P U B L I C A T I O N S CODON ISSN 1120-1770 online, DOI 10.15586/ijfs.v33iSP1.2059 117 P U B L I C A T I O N S CODON Effect of natural antioxidants and vegetable fiber on quality properties of fish sausage produced from Silver carp (Hypophthalmichthys molitrix) Ebrahim Mahdavi1, Peiman Ariaii2,* 1Department of Food Science & Technology, Nour Branch, Islamic Azad University, Nour, Iran; 2Department of Food Science & Technology, Ayatolla Amoli Branch, Islamic Azad University, Amol, Iran *Corresponding Author: Peiman Ariaii, Department of Food Science & Technology, Ayatolla Amoli Branch, Islamic Azad University, Amol, Iran, Email: p.aryaye@yahoo.com Received: 21 April 2021; Accepted: 27 July 2021; Published: 23 August 2021 © 2021 Codon Publications OPEN ACCESS PAPER Abstract In this study, production of low-fat, nitrite-free sausage based on Silver carp surimi was performed. In order to replace oils, inulin fiber (IF) was used; natural preservatives such as grape pomace extract (GE) and nisin (NI) were used as nitrite replacements with modified atmosphere packaging (MAP: 70% CO2 + 30% N2). For this purpose; five treatments including, T1: control, T2: control + MAP, T3: IF 5% + MAP, T4: IF5% + GE0.5% + MAP, and T5: IF5% + GE0.5% + NI 0.5% + MAP, were prepared. The physicochemical and texture properties of sausage at the beginning of storage and chemicals (Peroxide value, pH, and color index) and microbial index (Total count and psychrotrophic bacteria) during 42 days’ storage in the refrigerator (4 ± 1°C) were evaluated. The results showed that the use of natural preservatives had no effect on the physicochemical properties of the sausage (P > 0.05), but the FI had a positive effect on the texture characteristics (the firmness increased and elasticity decreased), increased moisture and ash, and reduced the fat content of sausages (P < 0.05). All in all, the best results among the treatment containing natural preservative was T5, in all microbial and chemical tests there was no significant difference with T1 (P > 0.05). All in all, a functional product with properties such as fiber and natural antioxidants, low fat and nitrite-free can be produced. Keywords: fat replacement; inulin fiber; nitrite; plant extracts; nisin; fish sausage Introduction Seafood is a valuable source of protein for humans and plays an important role in healthy nutrition. Fish have a very high nutritional value and provide most of the essential nutrients for humans, but what makes fish a specifically important food item is the presence of fatty acids. The human body is not capable of producing some unsaturated fatty acids such as omega-3 (Kwasek et al., 2020). Hypophthalmichthys molitrix is one of the warm water fish species that is widely cultivated in many countries of the world for its rapid growth, high feed conversion ratio, and good nutritional value (Fan et al., 2008; Stepien et al., 2019). Despite intermuscular bones and unpleasant odor (mud), this fish, like other fish, contain high-quality pro- tein and unsaturated fatty acids. To solve the abovemen- tioned problems, as well as produce high value-added products from Silver carp, it needs to be processed. On the other hand, with a glance at people’s livelihoods, mechanical life, and lack of time to prepare food, the idea of producing or supplying ready-to-eat or semi-prepared food, such as fish paste, fish ball, fish cake, fish sausage, and burger from seafood, seems to be a good solution (Fan et al., 2008; Ozpolat and Patir, 2015). Nitrite is used to prevent spoilage (undesirable physi- cochemical and biological changes) in sausage and ham (from production to consumption). Nitrate and nitrite Italian Journal of Food Science, 2021; 33 (SP1): 117–126 mailto:p.aryaye@yahoo.com 118 Italian Journal of Food Science, 2021; 33 (SP1) Mahdavi E and Peiman A So far, no study has been done on sausages with these preservations along with the MAP. Overall, according to the cases stated in this study, we evaluated the effect of natural antioxidants as a nitrite replacement and the use of fat substitutes to reduce the use of oil in sausage for- mulation produced by Silver carp surimi. MAP was also used for packaging of samples in different treatments. Material and Methods Raw materials At first, 30 kg of silver carp were caught from the farms and transported to the laboratory in ice boxes, followed by washing and removing the skin. After this step, the underlying meat was removed without contact with the viscera and then it was ground first through a 10 mm plate and then through a 5 mm plate in a meat grinder (MKG1300P, Panasonic, Japan). All chemicals used were purchased by Merck Germany and were of an analytical grade. Preparation of treatments To prepare the surimi, minced meat was washed with drinking water at a temperature below 10°C for 10 min in three stages. Rinsing was performed in 0.2% brine in the third step. Surimi obtained during experiments was stored at refrigerator temperature (Shabanpour et al., 2017). The sausages contained crushed ice 10%, liq- uid sunflower oil 10%, extenders and binders 7%, NaCl 1.5%, spice mixture 1%, monosodium glutamate 0.1%, polyphosphate 0.3%, and 70% mince. Sausage was pre- pared by mixing the ingredients in sequence in a food processor. The mix was stuffed into five-layer synthetic casings and cooked. Diameter and length of sausages were 25 mm and 20 cm, respectively. All fish sausages were packed separately in nine labeled polyethylene zip- bags and stored at 4°C prior to the measurements. Inulin fiber (IF) was