Effectiveness of the Red Dragon Fruit (Hylocereus Polyrhizus) Peel Extract as the Colorant, Antioxidant, and Antimicrobial on Beef Sausage

This study aimed to evaluate the effectiveness of red dragon fruit (Hylocereus polyrhizus) peel extracts addition on beef sausages. Red dragon fruit peel extracts were obtained by maceration using solvent at pH 5. Phytochemical characteristics, total phenols, antioxidant, and antimicrobial activity of the peel extracts were observed. Antioxidant and antimicrobial activities of the extracts were associated with high phytochemical compounds and total phenols contained in the extracts. Red dragon fruit peel extracts with various percentages (0%, 20%, 30%, and 40%) were added on beef sausages, and their physicochemical characteristics, nutrients, antioxidant activity, and microbiological profile were analyzed. The data were analyzed using analysis of variance and Duncan's multiple range test. Results showed that the addition of red dragon fruit peel extracts significantly reduced texture values, but increased intensity of luminosity, intensity of red color, and intensity of yellow color (P<0.05) beef sausages. It could be concluded that red dragon fruit peel extract containing phytochemical compounds was effective as an antibacterial agent and natural antioxidant. The addition of red dragon fruit peel extracts was effective in increasing the antioxidant activity and decreasing TBARS values. The addition of red dragon fruit peel extract did not affect the reddish colorization of beef sausages, but it was capable of increasing the yellowish colorization on beef sausage.


INTRODUCTION
The use of food additives in sausage processing is to provide some advantages such as reddish colorization, antioxidant, and antibacterial activity. In order to control pathogenic bacteria, nitrite and nitrate salts are commonly used in processed meat. These additives may also contribute to produce the red color and antioxidant activity. However, their presence is also associated with formation of carcinogenic compound, nitrosamine. Nitric oxide (NO), formed by nitrites or nitrates, can react with secondary amines in meat to form nitrosamine under certain conditions (eg, high temperature) (Honikel, 2008). This carcinogenic compound is formed in acidic environments, especially in the gastrointestinal tract of human gut (Honikel, 2008). When consumed continuously and exceeds the maximum levels, it can cause health problems such as cancer. Thus, the use of nitrite in food products should be limited.
The use of nitrite or nitrate can be replaced with natural ingredients derived from plants source which extensively studied both function and composition. One of the candidates is dragon fruit peel which is not generally utilized. The dragon fruit peel is potential as a natural colorant (Harivaindaran et al., 2008), an antibacterial against nine bacteria of food pathogen (Nurmahani et al., 2012), and natural antioxidant (Luo et al., 2014). The addition of red dragon fruit peel extracts were expected to give better characteristics based on color, antioxidant, and antibacterial on beef sausage. This study aimed to analyze the effectiveness of red dragon fruit peel extracts (Hylocereus polyrhizus) as antibacterial agent and natural antioxidant, as well as to evaluate the effectiveness of the extracts on beef sausages. Other analyses such as physicochemical and nutrient analysis were conducted to obtain the supporting data.

Experimental design
This experiment used a completely randomized design with 4 treatments of different levels of red dragon fruit peel extract addition (0%, 20%, 30%, and 40%) and three replications.

Procedure
Extraction of red dragon fruit peel. The red dragon fruit peel was extracted by modified maceration from Lourith & Kanlayavattanakul (2013) using distilled solvent at pH 5 adjusted by adding citric acid. Dragon fruit was cleaned and peeled manually. The peels were cut in small size (2 mm), dried at 50 o C, and powdered. The powder was macerated with distilled water solvent (1:50) for 60 min and filtered. The solution was evaporated at vacuum evaporator at 60 o C. The extract was stored at -20 o C. Sausage processing. The sausage was prepared by following the procedure conducted previously by Arief et al. (2014). The meat was added with 15% fat, 3.9% salt, 0.8% sodium tripolyphospate, and 20% ice and ground in a food processor for 30 s. Furthermore, the dough was added with 12% skim milk, 1% garlic powder, 0.5% white pepper powder, 0.5% ginger powder, 0.4% coriander, 0.2% nutmeg, 30% starch, and 40% ice. Percentage of each ingredient was calculated based on the amount of meat used in the dough. Different levels of red dragon fruit peel extracts (0%, 20%, 30%, and 40%, followed by reduction of ice percentage) were added into the dough. The dough was mixed for 90 s, and transferred into casing using a stuffer, then boiled at a temperature of 60-65 o C for 60 min.

