Ethanolic Sisyrinchium palmifolium L. Extract as Natural Preservative for Indonesian Tofu Preparation

Tofu is a food product that is easily contaminated by microbial due to its water content. Some bacteria that usually grow in tofu are Escherichia coli, Bacillus cereus, or Staphylococcus aureus . Preservatives are added to solve the common storage problem. However, some manufacturers use hazardous substances, such as formalin or other chemical substances, as a preservative. Tiwai onion ( Sisyrinchium palmifolium L.) is a plant that grows in Borneo and has a broad range of antibacterial activity. This study aimed to examine the activity and effectiveness of Sisyrinchium palmifolium extract as a preservative in tofu. Sisyrinchium palmifolium was extracted using the maceration method with ethanol three times. The concentrated ethanol agar and microdilution The 5% tetracycline HCl positive control diluted using NaCl 0.9%. Agar diffusion method was done with Sisyrinchium palmifolium ethanol e xtract 10000 μg/mL and tetracycline HCl 50 μg/mL. The microdilution method was done with Sisyrinchium ethanol extract with an initial concentration of 40000 μg/mL and tetracycline H Cl 2000 μg/mL The results showed that the minimum μg / mL. the effectivity of concentrated tested determining Total incubation 37, 25, and 4 sorbate as control. Furthermore, organoleptic evaluation was observed at 25 and 4 °C. The results showed that Sisyrinchium palmifolium ethanolic extract was effective as an alternative preservative for tofu at a concentration of 5 000 μg/mL. In c onclusion, ethanolic extract of Sisyrinchium palmifolium could serve as a novel candidate and effective preservative in tofu. pengujian dilakukan dengan metode difusi agar dan metode mikrodilusi kaldu. Larutan uji diencerkan menggunakan DMSO 5% sebagai pelarut dan larutan tetrasiklin HCl sebagai kontrol positif diencerkan menggunakan NaCl 0,9%. Metode difusi agar dilakukan dengan ekstrak etanol bawang tiwai 10000 μg /mL dan tetrasiklin HCl 50 μ g/mL. Metode mikrodilusi dilakukan dengan ekstrak etanol bawang tiwai dengan kon sentrasi awal 40000 μg /mL dan tetrasiklin HCl 2000 μg /mL. Hasil penelitian menunjukkan bahwa ekstrak etanol bawang tiwai memiliki aktivitas antibakteri dengan nilai konsentrasi hambat minimal 5000 μg /mL. Kemudian, efektifitas ekstrak etanol pekat bawang tiwai sebagai pengawet tahu diuji dengan menentukan Total Plate Count pada suhu inkubasi 37, 25, dan 4° C dibandingkan dengan kalium sorbat sebagai kontrol. Kemudian dilakukan evaluasi organoleptic pada suhu 4 dan 25 °C. Hasil penelitian menunjukkan bahwa ekstrak etanol bawang tiwai efektif sebagai pengawet alternatif tahu pada konsentrasi 5000 μg /mL. Kesimpulannya, ekstrak etanol bawang tiwai dapat menjadi kandidat pengawet yang baru dan efektif pada tahu .

sorbate as control. Furthermore, organoleptic evaluation was observed at 25 and 4 °C. The results showed that Sisyrinchium palmifolium ethanolic extract was effective as an alternative preservative for tofu at a concentration of 5000 μg/mL. In conclusion, ethanolic extract of Sisyrinchium palmifolium could serve as a novel candidate and effective preservative in tofu.
Though its effectiveness as preservative, it has been showed that exposure to formaldehyde may cause adverse human health effect such as skin irritation, allergy, pneumonia, and cancer (Norliana et al, 2009). Another preservation method widely used for tofu handling and processing are immersion of tofu in solution containing salt such as potassium sorbate or sodium chloride, or immersion of tofu in traditional mixture such as turmeric and lime solution or lime and kitchen salt solution (Jubayer and Faruque, 2013).
Tiwai onion (Sisyrinchium palmifolium L.) is a herbal plant whose traditionally used as natural product to prevent and cure hypertension, diabetic, and acnes (Febrinda et al, 2014). It has been reported the extract of Sisyrinchium palmifolium showed inhibitory activity against Staphylococcus aureus (Ifesan and Voravuthikunchai 2009;Ifesan et al, 2009) and other bacteria such as Shigella boydii (Panda et al, 2016). There was no reported research up to now regarding the use of the Sisyrinchium palmifolium as preservative and additive in tofu processing and preparation. We carried out this study to examine the ability of Sisyrinchium palmifolium extract as preservative in tofu.

Research Method Chemical and Reagents
Sisyrinchium palmifolium bulb was obtained freshly from Bandung, Indonesia, cleaned and characterized and dried prior to use. Tofu was freshly obtained from Yun Yi Industry, Escherichia coli (ATCC 8739), Bacillus cereus (ATCC 1178) were obtained from American Type Culture Collection. Nutrient Agar (NA), Nutrient Broth (NB), Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB), tryptone, yeast extract was used as medium for bacterial culture, tetracycline as standard material, sodium hydroxide, potassium sorbate, DMSO and all commercially available reagents were analytical grade and used as purchased (Merck, Sigma-Aldrich).