added to the primary sausage for- mulation as fat substitutes. In addition, a ranking test previously performed comparing sausage samples with IF at different concentrations showed significantly lower acceptability of the samples incorporating 6% or 7% of them when compared to the rest (5% or lower) (data not shown). After these, IF at 5% was chosen as optimal for the following study. Fat decreased by 5%. Also, extracts of orange peel, grape pomace, and NI in concentration of 0.5% (concentration approved by sensory evaluators unchanged in taste of fish sausage) were added separately and combined as a substitute for nitrite and then, the same steps were taken similar to control treatment and finally the treatments were packed into three multilayer flexible are preservatives that give a good pink color (Hernández- Hernández et al., 2009). Due to the potential dangers of using synthetic preservatives such as nitrite and consum- ers’ interest in using healthy and natural products, food industry researchers favor the use of natural antioxi- dants in food products. The use of natural preservatives, such as extracts, powders, and essential oils of plants, to increase the shelf-life of meat products is a promis- ing new technology, were . The active ingredients found in plants have antimicrobial and antioxidant properties (Hernández-Hernández et al., 2009; Odunayo Olatunde and Benjakul, 2018). The following are some of the com- pounds that have antioxidant and antimicrobial activity as nitrite substitutes. Grape pomace is one of the waste products (5000 tons a year) produced annually in juice factories, a rich source of several valuable compounds such as citric acid, tartrate, dietary fiber, and phenolic compounds. Anthocyanin (malvidin and penonidine), flavonol (quer- cetin and meristine), acetylbene, and phenolic acids are the major phenolic compounds, and flavan-3-L, catechin and epi-catechin, and gallic acid are the predominant phenolic compounds in the pulp (Drosou et al., 2015). Also, due to its red color, it seems to have a pink effect on the sausage. Nisin (NI) is a biphasic bacteriocin polypeptide con- taining 34 amino acids, produced by specific strains of Lactococcus lactis subsp. Lactis. Due to its antimicrobial properties and low toxicity to humans, it has long been regarded as a GRAS1 food preservative in the food indus- try. NI is known in two forms, A and Z, that are similar in structure and antimicrobial activity and differ in their amino acid in position 27; in type A, histidine and the Z variant is asparagine (Biswaro et al., 2018). Modified atmosphere packaging (MAP) is used in the storage of fresh (nonfrozen) foods or at least the process. The use of vacuum or modified atmosphere or high CO2 packaging is easily feasible for processed meats, but high levels of CO2 will have negative effects on product qual- ity, especially texture changes and increased blood loss (Hematian et al., 2012). Inulin is a nondigestible carbohydrate containing natural Fructooligosaccharides (FOS) and has dietary fiber prop- erties that are of great interest for its consumption due to its specific health and technological properties. Inulin in the mouth creates a fatty mouth feel. The characteristic of inulin as an imitative lipid relates to its ability to bind to water molecules and form a gel-like network (Menegas et al., 2013). 1Generally Recognized as Safe. Italian Journal of Food Science, 2021; 33 (SP1) 119 Fish sausage fortified with natural preservatives pH value Five grams of each sample were added to 45 mL of dis- tilled water and placed in a mixer for 30 s. Then, the pH of the samples was measured with a digital pH meter cal- ibrated to pH four and seven standards (Valipour et al., 2017). Color test The color of the sausage was measured using the Hunterlab colorflex colorimeter (Hunter Lab Inc). The color test results include three Hunter indices a*, b*, L*, where L* is a light symbol which is black (0) and white (100), a* is a green-to-red symbol, –a is green, and +a is red and b* are blue to yellow symbol, which + b is yellow and –b is blue. The experiment was performed in triplicates (Choi et al., 2009). Microbial analyses Ten grams of each sample were mixed and homoge- nized with 90 mL sterile sodium chloride solution and the required dilutions were prepared. One milliliter of each dilution was used for culture by pour plate method. Total count and psychrotrophic bacteria were counted on Plate Count Agar at 37°C for 2 days and 7°C for 10 days. The results were reported as log CFU2/g (Javadian et al., 2017). Mold and yeast test Dilution of the sample was first prepared in Peptone Water broth, then transferred to a plate containing DRBC3 medium. Plates were aerobically incubated at 25°C for 5 days (ISIRI, 2008) Inoculation and enumeration of Clostridium botulinum to ham samples Approximately 108 CFU/mL of Clostridium botulinum were added to the ham samples. The samples were then massaged to ensure complete mixing of the bacterium with the hams and placed in specially filled coatings in the baking chamber. At least 10 hams were considered for each treatment. All treatments were packed in zip- pered nylon bags and stored at refrigerator temperature (4 ± 1°C) during the experiment. For bacterial count at each sampling time, 1 g of sample was mixed with 9 mL of physiological serum and suspended for half an hour. Depending on the sample, the dilutions range from 102 to 104. One milliliter of diluted sample was poured into the petri dish and then 10–15 mL of SC agar medium 