Filtration and antioxidant activity of commercial fermented and non-fermented sausage

This study aims to screen peptides from commercially available fermented and non-fermented sausages in the local market, examine the chemical content of the products, and determine their antioxidant activities. The chemical content of the sausages was determined using proximate analysis and the Lowry assay. The antioxidant activity was measured using the DPPH method. The data were analyzed using ANOVA. The results showed that in the proximate analysis, only the water and fat content of the three sausages differed significantly (P<0.05). The concentration of protein in the fermented sausages was significantly higher than in the non-fermented sausages (P<0.05), with the highest protein content found in the fermented Pepperoni sausage. The non-fermented beef sausage extract had a higher IC50 value (log 2.07 ppm or 117 ppm) compared to Pepperoni and Salami sausages (P<0.05). The best IC50 value in inhibiting DPPH reactivity was found in the fermented Pepperoni sausage, followed by Salami and non-fermented sausages. This indicates that fermentation can increase the chemical content and antioxidant capabilities of the fermented sausage products.


Introduction
Free radicals are molecules that are unstable and highly reactive because they have one or more unpaired electrons [1].Free radicals will react with surrounding molecules to acquire electron pairs in order to achieve molecular stability.This reaction can cause damage to other molecules, both in terms of structure and function.Ultimately, the far-reaching effects of molecular damage within the body's cells due to these reactions can cause diseases such as cancer, stroke, heart disease, and premature aging.Free radicals can be formed naturally as by product from normal body metabolism.Even though the human body has the ability to form and neutralize free radicals, its capacity is limited.When free radicals outnumber the body's ability to neutralize them, certain diseases will start to emerge [2].Because of limitation from the body's capability to neutralize free radicals, human body needs something as an extra booster to neutralize free radicals.
Antioxidants are compounds that can inhibit the occurrence of oxidation processes [3].[1] added that antioxidants work by donating electrons to complete the electron deficienc ies needed by free radicals and by inhibiting the chain reactions of free radical formation.Antioxidants can be obtained from IOP Publishing doi:10.1088/1755-1315/1341/1/012041 2 vegetables, grains, legumes, and fruits that are rich in vitamins C, E, A, and β-carotene [4].In addition to these sources, [5] further state that antioxidants can also be found in animal-derived foods such as meat or protein hydrolysates from specific animal-derived sources containing bioactive peptide compounds.
Fermented sausages are one of the animal-based food products made from meat, where the meat is cured using the assistance of lactic acid bacteria (LAB) and air-dried.In the process of making fermented sausages, a mixture of fat, meat, spices, and seasonings is used, along with lactic acid bacteria [6].During the fermentation process, LAB will degrade the meat proteins, resulting in short protein fragments (peptides) or individual amino acids.[7] mentioned that the peptides generated from the fermentation process can exhibit different activities compared to their parent proteins.Hence, these peptides are referred to as bioactive peptides.One of the activities of bioactive peptides is their ability to act as antioxidants.
Antioxidants can serve as a preventive measure against the negative effects of free radicals that threaten human body.Nevertheless, the safety and effectiveness of antioxidants remain a challenge in the field of health.One approach to obtain safe and effective natural antioxidants is by using antioxidant peptides.The fermentation process of sausages is accompanied by the protein metabolism carried out by the fermentation starter bacteria, resulting in various types of peptides.The ability of these peptides to neutralize free radicals (as antioxidants) is still not fully understood.In addition, the variations in chemical composition across different types of fermented sausages produced through various fermentation processes and different starter types have yet to be investigated.The purpose of this study was to identify the chemical composition of commercial fermented sausages and analyze the antioxidant activity of peptide extracts from these fermented sausage products.

