STUDY THE PREVALENCE OF PSYCHROTROPHIC FOOD BORNE BACTERIA IN TILAPIA NILOTICA IN DAKAHLIA MARKETS AND ITS EFFECT ON FISH QUALITY

STUDY THE PREVALENCE OF PSYCHROTROPHIC FOOD BORNE BACTERIA IN TILAPIA NILOTICA IN DAKAHLIA MARKETS AND ITS EFFECT ON FISH QUALITY

SHEREEN S. MOUSTAFA; HATEM F.A. EL-DOSOKY and AZA. E.A. HASSAN

Animal Health Research Institute, Mansoura Branch

INTRODUCTION

Fish and fish products were incriminated as a cause of food poisoning, food intoxication as well as many infectious diseases (Lawsan, 1970). The microflora on fish is dominated by psychrotrophic Gram-negative rod shaped bacteria belonging to the genera Pseudomonas, Moraxella, Acinetobacter, Shewanella and Flavobacterium. Members of the Vibrionaceae (Vibrio and Photobacterium) and the Aeromonadaceae (Aeromonas spp.) are also common aquatic bacteria and typical of the fish flora. Gram-positive organisms as Bacillus, Micrococcus, Clostridium, Lactobacillus and coryneforms can also be found in varying proportions, but in general, Gram-negative bacteria dominate the microflora. Some psychrotrophic pathogens can grow in refrigerated food with little or no obvious change of sensory characteristics (Berrang et al., 1989) Pseudomonas species and Bacillus cereus are the most predominant psychrotrophic  microorganisms and their presence in food creates a great risk as they lead to food poisoning and / or spoilage of food products (Jay, 2000). Also Bacillus cereus causes health hazard to consumers as they cause spoilage and deterioration to meat and meat products and responsible for food poisoning outbreaks (Parry et al., 1993). Bacillus cereus can give rise to two distinct forms of food borne diseases, the emetic form and diarrheal form, the emetic form belived to be associated with an emetic toxin performed in food while diarrheal form is caused by an enterotoxin (Altayer and Sutherland, 2006). From the food safety point of view, proteins as normal conistituent of fish are exposed to degradation by microbial enzymes activity specially proteases (Gill, 1990), Decarboxylation of amino acids histidine is encountered among several species of bacteria including Bacillus cereus and Pseudomonas the significance important of histamine in fish has been discussed by several authors (Vidal-caroa et al., 1990 and Hod & Khalafalla, 1993). Encountered among several species of bacteria from another point of view we study the effect of psychrotrophic bacteria on the chemical changes of fish, it is well known that both enzymatic and microbiological activity are greatly influenced by temperature. So the aim of this study is the detection of the most common psychrotrophic food borne pathogens in Tilapia nilotica fish samples collected from different markets in Dakahlia Governorate and evaluate their effect on fish quality.

MATERIALS and METHODS

1 – Collection of samples

- A total of 100 random fish samples of Tilapia nilotica were collected from fish markets in Dakahlia Governorate.  

- Collected samples were transferred in ice box immediately to the laboratory under complete aseptic precautions without delay for bacteriological examination and quality tests.

2- Preparation of samples for bacteriological examination:-   

- 10 gram of each fish and 90 ml of sterile peptone water were thoroughly mixed by sterile blender for approximately 2.5 min., from which ten fold serial dilutions were prepared. 

- The prepared samples were subjected to the following examination:-

a- Determination of psychrotrophic bacterial count (ISO, 2004); using surface spread technique 0.1 ml from each dilution was transferred and evenly spread over a dry surface of previously prepared sterile plate count agar media, allowed to dry for 15 min., inoculated plates divided into two groups each containing one plate from each dilution, the first group incubated at 7º for 10 days and second incubated at 25º for 24 hr.