2colony-forming unit 3Dichloran Rose bengal Agar pouches (three and four layers) under modified atmo- sphere (MAP: 70% CO2 + 30% N2) (Rahmanifarah et al., 2013). The treatments were kept at refrigerator tempera- ture (4 ± 1°C) for 42 days. On 0, 7, 14, 28, 35, and 42 days of storage, three sausages from each section were ran- domly selected and tested to determine qualitative param- eters (physicochemical and microbiological). Proximate factors and texture analysis were examined at zero time. All experiments were performed with three replications. In total, five treatments were studied. Treatment 1: Control treatment (no additives) Treatment 2: Control treatment + MAP Treatment 3: 5% oil, 5% IF + MAP Treatment 4: 5% oil, 5% IF + 0.5% grape pomace extract (GE) + MAP Treatment 5: 5% oil, 5% IF + 0.5% GE+ 0.5% NI + MAP Proximate composition Moisture of samples using oven at 105°C, amount of pro- tein by the kjeldahl method, amount of fat using petro- leum ether solvent (boiling point of 40–60°C) by soxhlet extraction, and ash of samples using electric furnace at 550°C were measured (AOAC, 2005). Cooking loss test Thirty grams of samples were stuffed into screw top test tubes and were heated in a steam bath at 70°C for 30 min. The cooked samples were quickly immersed in cool water for 10 min. Cooking loss was determined by weighing individual sample before and after cooking, and the dif- ference was expressed as a percentage of the original weight (Choi et al., 2009). Texture analysis To measure the texture of the sausage, the cubic pieces were cut in 1 × 1 × 1 cm3 dimensions and subjected to com- pression test by a texture analyzer with a flat probe profile of 40 × 40 mm and a load of 10 kg. The force required to compress the samples to 70% of their initial height was measured at a constant rate of 200 mm/min (Vural, 2003). Chemical analyses Peroxide value Peroxide value of the samples was determined accord- ing to the Pearson method (Bagheri et al., 2016). Results were expressed in meq oxygen kg–1 lipids. 120 Italian Journal of Food Science, 2021; 33 (SP1) Mahdavi E and Peiman A at 44–47°C was added to the petri dish and thoroughly mixed. After solidification of the medium, about 10 mL of the same medium was poured into the petri dish. Plates were then placed in an anaerobic jar and incu- bated at 37°C for 22 h. At the end of incubation, all plates containing less than 150 colonies were selected and the black colonies on each plate indicating the probability of C. botulinum were counted (ISIRI, 1994). Statistical analysis All experiments were performed in a completely ran- domized design as triplicates and the result was reported as mean ± SD. Statistical analysis of treatments was per- formed by ANOVA using SPSS software. Significant mean differences were determined by the Duncan test at 0.05 level and charts were plotted using Microsoft Excel software. Results and Discussion Proximate factors The results of moisture content (Table 1) in differ- ent treatments showed that replacement of oil with IF increased moisture content (P < 0.05). But, two treat- ments that used natural preservatives had no significant effect on moisture content (P > 0.05). The lowest values were observed in control treatments (1 and 2) and the highest moisture content was observed in treatments 3, 4, and 5 (treatments containing IF). This is due to the high water absorption properties of the fiber and also because the fiber improves the stability of the emulsion (Choi et al., 2010). Hydrocolloids are also very import- ant factors in bonding with water and keeping it in the product. So, the increase in moisture after adding fiber is obvious (Sahin et al, 2005). Results of fat content (Table 1) showed that replacement of oil with IF reduced fat levels. The highest fat content was observed in control (1 and 2) treatments and the lowest fat content was observed in treatments 3, 4, and 5 (treatments containing IF) (P < 0.05). Menegas et al. (2013) by replacing IF in chicken sausage fermented with corn oil (45% fat) produced a low-fat sausage with 25% fat. Results of protein content (Table 1) in different sausage treatments showed that replacement of oil with IF had no significant effect on protein content. All treatments had no significant difference (P > 0.05). Menegas et al. (2013) reported that the use of IF in fermented chicken sausage had no significant effect on protein content. Results of ash content (Table 1) in different sausage treat- ments showed that replacement of oil with IF increased the ash content. The lowest values were observed in control treatments (1 and 2) and the highest amounts of ash were observed in treatments 3, 4, and 5 (treatments containing IF). Increasing ash by adding IF may be due to the fact that IF has significant amounts of ash (P < 0.05). Cegiełka and Tambor (2012) reported that adding IF to Polish chicken burger increased burger ash content. Texture analysis According to the results (Figure 1a), replacement of oil with inulin increased the firmness values. The lowest val- ues were observed in control treatments (1 and 2) and the highest values were observed in treatments 4 and 5 (treatments containing IF) (P < 0.05). Adding fiber seems to increase the ability of the protein to bind to meat. Thus, it creates a firmer and coherent texture and pre- vents deformation of the sausage (Álvarez et al., 2012). By definition, elasticity refers to the rate of return of the sample to its original state after removal of the deforma- tion force. Elasticity is inversely related to the