Effect of the fruit aqueous extract of balloon pepper (Capsicum baccatum var. Pendulum) on lipid oxidation, microbiological quality and consumer acceptance of fresh pork sausage and smoked

The reduction of the quality of foods usually associates with the deterioration of their compounds. Lipid oxidation is the main reason for this deterioration (Georgantelis et al., 2007). The phospholipids, polyunsaturated fatty acid together with the oxygen, heme pigments, and some metallic ions influence the deterioration process of processed foods either directly, or indirectly (Devatkal & Naveena, 2010).


Introduction
The reduction of the quality of foods usually associates with the deterioration of their compounds. Lipid oxidation is the main reason for this deterioration (Georgantelis et al., 2007). The phospholipids, polyunsaturated fatty acid together with the oxygen, heme pigments, and some metallic ions influence the deterioration process of processed foods either directly, or indirectly (Devatkal & Naveena, 2010).
Oxidation processes affect the nutritional, sensorial values of the products, and the level of thiobarbituric acid reacting substances (TBARS), which is the main parameter to assess the lipid oxidation (Almeida et al., 2015). Lipid peroxidation or lipoperoxidation (LPO) is the leading cause of chemical deterioration of food, a process that generates characteristic flavors and odors known as rancid (Campbell, 2015). The reaction mechanism involves the generation of free radicals (FR) that are highly reactive to atmospheric oxygen and form peroxyl radicals (OOR), activating a chain reaction that culminates in the formation of compounds called reactive oxygen species (ROS). ROS are highly unstable oxidizing substances, as they have one or more unpaired electrons (Campbell, 2015). ROS are the bases for the production of end products of lipid oxidation, which comprise the derivatives of the decomposition of hydroperoxides, such as alcohols, aldehydes, ketones, esters, and other hydrocarbons. High temperatures and small concentrations of pro-oxidant substances, such as metal ions, accelerate the LPO process (Campbell, 2015).
Brazilian legislation provides limits to the use of synthetic antioxidants, and, even the efficiency in food preservation, several studies demonstrate their risks for human health (Lima et al., 2010;Silva et al., 2010).
The carcinogenic potential associated with the use of synthetic antioxidants and other potential damages to health increased the interest in the use of natural antioxidants (Fratianni et al., 2010;Franco et al., 2012;Radha Krishnan et al., 2014) significantly. The literature also suggests the reduction of the use of natural antioxidants to preserve the sensorial features of foods (Rosa et al., 2013).
Meat products are particularly complex food systems, due to their composition, displaying high levels of protein, lipids, water, in addition to the presence of minerals and other constituents (Weiss et al., 2010). The complexity of this food matrix and the variability among the different meat products justifies the need for studies on its extract. Studies involving the effect of time and storage conditions of meat products are essential factors in meat processing since problems related to storage stability are common (Kozačins et al., 2012).

Effect of the fruit aqueous extract of balloon pepper (Capsicum baccatum var. Pendulum) on lipid oxidation, microbiological quality and consumer acceptance of fresh pork sausage and smoked
The present study evaluated the shelf life of frozen fresh pork sausage and smoked prepared with aqueous extract of balloon pepper fruits (Capsicum baccatum var. pendulum) as a natural ingredient with antioxidant properties in foods.

Materials and methods
Food processing experiments were performed in the Laboratory of Meat Processing at the Federal Institute of the state of Espirito Santo (IFES -Campus Alegre) in the municipality of Alegre, state of Espírito Santo, Brazil.
The male pigs were castrated surgically at seven days of age; all pigs started the experiment with 69 days of age and an average weight of 26.24 ± 5.44 kg.
Pork meat derived from hybrid (crossbred Landrace × Large White × Pietran) breeds of swine males, bred in confinement (feedlots) in the swine-breeding sector of the IFES-Campus Alegre in the municipality of Alegre, Espírito Santo, Brazil. The animals were slaughtered at 145 days of age and an average weight of 100.24 ± 5.44 kg. The longissimus dorsi muscles of 30 male pigs were sampled in six animals per treatment. Veterinarian food inspectors of the State Inspection System (SIE/IDAF) inspected and certified the meat from the slaughtered animals.
