BENHA VETERINARY MEDICAL JOURNAL, VOL. 34, NO. 2: 234-245, JUNE 2018

BENHA UNIVERSITY FACULTY OF VETERINARY MEDICINE

Effect of Marination on Vibrio Parahaemolyticus in Fillets

Hemmat M. Ibrahim, M. 1, Reham A. Amin1, Nesreen Eleiwa, Z.2, Hanan R. M. Ghanaym, 2 1 Food Hygiene Department, Faculty of Veterinary Medicine, Benha University ,2 Food Control Department, Animal Health Research Institute, Tanta Branch.

A B S T R A C T

Foodborne diseases affect millions of people each year. To reduce the incidence of bacterial foodborne pathogens such as V. parahaemolyticus, more effective treatment methods are needed. Accordingly, the effect of marinating fish fillets with lemon juice 50% , thyme powder 4g/kg and peel extract (PPE) (1% v/w) against V. parahaemolyticus were studied . The prepared marinades were artificially added to fish samples inoculated with V. parahaemolyticus then hold in refrigerator at 4oC and examined every day. Results indicated that the lemon juice decreased the count of V. parahaemolyticus from log 8.99.±0.02 cfu/g to log ≤1 cfu/g at fourth day by % 100, while thyme powder reduced the count to 4.86±0.51 log cfu/g at fifth day by reduction % 88.5. Finally, PPE decreased the count to log 4.89±0.38 cfu/g at fifth day by reduction % 76.3 . Thus, lemon juice was the most effective agent against V. parahaemolyticus as killing all the within four days of incubation. Keywords: Vibrio parahaemolyticus., marination, lemon, thyme, Pomegranate

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1.INTRODUCTION: chemicals are considered as carcinogenic Fish and constitute an and may be dangerous for public health. In important food component for a large section order to prevent the occurrence of this of world population. They come after carcinogenic substances in fish meat, it can be and as stable animal protein food processed with organic acids and either where fish forms a cheap source of protein during at reduced cooking tempera- (Bakr et al., 2011). tures or without any heat application. This Long cooking periods ( and ) widely accepted hygienic preservation method can cause the formation of is known as marination (Collignana et tricyclic amines in the foodstuffs. These al.,2001)

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Effect of Marination on Vibrio Parahaemolyticus in Tilapia Fillets

The marinating process is one of the Pomegranate was reported in numerous oldest methods of preservation of fish and studies to have excellent antifungal, other sea products (Giuffrida et al., 2007). antiprotozoal, antioxidant, anticarcinogenic, Marinades are solutions, including sugar, anti-inflammatory and antibacterial properties , oil, acids (from , fruit juice, (Wang et al., 2011). Pomegranate (Punica wine) and they are used to improve tenderness, granatum L.) is known as one of the healthiest juiciness, flavour and aroma and to extend fruits due to its high phenolic content and shelf life of seafood, meat, poultry and antioxidant activity (Tabaraki et al., 2012). vegetables (Cadun et al., 2005). Pomegranate have been reported in Marinated fish are typically inspired many studies that have antimicrobial activity as ready-to-eat products with no heat treatment against a wide range of microorganisms (Gram and Huss, 1996). Marinades are semi- including Gram positive and Gram-negative preserves; the preserving principal is the bacteria (Naz et al., 2007). combination of acetic acid and salt. The The present work was planned out to inhibitory effects of these substances on study the effect of different marinades (lemon bacteria and enzymes increase with juice, thyme powder and pomegranate peel concentration. The aim is not only to hold up extract) on safety and shelf life of tilapia fillets the action of bacteria and enzymes, but also to samples artificially inoculated with Vibrio tenderize or to change the taste, textural and parahaemolyticus. structural properties of raw material, resulting in a product with a characteristic flavour and an extended shelf life. Marinades are stored at 2. MATERIAL AND METHODS: cooler temperatures (4-6°C) keep for a long 2.1. Preparation of bacterial strain: - time (Gokoglu et al., 2004). Vibrio parahaemolyticus Lemon juice was very effective in (NCTC10885) pure strain was obtained from inhibiting Vibrio, Enterobacter, Citrobacter the Food Microbiology Laboratory, Animal spp. and E. coli (Jayana et al., 2010). Lemon Health Research Institute, Dokki, Egypt. The essential oils also showed antioxidant activity strain was maintained on tryptic soy agar (Kim et al., 2008). slants contain 3% NaCl at 40C. Loopful of Thyme oil is a tonic stimulant and Vibrio parahaemolyticus was transferred stomachic and digestive relieves gastritis, aseptically into 10 ml of sterile alkaline enterocolitis and mouth thrush. It is useful for peptone water with3% NaCl and then respiratory infections, asthma and bronchitis. incubated at 37oC for 24hrs.After that Vibrio It is effective for treating swelling provoked parahaemolyticus was counted by using by gout or rheumatic problems, for joint pains, spread plate method (FDA, 2001) to determine backache and sciatica. Thyme oil is also useful the concentration. The count was adjusted to for urinary and vaginal infections, 107cfu/ml (Shirazinejad and Ismail, 2010) with endometritis (candida), prostrates and tube dilution methods. The number of cfu/ml vaginitis (Ratan, 2006). Thyme oil exhibits was considered as infective dose to be antibacterial activity and has been useful in inoculated into fish fillet samples. dental practice.

