AQUEOUS LEAF EXTRACT OF SENDUDUK (Melastoma malabathricum L.) COULD IMPROVE THE PHYSICOCHEMICAL PROPERTIES OF BEEF SAUSAGE DOUGH

Improving comminuted meat products characteristics using a natural agent, such as phytochemicals, in order to replace the use of nitrite, have become a need due to the health reason. The quality of the sausage is also affected by the initial characteristics of the dough. Therefore, this research was conducted to investigate the effect of aqueous leaf extract of senduduk (Melastoma malabathricum L.) on the physicochemical properties of beef sausage dough. Different four formulas as treatment were employed to form the dough: formula A was as a control consisted of beef meat, vegetable oil, skim milk powder, tapioca, salt, phosphate, and seasoning; formula B was control added with extract 0.55%; formula C was control added with sodium nitrite 0.0011%, and formula D was control added with extract 0.55% and sodium nitrite 0.0011%. All ingredients were blended to be the dough. The result of the study denoted that the extract (B and D) significantly decreased (P<0.05) pH, and aw value with no difference in water content among the dough. The total phenolic content of the dough containing extract (B and D) was markedly higher (P<0.05) than were others. It increased significantly on antioxidant capacity, scavenging activity, and reduced the thiobarbituric acid reactive substances (TBARS) value of the dough. There was also no nitrite residual detected in all dough. In conclusion, the extract could improve the physicochemical properties of beef sausage dough and replace the use of nitrite in the dough.


INTRODUCTION
Sausage has been becoming a popular food in Indonesia. It can be found in an abundance of places and levels of society. Sausage has also come to be a daily diet menu of Indonesian. It is a positive circumstance since it can intensify the protein consumption of Indonesia people. Unfortunately, most of the commercial sausages were manufactured by including the synthetic food additive, which has negative side effects. Some are carcinogenic (De Mey et al., 2014), inducing metabolic disorders (Shalaby and Shanab, 2013), and triggering colorectal disease (Herrmann et al., 2015;Zhu et al., 2014).
Recently, the use of natural agents such as phytochemicals as preservative and antioxidant have been rising for food purposes. The increased use of natural agents is due to awareness of the health of consumers. Accordingly, it is critical to shift to a natural agent in which one of them is senduduk (Melastoma malabathricum L.) leaf extract (SLE). Traditionally, senduduk leaf is utilized for healing diarrhea, dysentery, wounds, sore legs, and thrush (Susanti et al., 2008). Therefore, the exploration of senduduk leaf is focalized on the pharmacological aspect. Many researchers reported that the SLE contains the phenolic compounds which be able to scavenge free radical and inhibit the oxidation process, as well as anti-bacteria both in vitro and in vivo assay (Alnajar et al., 2012;Alwash et al., 2014;Wong et al., 2012;Zakaria et al., 2011). Even, SLE does not cause any acute toxicity and safety (Alnajar et al., 2012;Alwash et al., 2014;Kamsani et al., 2019). For food purposes, Suharyanto et al. (2019) noted that the maceration using water as a solvent also yielded SLE containing phenolic substances and could inhibit the bacteria growth. This result can promote the intensified efforts of SLE utilization in food and magnify safety and healthy food. The necessity of using aqueous extract is to evade consumers from the negative effects of organic solvent residues in the extract (Anyanwu et al., 2017).
On the other hand, the characteristics of the sausage are also determined by the dough quality. The use of SLE in the dough will in turn influence the final product of the food. Therefore, examining the effect of SLE on the sausage dough is fundamental to provide an impact on the sausage product. The aim of the study was to assess the effect of aqueous leaf extract of senduduk (Melastoma malabathricum L.) on the physicochemical properties of beef sausage dough.

Extract preparation
The senduduk leaf collected from shrub in Bengkulu were purified from undesired materials or substances. The clean leaves were air-dried for 5-6 h at 45°C. The dried leaves were then powdered and sieved into a 35 mesh. The technique of extraction adopted from Doughari and Manzara (2008). The powder (40 g) were macerated using distilled water (400 mL) in 1000 mL Erlenmeyer flask in the dark and room temperature for 24 h. The macerate was filtered using whatman No. 1 filter paper. The filtrate solvent was evaporated using a rotary evaporator (Heidolph, Antrieb-W-Mikro, Germany) at 40°C. The viscous raw extract obtained was freeze-dried (Snijders Scientific, LY5FME, the Netherlands). The extract was stored at -25°C until use.

