EFFICIENCY OF BIFIDOBACTERIUM WITH/OR SALTS OF SORBIC ACID ON THE QUALITY OF CHILLED AND FROZEN FISH FILLET

DISCUSSION

Chilled fish fillets at 5^oC±1:

I- Sensory evaluation:

Results in Table (1) show that fillets retained good quality up to the third day of storage at 5^oC in untreated sample; gradual decline in sensory quality has been noticed. Fillets showed good quality for the 4^th day and just acceptable till the 6^th day. These results were in agreement with that recorded by Ravn et al., (1988); Laskshmanan and Gopakumar, (1996); Badr et al., (2001) and El-Mossalami et al., (2004).

Samples treated with 1.5% potassium sorbate and Bifidobacterial culture alone showed higher score till the 6^th day than control one (Chang and James, 1994; Farid et al., 1998; Badr et al., 2001; Kim et al., 1995a). While those samples treated with combination of potassium sorbate and Bifidobacterial culture were quite close in flavour, odor and appearance to fresh controls with higher scores over a period of 14^th day's storage. These results were in harmony with that recorded by Chang and James (1994) and Mosffer et al., (1999).

II- Chemical parameters of chilled treated fish fillets:

     1- pH-Value:

Table (2) and Figure (1) show a decrease in pH values in all treated samples during storage, compared with the control sample. Maximum decrease in pH values was observed in samples treated with 1.5% potassium sorbate and Bifidobacterial culture mixture. Decrease in pH values may be due to the microbial enzyme activity and autolysis producing organic acid or treatment of fillets with potassium sorbate alone or in mixture of Bifidobacterial culture. These results agree with those reported by Mendonca et al., 1989; Kim et al., 1995a; Zhuang      et al., 1996; Marshall and Jindal, 1997; Badr et al., 2001; Mosffer et al., 1999).

      2- Total Volatile Basic Nitrogen (TVBN) (mg/100gm):

Results represented in Table (2) and Figure (2) indicate the formation of total volatile basic nitrogen was affected by all treatments than the control one throughout storage, a gradual increase occurred. The lowest value of TVBN occurred in samples treated with combination of 1.5% potassium sorbate and Bifidobacterial culture, while maximum TVBN was found in control samples followed by samples treated with Bifidobacterial culture, 1.5% potassium sorbate and combination of 1.5% potassium sorbate and Bifidobacterial culture.

Connel (1990) reported that the content of TVBN is a useful indicator of freshness of lean fish and suggested 30-40 mgN/100g. However, the increment in TVBN during storage in ice could be the result of decomposition and degradation of nitrogen substance which may be due to the activity of microorganisms. These results are in line with those obtained by Woyewoda and Bilgh (1986); Khuntia et al., (1993); Badr et al., (2001).

III- Microbiological examination of chilled treated fish fillets:

Results in Table (3) and Figure (3) indicate maximum APC was observed in control samples followed by the fish fillets treated with Bifidobacterial culture, potassium sorbate 1.5% alone and combination of potassium sorbate 1.5% and Bifidobacterial culture respectively.

It is worthily to mention that the decrease of APC in those treated with 1.5% potassium sorbate may be due to its bacteriostatic effect (Buncic et al., 1995; Badr et al., 2001)and extend shelf life of fish during storage at 5^oC±1.

APC was higher in control and treated samples with Bifidobacterial culture and 1.5% potassium sorbate during storage for 12 days at 5^oC±1. These results coincide with those given by Mendonca     et al., (1989a); Khuntia et al., (1993); Buncic et al., (1995); Kim et al., (1995a); Zhuang et al., (1996); Marshal and Jindal, (1997); Badr et al., (2001).

Chang et al., (1995)reported that APC in refrigerated (4^oC) Tilapia fillets were directly affected by potassium sorbate.

With respect to those treated with Bifidobacterial culture, the observed decrease was due to the antimicrobial property of Bifidobacterial culture which produce lactic, acetic acid, hydrogen peroxide and possibility unknown compounds.

