EFFECTIVENESS OF GARLIC, ROSEMARY AND GINGER ESSENTIAL OILS ON IMPROVE THE QUALITY AND SHELF LIFE OF BAGRUS BAYAD FISH SAUSAGE PRESERVED BY COLD STORAGE

Shaban Abd El-Halim El-Sherif and Safwat Abd El-Ghafour. Fish Processing Technology Laboratory, National Institute of Oceanography and Fisheries, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

This research was carried out to evaluate the antioxidant activity and antimicrobial effects of individually (1.0%) garlic (Allium sativum), ginger (Zingiber officinal) and rosemary (Rosmarinus officinalis) essential oils (EOs) as natural preservatives to extend the shelf-life of raw Bayad (Bagrus bayad) fish sausage. Fish sausage treated with tested essential oils and untreated (control sample) were packed in polyethylene bags and stored in refrigerator at 4±1ºC for 25 days. The analysis of physico-chemical quality criteria; total volatile basicnitrogen (TVB-N), thiobarbituric acid (TBA) and pH value, microbiological quality indices; total bacterial count (TBC), psychrophilic bacteria (PCB) and yeast & mould (YM) counts were carried out on treated and untreated raw fish sausage periodically every 5 days during storage period and sensory analysis performed on fish sausage periodically with chemical and microbiological analysis after frying raw sausage. The obtained results showed that all investigated analysis parameters; (TVB-N), (TBA) and pH value, (TBC), (PCB) and (YM) counts were gradually increased during storage period in different ratios depending on the kind of tested essential oil, the increases of these parameters were significantly higher (p<0.05) in control sample than samples treatment with (EOs).The obtained results also showed that there was a significant (p<0.05) enhancement in sensory quality attributes of fish sausage; samples treated with ginger, rosemary and garlic essential oils, respectively. In addition, the shelf-life and the safety storage period for these products were found to be less than 20 days for control sample and more than 25 days of samples treated with EOs under cold storage. In general, the investigated essential oils caused a significant (p<0.05) extending of the shelf-life through retarded the spoilage and enhancement the physico-chemical, microbiological and sensory quality attributes for raw Bayad fish sausage during cold storage. Therefore, The tested EOs had a high effectiveness as antioxidative , antimicrobial and should be utilized for extending the shelf-life and enhancing quality attributes of raw fish sausage during cold storage as follows; sausage samples treated with1.0% ginger as the best treatment, followed by samples treated with1.0% rosemary and treated with1.0% garlic compared with untreated sample.
Fish is considered as a valuable part of human nutrition because of its high content of polyunsaturated fatty acids (PUFAs) and protein (Kykkidou et al., 2009); long-chain polyunsaturated fatty acids of these products have gained attention because of their prevention of human cardiovascular diseases (Ozogul et al., 2006). Fish sausage are considered better than beef, pork and chicken sausage because of high marine protein efficiency rate (PER) with low fat content containing long chain n-3 fatty acids, beneficial for health (Churi et al., 2016). Fish sausage can be produced by using similar methods and process of meat sausage. Due to changing consumer preferences toward healthier life styles and safer and cheaper foods, sausage manufacturers have turned to meat alternatives such as fish. The addition of fish meat in sausage formulation could improve nutritional quality of the product and can be a way to insert fish meat in human diet. The expansion of the fast-food industry and the increased consumption of processed-meat products make it necessary to reevaluate the quality characteristics of fish sausage products and to be available in the Egyptian market.
Despite fish and fish products have the rapeutic and nutritional value, these products have a short shelf-life, this is due to the vast amounts of free amino acids and volatile nitrogen bases and higher final pH that limit the useful life of the product being highly susceptible to oxidation of unsaturated fatty acids and this can affect the flavor, taste, texture, aroma, and shelf-life of fish and fish products (Mexis et al., 2009).
To prevent and delay the quality changes caused by lipid oxidation in foods and seafood, various synthetic antioxidants have been used (Benjakul et al. 2005). However, with growing concerns regarding the safety of synthetic antioxidants, natural antioxidants have been suggested as safe alternative to synthetic antioxidants to retard oxidative processes and to improve the keeping quality of fish. This ability is due to their potential as free radical scavengers which may terminate radical chain reactions and display antibacterial effects against bacterial pathogens (Singh et al., 2005). The bacteriostatic effects of spices may occur by two mechanisms: the delay and partial inhibition of DNA and proteins synthesis (Feldberg et al., 1988).
Spices and herbs have been added to food since ancient times, not only as flavoring agents, but also as folk medicine and food preservatives (Nakatani, 1994). Furthermore, certain spices and herbs were used as green materials, plant extracts; essential oils (EOs) and powders for prolong the storage life of foods by preventing rancidity through their antioxidants activity or through bacteriostatic or bactericidal activity, also to food-borne pathogenic bacteria (Shelef et al., 1980). Essential oils (EOs) contain a mixture of compounds, which includes terpenes, alcohols, acetones, phenols, acids, aldehydes, and esters and mainly used as food flavorings or functional components in pharmaceuticals (Corbo et al., 2009).
Garlic (Allium sativum) oil one of the commonly use spices to enhance flavor in food. Apart from it, Allium sativum has a wide spectrum of actions which include antibacterial, antifungal, antioxidant and beneficial effects on the cardiovascular and immune system of human. The presence of allicin, a sulfur-containing compound found in garlic, that enables the antimicrobial effects (Sallam et al. 2004 andGafar et al., 2012).
Rhizome of the ginger species, (zinger officinale) is widely used as a spice and food seasoning due to its sweet aroma and pungent taste. It has been known to have antioxidant activity and effective as antimicrobial (Zia-ur-Rehman et al., 2003).Ginger oil inhibits Aspergillus, a fungus known for production of aflatoxin, a carcinogen and contains spectra of biologically active compounds, such as curcumin, 6-gingerol, 6-shagaols, zingiberene, bisabolene and several other types of lipids that confer on it, the properties of being pungent and a stimulant. These compounds are responsible for the unique aroma and flavor of ginger (Akram et al., 2011). Many studies were carried out to prove the effect of combination of refrigeration and essential oils on extending the shelf-life of fish and fish products (Zakipour and Divband, 2012).
278 Therefore, this study was carried out to investigate the effect of individually (1%) garlic, ginger and rosemary essential oils on improve the quality and extending the shelf-life of raw Bayad fish sausage during storage at 4±1ºC for 25 days by determination of physico-chemical, microbial and sensory quality criteria so, shelf-life periodically during cold storage. Ingredients:sunflower oil, spices mixture, corn starch, NaCl and sugar were obtained from local market and sodium polyphosphate (Merck Co., Germany).The essential oils (EOs); garlic (Allium sativum), ginger (Zingiber officinal) and rosemary(Rosmarinus officinalis) were obtained from Kato Aromatic Co., Giza. Casings; mutton casings used for stuffing sausage were purchased from local market in Atabba, Cairo.