Dough preparation
The round meat of Brahman Cross was separated from connective and fat tissue and then was cut into small pieces. The cut meat was minced using a meat mincer and further was formulated with the ingredients as shown in Table 1. Four formulas were employed in the research: formula A as a control consisted of beef, vegetable oil, skim milk powder, tapioca, salt, phosphate, and seasoning; formula B was control added with SLE 0.55%; formula C was control added with sodium nitrite 0.0011%, and formula D was control added with SLE 0.55% and sodium nitrite 0.0011%. All ingredients were blended to be the dough for each formula.

Measurement of pH, aw, and water content
The dough pH was determined by adopting AOAC (2005) procedure. A total of 10 g of sausage dough was dissolved and homogenized in 100 mL of distilled water. The solution was filtered using filter paper and the pH filtrate was measured using pH meter (Schott Instrument Lab 850). The measurement of aw was performed using awmeter (Novasina Ms-1) calibrated previously (Lorenzo et al., 2014). The dough was put in the aw-meter holder and measured. Water content was determined using AOAC (2005) method.

Total phenolic content
Sample preparation was carried out by dissolving 1 g dough into 5 mL of absolute methanol for 24 h (Sukisman et al., 2014). The solution was then filtered and the filtrate was used for determining the total phenolic content by using Al-Saeedi and Hossain (2015) procedure with a slight modification. Briefly, 0.4 mL of the filtrate was reacted with 3 mL of 20% Folin-Ciocalteou (Merck KGaA, Germany) solution and let stand for 5 min. 1.5 1.5 1.5 1.5 NaNO2 (g) (0.0011%)* --0.01 0.01 Extract (g) (0.55%)* -5 -5 Description: * Based on the total ingredient (908.75 g).
The mixture was added with 3 mL of 10% Na2CO3 solution, and then incubated for 60 min in a dark and room temperature. The mixture was absorbed using a spectrophotometer (Agilent, UV-Vis 8453, USA) at a wavelength of 760 nm. A similar procedure was applied to various standard gallic acid concentrations (0-16 mg/mL). The total phenolic content was calculated using a linear regression equation of the gallic acid absorbance and expressed in mg equivalent gallic acid/100 g dough dry matter.

Antioxidant activity
Sample preparation was employed using Sukisman et al. (2014) procedure. One-gram dough was dissolved into 5 mL of absolute methanol for 24 h. The solution was filtered and the filtrate was used for antioxidant capacity determination (Mahmoudi et al., 2016). As much as 0.2 mL of sample-methanolate solution was mixed with 1.8 mL of DPPH-methanolate solution (Sigma-Aldrich, D9132-1G, Germany) at a concentration of 6 × 10 -5 mol/L and shaken for 20 s.
The mixture was incubated in a dark and room temperature for 60 min. It was then measured for absorbance using a spectrophotometer (Agilent, UV-Vis 8453, USA) at a wavelength of 517 nm. In the same way, standard butylated hydroxytoluene (BHT) (Himedia, GRM797-500G, India) solutions in various serial dilutions (0.0-4.5 mg/100 mL) were employed.
Percent scavenging activity was calculated by the formula [(Acontrol-Asample)/Acontrol] ×100 where Acontrol was the absorbance of the DPPH solution without dough and Asample was the absorbance of the dough. The antioxidant capacity of the dough was calculated using the linear regression equation of BHT as a standard and expressed as mg equivalent BHT/100 g dry matter.

Thiobarbituric acid reactive substances (TBARS) value
Thiobarbituric acid reactive substances (TBARS) assay was employed for measuring malondialdehyde content by using Turgut et al. (2016) procedure. A-5 g dough was homogenized in15 mL of distilled water and centrifuged at 2000 × g for 15 min. As much as 1 mL of the supernatant of the mixture was added with 2 mL of 0.25 M HCl containing thiobacbituric acid (TBA) (0.375%, w/v) and trichloroacetic acid (TCA) (15%, w/v) and then added with 3 mL of BHT 2%.
The tube was vortexed and incubated at 100ºC for 15 min. The mixture was cooled at room temperature and then centrifuged at 1000 × g for 10 min. Finally, it was measured for absorbance using a spectrophotometer (Agilent, UV-Vis 8453, USA) at a wavelength of 531 nm against the blind. TBARS were calculated using 1, 1, 3, 3-tetraethoxypropane standard curve (concentration 2×10 -6 to 10×10 -6 M) and expressed as mg malondialdehyde (MDA)/kg of dough.