After slaughter, the meat was stored at -18 °C until the beginning of the sausage preparation process (end of rigor mortis) 24 hours after slaughter, according to Dokmanovic et al. (2015). The natural antioxidant from the balloon pepper was prepared at the sector of Food Chemistry of the Laboratory of Food Technology of the State University of Northern Rio de Janeiro (UENF: Universidade Estadual Norte Fluminense), after planting and harvesting of the fruits. The commercial synthetic additives (Sugar, Salt (NaCl), Sodium nitrite, Sodium Erythorbate) derived from commercial enterprises specialized in chemical additives for the production of meat products.
The genotype used was the access 1613 of Capsicum baccatum of the germplasm collection of the Center for Agricultural Sciences and Technologies of the UENF. The ripened fruits were collected from the plant, stored in duly identified paper bags, and transported for the analyses to the Food Technology Laboratory (LTA) of the Center for Agricultural Sciences and Technologies (CCTA). The vegetal samples were deposited at the University herbarium under the number HUENF 9607.
The extraction method was adapted from Zimmer et al. (2012), choosing for the production of oleoresins. The oleoresin is an extract obtained by the use of an organic solvent that has substances that are responsive for the pungency, and that may contain substances that may act as antioxidants, besides being stable during the storage. The application of these oleoresins to the foods is justified for adding flavor and increasing the oxidative stability of the lipids, extending the shelf-life of these foods.
In the beginning, the fruits were cut to extract the seeds and washed with tap water, weighed, and placed on an air-circulation oven at 40 °C, until reaching a constant mass. The ethanol was the solvent used for the preparation of the extraction. After the drying, fruits were minced and submitted to extraction in the Soxhlet equipment, using ethanol 70% (Fruit-solvent ratio: 1:10 weight/volume) for four hours.
A rotary evaporator evaporated the extracts at 79 °C. After this process, the antioxidant extract of the balloon pepper was obtained. The antioxidant extract of the balloon pepper was the one used in the present work due to the antioxidant activity and concentrations of total phenol, chlorogenic acid, caffeic acid, quercetin and rutin ( Table 1).
The evaluation of the antioxidant activity was carried out by the photo-colorimetric method of stable free radical DPPH (2,2-Diphenyl-1-picrylhydrazyl). The technique consists of adding 1 mL of the ethanol extract at concentrations ranging from 10 to 1000 µg mL -1 . 1 mL of a methanol solution of DPPH (0.1 mmol L -1 ) was added to this, followed by the reaction which occurred at room temperature for 1 hour. The absorption of DPPH was immediately checked in a UV-Vis spectrophotometer at 515 nm.
The ability to scavenge free radicals was expressed as a percentage of radical oxidation inhibition and it is calculated as follows (Zimmer et al., 2012;Equation 1): (1) ADPPH -absorbance of DPPH solution; ASAMPLE -absorbance of the sample solution.
The total phenolic content was determined using the FolinCiocalteu method as described Singleton et al. (1999) which was based on the reduction of the phosphotungstic (H 3 PW 12 O 40 ) and phosphomolybdic (H 3 PMo 12 O 40 ) acids. To prepare the samples samples, 1 mg was weighed and solubilized with 1 mL of methanol, 30 µL of these solutions were drawn transferred to a 96 well plate. After that, they were added 50 µL of Folin-Ciocalteu reagent (1N) and added 100 µL of sodium carbonate (Na 2 CO 3 ) 7.5% after 10 min. The absorbance was obtained at 760 nm after 2 h of incubation at room temperature. The total phenols' results were expressed as gallic acid equivalents using a gallic acid standard curve (5.0 to 200 µg µL -1 ).