Fish mince preparation:-
The fish samples were beheaded, gutted and washed with tap water to remove all viscera, black membranes, swim bladder and blood. Skin and bones were manually removed to produce pure fillets. The yield of flesh was about 48%.The fillets were dipped in 1.0% chilled brine solution contained 0.5% acetic acid for 5 min. (to clean and remove any fishy odor) and fillets were drained. The drained fillets were cut and minced by a kitchen meat mincer using a 3 mm diameter holes plate.

Preparation of fish sausage:-
The formulation used in the production of fish sausage is given in Table 1. The minced fish meat was mixed with all other ingredients till obtained homogenized sausage batter; the batter was subdivided into four parts;(1) control without treatment with EOs (2) blended with 1.0% garlic, (3) blended with1.0% ginger and (4) blended with 1.0% rosemary EOs (EOs were substituted with sunflower oil), then every treatment was mixed well and stuffed into natural casings (from sheep) with diameter 1.5 cm, by using manual filler and punched to pieces of about 10 cm long. All fresh (raw) fish sausage samples were packed in polyethylene bags and kept in a refrigerator at 4±1°C for 25 days and physico-chemical, microbial and sensory attributes analysis were carried out immediately after preparation (0), 5, 10, 15, 20 and 25 days of storage. Quality criteria:-Total volatile basic nitrogen (TVB-N) was determined by the Macro distillation method proposed by Pearson (1991). Thiobarbituric acid values (TBA) were determined spectrophotometrically according to the procedure described by Siu and Draper (1978). TBA content was expressed as mg of malonaldehyde (MDA)/kg sample. The pH value was measured by using standard methods, AOAC (2006).

Microbiological analysis:-
The samples of raw fish minced and fish sausage were analyzed for microbial profile using standard procedures (APHA, 1992) for total bacterial count(TBC) (30°C, 3 days) and Psychrophilic bacteria (PCB) (7°C, 10 days) on plate count agar, yeast & mold (YM) counts on potato dextrose agar (21°C, 5 days). The results were expressed as log 10 cfu/g of sample.
Sensory evaluation:-Sensory properties including color, taste, odor, texture and overall acceptability of fried fish sausage by deep-frying in sunflower oil at 160°C for 7 min. (frying was carried out immediately during evaluation) were evaluated at zero time immediately after preparation and periodically every five days with chemical and microbiology analysis of raw sausage according to El-Sherif (2001) using the following numerical system: excellent 8.6 -10,very good 7.6 -8.5, good 6.6 -7.5, accepted 5.0 -6.5, poor 4 -4.9 and very poor 0-3.9.
Statistical Analysis:-Least significant difference test (L.S.D) at (p<0.05) and Standard Error (Mean ± SE) were carried out on the obtained data of the physico-chemical, microbiological and sensory evaluations as the mean of three replicates (Gomez and Gomez, 1984).

