ASSESSMENT OF THE PRESERVATIVE EFFECT OF SOME ANTIBACTERIAL AGENTS ON SHELF-LIFE OF FISH

ASSESSMENT OF THE PRESERVATIVE EFFECT OF SOME ANTIBACTERIAL AGENTS ON

SHELF-LIFE OF FISH

Elsaid A. Eldaly; Mohamed A.M. Hussien

and Asmaa M.R. Abo-Sleem

Food Control Department, Faculty of Veterinary Medicine,

Zagazig University, Zagazig 44519, Egypt.

Key Words: marinades, Fresh lemon juice , thyme , pomegranate peel extrac , tilapia fish fillets , refrigeration.

ABSTRACT:

The recent approach in food technology focus on finding not only safe and natural biocide, but also being an alternative for the chemical preservatives. marinades have the ability to act as bactericidal, antitoxigenic, virucidal, fungicidal, antiparasiticbesides being flavouring agents. So, it considers a promising new concern to fulfill this purpose. The present study designed to evaluate the effectof some marinades on safety and shelf life of tilapia fish fillets stored by chilling at (4 ± 1ºC) by using  a-Fresh lemon juice (100 ml). b-thyme (4 g/kg). c-pomegranate peel extract(PPE)(1%v/w). and to screen the bioactivity of the aforementioned marinades .The  mean count of the APC in the untreated groups was 5.96±0.02, 6.85±0.02 and 7.63±0.02 log₁₀ cfu/g at zero day,3^rd and 6^th day, respectively. WhileThe  mean count of the APC in thegroups treated withFresh lemon juice (100 ml) was  5.01 ± 0.02, 5.38 ± 0.02, 6.04 ± 0.02, 6.51 ± 0.02, 7.05±0.02 and 7.59 ± 0.02 log₁₀ cfu/g at zero day, and 3rd, 6th, 9^th, 12 ^th and ^15th day, respectively, While the mean count of APC of thyme (4 g/kg) was 5.06 ± 0.02, 6.01 ± 0.02,6.26 ± 0.02, 6.86 ± 0.02,7.62 ± 0.02log₁₀ cfu/g at zero day, 3rd, 6th, 9^th,12th and 15^th day,respectively.The mean count of APC in groups treated with pomegranate peel extract(PPE)(1%v/w) was 5.15±0.02, 6.13±0.02, 6.95±0.02 and 7.59±0.02, respectively.In this study the antibacterial effect of Fresh lemon juice (100 ml), thyme (4 g/kg) and pomegranate peel extract(PPE)(1%v/w)was recorded and proved with varying degree of potency leading to extent the shelf life of the tilapia fish fillets. So, it could be utilized effectively as bioactive agents.

INTRODUCTION:

Fish considers about 60% of the world source of protein ,that it has a high biological value due to high protein retention in the body. The low fat content of most fish products and low cholesterol level constitute essential aspect of health of people, especially in countries where cardiovascular disease mortality is high and presence of essential amino acids (Emikpe et al., 2011).

The shelf life and quality of fish degrades, due to a complex process in which physical, chemical and microbiological forms of deterioration are implicated.Enzymatic and chemical reactions are usually responsible for the initial loss of freshness whereas microbial activity is responsible for the obvious spoilage and thereby establishes product shelf life (Gram and Huss,1996).

Oxidative rancidity is an important organoleptic characteristic for rejection or approval of fish after prolonged storage (Amanatidou et al., (2000).

Mesophilic aerobic plate count (APC) has been  used for  to determine shelf life, organoleptic acceptability, sanitary conditions and adherence to good manufacturing practices. Initial mesophilic bacterial count gives valuable information regarding the bacteriological quality of raw materials and conditions surrounding food processing,handling and storage (Omojowo and Sogbesan, 2005).

There has been an increased interest in looking at antimicrobial properties which of extracts from aromatic plants. Hence, the extract of spices or bio preservatives have the greatest advantage to achieve the demands of the consumer for the natural preservatives (Pesavento et al.,2015).

The marinating process is one of the oldest methods of preservation of fish and other sea products (Giuffrida et al.,2007).

Marinades are solutions , including sugar , spices, oil, acids (from vinegar and fruit juice) and they are used to improve tenderness, juicness, flavour and aroma and to extend shelf life of seafood, meat, poultry and vegetables(Cadun et al.,2005). Marinated fish are typically inspired as ready-to-eat products with no heat treatment (Gram and Huss,1996).