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2.2. Preparation of fish fillet Samples: al., 2004). The contaminated samples were o A grand total of 1Kg of fresh Tilapia fillets stored at ambient temperature (30±2 C). V. were purchased directly from local markets in parahaemolyticus count in the sample was Tanta, Gharbia governorate, Egypt. The enumerated to get the initial load before samples were taken and transferred directly to addition of marinades (Terzi and Gucukoglu, the laboratory in an ice box under complete 2010). aseptic conditions without delay. The samples 2.5. Challenge trials: were cut into pieces and divided into five equal groups (200g of each) and placed in The samples were divided into five aseptic polypropylene trays designed for groups. Group one (control –ve) in which the disposable food packaging. samples were immersed in 300ml distilled water only. Group two (control +ve) the 2.3. Preparation of Marinades: samples were inoculated with107cfu/ml V. Marined consists of mixture of (100ml parahaemolyticus strain only and immersed in fresh lemon juice, 5.9g powder, 9.23g 300ml distilled water. Group three the samples 7 table salt,0.5g , 0.15g hot chili were inoculated with10 cfu/ml V. powder, 0.5g and 200ml distilled parahaemolyticus strain and immersed in water) were mixed well according to Siavash 300ml of marinade I at a ratio of 1.5:1 et al. (2016). (marinade: fish fillet, ml/g). For group four, the samples were inoculated with107cfu/ml V. Marined II as Marined I but addition of parahaemolyticus strain and immersed in thyme powder 4g/kg instead of lemon juice 300ml of marinade II at a ratio of 1.5:1 according to Daniela Istrati et al (2011). (marinade: fish fillet, ml/g). For group five, Marined III as Marined I but addition of the samples were inoculated with107cfu/ml V. pomegranate peel extract (PPE) (1% v/w) parahaemolyticus strain and immersed in supplied from National Research Center, 300ml of marinade III at a ratio of 1.5:1 instead of lemon juice according to Unalan et (marinade: fish fillet, ml/g). Marinades al., (2011). covered all surface of tilapia fillets samples. The tilapia fillets samples were gently agitated or pressed with a sterile spatula to ensure 2.4. Artificial contamination of fish fillet immersion in the marinade. Care was taken to samples with Vibrio parahaemolyticus: ensure that the solid components of the The tilapia fillets samples (200g) were marinades were distributed as evenly as inoculated with V. parahaemolyticus possible both within and between trays. 7 10 cfu/ml (Shirazinejad and Ismail, 2010) by All the trays were properly labelled, pipetting the inoculum drop by drop as evenly stored at 4oC and examined every day for as possible across the samples and mixed well sensory analysis (colour, odour and texture) with sterile glass rod for distribution of the according to (Hemin, 2013) and Vibrio inoculum and gentle rocking the samples parahaemolyticus count until spoilage of the immediately after inoculation. The inoculated examined samples occurred, according to tilapia fillets samples were left for 30 min. at (FDA, 2001). All the above experiment was room temperature to allow attachment and performed in triplicate (three times). absorption of the inoculated bacteria (Dubal et