Nitrite residual
The nitrite residual of the sausage dough was determined by AOAC (2005) procedure. A-5 g of sausage dough was added with 40 mL of distilled water and heated to reach 80ºC. The solution was transferred to a 500 mL volumetric flask and hot distillate water was added until the volume reached 300 mL. The flask containing the solution was put into the steam bath for 2 h and stirring occasionally. The solution was cooled at room temperature and then was filtered.
Twenty-five mL of filtrate was put in the 50 mL volumetric flask, added with 2.5 mL of sulphanolate and stand for 5 min. Then, a reagent of 2.5 mL N-(1-Naphthyl) ethylenediamine (NED) 2 HCl is added and homogenized and reached to the mark. The solution was allowed to stand for 15 min to form pink and then its absorbance was measured using a spectrophotometer (Agilent, UV-Vis 8453, USA) at a wavelength of 540 nm.
An equivalent procedure was applied for blanks of 45 mL of distilled water, 2.5 mL of sulphanilamide solvents, and 2.5 mL of NED solvents. Standard curves from NaNO2 solution (Sigma-Aldrich, Germany) with a serial concentration of 0.2; 0.4; 0.6; 0.8 μg/mL was applied like the sample. Nitrite content was calculated based on the standard curve regression equation and expressed in mg/kg of dry matter.

Statistical analysis
The experiment was designed by using a completely randomized design with three replications of each treatment. The data presented as mean with standard deviations. The data were analyzed by oneway ANOVA and the differences of treatments were continued with multiple comparisons Tukey test. The significant difference was set at p<0.05. The statistical analysis using the general linear model of SAS, version 9.3.

Value of pH, aw, and moisture content
The addition of SLE improved physical characteristics. The data in Table 2 depict that the incorporation of SLE and nitrite in the dough decrease the value of pH, aw, and water content of the dough. The SLE in the dough was a factor determining the physical properties of the dough. Formula B which is a dough containing SLE had a lower value than control (formula A) in pH and aw variables. The nitrite addition just only affected on the dough if it was mixed with the SLE (formula D). It was supported by the data Formula C which statistically not different from Formula A. The capability of SLE to lower the pH value of the dough was likely influenced by the phenolic compounds of the SLE. Aqueous extract of senduduk leaf hold phenolic compounds (Suharyanto et al.,, 2019) which capable of decreasing pH value of extract (Wang et al., 2015). The lessen of pH value is contributed by the hydroxyl group of phenolic compounds of the extract (Pereira et al., 2009). This result supported other studies of plant extracts that decrease the pH value of food product (Devatkal et al., 2010;Jung and Joo, 2013). It was also confirmed that the SLE pH measured was 4.1 at a concentration 500 mg/mL (data not shown).
The addition with nitrite to formula C also indicated the lower pH than control. Nitrite in meat products dissolves and form HNO2 (nitrous acid) to produce NO (nitric oxide) and NO2 (nitrite) (Honikel, 2008). The NO molecule reacts with myoglobin or amino acids, while NO2 reacts with water to re-form HNO2 and HNO3 (nitric acid) (Honikel, 2008). These compounds make the product more acidic (Suryati et al., 2014). Water activity (aw) of the product is one of the essential factors affecting the shelf life. The lower the value of aw, the better the quality of the food product is obtained. This study elucidated that SLE improved the aw value of the sausage dough. The inclusion of SLE with or without nitrite combination reduced aw value of the dough. The control (formula A) gained similar aw value from nitrite addition (formula C). It indicated that SLE as a determinant of the decline aw value. The low aw value of dough added with SLE was due to the addition of extracts. The extract reduced the presence of free water in the dough that most probably caused by phenolic compounds forming hydrogen bonds with water molecules (Andarwulan and Faradilla, 2012). The water content of the dough added with SLE (Formula B and D) and control (Formula A) had similar value. However, the dough containing SLE also gave similar value to the nitrate-containing dough. Generally, the addition of SLE could not affect the water content of the dough.
In this case, nitrite decreased the water content of the dough. This phenomenon revealed that the quality of the dough added with SLE had better physical quality, which indicated by the lower pH and aw value. Reducing water content economically will result in the loss of production.

Total phenolic content
The addition of SLE in the sausage dough increased the total phenolic content. The mean of total phenolic content of all dough are shown in Table 3. The result exhibited that formula B reached the highest phenolic content. It is most likely due to the contribution of SLE in the dough. Suharyanto et al. (2019) extracted senduduk leaf using water as a solvent and the result exerted phenolic compound in the extract. Other researchers were also found that water extracts of plants yielded plant extract with phenolic compounds held (Anggraini, 2017;Mariem et al., 2014;Nurdiana and Marziana, 2013).
Formula A and C that were not be added with SLE contained comparable phenolic compounds but they were lower than the formula blended with SLE (formula B and D). Although not added with SLE, formula A and C contained phenolic compounds. Those were most likely contributed by the seasons. Suryati et al. (2014) stated that seasons also play a vital role by contributing the phenolic compounds to the product.
The combination of senduduk leaf extract and nitrite reduced precisely extract role in contributing phenolic compounds. The decrease in the total phenolic content of formula D was most probably caused by nitrosation of the phenolic compound (González-Mancebo et al., 2002) and a little water proved to be important in the initial period of the reaction (Ji et al., 2011). Nitrosation might be mediated by the lower pH of the obtained result. González-Mancebo et al. (2002) stated that nitrosation could be inhibited by the rising pH value. The study exhibited that the combination of SLE and nitrite exerted the lowest pH value of the dough (as shown in Table 2). This condition promoted the lower of existing phenolic compounds through a nitrosation process.

