The correlation among residual nitrites, biogenic amines, N‐nitrosamine formation, and degradation occurrence of punicalagin α/β, rosmarinic acid, carnosol, and carnosic acid in extract‐treated sausage during storage

Abstract The aim of this study was to investigate the relation between residual α‐ and β‐punicalagin in Punica granatum L.; PPE and rosmarinic acid, carnosol, and carnosic acid in Salvia eremophila (SE) with residual nitrites, biogenic amines (cadaverine, putrescine, and histamine), N‐nitrosodimethylamine (NDMA), microbial counts, lipid oxidation indices, and color values in extract‐treated sausage over 14 days of storage. Sausage containing SE + nitrite 60 ppm (SSN) showed minimum levels of the residual nitrites (13.14 mg/kg), NDMA (0.74 ± 0.05 μg/kg), and biogenic amine (histamine, 1.8 mg/kg; cadaverine, 3.7 mg/kg; and putrescine, 4.3 mg/kg) due to retarded degradation rate of 285.84–216.44 mg/kg; rosmarinic acid, 41.62–33.16 mg/kg; carnosol, and 88.70–76.73 mg/kg; carnosic acid over storage time. The first‐order kinetic model fitted well for the degradation of rosmarinic acid and carnosol acid in SSN sample. TBA value remained below the threshold limit (0.32 mg kg−1) through 14 days for SSN. Second‐order and zero‐order reaction models had the best agreement with sausages' PV and TBA values, respectively. After 2 weeks of storage, E. coli and Cl. perfringens counts in the SN120 (sausage containing 120 ppm nitrite) and SSN were significantly lower than the other samples (p < .05), with the values 2.1 and 1.5 log cfu/g for SN120 and 2.2 and 1.6 log cfu/g for SSN formulation. Conversely, oxidation indices, residual nitrites, NDMA, and biogenic amine increased in sausage samples containing PPE extracts (SPN) owing to total degradation of α‐ and β‐punicalagin during storage. The results indicated that SE can be used as potential co‐preservative by reducing the levels of required nitrite in food industry.

The use of plant-based extracts as antimicrobials and antioxidants in food products has gained great attention (Ayaseh et al., 2022;Mahajan et al., 2015;Yaghoubi et al., 2023).In this regard, replacing synthetic antioxidants with natural compounds in various products causes to postpone oxidative degradation of lipids and microbial growth and improves nutritional value of foods (Mahajan et al., 2015).
Moreover, numerous studies stated that pomegranate and sages as natural antioxidants have been used to prohibit lipid oxidation and discoloration and extend shelf life of meat products (Kumar et al., 2015;Shah et al., 2014).Furthermore, natural antioxidants act as the nitrite scavenging and had an important role to inhibit Nnitrosamine formation (Yao et al., 2016).
The use of natural antioxidants in meat products has been extensively studied.Therefore, the objective of this study was to assess the effect of using Punica granatum L. and Salvia eremophila extracts with and without the use of nitrite in sausage formulation on the residual nitrites, biogenic amines (cadaverine, putrescine, and histamine), N-nitrosamine, oxidative indices (PV and TBA), microbial count, and color values (L*, b*, c*, oxymyoglobin and metmyoglobin).
In addition, the residual amount of punicalagins, rosmarinic acid, carnosol, and carnosic acid were determined in the sausages formulated with extracts during storage time.

| Plant material extraction
The peel of P. granatum was collected from a garden in Ghasrodasht of Shiraz (Iran, September 2019) at the fruit ripening stage.The aerial parts of the S. eremophila were also collected from Shiraz surrounding during the flowering stage of the plants.After grinding, the fresh pomegranate peels and aerial part of sage were subjected to solvent extraction using aqueous methanol (80% v/v) in the absence and presence of acetic acid (HOAc, 5% v/v).The PPE and SE were filtered and concentrated in vacuum oven at 40°C by following the method of Hashemi et al. (2018).

| Total phenolic content
Folin-Ciocalteu was used to measure total phenolic compounds in the resulting extracts as described by Pakfetrat et al. (2020).In order to estimate the total content of phenolics in plant compounds, a standard curve was plotted using various concentrations of gallic acid as mg/g.

| Radical-scavenging activity using DPPH assay
The antioxidant capacity of sage and pomegranate extracts was evaluated using the modified method conducted by Zare et al. (2021).The percentage of inhibition was calculated from the following Equation (1): A sample represents the absorbance of DPPH radical solution mixed with sample extract, while A Control represents the absorbance of DPPH radical solution in methanol.Quercetin was used as standards.Based on linear regression equations relating to levels of extract versus DPPH inhibition percentage, the IC 50 values were calculated using Curve Expert and Microsoft Excel.

