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Food Control 40 (2014) 46e49

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Food Control

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Inactivation of Anisakis larvae in salt-fermented squid and pollocktripe by freezing, salting, and combined treatment with chlorine andultrasound

Se-Ra Oh a, Cheng-Yi Zhang a, Tea-Im Kim b, Sung-Jong Hong b, In-Sun Ju c, Sun-Ho Lee c,Soon-Han Kim c, Joon-Il Cho c, Sang-Do Ha a,*

aDepartment of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daeduk-myun, Ansung, Gyunggido 456-756, Republic of KoreabDepartment of Medical Environmental Biology, College of Medicine, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of KoreacMinistry of Food and Drug Safety, Republic of Korea

a r t i c l e i n f o

Article history:Received 19 April 2013Received in revised form8 November 2013Accepted 12 November 2013

Keywords:Anisakis larvaeFreezingSalt-fermented seafoodSaltingUltrasoundChlorine

* Corresponding author. Tel.: þ82 31 670 4831; faxE-mail address: sangdoha@cau.ac.kr (S.-D. Ha).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.11.023

a b s t r a c t

The effects of freezing (�20 �C and �40 �C for 6 h, 12 h, and 1e21 days), salting (5, 10, 15, and 20% NaClfor 3 h, 6 h, 12 h, and 1e7 days), and a combined treatment with chlorine (500, 1000, 1500, and2000 ppm) and ultrasound (37 kHz frequency and 1200W for 5, 10, 15, 20, and 30 min) were investigatedto observe the inactivation of Anisakis larvae in salt-fermented squid and pollock tripe or in the test tube.All larvae inoculated in squid and pollock tripe were inactivated after 48 h at �20 �C and 24 h at �40 �C.The average recovery rates of the larvae inoculated in squid and pollock tripe were 94.4% and 95.2%,respectively. The viabilities of larvae were 81.7% in 5% NaCl and 26.7% in 10% NaCl after 7 days of storage.However, all larvae were inactivated when submerged in 15% NaCl after 7 days of storage and 20% NaClafter 6 days of storage. Viability was reduced from 43.3% to 13.3% when ultrasound alone was used totreat live larvae in test tubes for 15e20 min. Furthermore, although no reduction effect on viability oflarvae was observed when chlorine alone was used for treatment, 0% viability was observed using thecombined treatment of 1500 ppm chlorine and ultrasound for 30 min. Interestingly, when the viscera ofheavily parasitized conger eels were treated with chlorine and ultrasound, there was no reduction effecton viability of the larvae on the viscera. These results could be used to provide more specific guidelinesfor manufacturers and consumers about the freezing and salting conditions necessary to kill Anisakislarvae in salt-fermented squid and pollock tripe.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Salt-fermented seafood, called Jeotkal, is a popular traditionalfood in Korea. To preserve Jeotkal for long time periods, 15e20% saltis added to raw fish, cuttlefish, and shellfish during themanufacturing process of salt-fermented seafood, and the saltedseafood is typically fermented for about two months (Fig. 1).Although Jeotkal is mainly made in small-scale manufacturingplants or on home-made scale, about 30 kinds are industriallyproduced and distributed to consumers (Chae, 2011). Jeotkal is alsoused as a minor ingredient of Kimchi, a typical Korean traditionalfood, as well as a side dish (Lee et al., 1987, p. 14).

The high salt content used in the preservation of seafood cancause health problems such as hypertension and nephritis, and

: þ82 31 675 4853.

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there are concerns regarding parasites, especially Anisakis (Nema-tode: Anisakidae) third-stage (L3) larvae. The presence of AnisakisL3 larvae in fish and cephalopods has long been recognized as apotential human health risk (Agersborg, 1918; Kahl, 1936). Theconsumption of raw or undercooked fish or cephalopods can lead toa fish-borne parasitic disease called Anisakiasis, which causesdiarrhea, vomiting, abdominal pain, and nausea (Adams, Murrell, &Cross, 1997; Adams, Ton, Wekell, Mackenzie, & Dong, 2005), and toallergic sensitization. Therefore, salt-fermented seafood such assalted squid and pollock tripe has known health risks, since they donot undergo thermal processing.