536 Effect of Marination on Vibrio Parahaemolyticus in Tilapia Fillets

2.6. Sensory examination (colour, odour and the storage period at 4oC, respectively. While, texture): - in case of using thyme powder the scores It was conducted during storage was5; 4.33±0.33; 3.66±0.33; 3.33±0.33; according to Hemin (2013). 2.83±0.16; 2.50±0.50and1.66±0.33at the same period of storage, respectively. In case of 2.7. Bacteriological Analysis: using PPE, the score was 5; 3.66±0.33; Ten grams from each sample were 3.33±0.33; 2.66±0.33; 1.66±0.33and taken under aseptic condition to sterile 1.33±0.33 at the same period of storage, homogenizer containing 90 ml peptone water respectively. Comparing with the score of with 3% NaCl then the contents were overall acceptability in the control sample st homogenized at 4000rpm for 2.5 min. after which was 5, 3and 1 after zero day, 1 day and nd o that the mixture was allowed to stand for 6 2 day of the storage period at 4 C min. at room temperature under aseptic respectively. condition. The content of the flask was Table (2) revealed V. parahaemolyticus thoroughly mixed and 1ml was transferred into counts (log cfu/g) in tilapia fillet samples separated tubes containing 9ml of sterile treated with different marinades. The initial alkaline peptone water with3% NaCl, from counts of V. parahaemolyticus in tilapia fillet -10 which tenfold serial dilutions up to 10 were samples was 8.99± 0.02 log cfu/g. Such count prepared. From the prepared samples 0.1 ml of V. parahaemolyticus slightly decreased to from each prepared serial dilutions were 6.78±0.78; 7.94±0.53 and 8.37±0.11 log cfu/g spread plated with a glass spreader on respectively, after treatment with lemon juice, Thiosulfate citrate bile and sucrose agar thyme powder and PPE, respectively. o (TCBS) then incubated at 37 C for 24hrs Comparing with V. parahaemolyticus count in (Thatcher and Clark, 1978). The rounded the control-ve and +ve samples which were colonies 2-3mm in diameter, with green and 3.28. ±1.76 and 9.14±0.49 log cfu/g, /or blue centers were counted and expressed as respectively. On the 1st day at refrigeration colony forming units (log cfu/g). storage (4±1˚C) the mean value of V. 2.8. Statistical analysis: parahaemolyticcus in control –ve and +ve samples was 2.9±1.45 and 9.58±0.34 log The data was statistically treated by cfu/g, respectively. The mean value of V. one-way ANOVA using SPSS program for parahaemolyticus were 4.33±0.33; 6.54±0.53 windows (Version 17) (SPSS Inc. Chicago, IL and 6.99±0.19 log cfu/g, respectively. On the and USA) and Duncan’s post hoc test with p < 2nd day the mean counts of V. 0.05 considered to be statistically significant parahaemolyticus were 3.30±0.30; 6.19±0.41 (Knapp and Miller 1992). and 6.31±0.17 log cfu/g, respectively. On the 3rd day the mean value of V. 3.RESULTS: parahaemolyticcus were 3.20±0.20; 5.03±0.22 and 5.40±0.11 log cfu/g, respectively. On the The results obtained in table (1) showed 4th day the mean counts of V. that the scores of overall acceptability in case parahaemolyticus were not detected; of using lemon juice 4.86±0.51 and 4.89±0.38 log cfu/g, 5;4.66±0.33;4.33±0.33;3.66± 0.33;3.33±0.33; th st respectively. By the 5 day the mean value of 3.16±0.16 and2.66±0.33 at zero day, 1 day, V. parahaemolyticus were not detected; 2nd day, 3rd day, 4th day, 5th day and 6th day of

537 Ibrahim et al., 2018 (BVMJ-34(2): 234-245

1.03±0.37 and 2.13±0.81log cfu/g for samples reduced the growth of V. parahaemolyticus treated with lemon juice, thyme powder and by11.67%; 27.20%; 31.14%; 44.04%; 45.93% PPE, respectively. and 88.50% after zero day, 1st day, 2nd day, rd th th Table (3) showed that the reduction 3 day, 4 day, 5 day of refrigerated storage, percent of V. parahaemolyticus in tilapia fillet respectively. Meanwhile fish fillet samples were 24.58%; 51.83%; 63.29%; 64.40%; treated with PPE recorded reduction in V. 100% and 100% during refrigerated storage, parahaemolyticus by 6.80%; 22.24%; 22.90%; 39.45%; 45.60% and 76.30% after zero day, respectively in case of treatment with lemon st nd rd th th juice. While marinating with thyme powder 1 day, 2 day, 3 day,4 day ,5 day of refrigerated storage, respectively.

