Inactivation of anisakis larva using pulsed power technology and quality evaluation of horse mackerel meat treated with pulsed power

Anisakis is a parasite that is found in many marine products and can cause anisakiasis when present in fish consumed raw. The most common way to prevent anisakiasis is to freeze the fish, but this causes a noticeable decrease in the quality of the fish when eaten as sashimi. Although no practical method of killing anisakis other than freezing has been found, we have now succeeded in inactivating anisakis inside the fish meat by repeatedly and instantaneously applying electric current to the fish meat using pulsed power technology. The fish meat was placed in buffer saltwater, and pulsed power was applied multiple times. The immobilization rate was highest when the buffer saltwater was 5 mS/cm. The immobility ratio increased as the number of shots increased. Sensory evaluation of the fish meat after the pulse treatment confirmed that it retained its quality as sashimi. Breaking tests and color measurements were also conducted. We believe that this pulsed power treatment is a useful alternative to freezing as a method for killing anisakis.


Introduction
Anisakis is a nematode parasite found around the world and present in many marine products. It causes a parasitic infection in humans called anisakiasis when fish with anisakis in it is eaten raw, as with sashimi. Anisakiasis is most commonly caused by the third-stage larvae (L3) of A. simplex, A. physeteris, and P. decipiens varieties (Japan Food Hygiene Association 2013). The most common method of preventing anisakiasis is freezing the raw fish. A specific length of freezing time is necessary to kill anisakis (Wharton et al. 2002), with European Food Safety Authority (EFSA) stating that freezing at -20 °C for 24 h is effective at killing anisakis (EFSA 2010).
However, the quality of fish meat deteriorates when frozen. Ice crystals formed in the meat during freezing and storage cause dripping and softening of the texture when the fish meat is thawed (Nakazawa et al. 2020). In addition, the formation of metmyoglobin during storage causes dark meat to turn brown.
Unfortunately, freezing is still the only useful method for killing anisakis that only moderately affects raw fish quality. Inactivation of anisakis by low-voltage current has been reported, but no detailed studies have been conducted (ES 2 213 486 B1, in Spanish).
Pulsed power uses stored energy to instantaneously generate extremely high peak power. Biological applications include sterilization via pulsed electric fields (PEF), and research has been done on inactivation of Caenorhabditis elegans nematodes in multicellular organisms (Tanino et al. 2014). Research has also been done on using pulsed current to kill Nymphonella tapetis sea spiders (Wang et al. 2011). However, in both reports, the inactivated organisms were in the liquid, and there are no reports on the effects of electric energy on organisms not in direct contact with liquid, e.g., anisakis in fish meat.
In this study, pulsed power with a large electric current was applied to fish fillets containing anisakis, and we were successful in rendering the anisakis inside the fish meat inactive. The treated fillets were evaluated by sensory evaluation to ensure that they retained their quality as sashimi. The texture was measured by a breaking test, and the color was measured by spectrophotometry.

Sample fish and anisakis
Fillets of Trachurus japonicus horse mackerel caught in the waters around Japan were used. The average size of the fillets was 103 mm long [standard deviation (SD) 5.7], 45 mm wide (SD 2.1), and 9.6 mm thick (SD 0.54). Anisakis were collected from the internal organs of Scomber japonicus chub mackerel caught off the coast of Nagasaki, Japan, and were morphologically considered to be anisakis type I (Oishi et al. 1971). Most of the anisakis parasites in mackerel caught in Nagasaki are known to be Anisakis pegreffii (Suzuki et al. 2011), and it is presumed that the anisakis used in this experiment were also Anisakis pegreffii. The collected anisakis were stored in 0.9% NaCl in a refrigerator at 4 °C until the experiment.
The fillets were partially cut, and ten anisakis were inserted and glued with transglutaminase (Activa, Ajinomoto) into each fillet to make fillets containing anisakis (Fig. 1a). The anisakis were placed in the middle of the fillet (Fig. 1b). Although it would be ideal to use fish meat that had naturally become inhabited with anisakis, finding such fish meat in usable quantities would be difficult. Therefore, fillets were artificially embedded with anisakis in this research. In the experiments, the untreated anisakis were kept in the same conditions as the treated anisakis, and it was confirmed that the untreated anisakis were alive during activity evaluation. The conductivity of the fillets at 100 kHz was measured using a frequency generator, amplifier, plate-to-plate electrode, and oscilloscope, and was approximately 8 mS/cm.

