Animal Health Research Institute,

Damanhour Branch

Bacteriological study on some canned fish in Bohaira province

(With 6 Tables)

By

Omaima A. Saleh; Seham Gorgy and A.A. Bkheit

(Received at 18/12/2006)

دراسة بکتريولوجية على بعض الأسماک المعلبة بمحافظة البحيرة

اميمه عبد الفتاح صالح ، سهام فؤاد جورجى ، احمد ابو المجد بخيت

أجريت هذه الرسالة علي 105 عينة عشوائية من منتجات الأسماک المعلبة شملت 30 تونة و25 من کل من السالمون والسردين والأنشوجة تم جمعها من أماکن مختلفة من محافظة البحيرة وبعد الفحص الظاهري تم إجراء الفحوص البکتريولوجية والکيميائية أوضحت الفحوص البکتريولوجية أن نسبة توجد البکتريا الهوائية 53.3، 60، 52 و 36% في کل من التونة والسالمون والسردين والأنشوجة وکان متوسط العد البکتيري 2.16 × ²10 ± 7.2 × 10 ، 4.6 × ²10 ± 1.62× ²10، 2.62 × ²10 ± 5.9 × 10 و 1.63 × ²10 ± 5.3 × 10 علي التوالي. وکانت نسبة تواجد العصيات القولونية 10 ، 12 ، 4 و 12% في کل من التونة والسالمون والسردين والأنشوجة ومتوسطات العد البکتيري لها 1.57 × ²10 ± 3.2 × 10، 3.2 × ²10 ± 4.5 × 10 ، 4 × 10 و 8.3 × 10 ± 1.5 × 10 في المعلبات المذکورة علي التوالي. أما عن المکورات العنقودية فقد تواجدت بنسبة 36.6 ، 44 ، 28 و20% في نفس المعلبات ومتوسطات العد البکتيري لها کان1.33 × ²10 ± 5.9 × 10 ، 1.83 × ²10 ± 7.5 × 10 ، 1.79 × ²10 ± 7.2 × 10 و 8 × 10 ± 1.64 × 10 علي التوالي. کان تواجد العصيات الهوائية المکونة للأبواغ 20 ، 24 ، 20 و28% في کل من التونه والسالمون والسردين والأنشوجة واختلفت متوسطات العد البکتيري بين العينات الغير معاملة حرارياً والمعاملة حرارياً في ⁵80م لمدة 1/3 ساعة فکانت في الأولي 1.4 × ²10 ± 6.4 × 10 ، 8 × 10 ± 3.3 × 10 ، 9.2 × 10 ± 4 × 10و5.4 × 10 ± 1.7 × 10 بينما في المعاملة حرارياً فکان المتوسطات 7.29 × 10 ± 2.28 × 10 ، 1.43 × ²10 ± 5 × 10 ، 9.2 × 10 ± 3.3 × 10 و 5.57 × 10 ± 0.99 × 10 في التونة والسالمون والسردين والأنشوجة هذا وقد تم إجراء التحليل الإحصائي لجميع العينات. تم التصنيف البيوکيميائي للعزول المختلفة ففي العصيات القولونية تم عزل الکليبسيلا الرءوية نوع اوزاني وکذلک الانتيروباکتر نوع ايروجينس ونوع الانتر ميديا کما تم عزل ثلاث معزولات من عصيات السودوموناس ايروجينوسا عرضياً أما في المکورات العنقودية فقد تم  عزل المکورات العنقودية الزهبيه والمکورات العنقودية القشرية بنسب مختلفة. تم تصنيف العصيات المکونة للابواغ الهوائية في المعلبات المختلفة وکانت في التونة من أنواع مارينس وسيرکولانس وبوميلس ولينکيفورمس ومايکويدس وميجاتيريم وستيروثيرموفيلسي وفي السالمون تم عزل أنواع ماکواريم ومارينس وفي السردين تم عزل نوع نوع مايکويدز ومانييز وميجاتيريم وفى عينات الأنشوجة تم عزل نوع البوميلس والمارينز ومايکويدز وماکوريم وليکنيقورمس بمعدلات عزل مختلفة وقد تم مناقشة الأهمية الصحية للأنواع المختلفة. تم قياس نسبة النيتروجين القاعدي المتصاعد وکان بمتوسطات تواجد 39.82 ± 2.54 ، 46.02 ± 1077 ، 65.26 ± 4.63 و 33.24 ± 1.91 في کل من التونة والسالمون السردين والأنشوجة علي التوالي هذا وقد تمت مناقشة النتائج واثر المعزولات البکتيرية علي الصحة العامة والاشتراطات الصحية إضافة إلي المقترحات اللازمة لتحسين الصورة الميکروبية والکيميائية لتلک المنتجات السمکية المعلبة.  