APC of fillets with Bifidobacterial culture alone rapidly increased after 3 days of storage, but fillets were not considered spoiled by the sensory panel until after 6 days.

Aerobic food spoilage organisms in refrigerated food can reduce shelf life and microbiological quality (Reddy et al., 1970; Shewan, 1971; Gilliland and Specks, 1975; Post et al., 1985; Ingham, 1989; Berry et al., 1991). The combination of lactic acid producing bacteria (BIfidobacterial culture) and food additives could be considered as a food preservative to repress growth of such microorganisms (Reddy      et al., 1970; Gilliland and Ewell, 1983; Lindgren and Dobrogoze, 1990).

 Table (3) revealed that APC in untreated fish rapidly increased for 14 days. While counts from a treatment with combination of potassium sorbate and lactic acid culture caused a significant decrease during the 6 days of storage at pH 5.8. However, undesirable microorganisms grew visibly after 9 days. This was in agreement of Kim and Hearnberger (1994) and Chang et al., (1995) who stated that combination of Bifidobacteria with potassium sorbate efficiency inhibited growth of Gram negative bacteria in refrigerated catfish fillets.

From the achieved results in Table (3) and Figure (4), Enterobacteriaceae count at zero time were 10³ increased after 2, 4, 6, 8, 10, 12 and 14 days of storage at 5^oC, which become 3.9×10⁵ (signs of rejection was noticeable), Coliforms could be detected with slight increase from 92 till reached 210 CFU/ml at the end of storage period (Table 3 and Figure 5). E. coli failed to be detected in control and treated samples. These findings were in agreement with those reported by Rose (1968); Abd Almenom (1986) and Daoud and El-Mossalami (2002) who stated that Gram negative bacteria are more susceptible to cold. There is a marked decrease in total bacterial, Enterobacteriaceae, Coliforms and Staphylococcal counts in samples treated with 1.5% potassium sorbate than other control samples during storage at 5^oC for 18 days. These results coincide with those given byMendonca et al., (1989b); Khuntia et al., (1993); Buncic et al., (1995); Kim et al., (1995b); Zhuang et al., (1996); Marshal Jindal (1997) and Badr et al., (2001).

It is worthily, to mention that in samples treated with Bifidobacterial culture, there was a marked decrease in all bacterial counts (plate count, Enterobacteriaceae, Coliforms and Staphylococcal count), as Bifidobacterial culture having a pronounced inhibiting effect on Gram negative and Gram positive bacteria (Reddy et al., 1984; Schaack and Marth, 1988; Wijsman et al., 1989; Harris et al., 1991; Okereke and Montville, 1991; Kebbary, 1995 Badawi and El-Sonbaty, 1997; Abou-Dawood, 2002).

Bifidobacteria may control food spoilage bacteria and foodborn pathogens through production of lactic and acetic acids as well as other antibiotic substances (Laroia and Martin, 1990; Modler et al., 1990; Hughes and Hoover 1991; Ray 1992).

These results agree with Martin and Chou (1992); Biavati et al., (1992) and Mehanna et al., (2002) who observed that 88% of Bifidobacterial strains tested decrease their viability after 7 days of storage under acidic conditions (skim milk at pH 4.0) and less than 10% survived after 15 days. Others recorded a decrease in 0.5 log cycles after 4months at refrigeration (Robinson, 1990; Gomes et al., 1995; Mehanna et al., 2002).

Blanchette et al., (1996) and Effat (2002)found that the presence of lactic and acetic acids inhibit the growth of Bifidobacteria after 4 weeks of refrigeration storage.

A previous report on treated fillets with potassium sorbate or combined with Bifidobacteria attributed antimicrobial effects primarily to potassium sorbate (Kim and Hearnsbergers 1994).