Activity evaluation of anisakis
Parasite survival is generally judged on the basis of whether they move spontaneously or not (Japan Food Hygiene Association 2015). Anisakis were placed in warm saline solution in a water bath and stimulated with tweezers; those that showed no reaction were judged to be "immobile." Immediately after pulse treatment, most of the anisakis were in a stunned state and could not be judged accurately. Therefore, observations were made after 24 h and 48 h, and the immobility ratio (ratio of immobile anisakis to the total number) was calculated. Anisakis were removed from the fillets immediately after treatment and kept in a refrigerator at 4 °C in 0.9% saltwater until observation.

Experimental equipment
The pulsed power was generated using a capacitor-bank-type circuit with a capacity of 40 μF or 80 μF. The charging voltage for both is 15 kV, and the stored energy per pulse was 4.5 kJ and 9.0 kJ, respectively. To apply the pulsed current to the fillets, stainless-steel parallel plate electrodes (diameter 40 cm, distance between electrodes 11 cm) were submerged in a cylindrical plastic barrel filled with 180 l of buffer saltwater (Fig. 1c). A plastic mesh basket containing the fillets was inserted between the electrodes, and a pulsed current was applied. During the process, saltwater was pumped into the basket under the center of the lower electrode to agitate the saltwater and the fillets (Fig. 1d).

Optimization of water conductivity for anisakis treatment
Pulsed currents were applied to 5.5 kg of fillets (about 250 fillets), including five fillets with anisakis (50 anisakis in total). The buffer saltwater conductivity was adjusted to 0.2 (tap water), 2, 5, 8.5, and 11.5 mS/cm, respectively (0.6% NaCl at 11.5 mS/cm). The charging voltage to the capacitor and capacitance was fixed at 15 kV and 40 μF, the operating frequency was 1 Hz, and 500 shots were applied for each experiment. The total immersion time of the fillets in the saltwater during treatment was about 10 min. Immobility ratios were determined for each conductivity level. The initial saltwater temperature was 5 °C.

Dependence of shot number on immobility ratio of anisakis
Pulsed currents were applied to 7 kg of fillets, including five fillets with anisakis (50 anisakis in total). The buffer saltwater conductivity was adjusted to 5 mS/cm (about 0.3% NaCl) for all experiments. The charging voltage to the capacitor and capacitance was fixed at 15 kV and 80 μF, the operating frequency was 1 Hz, and 100, 200, 300, 400, and 500 shots were applied. Immobility ratios were determined for each number of applied pulses. The initial saltwater temperature was 6 °C.

Sensory evaluation of fish
Sensory evaluation of the pulse-treated fillets was conducted using the method of Okazaki et al. (2009). For the pulse-treated fillets, 450 shots were applied at 80 μF-15 kV-2.5 Hz, with saltwater conductivity at 5 mS/ cm. Initial saltwater temperature was 6 °C, and pulsed currents were applied to 3 kg of fillets in the experiment. The total immersion time of the fillets in the saltwater during treatment was about 5 min.
The pulse-treated fillets were evaluated using a sevenlevel evaluation method (+ 3: very good, + 2: good, + 1: slightly good, 0: neutral, −1: slightly bad, −2: bad, −3: very bad) based on a paper by Okazaki et al. (2009). The control fillets were also immersed in the same saltwater and for the same time as the pulse-treated fillets.
Parameters evaluated included: appearance (dark meat, light meat), smell, texture (moisture, elasticity), taste (fishy, umami), and general quality. For texture, "good" was defined as having considerable elasticity and firm flesh, which is generally preferred in horse mackerel sashimi. The evaluations were conducted by eight Japan Seafoods employees. Fillets were stored in a refrigerator at 5 °C until evaluation, and were evaluated 1 day and 3 days after pulse treatment. The untreated fillets (control) were stored under the same conditions.