SUMMARY

105 random sample of canned fishes included (30 tuna, 25 of each salmon, sardine and anchovies) were collected from different localities and retailed shops in Al-Bohaira province. Samples were submitted for bacteriological and chemical examinations. The aerobic bacteria were detected in 53.3, 60, 52 and 36% of tuna, salmon, sardine and anchovies respectively with mean values of 2.16 x 10² ± 7.2 x 10, 4.6 x 10² ± 1.62 x 10, 2.62 x 10 ² ± 5.9 x 10 and 1.63 x 10² ± 5.3 x 10/g, respectively of same products. Coliformes could be detected in 10, 12, 4 and 12 % with mean values of 1.57 x 10² ± 3.2 x 10, 3.2 x 10² ± 4.5 x 10 , 4x 10, 8.3 x 10 ± 1.5 x 10 for tuna, salmon, sardine and anchovies respectively. The mean counts for staphylococci were 1.33 x 10² ± 5.9 x 10, 1.83 x 10² ± 7.5 x 10, 1.79 x 10² ± 7.2 x 10, 8 x 10 ± 1.6 x 10 respectively. The biochemical identification of the gained isolates revealed only 10 isolates of coliforms in all samples (9.5 %) included Klebsiella pneumoniae sub ozaenae, Enterobaeter aerogens and Enterobacter intermedium in incidences  3.8, 4.8 and 0.1 % respectively. Three isolates of Psudomonals aeruginosa were detected in tuna. Staphylococci were detected in 36.6, 44, 28 and 20 % in tuna, salmon, sardine and anchovies. Staphylococcus aureus and Staphylococcus epedermidis were detected with variable incidence. Bacillus spp. were detected in heat treated samples and non heat treated in variable incidences. Its mean counts varied from crude unheated treated samples and those heat treated at 80 c for 1/3 hour. In the first it were 1.4 x 10² ± 6.4 x 10 , 8 x 10 ± 3.3 x 10 9.2 x 10 ± 4 x 10 and 5.4 x 10 ± 1.7 x 10 while in heat treated it were 7.29 x 10 ± 2.28 x 10, 1.43 x 10² ± 5 x 10, 9.2 x 10 ± 3.3 x 10 and 5.57 x 10 ± 0.99 x 10, in tuna salmon sardine and anchovies respectively The identification of isolates revealed in tuna Bacillus lichniforms,  Bacillus. mycoides, Bacillus migatarium and Bacillus sterothermophilus in salmon. Bacillus miquerium, Bacillus marins and Bacillus migatarium in sardine Bacillus mycoides, Bacillus marins and Bacillus pumilus, marines, mycoides, muquaruim and Bacillus lichniforms in variable incidences among heat treated and non heat treated samples. The total volatile nitrogen base were estimated as indicator for the quality of the tissue protein. The mean values were 39.82 + 2.54, 46.02 ± 1.77, 65.26 ± 4.63 and 33.24 ± 1. ± in tuna, salmon, sardine and anchovies respectively. Public health hazards and recommended measures to improve the quality and the recommended measures for improving canned fish products were discussed.                         

Key words: Canned fish, tuna, sardine, salmon, anchovies

Introduction

Fish canning industry become well established in Egypt as widely distributed under different market names. The used fish must be first quality frozen, fresh or chilled (Badr, 1982; Akande et al., 1990 and Fava, 1996).Sea foods are low acid foods and have slow rate of heat penetration so are difficult to heat processed. Besides, products such as fish packed in oil must undergo a more severe thermal process than the same food products with different formulation in order to account for the protective effect of oil (Ababouch et al., 1986 and NFPA, 1982). 