| Antibacterial minimum inhibitory concentration
Escherichia coli strain (ATCC 25922) as gram-negative and Clostridium perfringens strain (RITCC 2752) as gram-positive bacteria were purchased from the Iranian Research Organization for Science and Technology.Stock cultures including Mueller-Hinton Broth (MHB; Merck, Darmstadt, Germany) for E. coli and Fluid Thioglycollate Media (FTG; Gibco, Paisley, Scotland) for C. perfringens were prepared and kept at 4°C until further use (Moarefian et al., 2012).Antimicrobial effect of sage and pomegranate extracts was evaluated by MIC (minimum inhibitory concentration) determinations.In order to determine MIC, a broth dilution method was used with some modifications (Moarefian et al., 2012).E. coli suspension was obtained by microbial count of 1.5 × 10 8 cfu/mL and turbidity equivalent to 0.5 McFarland standard; 100 mL of SE and PPE dispersion in Tween 20 was mixed to 100-mL MHB medium in microtiter plate wells and inoculated with 10-mL E. coli suspension at 37°C for 24 h.Bacterial growth was evaluated by turbidity developed in broth culture and for determining MIC, cell suspensions were subcultured on MacConkey medium (Merck, Darmstadt, Germany) in triplicate.The same method was applied to measure MIC concentrations of the SE and PPE against C. perfringens.
Bacterial suspension was prepared in FTG with microbial count of 10 8 cfu/mL.SE and PPE dilution was done in 1 mL of FTG medium in test tube, and 100 mL of C. perfringens was inoculated.Liquid paraffin (0.5 mL) was added on top of the culture medium to maintain anaerobic conditions in test tubes and incubation was done at 37°C for 24 h.Turbidity was used as indicator of growth to determine MIC content.

| Sausage production
Sausage formulations were produced from ground beef.Three sources of ground beef from chuck section were mixed together, thereafter divided into three groups and three sausage formulations were prepared.Sausage samples were manufactured by using the following formula: 80% beef meat, 1% soybean powder, 2% gluten, 4% wheat flour, 0.4% Na 5 P 3 O 10 , and 0.04% ascorbic acid.

| Sausage preparation for HPLC and LC-DAD-ESIMS analysis
First, weighing (5 g) of the samples and rinsing with n-hexane (15 mL) were executed, followed by their transfer to a separatory funnel having 5-mL n-hexane and then extraction using nhexane-saturated acetonitrile (150 mL).The organic solvent layer was moved to a different funnel for the residual removal.A roundbottom flask was employed for the collection of acetonitrile phase and an SB-1200 water bath (Eyela, Japan) along with an N-1200A vacuum rotary evaporator (Eyela, Japan) was applied for the evaporation of this phase to the final 3-to 4-mL volume.Small solvent portions (1:1 ratio of acetonitrile: iso-propanol) were used for rinsing the flask, succeeded by its transfer to a 10-mL flask and collection of the exact amount through repeating the rinsing process (Choi et al., 2019).
A Shimadzu LCMS-2010EV equipped with an ESI mass column and SPD-M20A diode array detector was used for the LC-DAD-ESI-MS analyses.Column's Shim Pack XR-ODS C 18 column (75 × 3.0 mm, 2.2 μm) and LC pump (LC 20 AD) with flow rate 0.25 mL/min was used.Injection volume was 5 μL of the 10 timesdiluted the sample solutions.In negative modes, the ESI was used as an ionizing source.The MS parameters include as: MS detector voltage: ±1.5 kV, interface: ±4.5 kV, CDL: ±10 V, and Q-array (Rf: ±150 V) voltages uploaded from the tuning file.There were three parameters that were scanned: mass range 100-1000 units, nebulizer gas N 2 , flow rate 1.5 L/min, and heat block and CDL temperatures 230 and 275°C, respectively.On a DAD detector, the Ultraviolet spectrum was recorded between 190 and 600 nm, and a temperature of 40°C was set for both the cell and the column.The HPLC apparatus was a Knauer analytical HPLC with an Azura (p 6.1 L) pump and a Knauer Ultraviolet detector (2.1 L).The analytical column was a Eurospher-100 C18 (Eurospher-100 C18, 250 × 4.6 mm, Knauer, Germany).
A 0.45-m membrane filter (Whatman, Amersham, UK) was used to filter the mobile phase and degassed under vacuum.The flow rate and injection volume were set at 1.0 mL/min and 20 μL.

| Residual nitrite determination
Nitrite level was identified concerning to the previous report of Wang et al. (2021).The dry sausage (5 g) was minced by meat grinder and placed into conical flask, saturated borax solution (12.5 mL), and then distilled water (150 mL; 70°C) was added.The resulting mixture was placed in boiling water bath (15 min), then cooled to room temperature.Reagent K 4 Fe(CN) 6 •3H 2 O (5 mL; 106 g/L) was added to the mixture and shaken.Afterward, Zn (CH 3 COO) 2 •2H 2 O solution (5 mL; 220 g/L) was added.Subsequently, distilled water was incorporated into the mixture to reach the final volume of 200 mL.
Using filter paper (20 μm pore size, Whatman, Mosu Scientific Equipment Co., China), the mixture was filtered and collected.paminobenzenesulfonic acid (2 μL; 4 g/L) was added to the sausage filtrate (40 mL).After standing for 5 min, N-ethylenediamine standard solution (1 mL; 2 g/L) was inserted into the mixture and stood at room temperature for 15 min.Absorbance of unknown sample and standard nitrate were measured at 538 nm by spectrophotometer (AA-680, Shimadzu, Kyoto, Japan).