Analysis of Variables
Analysis of phytochemical components. The phytochemical component test was performed to determine the type of active compounds contained in the extract according to Baxter et al. (1998). The tested compounds were alkaloids, flavonoids, phenols hydroquinone, steroids, triterpenoids, tannins, and saponins.
Analysis of total phenolic content. Total phenol content of extracts was determined using spectrophotometric method according to Adnan et al. (2011). A total of 100 mL extracts was mixed with 7.9 mL of distilled water and 0.5 mL of Folin-Ciocalteu reagent (Sigma-Aldrich Co., USA). After 2 min, 1.5 mL of 7.5% sodium carbonate reagent was added and homogenized. Samples were measured using spectrophotometer at 765 nm (Genequant 1300, Sweden) after being incubated for 2 h. The content of total phenol was expressed as gallic acid equivalent.
Analysis of antioxidant activity. Analysis of antioxidant activity of extracts was conducted by measuring free radicals inhibition against 1,1-diphenyl, 2-picrylhydrazil (DPPH) according to Adnan et al. (2011). A total of 150 mL extract with different DPPH concentrations was mixed with 0.9 mL (25 mg L -1 ) in methanol solution. The mixture was left for 20 min and the absorbance was measured in spectrophotometer with a wavelength of 517 nm. Pure methanol was used as a control. The control was prepared with 100 mL of methanol and dissolved with DPPH solution. Absorbances of samples were measured in a spectrophotometer with a wavelength of 517 nm (Genequant 1300, Sweden). The antioxidant activity was expressed as a percentage of free-radical inhibition which calculated based on the following formula: % Inhibition = (A control -A sample) × 100 / A control The percentage of inhibition indicates the antioxidant activity level. Vitamin C (Merck, KgaA, Germany) was used as a standard.
Analysis of antimicrobial activity. Analysis of antimicrobial activity of extracts was performed by the well diffusion method to determine the pattern of inhibition of the extract against pathogenic bacteria according to Rohin et al. (2012). Bacterial culture was inoculated in NaCl 0.85% to obtain the bacteria concentration of 10 8 cfu mL -1 (compared with 0.5 Mc. Farland standard solution).
Bacterial culture was diluted to obtain a culture concentration of 10 6 cfu mL -1 . The other culture was grown in Mueller-Hinton Agar medium (Difco TM , USA) and provided with holes as well as a predetermined diameter. The extracts were inserted into the well and covered with filter paper. Grail was stored in the refrigerator for 2-3 h, followed by incubation at 37 o C for 24 h and 48 h. The antimicrobial activity was characterized by the formation of clear zones around the wells and measured for its diameter (mm).
Nutritional composition. The proximate analysis of sausages was performed using AOAC (2005) standard, including moisture, protein, fat, and ash contents. Carbohydrate content was determined by difference from the proximate analysis.
Water activity and pH measurement. The pH value of the sausages was measured using pH meter (Hanna HI 99163, HANNA Instruments, USA). Water activity of sausages was measured using a w meter (Novasiana, Switzerland).
Color measurement. The color of sausages was measured using a Minolta Chroma meter CR 300 (Minolta Co., Ltd. Osaka, Japan). The sample was measured on three different surfaces. L, a, and b values refer to the intensity of luminosity, the intensity of red color, and the intensity of yellow color. A and b values were used to determine °HUE of samples, with the following formula.°H UE = arc tg (a/b) Texture measurement. Textural analysis of sausages was performed using texture analyzer Steven-LFRA. The sausages at 2.54 cm in diameter were selected prior to the tests. Texture measurement procedure was carried out in accordance with the manufacturer's instructions.
Emulsion stability. Emulsion stability of sausages was measured by measuring the volume of oil and water emulsion according to Zorba et al. (1993). The sample was heated in water bath at 80 °C for 30 min. The samples were centrifuged at 2000 rpm for 15 minutes. The volumes of oil and water were measured to determine the emulsion stability (ES) of samples, with the following formula.
Antioxidant activity. Sausage samples were extracted to obtain a supernatant prior to the determination of antioxidant activity based on the procedure performed previously by Tangkanakul et al. (2009). The sausage was extracted with absolute methanol at room temperature with ratio of 1:5. The filter paper was used to separate the supernatant. The supernatant was stored in sealed bottles and stored at -20 o C. Antioxidant activity was determined using the same procedure with Adnan et al. (2011).

