Prevalence of ِِِِِAeromonas Species in fresh and marine water fishes with a Trial to improve their shelf life at refrigeration
Prevalence of ِِِِِAeromonas Species in fresh and marine water fishes from the seafood market of Zagazig ,Egypt with a Trial to improve the their shelf life
Eldaly, E.A.; Saleh, E.A. and Abd El- Hafeez, M.M.
Food Control Dept.,Faculty of Veterinary Medicine,Zagazig University, Zagazig,Egypt.
INTRODUCTION
Fish is a rich source of easily digestible protein that also provides polyunsaturated fatty acids, vitamins and minerals for human nutrition. Although some species of fish are used industrially for fish meal manufacture, a need for their preservation and utilization for human consumption has been recognized in order to prevent post-harvest fishery losses (Venugopal and Shahidi, 1995 and Koffi-Nevry et al., 2011).
Aeromonas species are widely spread in waters, water habitants, and many food products (seafood, shellfish, and raw foods of animal origin like poultry, ground meat, raw milk, and raw vegetables). (Daskalov,2006). The high environmental prevalence of these bacteria should be regarded as an important threat to public health, since Aeromonas infections are generally acquired through consumption of water and food (Borrell et al., 1998)
According to Adams and Moss (2000) and Kirov (2001) Aeromonas (principally A. hydrophila) currently has the status of a foodborne pathogen of emerging importance. It has attracted attention primarily because of its ability to grow at cold temperatures. Aeromonas spp. were first considered as possible causative agents of human gastroenteritis more than 30 years ago (Lautrop, 1961).
Genus Aeromonas has emerged as an important human pathogen because of suspected food-borne outbreaks (Altwegg et al., 1991; Kirov et al., 1993) and the increased incidence of its isolation from patients with traveler's diarrhea (Hanninen et al., 1995; Yamada et al., 1997). Among the 14 species of Aeromonas known to date (Carnahan and Altwegg, 1996),A. hydrophila, A. caviae, and A. veronii biotype sobria have most commonly been involved in human infections and have been found to produce a variety of virulence factors such as hemolysins, cytotoxins, enterotoxins, proteases, leukocidin, phospholipases, endotoxins, outer membrane proteins, and fimbriae (Chopra and Houston, 1999 ).
According to Kirov (1993, 2003),Kirov and Sanderson (1995), and Isonhood and Drake (2002), Aeromonas species have been recognized as pathogens which can cause a number of serious extraintestinal infection including bacteraemia, meningitis, pulmonary and wound infections. Aeromonas spp. may play a significant role in ‘‘summer -diarrhoea’’, a worldwide problem particularly in children under five years old, the elderly, and travellers. The role of these bacteria in foodborne incidences is not firmly established, but Aeromonas spp.has the potential to emerge as significant foodborne Pathogens. A. hydrophila were reported by Janda and Duffey (1998) to predominate in cases of Aeromonas-associated gastroenteritis. Kirov (2003) noted that the disease spectrum of A. hydrophila included gastroenteritis, septicemia, traumatic and aquatic wound infections, and infections.
Therefore,the current study aimed to detect, count and identify Aeromonas spp. in some fresh and marine water fishes as well as to impose a trial to improve and extened their shelf life,
Material and methods
A total number of 120 fishes samples of Tilapia nilotica, Cat fish and Carp fish which is fresh water fishes (20 of each) and Blue spot fish, Saurus and Pagrus which is marine water fishes (20 of each) were collected from different fish markets in Zagazig city. The collected samples were well identified and packaged separately in a sterile plastic bag; then directly transferred with a minimum of delay to the post-graduate laboratory of food Control Dept.Faculty of Veterinary Medicine, Zagazig University under aseptic conditions, where they were subjected to sensory and bacteriological examination
A-Sensory examination
This examination was adopted according to the procedure recommended by (Braumuller, 1958).
B-Bacteriological examination:
Muscle samples of fish under examination were prepared according to ICMSF, (1978),enumeration, Isolation and identification of Aeromonas spp. according to the method recommended by ICMSF (1978);Roberts et al., (1995) andMacFaddin, (2000)using Glutamate starch phenol-red agar medium (GSP Agar) which is (Pseudomonas Aeromonas Selective Agar Base)proposed by ICMSF (1978)
C-Trails to improve the shelf-life of fish:-
Antibacterial agents used
* Tap water. * Lemmon extracts 120 PPM.
* Thyme oil 0.2 % (V/W). * Acetic Acid 1 % (V/W).