From our results, an additive interaction occurred when Bifidobacteria were combined with potassium sorbate, the initial pH values of fillets treated with 1.5% potassium sorbate and Bifidobacteria, either alone or combined were 5.7, and 5.6 Units, lower than untreated control fillets pH (6.0). Hence, inhibitory effect results in our study were likely due to changes in pH and to the specific action of potassium sorbate. These results were in agreement of Chang et al., (1995) and disagreed with Kim and Hearbsberger (1992b) who stated that treated catfish fillets with organic acid didn’t necessarily have lower pH than controls, but remained inhibitory to growth of aerobes (Kim and Hearnsberger, 1994). Quality and safety of refrigerated foods have been enhanced by preventing growth or destroying aerobic spoilage bacteria and foodborne pathogens during storage and handling using food additives and biopereservatives (Gilliland and Ewell, 1983; Lindgren and Dobrogosz, 1989; Kim and Hearnsberger, 1994). However, biopreservatives such as lactic acid bacteria suppress aerobic bacteria that cause food spoilage (Raccach and Baker, 1978; Gilliland and Ewell, 1983; Schaack and Marth, 1988).

Frozen fillets at -18^oC±1:

I- Sensory evaluation:

The data obtained during experiment in Table (4) showed that organoleptic scores had occurred in frozen samples during the successive weeks of storage at home freezer (-18^oC) where it reached a suggestive limit of 3 at the 14^th week of storage.

The organoleptic scores of treated fillets with Bifidobacterial culture decreased gradually to suggestive limits 2.9 at the 22^nd week of storage at home freezer (-18^oC), while reached 2.3 at the end of experiment 24^th week of storage.

The organoleptic scores of treated Tilapia fillets with 1.5% potassium sorbate gradually decreased till reached a suggestive limits 3.0 at the 22^nd week of storage at home freezer, while scores of 2.5 at the 24^th week of storage (end of the experiment).

Within each treatment, acceptability decreased with increasing storage time with the exception of combination of 1.5% potassium sorbate and Bifidobacterial culture treatment was the best in maintaining the acceptability till the end of storage period (24 weeks). These values agreed well with the microbiological shelf life. These results agreed with those of Mocking and Machava (1986); Benner et al., (1994) and Kim  et al., (1995b).

The difference in keeping quality time is due to the nature of the initial microflora present on fish at the time of capture as well as the effect of freezing on the microbial load. Moreover, the use of 1.5% potassium sorbate, Bifidobacterial culture and/or combination of them on treated samples had antimicrobial effect on the surface contaminations of fish samples. This held the view reported by Jadhav and Magar (1970); Choi et al., (1986); Joseph et al., (1989) and Cano-Munoz (1991).  

II- Chemical parameters of frozen treated fish fillets:

     1- pH-Value:

The data obtained during this investigation recorded in Table (5) and Figure (7) regarding the pH values of the fresh samples was 5.9. Gradual increase in the pH values of frozen samples occurred during successive weeks of storage at home freezer (-18^oC), where it reached a limit of 6.23 at the 10^th week and 6.7 at the 18^th week.

The mean pH value of Tilapia fish fillets samples treated with Bifidobacterial culture subjected to a slight decrease (5.6) immediately after treatment then showed a gradual increase where it arrived a level of 6.1, 6.6 and 6.7 at the 12^th, 22nd and 24^th weeks of storage respectively at home freezer (-18^oC).

The mean pH values of Tilapia fillets samples treated by 1.5% potassium sorbate (Table 5 and Figure 7) subjected to a slight decrease (5.6) immediately after treatment, then showed a gradual increase where it arrived a level of 6.0, 6.5, and 6.6 at 12^th, 22^nd and 24^th weeks of storage respectively at home freezer (-18^oC).

It is of importance to recognize that the maximum pH limit given by EOS (1991) had been encountered in the frozen samples stored for 10-12weeks at -18^oC is 6.2. On the other hand, such limit had been observed at 10, 14, 16, and 20 weeks of storage at home freezer for control, treated samples with Bifidobacterial culture, 1.5% potassium sorbate and combination of them respectively at the same temperature.