| Determination of biogenic amines
Biogenic amine content was measured according to Wang et al. (2021).

| N-nitrosamines analysis
Ten grams of minced sausages was mixed with NaOH solutions (10 mL; 0.1 mol/L) and sonicated for 15 min at 25°C.Then, methanol (20 mL) was added and homogenized for 3 min.After centrifugation at 10,000 g for 10 min at 4°C, homogenates were filtered with filter paper.The methanol-water extract (15 mL) was combined with NaCl solution (5 mL; 20%) and loaded on a ChemElut column with a capacity of 20 mL.A 50-mL solution of dichloromethane was used to elute the column after it had been equilibrated for 20 min.After being concentrated to 1 mL under reduced pressure and evaporated in a water bath under nitrogen conditions (40°C), the eluent was analyzed by GC coupled to a quadrupole mass selective spectrometer (Agilent Technologies, Palo Alto, CA, USA).Agilent DB-5MS column (30 m × 0.25 mm × 0.25 μm) with injector temperature 230°C was used for chromatographic separation.Specifically, the oven was programmed to run for 2 min at 50°C, 2 min at 150°C, 5 min at 250°C, 1 min at 20°C/min, and 1 min at 280°C.
There was a velocity of 1.0 mL/min for the helium carrier gas.Using electron-impact ionization, ions were produced.There were temperatures of 230°C and 250°C for the ion source and transfer line.The injection volume was 1 μL.By comparing retention indices and MS spectra with pure standards, the compounds were identified.

| Microbial analysis
A portion (10 g) of sausages aseptically transferred into individual stomacher bag containing 90 mL of Ringer's solution.Samples were homogenized using stomacher for 2 min to prepare the initial dilution.E. coli was counted following 48-h incubation at 37°C on Baird Parker agar supplemented with sorbitol McConkey agar (Difco) (Ghaderi-Ghahfarokhi et al., 2016;Ibrahim et al., 2011).The stock culture of the Cl.Perfringens strain was prepared in Duncan and Strong medium.Spore suspension was moved to microtubes and centrifugated.The spores were resuspended in sterile peptone water (0.1%), heat shocked for 20 min at 75°C, and manually blended ( 2) with meat paste.The paste was cooked in water bath for 1 h at 75°C and then cooled at 20°C.For enumeration of Cl.Perfringens, sample (10 g) was homogenized in sterile salt solution (90 mL, NaCl, 0.85%) using a sterile stomacher (Sanyo, Japan) and kept in anaerobic culture in sulfite polymyxin sulfadiazine agar.

| Kinetic modeling
Zero-order, first-order, and second-order kinetic models were used to determine the reaction rates of various parameters including rosmarinic acid, carnosol, and carnosic acid, as well as oxidative indices (PV and TBA).For the zero-order reaction, the equation can be expressed as follows: where C 0 is the initial concentration, C is the concentration at time t, and k is the reaction rate constant of the zero-order reaction.
Assuming first-order kinetics, the equation can be expressed as follows (Zhang et al., 2012): where k is the reaction rate constant of the first-order reaction.
For the second-order kinetics, the rate of reaction can be determined as follows: where k is the reaction rate constant of the second-order reaction.

| Statistical analysis
The statistical analyses were carried out using IBM SPSS statistics, version 22.0 (IBM Corp, Armonk, NY, USA).Statistical assumptions such as normality of data distribution and homogeneity of variances were tested by Shapiro-Wilk and Levene's tests, respectively, before conducting one-way ANOVA to check for differences regarding the effect of different extracts (PPE and SE) and the effect of different storage time on the oxidation indexed, biogenic amines, residual nitrites, NDMA, color, and microbial counts.Duncan's multiple range test was used for post hoc analyses.Statistical significance was set at 5%.The results were presented as mean ± SD.