Materials and Method
The research was conducted at the Integrated Laboratory, Department of Animal Production and Technology, Faculty of Animal Science, IPB University.The materials used in this study were commercial fermented and non-fermented sausage, phosphate buffer 20 mM pH 7, sodium carbonate (Na2CO3) 2%, sodium hydroxide (NaOH) 2 N, CuSO4 1%, sodium potassium tartrate (NaK Tartrate) 2%, Bovine Serum Albumin solution, Folin-Ciocalteu 1 N, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid (vitamin C), methanol 100%, and distilled water.Study were performed using a single factorial complete randomization design (CRD), which involves different types of sausages, each with 3 replications.The data were analyzed using Analysis of Variance (ANOVA) followed by the Tukey post hoc test.
The methods used in this study were peptide filtration using centrifugal filter unit cut-off 3 kDa, pH test using pH meter, Lowry method, proximate analysis that contains moisture content; ash content; protein content; fat content, total phenol analysis using the Folin-Ciocalteu reagent based on the reducing power of phenolic hydroxyl groups, total flavonoid analysis using calorimetric method, and antioxidant activity analysis using DPPH method.The entire proximate analysis procedure followed the guidelines set by AOAC (Association of Official Analytical Chemists) [8].
The DPPH method is based on the reduction of the colored DPPH free radical by free radical inhibitors.This procedure involves measuring the decrease in DPPH absorbance at its maximum wavelength, which is proportional to the concentration of the free radical inhibitor added to the DPPH reagent solution.The procedure began by preparing a DPPH solution by dissolving 5 mg of DPPH solid in 100 mL of methanol.An amount of 0.3 mL of the sausage extract is added to 0.7 mL of distilled water and 3 mL of the DPPH solution that has been dissolved in methanol.The solution is then homogenized using a vortex mixer and left to stand for 30 minutes at room temperature, shielded from light exposure.The homogenized solution is transferred to cuvettes, as well as for the blank and standard measurements.The blank used is the reagent (buffer) without the addition of extract.The standards used are ascorbic acid with concentrations of 10 and 60.Antioxidant measurement was performed using a UV-Vis spectrophotometer at a wavelength of 517 nm.The quantification of DPPH inhibition was determined by calculating the Inhibitory Concentration (IC50) value.

3.
Results and Discussion

pH level of commercial fermented sausages
The pH values of fermented and non-fermented sausages available in the market can be observed in Table 1.The results showed that the pH values of these three sausages differ significantly (P<0.05), with the lowest pH value obtained from the Pepperoni sausage extract.Meanwhile, the Salami sausage extract used in this study has the highest pH among the three sausages.Pepperoni and Salami are both types of dry fermented sausages.Dry fermented sausages typically have pH values ranging from 5.0 to 5. 3 [9].Different letters following the mean values in the column indicated significant differences (P<0.05) The low pH value of the Pepperoni sausage extract was an implication of the fermentation process that occurred in the sausage.[10] stated that during the fermentation stage, the pH of sausages decreases due to the activity of starter bacteria (typically lactic acid bacteria) that produce lactic acid from the metabolism of sugars in the meat (especially glycogen).The produced lactic acid can inhibit the growth of pathogenic bacteria, thus extending the shelf life of the sausage.Another perspective suggested that pH value is related to the microbial survival, as the pH decreased, food products can have longer shelf lives because spoilage microbes cannot thrive [11].[12] also mentioned that ideally, fermented sausages should achieve a pH value of 5.3 or lower to control the growth of pathogenic bacteria such as E. coli and Staphylococcus aureus.
Meanwhile, Table 1 showed that the pH value of the non-fermented sausage extract and the fermented sausage extract of Salami were not significantly different (P>0.05).This could be attributed to several possibilities.First, the Salami fermented sausage might be using meat with an initial pH value that is almost the same or even higher than the meat used in the non-fermented sausage.Second, the storage process could have led to an increase in pH values in the fermented sausages.According to [13], the increase in pH during storage is caused by the depletion of the substrate (sugar) concentration for fermentation, led to the domination of alkaline metabolite products.Various fermented sausage products stored at room temperature or in the refrigerator, using different types of pac kaging, experience pH increase albeit with varying intensities.A similar pattern is observed in other meat products, although the rate of increase differs depending on the type of meat and the processing method [14].Unfortunately, in this study, detailed information about the type of meat used and the storage period for the products is not provided.