degree of firmness of the treatment. In fact, the formation of a sta- ble network structure increases the elasticity (Zhou et al., 2010). According to the results (Figure 1b), the highest values were observed in control treatments (1 and 2) and the lowest values were observed in treatments 3, 4, and 5 Table 1. Proximate factors of different treatments of sausage. Treatment/Proximate factors Moisture (%) Fat (%) Protein (%) Ash (%) 1 61.94 ± 0.10b 19.64 ± 0.37a 18.29 ± 0.32a 1.73 ± 0.02b 2 62.00 ± 0.43b 19.78 ± 0.40a 17.94 ± 0.43a 1.78 ± 0.02b 3 67.89 ± 0.36a 14.70 ± 0.40b 18.17 ± 0.32a 2.09 ± 0.02a 4 67.75 ± 0.43a 14.95 ± 0.30b 18.18 ± 0.32a 2.11 ± 0.03a 5 67.54 ± 0.44a 15.11 ± 0.14b 17.91 ± 0.31a 2.03 ± 0.03a a,b,cDifferent small letters in the same column represents significant difference (P < 0.05) (T1: control, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). Italian Journal of Food Science, 2021; 33 (SP1) 121 Fish sausage fortified with natural preservatives Peroxide value According to the results of the present study (Figure 3a), the peroxide value increased in all treatments with increasing time (P < 0.05); the increase of peroxide value in meat products was also reported by other researchers (Javadian et al., 2017; Valipour et al., 2017). The use of MAP slowed the increase in peroxide value, so that the lowest values of peroxide were observed in the nitrite-containing treatments with the modified atmosphere (P < 0.05). The high level of carbon diox- ide used in MAP prevents the growth of aerobic bac- teria. The highest level of carbon dioxide in MAP has been reported up to 50% (Silbande et al., 2018). The lowest values were observed on all days of treatment after treatment 2, in treatments 1 and 5 (P < 0.05). The lower peroxide value in the inulin treatment is due to the lower fat content in these treatments and thus to the reduction of oxidative spoilage. Also, using two preservatives together more effectively decreased the peroxide value. Plant extracts contain phenolic com- pounds. Polyphenols are capable of trapping free radi- cals, especially proxy radicals, which are one of the key intermediate chain reactants, thereby terminating the cycle of oxidative spoilage reactions. Thus, the effect of treatments containing extracts had a higher effect on increasing the peroxide value (Fidan et  al., 2019). The reason for the lower peroxide value after addition of NI can be attributed to the effect of bacteriocin on the decrease in lipolytic bacteria (such as Pseudomonas species) (Dehbandi et al., 2014). Dehbandi et al. (2014) also reported that the use of NI slowed the peroxide value in the Kilka fish surimi during storage. The acceptable level of peroxide in meat for human con- sumption is 5 (Yanar, 2007). In relation to sausage at the (treatments containing IF). Amina et al. (2014) examined the effect of adding 5%, 10%, and 15% apple waste fiber to meat nugget texture. Apple fiber increased texture firm- ness and reduced nugget elasticity. Cooking loss Cooking sausage efficiency depends on cooking tempera- ture, cooking time (Kim and Chin, 2007), ingredients, and fat content in the product (Huang et al., 2005). The results of cooking loss (Figure 2) in the present study showed that with increasing time the values of cook- ing loss increased in all treatments. Replacing the oil with IF reduced the cooking loss. The highest values were observed in control (1 and 2) on all days of stor- age and the lowest values were observed in treatment  3 (treatments containing IF) (P < 0.05). Loss of cooking is associated with the amount of fat and water-holding capacity (Eldemery, 2010). Adding dietary fiber to meat products appears to improve water-holding capacity and improve emulsion, thereby increasing cooking efficiency (Choi et  al., 2010). Various researchers have suggested this as the capacity to bind to water in dietary fiber that improves tissue properties (Choi et al., 2010, 2015). Eldemery (2010) also reported that adding orange fiber to meat burger formulation decreased burger cooking loss and with increasing concentration, lower cooking loss were observed. c 1 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 2 4 6 8 10 12 (A) (B) 2 3 Treatment F irm ne ss ( kg ) E la st ic ity ( cm ) 4 5 1 2 3 Treatment 4 5 c b a a a a b b b Figure 1. Texture analysis of different treatments of sau- sage [firmness (A) elasticity (B)] (T1: control, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). 1 0 1.5 2 2.5 3 3.5 4 7 14 21 Storage time (day) C oo ki ng lo ss ( % ) 28 35 42 2 3 4 5 Figure 2. Cooking loss of different treatments during stor- age) T1: control, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). 122 Italian Journal of Food Science, 2021; 33 (SP1) Mahdavi E and Peiman A 1 0 0 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 1 2 3 4 5 6 7 (A) (B) 7 14 21 Storage time (day) P V ( m eq /k g) pH 28 35 42 0 7 14 21 Storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 Figure 3. Changes in peroxide value (PV) (A) and pH (B) of different treatments during storage (T1: control, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). end of the storage period, the peroxide value was at an acceptable level in all samples except treatments 3 and 4. pH value The pH value of food affects microbial flora composi- tion. In fish, gram-negative and proteolytic microorgan- isms are dominant. In many foods of animal origin, such as poultry, meat, fish, and dairy products, pH reduction occurs during storage due to chemical changes such as post-slaughter changes