| Phenolic compounds, radical scavenging, and antimicrobial activities of PPE and SE
The level of phenolic compounds (145.43 ± 3.82 mg gallic acid/g plant) and (123.37 ± 2.83 mg gallic acid/g plant) which was in accordance with lower DPPH IC 50 (19.28± 1.35 μg/mL) and (27.09 ± 1.65 μg/mL) in acidic and nonacidic crud alcoholic of pomegranates resulting fractions, respectively (Table 1 and   Figure 1).Pomegranate peel extract was mostly discovered as a wealthy source of antioxidants because hydrolyzable tannins such as punicalagin and ellagitannins are predominantly located in the fruit mesocarp and peel of pomegranates which provide 92% of antioxidant activity (Aloqbi et al., 2016;Feng et al., 2017).The radical scavenging activity of extracts in acid was markedly (p < .05)more than nonacid extraction.Acidifying the extraction solution affects the dissociation of phenols and acid-induced dissociation, the gallotannins, ellagitannins, gallagic acid, and gallagyl esters are hydrolyzed to several ions and numerous compounds such as gallic acid, ellagic acid, quercetin, punicalagin, etc.As a result, the phenolic contents and antioxidant potential of the PPE extracts increased (Table 1).Obtained IC 50 in the present study was highly greater than those previously reported for pomegranate peel extract in a DPPH test (IC50 4.9 μg/mL) (Hugo & Hugo, 2015).In previous literatures, aqueous pomegranate peel extract exhibited high phenolic level as 161.25 and 140 mg/g in terms of catechin equivalent (Hugo & Hugo, 2015).SE showed DPPH free radical scavenging activity with an IC 50 (59.76± 2.51 μg/mL and 62.26 ± 4.89 μg/mL) with total phenolic content (79.08 ± 4.26 mg gallic acid/g plant and 77.67 ± 4.83 mg gallic acid/g plant) under acidic and nonacidic conditions, respectively (Table 1).No significant difference was observed between acidic and nonacidic treatments.Significantly (p ˂ .05)lower antioxidant activity was demonstrated for SE compared to quercetin (20.18 μg/ mL) and pomegranate.Three major compounds in S. eremophila, namely, rosmarinic acid, carnosol, and carnosic acid were identified (Figure 1).Rosmarinic acid is characterized as the major hydrophilic compound while carnosol and carnosic acid are main lipophilic constituents.Rosmarinic acid as phenylpropanoid compound comprises an ester of caffeic acid and 3,4-dihydrophenyl lactic acid.Carnosic acid is a labdane-type diterpene and carnosol is a major oxidation product of carnosic acid which are proven with high antioxidative (4) capacities (Birtić et al., 2015).Concerning data obtained by Table 1, from the antioxidative point of view, the pomegranate is depicted higher antioxidant activity.
Bioactive compounds derived from pomegranate and sage have been represented to be a good alternative to prevent growth of various pathogenic bacteria.The antimicrobial activity of PPE and SE against common food spoilage and pathogenic bacteria such as E. coli and Cl.perfringens was examined (Table 1).In the case of gram-negative bacteria (E.coli), there was no significant difference (p > .05) in MIC of pomegranate and sage extracts under acidic and nonacidic conditions while in the case of gram-positive bacteria (Cl.perfringens), the antibacterial effects of the SE extract (0.35 mg/ mL media) are higher than PPE extracts (2.5 mg/mL media; Table 1).Kanatt et al. (2010) stated that pomegranate peel extract at concentration of 0.01% could sufficiently prevent gram-positive bacteria such as Staphylococcus aureus and Bacillus cereus; however, concentration of 1% could not inhibit gram-negative bacteria growth, namely, E. coli, Salmonella enteritidis, and Pseudomonas spp.Hugo and Hugo (2015) revealed that pomegranate extract was less effective against E. coli and P. aeruginosa.

| LC-MS of sausage containing PPE and SE
HPLC analysis showed that the concentration of methanol extracted α-and β-punicalagin was 67 and 232 mg/kg, respectively, before being added to the sausage (Figure 1).As evidenced by the LC-MS analysis, there were no punicalagins in the sausage prepared with pomegranate extract from the first to the 14th day of storage time, implying all of them had been converted or decomposed.
Furthermore, the new spectra concerning the derivatives of quinic acid and the derivatives of gallic acid and sucrose are found as major compounds in pomegranate peel extract-treated sausage (Figure S1).
The concentration of rosmarinic acid, carnosol, and carnosic acid compounds in sage extracted with methanol was 313.13, 41.61, and 86.94 mg/kg, before adding to sausage formulation which reduced to 258.06, 36.08, and 82.46 mg/kg (Table 2), respectively, in SS sausage during 14-day storage time.The same reduction pattern was observed by rosmarinic acid (216.44 mg/kg), carnosol (33.15 mg/kg), and carnosic acid (76.73 mg/kg) in SSN sausage further 14th day of storage time (Figure S1).
In SS sausage formulation, rosmarinic acid concentrations declined during storage while the ratio of carnosic acid to carnosol is not significantly affected in SS-treated sausages (Table 2).
Notwithstanding, the addition of nitrite to the SS-treated sausages (SSN) formulation appears to accelerate the decaying of the three antioxidative substances rosmarinic acid, carnosol, and carnosic acid.These results implied that these compounds might have the ability to interact with sausage ingredients including probably nitrite to prevent its conversion to nitrosamine.

| Degradation modeling of rosmarinic acid, carnosol, and carnosic acid in the sausage samples
Experimental results of changes in rosmarinic acid, carnosol, and carnosic acid over time are shown in Figure 2. According to Figure 2a-c, the amounts of rosmarinic acid and carnosic acid in both SS and SSN sausage samples decrease during the storage time.Figure S1d,e exhibits that the amount of carnosol in both sausage samples rapidly decreased on the first day, after that, decreasing pattern proceeds at a slower rate and the changes are very small, so that its concentration is almost constant on day 14 compared to day 7.
In order to fit the data to the first-and second-order reaction models, plots of ln (C/C 0 ) and 1/C versus t were drawn, and R 2 and k values were calculated (Table 2).The comparison of R 2 values for rosmarinic acid and carnosol acid shows that both the first-and second-order reaction models are in good agreement with the experimental data, while the carnosol values in both sausage samples do not fit to any of the first-and second-order models.The results of fitting the data to the first-order kinetic equation are also shown in Figure 2. The first-order reaction rate constants (k) for rosmarinic acid and carnosol acid in SSN sample are greater than SS sample, which indicates that these two compounds degrade faster in SSN sample.