National academy of science (1985) stated that fish, mollusks and crustaceans acquired pathogenic micro-organisms or toxins from the natural aquatic environment from sewage contaminated harvesting areas and from contamination by workers, utensils and equipment during harvesting, processing and preparation. In addition, Bryan (1988) claimed that, the causes of bacterial foodborne illness transmitted by fish could be attributed to fishing from polluted waters, improper practices of strict sanitation procedures in fishing vesicles, processing plants and storage facilities as well as diseased food handlers

Schwab et al. (1982) found that the aerobic plate count, E coli and Staph. aureus counts were the useful indicators to evaluate faulty processing and /or handling practice.

Bacillus species are ubiquitous in nature, some species are of importance in spoilage of heat treated foods. Five species have been recognized as causative organisms of food poisoning including Bacillus cereus, Bacillus brevis, Bacillus subtilis, Bacillus lichniforms and Bacillus sphericus. Other members of Bacillus spp. may also be associated with food poisoning, diarrhea and emetic types are predominant symptoms (Cilbert et al., 1981; Robert et al., 1996 and Faille et al., 2002)

Total volatile basic nitrogen (TVB-N) was served only as an indicator of spoilage and did not correlate with storage period (Barile    et al., 1985). On the other hand, Srikar et al. (1993) found that TVB-N increased throughout the period of storage, indicating deterioration in the quality of the tissue protein. Also they observed that the lower TVB-N content in products stored at 2.5^o C may be attributed to the slow rate of hydrolyses. The aim of this study was to investigate and evaluate the bacteriological quality of some canned fish namely tuna, salmon, sardine and anchovies through the aerobic bacterial count, detection and isolation of coliforms, staphylococci and Bacillus spp with studying the public health importance as well as the recommended measures for improving the quality of canned fish.

Materials and Methods

105 random samples of canned fishes included 30 tuna, 25 of each salmon, sardine and anchovies were collected from different localities, retail shops and supermarkets in Al-Bohaira province.  Samples were dispatched directly to the laboratory with minimum of delay where they prepared and examined chemically and bacteriologically

Preparation of samples for bacteriological examination were done according to A.P.H.A. (1985)                      

Bacteriological examination:

The prepared samples were subjected to the following examinations.

1-   Aerobic plate count (APC) according to APHA (1992)

2- Coliform count using Most Probable Number (MPN) according toA.O.A.C.(1975)

3- Staphylococcus aureus count was carried according to Baird Parker (1962)

4- Enumeration  of aerobic spore former according to APHA (1985)

5- Enumeration of aerobic spore former of heat treated samples  according to APHA (1985)

 6- Identification of isolates was done according to Willis (1977); Macfadian (1980) and Kreg and Holt (1984).

Chemical examination:

Total volatile basic nitrogen (TVB–N)was carried according to FAO (1992)

Table 1: Statistical analytical results of aerobic plate, coliforms, staphylococci and Bacillus spp. (c.f.u. /gm) of the examined canned fish.

B.C .un H        : Bacillus cereus of unheated sample

BC H               : Bacillus cereus of heated samples

Table 2: Means and standard error for aerobic plate count, coliforms, staphylococci and Bacillus spp. of the examined fish.

B.C un H               : Bacillus cereus unheated count.

B.C H                      : Bacillus cereus heated count.

* Counts                : Calculated as Mean + Standard Error

* Means                :  with the same letters (A, B are not significantly different (P < 0.01)  

Table 3: Incidence of the identified Gram-ve bacilli isolates 

Table 4: Incidence of the identified staphylococci isolates. 

Table 5: Incidence of identified Bacillus spp. isolated from the examined tuna, salmon, sardine and anchovies.

Table 6: The statistical analy tical results of total volatile basic nitrogen (T.V. B- N) / 100g of the examined canned fish.