Microbiological analysis.
The procedure of microbiological analysis was performed according to FDA (1998). Sausage (25 g) was mixed with 225 mL of buffered peptone water (BPW), and homogenized for 1-2 min to obtain a 10 -1 solution. Then, 1 mL suspension was transferred with a sterile pipette to 9 mL of BPW solution to obtain a 10 -2 dilution. The dilution with the same ways was continued until 10 -4 dilution was obtained. Microbiological analysis consisted of total plate count, Escherichia coli, Salmonella sp., and Staphylococcus aureus analysis.

Statistical Analysis
Data were analyzed using the General Linear Model of Statistical Analysis System's Procedures (SAS Institute Inc., Cary, NC, USA, 2002) and the Duncan's multiple range test was conducted as further analysis. The addition of red dragon fruit peel extracts was set as treatment. Significance range was set at 0.05.

Characteristics of Red Dragon Fruit Peel Extracts
The results indicated that flavonoids, phenols hydroquinone, steroids, triterpenoids, saponin, and tannins were the type of phytochemical compounds contained in red dragon fruit peel extract (Table 1). The antioxidant test was indicated by the phenolic, DPPH scavenging activity, and antioxidant capacity ( Table 2). The inhibition zone produced by red dragon fruit peel extracts showed the antibacterial activity (Table 3).

Characteristics of Beef Sausages with Red Dragon Fruit Peel Extract Addition
Nutritional composition (water content, ash content, protein content, fat content, and carbohydrate content) of beef sausage were presented in Table 4. The results revealed that water content, ash content, fat content, and carbohydrate content of sausage were unaltered by incorporation of red dragon fruit peel extracts.
However, we found that the treatments affected protein content of beef sausage. Physicochemical characteristics (pH value, water activity, emulsion stability, texture, and color intensity) of beef sausages were presented in Table 5. The data indicated that pH value, water activ-ity, emulsion stability, red color stability, and ºHUE were unaffected by incorporation of red dragon fruit peel extracts, but the treatments significantly influenced texture, lightness intensity, and yellowness degree of beef sausage. The result of antioxidant activity and malondialdehyde (MDA) content in beef sausages was presented in Table 6. Significant effects of red dragon fruit peel extracts addition were observed in antioxidant capacity, DPPH scavenging activity, as well as TBARS value. Microbial evaluation including total plate count and some specific bacteria such as E. coli, Salmonella sp., and S. aureus was outlined in Table 7. Different levels of red dragon fruit peel extract unaltered the total plate count on beef sausages.