- Average number of colonies per gram was determined and the psychrotrophic count / gram was calculated and recorded.

b- Enumeration and identification of Pseudomonas species (ISO, 2004)  

- 0.1 ml of each dilution was separately inoculated into duplicated Petri-dishes of Pseudomonas selsctive agar media supplemented with glycerol. The inoculated plates were incubated at 25º c for 48hr after which all developed colonies with greenish yellow pigment were enumerated and the average count / gram was calculated and recorded.

c- Enumeration and identification of Bacillus cereus (ISO, 1987)

- The spreading technique was applied on the surface of Bacillus cereus selective agar media which incubated at 30º c for 24hr, then count was recorded. Suspected Colonies were identified by microscopical and biochemical examination.

3- CHEMICAL EXAMINATION OF FISH  

a- Quantitative Assessment of Histamine: was done by using thin layer chromatography method (TLC), (INFO SAMAK, 1989)

b- Determination of total volatile basic nitrogen (TVBN) mg/100gm: It was done according to FAO (1980).

c– Quantitative Assessment of Thiobarbituric acid (TBA) MD/kg:- According to Pikul et al. (1983)

RESULTS

Table 1: Total Psychrotrophic count, Pseudomonas and Bacillus cereus counts of the examined Tilapia nilotica samples (n= 100)

Table 2: Frequency distribution of examined Tilapia nilotica samples based on their total Psychrotrophic & Pseudomonas and Bacillus cereus count. (n= 100):

Negative < 10²  (according to Amal F.A & Shaboury, 2009)             

Table 3:Results of biochemical examination of fish samples:-

                N. of samples = 25 of each

Table 4: Incidence of Histamine level Histamine, TVB-N and TBA in T. nilotica fish samples in relation to the level pointed by Egyptian standard:

DISCUSSION

Fish are regarded as being most popular and more perishable than other high protein foods. The flesh of healthy fish is considered bacteriologically sterile, However they are sometimes contaminated with bacterial pathogens and thus can inflect heavy losses in fish all over the world. Fish and fish products were incriminated as a cause of food poisoning, food intoxication as well as many infectious diseases (Lawsan, 1970). The Psychrotrophic bacteria have been received an increased attention by several investigators during recent years due to modern developments in fish production which results in fish must be held for longer period at low temperature before transportation, processing, manufacture or consumption. Psychrotrophes are those organisms that grow well at or below 7º c and have their optimum between 20ºc - 30ºc. Some psychrotrophic pathogens can grow in refrigerated food with little or no obvious change of sensory characteristics   (Berrang et al., 1989) Pseudomonas species and Bacillus cereus are the most predominant psychrotrophic micro organisms and their presence in food creates a great risk as they lead to food poisoning and / or spoilage of food products (Jay, 2000). The results in table (1) revealed that the total Psychrotrophic count of examined Tilapia nilotica samples ranged from 2.0 × 10³ as a minimum count to 4.2 × 10⁴ as a maximal count with a mean 3.1 × 10² ±1.8 × 10² SE, variable results were reported by (Mousa and Mahmoud, 1997), who recorded that the mean values of total Psychrotrophic count of examined tilapia nilotica was 0.56 × 10² ± 0.028 × 10² /g. While (Mahmoud, 1994). Recorded that the Mean value was 5.84 × 10³ ± 0.090. (Amal F.A El. Shaboury, 2009). mentioned that the total Psychrotrophic count of examined Tilapia nilotica samples ranged from 1.7 × 10³ to 3 × 10⁴ with  a mean value of 3.2 × 10² ± 1.3 × 10². Results in table (2) mentioned that the frequency distributions of total Psychrotrophic count of examined Tilapia nilotica samples were 45 % out of the examined samples considered negative (< 10²). 7% of samples lied between 10² to < 10³, 13 % lied between 10³ to < 10⁴ while the majority of positive samples (35%) lied between 10⁴ to< 10⁵. The obtained results agreed with that mentioned by (Hayes, 1992), and (Amal F.A & El-Shaboury 2009) as they recorded that the majority of examined Tilapia nilotica samples lied between 10⁴ to 10⁵. (Lawsan, 1970), stated that fish, fish products were incriminated as acause  of food poisoning, food intoxication as well as many infectious diseases. Pseudomonas species and Bacillus cereus are the most predominant Psychrotrophic bacteria which their presence in food creats a great risk as they lead to food poisoning and / or spoilage of food products (Jay, 2000). Pseudomonas species was isolated from fresh water fish by Gram 1993, (Mousa and Mahmoud, 1997), in this study results in table (1) mentoned that the minimum Pseudomonas species count of examined Tilapia nilotica samples was 8.2 × 10² & maximal count was 2.1× 10 ⁴ with means count of 5.1 × 10³  ± 1.1 × 10². Lower Pseudomonas count was recorded by (Lamada – Hanan, 1999), but this above results agreed with that recorded by (Amal F.A and El-Shaboury, 2009) as they recorded that the mean value of pseudomonas count in the examined Tilapia nilotica samples was 4.5 × 10³. The frequency distribution of examined tilapia nilotica samples based on their pseudomonas count was recorded in table (2) and revealed that the majority of samples lied between 10⁴ < 10⁵ with the percentage of 36% from total examined tilapia nilotica samples, a same results were recorded by (Hassan 1991 and Amal F.A & El- Shaboury 2009). Bacillus cereus was widely distributed in nature, water, soil, air and can be isolated from a wide variety of foods, (Parry et al., 1993) mentioned that Bacillus cereus cause health to consumers and also responsible for food poisoning outbreaks. The results in table (1) illustrated that the minimal Bacillus cereus count of examined Tilapia nilotica samples was 7.0 × 10², maximal was 8.2 × 10³ with mean value 4.0 × 10³ ± 2.6 × 10². The frequency distribution of Bacillus cereus count in tilapia nilotica samples was recorded in table (2) which revealed that the majority of samples lied between 10³   < 10⁴, a resembling results were mentioned by (Amal F.A & El–Shaboury, 2009). In this study the quality outlines of examined Tilapia nilotica were detected by determining the effect of Psychrotrophic bacteria on biochemical characters of examined fish, a quantitative assessment of Histamine (H), Total volatile basic nitrogen (TVBN) and Thiobarbituric acid (TBA) was made to determine the effect of Pseudomonas species and Bacillus cereus on fish quality. Results in table (3) revealed that minimal and maximal level of histamine were 5.7 and 24.6 mg% with11.2 ± 1.3SE mean value respectively, these were nearly in agreement with results registered by (Park et al., 1980), (Vidal –Caroa et al., 1990); (Hoda, H.A. and Khalafala, 1993), as they recorded that histamine level in naturally spoiled fish ranged from 22 mg/Kg up to 25 mg/kg. (Hosseini et al., 2009)mentioned that histamine amounts depend on production date of samples.Table 3 showed that  minimal and maximal values of TVBN were 10.2 & 25.1 mg/100gm respectively with a mean value 16.3 ± 3.2 mg/100gm these results with in agreement with (Acuff et al., 1984). Increasing of TVBN during storage indicated possible spoilage of fish, such increase may be attributed to the production of volatile basic compound such as ammonia as reported by (Putro et al., 1985; Galli et al., 1993). Also table (3) revealed that the minimal and maximal thiobarbituric acid (TBA) values of examined fish samples were 0.534 mg MD/Kg and 0.988 mg MD/Kg with a mean value 0.701 ± 0.04mg DM/Kg and these results were in agreement with (Undeland and Lingnert 1999). However these results were lower than (Sohad et al., 2008)who recorded an increase in thiobarbituric acid value (TBA). From all above results fresh fish should be consumed as soon as possible and surely within 3 days from purchased asrecorded by (Ahmed and Yassien 2000), because of longer storage may result in rapid rise in histamine content, volatile nitrogen basic compound and thiobarbituric acid. Also growth of Psychrotrophic bacteria in fish has become a significant problem due to wide spread use of refrigerated storage of fish, that cause a potential threat to consumers health. According to the results recorded in table (4), Histamine, TVBN and TBA, were higher than the permissible limits obtained by Egyptian standard (2005) with percentages 16.0, 12.0 and 0.0. respectively.

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