Lemon juice was very effective in inhibiting Vibrio spp (Jayana et al.,2010) .

Pomegranatehave excellent antifungal, antiprotozoal, antioxidant, anticarcinogenic, antiinflammatory and antibacterial properties against a wide range of microorganisms including Gram positive and Gram negative bacteria (Naz et al.,2007andWang et al.,2011).it  known as one of the healthies fruits due to its high phenolic content and antioxidant activity(Tabaraki et al.,2012).

Vibrio spp. are Gram –negative rod shaped, oxidase positive, non-spore forming bacteria and halophilic bacteria that generally widespread in the coastal and estuarine environments(Austin,2010).

Therefore, the aim of the study  were planned to evaluate the effectof some marinades on safety and shelf life of tilapia fish fillets stored by chilling at (4 ± 1ºC) .

2. MATERIALS AND METHODS:

Preparation of Marinades:

The three marinades used in this study; Marined I consists of mixture of 100 ml fresh lemon juice, 5.9 g garlic powder, 9.23 g table salt, 0.5 g turmeric, 0.15 g hot chili powder, 0.5 g black pepper and 200 ml distilled water were mixed well according to Siavash Maktabi et al. (2016).Marined II as Marined I but addition of thyme 4 g/kg instead of lemon juice according to Daniela et al. (2011).Marined III as Marined I but addition of pomegranate peel extract (PPE) (1% v/w) which supplied from National Research Center instead of lemon juice according to Ünalanet at. (2011).

Preparation of sample:

 About 60 fish samples (of average weight 1200 - 1700 g) of Oreochromis niloticus   were purchased from fishmarkets in Tanta, El-Gharbia governorate, Egypt. The samples were wrapped in a separate, sterile and labeled polyethylene bags and directly transferred to the laboratory, Animal Health Research Institute, Tanta branch, in a sterile icebox for further preparation within 2h after purchasing. Fish samples were washed thoroughly with potable water then each sample was gutted, deheaded, cleaned and filleted into pieces and divided into four equal group (control, Marined I , Marined II , Marined III )( (250 g of each) and placed in aseptic polypropylene trays designed for disposable food packaging.Chemical and bacteriological examinations are examine within 2hrs after dipping and then periodically every three days until decomposition apper in each group . the scheme replicates three times.

1- Sensory examinations:

Sensory evaluation was carried out by a nine-member well trained panel. Panel members were from Food Hygiene Department, Animal Health Research Institute, Tanta Branch. Panelists were asked to evaluate each group for overall acceptability according to Kanatt et al.,(2010).

2-Keeping quality tests:

1. Determination of pH (ES63/ 11, 2006):

In a blender, Ten grams of the examined fish fillet samples were homogenized with 25 ml if neutral distilled water, left to stand for 10 min, then filtered. The pH was determined by using a pH meter (Digital, Jenco 609).

2. Determination of Total Volatile Basic Nitrogen (TVN):

The technique applied for determination of total volatile nitrogen (TVN) was recommended by Malle and Tao (1987)as follows:                                                                                              

Fish extracts for determination of (TVB-N) values prepared by homogenizing 100 gm of each frozen fish sample with 200 ml of 7.5% (w/v) aqueous trichloroacetic acid (TCA) solution in a laboratory homogenizer for 1 min at a high speed. The homogenate was centrifuged at 3000 rpm for 5 min and the supernatant liquid was then filtered through Whatman No.1 filter paper. TVB-N was measured by steam-distillation of the TCA-fish extract, Twenty five ml of the filtrate was added to a Kjeldahl-type distillation tube, followed by 5 ml of 10% (w/v) aqueous NaOH solution. Steam-distillation was performed using a vertical steam distillation unit and the distillate was received into a beaker containing 25 ml of 4% (w/v) aqueous boric acid and 0.04 ml of methyl red and bromo-cresol green indicator solution up to a final volume of 100 ml. the boric acid solution turned green when alkalinized by the distilled TVB-N. The titration was allowed to run against aqueous 0.025 N sulfuric acid solutions to the endpoint indicated by a green to pink color change. Complete neutralization was obtained when the color turned pink on the addition of a further drop of sulfuric acid.

            The TVB-N content was calculated by the following formula:

            TVB-N (mg N per 100 gm) = (14 mg/mol ×a×b×300) ÷ 25 ml

            Where, a = ml of sulfuric acid

                          b =Normality of sulfuric acid

3. Determination of Thiobarbituric Acid Number (TBA):

The method adopted for estimation of TBA by Satoh, 1978 and Ohkawa et al 1979 was applied as follow:  

  TBA test which depends on determination of malonaldehyde (MD) as an end product of lipid peroxidation. The extent of oxidative rancidity is normally reported as TBA number or values and expressed as milligrams of malonaldehyde equivalents per kilogram of the samples.