in meat or processing. If the food storage time is increased and the microorganisms prolif- erate, the pH will rise and will be contaminated with the infection and food born microorganisms. Results of sau- sage pH values (Figure 3b) in the present study showed that pH values first decreased and then increased. The initial decrease in pH may be due to the activity of lactic acid bacteria and acidification of the environment. But, the increase in pH after storage time is due to the natural pattern of spoilage of meat products that is related to the activity of spoilage by microorganisms (Kim et al., 2011). The use of modified atmosphere slowed the pH value. High levels of carbon dioxide reduce bacterial growth and result in slowing the increase in pH compared to the control treatment. Overall, the lowest pH values were observed in nitrite + modified atmosphere treatment. Using two preservatives more effectively decreased the pH value. Overall, the lowest values were observed on all days of treatment after treatment 2 in treatments 1, 5 (P < 0.05). The latter treatments had no significant dif- ference (P > 0.05). The low pH of the sausages treated with the extract is due to its antioxidant properties and the phenolic compounds present in the extract that can protect the sausages from the function of the internal proteases and thus inhibit protein breakdown and pro- duction of amines (Fan et al., 2008). The mechanism of NI is due to its antibacterial properties and reduction in the capacity of bacteria for oxidative deamination of nonprotein nitrogen compounds (such as ammonia and trimethylamine), thereby reducing the pH-increasing process (Dehbandi et al., 2014). Color test The color index L indicates the brightness symbol (black to white). So, more the L, lighter the meat. The color index a is the indicator of the color change from green to red. The color index b represents the color change from blue to yellow. Results of color index showed that replace- ment of nitrite with preservative reduced color index L and a, and increased color index b. Sodium nitrate and nitrite are used to create a bright red (pink) and prevent turbidity of meat products, as well as antimicrobial pre- servatives, to create a special flavor. So, it seems natural to replace nitrite with other com- pounds and change its color. According to the results of using modified atmosphere, the color index L decreases (Figure 4A). Overall, the highest values were observed at the end of the storage period after the control treatments in treatment 5. There was no sig- nificant difference between the two latter treatments (P < 0.05). This indicates that the oxidation process is decreased. In fact, the brightness values of the sau- sage samples are correlated with the peroxide values. As the number of peroxides increases, the brightness decreases and the samples darken. In fact, it can be stated that the use of three preservatives, prevents oxi- dation of pigments and act as a chemical preservative such as nitrite. The color index a (Figure 4B) also decreased in all treat- ments. Generally, one of the causes of oxidation in meat products is the presence of compounds such as myoglo- bin and hemoglobin, which in the presence of metals, such as iron, act as peroxidants. Thus, it is one of the fac- tors affecting the oxidation of oxymyoglobin (light red) to metmyoglobin (brown color) during the storage of meat products. Therefore, as a result of redox oxidation, color index a is reduced (Jin et al., 2007). Italian Journal of Food Science, 2021; 33 (SP1) 123 Fish sausage fortified with natural preservatives 1 0 35 37 39 41 43 45 47 (A) (B) (C) 7 14 21 Storage time (day) C ol or in de x (L ) C ol or in de x (a ) C ol or in de x (b ) 28 35 42 0 5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 5.5 6 6.5 7 7.5 8 7 14 21 Storage time (day) 28 35 42 0 7 14 21 Storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Figure 4. Changes in color index L, b, and a (A, B, and C, respectively) of different treatments during storage (T1: con- trol, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). 1 0 0 1 2 3 4 5 6 7 8 7 14 21 Storage time (day) T V C ( lo g C F U /g ) 0 1 2 3 4 5 6 7 8 P T C ( lo g C F U /g ) 0 1 2 3 4 5 6 7 8 9 C lo st rid iu m b ot ul in iu m ( lo g C F U /g ) 28 35 42 0 7 14 21 Storage time (day) 28 35 42 0 7 14 21 Storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 1 2 3 4 5 (A) (B) Figure 5. Changes in total viable count (TVC) (A), psychro- trophic bacteria count (PTC) (B), and Clostridium botulinum (C) of different treatments during storage (T1: control, T2: control +MAP, T3: FI 5% +MAP, T4: FI 5% +GE 0.5%+ MAP, T5: FI 5% +GE 0.5% +N 0.5%+ MAP). The color index b (Figure 4C) decreased and increased during the storage period, which was consistent with the results of Giatrakou et al. (2010), who also reported that yellow index changes in meat products did not have a specific pattern and can change during storage by prod- uct type, formulation, and form of packaging. Total and psychrotrophic bacteria The results of total count bacteria (TVC) (Figure 5A) and psychotrophic bacteria (PTC) (Figure 5B) were somewhat consistent (P < 0.05) and were increased during storage time (P < 0.05). MAP decreased the growth of TVC and PTC so that the lowest values were observed in nitrite + modified atmosphere treatment (treatment 2). Gases used in modified atmosphere, such as carbon dioxide, have anti- microbial activity, and their mechanism is to dissolve in the water content of the food and produce carbonic