Table (1): The effect of different marinades (lemon, thyme and pomegranate peel extract) on overall acceptability of artificially inoculated fish fillet samples with V. parahaemolyticus during cold storage at 4 0C.

Group Marined1 Marined II Marined III Period Control (lemon juice) (Thyme (PPE) powder)

Zero day 5 5 5 5

1st day 3.33±0.43 4.66±0.33 4.33±0.33 3.66±0.33 2nd day - 4.33±0.33 3.66±0.33 3.33±0.33 3rd day - 3.66±0.33 3.33±0.33 2.66±0.33

4th day - 3.33±0.33 2.83±0.16 1.66±0.33

5th day - 3.16±0.16 2.50±0.50 1.33±0.33

6th day - 2.66±0.33 1.66±0.33 -

5 = very acceptable 4 = Acceptable 3 =Middle 2 = Unacceptable 1 = Rejected - =spoilage The values represent Mean ± SEM of three experiments.

538 Effect of Marination on Vibrio Parahaemolyticus in Tilapia Fillets

Table (2): The effects different marinades (lemon, thyme and pomegranate peel extract) on V. parahaemolyticus count (log cfu/g) artificially inoculated in fish fillet samples Groups Zero day 1st day 2nd day 3rd day 4 th day 5th day

Control -ve ab 2.9±1.45 - - - 3.28±1.76 -

ab 9.58±0.34 Control +ve 9.14±0.49 abc - - - -

a 4.33±0.33 b bc Marined1(lemon) 6.78±0.78 abc 3.3±0.3 3.2±0.2 ND ND

ab b b a Marined 2(Thyme) 7.94±0.11 6.54±0.53 6.19±0.41 5.03±0.22 4.86±0.51 1.03±0.37

a ab b c a Marined3(PPE) 8.37±0.53 6.99±0.19 6.31±0.17 5.40±0.11 4.89±0.38 2.13±0.81 Initial load of V. parahaemolyticus 8.99± 0.02 log cfu/g The values represent Mean ± SEM of three experiments. Means within a column followed by different letters are significantly different (P < 0.05). N.B.: (-) means samples rejected

(ND) means microorganism not detected.

Table (3): Reduction % of V. parahaemolyticus count artificially inoculated into fish fillet samples treated with different marinades (lemon, thyme and pomegranate peel extract) Zero th th Groups 1st day 2nd day 3rd day 4 day 5th day 6 day day Marined I (lemon juice) 24.58 100 - 51.83 63.29 64.4 100

Marined II (Thyme powder) 11.67 27.2 31.14 44.04 45.93 88.5 -

Marined III (PPE) - 22.24 22.9 39.45 76.3 6.8 45.6

N.B.: (-) means samples rejected.