The present data reported here, indicated that maximum pH limit stipulated by EOS (1991) has been recognized in the frozen untreated fillets samples after 10 weeks of storage at home freezer (-18^oC) whereas treated samples showed such limit within 14^th, 16^th and 24^th week with Bifidobacterial culture, potassium sorbate, or mixture of 1.5% potassium sorbate and Bifidobacterial culture of storage at the same degree of temperature.

In this regard, Eitenmiller et al., (1982) considered that, the pH value was not a good diagnostic indicator for the quality of fish as well as unsatisfactory to indicate early stages of spoilage in frozen fishes.

Meanwhile, Galli et al., (1993) held the opinion that gradually increased pH values of fishes during freeze storage indicated bacterial growth and possible spoilage of fish. A pH value of more than 6.6 had been reported for spoiled fish and could be attributed to the decomposition of fish protein to the level of amino acids followed by their decarboxylation and deamination and production of volatile basic compounds such as ammonia.

From the above mentioned discussion, it could be safely concluded that the treatment of fish by 1.5 potassium sorbate and/or Bifidobacterial culture were the best treatment lead to extending the keeping quality of frozen fishes comparison with the untreated frozen fishes.

2- Total Volatile Basic Nitrogen (TVBN) (mg/100gm):

It is evident from the results recorded in Table (5) and Figure (8) that the estimated TVBN values reached 32.92 mg/100gm of the examined control samples at 16^th week of storage whereas in treated samples they were 31.6, 31 and 30 mg/100gm of samples treated with Bifidobacterial culture, potassium sorbate 1.5% and combination of both at 24 week of storage respectively.

Meanwhile, EOS (1991) stipulated the maximum limit of TVBN for frozen fish to be not more than 30 mg/100g of fish flesh. The gradual increase of TVBN in present data may be attributed to several volatile odour bearing compounds like the volatile basic nitrogenous compounds produced in fish as a result of bacterial spoilage which are not normally found in live muscles. The increase TVBN is logarithmically parallel with microbial growth; therefore it may provide useful data for the evaluation of fish freshness. This substitutes the finding reported byBotta et al., (1984).

In this respect, Ehira et al., (1984)andPerson (1984) also stated that the fish at the point of incipient deterioration contain 30 mg of TVBN per 100 g fish.

Such findings spot light on the efficiency of treatment with a mixture of 1.5% of potassium sorbate and bifisobacterial culture in extending the keeping quality of treated fish up to 24 weeks in comparison to untreated samples.

III- Microbiological examination of frozen treated fish fillets:

From the results obtained in Table (6) and Figure (9), it is evident that the minimum aerobic plate counts of experimentally control frozen fish fillets at zero time was 8×10⁴ CFU/g. The storage of such fish at home freezer for 18^th weeks caused slight decrease in aerobic plate count during the first 4 weeks constituting 3.5×10³ CFU/g.

Nevertheless, treated fish fillets with Bifidobacterial culture, the aerobic plate counts decreased reaching 6×10⁴ at zero time of the experiment, followed by the decrease of such counts to 7.4×10² CFU/g after 24 weeks of storage.

Concerning the treatments of fish fillets with potassium sorbate, the aerobic plate counts decreased reaching 4×10⁴ CFU/g at zero time of experiment followed by the decrease of such counts to 5.2×10² CFU/g, after 24 weeks of storage at home freezer.

In comparison of the aerobic plate counts of experimentally frozen fish with 1.5% potassium sorbate or Bifidobacterial culture at home freezer (-18^oC), the reduction in total bacterial numbers during the first 4 weeks of freezing storage period could be attributed to the disappearance of mesophilic organisms that could not adapt to the cold environment. At this point psychrotrophic spoilage flora had been established and began to multiply, as well as the total plate count decreased rapidly until signs of spoilage appear (18 weeks). This substitutes the finding reported by Acuff et al., (1984). In this respect, Frazier (1967) reported that, freezing kills some but not all microorganisms present in fish where psychrotrophs can survive freezing and are ready to grow on thawing.