Actually, 10 grams of the sample were blended with 50 ml of distilled water in warring blender for 2 minutes. The mixture was transferred quantitatively into Kjeldahl flask by washing with an additional 47.5 ml of distilled water. However, 2.5 ml of HCL were added to 125 ug/g fat and then a small amount of stones was placed to prevent pumping. The flask was heated at the highest heat obtainable in the Kjeldahl distillation apparatus.    

Approximately, 10 minutes after the boiling were required to collect 50 ml of the distillate.  Therefore, 5 ml of the distillate were mixed to 5 ml of TBA reagent in 50 ml glass stoppered tube. The contents were mixed and immersed in a boiling water bath for 35 minutes.                                          

A distilled water TBA reagent blank was prepared and treated like samples.  After heating, cooling under tape water for 10 minutes was applied. The optical density of sample against the blank was measured by using Spectrophotometer (UNICAM969AA Spectronic, USA) at a wave length of 538 nm.                                                                                                         

TBA value= R x 7.8 (mg malonaldehyde /Kg)

Where,

R = Reading of sample against blank

3-Bacteriological analyses

Preparation of samples for bacteriological examination

according to(ISO 6887-1(2003))Ten grams of the examined samples were weighted into sterile stomacher bags diluted with 90 ml sterile buffered peptone water (BPW 0.1%) and homogenized in a stomacher (Seward 400) for 2 min. To give dilution of 1/10.One ml of homogenate was mixed with 9ml of BPW (0.1%) and then decimal serial dilutions were prepared up to 10^⁶  to cover the expected range of samples contamination, then aimed for bacterial enumeration of the aerobic plate count  according to ISO (2002)and total psychrotrophic count according to ISO17416:2001.

Pseudomonas spp count, according to (Kreig and Holt,1984)Hundred microlitre was pipetted into Pseudomonas Agar base (CM 559; Oxoid) supplemented with cetrimide, fucidin, and cephaloridine (CFC) supplements (SR 103; Oxoid, Basingstoke, Hampshire, UK) providing a selective isolation medium for Pseudomonas spp. Colonies were counted after 2-days incubation at 25^OC. The bluish-green colonies were recorded as the total count of pseudomonas.

vibrio spp count, according to (FDA,2004) Loopfuls from the orginal homogenate were separately steaked onto Thiosulfate citrate Bile and sucrose agar (TCBS) then incubated at 37 ºC for 24hrs.

1. RESULTS AND DISCUSSION:

Sensory evaluation is the most common quality criteria used for evaluating fish freshness and determination of shelf life of seafood In addition, It is considered fast, simple, costless method that provides immediate quality information (Reineccius, 1990).

Sensory evaluation on Orechromis niloticus fish fillets along the time of storage after treatment was presented in table(1) and indicated that sensory characteristics of Orechromis niloticus fish fillets were enhanced by different treatments. It is obvious from results mentioned above in the same table (1) That in the beginning of the storage, there wasn’t a clear effect between the comparable groups on the colour score especially at zero day. As the period storage advanced, the effect of different marinades could be noticed.   

Generally, Fresh lemon juice (100 ml) remained the highest on all the storage interval till the end, approving that Fresh lemon juice (100 ml) has a potent activity in delaying the sensory changes in of the treated  fish fillets samples and extending its shelf life to 12^th days and decomposed at 13^th days,  followed by the thyme (4 g/kg) then pomegranate peel extract(PPE)(1%v/w) .

The post mortem pH is between 6.0 and 6.8 (Khalafalla et al., 2015) then rises again with spoilage (Bilgin, 2003).

Table (1): Effect of different natural marinades on colour and odour scores of tilapia  fish fillet  stored at 4 ± 1°C.

The data recorded in table (2) Revealed that the intial pH values were nearly the same in all groups. On the 3^rd  day the PH values gradually increased during refrigeration storage at  4 ± 1°C . A gradual increase in pH values in samples has been observed as the storage time increased with a significant (p<0.05) decrease in pH values of treated samples (pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice)compared with control group at 6^th day of storage period mean while no significant difference between control and  treated sample (pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice) until became 6.91±0.59, 6.72±0.58, 6.44±0.52 and 6.38±0.48 for (control(C), pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice) groups of fish fillets, to reach the maximum acceptable limit of pH value (6.5) in fish and fish products recommended by EOS (2005) at 9^th, 9^th, 9^th,12^th and 15^th day of storage, respectively. Only lemon treated samples were considered as acceptable until the end of the storage period (day 15). These results showed in accordance with sensory evaluation results.