Standards (phenolic and flavonoid acids) and the samples extracts were analyzed on Shimadzu Model LC-20A, with LC20AD pumps. The chromatograms were monitored at a wavelength of 254 and 350 nm for phenolic compounds (detector spectrum scanned by ultraviolet photodiode array SPD-M20A) and an injection volume of 20 µL. Reverse phase column RP-18 from Macherey-Nagel (5 µm, 4.0 × 250 mm) was used. To prepare the samples to be injected, 10 mg of the extract was weighed and solubilized in 1 mL of mobile phase: 500 µL was pipetted of mobile phase contained in the pump A and 500 µL of mobile phase contained in the pump B, following this procedure for the two gradients. The extract was filtered using of microfilters (Sartorius®) and syringes (BD Plastipak®) were then applied three times to each method, in the liquid chromatograph. Chlorogenic acid (Sigma-Aldrich), caffeic acid (Tedia Brazil), rutin (Merck) and quercetin (Merck) standards were used to measure the standard curves. To identify and quantify the compounds in samples, comparisons were made of the retention times of the samples with retention times of pure commercial standards. After this preliminary analysis were made co-injections of pure standards and samples were made for further analysis of their retention times. Comparisons between the UV spectrum of the compounds and the commercial standards were also carried out.
After thawing, all fat in muscle was removed, and the meat was diced and stored at approximately 4 °C. Pork fat was also thawed, diced, and stored at the same temperature. Next, longissimus dorsi muscles were ground in a meat grinder (20, Talleres Ramon). Pork fat was ground in the same equipment. All animal raw material was placed in a tray when seasoning and additive were added and hand-mixed to meat and fat.
After adding a mixture of curing salts and aqueous extract of balloon pepper fruits (Capsicum baccatum var. pendulum), fresh sausage material was placed in other trays, labeled, and refrigerated for 1 h at 2 °C to 4 °C. Sausages were manually encased using 36-mm natural sheep casing. The sausages prepared were vacuum-packaged (TM720, TecMaq) in polyethylene bags, labeled, stored at -18 °C, and thawed at 4 °C before analyses.
Four different formulations of fresh pork sausage and smoked sausage were produced for the study of lipid oxidation: Control, F1, F2 and F3. The formulations promoted the substitution of the synthetic antioxidant with the natural one. A control using sodium erythorbate was included in the experiment (Table 2).
After preparation, a portion of the samples were taken to the smoker, where they remained for approximately 3 hours until they reached an internal temperature of 75 °C. They were removed from the smoker and placed in a protected place for a period of 24 h for natural cooling. Then, the samples were vacuum packed and identified. Finally, stored in a cold room at -18 °C, until the moment of analysis.
Samples from different formulations were grounded and homogenized. Moisture (direct drying in stove at 105° C), dry ash (residue after incineration in muffle at 550° C), proteins (classic Kjeldahl's method) and crude ethereal extract (direct extraction in Soxhlet) were determined according to the described by Cechi (1999) and Association of Official Analytical Chemists (2000) ( Table 3).
The microbiological analyses of the samples were performed at the microbiology laboratory of the IFES Campus Alegre. The sensorial analysis was performed at the Food Technology Laboratory, at the UENF University.
Preliminary tests were performed to verify the concordance with the Brazilian legislation on the standard for the production of fresh sausage as refers to the percentage of fresh material, and feed additives. Table 2 displays the percentage of fresh matter and additives of the different formulas.
The analyses described next were performed on non-stored samples [on the fabrication day; n=20 for each formulation and control group (total n=100)] and after 15, 30, 45 and 60 days of storage [n=20 for each formulation and control group (total n=100), for each period].

Lipid oxidation
Five grams of fresh and smoked pork sausages were homogenized and placed in a 30 × 200 mm glass tube, without border, and with a rounded bottom. 30 mL of 5% trichloroacetic acid (TCA) and 1 mL of 0.15% synthetic antioxidant butylated hydroxytoluene (BHT) were added. The material was homogenized in an Ultra-Turrax for 60 seconds. Later the sample was filtered using qualitative filter paper (12.5 mm) to a 50 mL volumetric flask. The volume was completed with 5% TCA solution. Five mL of the flask content were withdrawn, transferred to a sample tube, and added with 5 mL of 0.08 M thiobarbituric acid. Tubes were incubated in a boiling water bath for 40 minutes, to allow the formation of the colored complex. After this period, the tubes were placed in running water until reaching room temperature, and the solution was measured in a Kasuaki (UV-5100 model) spectrophotometer at 532 nm wavelength. The results were subtracted from the results of the blank sample.