acid, which enters the cell membrane of the microorganism and after ionization disrupts intracellular electrical balance and ultimately causes bacterial death (Hematian et al., 2012). Use of two preservatives together more effectively slowed down the growth of TVC. The lowest values were observed on all days of treatment 2 and then in treatment 1 and treat- ment 5 (P < 0.05), and similar results were observed with PTC bacteria, indicating a positive effect of the two pre- servatives as a substitute for nitrite in sausages. The lower TVC and PTC in the extract-containing treatments can be 124 Italian Journal of Food Science, 2021; 33 (SP1) Mahdavi E and Peiman A chemical and microbial properties to conventional sau- sage treatment (containing nitrite). Among the treat- ments containing natural preservatives, the best results were observed in treatment 5 (5% oil, 5% IF + 0.5% GE + 0.5% NI + MAP). Based on the results, a functional prod- uct with properties, such as fiber and natural antioxi- dants, low fat, and nitrite-free, can be produced. References Álvarez, D., Xiong, Y.L., Castillo, M., Payne, F.A. and Garrido, M.D., 2012. Textural and viscoelastic properties of pork frankfurt- ers containing canola–olive oils, rice bran, and walnut. Meat Science 92: 8–15. https://doi.org/10.1016/j.meatsci.2012.03.012 Amina, B., Parveen, S., Rather, S., Akhter, R. and Hassa, M., 2014. Effect of incorporation of apple pomace on the physic-chemical, sensory and textural properties of mutton nuggets. International Journal of Advanced Research 2(4): 974–983. AOAC, 2005. Official method of analysis (17th ed.). Washington, DC: Association of Official Analytical Chemists. Bagheri, R., Izadi Amoli, R., Tabari Shahndash, N. and Shahosseini,  S.R., 2016. Comparing the effect of encapsu- lated and unencapsulated fennel extracts on the shelf life of minced common kilka (Clupeonella cultriventris caspia) and Pseudomonas aeruginosa inoculated in the mince. Food Science and Nutrition 4(2): 216–222. https://doi.org/10.1002/fsn3.275 Biswaro, L.S., da Costa Sousa, M.G., Rezende, T.M.B., Dias, S.C. and Franco, O.L., 2018. Antimicrobial peptides and nanotechnology, recent advances and challenges. Frontiers in Microbiology 9: 855. https://doi.org/10.3389/fmicb.2018.00855 Burt, S., 2004. Essentialoils: their antibacterial propertied and potential application in foods – a review. International Food Mashinicrobiology 94(3): 223–253. https://doi.org/10.1016/j. ijfoodmicro.2004.03.022 Cegiełka, A. and Tambor, A., 2012. Effect of inulin on the physi- cal, chemical and sensory quality attributes of polish chicken burgers. Journal of Food Research 1: 161–178. https://doi. org/10.5539/jfr.v1n1p169 Choi, Y.S., Choi, J.H., Han, D.J., Hack-Youn, Y., Lee, M.A., Jeong, J.Y., et al. 2010. Effects of replacing pork back fat with vegetable oils and rice bran fiber on the quality of reduced-fat frank- furters. Meat Science 84: 557–563. https://doi.org/10.1016/j. meatsci.2009.10.012 Choi, Y.S., Choi, J.H., Han, D.J., Kim, H.Y., Lee, M.A., Kim, H.Y., et al. 2009. Characteristics of low-fat meat emulsion sys- tems with pork fat replaced by vegetable oils and rice bran fiber. Meat Science 82: 266–271. https://doi.org/10.1016/j. meatsci.2009.01.019 Choi, Y., Kim, H.-W., Hwang, K.E., Song, D.-H., Jeong, T.-J., Kim,  T.-B., et al. 2015. Effects of fat levels and rice bran fiber on the chemical, textural, and sensory properties of frankfurters. Food Science and Biotechnology 24(2): 489–495. https://doi. org/10.1007/s10068-015-0064-5 Dehbandi, A., Motallebi, A.A., Razavilar, V. and Pourgholam, R., 2014. Effect of nicin z on some of spoilage chemical and due to phenolic compounds such as cineol. Phenolic com- pounds in plant extracts destroy the microorganisms and cause liposaccharides to exit and increase the permeability of the cytoplasmic membrane to ATP4. ATP release leads to the termination of cell energy storage and cell death (Burt, 2004). NI causes a gap in the plasma membrane and the cytoplasmic components secreted in the surrounding plasma space, and the ability of the compounds to transfer to the plasma membrane is impaired, and after ATP hydro- lysis, cell death occurs. The anionic lipid present in the membrane is an active site for NI binding (Li et al., 2016). In the present study, no mold or yeast was observed. Clostridium botulinum Clostridia are bacteria that occur in the environment and on the flora of the intestines of humans and animals (Wagner et al., 2006). Results of C. botulinum (Figure 5C) showed that with increasing time, C. botulinum decreased in all treatments. According to the results of the MAP, the process of growth of C. botulinum was slowed so that on day 14 the lowest value of the bacterium was observed in control + MAP treatment (T2). The use of the preserva- tive effectively inhibited the bacterium as no treatment was observed on day 21 due to the antimicrobial activ- ity of the extracts. Phenolic compounds in plant extracts destroy microorganisms, resulting in the release of lipo- saccharides and increased permeability of the cytoplas- mic membrane to ATP. ATP withdrawal results in the depletion of cell energy storage and cell death (Burt, 2004). C. botulinum is a gram-positive bacterium, and the fact that NI is effective against gram-positive bacteria but not effective against gram-negative bacteria has been well established. NI is specifically active against vegetative cells and heat-resistant spores of Bacillus, Clostridium, and Listeria monocytogenes. The mechanism of NI antimicro- bial