Freezing, thermal processing, salting, and high hydrostaticpressure (HHP) treatments can be used to kill Anisakis larvae. Ofthese methods, freezing and thermal processing are effectivemethods; according to the United States Food and Drug Adminis-tration, raworundercookedfishproducts shouldbe frozen at�35 �C(�31 �F) for 15hor�20 �C (�4 �F) for 7 days in ablast freezer and the

Fig. 1. The manufacturing process of salt-fermented seafood.

Table 1Viability of Anisakis larvae inoculated in squid and pollock tripe after freezing.

Freezertemperature (�C)

No. oflarvae

Storagetime (day)

Meana recoveryrate (%)

Meanb viability(%)

Squid Pollocktripe

Squid Pollocktripe

4c 20 6 h 100 10012 h 100 100

1 100 1002 100 1003 100 1007 100 100

14 100 10021 100 75

�20 20 6 h 98.3 96.7 55.8 53.612 h 93.3 93.3 9.1 9.2

1 88.3 95.0 3.9 1.72 96.7 95.0 0 03 91.7 93.3 0 07 90.0 91.7 0 0

14 98.3 100 0 021 100 100 0 0

�40 20 6 h 96.7 100 28.0 26.712 h 96.7 98.3 3.5 1.7

1 90.0 90.0 0 02 93.3 91.7 0 03 93.3 91.7 0 07 88.3 93.3 0 0

14 95.0 96.7 0 021 100 96.7 0 0

a Average of 3 recovery rate values at each condition.b Average of 3 viability values at each condition.c Larvae in saline as a control to verify that larvae stored at 4 �C are alive.

S.-R. Oh et al. / Food Control 40 (2014) 46e49 47

European Community recommends cooking fish at 60 �C for at least10 min (FDA e Food and Drug Administration, 2001; Huss, 1994;Pravettoni, Primavesi, & Piantanida, 2012). Gustafson (1953) re-ported that most live Anisakis larvae are killed within the first weekwhen herring is frozen at�30 �C for 16 h and then stored at�12 �C.Salting may be carried out by brining, dry-salting, or wet-salting(Helena, Sónia, Maria, Rui, & Paulo, 2012). Wootten and Cann(2001) showed that brining with 21% salt for 10 days killed alllarvae. Recently, high hydrostatic pressure (HHP) treatment wasused to demonstrate the effect of HHP on the inactivation of Anisakislarvae (Dong, Cook, & Herwig, 2003; Molina & Sanz, 2002). Andreaet al. (2010) reported that HPP treatment for 5 min at 300 Mega-pascal (MPa) was sufficient to completely inactivate Anisakis larvaelodging in mackerel (Scomber scombrus).

Chlorine has been widely used to reduce foodborne pathogenson various foods during the washing process (Sagong et al., 2011;Wan Norhana, Poole, Deeth, & Dykes, 2010). Because the Anisakisparasite is resistant to chlorine, seafood infected with Anisakislarvae can be simultaneously treated with chlorine water and ul-trasound. Ultrasound has various applications in the food pro-cessing industry (García, Burgos, Sanz, & Ordo�nez, 1989).Microorganisms on the surface of fresh produce can be detached,and cells can be destroyed by treating produce with ultrasound inthe 20e100 kHz frequency range (Scouten & Beuchat, 2002;Seymour, Burfoot, Smith, & Cox, 2002). However, there are nostudies on the combined effect of chlorine and ultrasound treat-ment on inactivation of Anisakis larvae.

This study was conducted to observe the effects of freezing,salting, and combined treatment with chlorine and ultrasound onthe inactivation of Anisakis larvae and to determine the specificconditions of freezing, salting, and combined treatment necessaryfor killing Anisakis larvae in fresh salt-fermented squid and pollocktripe or in test tube.