| Lipid oxidation
Lipid oxidation causes sensory disorders including loss of nutrients, off-flavor, rancid odor, drip losses, discoloration, shelf life shortening of meat product, and the accumulation of carcinogenic and/or mutagenic compounds (Aliakbarlu & Khalili Sadaghiani, 2015;Mancini et al., 2015).The oxidative quality of the products was evaluated by the determination of PV and TBA (Table 3) which are known indicators to demonstrate the amount of the primary and secondary lipid oxidation products.was shown between PV of SN120 (5.2 ± 0.5) and SSN (6.2 ± 0.4), both PVs are acceptable for sausage formulations (˂25 meq O 2 /kg oil), indicating that reducing nitrite around 50% and using SE could progressively postpone the lipid oxidation.
Sausage curing might completely or partially denature myoglobin.
The supportive effect of globin on Fe +2 is disappeared.Ferrous metal (Fe 2+ ) ions and free radical as reactive compounds can initiate lipid peroxidation.It is proved that antioxidant components can bind with metal ions such as Fe 2+ and inhibit free radical formation.As a consequence, the propagation of free radical reactions can be prevented using the chelation of transition metal ions (Aloqbi et al., 2016).In line with our study, Hugo and Hugo (2015) stated that TBA of the samples treated with or/and without PPE enhanced during 20 days of chilled storage.However, Aloqbi et al. (2016) reported that the amount of Fe 2+ -ferrozine complex significantly reduced in a dose-dependent manner in the presence of punicalagin in pomegranate juice compared with BHT at the same concentration.In the present study, the rising pattern of PV in SP formulation is ascribed to completely decompose or/and convert α-and β-punicalagin as potential antioxidant to other compounds, showing no peak bands in LC-mass spectra in the sausages (Figure S1).High antioxidant activity of SE has been identified to be not only based on the main phenolic compounds such as rosmarinic acid, carnosic acid, and carnosol but also some minor constitutes such as monoterpenoids, labdane, sesquiterpenoid, ent-kaurane, rearranged abietane, abietane, tanshinone, icetexane, clerodane, and pimarane diterpenoids (Asadollahi et al., 2019;Jassbi et al., 2016).
The antioxidant properties of carnosol and carnosic acid presumably owing to the presence of a catechol moiety.They act in a hydrogen donator system to interfere with the free radical propagation process (via circumvents oxidation), metal chelators, and singlet O 2 quenchers which are able to prevent or postpone unsaturated fatty acids and triglycerides against oxidation in bulk and emulsified lipid systems (Birtić et al., 2015;Naveena et al., 2013).to the water-soluble extract (rosmarinic acid) (Naveena et al., 2013).
These findings indicated that higher amount of phenols does not necessarily mean higher antioxidant activity.Both carnosic acid and carnosol can potentially prevent lipid oxidation against reactive oxygen species (ROS)-induced lipid peroxidation, however, behaving differ route in their reaction with ROS.Carnosol showed resistance to oxidation and its concentration remained constant in the presence of hydroxyl radical or 1 O 2 compared to carnosic acid because carnosic acid represents great reactivity toward ROS and is easily oxidizable (Loussouarn et al., 2017).However, loss of carnosol also occurs when subjected to ROS in a lipid medium.This could suppose that carnosol has the potential to interact directly with lipid oxidation-derived  products and can be degraded.Oxidation of carnosic acid was proved by a reduction in the carnosic acid peak and a concomitant production of carnosol (Loussouarn et al., 2017).Consistent with present study, Doolaege et al. (2012) noted that rosmarinic acid, carnosic acid, and carnosol from SE could efficiently retard lipid oxidation in liver pâté.
Therefore, it was advisable that sausage samples containing 60 ppm nitrite and SE displayed lower TBA quantities than threshold which can be considered acceptable samples.2. According to Table 2, the R 2 values of the second-order kinetic model have the highest value for most sausage samples, indicating that the changes in PV is better fit to the second-order model.

| Modeling of PV and TBA values of sausage samples
The results show that the reaction rate constants, k, in SN60, SS, and SPN samples are almost equal, and as can be seen, the final values of PV after 14 days are almost the same for all three samples (Figure 2).
SSN and SN120 samples have the highest amount of k, respectively, after the above three samples.As a result, PV of 14 days for SN120 sample has the lowest value.
On the other hand, in all six sausage samples, the zero-order reaction model fits the TBA values well.So that the R 2 value in all samples TA B L E 3 Peroxide values (meqO 2 /Kg), TBA (mg MDA/Kg), and kinetic parameters for PV and TBA of the sausage treated with pomegranate and salvia extract after first, 7th, and 14th day of storage.

