic systems, overboard waste discharge, or even as a result of a vomiting event [
53]. Enteric viruses are shed at high levels, perhaps billions of particles per illness, and are typically highly infectious, with only a handful of particles required to establish an infection [
54]. Viruses are very stable in the environment, and can persist in shellfish growing waters and even within shellfish for extended periods [
55,
56]. Because these viruses require a human gut to replicate, they do not grow, or amplify, within shellfish as a result of temperature abuse as can occur for bacteria. Rather they simply contaminate shellfish as a result of filter feeding activities, typically sequestering themselves at relatively low levels. Because direct testing for viruses is currently impractical, classification of shellfish harvest areas are regulated based on the levels of fecal bacteria in growing waters or directly within the shellfish meats, as described previously. While this classification system prevents a great deal of unsanitary shellfish from potentially reaching the dinner table, it is now recognized that low FC levels in growing waters, or within shellfish meats, do not necessarily indicate that there are no viruses present [
57]. This is principally due to the fact that viruses can persist within shellfish tissues for longer periods than FCs [
55,
56,
58]. Presumably this is due to virus’ resistance of the acidic digestive processes of shellfish [
56]. Thus, a suitable intervention for viruses potentially sequestered within raw shellfish would be of key significance.
Cooking and depuration, two traditional means of sanitizing shellfish, are of limited effectiveness against viruses. Depuration is a method in which live shellfish are placed in clean water for 2–3 days to permit the bivalves to pump and purge pathogens. This method is generally effective against fecal bacteria which can be reduced by several orders of magnitude, but it is accepted that pathogenic human viruses do not purge efficiently enough to make the process a viable intervention for virus contamination. Cooking is thought to inactivate viruses to a substantial degree, but it is unclear what temperatures and cooking times are completely effective against these viruses within shellfish. In fact, several documented outbreaks have been associated with “properly cooked shellfish” [
59,
60,
61]. Furthermore, consumers often prefer uncooked or lightly-cooked shellfish.
Although there are many different types of fecal viruses that can be potentially transmitted by shellfish, the two principal shellfish-borne virus threats are recognized to be human norovirus (HuNoV) and hepatitis A virus (HAV). Human norovirus is now arguably considered to cause the majority of food-borne incidents worldwide [
19,
62]. Approximately half of the food-borne incidents associated with shellfish are due to norovirus and the overall fraction of food-borne noroviruses attributed to molluscan shellfish is approximately 13% [
63,
64].
Initial research on the potential of HPP to inactivate norovirus focused on genetically-related surrogate research viruses, such as feline calicivirus [
65,
66,
67,
68] and murine norovirus [
69], because human norovirus strains have not been reproducibly propagated in the laboratory [
70,
71] and there are no suitable small animal research models for the virus. Surrogate work pointed to reasonable prospects for inactivation of norovirus. Feline calicivirus was found to be highly sensitive to HPP with 5 min room temperature treatments of 275 MPa being sufficient to inactivate 7-log
10 of the virus [
68,
72]. The subsequent isolation and discovery of murine norovirus, which was propagable, made it possible to evaluate a closer genetic relative of human norovirus [
73]. Results indicated that higher pressures, on the order of 400 MPa, were needed to inactivate substantial quantities of this virus [
69]. However successful inactivation of murine norovirus within oysters was demonstrated at this pressure level [
69,
74]. Also work with immunocompromised mice confirmed that inactivation by HPP
in vitro, as assessed by tissue culture and
in vivo, as assessed using mice were essentially equivalent [
75].
More recent work has looked at the potential of HPP to inactivate human norovirus. A human volunteer study evaluated conditions required to inactivate the prototype norovirus strain (GI.1 Norwalk). Four-log
10 PFU of Norwalk virus was injected into oysters and three 5-min pressure treatments were performed at 400 MPa, 22 °C; 400 MPa, 6 °C; and 600 MPa, 6 °C. Unfortunately only the 600 MPa treatment was sufficient to protect all volunteers [
76]. Based on a reduced illness frequency, it was postulated that the 400 MPa, 6 °C treatment may have inactivated some norovirus virus. Subsequent investigations using the newly developed porcine gastric mucin binding assay (PGM-MB) binding assay, which can assess norovirus inactivation, have confirmed that norovirus is sensitive to HPP at about 400 MPa [
77,
78,
79].