Measn in the same letter do not differ significantly at p = 0001

Discussion

The results in Tables (1, 2) denoted the bacterial counts in different canned fish products under investigations. The incidence of aerobic plate count was. 53. 3, 60. 52 and 36 % for tuna, salmon, sardine and anchovies respectively with means of 2.16 X10² ± 7.2 x 10, 4.6 x 10² ± 1.62x 10², 2.62x 10² ± 5.9 x 10 and 1.63 x 10² ± 5.3 x 10 respectively.These results are nearly similar to that recorded by Davidson et al. (1981) who reported that 63 % of cans conductive to microbial survival. Higher results were recorded by Ababouch et al. (1986) who claimed that canned fishes packed in oil more are susceptible for holding bacterial contamination due to the fat protection effect which minimizes the thermal effects on preprocessing bacterial loads. Lower incidence was recorded by Paterno and Gopez (1970) who claimed 21.53 % of the examined locally canned food after incubation at 55°C contained viable organisms. Foster et al. (1977) recorded higher count for fish fillets, flounder fillets and salmon when ranged from 3.5 x 10³ to 9.3 x 10⁴ / gm. The lower count in our study could be attributed to the fat and oil protection effects through thermal processing of canned fishes as claimed by NFPA (1982) and Ababouch, et al. (1986). Our results were in accordance with Akande, et al. (1990)who claimed that total viable counts of canned tuna were generally low as ranged from 1.5 X10² to 3.5x 10^{2 .}

Table (2) denoted that there were no significance variation between the different studied products that could be turned to the lower count of aerobic plate count

Coliform as recorded in Tables (1&2) for tuna, salmon, sardine and anchovies could be detected in 10, 12, 4 and 12 % respectively. These results abroached to the results of Silverman et al., (1964) on frozen fillets as 19 % of samples were positive. The mean values of coliforms were 1.57 x10² ±3.2 x 10, 3.2 x 10² ±4.5 x 10, 4 x 10 and 8.3 x 10 ±1.5 X 10 in tuna, salmon, sardine and anchovies respectively. Salmon yield significant variation that could be turned to the spices and additives. The presence of coliforms is an index of hygienic quality of fish and shellfish originated from environmental sources and low sanitary precautions during production. and processing   (Freeman, 1960; Silvermen, et al. 1964; El-Morshedy et al., 1981 and A.O.A.C., 1975). Table (3) denotes the identified coliform isolates from different examined canned fishes. Enterobacter species including Enterobacter aerogens (4.8 %) and Enterobacter intermedium (1%) are the predominant followed by Klebsiella pneumoniae sub ozanae (3.8 %). The summated coliforms in canned fish products were 9.5 %. These results are in accordance with Mousa (1986) who could isolate Enterobacter spp, Klebsiella spp in addition Proteus spp and Citrobacter as coliform contaminant of fishes. Our results agreed also with Nambiar (1980) who concluded that coliform exceed 4.5 %. Pseudomonas aeruginosa were accidentally isolated only 3 isolates were detected in Tuna (2) and one in salmon. The presence of Pseudomonas occur in proceeded beyond the 3 ^rd day of storage.

Table (1) denoted the frequency of staphylococci isolated from tuna, salmon, sardine and anchovies which were 36.6, 44, 28 and 20 % respectively  with mean values of 1.33 x 10²- ±5.9 x 10 , 1.83 x 10 ² ±7.5 x 10 , 1.79 x 10² ±7.2 x 10 and 8 x 10 ±1.6 x 10. Table (2) showed no significance variation between the different products. The identification of the gained isolates revealed 26.6, 36, 12 and12 % Staphylococus aureus in each of tuna. salmon, sardine and anchovies respectively. These results agreed with Lawson (1970) who recorded that Staphylococcus aureus was rarely recovered from freshly caught fish, but after handling and processing reached from 10 – 30 %. Moreover, presence of Staphylococcus aureus is useful indicator to evaluate faulty processing and / or handling practice.