2. Materials and methods

2.1. Materials

Live Anisakis larvae were collected from the viscera of heavilyparasitized conger eels (Conger myriaster) obtained at the Nor-yangjin Fishery Market in Seoul, Korea. The viscera of conger eelswere carried to the laboratorywithin 2 h using an ice box. Followingtransport, live larvae adhering to the viscera tissue were collected,immediately placed in saline (0.85% NaCl), and stored at 4 �C.

Forty-eight frozen squids (Sepioteuthis sepioidea) and pollocktripe separated from frozen pollock (Theragra chalcogramma) werepurchased in Seoul, Korea from Garak Market and NoryangjinFishery Market, respectively. Viscera of conger eels naturallyinfected with live larvae were purchased for ultrasound and chlo-rine treatment instead of squid and pollock tripe due to difficulty ofobtaining naturally infected specimen.

2.2. Sample preparation and inoculation

To standardize the conditions of each freezing treatment, 20 livelarvae were inoculated between the thawed squid muscle andviscera (20 larvae per sample). Each squid sample was sealed in ahigh density polyethylene (HDPE) bag. Two thawed pollock tripewere placed in a Petri dish and inoculated with 20 live larvae; acover was put on each Petri dish, and each dish was sealed in anHDPE bag. Following inoculation, samples were stored at 4 �C for1 h to let the larvae adapt to the changed conditions. Live larvaeisolated from viscera of conger eels were kept in saline and storedat 4 �C for use in salting and combined ultrasound and chlorinetreatment experiments. Viscera of conger eels naturally infectedwith live larvaewere also stored at 4 �C until shortly before the startof the experiment.

2.3. Freezing and salting

Since �20 �C is the most commonly used freezer temperature,and several manufacturers of salt-fermented seafood use freezersat �40 �C, we set �20 �C and �40 �C as the freezing conditions.Samples inoculated with live larvae were placed in a �20 �C front-loading freezer (to simulate a domestic freezer) and a �40 �C chestfreezer (to simulate an industrial freezer). Three samples wereremoved from each freezer at storage times of 6 h, 12 h, and 1e21 days (Table 1). After removal from the freezers, samples were

Fig. 2. Viability of Anisakis larvae in salt solution (brine). Experiments were conductedin triplicate. Data are mean values with standard deviation.

S.-R. Oh et al. / Food Control 40 (2014) 46e4948

thawed for 1e2 h at room temperature (20 � 2 �C) while still inHDPE bags. Following thawing, larvae inoculated in each samplewere recollected, promptly moved to saline, and analyzed. Twentylive larvae in saline stored at 4 �C were set as the control at everystorage time to verify that larvae stored at 4 �C were still alive.

Saltingwas carried out by immersing live larvae isolated from theviscera of conger eels into a salt solution (brine). Live larvae weresubmerged in salt solutions of 5,10,15, or 20%NaCl at 4 �C (20 larvaeper solution). Generally, 15e20% NaCl is used for preparation of salt-fermented seafood. Presently, low salt products are manufacturedbecause of health concerns, which is why we chose our saltingconditions. Storage times were 3 h, 6 h, 12 h, and 1e7 days. Aftertreatment, larvaewere immediatelymoved into saline and analyzed.

2.4. Combined treatment with ultrasound and chlorine

Live larvae isolated fromviscera of conger eels were treatedwith25 ml of 500, 1000, 1500, and 2000 ppm chlorine solutions insideglass tubes for 5 min (30 larvae per solution). Chlorine in the formof sodium hypochlorite (NaOCl, 12%, Shimadzu Co., Kyoto, Japan)was used in this study due to its wide use in processing plants ofvarious foods. Shortly after chlorine treatment, the larvae weremoved into 25 ml tap water inside glass tubes and treated by ul-trasound (Elmasonic P 300 Hmodel, 230 V, Germany) at 37 kHz and1200 W for 5, 10, 15, 20, and 30 min. The treated larvae weretransferred to saline and analyzed. Similarly, the viscera of congereels naturally infected with live larvae were treated with a com-bination of chlorine and ultrasound. The viscera from conger eelswere submerged in 300 ml of 500, 1000, 1500, and 2000 ppmchlorine solutions inside zipper bags for 5 min (3 viscera per bag);the samples were transferred to 300ml tap water places in a zipperbag and treated by ultrasound at 37 kHz and 1200 W for 5, 10, 15,20, and 30 min. Following treatment, larvae were collected, trans-ferred to saline, and their movement was observed.