A. Sampling:
30 random samples were collected from fishes markets in Zagazig city. The collected samples were identified and packaged separately in a sterile plastic bag under aseptic conditions; then directly transferred with a minimum of delay to the laboratory of food control department, Faculty of Vet. Medicine, ZagazigUniversity, where they were subjected for treatment and examination protocols.
B. Treatment protocols:
In the laboratory, fresh Tilapia nilotica samples were divided into (6) groups, each group contains 5 fishes:
First group untreated.
Second group dipped in previously isolated and identified Aeromonas hydrophilabroth.
Third group dipped in previously isolated and identified Aeromonas hydrophilabroth then treated by Tap water for 10 minutes.
Fourth groupdipped in previously isolated and identified Aeromonas hydrophilabroth then treated by dipping in lemon extract 120 ppm for 10 minutes.
Fifth group dipped in previously isolated and identified Aeromonas hydrophilabroth then treated by dipping Thyme oil 0.2 % (V/W) for 10 minutes.
Sixth group dipped in previously isolated and identified Aeromonas hydrophilabroth then treated by dipping acetic Acid 1 % (V/W) for 10 minutes.
The control as well as treated groups will be subjected to the following examinations at zero time and after storage in domestic refrigerator (4º C + 1º C) then analyzed periodically every 48 hrs till appearance of spoilage signs:
1-Organoleptic examination which was carried out according to the method recommended in part I.
2-Measurement of PH (EOS/ 2760-1:2006):
The PH meter (steward) was calibrated by buffers (4.00 - 7.00 - 10.00), 10 gm of the prepared sample was weighted in a conical flask then 100ml of distilled water at 25°C was added. The mixture was shacked gently for 30 minutes until good homogenecity then left for 10 minutes. A part of the clear fluid was transferred to a clean beaker to be measured by pH meter.
3-Bacteriological examination which was carried out according to the method recommended in part I.
Results and Discussion
Table (1): Organoleptic examination in examined samples of freshand marine water fishes (No. = 20 for each):-
Fish / Fishes / Min / Max / Mean ± SEMFresh water fishes / Tilapia nilotica / 61.00% / 89.00% / 72.00 ± 2.00%
Cat fish / 57.00% / 89.00% / 72.00 ± 2.00%
Carp fish / 64.00% / 89.00% / 74.00 ± 2.00%
Marine water fishes / Blue spot fish / 64.00% / 76.00% / 70.00 ± 2.00%
Saurus / 51.00% / 86.00% / 68.50 ± 2.00%
Pagrus / 50.00% / 89.00% / 69.50 ± 3.00%
Min = Minimum SEM = Standard Error Mean
Max = Maximum No = Number of each fishes sample.
Table (2): Incidence of Aeromonas in examined samples of fresh and marine water fish. (No. = 20 for each):-
Fish / Fishes / No. of positive samples / %Fresh water fishes / Tilapia nilotica / 13 / 65
Cat fish / 20 / 100
Carp fish / 20 / 100
Marine water fishes / Blue spot fish / 18 / 90
Saurus / 8 / 40
Pagrus / 15 / 75
Table (3): Aeromonas count (CFU/g) in examined muscles of fresh and marine water fish samples (No = 20 for each):-
Fish / Fishes / Min / Max / Mean ± SEMFresh water fishes / Tilapia nilotica / ≤100 / 12x105 / 21.1x104 ± 7.7x104
Cat fish / 6x106 / 104x106 / 36x106 ± 6.5x106
Carp fish / 2x106 / 19x106 / 7.7x106 ± 1.2x106
Marine water fishes / Blue spot fish / ≤100 / 17x105 / 36.9x104 ± 0.95x105
Saurus / ≤100 / 11x106 / 13x105 ± 6.5x105
Pagrus / ≤100 / 4x106 / 13.7x105 ± 2.7x105
Table (4): Frequency distribution of isolated Aeromonas organisms recovered from examined fresh and marine water fish samples.