Texture analysis of fillets
Breaking tests were conducted on fillets using universal testing instruments (EZ Test, Shimadzu Corporation). For the pulse-treated fillets, 300 shots were applied at 80 μF-15 kV-2.5 Hz in buffer saltwater with a conductivity of 5 mS/cm. Initial saltwater temperature was 6 °C, and pulsed currents were applied to 3 kg of fillets. Since there is a large difference in quality depending on where the fish is sourced, the control, pulse-treated, and thawed (quickly frozen, then stored frozen for 1 month at −20 °C) fillets were measured using the same lot of horse mackerel, and nine fillets of each condition were measured. The control and pulsetreated products were measured after 2 days of refrigerated storage at 5 °C. Seafood products are most commonly eaten 1 or 2 days after packaging at a fish processing factory, so measurements were done at similar timing. Thawed fillets were measured immediately after thawing. A plate-shaped jig with a width of 10 mm and a thickness of 1.5 mm was used to press the fillet. The fillets were placed on a stage, and the dorsal meat was pressed from the skin side; two measurements were taken per fillet (Fig. 2a).
The jig pressed the meat at a speed of 10 mm/min, and the elastic modulus and breaking load were read from the measurement waveform. A typical waveform is shown in Fig. 2b.

Color analysis of fillets
The L*a*b* values of untreated (control), pulsed, and thawed fillets were measured using a spectrophotometer (SA5500, Nippon Denshoku Industries). The same lot of fillets was used for all measurements. For the pulse-treated fillets, 400 shots were applied at 80 μF-15 kV-2.5 Hz in buffer saltwater with a conductivity of 5 mS/cm. Untreated (control) and pulsed fillets were packaged and stored at 5 ℃ and measured 1 day after treatment. Thawed fillets were packaged, quickly frozen, and stored frozen (−20 °C) for 3 days before measurement. Light meat (A) and dark meat (B) were measured (Fig. 2c). For B, the skin was shaved off to expose the dark meat. All measurements were taken after opening the packages and leaving them at 5 °C for 4 h (n = 10).

Pulsed current and inactivation of anisakis
The typical voltage/current and power/energy waveforms of the pulsed power used in the experiment are shown in Fig. 3. These waveforms were recorded with a capacity of 80 μF, a charging voltage of 15 kV, and a buffer saltwater conductivity of 5 mS/cm.
The peak values of voltage, current, and power reached 15 kV, 6 kA, and 100 MW, respectively, and the time duration (10% of the peak) was about 380 μs. The energy input between the electrodes was 7 kJ, which means that the energy transfer efficiency from the capacitor to the electrodes was about 78%. In the experiment, this pulsed power was repeatedly applied between the electrodes containing the fillets.
Inactivation of anisakis in the fillet was demonstrated by applying repetitive pulsed current. Most of the anisakis that were judged to be immobile turned white and cloudy, indicating that the anisakis had died (Fig. 4).

Water conductivity and immobility ratio
The relationship between buffer saltwater conductivity and immobility ratio is shown in Fig. 5a.
The highest immobility ratio was observed at buffer saltwater conductivity of 5 and 8 mS/cm after 24 h in pulse current application, and at 5 mS/cm after 48 h.

Shot number and immobility ratio
The relationship between number of pulses and the immobility ratio is shown in Fig. 5b.
As the shot number of pulses increased, the immobility ratio increased: 400 shots resulted in a 98% immobility rate after both 24 h and 48 h, and 500 shots resulted in a 100% immobility rate after both 24 h and 48 h.

Sensory evaluation of fish
The anisakis immobility rate was 100% when 3 kg of fillets were treated with 450 shots of pulsed current. The temperature of the fillets after 450 shots was about 20 °C, and the buffer saltwater temperature was about 11 °C as measured by a fiber thermometer.
The results of sensory evaluation are shown in Fig. 6. Although slightly worse than the control, the average score of pulse-treated fillets for each item was between −1 (slightly bad) and 0 (neutral), indicating that the quality of the sashimi was maintained.