2.5. Measurement of mobility, recovery rate, and viability

All experiments were carried out in triplicate. Live larvaerecollected from each sample in saline were counted, the recoveryrate was calculated, and the larvae were stored at 37 �C for 10 min.In the absence of spontaneous movement, larvae in saline wereremoved from the 37 �C incubator and gently touched with a sharpwooden stick approximately 5 times to observe movement. Ifmovement of the larvae was not observed shortly after touching,the larvae were again stored at 37 �C for 10 min, and the experi-ment was repeated. All larvae that showed movement eitherspontaneously or by the stimulating tool were recorded as “live.” Ifthere was no movement after being stimulated 5 times, the larvawas recorded as “dead.” Following the observation, viability wascalculated. Values of recovery rate and viability were shown as %.

3. Results and discussion

3.1. Viability of Anisakis larvae after freezing in squid and pollocktripe

The viabilities of the larvae collected after freezing at �20 �Cand �40 �C are presented in Table 1. The average recovery rates ofthe larvae inoculated in squid and pollock tripe were 94.4% and95.2%, respectively. At 4 �C, 100% viability was observed after 6 he14 days, and 75% viability was observed after 21 days. Gibson (1970)stated that 75% of Anisakis larvae showedmovement after 20 days ofstorage at 4 �C. All larvae inoculated in squid and pollock tripe wereinactivated after 48 h at �20 �C and 24 h at �40 �C. Although theinactivation times of larvae inoculated in squid and pollock tripe

were the same, there were differences in viabilities of the larvae insquid and pollock tripe. In�20 �C freezer, the viabilities of the larvaein squid were 55.8, 9.1, and 3.9% after 6 h, 12 h, and 1 day, respec-tively, whereas the viabilities of the larvae in pollock tripewere 53.6,9.2, and 1.7% after 6 h, 12 h, and 1 day, respectively. Similarly,at �40 �C, the viabilities of the larvae in squid were 28.0 and 3.5%after 6 h and 12 h, respectively, whereas the viabilities of larvae inpollock tripe were 26.7 and 1.7% after 6 h and 12 h, respectively.

Our results indicate that larvae viabilities in squid were slightlyhigher than those in pollock tripe. These differences may be due todifferences between the two samples including the thickness,weight, size, and existent portion of the larvae. Dailey (1975) re-ported that freezing temperature, freezing time, and sample size arevery important factors for killing Anisakis larvae; the United StatesFood and Drug Administration recommends�35 �C (�31 �F) for 15 hor �20 �C (�4 �F) for 7 days in a blast freezer. However, Deardorff,Raybourne, and Desowitz (1984) stated that Hawaiian snappersmust be stored at�20 �C for 1 day, and imported rockfishes (Sebastes.spp.) in the round must be stored at�20 �C for 5 days. Most herringlarvaehave also been reported to be inactivatedwithin thefirstweekof freezing at �30 �C for 16 h followed by storage at �12 �C(Gustafson, 1953). Therefore, there are many mitigating factors thatcan affect the FDA recommendations; the differences in freezingconditions necessary to kill the larvae depend on fish species.

3.2. Viability of Anisakis larvae after treatment with salt solution(brine)

The viability of larvae in salt solution as a function of storagetime is shown in Fig. 2. The viabilities of the larvae were 81.7% in 5%NaCl and 26.7% in 10% NaCl after 7 days of storage. After 7 days, alllarvae were inactivated when submerged in 15% NaCl, while inac-tivation took, 6 days in 20% NaCl. After storage for 3e12 h at eachconcentration of salt solution, most of the larvae were alive. Intypical processing of salt-fermented squid and pollock tripe, 15e20% salt is usually added to the rawmaterial, and the salting periodis between 1 and 2 days. Using these concentrations as guidelines,we incubated larvae in 15% or 20% NaCl; after 2 days, the viability ofAnisakiswas reduced approximately 20% and 50%, respectively. Thisstudywas performed in brine; in cases where raw seafood productsare naturally infected with live larvae, the effect of salting oninactivation of the larvae might be less significant. The diffusion of