activity against the vegetative walls of cells is binding to the cytoplasmic membrane. NI is a cationic antimicro- bial that binds to electrons with a negative charge through electrostatic bonding. After binding, NI enters the mem- brane and creates a temporary small cavity. This process causes the rapid release of ions, amino acids, and cellular ATP (Vongsawasdi et al., 2012). Conclusion The results of the present study showed that addition of IF had a positive effect on a physicochemical and texture of sausages. Also, the use of two natural preservatives combined with modified atmosphere had almost similar 4Adenosine triphosphate https://doi.org/10.1016/j.meatsci.2012.03.012� https://doi.org/10.1002/fsn3.275� https://doi.org/10.3389/fmicb.2018.00855� https://doi.org/10.1016/j.ijfoodmicro.2004.03.022� https://doi.org/10.1016/j.ijfoodmicro.2004.03.022� https://doi.org/10.5539/jfr.v1n1p169� https://doi.org/10.5539/jfr.v1n1p169� https://doi.org/10.1016/j.meatsci.2009.10.012� https://doi.org/10.1016/j.meatsci.2009.10.012� https://doi.org/10.1016/j.meatsci.2009.01.019� https://doi.org/10.1016/j.meatsci.2009.01.019� https://doi.org/10.1007/s10068-015-0064-5� https://doi.org/10.1007/s10068-015-0064-5� Italian Journal of Food Science, 2021; 33 (SP1) 125 Fish sausage fortified with natural preservatives Kwasek, K., Thorne-Lyman, A. and Phillips, M., 2020. Canhuman nutrition be improved through better fish feeding practices? A review paper. Critical Reviews in Food Science and Nutrition 60(22): 3822–3835. https://doi.org/10.1080/10408398.2019.170 8698 Kim, I.S., Jin, S.K. and Mandal, P.K., 2011. Quality of low-fat pork sau- sages with tomato powder as colour and functional additive during refrigerated storage. Journal of Food Science and Technology 48(5): 591–597. https://doi.org/10.1007/s13197-010-0182-2 Kim, S.H. and Chin, K.B., 2007. Physico-chemical properties and changes of sarcoplasmic protein bands of chicken meat cuts with or without salt during cooking temperature. Korean Journal of Animal Science and Technology 49(2): 269–278. https://doi. org/10.5187/JAST.2007.49.2.269 Li, H., Xu, Z., Zhao, F., Wang, Y. and Liao, X., 2016. Synergetic effects of high-pressure carbon dioxide and nisin on the inacti- vation of Escherichia coli and Staphylococcus aureus. Innovative Food Science & Emerging Technologies 33: 180–186. https:// doi.org/10.1016/j.ifset.2015.11.013 Menegas, L., Colombo Pimentel, T., Garcia, S. and Helena Prudencio, S., 2013. Dry-fermented chicken sausage pro- duced with inulin and corn oil: physicochemical, microbio- logical, and textural characteristics and acceptability during storage. Meat Science 93: 501–506. https://doi.org/10.1016/j. meatsci.2012.11.003 Odunayo Olatunde, O. and Benjakul, S., 2018. Natural preservatives for extending the shelf-life of seafood: a revisit. Comprehensive Reviews in Food Science and Food Safety 17: 1595–1512. https://doi.org/10.1111/1541-4337.12390 Ozpolat, E. and Patir, B., 2015. Determination of shelf life for sau- sages produced from some freshwater fish using two different smoking methods. Journal of Food Safety 36(1): 69–76. https:// doi.org/10.1111/jfs.12214 Rahmanifarah, K., Shabanpour, B. and Shabani, A., 2013. Effect of thermal microbial inactivation and washing on quality proper- ties of fish sausage during cold storage (4°C). Journal of Aquatic Food Product Technology 24(4): 386–396. https://doi.org/10.10 80/10498850.2013.781723 Sahin, S., Sumnu, G. and Altunakar B., 2005. Effects of batters containing different gum types on the quality of deep-fat fried chicken nuggets. Journal of the Science of Food and Agriculture 85: 2375–2379. https://doi.org/10.1002/jsfa.2258 Silbande, A., Adenet, S., Chopin, C., Cornet, J., Smith-Ravin, J., Rochefort, K., et al. 2018. Effect of vacuum and modified atmo- sphere packaging on the microbiological, chemical and sensory properties of tropical red drum Sciaenopsocellatus fillets stored at 4°C. International Journal of Food Microbiology 266: 31–41. https://doi.org/10.1016/j.ijfoodmicro.2017.10.015. Shabanpour, B., Neirizi, M., noori hashem abad, Z. Study the qual- ity of silver carp (Hypophthalmichtys molitrix) Surimi during the ice storage. Iranian Food Science and Technology Research Journal, 2017; 13(1): 202–213. https://doi.org/10.22067/ifstrj. v1395i0.40216 Stepien, C.A., Snyder, M.R. and Elz, A.E., 2019. Invasion genetics of the silver carp Hypophthalmichthys molitrix across North America: differentiation of fronts, introgression, and eDNA bacterial properties in surimi of kilka (Clupeonella cultriventris caspia) stored in 4°C. Iranian Scientific Fisheries Journal 23(3): 41–56. https://doi.org/10.22092/isfj.2014.103540 Drosou, C., Kyriakopoulou, K., Bimpilas, A., Tsimogiannis, D. and Krokida, M.A., 2015. Comparative study on different extraction techniques to recover red grape pomace polyphenols from vini- fication by products. Industrial Crops and Products 75: 141– 149. https://doi.org/10.1016/j.indcrop.2015.05.063 Eldemery, M.E., 2010. Effect orange albedo as a new source of dietary fiber on characteristics of beef burger. In The 5th Arab and 2nd International Annual Scientific Conference on: Recent Trends of Developing Institutional and Academic Performance in Higher Specific Education Institutions in Egypt and Arab World. Mansoura University, Egypt. Fan, W., Chi, Y. and Zhang, S., 2008. The use of a tea polyphenol dip to extend the shelf life of silver carp (Hypophthalmicthys molitrix) during storage in ice. Food Chemistry 108: 148–153. https://doi.org/10.1016/j.foodchem.2007.10.057 