SURFACE CONTAMINANTS OF SOME FISH PRODUCTS

SURFACE CONTAMINANTS OF SOME FISH PRODUCTS

Hafez, A.E.;Rasha M.El-Bayomi and NohaE.A.Badr

Food Control Department, Faculty of Veterinary Medicine,

Zagazig University, Zagazig 44519, Egypt.

Key Words: Fish, Cumin, Rosemary, Chitosan.

ABSTRACT

Fish is considered one of the most nutritive and highly desirable foodstuffs as fish meat has excellent nutritional value being rich in proteins, vitamins and unsaturated fatty acids. However, this high nutritional value, fish was incriminated as a cause of food poisoning, food intoxication, allergic and skin disorders as well as other many infectious diseases as a result of rapid deterioration. Thus, the present study was planned to throw a light on the quality of fresh fish sold at Zagazig markets and to investigate the effect of some natural additives on the quality and shelf life. Results revealed contamination of the examined fish by of enterococcus, Psychrotrophicand pseudomonas as well as different species of salmonella.Furthermore, treatment of fish by cumin oil 1%, rosemary and chitosan, separately, was effective method in fish preservation and shelflife extension during the cold storage. Chitosan was the best additive compared with cumin and rosemary.

1. INTRODUCTION

Nowadays, fish constitute one of the most important food stuffs; it provides the man with the animal protein of high biological value besides their high palatability and good digestibility. Fish has long been regarded as nutrition and highly desirable food due to its contribution of high quality animal protein, its richness in phosphorus and generous supply of vitamins are low in saturated fats. Fish in their natural environment have their own micro-flora in the slime, on their body, in their gut and gills. These micro-organisms, as well as enzymes in the tissues of the fishes, bring about putrefactive changes in fish when died. Fish is subjected to many risks of contamination from different sources either during their aquatic environment, sewage pollution of harvesting areas and/or after being harvesting by workers, utensils and equipment during transportation, distribution and food preparation (El-Leboudi, 2002).Enterococci are of significance in food and clinical microbiology. They are everywhere microorganisms, and have a main habitat in the gastrointestinal tracts of humans and warm blooded animals (Giraffa, 2002).Psychrotrophic bacteria are able to grow relatively rapid at chilling temperature, room temperature and responsible for many undesirable changes in flavor, odor, texture and color of the refrigerated chicken meat (APHA, 2001).Pseudomonas species are Gram negative rods, nonsporing which are motile through polar flagellae and widely distribute in soil and water. Pseudomonas species may cause food spoilage and reported to have lipolytic and proteolytic activities (Carter et al., 1990).Salmonellosis is a worldwide problem responsible for food poisoning outbreaks but it is mainly difficult to determine whether the contamination of fishes occurred in their aqueous habitat or during their handling and marketing (Etzelet al., 1998).

Essential oils are aromatic and volatile oily liquids derived by plant material and served as flavoring agents with wide spectra of antimicrobial action (Aktharet al., 2014).Chitosan has been proved to be non-toxic, biocompatible and biodegradable and it has a broad-spectrum antimicrobial activity against both, gram-negative and gram-positive  bacteria as well as fungi (Yuan et al., 2016).Rosemary (Rosmarinusofficinalis L.) extracts are most active as natural antioxidants and antibacterials in seafood; it can reduce the microbial growth, keep the sensory characteristics, and extend the shelf-life of seafood during preservation(Pezeshket al., 2016).Cumin (CuminumcyminumL.) has characteristic flavor and strong odor due to its essential oils content that may be considered as a source of antibacterial, antifungal and antioxidant components, which are used in food preservation. Its main constituent and important aroma compound is cuminaldehyd(Hajlaouiet al., 2010).

Therefore, the present study was planned to throw a light on the quality of some fresh fish sold at Zagazig markets and to investigate the effect of some natural additives on the quality through following points.