Total volatile basic nitrogen (TVB-N) is an important characteristic for the quality evaluation in sea food products.

Tables (2) Effect of different natural marinades on pH on tilapia  fish fillet stored at 4±1°

From the results achieved in table (3), it could be noticed that TVB-N values was 5.41±0.56mg/100g for control samples of Orechromis niloticus fish fillets,while results were 5.34±0.48,5.13±0.47 and  4.28±0.49mg/100g for samples treated with  pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice, respectively in Orechromis niloticus fillets. With no significant difference between control and all treated groups at zero day of a storage period.

Table (3): Effect of different naturalmarinades on TV B-N oftilapia  fish fillet stored at 4 ± 1°C

The data recorded in table (4) Revealed that at zero days, the initial mean values of TBA in Orechromis niloticus fish fillets  was 0.48±0.08, 0.47±0.09, 0.45±0.05 and 0.45±0.06 mg MAD/kg for control, pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice treated groups, respectively. There were no significance differences between all groups. Low value of TBA represents the good quality of fish.

Table (4):  Effect of different natural marinades on TBA of tilapia  fish fillet stored at 4 ± 1°C

Gradual increases were observed in control and all treated groups as storage time increases till reach at 3^rd day 2.73±0.19,2.4±0.13,2.14±0.14 and 1.62±0.11 mg MAD/kg for control, pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice treated groups, respectively and reach at 6^th day  4.28±0.34,3.44±0.36,3.11±0.35 and 2.84±0.28, respectively with no meaningful difference between control and all treated groups. The increase in TBA value during the chilled storage may be attributed to the partial dehydration of fish and to the increased oxidation of unsaturated fatty acids (Mendes et al.2008).

Regargless to the increasing pattern in the TBA in all tested fish samples, the TBAvalues of all tested groups were much lower than the maximum permissible value (4.5 mg malondialdehyde /kg fish flesh) reported by EOS (2005) for chilled fish. A Correlation between sensory evaluation and chemical parameters (PH, TVN and TBA) were observed in all treated and control groups suggestion that the use of natural marinade improve the quality of fish fillets to an extent .but studying their effects on bacteriological quality on fish fillets as an example for meat products still needed.

Bacteriological profile:

Microbial activity is responsible for spoilage of most fresh and several lightly preserved seafoods .About one-third of the world's food production is lost annuallyas a result of microbial spoilage(Tingting et al., 2012).

From the results in table (5) it is evident that The initial APC (log₁₀CFU/g) was 5.96±0.02 ,5.15±0.02 ,5.06±0.02 ,5.01±0.02 log₁₀CFU/g for control, pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice groups in Orechromis niloticus fish fillets ,that increased progressively (p<0.05) with a storage time to final values of 7.63±0.02 ,7.59±0.02 ,7.62±0.02 and 7.59±0.02 log₁₀CFU/g at  6^th,9^th, 12 ^th and 15 ^th day of refrigerated storage (time of production of off odour and taste by the sensory evaluation) in Control, pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice groups, respectively in Orechromis niloticus fish.

Tables (5): Effect of different natural marinades on APC of tilapia  fish fillet stored at 4 ± 1°C

APC in treated group was significantly (p<0.05) lower than control groups in each occasion of  examination which indicates the high antimicrobial activities of essential oils.

Also, it could be detected that a significant differences (P<0.05) between the different marinades were noticed, lemon juice group have strong inhibitory effect against the growth of APC followed by thyme 4g/Kg group    pomegranate peel extract (PPE) (1% v/w) group .

According to the permissible limit of APC in chilled fish(7 log₁₀CFU/g) recommended by ICMSF (1998), it was evident that all groups exceeded such limit at the day when it became unacceptable by the sensory evaluation results .it could be observed that APC in control, pomegranate peel extract (PPE) (1% v/w) and thyme 4g/Kg  groups exceeded the maximum recommended limit of 7 log₁₀CFU/g for APC in chilled fish (EOS 2005)at 6^th ,9^th and 12^thday of storage, respectively, while such limit has not exceeded by lemon juice treated groups until end of storage period and remained acceptable microbiologically and safe for consumption up to 15 days storage at 4±1ºC because the values were below the MPL.