Characteristics of Red Dragon Fruit Peel Extracts
Flavonoids, phenols hydroquinone, steroids, triterpenoids, saponin, and tannins were the type of phytochemical compounds contained in red dragon fruit peel extract. Flavonoids and phenols hydroquinone in red dragon fruit extract had the correlation with antioxidant activity. Flavonoids (Nurliyana et al., 2010) and phenols (Wu et al., 2006) found in dragon fruit peel extract were responsible for antioxidant activity in peel extracts. Steroids and triterpenoids compounds in the red dragon fruit peel had the correlation with antibacterial activity. Amalia et al. (2015) also found similar compounds in the screening, which were suspected to have antibacterial activity through reaction with cell wall proteins.
Phytochemicals qualitative test in red dragon fruit peel extract showed positive results in saponins. These results were in contrast to the finding of Amalia et al. (2015) using n-hexane as the solvent. This difference could be due to differences in solvents, where polar solvents (e.g water and ethanol) may have more soluble property in comparison with non-polar solvents (e.g n-hexane) (Baxter et al., 1998). Red dragon fruit peel extract also contained tannin, while the alkaloids were undetected.
The total phenolic of red dragon fruit peel extract was higher than the results of Nurliyana et al. (2010). Differences in solvents for extraction process may account for these different results. This study employed the distilled water, a solvent that is recommended for the food industry because of the extraction contains no residue that makes it is safe for consumption (Kumar et al., 2015).
The antioxidant activity of the red dragon fruit peel extract was identified by DPPH scavenging activity and antioxidant capacity. The DPPH scavenging activ-ity (50.14%-52.15%) is not much different from those reported by Fidrianny et al. (2014) using n-hexane, ethyl acetate, and ethanol. Antioxidant capacity obtained is 321.78±6.29 mg VCE/100 g. Lourith & Kanlayavattanakul (2013) stated that the red dragon fruit peel extract with solvent water had better antioxidant activity than using ethanol. Harivaindaran et al. (2008) suggested that the DPPH scavenging activity and antioxidant capacity was proportional to the total phenol antioxidants in red dragon fruit peel.
The inhibition zone produced by red dragon fruit peel extracts showed the antibacterial activity (Table 3). Gram-positive bacteria, S. aureus ATCC 25923, was more sensitive to the antibacterial activity of the red dragon fruit peel extract. High inhibition of Gram-negative bacteria, P. aeruginosa ATCC 27853, by red dragon fruit peel extract resulted from antibacterial compounds content such as phenolic compounds. This assumption was supported by Arief et al. (2015) which stated that the Gram-positive bacteria were more susceptible to antibacterial activity due to the absence of a lipoprotein wall that capable of preventing antimicrobial compounds.
The bacterial inhibition is also observed in the pathogenic bacteria S. enterica ser. Typhimurium ATCC 14 028, according to research conducted by Nurmahani et al. (2012) which demonstrates the inhibition of pathogenic bacteria S. thypii. Phenolic compounds in the extracts of pomegranate peel studied by Choi et al. (2011) were able to inhibit the growth of Salmonella. The B.
cereus and E. coli ATCC 25 922 was also inhibited by red dragon fruit peel extracts. Antibacterial activity against both bacteria was also observed by Tahera et al. (2014).