2. MATERIALS AND METHOD

Part I

2.1.Collection of samples:

A total of 120 random samples of fish (Nile Tilapia, Blue Runner, Bagrus, Cat Fish, Mullet, and Striped Red Mullet), 20 of each, were randomly collected from supermarkets and shops from Sharkia Governorate, Egypt. The collected samples were transferred to the Food Control laboratory, Faculty of Veterinary Medicine, Zagazig University.

2.2 Preparation of fish samples:

It was carried out according to ICMSF (1978).

2.3.Determination of Enterococci count:

It was carried out on a bile esculin ager (ISO, 2000)

2.4.Determination of total psychrotrophic count:

 On standard plate count agar (APHA, 2002)

2.5.Enumeration of Pseudomonas species:

On Pseudomonas isolation agar (Kreig and Holt, 1984)

2.6.Isolation and identification of Salmonellae(ISO, 2002):

 Serological identification of Salmonellae:according to Kauffman – White scheme (Kauffman, 1974) for the determination of Somatic (O) and flagellar (H) antigens

Polymerase chain reaction (PCR) identification of Salmonellae:

Primer sequences of Salmonellae used for PCR system:

The primers for detection of virulence factors including invasion A (invA)(Shanmugasamyet al., 2011), hyper-invasive locus (hilA) (Guoet al., 2000) and fimbrial (fimH) genes (Menghistu, 2010)of Salmonella species.DNA preparation from bacterial culture (Shahet al.,2009).DNA amplification of Salmonella (Singh et al., 2013).

Part II:Trials to improve fish quality by some natural additives:

Selected additives:

Cumin oil 1% (Cuminumcyminum L.), Rosemary (Rosmarinusofficinalis L.) and Chitosan.These additives were obtained from National Research Center, Dokki, Giza.

Design of the experiment:

Sampling:

Nile tilapia fishwere collected from differentfish markets in ZagazigCity.The samples were taken and transferred directly to the laboratory using an ice box under complete aseptic conditions without undue delay for preparation of tilapia fillets.

The experiment:

In the laboratory, tilapia fillets were divided into four equal groups; control group without additives, cumin oil 1% treated group, rosemary treated group and chitosan treated group.  All the groups were sampled immediately after treatment (zero time) and every 48 hours. All groups were kept in fridge at 4±1°C. Bacteriological examination (Enterococci, Psychrotrophic) was conducted as mentioned in part I of study.

Statistical analysis:

        Statistical analysis of data was done by using the statistical package for social sciences (SPSS Inc.; Chicago, IL, USA) software. One Way ANOVA at 95% level of confidence was done and differences among individual means were compared by Duncan Multiple Range test.

3. RESULT AND DISCUSSION

3.1.Bacteriological evaluation of the examined fish samples:

Enterococci are a group of bacteria that are a part of the normal intestinal flora. Enterococci are used as fecal indicator bacteria indicate fecal contamination of fish.

Table (1): Statistical analytical results of bacteriological examination in surface of the examined fissh samples (log₁₀ cfu/g)

No: number of examined samples (10 of each fish), cfu/g: Colony forming unit per gram

S.E: Standard error of mean

 Means within the same column with different superscript letters are significantly different (P< 0.05).

Results illustrated in table (1) revealed that, the mean counts of enterococci in surfaces of the examined Nile tilapia, blue runner, bagrus, cat fish, mullet and striped red mullet samples was 3.3 ± 0.2897, 3.6 ± 0.1946, 4.7 ± 0.0557, 3.4 ± 0.1944, 3.7 ± 0.3069 and 3± 0.1909 Log₁₀ CFU/ g, respectively. It was found that the external surface of bagrus samples had the highest count of enterococci compared with other fish species, while the external striped red mullet samples had the lowest count of enterococci. High significance difference at P< 0.05 was detected between the examined samples without individual variations between Nile tilapia, blue runner, catfish and mullet.