Microbiological analysis
Microbiological analysis of the samples was performed just after thawing the sausage, according to Silva (2010). The results were compared with the standards recommended in the RDC 12 (Brasil, 2001), for coliforms at 45 °C (Most Probable Number method), Coagulase-positive Staphylococcus (Presence or absence), and Salmonella spp. (presence or absence).

Sensory analysis
An acceptance test with hedonic scale was used for the sensorial evaluation, using a scale of 9 points (9-liked immensely; 8-liked a lot; 7-liked moderately; 6-liked lightly; 5-indifferent; 4-disliked lightly; 3-disliked moderately; 2-disliked a lot and 1-disliked extremely). The sausage were analyzed for sensory attributes by 51 untrained participants recruited among students and employees of the UENF University, at the Campus dos Goytacazes (RJ-Brazil) Campus. Among them, 53% were women, 47%, men. The age ranged from 18 to 45 years. Briefly, 1.0 cm-thick slices were cut starting from the middle of the sausage cooked electric oven PHILCO PFO32L at 180 °C for 10 minutes. One slice of cooked sausage of each formulation were placed in labeled plates and randomly presented to participants. Water and unsalted crackers were provided for cleansing the palate between evaluations. Participants were given an evaluation form with the nine-point hedonic scale to assess color, aroma, taste, texture and general impression. The evaluations were carried out in individual booths.
For the attributes associated to the will to buy, the evaluation sheet had five points (5-definitively, I would buy; 4-probably I would buy; 3-maybe I would buy, maybe not; 2-probably I would not buy; 1-definitively I would not buy). In these sheets, the judges expressed numerical values according to their preferences.
Sensory analysis was performed after the microbiological analysis. All samples satisfied the Brazilian microbiological requirements as RDC 12/2001 (Brasil, 2001). The ethical in health committee endorsed this project under the statement 2,066,829.

Statistical analysis
The results were submitted to Analysis of Variance (ANOVA) at 5% probability. The treatments were compared by the test Student-Newman-Keuls.
Sensory analysis results were submitted to statistical analysis by the analysis of variance (ANOVA) and Tukey test at 5% probability. All statistical analyses were performed using the statistical program SAS Institute (2009) version 9.3.

Results and discussion
The natural antioxidant (the extract of balloon pepper) was added at different concentrations in the different formulations in order to delay lipid oxidation and extend the shelf life of the fresh sausage. Table 4 present the values of TBARS obtained (Table 4).
TBARS values in the fresh sausages did not display significant differences (P>0.05) among the treatments, and at different times of analysis (storage), but varied along the experimental period. TBARS values ranged between 0.015 mg MDA/kg of sample and 0.065 mg MDA/kg of the sample, respectively at 15, and 60 days of storage. The data of TBARS point out the similarity between the action of the natural antioxidant derived from the balloon pepper, and the synthetic sodium erythorbate. The lack of differences along the storage time might associate with the vacuum-storage. In these conditions, the degradation of TBARS might have produced other substances, but TBARS.
Smoked sausage samples also did not display significant differences (P>0.05) associated neither to the treatment nor the storage time. TBARS values ranged between 0.013 and 0.069 mg MDA/kg of sample along the experimental period. The results of TBARS of smoked sausage samples may be associated with MDA degradation due to the action of phenolic compounds produced during the smoking process.
Neither the TBARS values of fresh nor smoked sausage displayed a significant increase (P>0.05) during the experimental period. The results of TBARS during the experimental period highlighted the satisfying antioxidant action of the pepper extract since no differences with the control treatment could be observed along the experimental time. Even the same, it is possible to affirm that the storage by freezing and vacuum packaging contributed positively, as comparing the treatments with antioxidants (both synthetic and natural). These observations highlighted the delay in the lipid oxidation of the products along the 60 days of the experiment.
During processing and storage of the sausages, physical and chemical alterations caused the production of free radicals, which caused the oxidation of the unsaturated fatty acids of meat (Falowo et al., 2014;Hygreeva et al., 2014).