Fish / Fishes / No. of Aeromonas spp. isolates / Aeromonas isolates / No. / %Fresh water fishes / Tilapia nilotica / 13 / A. hydrophila / 8 / 61.5
A. sobria / 4 / 30.8
A. caivae / 1 / 7.7
Cat fish / 20 / A. hydrophila / 13 / 65
A. sobria / 6 / 30
A. caivae / 1 / 5
Carp fish / 20 / A. hydrophila / 13 / 65
A. sobria / 4 / 20
A. caivae / 3 / 15
Marine water fishes / Blue spot fish / 18 / A. hydrophila / 14 / 77.8
A. sobria / 1 / 5.6
A. caivae / 3 / 16.6
Saurus / 8 / A. hydrophila / 8 / 100
A. sobria / 0 / 0
A. caivae / 0 / 0
Pagrus / 15 / A. hydrophila / 10 / 66.7
A. sobria / 0 / 0
A. caivae / 5 / 33.3
Table (5): Organoleptic examination of untreated samples and treated samples of Tilapia nilotica fishes:-
Day / Control group / In vitro-contaminatedgroup / Tape water
treated group / Lemon extract
treated group / Thyme oil
treated group / Acetic acid
treated group
1 / 87.86±0.61% / 87.14±1.82% / 87.14±1.82% / 87.14±1.43% / 86.43±1.32% / 90.71±1.82%
3 / 61.43±1.32% / 30±1.82% / 60±2.07% / 73.57±1.43% / 77.14±1.82% / 83.57±1.82%
5 / 30.71±1.82% / AD / 31.43±1.32% / 62.14±1.82% / 65±1.75% / 75±2.25%
7 / AD / AD / 30.71±1.82% / 53.57±1.14% / 67.14±2.07%
9 / AD / 31.43±1.32% / 61.43±1.32%
11 / AD / 52.86±1.32%
13 / 34.29±1.82%
15 / AD
AD = Apparently Decomposed
Table (6): pH in examined samples of untreated and treated samples of Tilapia nilotica fishes:-
Day / Control group / In vitro-contaminatedgroup / Tape water
treated group / Lemon extract
treated group / Thyme oil
treated group / Acetic acid
treated group
1 / 6.82±0.04 / 6.84±0.07 / 6.64±0.04 / 6.14±0.18 / 6.54±0.09 / 5.09±0.07
3 / 6.24±0.07 / 6.48±0.07 / 6.34±0.02 / 6.08±0.21 / 5.54±0.06 / 5.38±0.11
5 / 6.34±0.17 / * / 6.44±0.11 / 6.22±0.09 / 5.82±0.06 / 5.64±0.07
7 / * / * / 6.52±0.16 / 6±0.09 / 5.92±0.05
9 / * / 6.42±0.13 / 6.1±0.04
11 / * / 6.32±0.05
13 / 6.44±0.07
15 / *
* = pH not measured
Table (7): Aeromonase hydrophila count (CFU/g) in examined samples of untreated and treated samples of Tilapia nilotica fishes
Day / Control group / In vitro-contaminatedGroup / Tape water
treated group / Lemon extract
treated group / Thyme oil
treated group / Acetic acid
treated group
1 / 2.5x104±
0.2x104 / 6x106±
1.1x106 / 9.5x104±
0.6x104 / 2.2x104±
0.2x104 / 8.4x102±
1.7x102 / 4x102±
0.7x102
3 / 2.2x105±
0.33x105 / 12x106±
1.2x106 / 7.2x105±
0.39x105 / 2.2x105±
0.33x105 / 2.3x104±
0.29x104 / 2.4x103±
0.51x103
5 / 41.2x105±
6.2x105 / * / 45.2x105±
4.2x105 / 9x105±
1.8x105 / 22.4x103±
2.3x103 / 15x103±
1.2x103
7 / * / * / 2.9x106±
0.43x106 / 2.3x105±
0.3x105 / 32.4x103±
3.1x103
9 / * / 17.8x105±
3.1x105 / 4.8x104±
0.8x104
11 / * / 22.8x104±
2.3x104
13 / 15.6x105±
4.8x105
15 / *
* = Not counted
Discussion
I-Organoleptic examination:
Consumers are the ultimate judges of quality and acceptance depends on satisfying their sensory requirements. Freshness declines as the storage life passes, until the product is no longer acceptable to the consumers. Therefore, Freshness implies acceptability; loss of freshness indicates loss of storage life when the product is unacceptable (Hall, 1992).
A-Fresh water fishes:
It is evident from the results recorded in table (1) that the organoleptic examination of the collected samples of Tilapia nilotica, ranged from 61% to 89% with a mean value of 72.00 ± 2.00, while in cat fish (Clarias lazera) ranged from 57% to 89% with 72.00 ± 2.00 as an average and from 64% to 89% with an average of 74.00 ± 2.00 in Carp fish. All the examined samples were considered fresh and fit for human consumption. Nearly similar results were recorded by Sallam (1990), Abo Samara (2001)and Morshdy et al., (2002). The results showed that physical examination must be associated with bacteriological examination to give the accurate judgment.