PV's kinetic parameters
Zero-order kinetic First-order kinetic Second-order kinetic

TBA's kinetic parameters
Zero-order kinetic First-order kinetic Second-order kinetic is greater than The comparison of the values of the reaction rate constant, k, of the samples shows that the SP sample has the highest value of k, followed by the SS, SN60, SPN, SN120, and SSN samples, respectively.However, compared to SP, the values of k of these five samples are very close to each other.In general, SP and SSN samples have the highest and lowest 14-day TBA value, respectively.Color measurement of meat products and their appearance is considered as essential part of meat researches, implying freshness and wholesomeness, which, in turn, the customer's decisions to accept or reject foodstuff.Thus, the inhibition of detrimental changes in meat pigment is crucial (Ghaderi-Ghahfarokhi et al., 2016).L*, a*, and b* of treated sausages with sage and pomegranate during chilled storage are exhibited in Table 4.

|
L* values of sausages illustrated decreasing trend during storage.
As can be seen, L* value of SN120 (58.2 ± 1.7), SN60 (58.0 ± 0.7), and SSN (58.5 ± 0.5) were obviously more than others, after 14-day storage time.The lowest L* value of sausage was measured in SP (46.2 ± 1.3).Brown color development occurs by Maillard reaction (He et al., 2012).The lipid oxidation process results in increased myoglobin oxidation (metmyoglobin) and meat discoloration.Regarding the results obtained by Gardeli et al. (2019), changes in color of sausage containing pomegranate can be induced by anthocyanins decomposition as well as the interaction between anthocyanins and condensed tannins that could favor the formation of polymeric anthocyanins.He et al. (2012) and Naveena et al. (2013) reported that the addition of pomegranate juice and rind pomegranate powder extract changed the chicken meat patties from pale raw to grayish color (He et al., 2012;Naveena et al., 2013).
In the case of a* values, the surface color of sausages was influenced by formulation at any storage time (p < .05).A substantial reduction of a* value in differently treated samples was found through the storage period (p < .05)(Table 4).This was associated with the conversion of red oxymyoglobin to brown metmyoglobin.
The reducing a* parameter is in line with results obtained by Hugo 3.4.2| Oxymyoglobin (%) and metmyoglobin (%) content The desirable red color of the meat is commonly related to the ratio proportion of red myoglobin, bright-red oxymyoglobin, and graybrown metmyoglobin (Ghaderi-Ghahfarokhi et al., 2016).According to consumer's preference, the percent of MetMb should not exceed greater than range 30%-40% of total pigments on the surface of fresh meat.OxyMb of all sausages decreased when the storage time increased (Table 4) which was accompanied by the increase in for raw pork burgers incorporated with Moringa oleifera leaf extract during storage at 4°C (Falowo et al., 2017;Muthukumar et al., 2014;Shah et al., 2015).Liu et al. (2015) stated that beef patties treated with several natural antioxidants led to increase in the content of MetMb after 6 days of storage and then reduce MetMb concentration after 8 days of storage.

| Biogenic amine accumulation
Biogenic amines are organic bases with aliphatic, aromatic, or heterocyclic structures that are mainly produced from decarboxylation of amino acid by microbes in food, especially in meat and fish products.As shown in Figure 3a-c    biogenic amine in this study, followed by cadaverine and histamine.

| Residual nitrites and NDMA content
As seen in When the meat pH reduces to less than 6.0, the nitrite can be turned into nitrous acid or nitric oxide, which can react with polyphenols or endogenous substances, reducing nitrite residues (Wang et al., 2015).Such reductions in residual nitrite were probably due to the reaction of the bio-compounds present in plant polyphenols.As found in Table 2, the reduction pattern in the amount of rosmarinic acid, carnosol, and carnosic acid compounds in sage extract was lowered from 313. 13-258.06, 41.61-36.08, and 86.94-82.46mg/ kg, before adding to sausage formulation, respectively, to 297.53-216.44, 40.62-33.15, and 81.78-76.73mg/kg in SSN sausage further 14th day of storage time.It could be implied that SE had nitrite scavenging ability due to bioactive compounds such as rosmarinic acid, carnosol, and carnosic acid.
However, the use of the PPE (SPN) had no statistical influence on the residual nitrite with SN60 owing to total decomposition of α-and β-punicalagin in PPE during storage.Meanwhile, nitrogen compounds in muscle matrix are dynamically transformed during storage, which may explain the gradual decrease in residual nitrite content regardless of treatment (De Mey et al., 2014).A similar trend was also reported by Li et al. (2013) and Wang et al. (2015) for dry-cured sausages, indicating that green tea and grape seed polyphenols substantially declined residual nitrite.In line with the present study, Deng et al. (2022) incorporated tea polyphenol, apple polyphenol, and cinnamon polyphenol into dry-fried bacon and concluded that plant polyphenols potentially can be used as natural antioxidants for reducing nitrite application level while also ameliorating the safety of bacon.
The amount of NDMA gradually augmented during the storage regardless of treatment (Table 4).The highest NDMA content was observed in the SN120 and the lowest in those treated with SSN throughout the whole processing stages.After storage for 2 weeks, NDMA concentration reached 2.12 and 0.74 mg/kg in SPN and SSN, respectively.The changes in NDMA levels over storage might depend on chemical reactions between NDMA precursors (residual nitrite) in meat products (Wang et al., 2015).