The count of enterococci in muscles of the examined Nile tilapia, blue runner, bagrus, catfish, mullet and striped red mullet samples ranged from 2 to 4.3, 2 to 4, 2.5 to 5, 3 to 4.3 and 3 to 5.5 Log₁₀ CFU/ g, respectively with mean counts of 3.1 ± 0.2520, 2.9 ± 0.2084, 3.6 ± 0.2168, 3.7 ± 0.1574, 4.4 ± 0.2961 and 3.8 ± 0.1208 Log₁₀ CFU/ g, respectively. (Table 2). It was found that the counts of enterococci varied between the different species of fish. Muscles of the examined blue runner fish had the lowest count of enterococci compared with other samples, while the examined mullet samples had the highest count of enterococci; this result attributes to the high fat content of mullet than other species.

Table (2): Statistical analytical results of bacteriological examination in muscles of the examined fish samples (log₁₀ cfu/g)

No: number of examined samples (10 of each fish), cfu/g: Colony forming unit per gram

S.E: Standard error of mean

Means within the same column with different superscript letters are significantly different (P< 0.05)

High significance difference at P< 0.05 was detected between the examined samples.This result was in line with many other investigations which reported contamination of fish by different species of Enterococci;Abdel-Aziz et al.(2003) reported that Enterococcus reached 85%, 60%, 5% in extensively, semi intensively and intensively tilapia fish farms, respectively . While, Abd El-Sattar (2009) found that 31% of the examined fish samples were contaminated by enterococci; Khafagyet al. (2009) reported that23.76% ofthe examined Nile tilapia were positive forEnterococcus Faecalis; Hammadet al. (2014) isolated enterococci from 45% of the examined fish samples and Osman et al. (2016) found that 10% of the examined fish samples were contaminated by enterococci.Concerning to enterococci count, Lanet al. (2007)detected higher count of enterococci (4.1 Log₁₀ CFU/ g) in the examined tilapia fish samples. Meanwhile, lower count of enterococci (1.41 Log₁₀ CFU/ g) was reported by Boariet al. (2008).

As shown in table (1), psychrotrophic count in surfaces of the examined Nile tilapia, blue runner, bagrus, cat fish, mullet and striped red mullet samples ranged from 3.7 to 6.7, 4.5 to 6.8, 5.5 to 6.3, 4.5 to 6.6, 5.7 to 7.3 and 5.8 to 6.3 Log₁₀ CFU/ g, respectively.The highest count of psychrotrophic bacteria was detected in the surface of mullet samples, while, the lowest count of psychrotrophic bacteria was detected in the surface of Nile tilapia.  High significance difference at P< 0.05 was detected between the examined samples without individual variation between the different species.Meanwhile, the meanpsychrotrophic counts in muscles of the examined Nile tilapia, blue runner, bagrus, cat fish, mullet and striped red mullet samples were 4.9 ± 0.3459, 5.7 ± 0.1918, 6.2 ± 0.1126, 6.3 ± 0.1594, 6.8 ± 0.1093 and 6.4 ± 0.0840 Log₁₀ CFU/ g, respectively. The achieved results revealed that Nile tilapia had the lowest count of psychrotrophic bacteria compared with other fish species, while mullet samples had the highest count of psychrotrophic bacteria.  High significance difference at P< 0.05 was detected between the examined samples with some variations in the count of psychrotrophic bacteria between the different species of examined fish samples.Counts of psychrotrophic bacteria in fish muscles recorded in the present study differed from other investigations reported lower counts of psychrotrophic in the examined fish samples, conducted by Mansour and El-Shaboury (2009), Dumanet al. (2015) and Ibrahim et al. (2016), respectively.However, higher counts of psychrotrophic bacteria were reported by Mahmoud (1994) (5.8 Log₁₀ CFU/ g for tilapia, respectively).High psychrotrophic count in the examined fish samples may be attributed to cross contamination from environment around the fish, unsatisfactory sanitation during handling, processing and distribution as well as inadequate chilling and/or freezing which increase the count of psychrotrophic microorganisms.In addition to,equipment, workers, containers, boxes, as well as using polluted water during transportation play an important role for increase counts of psychrotrophic bacteria.