B- Marine water fishes:
The results reported in table (1) revealed that the organoleptic examination of the collected samples of Blue spot fish, ranged from 64% to 76% with a mean value of 70.00 ± 2.00, while in Saurus ranged from 51% to 86% with 68.50 ± 2.00 as an average and from 50% to 89% with an average of 69.50 ± 3.00 in Pagrus. All the examined samples were considered fresh and fit for human consumption.
II-Microbiological examination:
(A)-Incidence and count of Aeromonas species in the examined fresh water fishes samples:
The obtained results in tables (2 &3) revealed that the incidence of Aeromonasspecieswas 65% in examined Tilapia nilotica samples with a total count/g ranged from ≤100 to 12x105and with a mean value 21.1x104 ±7.7x104 CFU/g, while in Cat fish was 100% of examined samples with a total count/g ranged from 6x106 to 104x106with a mean value of 36x106 ± 6.5x106CFU/gas well as in Carp fish was 100% of examined samples and with a total count/g ranged from 2x106 to 19x106 with an average of 7.7x106± 1.2x106CFU/g.
The obtained results were coincidence with that reported by El-Kelish (1995), El-Atabany (1995) and Lamada (1999), higher findings were reported by Bastawarows and Mohamed (1999) and lower findings were reported by Naser (1991). Such variations may be attributed to presence of aeromonas spp. in mud and cat fish as a bottom feeder most contaminated species.
Comparatively, the obtained results declared that the Cat fish had a higher count of Aeromonas spp. than that of Tilapia nilotica and Carp fish. This could be attributed to dirty habitat in which Cat fish live and prolonged exposure of such fishes to various types of contamination during marketing where the fishes live for a relatively long time after catching while other fish species dead shortly after harvesting and this result agrees with Abo Samara (2001).
(B)-Incidence and count of Aeromonasspecies in the examined marine water fishes samples:
It is evident from the results recorded in tables (2&3) that the incidence of Aeromonasspp.was 90% in examined Blue spot fish samples with a total count/g ranged from ≤100 to 17x105and with a mean value 36.9x104± 0.95x105 CFU/g, while in Saurus fishes was 40% of examined samples with a total count/g ranged from ≤100 to 11x106with a mean value of 13x105 ± 6.5x105CFU/g as well as in Pagrus fishes was 75% of examined samples and with a total count/g ranged from ≤100 to 4x106 with an average of 13.7x105± 2.7x105CFU/g.
C-Incidence of isolated Aeromonas organisms recovered from examined fishes samples:
The identification of Aeromonas species (A. hydrophila, A. sobriaand A. caviae) was demonstrated in table (4)which showed that these strains were recovered from the examined samples of Tilapia nilotica in percentages of 61.5, 30.8 and 7.7%, respectively. While in Cat fish they were recovered from 65, 30 and 5%, respectively. On other hand, these strains were recovered from 65, 20 and 15% of examined Carp fish samples, respectively.
In Egypt, (Mousa, 1986 and Khalil et al., 1990)isolated Aeromonas from the muscles of Tilapia nilotica and Mugil cephalus. The widespread occurrence of A. hydrophila has been proved by many investigators, who isolated this organism from human beings, fowls and sea-foods (Fehlhaber et al., 1985 and Palumbo et al., 1989). Its wide spread in the environment causes food related illness and becomes of concern to food microbiologist as has been isolated from a variety of foods (Palumbo et al., 1989).
Data in table (4) revealed that the incidence of identified Aeromonas spp. (A. hydrophila, A. sobria and A. caviae) isolated from examined marine water fishes samples was 77.8, 5.6 and 16.6% for Blue spot fish samples, 100, 0 and 0%, for Saurus fish samples and 66.7, 0 and 33.3% for Pagrus fish samples, respectively.From the aforementioned results it can be concluded that A. hydrophila was the most predominant identified Aeromonas spp. isolated from both freshwater fish and marine water fish samples. Conclusions regarding the significance of A. hydrophila in foods should be made with circumspection. These findings substantiate what have been reported byGarcía-López et al (2011). Who was able to isolate five strains of Aeromonas species during a three-year period in the same geographic in Spain.
IV. Trials for improve the shelf life of fresh water fishes:
The basic goal of all decontamination trails is to reduce the risk of pathogenic microorganism and decrease the microbial load as well as to prolong shelf life of fishes. Advantages and disadvantages of the chemicals used in decontamination must be taken in to account on choosing the best one for commercial uses.