Concerning aerobic sporeformers they were present in 26.6, 24, 20 and 28 % with means of 1.4 x 10²± 6.4 x 10, 8 x 10 ±3.3 x 10, 9.2 x 10 ± 4 x 10 and 5.4 x 10 ± 1.7 x 10 in unheated samples, 7.29 x 10± 2.28 x 10, 1.43 x 10² ±5 x 10, 9.2 x 10 ±3.3 and 5.57x10 ± 0.99x10 in heat treated tuna, salmon, sardine and anchovies respectively. Statistically, there were no significance variations between products at p > 0.01. These results agreed with Ababoush et al. (1986) who found 28 % of canned fishes contained typical under processing spoilage organisms pure culture of aerobic and anaerobic spore former bacilli. Lower results were recorded by Pavlov (1989) who found 16 %in canned meat. The incidence in anchovies could be attributed to the use of solar salts which may be rich with Bacillus species as recorded by Shewan, (1971). Higher incidence was recorded by Davidson et al. (1981) who claimed that, 63 % of high protein food products such as fish are very conductive to microbial survival .Also they found pure cultures of aerobic and anaerobic spore forming bacilli. Lowest count of aerobic spore former was detected by khalaf and Marth (1984). It was 2 c .f. u / gm for cans of sardine and tuna in U.S supermarkets. The sources of contamination as recorded by National Academy of Science (1985) in fishes could be attributed to natural aquatic environment from sewage, contaminated harvesting areas and from contamination by workers, utensils and equipment during harvesting, processing distribution and food preparation. Table (5) declared the identified aerobic spore forming bacilli in all the examined canned fishes in tuna 23 were recovered. 47.7 % were isolated from un-heat treated and 52.3% from heat treated samples. These isolates were Bacillus marins 4 (17.2 %), 1(4.3 %) Bacillus. circulans. 2 (8.6 %) Bacillus Pumilus 4 (17.2) % Bacillus lichnforms, 5 21.0% Bacillus. mycoides 3 Bacillus migatariam 2 and Bacillus. mequarium out of 25 sample from heated and unheated samples. Sardine yielded 10 isolates 2 Bacillus marins 4 Bacillus mycoides. 4 Bacillus migatarium. Finally anchovies yielded 19 isolates (6 Bacillus marins, 2 Bacillus pumilis, 5 Bacillus lichniformes, 3 Bacillus mycoides and 3 Bacillus maquarium). These results agreed with Herson and Hulland (1980). In addition Rodel and lucle (1990) reported that storage with inadequate refrigeration of commercially pasteurized sealed cans caused spoilage of product where Bacillus lichniforms and Bacillus subtilis frequently isolated. Many of the gained isolates were recorded as food poisoning organisms as recorded by Hadlock (1983).

Table (6) shows the statistical analytical results of total volatile basic nitrogen (T.V.B.– N / 100 gm) of the examined tuna, salmon sardine and anchovies the minimal values were 16.13, 31.22, 32.13, 16.13 respectively the maximal reached 63.12, 65.19, 126.19 and 52.18 /100gm  respectively. The component of T.V. B- N found in small quantities in fresh fish flesh as increased with time of storage as correlation between increase spoilage proceeds and increase fishy odor. The mean values of TVB–N as in Table (6) were 39.82 ± 2.54, 46.02 ± 1077. 65.26 ± 4.63 and 33.24±1.91 in tuna, salmon, sardine and anchovies respectively. These results agreed with El–Wakeil et al. (1975) who found an increase in both T V B N from 17.2 in fresh fish to 18.4, 44,75, 120, 13 176.8 and 115 mg / 100g in salted fishes stored for 0,30, 60, 90 and 120 days respectively .In additions El-Sharnouby et al., (1989) found an increase in TV B- N from 28.44 up to 81067 mg % in finished product of salt cured gray mullet while in fresh fish increased from 20.75 mg % to 121.2 mg % in salted cured conman crap. The statistical analysis revealed significant decrease in anchovies than other products. These results are in accordance with Hernandez - Herrero (1997) who supposed that, a part of T.V.B. N content diffused into the brine with other nitrogen fractions. Our gained results are within limit of acceptance as Connell (1990) who reported that the content of TVBN used as indicator for estimating the freshness of lean fish and suggested that 200 mg / 100gm are the maximal limit for marine fish.

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