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Furthermore, the obtained results 4. DISCUSSION: indicated that only marination by lemon juice combated against the artificially inoculated V. The demand for safe foods, coupled th with the preference by consumers for foods parahaemolyticus till the 6 day of the study free of synthetic additives, has increased the and revealed the highest improvement of interest for natural preservatives in sensory attributes, this is because lemon recent years (Moreir, et al.2007). contain organic acid which have antioxidative Improvements in the shelf-life of action, acid taste production, decrease of the product have an important economic impact protein content and the increase of the acidity by reducing losses attributed to spoilage and and the free amino-acid content give the marinated fillets a characteristic texture and by allowing the products to reach distant and new markets. In a broad sense the ultimate aroma. goal of sensory evaluation was to predict its So, this could be considered of highly acceptance. Sensory analysis has become importance indication and also a solution for popular marketing research as well as in the rapid decomposition of fresh fish and meat quality assurance and in research and products especially those preserved by development (Gouin, 2004). refrigeration While the sensory quality obtained by thyme powder indicated that shelf The results obtained in table (1) showed th that the scores of overall acceptability in case life prolonged to 5 day, this may be due to of using lemon juice was thyme is traditionally used as flavouring agents in fish and thyme essential oil contains 5;4.66±0.33;4.33±0.33;3.66 ±0.33;3.33± 0.33; 3.16±0.16 and 2.66±0.33 at many flavonoids, like apigenin, aringenin, zero day, 1st day, 2nd day, 3rd day, 4th day, 5th luteolin and thymonin (Penalever et al., 2005). day and 6th day of the storage period at 4 oC On the other hand, Fathy et al. (2015) respectively. While, in case of using thyme reported that thyme extract has strong powder the scores was5; 4.33±0.33; antimicrobial and antioxidant activity and can 3.66±0.33; 3.33±0.33; 2.83±0.16; 2.50±0.50 maintain the quality parameters and extend the and1.66±0.33 at the same period of storage, shelf life of refrigerated Nile tilapia fillet for 9 respectively. days longer than control one. Moreover, concerning PPE, the score While, fish fillet group treated with was 5; 3.66±0.33; 3.33±0.33; 2.66±0.33; PPE showed somewhat acceptable 1.66±0.33 and 1.33±0.33 at the same period of improvement of sensorial characters and storage, respectively. Comparing with the extended the shelf life till the 4th day score of overall acceptability in the control compared with control group during storage sample which was 5, 3and 1 after zero day, 1st period. nd o day and 2 day of the storage period at 4 C, Generally, sensorial characters of respectively. From the obtained results there different treated tilapia fillet samples during was a decline of sensorial characters after the cold storage (4ºC) were improved by using first day of storage with clear reduction of lemon juice then thyme powder and at least overall acceptability values in the control nd PPE as compared with the control ones during samples and showed complete spoilage at 2 the same storage period. day of the storage period at 4oC.

542 Effect of Marination on Vibrio Parahaemolyticus in Tilapia Fillets

Spoilage characteristics develop in who found that reduction levels of V. food as microorganisms digest the sugars, parahaemolyticus inoculated into tilapia complex carbohydrates, proteins and fats of (Oreochromis niloticus) fillet samples and food producing undesirable effects in the food marinated with lemon juice and incubated 4°C if the spoilage microorganisms grow to for 30 or 120 min from >7 log cfu/g to 5 log significant levels. Typically, the threshold cfu/g. level for observation of food spoilage by By the 1st day at refrigeration storage odour, taste or sight is not reached until the 7 (4±1˚C) the mean value of V. spoilage microflora exceeds about 10 parahaemolyticcus in control –ve and +ve organisms/g of food. Fish microflora includes samples was 2.9±1.45 and 9.58±0.34 log bacterial species such as Vibrio species (Gram cfu/g, respectively. The mean value of V. and Huss, 2000). Microbial growth and parahaemolyticcus were 4.33±0.33 metabolism is a major cause of fish spoilage ;6.54±0.53and6.99±0.19 log cfu/g for samples which produce amines, biogenic amines such treated with lemon juice, thyme powder and as putrescine, histamine and cadaverine, PPE, respectively, the means within a column organic acids, sulphides, alcohols, aldehydes followed by different letters are significantly and ketones with unpleasant and unacceptable different (P < 0.05). off-flavours (Dalgaard et al., 2006). On the 2nd day of refrigeration storage Many extracts and essential oils have at (4±1 0C) of treated tilapia fillets samples, been derived from plants and found to have the mean counts of V. parahaemolyticus were antibacterial, fungicidal and insecticidal 3.30±0.30; 6.19±0.41 and 6.31±0.17 log cfu/g properties (Hänsel et al., 1999). respectively, as well as they were acceptable Table (2) revealed V. from aesthetic points without off odour or parahaemolyticus counts (log cfu/g) in tilapia discoloration. compared with the control –ve fillet samples treated with different marinades. and +ve. samples which showed extreme The present data exhibited the potential of discoloration and off-odour in this day of marinades as natural food preservative against storage indicating complete spoilage. V. parahaemolyticus in tilapia fillet samples. By the 3rdday of refrigeration storage The initial counts of V. parahaemolyticus in of treated tilapia fillets samples with different tilapia fillet samples was 8.99. ±0.02 log cfu/g. marinades (lemon juice, thyme powder and Such count of V. parahaemolyticcus slightly PPE), the mean value of V. parahaemolyticcus decreased to 6.78±0.78; 7.94±0.53 and were 3.20±0.20 ;5.03±0.22and 5.40±0.11 log 8.37±0.11 log cfu/g respectively, after cfu/g for samples treated with lemon juice, treatment with lemon juice, thyme powder and thyme powder and PPE, respectively, the PPE, respectively. Comparing with V. means within a column followed by different parahaemolyticus count in the control -ve and letters are significantly different (P < 0.05). +ve samples which were 3.28. ±1.76 and At the 4th day of refrigeration storage 9.14±0.49 log cfu/g, respectively. The means at (4±1 0C) of treated tilapia fillets samples the within a column followed by different letters mean counts of V. parahaemolyticus were not are significantly different (P < 0.05). detected; 4.86±0.51 and 4.89±0.38 log cfu/g, These finding was nearly similar to respectively. that reported by Prateek and Donald (2013)