Texture analysis of fillet
The results of elastic modulus and breaking load measurements for untreated (control), pulsed, and thawed fillets are presented in Table 1.
The elastic modulus and breaking load of the thawed fillets were significantly lower than those of the control, while those of the pulsed fillets were almost the same.

Color analysis of fillets
The L*a*b* color system is commonly used in many fields to describe the color of objects, and is standardized by the Commission internationale de l'éclairage (CIE). In The results of spectrophotometer measurements of the fillets are shown in Fig. 7. The bottom and the top ends of the whiskers represent the minimum and maximum values, respectively. The bottom of the box, the line inside the box, and the top of the box represent the first quartile data, the median, and the third quartile data, respectively. The circle plot indicates an outlier. For light meat, pulsed and thawed fillets had higher L* values and were thus whiter than the control. Values for a* of the control, pulsed, and thawed fillets decreased in that order, with the thawed fillets being the least red.
For dark meat, there was no significant difference in L* value between the control and pulsed fillets, but the thawed fillets had a significantly lower L* value. There was a significant difference between the pulsed and thawed a* values, with the pulsed fillets having a higher value, meaning the pulsed fillets were redder.
There were no significant differences in the b* values for both light meat and dark meat.

Discussion
Anisakis were inactivated through the application of a pulsed current. The conductivity of buffer saltwater with the highest anisakis immobilization ratio was 5 mS/cm. From the impedance ratios obtained from pulses with only saltwater compared with pulses with fillets, it was inferred that the conductivity of the fillet was about 8 mS/cm. This agrees with the results of conductivity measurements of fillets using the voltage and current method (see "Materials and methods").
The immobility ratio was the highest when the conductivity of the buffer saltwater was 5 mS/cm because the current flowed to the fillets more readily when the conductivity of the fillet was higher than that of the saltwater. If the conductivity of the buffer saltwater was too low, however, resistance would increase and current level would be low. The current flowing between the electrodes is highly dependent on the conductivity of the buffer saltwater, but the current is distributed depending on the conductivity ratio of the saltwater and the fillets. Therefore, in this experiment, the highest current flowed to the fillet (and Fig. 6 The results of sensory evaluation. Error bars represent standard deviations (n = 8) 3.04 ± 0.54 3.15 ± 0.46 n.s 2.61 ± 0.41 * the anisakis) when the conductivity of the buffer saltwater was 5 mS/cm and the fillet was 8 mS/cm. The immobilization ratio of anisakis increased as the number of shots increased, and reached 100% immobilization at 500 shots when saltwater conductivity was 5 mS/cm and shots were 80 μF-15 kV-1 Hz. It was confirmed that the inactivation effect of pulsed current on anisakis is the result of a buildup of damage in the anisakis over the duration of the repeated shots until the anisakis becomes immobilized as the damage reaches a fatal level.
The mechanism of death for anisakis by pulsed current is still unknown, but we suppose that the instantaneous application of large amounts of energy to the anisakis causes degeneration of various substances that make up the cells, leading to death.
Sensory evaluation confirmed that the pulse-treated horse mackerel fillets retained their quality as sashimi. The results of the breaking test showed that the elastic modulus and breaking load of the thawed product were significantly lower than those of the control, while those of the pulsed fillets were almost the same, suggesting that the pulse-treated fillets retained a texture closer to that of the control than the thawed product. The results of the spectrophotometer showed that the pulse-treated fillets retained a color closer to the control than the thawed fillets, especially for the dark meat. Since the color of dark meat has a significant impact on the appearance of freshness, the pulsed fillets have a higher commercial value as sashimi than the thawed fillets. From the above, it can be said that the quality of the fillets is better when pulsed power is used to kill anisakis instead of freezing, and pulsed power is a useful alternative to freezing for killing anisakis.
In the future, we plan on studying in detail which electrical parameters affect the inactivation of anisakis. It is also believed that the quality of fish meat can be further improved by increasing anisakis killing efficiency. In addition, studies on other species of fish, including chub mackerel, are planned for the future.
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