Preparation of Ethanolic Sisyrinchium palmifolium Extract
The powdered and dried Sisyrinchium palmifolium bulb was extracted using maceration method using three times of ethanol (1:5). The extract was filtered using filter paper and the filtrates were evaporated under reduced pressure in a rotary evaporator. The extract was dissolved in 5% DMSO (in 0.9% NaCl) before use. The end concentration of extract is 40000 μg/mL and 10000 μg/mL.

Preparation of Suspended Microbe
The preparation of suspended microbe was adapted from CLSI (CLSI, 2012). There are two microbes used in this study, Eschericia coli and Bacilus cereus. The microbes grown on the surface of agar plate was slowly taken and suspended in nutrient agar media and incubated at 37°C. The suspended microbe was made by selected the microbe of interest and suspended in nutrient broth media (1.8% in water). Dilution was made using NaCl solution (0.9% in water) until it reaches the 0.5 McFarland value or the UV-Vis spectrophotometer absorbance range between 0.08-0.13 at 625 nm. The total colony forming unit was measured before the antimicrobial activity determination.
Antimicrobial Activity of Ethanolic Extract Determination 1. Agar diffusion method Antimicrobial activity was evaluated using paper disc agar diffusion. Ethanolic Sisyrinchium palmifolium extract 40000 μg/mL was diluted in 5% DMSO until the final concentration of the test solution are 50, 100 and 200 µg/ml, respectively. Tetracycline HCl (50 µg/ml in 0.9% NaCl) was used as a negative control meanwhile 0.9% NaCl was used as positive control. Ten microliters of the test solution (extracts, tetracycline HCl and 0.9% NaCl) were then added to sterile filter paper disc and placed on the dishes containing microbe of interest. The dishes were incubated for 18-24 hours at 37°C.

Determination of Antimicrobial Activity of Ethanolic Extract in Tofu
Total plate count was used to determine the antimicrobial activity of the extract. Potassium sorbate (1% in sterile water solution) was used as control. The tofu was immersed in water, after several hours, the water was carefully taken and used as samples for determining the antimicrobial activity. Ethanolic Sisyrinchium palmifolium extract which produce activity on the previous assay was used as test substance. Plate count agar (PCA) was used for total plate count. Test substance and the control were added in comparison of 15:1:1. All the plates were incubated at different condition to mimic the storage of tofu, 18-24 hours (37 o C), 72 hours (25 o C), 168 hours or 7 days (4 o C). Organoleptic evaluation was carried out every single day of the assay for storage in 25 o C and 4 o C.

Results and Discussion
Sisyrinchium palmifolium bulb is herbal plant that may be produced and cultivated under different condition. Characterization and phytochemical screening were applied to convince standardize bulb was used in this study. Characterization and phytochemical screening of dried Sisyrinchium palmifolium bulb is given in Table 1 and   Table 4 for Escherichia coli and Table 5 for Bacillus cereus. It was showed that ethanolic Sisyrinchium palmifolium extract has MIC value of 5000 µg/mL for Escherichia coli and 10000 µg/mL for Bacillus cereus. We then conclude to use 5000 µg/mL as the minimum concentration for antimicrobial activity evaluation on tofu products.
Total plate count (TPC) was used to evaluate the effectiveness of the extract as preservative because the reliability and the reproducibility of the method (Table 6-8). Result showed that the addition of extract decreases the number of total colonies. Three temperatures used in the experiment to count the TPC of tofu (37, 25, and 4°C). The results showed that Sisyrinchium palmifolium extract was able to suppress the growth of microbial in tofu either in reduced temperature as well as in room temperature. Tofu stored in reduced temperature showed lower total colony count compared to tofu stored in room temperature during observation time. However, there is a limitation storage time for treated tofu in refrigerator. The Sisyrinchium palmifolium extract showed good activity on tofu compared to potassium sorbate.    ---+ + + + + + + + Note: (-) = negative control, (+) = positive control µg/mL, -= no bacteria suspension observed, + = bacteria suspension observed, i.e., 1 = 20000 µg/mL, 2 = 10000 µg/mL, 3 = 5000 µg/mL, 4 = 2500 µg/mL, 5 = 1250 µg/mL, 6 =625 µg/mL, 7 = 312 µg/mL, 8 = 156 µg/mL, 9 = 78 µg/mL, 10 = 39 µg/mL   Total plate count (TPC) was used to evaluate the effectiveness of the extract as preservative because the reliability and the reproducibility of the method (Table 6-8). Result showed that the addition of extract decreases the number of total colonies. Three temperatures used in the experiment to count the TPC of tofu (37, 25 and 4 °C). The results showed that Sisyrinchium palmifolium extract was able to suppress the growth of microbial in tofu either in reduced temperature as well as in room temperature. Tofu stored in reduced temperature showed lower total colony count compared to tofu stored in room temperature during observation time. However, there is a limitation storage time for treated tofu in refrigerator. The Sisyrinchium palmifolium extract showed good activity on tofu compared to potassium sorbate.   To obtain deeper insight into the acceptability of tofu as food products, the organoleptic evaluation was conducted for storage in 25 and 4 °C (Table 9-10). It showed that the Sisyrinchium palmifolium extract capable to maintain the texture and odor of tofu during the storage and therefore may increasing the self-life of tofu.

Conclusion
In summary, Sisyrinchium palmifolium extract was found to be a good candidate as substitution of chemical preservative in tofu processing and preparation.