3.2
Proximate analysis on commercial fermented sausage Table 2 illustrates the differences in proximate analysis (moisture content, ash content, protein content, fat content) conducted among non-fermented sausage, Salami, and Pepperoni.Moisture content.Moisture content is the percentage of water content in a substance or product.Table 2 showed that the highest moisture content was found in Pepperoni sausage with a moisture content of 69.96% (P<0.05), while non-fermented sausage and Salami sausage have moisture contents that were not significantly different (P>0.05).Although [15] stated that fermented sausages generally have lower moisture content due to the ripening process, the moisture content can deviate due to storage conditions, especially in the freezer.According to [16], during the storage process, there is a possibility of condensation occurring in the product, which subsequently leads to moisture formation within the product.Moisture within the product can result in undesirable effects on food quality, such as the formation of ice crystals in frozen food or the softening of food texture.Based on this, Pepperoni sausage might undergo relatively extreme conditions during storage, leading to an increase in its moisture content to a level similar to that of non-fermented sausages.Another possibility is that Pepperoni sausage initially has a relatively high moisture content.Generally, the moisture content on Table 2 was still below the maximum moisture content of meat sausages according to [17], which falls within the range of 45%-80%.Based on this, the results of the moisture content obtained in this study comply with the standards for sausages were still suitable for consumption.
Ash content.Ash content indicates the mineral content in a substance [18].[19] added that about 96% of food materials consist of organic substances and water, while the remaining 4% comprises 30 mineral elements.The results of ash content analysis in fermented and non-fermented sausages showed that the ash content of these three products were not significantly different (P>0.05).[20] stated that the fermentation process of meat led to an increase in minerals (especially iron) and vitamin B. In general, fermented meat is a good source of vitamins and minerals, including vitamin B12, B6, and niacin, as well as iron, phosphorus, and zinc.However, the absence of differences in minerals and vitamins (ash content) on Table 2 was suspected to be due to differences in the quality of raw materials used, especially in terms of the initial mineral content of the raw materials.This was consistent with the report by [21], which stated that the raw material conditions and sausage processing influenced the mineral content in the final product.Based on this, it's possible that the non-fermented sausage raw material used in this study may have a relatively high ash content, thus offsetting the ash content of fermented Salami and Pepperoni products.
Fat content.Based on the research results, the fat content of fermented and non-fermented sausages were different significantly (P<0.01).The fat content of fermented salami sausage was the highest compared to both fermented pepperoni and non-fermented sausage.The high fat content in Salami was due to several factors, one of them was the drying process that can increase the fat content in the sausage.[22] mentioned that during the curing process, lipid oxidation occurs, leading to an increase in fat content.This increase encourages the production of free fatty acids and reactive substances like 2-thiobarbituric acid, resulting in a final product with 82-95% total free fatty acids after curing.Traditional fermented sausages typically have around 32% fat content right after production, but due to drying, the fat content increases to about 45-60% [23].Another factor contributing to higher fat content in salami sausage was likely the use of meat fat as one of the ingredient in sausage mixture.Fermented sausages are composite products made from a blend of meat, fat, seasonings, and bacterial cultures.The addition of fat to sausage products naturally increases the percentage of fat content in salami.[24] stated that dry fermented sausages are processed meat products with a high content of saturated fat.According to [25], the fat content of fermented sausages can be influenced by the amount of fat added to the sausage mixture and by certain bacteria that break down fat during their growth.
Protein content.Protein is defined as a complex organic compound containing amino acids bound to each other through peptide bonds.Proteins consist of carbon, oxygen, nitrogen, and sulfur atoms [26].
Based on the research results, the protein content of fermented and non-fermented sausages showed no significant differences among all sausage products used in this study (P>0.05).Generally, the protein content in sausages comes from the raw materials used, which is Food sources with high protein content include red meats such as beef, buffalo, and goat [27].[28] mentioned that red contains an average of 20-25 g of protein per 100 g (when raw), making it a high-protein source.The similarity in protein content among the three products used in the study was likely due to the protein content in the raw meat used.Another possibility was that the synthesis during fermentation in pepperoni or salami sausages was not significantly different.Additionally, this could also be attributed to protein degradation (catabolism) during the storage of fermented sausages, which was much higher compared to non-fermented sausages.

Protein concentration in fermented sausages
The results in Table 3 indicated that fermented sausages (Pepperoni and Salami) have a higher protein concentration compared to non-fermented sausages (P<0.05).Additionally, the protein concentration in Salami extract was higher than in Pepperoni sausage extract (P<0.05).The protein concentration in Table 3 was different from the protein content obtained from proximate analysis (Table 2).The proximate analysis results in Table 2 showed that the protein content in the three sausages was not different (P>0.05),while Table 3 showed the opposite pattern.This was due to the fact that protein content obtained from proximate analysis was determined using the Kjeldahl method, which is based on the total nitrogen content in the sample [29].Meanwhile, the protein concentration in Table 3 was determined using the Lowry method, which specifically targets protein molecules through their reaction with the Folin-Ciocalteu reagent.Different letters following the mean values in the column indicated significant differences (P<0.05) The higher protein concentration in fermented sausages compared to non-fermented sausages was likely due to the influence of the fermentation process.[30] stated that during fermentation, microorganisms produced protease enzymes that hydrolyze proteins into smaller peptides and amino acids.The breakdown of proteins into simpler forms can make them more accessible and easier to digest.As a result, the protein content became more concentrated in the final product, which was likely to lead to an increase in protein concentration.In addition, the increase in protein concentration due to the fermentation process is also believed to be caused by protein synthesis by microbes during fermentation.
[31] also added that fermented foods tend to increase protein concentration and serve as a source of high-quality bioactive peptides.