Results recorded in table (3) and figure (3) revealed that, the mean pseudomonas count was 5 ± 0.3015, 6 ± 0.1844, 6.7 ± 0.2238, 5.7 ± 0.1735, 6.2 ± 0.0645 and 5.5 ± 0.2798 Log₁₀ CFU/ g in the surface of the examined Nile tilapia, blue runner, bagrus, cat fish, mullet and striped red mullet samples, respectively. It was found that bagrus samples had the highest count of pseudomonas (6.7 Log₁₀ CFU/ g) compared with other fish species, while Nile tilapia had the lowest count of pseudomonas (5 Log₁₀ CFU/ g).

High significance difference at P< 0.05 was detected between the examined samples without individual variations between blue runner, cat fish, mullet and striped red mullet samples. Results recorded in table (2) revealed that pseudomonas count in the muscles of  the examined Nile tilapia, blue runner, bagrus, cat fish, mullet and striped red mullet samples ranged from 3 to 6.9, 4.8 to 6.5, 6 to 7.6, 4.5 to 6.3, 5.4 to 6.7 and 5.3 to 6.49 Log₁₀ CFU/ g, respectively.It was found that bagrus fish had the highest count of pseudomonas (6.8 Log₁₀ CFU/ g) compared with other fish species, while Nile tilapia had the lowest count of pseudomonas. High significance difference at P< 0.05 was detected between the examined samples but there were no individual variations between blue runner, cat fish, mullet and striped red mullet samples.This result was in accordance with El-Noby (2002) and Abd El-Aziz (2015).Lower counts of pseudomonas were recorded by Bahurmizet al. (2016),Khidhiret al.  (2014) and Arefet al. (2018) (3.1, 3.8 and 2.1 for cat fish Log₁₀ CFU/ g, respectively). But, higher count of pseudomonas was recorded by Begum et al. (2010) (5.9 Log₁₀ CFU/ g).High counts of Pseudomonas in the examined fish samples could be attributed to Pseudomonas spp. are widely distributed in nature, unsanitized equipment, pouted water and fishermen hands especially during harvesting, transportation and storage. In addition to, heavily contaminated boats and boxes which transfer the organisms to fish during cleaning.

Table (3): Incidence of Salmonella spp. in the examined fish samples

O: Somatic antigen, H: Flagellar antigen

As found in table (3)Salmonella spp. failed to be detected in the examined Nile tilapia, blue runner, cat fish and striped red mullet, while Salmonella spp. were isolated from the examined bagrus and mullet samples only with a prevalence 2 (10%) and 1 (5%), respectively.Serological identification of the isolated strains revealed that S. essen, S. enteritidisand S. saintpaulwere the isolated strains.The obtained results in this study agreed with other studies isolated Salmonella spp. from the examined fish samples; as Zhang et al.(2015) and Budiatiet al. (2016).Meanwhile, this result disagreed with Davies et al. (2001) who revealed that all examined fish samples were free from Salmonellae.Results in figure (1) illustrated that the isolated strains harbored virulence genes such as invasion A gene (invA),hyper-invasive locus gene(hilA)and fimbrial gene(fimH). Invasion A gene (invA) was detected in all identified Salmonella strains, while, hyper-invasive locus gene(hilA) was detected in S. enteritidisand S. saintpaul but fimbrial gene(fimH) was detected in S. enteritidisonly.

Figure (1):Agarose gel electrophoresis of multiplex PCR of invA (284 bp), hilA (497 bp) and fimH (1008 bp) virulence genesfor characterization of Salmonella species. Lane M: 100 bp ladder as molecular size DNA marker. Lane C+: Control positive strainforinvA, hilA and fimH genes. Lane C-: Control negative. Lane 1 (S. Essen): Positive strain for invA gene. Lane 2 (S. Enteritidis): Positive strainforinvA, hilA and fimH genes.Lane 3. S. Saintpaul): Positive strain for invAand hilA genes.