Regarding the results mentioned in the same table (6), it was demonstrated that the initial count of total psychrotrophic  had the highest count of psychrotrophic bacteria along cold storage period compared to other treatments. Also, it could be detected that lemon juice group followed by thyme 4g/Kg , pomegranate peel extract (PPE) (1% v/w), respectively, have strong inhibitory effects against the growth of psychrotrophic bacteria which reduced significantly (P≤0.05) immediately at zero time from 4.92±0.02log₁₀cfu/g for the control sample to 4.13±0.02 ,3.93±0.02 and 3.62±0.02 log₁₀CFU/g for fish fillet samples treated with pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juice, respectively for Orechromis niloticus fish.

Table (6): Effect of different naturalmarinades on Psychrotrophic count oftilapia  fish fillet stored at 4 ± 1°c

At 3^rd day of storage period till end of cold storage period, all different treatments significantly (P≤0.05) reduced psychrotrophic bacterial count in compare with control group.

Also as reported in APC, PTC counts were gradually increased for all fish fillet samples (the control and treated samples) during cold storage period.

In this study,  the information given by the obtained results revealed that using marination by lemon juice combined with garlic powder, table salt, turmeric, hot chili powderand black pepperon fish fillets can improve its safety and extend its  shelf life by enhancing sensory characters and antibacterial effect against Gram negative bactria especially Vibrios pecies.

1. CONCLUSION

Pomegranate peel extract (PPE) (1% v/w),thyme 4g/Kg and lemon juiceare natural preservatives with a remarkable antibacterial effect. The result of this study confirmed their role during the storage in maintaining the fish quality and extending its shelf life depending on the sensory evaluation and the values of pH, APC and PTC. Therefore, there is a possibility of using them in food as natural preservatives instead of the chemicals without compromising the sensory attributes of the food.

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تقییم التاثیر الحفظی لبعض مضادات البکتیریا علی فتره حفظ الاسماک

السعید ابو زید الدالی  ومحمد عبدالله فکرى محمد حسین و اسماء محمد رجب ابوسلیم

قسم مراقبة الأغذیة –کلیة الطب البیطرى – جامعة الزقازیق – مصر – صندوق برید 44511

   اتجهت الدراسات الحدیثة فى مجال تکنولوجیا الاغذیة الى استخدام المواد الحافظة الطبیعیة کبدائل آمنة للمواد الحافظة الاصطناعیة. تمتلک التتبیلات القدرة على التأثیر على العدید من المیکروبات بالاضافة الى استخدامها کعوامل لتحسین نکهة الاسماکلذلک فهى تعتبر وسیلة جیدة تستخدم فى حفظ الاسماک. أجریت هذه الدراسة لاطالة مدة حفظ فلیه سمک البلطی باستخدام  عصیر اللیمون الطازج و الزعتر ومستخلص قشر الرمان لدراسة مدى تأثیرها على الحالة المیکروبیة لفلیه السمک بترکیزات مختلفة أثناء التبرید عند درجة حرارة درجة 4مئوبة.وکان العدد الکلى للمیکروبات المعویة باستخدام عصیر اللیمون الطازج هى5.01±0.2و5.38±0.02و6.04±0.02 و6.51±0.02و7.05±0.02و 7.59±0.02لوغاریتم مستعمرة بکتیریة/جرام فى الیوم الاول والثالث والسادس والتاسع والاثناعشر والخامس عشر  على التوالى وباستخدام الزعتر  کان العد الکلى للمیکروبات المعویة 5.06±0.02و 6.01±0.02و6.26±0.02و6.86±0.02و7.62±0.02لوغاریتم مستعمرة بکتیریة/جرامفى الیوم الاول والثالث والسادس والتاسع والثانی عشر  على التوالى بینما کان العدد الکلى للمیکروبات المعویة باستخدام مستخلص قشر الرمان 5.15±0.02و6.13±0.02و6.95±0.02و7.59±.02 لوغاریتم مستعمرة بکتیریة/جرام فى الیوم الاول والثالث والسادس والتاسع على التوالى.أدى استخدام التتبیلات الى تحسین جودة فلیه سمک البلطی بدرجات مختلفة. لذلک توصى الدراسة باضافهالتتبیلات الطبیعیة الى الاسماک لما لها من تأثیر مثبط على البکتریا وتحسین جودة اللحوم.