| Antimicrobial activity
The antimicrobial activity of PPE and SE incorporated into sausage in the absence and presence of nitrite against common food spoilage and pathogenic bacteria including Cl. perfringens and E. coli were investigated.E. coli and Cl.perfringens were around 4.4 and 3.2 log cfu/g in raw material.After 2 weeks of storage, E. coli and Cl.perfringens counts in the SN120 and SSN were significantly lower than the other samples (p < .05),with the values 2.1 and 1.5 log cfu/g for SN120 and 2.2 and 1.6 log cfu/g for SSN formulation (Figure 4).Our results discovered that adding antioxidants for sausage curing can effectively reduce E. coli and Cl.perfringens populations during ripening and storage.Different studies revealed that most plant extracts are inefficient against gramnegative organisms.Compared to previous results gained with diverse aqueous extracts, the obtained data are similar in some cases and even better (Kanatt et al., 2010;Yin et al., 2016).The most probable mechanism of antimicrobial activity by phenolic compounds has been assumed to be owing to the disruption of the cell membrane and increased membrane permeability (Ibrahim et al., 2011).The changes in rosmarinic acid and carnosic acid with time were evaluated by the first-order kinetic model.According to the k values, these compounds are degraded at a higher rate in SSN sample than in SS sample.Moreover, PV changes with time were in good agreement with the second-order kinetic model, while the zero-order reaction model had the best fit with the TBA values.Furthermore, it was manifested that the addition of sodium nitrite dose to sausage formulation could be declined from 120 to 60 mg/kg when SE (1%) was utilized.

| CON CLUS IONS
DPPH radical scavenging activity ( % ) = A Control − A Sample ∕ A Control × 100 2.2.3 | Determination of peroxide value (PV) and thiobarbituric acid (TBA) value PVs (primary lipid oxidation indices) and TBAs (secondary lipid oxidation indices) were determined by the method of Naveena et al. (2013).

F
I G U R E 1 HPLC analysis of pomegranate and salvia; pomegranate crude extract (a), pomegranate crude extract + acetic acid (b), salvia crude extract (c), salvia crude extract + acetic acid (d), LC-TCI of the pomegranate extract (e), and salvia (f) before addition to the sausage.LC-TCI of the pomegranate extract before adding to the sausage Contrary to TBA data, PV of all samples (except SP) represents a reducing pattern that corresponds with the rising trend for TBA values for all sausages.It supposes owing to two reasons: (A) primary oxidation compounds were converted to secondary ones, (B) somewhat decomposition of rosmarinic acid, carnosol, and carnosic acid (in sage) occurred during the storage time at 4°C.Carnosic acid and carnosol were indicated as a scavenger of hydroxyl, DPPH radicals, reactive oxygen species (ROS), or lipid radicals.Mode of action of diterpenes in different in-vitro systems indicated that the non-water-soluble extract (i.e., carnosic acid and carnosol) displayed greater antioxidant capacity and electron donor properties compared Changes in the (a) rosmarinic acid, (b) carnosol, (c) carnosic acid, changes in the PV (d), and TBA (e).

Figure
Figure 2d,e show the changes in PV and TBA values as a function of time for six sausage samples.The values of PV in the SP sample increase with time, while in the remaining samples, a decreasing trend

and
Hugo (2015) in pomegranate-treated raw pork upon 9 days of refrigerated storage time andHugo and Hugo (2015) in rosemary extracts-treated pork meat.The increased rate pattern observed for b* values during refrigerated storage time was as follows; SN120 (10.5-11.6)˂ SSN (12.0-13.2) ˂ SN60 (12.3-15.8)˂ SPN (14-17.5)and SS (14-17.3)˂ SP (15.2-23.5)(Table 4).Although b* values of the samples treated with PPE and SE rapidly faded (p < .05),yellowness decreasing rate was displayed in those treated with combination of SE and nitrite during storage time.The increase in b* parameter may be ascribed to facilitate polyphenol degradation, lipid oxidation, and formation of metmyoglobin.Similarly, Hugo and Hugo (2015) stated increased b* values in pomegranate and rosemary-treated raw meat.
MetMb content.The order of OxyMb content of sausage formula was as follows SN120 > SN60 > SSN > SPN > SS > SP.Meanwhile, the decline of OxyMb level was more considerable within the first 7 days of storage for all treated samples.OxyMb content of SN120 and SN60 was significantly (p ˂ .05)greater than other formulations.The reduction of ferric-metmyoglobin (Fe 3+ ) to ferrous-oxymyoglobin (Fe 2+ ) simplifies by adding nitrites.The pomegranate extract represented lowest effect on protection of OxyMb because of the degradation of punicalagin α and β while the bioactive compounds of sage (rosmarinic acid, carnosic acid, and carnosol) cause to protect of OxyMb.Therefore, higher protection of OxyMb was exhibited in sausages incorporated with sage extract and nitrite compared with sausage containing sage extract and this impact was conspicuous from 7 days of storage time onwards.Besides, a significant negative correlation was observed between OxyMb pigment and lipid oxidation.In the present study, TBA data were in line greatly with OxyMb content (r 2 = −.76) and a* (r 2 = −.85),implying a reduction in OxyMb content and a* with enhancement in TBA value during refrigerated storage time.As a result, SN120, SN60, and SSN can be assumed to have more OxyMb content (>40%) at the final period of storage.Similarly, thyme essential oils + ascorbic acid (Ghaderi-Ghahfarokhi et al., 2016) could postpone reduction of OxyMb in minced beef burgers during cold storage.In contrast, Michalczyk et al. (2012) stated neither addition of essential oils of hyssop and coriander nor the storage temperature substantial influence the amount of OxyMb of ground beef following 15-day storage at 6°C.However, the gradual enhancement in the amount of MetMb during storage was accompanied by the increase in the TBA in sausage formulations.A negative relation between a* and MetMb content was also reported , putrescine is the most abundant TA B L E 4 L*, a*, b*, oxymyoglobin (%), metmyoglobin (%) of sausage treated with pomegranate and salvia extract after first, 7th, and 14th day of storage.