The analysis of lipid oxidation through the TBARS assay quantified the malonaldehyde compound (MDA), which is one of the leading products of decomposition of hydroperoxides of the fatty acids produced during the oxidation process. According to Gray & Pearson (1987), lipid oxidation in meat products begins as TBARS levels are between 0.5 and 2.0 mg MDA/kg of the sample. Nam et al. (2001) observed that packaging raw meat under vacuum conditions was enough to protect the cholesterol and fatty acids from oxidation. In this study, all the samples used to study the maturation process were individually vacuum-packed and, possibly, this process protected cholesterol and fatty acids, especially the unsaturated ones, from adverse conditions that could have promoted oxidation. However, TBARS is a relatively stable compound. Ham et al. (2020) evaluated the efficacy of ascorbic acid on processing characteristics and lipid oxidation of pre-rigor salted chicken breasts during seven days of vacuumpackaged storage, and suggested that pre-rigor salting with ascorbic acid could be useful in improving lipid oxidation stability during refrigerated vacuum storage, without negative impacts on processing characteristics.
The treatments and the interactions treatment × type did not display significant differences (P>0.05). The only significant differences were between the different types of sausages: fresh and smoked, Table 5 describe. Figures 1 and 2 display the average MDA/kg (expressed as mg) during the storage period in the samples of fresh and smoked sausage, respectively.
Fresh sausages displayed higher MDA values than the smoked ones. These higher values may have been due to the degradation of MDA, and other compounds during the vacuum storage and to the action of phenolic compounds produced during the smoking process when partial dehydration of the product occurs. Smoke components include halophilic and lipophilic substances that react differently in meat products. Among the preservative properties of the smoking processes can also be mentioned the antioxidant effect that the smoking compounds exert, mainly the monoglyd and dimethyl ether of pyrogallol capable of delaying the oxidative and hydrolytic rancification of fats (Pardi et al., 2007). These results corroborate those presented by Schwert et al. (2011Schwert et al. ( , 2020 where they concluded that, the use of liquid and traditional smoke with temperature   control ~ 300 °C for a smoke generation are promising and safe approaches for obtaining low levels of benzo (a) pyrenes, lipids and protein oxidation in Sausage type Calabrese.
As observing the values found in this study, TBARS values are within quality values. Studies display that TBARS values up to 1.59 mg MDA/kg of the sample are considered low, are not perceived as rancidity by sensorial analyses, and do not cause any harm to the consumers' health (Torres & Okani, 1997). According to Ahmad & Srivastava (2007) no rancidity smell in meat is detectable as TBARS values span between 0.5 and 1.0 mg MDA/kg of the product. For this reason, the average values found are within standards of acceptability, defining that sausages may be appropriate for consumption.
As defining the effect of the addition of oregano and marjoram extracts in "Toscana" sausage, Bussata (2010) described 0.806 mg MDA/kg after 35 days of storage. Almeida et al. (2015) observed that TBARS values differed at the same storage time in sausages prepared with the extract of the peel of the Brazilian grape (Plinia cauliflora). According to the same authors, the lower lipid oxidations are due to the phenols of the Plinia cauliflora peel.
Abu Salem & Ibrahim (2010) assessed the addiction of salvia oil extract at 0.05% and 0.025% in buffalo sausages and found values of 0.91 mg MDA/kg of product in sausages without natural antioxidant, and 0.51 and 0.46 mg MDA/kg in sausages treated with the salvia extract. Jayawardana et al. (2011) used Adzuki bean at 0.2% in smoked and not smoked sausages. The authors described positive results in the control of lipid oxidation in both products and associated these results to the presence of the polyphenols in the extract of the Adzuki bean, which are rich in anthocyanins.
The antioxidant effect of the phenols derives from the hydroxyl group linked to the aromatic ring. The aromatic ring can donate electrons to hydrogen atoms, neutralizing the free radicals. According to Almeida et al. (2015), this mechanism blocks the degradation of the oxidative form, as observed for the MDA.
The results of our study corroborate those of the other works presented, as we did not observe an increase of lipid oxidation, keeping TBARS values relatively constant until the end of the experiment (60 days). Table 6 describes the microbiological quality of the fresh sausage, as it refers to coliforms (MPN/g), coagulase-positive Staphylococcus (UFC/g), Salmonella sp., and sulfate-reducing Clostridium at 46 °C (UFC/g).