Fidan, H., Stefanova, G., Kostova, L., Stankov, S., Damyanova, S., Stoyanova, A., et al. 2019. Chemical composition and antimi- crobial activity of Laurus nobilis L. essential oils from Bulgaria. Molecules 24: 804. https://doi.org/10.3390/molecules24040804 Giatrakou, V., Ntzimani, A. and Savvaidis, I., 2010. Effect of chitosan and thyme oil on a ready to cook chicken product. Food Microbiology 27: 132–136. https://doi.org/10.1016/j.fm.2009.09.005 Hematian Sourki, A., Ghiafeh Davoodi, M., Tabatabaei Yazdi, F., Mortazavi, S.A., Karimi, M., Razavizadegan Jahromi, S.H., et al. 2012. Studies on the effects of packaging type and modified atmosphere on properties of Barbari bread fortified with whole soy flour. Food Science and Technology 9(36): 77–85. Hernández-Hernández, E., Ponce-Alquicira, E., Jaramillo-Flores, M.E. and Guerrero Legarreta, I., 2009. Antioxidant effect rosemary (Rosmarinus officinalis L.) and oregano (Origanum vulgare L.) extracts on TBARS and color of model raw pork batters. Meat Science 81: 410–417. https://doi.org/10.1016/j. meatsci.2008.09.004 Huang, S.C., Shiau, C.Y., Liu, T.E., Chu, C.L. and Hwang, D.F., 2005. Effects of rice bran on sensory and physico-chemical properties of emulsified pork meatballs. Meat Science 70: 613–619. https:// doi.org/10.1016/j.meatsci.2005.02.009 Institute of Standards and Industrial Research of Iran (ISIRI), 1994. Searching and enumeration of Clostridium perfringens in food, (Standard No. 2197). Iran. Institute of Standards and Industrial Research of Iran (ISIRI), 2008. Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of yeasts and moulds – Part 1: Colony count technique in products with water activity greater than 0.95. (Standard No. 10899-1). Iran. Javadian, S.R., Shahoseini, S.R. and Ariaii, P., 2017. The effects of liposomal encapsulated thyme extract on the quality of fish mince and Escherichia coli O157: H7 inhibition during refriger- ated storage. Journal of Aquatic Food Product Technology 26(1): 115–123. https://doi.org/10.1080/10498850.2015.1101629 Jin, S.K., Kim, I.S., Choi, Y.J. and Hur, S.J., 2007. The development of sausage including meat from spent laying Hen Surimi. Poultry Science 86: 2676–2684. https://doi.org/10.3382/ps.2006-00451 https://doi.org/10.1080/10408398.2019.1708698� https://doi.org/10.1080/10408398.2019.1708698� https://doi.org/10.1007/s13197-010-0182-2� https://doi.org/10.5187/JAST.2007.49.2.269� https://doi.org/10.5187/JAST.2007.49.2.269� https://doi.org/10.1016/j.ifset.2015.11.013� https://doi.org/10.1016/j.ifset.2015.11.013� https://doi.org/10.1016/j.meatsci.2012.11.003� https://doi.org/10.1016/j.meatsci.2012.11.003� https://doi.org/10.1111/1541-4337.12390� https://doi.org/10.1111/jfs.12214� https://doi.org/10.1111/jfs.12214� https://doi.org/10.1080/10498850.2013.781723� https://doi.org/10.1080/10498850.2013.781723� https://doi.org/10.1002/jsfa.2258� https://doi.org/10.1016/j.ijfoodmicro.2017.10.015� https://doi.org/10.22067/ifstrj.v1395i0.40216 https://doi.org/10.22067/ifstrj.v1395i0.40216 https://doi.org/10.22092/isfj.2014.103540 https://doi.org/10.1016/j.indcrop.2015.05.063� https://doi.org/10.1016/j.foodchem.2007.10.057� https://doi.org/10.3390/molecules24040804� https://doi.org/10.1016/j.fm.2009.09.005� https://doi.org/10.1016/j.meatsci.2008.09.004� https://doi.org/10.1016/j.meatsci.2008.09.004� https://doi.org/10.1016/j.meatsci.2005.02.009� https://doi.org/10.1016/j.meatsci.2005.02.009� https://doi.org/10.1080/10498850.2015.1101629� https://doi.org/10.3382/ps.2006-00451� 126 Italian Journal of Food Science, 2021; 33 (SP1) Mahdavi E and Peiman A Wagner, K., Vorauer-Uhl, K. and Katinger, H., 2006. Nebulization of liposomal rhcu/zn Sod with a novel vibrating membrane neb- ulizer. Journal of Liposome Research 16: 113–125. https://doi. org/10.1080/08982100600680733 Yanar, Y., 2007. Quality changes of hot smoked catfish (Clarias Gariepinus) during refrigerated storage. Journal of Muscle Foods 18: 391–400. https://doi.org/10.1111/j.1745-4573.2007.00094.x Zhou, W.W., Meng, L., Li, X., Ma, L. and Dai, R., 2010. Effect of the interaction between carrageenan, gellan gum and flax- seed gum on quality attributes of starch-free emulsion-type sausage. Journal of Muscle Foods 21(2): 255–267. https://doi. org/10.1111/j.1745-4573.2009.00180.x metabarcode detection. PLoS One. 14(3): e0203012. https://doi. org/10.1371/journal.pone.0203012 Valipour Kootenaie, F., Ariaii, P., Khademi Shurmasti, D. and Nemati,  M., 2017. Effect of chitosan edible coating enriched with eucalyptus essential oil and α-tocopherol on silver carp fillets qual- ity during refrigerated storage. Journal of Food Safety 37(1): e12295. https://doi.org/10.1111/jfs.12295 Vongsawasdi, P., Nopharatana, M., Supanivatin, P. and Promchana, M., 2012. Effect of nisin on the survival of Staphylococcus aureus inoculated in fish balls. As Journal of Agricultural – Industry 5(01): 52–60. Vural, H., 2003. Effect of replacing beef fat and tail fat with interes- terified plant oil on quality characteristics of Turkish semi-dry fermented sausages. European Food Research and Technology 217(2): 100–103. https://doi.org/10.1007/s00217-003-0727-y https://doi.org/10.1080/08982100600680733� https://doi.org/10.1080/08982100600680733� https://doi.org/10.1111/j.1745-4573.2007.00094.x� https://doi.org/10.1111/j.1745-4573.2009.00180.x� https://doi.org/10.1111/j.1745-4573.2009.00180.x� https://doi.org/10.1371/journal.pone.0203012 https://doi.org/10.1371/journal.pone.0203012 https://doi.org/10.1111/jfs.12295 https://doi.org/10.1007/s00217-003-0727-y� _GoBack