S.-R. Oh et al. / Food Control 40 (2014) 46e49 49

salt into samples is affected by several factors. Salting conditions forkilling larvae depend on the type of seafood, the salting period, thethickness and size of sample, and the concentration of the salt so-lution. Rodrigues (2006, p. 205) reported that the number of liveparasites in fresh cod (Gadus spp.) is greater than that in salted codsince parasites are removed by washing with salt water and inac-tivated by salting. Most larvae in raw salt-fermented seafood can beinactivated by freezing before salting during the manufacturingprocess of products. However, specific salting conditions for killingparasite larvae may be necessary for promoting the safety of salt-fermented seafood products.

3.3. Viability of Anisakis larvae after combined treatment withultrasound and chlorine

Viabilities of in test tube larvae after a single treatment withchlorine or ultrasound and a combined treatment with both chlo-rine and ultrasound are shown in Table 2. When 500e2000 ppmchlorine was used in test tube for 5 min on live larvae, there was noeffect on reduction of larvae viability. On the other hand, ultra-sound treatment at 37 kHz and 1200W for 30 min or 40 min led toviabilities of 5.6% and 0%, respectively (data for the 40 min treat-ment time is not shown in Table 2). When live larvae in test tubeunderwent ultrasound treatment for 15e20 min, viability wasreduced rapidly from 43.3% to 13.3%. Ultrasound treatment wasshown to destroy the larvae by increasing intracellular pressure(Lee et al., 2012). In the combined treatment with 1500 ppmchlorine and ultrasound for 30 min, 0% viability was observed.Although there was no reduction effect on viability of the larvaewhen chlorine alone was used for treatment, combined treatmentwith chlorine and ultrasound had a slight synergistic effect on thereduction of larval viability. Ultrasound allows chlorine moleculeson the surface of larvae to penetrate into the larval tissues, whichaffects survival. However, when the viscera from heavily parasit-ized conger eels were treated with chlorine and ultrasound, 84e100% viability was shown under all conditions of combined treat-ment (data not shown). Since larvae firmly adhere to the visceralmembrane, they are difficult to detach and destroy by ultrasound,suggesting that the combined treatment of chlorine and ultrasoundon raw salt-fermented seafood including squid and pollock tripe isinadequate for removal and killing of larvae during washing.

4. Conclusions

The FDA recommends freezing times and temperatures to killAnisakis larvae in raw and undercooked seafood for seafood safety.However, the recommendations are quite limited because seafoodis very diverse, and specific conditions for inactivation the larvaecan vary depending on the kind of seafood. This study providesmore specific guidelines for manufacture of salt-fermented squidand pollock tripe with regard to freezing and salting conditions inorder to inactivate the larvae. Freezing at �20 �C for 48 h or �40 �C

Table 2Viability of Anisakis larvae after combined treatment with chlorine and ultrasound.

Concentration ofchlorine (ppm)

Treatment time of ultrasound (min)

0 5 10 15 20 30

0 100a 75.6 58.9 43.3 13.3 5.6500 100 58.9 47.8 34.4 6.7 3.31000 100 52.2 43.3 34.4 5.6 1.11500 100 51.1 41.1 33.3 5.6 02000 100 50.0 40.0 32.2 4.4 0

a Viability (%) of the larvae after the treatment, and average of 3 viability values.

for 24 h and salting in 15% NaCl for 7 days may be a promisingmeans to inactivate or reduce the larvae on raw salt-fermentedsquid and pollock tripe in seafood processing plants. Recently, theconsumption of seafood products and raw fish has increasedconsistently. For this reason, it is recommended that more studieson inactivation of Anisakis larvae as well as other parasites invarious seafood products are conducted.

Acknowledgment

This research was supported by a 2012 grant (12162KFDA012)from the Korea Food & Drug Administration for studies on haz-ardous microbes and microbiological safety management ofseafood.

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