The storage process is directly associated with the development and survival of microorganisms. The microbiological stability is crucial to the products' quality. According to Fellows (2006), food preservation is due to antimicrobial action addicted to the product, the presence of lactic acid derived from the fermentation processes, and the control of pH.
The enumeration of all the microorganisms searched was below the standard required by Brazilian laws (Brasil, 2001). The microbiological quality was due to the quality of the raw materials, good manufacturing practices, vacuum packaging of the product, and use of additives, which prevented the growth of some species of microorganisms, and the use of natural antioxidant, with its antimicrobial action.
Table seven displays the average notes of the sensorial analysis for the attributes color, scent, taste, texture, and impression (Table 7).
Sensorial analysis displayed that the attribute "color" did not display significant differences (p>0.05) between control and F2, and between F1 and F3. There was a significant difference (p<0.05) between control and F1, control and F3 and between F1 and F2. The consumers observed a color reduction with the inclusion of 0.5% of balloon pepper extract, compared to the formulas with 1.0 and 1.5% addiction. The red tone of the extract, influencing the final color of the sausages, can explain this reduction of the color. López-Vargas et al. (2014) observed this correlation in pork hamburger added with passion fruit flour. The product added with the lower concentrations displayed lighter colors than the remaining. Valle (2017) studied bovine restructured meat beef added with aqueous extract of pink  5.4 c 6.0 b 6.6 a 6.4 b 6.2 b F2 6.6 ab 6.9 a 7.3 a 7.0 ab 7.1 a F3 5.9 bc 6.5 ab 7.3 a 5.9 ab 6.8 a pepper (Schinus terebinthifolius, Raddi), and the results of the research disagree with those presented in the previous article.
The aroma did not show significant differences (p>0.05) between the control, F2 and F3, and between F1 and F3. This datum suggests that taster perceive the inclusion of the extract at low (0.5%) or high (1.5%) concentrations, but do not detect the product at intermediate concentrations (1.0%).
This observation differs as there is the inclusion of different kinds of ingredients as antioxidants, as displayed by Valle (2017). Maia et al. (2020) also points out that the tasters did not perceive the inclusion of passion fruit meal, potassium chloride (KCl), and calcium chloride (CaCl2) in smoked sheep meat sausage.
The attribute "taste" did not display any significant difference (p>0.05) among the formulas. The texture did not differ significantly between the control, F2, and F3, and between F1, F2, and F3. The reduction of water use was a crucial parameter in the results obtained in this study, as there was no significant difference among the different formulas that received the extract of balloon pepper and between the formulas that received, and the one that did not receive the extract.
The general impression of the product displayed significant differences (P<0.05) between F1 and the remaining treatments. This result is probably due to the higher perception of the tasters for the inclusion of the extract of the balloon pepper (Capsicum baccatum var. pendulum) at low concentrations (0.5%).
The results highlighted that the attributes color, aroma, texture, and general impression of the treatment F2 (addiction of 1% of pepper extract to substitute the antioxidant) received higher acceptance by the consumers. The average scores received are closer to seven. According to Dutcosky (2011), the acceptability index of 7.0 is the minimum that a product should reach to be considered of positive acceptability in the different sensorial parameters assessed. As refers to the buying intention, scores 4 to 5, 3 to 2, and 1 were associated respectively to "acceptation, " "indifference, " and "rejection. " The average scores of the buying intention of the F2 and F3 formulas were higher than the remaining, suggesting that consumers "definitely would buy" these products on the market. On the other side, F1 treatment received an average score of 1.9, indicating higher rejection. Consumers "definitely would not buy" this product.

Conclusions
The results point out that the balloon pepper extracts acted competently as antioxidants, preventing rancidity, and preserving the original qualities of the product during the 60 days of storage. The freezing and vacuum storage also contributed to delay the oxidative processes. The fresh sausages displayed higher lipid oxidative values as the smoked ones, but their responses after 60 days of storage were similar. The sensorial analysis pointed out the higher acceptability of the F2 formula by the consumers, and the higher willingness to buy. This paper clearly shows that the use of a 1% extract of the balloon pepper demonstrates good acceptability among the consumers.