The fitness of any article of food should be based on combined information obtained from organoleptic examination which includes smell, appearance and texture, chemical and microbial evaluation. Organoleptic examination:-
Spoilage is an objective measurement which includes changes in color, texture, odor, taste and single symptom or group of symptoms of overt microbial activity, manifested by changes in fish’s odor, flavors or appearance. The results obtained from this study indicated that treatment of fish samples by lemon extract, thyme oil and acetic acid were the best as increasing the tenderness and enhancing the sensory evaluation which include appearance, odor, color and over all acceptability.
Organoleptic examination of Tilapia nilotica over all the refrigeration of storage at 4+1º C is presented in table (5). The results revealed that Tilapia nilotica control group samples, in vitro-contaminated group samples, tape water treated group samples, lemon extract treated group samples, thyme oil treated group samples and acetic acid treated group samples were organoleptically rejected shown extreme discoloration and off odor after 7th, 5th, 7th, 9th, 11th and 15th days of refrigeration storage, respectively.
Moreover, it is evident that the mean values of (organoleptic examination) at 1st day in Control group samples, in vitro-contaminated group samples, tape water treated group samples, lemon extract treated group samples, thyme oil treated group samples and acetic acid treated Group samples in Tilapia nilotica fishes was (87.86±0.61%), (87.14±1.82%), (87.14±1.82%), (87.14±1.43%), (86.43±1.32%) and (90.71±1.82%) respectively.
By the 3rd day of refrigeration storage at 4+1º C the mean values of (organoleptic examination) of Tilapia nilotica fishes was (61.43±1.32%), (30±1.82%), (60±2.07%), (73.57±1.43%), (77.14±1.82%) and (83.57±1.82%) in control group samples, in vitro-contaminated group samples, tape water treated group samples, lemon extract treated group samples, thyme oil treated group samples and acetic acid treated group samples respectively.
At 5th day of refrigeration storage at 4+1º C for Tilapia fishes the mean values of(organoleptic examination)for untreated control group samples and treated samples with tape water, lemon extract, thyme oil and acetic acid was (30.71±1.82%), (31.43±1.32%), (62.14±1.82%), (65±1.75%) and (75±2.25%) respectively. The in vitro-contaminated group samples show signs of decomposition.
By 7th day of refrigeration storage at 4+1º C for Tilapia fishes the mean values of(organoleptic examination)for treated samples with lemon extract, thyme oil and acetic acidwas (30.71±1.82%), (53.57±1.14%) and (67.14±2.07%) respectively.
Also at 7th day of refrigeration storage at 4+1º C theuntreated control group samples of Tilapia fishes showed decomposition and treated samples with tape water show decomposition also.
Such results nearly similar to that of Abd Al-Rahman (2010) where the Tilapia nilotica control samples were still organolypticaly acceptable till 10th day of refrigeration storage at 4+ 1º C.
At 9th day of refrigeration storage at 4+1º C the mean values of (organoleptic examination)in treated samples of Tilapia fishes withthyme oil and acetic acidwas(31.43±1.32%) and (61.43±1.32%) respectively. The lemon extracttreated fish samples show signs of decomposition.
By 11th daythyme oiltreated samples of Tilapia fishes were decomposed while the mean value of the(organoleptic examination)of refrigeration storage at 4+1º C for Tilapia fishes for treated samples with acetic acidwas (52.86±1.32%).
At 13th day the mean value of (organoleptic examination) of acetic acidtreated samples of Tilapia nilotica fishes was (34.29±1.82%).
Finally at 15th day the acetic acid treated samples of Tilapia nilotica fishes were decomposed. Nearly similar findings were found by Sallam et al. (2007) and Abd Al-Rahman (2010) where the Tilapia nilotica acetic acid treated samples were still organolypticaly acceptable till 18th day. The use of 1% acetic acid could retard the microbial growth, delay chemical changes, and improve the sensory attributes and extend shelf life of fishes.
2-Measurement of pH:
The pH value of live fish muscle is close to 7.0, however post mortem pH can vary from 6.0 to 7.0 depending on season, species and other factors(Simeonidou et al., 1998).
From the results obtained in table (6), it is evident that the mean values of (pH in examined samples) at 1th day in control group samples, in vitro-contaminated group samples, tape water treated group samples, lemon extract treated group samples, thyme oil treated group samples and acetic acid treated group samples in Tilapia nilotica fishes was (6.82±0.04), (6.84±0.07), (6.64±0.04), (6.14±0.18), (6.54±0.09) and (5.09±0.07) respectively.