Characteristics of Beef Sausages with Red Dragon Fruit Peel Extract Addition
The water content and fat content of each beef sausage were in accordance with the established standard,   BSN (1995). The protein content of beef sausage did not meet the minimum standards set by BSN (SNI 01-3820:1995), as influenced by the low levels of non-meat protein content used in the sausages (Nurul et al., 2010). A higher level of red dragon fruit peel extracts addition was responsible for the lower pH value of beef sausage. The change in pH was in the range of 5.72-5.80, which could be caused by the same pH of distilled water used to dissolve the extract. The solvent was obtained by the addition of citric acid in distilled water according to Harivaindaran et al. (2008). The water activity of this sausage is due to the percentage of the addition of dragon fruit peel extract in sausages followed by the reduction of ice on the dough. Emulsion stability of a product is influenced by the degree of meat tenderness (Aminlari et al., 2009). The meat used in the study came from the same part i.e., topside.
A higher level of extract addition in the sausage associated with a reduction of its hardness, which could be linked with moisture and protein contents of sausages. The higher protein content resulted in higher harsh texture (Youssef & Barbut, 2010). Youssef & Barbut (2010) showed that the higher addition of protein (8% to 14%) increased the value of hardness because protein formed a denser complex.
HUE values of sausages in this study were 58.83-62.85, thus producing a yellowish red color of the product (ºHUE = 54º-90º). Totosaus (2009) stated that sausages with the addition of natural colorant resulted in the value of ºHUE which almost the same with sausage using nitrate and nitrite. Sausage with the extract addition had higher red intensity value than those without addition. This is a noticeable result, that addition of red dragon fruit peel extract could produce red intensity on the sausage due to the presence of natural pigments contained in dragon fruit peel. Jamilah et al. (2011) reported that red dragon fruit peel contained betacyanin pigment which contributes to a natural red dye. Betacyanins pigment is the extraction product of betalains compound (Harivaindaran et al., 2008). This pigment is capable of holding the red color in acid condition with the range of pH from 4 to 7.
However, the intensity of red color on sausages with the extract addition was quite low. Stability of the color pigment betacyanin may decrease as a result of the rising temperature and long enough heating process. Faridah et al. (2015) stated the stability of betacyanin pigmentin dragon fruit peel decreased along with the increased heating temperature (70°C and 100°C). Harivaindaran et al. (2008) stated that the red dragon fruit peel had the highest color stability by heating at 100°C for 5 min and would affect the colors produced when the heating carried out was long enough. The intensity of the yellow color in this study increased with the increasing level of the extract used. Beef sausages with the addition of red dragon peel extract as much as 40% have the highest value of yellow color. According to Woo et al. (2011), besides produces natural red color, betalain pigment also contains betaxantine which ca-pable of producing natural yellow color. In addition, the intensity of yellow color in sausages is higher compared to red color. This difference is due to the quite long heating time conducted in the processing which can affect betasianin pigment content. Herbach et al. (2004) stated that the effect of heating time with high temperature led to the degradation of betacyanin and production of yellow pigment.
The antioxidant activity increased along with the increased level of dragon fruit peel extracts addition. Nurliyana et al. (2010) found that the higher concentration of red dragon fruit peel extract resulted in a higher DPPH scavenging activity. The value of antioxidant activity found in the analysis was high. This result due to the polyphenolic compounds contained in the red dragon fruit peel extract (Harivaindaran et al., 2008) and the addition of spices of sausage as garlic, coriander, and pepper which have antioxidant activities .
Sausage with the addition of red dragon fruit peel extracts had smaller TBARS values than the sausage without the extract addition. This value was associated with the high antioxidant activity correlated with the phenolic in sausages, thus it could inhibit the oxidation of sausage (Wu et al., 2006). Phenolic compounds donated a hydrogen atom to the free radicals that enhanced the stability of radical phenolic derivative (Jongberg et al., 2013).
The addition of the red dragon fruit peel extract could not decrease the total plate count of the sausages (Table 7), eventhough the red dragon fruit peel extract could inhibit the pathogenic bacteria by in vitro methods (Table 3). The total plate count of all sausages were in 2 log 10 cfu/g. It was still in Indonesian National Standart for sausage /01-3820:1995 (BSN, 1995). Bacteria Escherichia coli, Staphylococcus aureus, and Salmonella sp. were not found in the results of microbiological analysis of beef sausage with the addition of red dragon fruit peel extract. The absence of E. coli on the sausage was caused by the heat treatment. The acceptable condition for E. coli bacteria and S. aureus growth was at 7-50°C and 7-48°C, respectively, but their optimum growth was at 37 o C (Adams & Moss, 2008). Salmonella sp. and S. aureus also were not found in the results of microbiological analysis of meat used in this study. Salmonella sp. was unable to grow at high temperature such as heating at 65-74 o C (Jay, 2012).

CONCLUSION
Red dragon fruit peel extract (Hylocereus polyrhizus) containing phytochemical compounds was effective as an antibacterial agent and natural antioxidant. The addition of red dragon fruit peel extracts on beef sausages effectively increased antioxidant activity and lowered TBARS values. Microbiological quality of the sausages met in Indonesian National Standard for sausage /SNI 01-3820:1995. Red dragon fruit peel extract at a concentration of up to 40% was ineffective in increasing the reddish intensity of beef sausages, but it was capable of improving the yellow color in beef sausage.