Results and Discussion:-
Proximate composition of raw Bayad fish minced and sausage treated and untreated with some essential oils (EOs):-Mean values for the proximate chemical composition of fresh raw fish minced and fresh (raw) sausage are given in  (Stansby, 1982) and the big size of fish gave high filleting yield (48%) indicating that fish sausage that healthier than traditional meat sausage particularly due to the presence long chain n-3 fatty acids and protein (Oliveira et al., 2014).
On the other hand, it could be noticed that the moisture content of raw fish sausage was decreased when compared to the mince fish meat, this reduction after processing may be due to the ingredients which were added to the mince fish. The relative increase in fat, ash and carbohydrates in fish sausage might be attributed to the addition of starch, fat and salt and other ingredients in the preparation of the sausage while the decrease in protein content may be due to the degradation of protein (Ahmed and Elhaj, 2011). It is clear from the present results that parameters chemical composition in raw fish sausage were not affected by inclusion of the plant essential oils as there were no significant different (p<0.05) between their means and those of control.        Res. 4(11), 276-289 283 Fig. 4 shows the changes of total bacterial count of different raw fish sausage samples during cold storage as affected by addition of 1.0% garlic, 1.0% ginger and 1.0% rosemary EOs individually. At zero time of storage, control samples (untreated with EOs) showed the highest mean counts of TBC comparing to other samples treated with essential oils of garlic, ginger and rosemary that caused a significantly (p<0.05) reduction in microbial count of treated fish sausage immediately after preparation. The TBC of control and treatment samples with EOs fish sausage were gradually increased significantly (p<0.05) during storage at 4±1ºC with a highly increase in control and depending on the kind of EOs. This increase may be due to the growth of psychrophilic bacteria under this storage condition as reported by Osheba et al. (2010).TBC (log cfu/g flesh) of control sample were 3.25 at zero time of storage increased to7.65 log cfu/g sausage sample at the end of storage period (25 days), while samples treated with garlic, ginger and rosemary EOs were 2.45, 2.60 and 3.02 at zero time increased to 4.65, 3.70 and 4.00 cfu g -1 respectively.This mentioned that garlic, ginger and rosemary EOs contain the phenolic compounds such as Timol, Carvacol, acid 1, 8 Cafeic and Transcinnam aldehyde responsible for the antibacterial properties (Salvat et al., 2001).