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By the 5thday of refrigeration storage 3rd day,4th day ,5th day of refrigerated storage, of treated tilapia fillets samples the mean respectively. value of V. parahaemolyticus were not Meanwhile, fish fillet samples treated detected; 1.03±0.37 and 2.13±0.81log cfu/g with PPE recorded reduction in V. for samples treated with lemon juice, thyme parahaemolyticus by 6.80% ; 22.24%; powder and PPE, respectively. The means 22.90%; 39.45%; 45.60% and 76.30% after within a column followed by different letters zero day, 1st day, 2nd day, 3rd day,4th day ,5th are significantly different (P < 0.05). day of refrigerated storage, respectively. Also, at the six day the treated So, the present results showed that samples with lemon juice become yellow in marinating with lemon juice showed colour while samples treated with thyme maximum activity against V. powder and PPE became darken showed parahaemolyticus compared with the control extreme discoloration, off-odor indicating on fifth day of refrigerated storage at 40C. complete spoilage. In this study, the information given by These results were nearly similar to the obtained results revealed that using those reported by Daniela et al., (2011) who marinating by lemon juice on tilapia fillets can found that marinating by lemon enhanced improve its safety and extended its shelf life shelf life at 40C by preventing protein by enhancing sensory characters and due to degradation and delay microbial spoilage. their antibacterial effect against Gram negative The results showed growth of V. bacteria especially Vibrio species. parahaemolyticus in marinated samples. As illustrated in table (3). lemon juice reduced the 5. REFERENCES: growth of V. parahaemolyticus by 24.58%; 51.83%; 63.29%; 64.40%; 100% and 100% Aibinu, I.; Adenipekun, T.; Adelowotan, T.; during refrigerated storage, respectively. Ogunsanya, T. and Odugbemi, T. (2006): Evaluation of the Lemon contain high concentrations of antimicrobial properties of different citric acid, the major organic acid present in parts of Citrus aurantifolia (lime these juices, which is partly responsible for the fruit) as used locally. Afr. J. antibacterial activity of these fruits due to Traditional, Complementary Altern. contain a group of flavonoids including Med., 4: 185-190. polymethoxy flavones, flavone glycosides and Bakr-Wafaa, M. K.; Hazzah-Walaa,A.and limonoids which enhance antimicrobial Abaza-Amani, F. (2011):Detection of activity (Ladaniya, 2008). Salmonella and Vibrio species in Another study in Nigeria also showed some seafood in Alexandria. J. that lemon juice (both aqueous and ethanol American Sci., 7(9): 663-668. extracts) killed most gram positive and gram- Burt, S. (2004): Essential oils: their negative bacteria at a concentration of 256 antibacterial properties and potential mg/mL (Aibinu et al., 2006). applications in foods-a review. Inter J While, marinating with thyme powder Food Microbiol; 94:223-53. reduced the growth of V. parahaemolyticus Cadun, A.S.; Cakli, D. and Kisla, D. (2005): by11.67%; 27.20%; 31.14%; 44.04%; 45.93% A study of marination of deep water and 88.50% after zero day, 1st day, 2nd day, pink (Parapenaeus

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