On the other hand, ESO (1991) stated that, the permissible limit for the total bacterial count for frozen fish was no more than 10⁶ CFU/g fish muscles. Comparatively, it is obvious that the incipient spoilage in fish fillets took place after 18 weeks as well as, the incipient deterioration in frozen fish fillets treated with potassium sorbate, Bifidobacterial culture occur after 24 weeks alone or in mixture.

The microbial activity is one of the main caused of quality deterioration of fish, so the spoilage pattern of fish depends upon the initial bacterial count, in addition to those acquired during handling and storage (Cobb and Vanderzant, 1971). However, Thatcher and Clark (1978) stated that, the high viable counts of frozen fish indicates contamination of materials from unsatisfactory sanitation during handling, processing as well as inadequate chilling and/or freezing.

From the present data it could be concluded that, the extended shelf life of treated frozen fillets with 1.5% potassium sorbate and Bifidobacterial culture had higher inhibitory effect that each of them, these results were in harmony with Harris et al., (1991); Okereke and Montville, (1991) and  Chang et al., (1995) who stated that combination of organic acids and Bifidobacterial culture was effective in suppressing aerobic spoilage bacteria on catfish fillets and should be considered as a potential method for shelf life extension.

The Enterobacteriaceae counts in experimentally control frozen fillets at zero time was 10³ CFU/g. This was followed by a gradual decrease in the Enterobacteriaceae counts to 10² CFU/g after 12 weeks storage (Table 6 and Figure 10)

With respect to Enterobacteriaceae counts in experimentally treated fish with Bifidobacterial culture a gradual decrease was observed till reached 10² CFU/g at 22 weeks till 24 weeks (end of the experiment). These results agree with that recorded by Kebary (1995) and Korshunove et al., (1999).

Dealing with the treated fillets with 1.5% potassium sorbate, the Enterobacteriaceae counts decreased to 10² CFU/g from 10 weeks till the end of the experiment (24 weeks). These results agree with that reported by Buncic et al., (1995) and Badr et al., (2001).

It is concluded, that the data present revealed the noticeable significant decrease of Enterobacteriaceae count till reach <10² at the end of experiment.

The counts of Enterobacteriaceae may have potential indicator for not only of health hazard but also as an indicator of spoilage (Gorczyca et al., 1985). Members of family Enterobacteriaceae are potential public health importance as it causes diseases for humans during lowering of their resistance. Also this group contains most members of food poisoning microorganisms (Edwards and Ewing 1972; Collins 1984).

 Freezing kills a proportion (usually 60-90% of the Enterobacteriaceae bacteria present) and cold storage results in a further less dramatic and gradual decrease in their numbers, in addition other organisms are injured (Weber and Schmidt, 1989).

From the present data, it could be concluded that the treatment of fish fillets with 1.5% potassium sorbate and Bifidobacterial culture lead to lower in Enterobacteriaceae count to a great extent. As the antimicrobial effect of organic acids (potassium sorbate) could be enhanced by the combination with lactic acid bacteria producing natural organic acids as lactic and acetic acid (El-Shenawy and Marth (1988); Chang and James 1994).

Korshounov et al., (1999) stated that Bifidobacterial strains capable of exhibiting the growth of all indicator bacterial strains (Escherichia coli, Klebsiella, Staphylococcus aureus, Enterococcus faecalis, Pseudomnas aeruginosa).

The given results in Table (6) and Figure (11) showed that the MPN of coliforms in frozen untreated Tilapia fillets during frozen storage at home freezer at zero time were 92 CFU/ml. The count gradually decreased and correlated the lowest levels after 18 weeks (9 CFU/ml). In general, the MPN of coliforms decreased gradually till reached <3 CFU/ml in all treated samples at the end of storage time (end of experiment) at home freezer (-18^oC). Nearly similar results were obtained by Wu and Chen, (1980); El-Sayed (1991) and Yehia (1996). But higher numbers were obtained by Abdel-Galil et al., (1988); Mahmoud (1990) and Mahmoud (1994).