These detected genes play an important role in Salmonella pathogenicity.Invasion A gene (invA) is well understood and this gene contributes significantly to virulence factor of Salmonella Pathogenicity Island (SPI); the virulence factor due to invA gene is reported to be responsible for invasion of gut epithelial tissue in human and animals (Van Asten and Van Dijk, 2005). The fimH gene encodes the major structural subunit of type I fimbrial protein, while this gene has been implicated in Salmonella pathogenicity because it is responsible for bacteria binding (Boddickeret al., 2003). The virulence regulator hilA is a key gene for colonization, and thus up regulation of this gene can lead to enhanced colonization or invasion (Kumaret al., 2015).

3.2. Improving the hygienic quality of fish using Cumin oil 1%, Rosemary and Chitosan:

Results illustrated in figure (2) revealed that themean enterococci count was 3.6 ± 0.2961, 3.5 ± 0.1151, 3.4 ± 0.0872 and 2.9 ± 0.2962 Log₁₀ CFU/ g, while, psychrotrophic count was 6.8 ± 0.1227, 6.7 ± 0.0901, 6.7 ± 0.2655 and 6.6 ± 0.0739 Log₁₀ CFU/ g in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the beginning of the experiment(Figure 3). The mean count of enterococci was 3.9 ± 0.2962, 3.1± 0.1198, 2.5 ± 0.4067 and 2.5± 0.4067 Log₁₀ CFU/ g(Figure 2), while, the mean count of psychrotrophic bacteria was 5.8± 0.2672, 5.3± 0.1103, 5.2 ± 0.1392 and 5 ± 0.3010 Log₁₀ CFU/ g(Figure 3), in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the 3^rd day of chilling. Enterococci counts ranged from 3.8 to 4.5, 3 to 3.6, 2.5 to 3.3 and 2.6 to 3 Log₁₀ CFU/ g (Figure 2), while, psychrotrophic counts ranged from 6 to 6.2, 5.2 to 6.1, 4.7 to 5.5 and 4.5 to 5.7 Log₁₀ CFU/ g in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the 5^th day of chilling(Figure 3).As shown in figure (2) the mean counts of enterococci were 4.6± 0.1476, 3.8 ± 0.4637, 3.8 ± 0.3082 and 3.1 ± 0.3206Log₁₀ CFU/ g.

While, the mean count of psychrotrophic bacteria was 6.6 ± 0.3333, 6.3 ± 0.5312, 5.7 ± 0.2593 and 5.7 ± 0.2347 Log₁₀ CFU/ g in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the 7^th day of chilling (Figure 3). Results found in figure (2) illustrated that, the mean count of enterococci was5.3 ± 0.3010, 4.7 ± 0.2608, 4.1 ± 0.1834 and 4 ± 0.0523 Log₁₀ CFU/ g, while, the mean count of psychrotrophic was 6.9 ± 0.3117, 6.4 ± 0.4729, 6.4 ± 0.4507 and 6 ± 0.6226 Log₁₀ CFU/ g in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the 9^th day of chilling(figure 3).As recorded in figure (2) the enterococci count ranged from 6 to 6.6, 5 to 5.4, 4.7 to 5.3 and 4.6 to 5.3 Log₁₀ CFU/ g, while, psychrotrophic count ranged from 6.6 to 8.5, 5.8 to 8.3, 6 to 8.3 and 5.9 to 6.4 Log₁₀ CFU/ g in control and treated samples by cumin oil 1%, rosemary and chitosan, respectively at the 9^th day of chilling (figure3).The achieved results in this study revealed that treatment of fish samples by spices (cumin and rosemary) reduced the bacterial count and extended the shelf life of fish. Rosemary was effective than cumin in fish preservation. The antibacterial effect of cumin may be attributed to the cumin aldehyde (p-isopropilbenzaldehyde) (De et al., 2003), while antibacterial effect of rosemary may occur by two mechanisms: the delay and partial inhibition of DNA and proteins synthesis, and the induction of intracellular ATP depletion (Oussalah et al.,2006). The obtained results in this study were in accordance with Kenar et al. (2010) who reported that rosemary extracts has extended the shelf life of vacuum packed sardine fillets by 7 days when stored at 3±1^oC,and Singhet al. (2016) who reported that treatment with 8.5 ml rosemary oil/kg fish could effectively retard microbial growth, delay chemical deterioration, maintain or improve sensory attributes, and extend the shelf life of fish samples for 14 days during refrigerated storage. Chitosan was the most effective treatment that decreased the bacterial counts in the treated fish samples and extended their shelf life (chitosan > rosemary > cumin) . This result could be attributed to its superior film-forming properties, ability to adsorb nutrients used by bacteria, and capacity to bind water and inhibit various bacterial enzyme systems (Darmadji and Izumimoto, 1994). Chitosan acts mainly on the outer surface of the bacteria and interacts with the cell membrane to alter cell permeability (Rabea et al., 2003).This result matched with what had been reported by Cao et al.(2009) and Duanet al. (2010)who reported that 5 g/L chitosan extended the shelf life from 8–9 days to 14–15 days.