HAV contamination of shellfish is now uncommon in most parts of the developed world due to improved hygienic standards and vaccination campaigns [
80] but it remains a problem in the developing world and the Mediterranean region [
81]. HAV illness can be quite serious, often resulting in hospitalization and occasionally in mortality. Morbidity and mortality due to HAV is often age related, with persons over the age of 50 being more prone to mortality and with young children often having only unapparent infections [
82,
83]. Considering that exposure induces immunity that is generally thought lifelong, HAV vaccination campaigns are often not given much emphasis in endemic regions since the local populations largely become immune at young ages. Unfortunately shellfish and uncooked fruits and vegetable products grown in endemic regions and sold in developed countries can become vectors for HAV outbreaks [
84,
85]. A tissue culture-adapted strain of HAV has been evaluated for sensitivity to HPP. Results indicate that a 5 min room temperature treatment at 450 MP is sufficient to inactivate 7-log
10 of HAV virus stock [
68]. Evaluation of HAV-contaminated oysters demonstrated a 3-log
10 reduction after a 1 min-400 MPa treatment at 9 °C [
86].
It was hoped that HPP would also inactivate other pernicious viruses which could potentially contaminate shellfish such as Aichi virus [
87], hepatitis E virus (HEV; [
88]), coxsackie viruses,
etc. HEV has yet to be evaluated but Aichi and a number of other members of the picornavirus family have proven more tolerant to high pressure, requiring either pressures well above 400 MPa, or even being completely as resistant to 600 MPa treatments [
89,
90].
3.6. Parameters
Research has shown that there are a number of considerations for inactivating bacteria and viruses with HPP. Of course the primary determinant for pathogen inactivation is the pressure level applied, which generally follows first-order kinetics since plotting log
10 pathogen reduction
versus increasing pressure applied gives a straight line. Beyond pressure levels applied, time under pressure and pre-pressurization temperature can have a considerable influence on inactivation levels. For all pressure-sensitive viruses tested to date, increased application time does increase the amount of virus inactivation observed but the amount of increase observed asymptotically decreases, matching log-logistic or weibull kinetics [
43,
52,
91]. Solutes, such as salt and sugar generally decrease the effectiveness of HPP inactivation for viruses and bacteria [
43,
51,
67,
68,
92]. Formally speaking the reason for this is unknown, but presumably, the presence of solutes may tend to prevent compression and addition of more water molecules into the solvation cage surrounding the protein. Although currently undefined, this may be an important consideration for shellfish grown in different salinities, since the salt content of bivalves mimics the waters from which they have been harvested. Also, generally speaking, bacteria that are actively growing in exponential phase are more sensitive to pressure than bacteria in stationary phase [
51].
Perhaps more intriguing is the concept that temperature has a substantial influence on inactivation. For vegetative bacteria, pressure applied above and below room temperature generally appears to enhance inactivation [
51]. For viruses, the temperature effect is variable since different viruses react differently for HPP at different temperatures. For noroviruses and all caliciviruses tested to date, refrigeration temperatures dramatically enhance inactivation, often by several logs [
52,
69]. Curiously, HAV is the reverse. Room temperature and above dramatically enhance inactivation by HPP as compared to refrigeration temperatures [
92,
93]. Unlike the
Caliciviridae, other picornaviruses have shown variable inactivation with respect to temperature [
90]. Why viruses behave differently under pressure at different temperatures is currently unknown.
Presumably, HPP-treated shellfish would have a neutral pH, but for other food matrices, pH is another important consideration. Low pH generally enhances HPP inactivation of vegetative bacteria, but viruses respond differently to low pH under pressure. For human norovirus, low pH appears to be inhibitory for HPP inactivation [
78]. For HAV, a virus that is known to be tolerant of pH 1, lower pH actually enhances HPP inactivation [
92,
94]. It is also important to note that weak organic acids, such as acetic acid, become stronger acids under pressure. Why HAV and HuNoV behave differently under pressure in acidic pH is currently unknown.
There can be a substantial temperature increase associated with the application of pressure. As anyone who has ever filled a scuba tank knows, when air inside the tank is compressed, heat is generated, making the tank warm. Likewise, anyone who has fully opened the valve of a pressured tank knows that as the pressure is released, the tank becomes quite cold. These effects are due to a principle called adiabatic heating and cooling [
95]. Although the incremental amount of heating/cooling does vary somewhat with the initial temperature at which pressure is applied, the temperature increase is approximately 3–3.5 °C per 100 MPa for water-based commercial units. Also, the degree to which this heat dissipates to the environment varies with the size of HPP units, with smaller units dissipating heat to the environment more rapidly due to increased surface area to volume ratio.
3.8. Organoleptic Considerations
The color, taste, texture, appearance and smell of raw oysters are of paramount concern to the industry and consumers alike. Currently there is a perception by industry that pressures above 300 MPa result in undesirable changes to oyster quality. It is true that the degree to which HPP changes a raw oyster’s characteristics is a function of the pressure applied and the temperature at which pressure is applied. Some whitening or blanching occurs when treating oysters at 600 MPa at room temperature but this is minimized when 600 MPa is performed at 5 °C. Overall appearance of oysters and clams on-the-half-shell is much better when shucked by HPP [
96], since the bivalve meat is completely intact (see
Figure 1). This differs from a hand-shucked oyster which can often be sliced by the shucking knife. There are reports that HPP can induce some firmness or chewiness in seafoods, but this change is relatively subtle and may be considered desirable, since firmness can be considered an attribute of freshness [
13]. Juiciness and flavor are enhanced by HPP, since shellfish take up liquid from the surrounding liquor within the shell [
44,
97,
98]. This attribute results in a “yield” increase since the shucked oyster becomes more voluminous due to absorption of liquor fluid. One drawback is that liquid taken up does not remain within the bivalve tissues over time. Thus an originally full jar of HPP-shucked oysters will be reduced in volume a week later. Also depending on what the oysters were feeding on when harvested, sometimes this liquid in the jar can be an unappealing yellow or greenish color.