Comparatively, the obtained results of the experimentally frozen fish and treated frozen fish were within the permissible limits (100 colonies/ml) recommended by ESO (1991) for frozen fishes. Used of the coliforms count as an index of pollution in frozen food has been criticized because of the susceptibility of this group of microorganisms to freezing injury resulting in gradual disappearance in their numbers in frozen food during continued storage (Licciardello and Hill, 1978). Presence of coliforms in frozen fish in the present study may be attributed to neglected sanitary measures during production and handling. This agrees with that reported by (Licciardello and Hill, 1978). In this respect, Farouk (1989); El-Sayed (1991); Seback (1998) stated that the presence of coliforms in fish serves as an index of sanitation and proper handling conditions.

E. coli could not be detected in this study. The present results were coincide with that of Raj Liston, (1963); Joseph et al., (1989) and Soliman and Shalaby (2001).They reported that E. coli was absent in fish after freezing. While samples treated with 1.5% potassium sorbate, Bifidobacterial culture alone or in combination there is noticeable decrease in coliforms count was due to the effect of bacteriocidal effect of potassium sorbate (Badr et al., 2001) and the antimicrobial substances and acid production of Bifidobacterial culture (Badawi and El-Sonbaty, 1997; Nour and Abosrea 2005).

From the achieved results in Table (6) and Figure (12), coagulase-positive Staphylococcus aureus in Tilapia fillets were present in count of 3×10² CFU/g at zero time of control samples. Similar findings were obtained by Mohamed (1990); Saad et al., (1991) and El-Shater (1999). The frequent contamination on examined fish samples with Staphylococcus aureus is undoubted imparted from skin, mouth and nose of fish handlers (Polledo et al., 1986), beside dirty utensils (Banwarl, 1989). From the recorded results, Staphylococcus aureus counts decreased by freezing till reach <10² CFU/g at the 18^th week of storage. It is evident that the freezing resulted in loss of Staphylococcus aureus viability as manifested by the drop of viable counts. This observation is consistent with that finding of Raj Liston (1963) who found that a temperature of -18^oC for 393 days decreased the number of Staphylococcus aureus contaminating sea food by ten folds. In this respect Jackson (1974) reported that certain function such as multiplication and cell division could obviously not occur below the minimum growth temperature. Moreover, Ingram and Mamckery (1967) recorded that during the process of freezing many organisms are mechanically crushed or injured by extracellular ice crystals. Similar findings had been recorded by Niazi et al., (1988) who recorded that the exposure of toxigenic Staphylococcus aureus to freezing storage resulted in gradual loss in its viability by time.

Regarding the effect of 1.5% potassium sorbate and Bifidobacterial culture on fish fillets stored at the same temperature, the viable counts at Zero time was 3×10² CFU/g, recording noticeable decrease in its count reaching <10² at the 10^th week of storage. These results are in agreement of Niazi et al., (1988) who reported that after 24 weeks of storage no viable organisms could be detected in minced meat. Joseph et al., (1989) reported that coagulase positive Staphylococci were absent in frozen fish after freezing.

Concerning, Staphylococcal counts in fish fillets treated with a mixture of Bifidobacterial culture and 1.5% potassium sorbate, there is a recorded decrease at zero time which was 3×10² CFU/g with a remarkable decrease till reach <10²at the beginning of 18^th week of storage. This reduction was due to the bacteriocidal effects of potassium sorbate and the effect of freezing with addition of the antimicrobial activity associated with Bifidobacterium (Anand et al., (1984); Lindgren and Dobrogosz (1990); Kurmann and Rasic (1991); Kebary (1995); Badawi, (1997); Korshunov et al., (1999); Ahmed et al., (2002); Vazquez et al., (2005).

The combination of potassium sorbate 1.5% and Bifidobacterial culture was shown to be effective on improving sensory, chemical, microbiological quality and increasing the shelf life of Bolti fish fillets during the storage period of both samples stored at 4^oC or those samples stored at -18^oC.

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