Figure (2): Effect of Cumin Oil 1%, Rosemary and Chitosan on Enterococcus counts (log₁₀ cfu/g) of chilled Nile Tilapia at zero, 3^rd, 5^th, 7^th, 9^th and 11^th day at 4±1^° C

Figure (3): Effect of Cumin Oil 1%, Rosemary and Chitosan on Psychrotrophic counts (log₁₀ cfu/g) of chilled Nile Tilapia at zero, 3^rd, 5^th, 7^th, 9^th and 11^th day at 4±1^° C

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التلوث السطحی لبعض منتجات الأسماک

عبدالسلام الدیدامونی حافظ ورشا محمد البیومی ونها البازعبد المحسن بدر

قسم مراقبة الأغذیة ، کلیة الطب البیطرى ، الزقازیق مصر.

تُعتبرالأسماک من أهم الأغذیة ذات القیمة الغذائیة العالیة فهی مصدر غنی بالبروتین والحموض الأمینیة اللازمة لجسم الإنسان. کما أنها أیضاً تحتوی العدید من الفیتامینات والأملاح المعدنیة کالفوسفور والکالسیوم وغیرها. ونظراً لسهولة هضمها وأسعارها المناسبة وسهولة تحضیرها فهی من أکثر الأغذیة إستهلاکاً. لذلک أجریت هذه الدراسة على ستة أنواع مختلفة من أسماک الیاه العذبة (البلطی والبیاض والقرموط) والمالحة (البوری والباغة والبربونی) المتداولة فى مدینة الزقازیق- محافظة الشرقیة- مصر، حیث تم تجمیع عدد120 عینة من کل من البلطی والباغة والبیاضوالقرموط و البوریوالبربونی بواقع عشرینعینة من کل نوع علی حدة لإجراء الفحص البکتیریولوجی علیها. کما تناولت الدراسة أیضاً بعض المحاولات لتحسین الحالة الصحیة لأسماک البلطی وزیادة فترة حفظها بإستخدام بعض الإضافات الطبیعیة. أظهرت النتائج أن بعض الأسماک المسوقة فی مدینة الزقازیق کانت ملوثة بکتیریاً ببعض میکروبات التسمم الغذائی وأیضاً المیکروبات المسببة لفساد الأسماک. کما أن إستخدام بعض الإضافت الطبیعیة (زیت الکمونوإکلیل الجبل والشیتوزان) کان له تأثیر فعال فی خفض العد الکلی للبکتریا وزیادة فترة حفظ الأسماک. حیث کان الشیتوزان هو الأفضل یلیه إکلیل الجبل ثم الکمون. لذلک توصی هذه الدراسة بوجوب إتباع الإجراءات الصحیة بدایة من عملیة الصید حتی الإستهلاک، وأیضاً ضرورة متابعة أسواق الأسماک من الجهات المختصة للتأکید على سلامة الأسماک وأتباع طرق العرض المناسبة من قبل البائعین للحد من التلوث البکتیرى. کما توصی أیضاُ بإستخدام زیت الکمونوإکلیل الجبل والشیتوزان لتحسین الحالة الصحیة للأسماک وزیادة فترة حفظها. کما یجب معاملة الأسماک حراریاُعند درجات حرارة مناسبة لقتل الملوثات البکتیریة الموجودة بها.