F I G U R E 3
Changes in the amount of cadaverine (a), histamine (b), and putrescine (c) in sausage treated with SE and PPE during storage time (0: black, 7th day: dark gray, 14th day light gray).

F I G U R E 4
Changes in the count of E. coli (a), and Cl.perfringens (b) in sausage treated with SE and PPE during storage time (0: black, 7th day: dark gray, 14th day light gray).nitrite) could progressively inhibit the formation of biogenic amines (putrescine, histamine, and cadaverine).On account of total degradation of α-and β-punicalagins of PPE, SP could not postpone the formation of biogenic amines.In several cases, cadaverine is caused by the activity of decarboxylase-positive bacteria, such as Enterobacteria (E.coli) that are not completely inhibited by curing.The amount of E. coli in sausage containing SE was significantly lower than PPE, causing reduction in decarboxylase activity and also resulting in cadaverine.Like cadaverine, the highest amount of putrescine was observed in sausage treated with PPE, while the lowest was in the SN120 and SSN at the end of 2-week storage.Similarly, it noteworthy that combined SE and nitrite (60 ppm) displayed the strongest impact on inhibiting the generation of histamine.Unlike putrescine and cadaverine, the change in histamine levels did not show variation during storage.The main reason behind this is the production of histamine depends greatly on histidine decarboxylase, Agro-industrial by-products including pomegranate peel or sage leaves are leading sources of phenolic compounds that possess high antimicrobial and antioxidant properties.Although nitrite exerts many positive influences as a curing agent, lower residual nitrite contents in meat products require to minimize the risk of nitrosamine generation as carcinogenic compounds.The addition of SE to sausage formulation could progressively postpone lipid oxidation and discoloration.Moreover, could reduce residual nitrites and NDMA content during refrigerated storage due to residual rosmarinic acid, carnosol, and carnosic acid in extract-treated sausage.However, oxidation indices, residual nitrites, NDMA, and biogenic amine increased in sausage samples containing PPE extracts on account of total degradation of α-and β-punicalagin during storage.
Comparison procedure according to SAS test was carried out for the data shown in each raw.Values are the average of triplicates ± standard deviation.Different capital letters in each row indicate significant statistical difference (p ≤ .05).

Table 3
Concentration (mg/kg) and kinetic parameters for rosmarinic acid, carnosol, and carnosic acid changes in sausage samples after first, 7th, and 14th day of storage.
TA B L E 2Note: Comparison procedure according to SAS test was carried out for the data shown in each column.Values are the average of triplicates ± standard deviation.Different capital letters in each column indicate significant statistical difference (p ≤ .05).Same letters in each column and row indicate no significant statistical difference (p ≥ .05).
Comparison procedure according to SAS test was carried out for the data shown in each row and column.Values are the average of triplicates ± standard deviation.Different lowercase letters in each row and capital letters in each column indicate significant statistical difference (p ≤ .05).Same letters in each column and row indicate no significant statistical difference (p ≥ .05).SN120 (sausage containing nitrite 120 ppm), SN60 (sausage containing nitrite 60 ppm), SS (sausage containing SE extract 1%), SP (sausage containing PEP extract 1%), SPN (sausage containing PEP extract 1% + NO 2 60 ppm), and SSN (sausage containing SE extract 1% + NO 2 60 ppm).
Values are the average of triplicates ± standard deviation.Different lowercase letters in each column and capital letters in each row indicate significant statistical difference (p ≤ .05).Same letters in Note:

Table 4 ,
the residual nitrite contents in the sausages treated with SE reduced more quickly than those with PPE and reached to 13.14 and 35.26 mg/kg at the end of ripening of sausage containing SE and PPE.It is demonstrated that numerous factors, mainly raw meat type, initial nitrite level, meat pH, storage temperatures, and the presence of reductants can affect nitrite depletion.