Phenol and flavonoid content
Phenolic compounds are the largest group of phytochemicals responsible for the majority of antioxidant activities, while flavonoids are the largest group of natural phenolic compounds [32].The content of both compounds in the sausages used in this study aims to observe the fundamental components that generally contribute to antioxidant properties.Table 4 showed that the flavonoid and phenolic compounds in the extracts of fermented sausages (Pepperoni and Salami) were different from the extracts of non-fermented sausages (P>0.05).Overall, the total phenolic flavonoid content in fermented sausages extracts was higher than in non-fermented sausages extracts.This difference is believed to occur due to the increase in phenolic and flavonoid compounds during the fermentation process, influenced by cooking and drying of the sausages.[33] stated that the ability to increase the amount of phenolic and flavonoid compounds during fermentation was a result of microbial hydrolysis According to these reactions involve the breakdown of macromolecules into simpler molecules through the addition of water.Different letters following the mean values in the column indicated significant differences (P<0.05)Table 4 showed that the phenolic compound content in Pepperoni extract was higher than in Salami extract(P<0.05).Meanwhile, the total flavonoid content in both sausages did not show a significant difference (P>0.05).This was likely due to the release of phenolic compounds during the fermentation process of Salami sausage being more pronounced compared to Pepperoni sausage.On the other hand, the tendency of starter microorganisms in salami and pepperoni sausages to release flavonoid components seemed to be similar.

Antioxidant activity
Antioxidants are compounds capable of donating electrons and have small molecular weights, yet possess the ability to halt oxidation reactions by preventing the formation of free radicals [35].The DPPH method was used to test the antioxidant activity in samples.The principle of antioxidant testing with DPPH involved the antioxidant's ability to stabilize DPPH by providing a hydrogen atom, causing a color change from purple to yellow in DPPH.This ability to provide a hydrogen atom to DPPH was referred to as scavenging activity of antioxidant component [36].
Figure 1 illustrated the inhibition pattern of the purple color intensity of DPPH by fermented and non-fermented sausages at different concentrations.The reduction in purple color intensity was assumed to represent the inhibitory ability (% inhibition) of the sausages against the DPPH free radical reaction.As the purple color diminishes, the antioxidant capacity of the sausages increases.Different letters following the mean values in the column indicated significant differences (P<0.05) According to [37], antioxidant activity can be categorized into five groups: very strong (IC50<50 ppm), strong (50 ppm<IC50>100 ppm), moderate (100 ppm<IC50>150 ppm), weak (150 ppm<IC50>200 ppm), and very weak (IC50>200 ppm).Based on this classification, the antioxidant activity of the non-fermented sausage extract was in the moderate category, while the Pepperoni and Salami extracts were categorized as very strong and strong, respectively.

4.
Conclusions In conclusion, the three sausage samples, both fermented and non-fermented, exhibit diverse chemical compositions.The Pepperoni extract has the lowest pH compared to Salami and non-fermented sausages.The highest moisture content was found in Pepperoni sausage, while the lowest was in Salami sausage.The highest fat content was observed in the Salami sausage.On the other hand, the concentration of protein in fermented sausages was significantly higher than in non-fermented sausages, with the highest protein content found in Pepperoni.The same trend was observed in the phenol content of the sausages, with the highest phenol content in Pepperoni sausage extract, while the highest flavonoid content was found in Salami sausage extract.Antioxidant testing using the DPPH method indicated that all three sausages possess antioxidant capabilities as they exhibit DPPH scavenging activity.The best IC50 values for inhibiting DPPH reactivity were found in fermented Pepperoni sausage, followed by Salami and non-fermented sausages.This suggests that the fermentation process enhanced the antioxidant abilities of the fermented sausage products.

Figure 1 .
Figure 1.The inhibition curve of DPPH activity by non-fermented sausage extract (A), Pepperoni (B), and Salami (C) with fitting curve.

Figure 1
Figure 1 showed an increase in DPPH inhibition of the three different sausage extracts.This phenomenon occurred due to the higher concentration of the sausage extracts, which consequently led to an increased concentration of bioactive antioxidant components in the extracts.From Figure 1, the IC50 values (the concentration required to inhibit 50% of DPPH activity) were obtained based on the curve fitting equation as presented in Table 5.The non-fermented sausage extract has a larger IC50 value (log 2.07 ppm or 117 ppm) compared to Pepperoni and Salami extracts (P<0.05).Meanwhile, the IC50 values of Pepperoni and Salami extracts were not significantly different (P>0.05), with log values of 1.64 ppm (43 ppm) and 1.83 ppm (67.63 ppm), respectively.

Table 1 .
The average pH of fermented and non-fermented sausages

Table 2 .
Proximate analysis of fermented and non-fermented sausages

Table 3 .
Protein concentration of fermented and non-fermented sausage

Table 4 .
Total phenol and flavonoid of fermented and non-fermented sausage

Table 5 .
IC50 value of fermented and non-fermented sausage