Figure 1.
A hand-shucked clam (left;
Mercenaria mercenaria) is compared to a high pressure processing (HPP)-treated clam (right). Note: this figure is reprinted with permission from [
9]. Copyright Elsevier B.V. 2014.
Figure 1.
A hand-shucked clam (left;
Mercenaria mercenaria) is compared to a high pressure processing (HPP)-treated clam (right). Note: this figure is reprinted with permission from [
9]. Copyright Elsevier B.V. 2014.
Studies clearly indicate that HPP-treated oysters are well received after treatments at pressures that are higher than are currently used commercially to shuck oysters and inactivate vibrio [
99]. For example, recent results (shown in
Table 1) from an organoleptic study evaluating the taste of oysters has shown that oysters treated at 400–600 MPa at 6 °C and 300–500 MPa at 22 °C are actually preferred to manually-shucked oysters [
100]. Another challenge for HPP-treated oysters is time between processing and consumption may be several days which may compromise their perceived quality as compared to a fresh-shucked oyster.
Table 1.
Organoleptic analysis of HPP-treated # oysters * using untrained volunteers.
Table 1.
Organoleptic analysis of HPP-treated # oysters * using untrained volunteers.
| Control | 300 MPa 22 °C | 400 MPa 22 °C | 500 MPa 22 ° C | 400 MPa 6 °C | 500 MPa 6 °C | 600 MPa 6 °C | F value (Sig p value) |
---|
Appearance | 4.11 ± 1.6 | 5.46 ± 1.5 | 5.39 ± 1.4 | 5.20 ± 1.6 | 5.39 ± 1.5 | 5.45 ± 1.4 | 5.22 ± 1.7 | 5.78 (0.000) |
Texture | 4.54 ± 1.9 | 5.23 ± 1.7 | 5.36 ± 1.8 | 5.55 ± 1.6 | 5.20 ± 1.6 | 5.47 ± 1.5 | 5.43 ± 1.7 | 2.46 (0.024) |
Flavor | 4.64 ± 1.7 | 5.04 ± 1.8 | 5.05 ± 1.7 | 5.13 ± 1.7 | 4.86 ± 1.6 | 5.35 ± 1.6 | 5.27 ± 1.6 | 1.24 (0.287) |
Aroma | 4.90 ± 1.4 | 5.27 ± 1.3 | 5.04 ± 1.2 | 5.30 ± 1.3 | 5.27 ± 1.4 | 5.33 ± 1.3 | 5.33 ± 1.4 | 0.94 (0.469) |
Acceptability | 4.64 ± 1.6 | 5.14 ± 1.6 | 5.13 ± 1.6 | 5.28 ± 1.6 | 5.02 ± 1.5 | 5.53 ± 1.4 | 5.38 ± 1.6 | 2.05 (0.058) |
3.9. Challenges and Future Directions
There are a number of challenges to the widespread application of HPP to the shellfish industry. First HPP is relatively expensive with the minimal cost for a commercial scale unit being several hundred thousand US dollars. Thus to be economically viable, shellfish harvesters and processors must be relatively large in scale. Currently most shell aquaculture and fishing operations are too small to successfully amortize this expense. Development of a low cost pressure unit for limited shellfish quantities that is suitable for use near or at the point of consumption would be a boon for the shellfish industry and consumers alike. Another challenge is consumer resistance and regulations regarding in shell-shellfish. A closed shell is often used to judge that shellfish are alive and fresh. HPP kills shellfish, so consumers must be educated to the concept that these are safe and good to eat. In most jurisdictions, there are no longer prohibitions against sale of in-shell HPP-treated shellfish but regulations against theses may remain in some places.
Another potential challenge is that shellfish must be HPP-treated relatively quickly after harvest. If the liquor inside the shell dries out or is reduced in volume permitting air within the shell, as can happen after a day or two of cold storage, the shells may be cracked, or even crushed when pressure is applied due to air pockets under the shell (personal observation). Lastly, should HPP treatment become mainstream as a pathogen intervention technique for shellfish, it will be important that current hygienic quality standards for safe harvest remain in place. HPP should only be applied in addition to current hygienic standards, not in lieu of these standards or as a way to utilize shellfish grow under non-hygienic conditions.