Antioxidant capacity and scavenging activity
The existence of phenolic compounds in the dough added with SLE affected the antioxidant capacity and scavenging activity of the dough. Antioxidant capacity expresses the concentration of antioxidants in the extract that equivalent to BHT. Scavenging activity shows the percentage of the capability of antioxidant in the extract to scavenge DPPH free radical. These data are shown in Table 3. The pattern of the antioxidant capacity and scavenging activity were similar to those phenolic compounds of the dough. The higher phenolic compounds held in the dough the higher the antioxidant capacity and scavenging activity of the dough. This study indicated that the addition of SLE significantly enhanced the antioxidant capacity and scavenging activity of the dough as shown by formula B and D. This result revealed that SLE in the dough played a role in increasing antioxidant activity.
This role was acted by phenolic compounds of the SLE. As mentioned above, the SLE contained phenolic compounds and these compounds could increase the antioxidant activity of plant extract (Alnajar et al., 2012;Zakaria et al., 2011). This research result also showed that nitrite had a low effect on the antioxidant activity (antioxidant capacity and scavenging activity) of the dough. The antioxidant activity was not different from control (formula A).
Nitrites can actually act as antioxidants (Karwowska et al., 2020). The addition of nitrite can form NO which in the lipid oxidation mechanism will form ROONO (Patel et al., 2000), but the role of these antioxidants is probably inhibited by the presence of phenolic compounds. This is because NO can react with phenolic compounds to form nitroso compounds from phenolic (González-Mancebo et al., 2002). These derived compounds apparently cannot act as antioxidants (Zubillaga et al., 1984).

TBARS value and nitrite residual
The result of the study showed that the TBARS value was significantly different lower of dough added with SLE from not added. The lower value of TBARS indicating the lower MDA production. MDA is one of lipid oxidation and this is a marker to determine the lipid oxidation. TBARS is indicated by the MDA production and expressed as mg MDA/kg of dough (Table 4).
The low TBARS value of dough added with SLE due to phenolic compounds in SLE acts as an antioxidant by preventing the oxidation process (Alnajar et al., 2012;Alwash et al., 2014;Zakaria et al., 2011). The TBARS value of the dough added with SLE confirmed that the phenolic compounds of the SLE capable of antioxidants role. The capability of phenolic compounds is due to the hydroxyl group structure of these compounds (Bendary et al., 2013). The phenolic compounds donate hydrogen and react to reactive species in the termination reaction to break down the new radical formation cycle (Pereira et al., 2009). However, TBARS values of all doughs were below the detectable threshold of rancidity, which is 5 mg MDA/kg (Insausti et al., 2001). Any nitrite residues in all doughs were not detected as shown in Table 4. This condition was possible due to the amount added was very little so that it was below the detectable threshold of tools and methods. Honikel (2008) stated that the nitrite concentration will decrease by 65% from the time of incorporation to the heating process finished. In formula D, it was thought to be due to the reduction process by phenolic compounds. Some previous research results suggested that phenolic compounds could reduce nitrites in Dendeng products (Suryati et al.,, 2014) and dry-cured bacon (Wang et al.,, 2015). The factors that caused the detection of nitrite residues in this study were quite complex. Detection of nitrite residues did not indicate that this product is safe from the negative effects of nitrites. The maximum limit of nitrite residual is 30 mg/kg product weight (BPOM, 2019).

CONCLUSION
The addition of aqueous extract of senduduk leaf improved the physical and chemical properties of the raw sausage dough. The addition of 0.55% senduduk leaf extract decreased the pH, aw, and water content of the dough; enhanced the total phenolic content, antioxidant activity of the dough; and reduced the TBARS value of the dough. The use 0.55% of senduduk leaf extract could replace nitrite 0.0011% in the dough physicochemical characteristics. The good characteristics of the dough will generate a good properties of final product.

CONFLICT OF INTEREST
We declare that there are no conflict of interest in this work.