Microbiological load changes of raw Bayad fish sausage during storage at 4 ±1 °C as affected by essential oil (EOs):-Total bacterial count (TBC):-
Also, it could be detected that a significant differences (P<0.05) between the different oils were noticed, ginger have strong inhibitory effect against the growth of TBC followed by rosemary and garlic EOs. In our study the raw fish sausage of control sample was considered accepted until 20 th day of storage and were rejected because TBC count was exceeded than the maximum permissible level MPL (10 6 numbers/g) was reported the acceptable limit of TBC for sausage (Sekin and Karagozoglu, 2004), also MPL recommended by the International Commission on Microbiological Specifications for TBC in all sausage products is below 7 log 10 cfu/g sample (ICMSF, 2002), while the other fish sausage samples treated with EOs remained acceptable microbiologically and safe for consumption up to 25 days storage at 4±1ºC because the values were below the MPL. This antimicrobial activity of these essential oils could be resulted from the hydrophobic nature which enables them to partition in the lipids of the bacteria cell membrane and mitochondria, disturbing the structures and rendering it more permeable (Sikkema et al., 1994). Therefore this study demonstrated that the use of (1.0%) essential oils of garlic, ginger and rosemary reduced the microbial load of raw Bayad fish sausage during cold storage period compared to the control. These results are in agreement with found by Badee   sausage sample untreated with any tested essential oil had the highest count of PCB along cold storage period compared to other treatments. The obtained results point to garlic followed by ginger and rosemary EOs have strong inhibitory effects against the growth of PCB which reduced significantly (p<0.05) immediately at zero time from 2.85 log cfu g -1 for the control sample to 2.40, 2.10 and 1.45 log cfu g -1 for fish sausage samples treated with rosemary, ginger and garlic EOs, respectively. Also as reported in TBC, PCB counts were gradually increased for all fish sausage samples during cold storage and the control sample was highest (p<0.05) count until the final of storage period. As demonstrated, the treated samples with ginger, rosemary and garlic oils respectively showed the lowest counts in PCB at the final of cold storage. Therefore, ginger oil was observed to possess high antimicrobial activity followed by rosemary and garlic oils in immediate reduction of bacterial population, where the low effectiveness of garlic oil in comparison with ginger and rosemary oils could be attributed to the losses of volatile sulfur compounds, which have high biological activity, during distillation, and also due to the nature of garlic oil itself, which is volatile and hydrophobic (Sallam et al., 2004). Also, Gonza´lez-Fandos (1996) reported that garlic could differentially inhibit bacteria; lactic acid bacteria are the least sensitive microorganisms, and garlic can stimulate the growth of these bacteria by providing them with a carbohydrate source. These results are in accordance with that of Osheba et al. (2010) and Özpolat et al. (2014). On the other hand, YM counts were gradually increased for all fish sausage samples during cold storage as noticed in Figure 6 and a highly significant differences (P<0.05) between the control sample and other samples treated with EOs were noticed thus, the control sample was highest (p<0.05) count until the final of storage period. At the end of storage period, YM counts reached to 4.00 log cfu g -1 of control sausage samples, while 2.15, 2.55 and 2.90 of samples treated with garlic, ginger and rosemary oils respectively. Therefore, the sausage sample treated with garlic oil was the lowest YM counts followed by samples treated with ginger and rosemary oils compared with control sample, this indicated that tested EOs especially garlic oil have stronger effects against the growth of yeast and molds than ginger and rosemary oils in reduction of yeast and molds population.   Table (3). It could be found that Bayad fish sausage treated with EOs had excellent scores for all sensory parameters immediately after preparation (at zero time of storage) with the presence of some garlic odor and no found significant (p<0.05) differences between them till 10 days of storage, then a significant (p<0.05) differences were found until the end of storage period. While, fish sausage control had excellent scores for color and texture then very good scores for flavor (taste and odor) immediately after preparation. After that, all sensory parameters scores for treated and untreated sausage samples were decreased gradually with prolongation the storage period.
Therefore, a noticeable significant (p<0.05) difference could be observed between samples treated with EOs and control in the case of taste and odor characteristics during the storage period. EOs was found to have an added advantage in terms of their synergistic effect against oxidation and in enhancing the taste and odor. The decline in overall acceptability of control samples as a result of this difference in taste and odor was more prominent during 15-20 days of storage and the off flavor formation was occurred. While, there was no marked difference (p<0.05) between the EOs treated samples and control in color and texture during zero time till 10 days of storage then, a significant (p<0.05) difference was found until the end of storage period.
On the other hand, the taste, color, odor, texture and overall acceptability scores of control sample reached to 4.0, 4.5, 4.5, 4.2 and 4.2, respectively by 20 days of storage which was below the acceptable limit of score (5) and was greatly deteriorated being undesired as softening of texture and off odors was appeared then, this product become rejected for consumers. In the case of the other samples treated with EOs, the scores were above of acceptable limit (5) even after the end of storage period (25 days). Hence, the scores of overall acceptability garlic, ginger and rosemary samples were 5.3, 6.5 and 5.8 respectively at the end of storage period (25 days) and was in the order samples, ginger > rosemary > garlic. Therefore, Bayad fish sausage samples treated with 1.0% ginger showed the significant (p<0.05) highest mean score of overall acceptability followed by samples treated with 1.0% rosemary and treated with 1.0% garlic compared with untreated samples (control) which rejected before 20 days of cold storage. Even through the added EOs delayed oxidation and extending the shelf-life, their antioxidant activity reduced during storage by increase significantly (p<0.05) differences in all individual tested samples during the end of storage periods. Mean values of 3 replicates ± standard error. Mean values are significant and insignificant different (p<0.05). Excellent (10-8.6), very good (8.5-7.6), good (7.5-6.6), accepted (6.5-5.0), poor (4.9-4) and very poor (3.9-0).

Conclusion:-
In conclusion, the results obtained in this study showed the tested essential oils (EOs) of individualy1.0% garlic, 1.0% ginger and 1.0% rosemary have antioxidative and antimicrobial properties that enhancement the physicochemical, microbiological and sensory quality attributes of raw Bayad fish sausage during cold storage thus, extending the shelf life. This is justified by the low TVB-N, TBA, and pH as well as bacteria, yeast and mold counts of fish sausage related with EOs than untreated control samples. Sensory evaluation revealed that shelf life of fish sausage samples was longest (P < 0.05) for samples treated with garlic, ginger and rosemary at1.0% (25 days) as compared to the control (20 days) under storage at4±1ºC.Therefore, the present findings recommended that the EOs of garlic, ginger and rosemary should be utilized for extending the shelf-life and enhancing quality attributes and sensory properties of fish sausage during cold storage as follows; sausage samples treated with 1.0% ginger, as the best treatment, followed by samples treated with1.0% rosemary and finally treated with1.0% garlic.