Nutrient Content, Active Compound, and Antibacterial Activity of Padina australis against Aeromonas hydropilla

Macroalgae belong to thallophytes group or plant-like organisms which generally live in coastal areas.1-3 Macroalgae known as seaweed have significant economic value since they can be utilized as vegetables, traditional medicines, organic fertilizers, and livestock fed.1,3 Even the phytocoloid compounds extracted from macroalgae as agar, carrageenan, and alginate3, it can be used as raw materials of various industries, such as medicine, cosmetics, food, etc. Based on the pigment content, macroalgae are classified as green, red, and brown macroalgae.4,5 Several studies has been revealed that green, brown and red algae contain different metabolites and have much biological activity such as antiviral, antibacterial and antifungal.6


INTRODUCTION
Macroalgae belong to thallophytes group or plant-like organisms which generally live in coastal areas. [1][2][3] Macroalgae known as seaweed have significant economic value since they can be utilized as vegetables, traditional medicines, organic fertilizers, and livestock fed. 1,3 Even the phytocoloid compounds extracted from macroalgae as agar, carrageenan, and alginate 3 , it can be used as raw materials of various industries, such as medicine, cosmetics, food, etc. Based on the pigment content, macroalgae are classified as green, red, and brown macroalgae. 4,5 Several studies has been revealed that green, brown and red algae contain different metabolites and have much biological activity such as antiviral, antibacterial and antifungal. 6 Padina australis is one of the brown macroalgae that possess numerous compounds that can be used for various properties, either nutrient content or active compounds. P. australis contains 1.05 ± 0.09% protein, 0.58 ± 0.01% fat, 8.78 ± 0.80% carbohydrate, 87.25 ± 0.86% water, 2.34 ± 0.16% ash 7 and minerals as calcium, magnesium, potassium, sodium, copper, zinc, iron 8 that enable to be developed as a food source and livestock fed. P. australis also contains various active compounds, such as steroid, terpenoid, flavonoid, tannin, and saponin 2,9,10 that can be used as medicinal drugs. P. australis found abundantly in Indonesia sea and distributed almost all stony coasts including East Nusa Tenggara waters. According to Salosso and Jasmanindar 11 , brown macroalgae recorded in 5 sampling sites of Kupang Bay and distributed in all locations and sampling sites are P. australis with up to 80% occurrence frequency. Nevertheless, this species has not been maximally utilized yet by the community. 10 To optimize the utilization of P. autralis collected from Kupang Bay, a study on nutrient content, active compounds, and antibacterial activity of P. australis were carried out in this paper.

P. australis collection
P. australis were collected at the lowest tide in Kelapa Lima coastal waters, Kupang Bay, by searching along the coast and taking all encountered P. australis. They were put into a plastic bag, cleansed, recorded the fresh weight, air-dried, and then ready for further analyses.

Chemical composition analysis
Chemical composition analyses of P. australis include water, ash, protein, and fat content. 12 Carbohydrate content was determined by difference as follows: 100% -(% water + % fat + % protein + % ash). Amino acid content was determined using a High-Performance Liquid Chromatography (HPLC). Mineral analyses on calcium (Ca), potassium (K), and iron (Fe) were assayed using Atomic Absorption Spectrophotometer (AAS).

Phytochemical analysis of P. australis
Phytochemical investigations of P. australis include alkaloid, saponin, flavonoid, tannin, terpenoid, and steroid. Alkaloid was examined using Culvenor-Fiztgerald method, saponin was analyzed using a foam test, tannin was analysed using FeCl, and terpenoid and steroid was analyzed using the Lieberman-Burchard method. 13

P. australis extraction
Water extraction was done by boiling 10 g of finely ground P. australis in 100 ml of distilled water (10%), and incubated for 24 h. The samples have then filtered and stored until further use. 14 Methanol extraction was done by macerating 10 g of finely ground P. australis in methanol for 48 h, filtered to separate the sediment and extract, then evaporated, and ready for the antibacterial test.

Antibacterial activity test
Water and methanol extracts of P. australis were examined against A. hydropilla. This examination used disc test method 15 by immersing the sterile disc paper into each extract. After 15-30 min, the disc paper was attached to the TSA media which have been inoculated with A. hydropilla. Measurement of the inhibition zone was carried out after 24 h inoculation at 37 0 C by observing the presence of clear area formed around the disc paper.
The amino acid and mineral content of P. australis P. australis containing 15 amino acids in different concentration ( Table  1). The highest content were aspartic acid (1.16% w/w) and glutamic acid (1.32 %w/w) and the lowest content were histidine (0.12%w/w) and methionine (0.2 % w/w).

Active compounds of P. australis
Qualitative test of active compounds indicated that P. australis contained alkaloid, saponin, flavonoid, tannin steroid, and terpenoid.

Antibacterial activity of P. australis
The antibacterial analysis showed that water and methanol extract of P. australis could inhibit the growth of A. hydropilla. It was indicated with the presence of inhibition zones of 10.5 mm for water extract and 10 mm for methanol extract of P. australis (Table 3).
This study provided information on the nutrient content of P. australis, such as protein, fat, carbohydrate, water, and ash. Protein and fat content of P. australis found in Kelapa Lima coast, Kupang Bay, was higher than in Tidung waters, Seribu Islands, only 1.05% protein and 0.58% fat. 7      in Pahuwato waters, Gorontalo, has only 4.78% protein and 0.52% fat 5 , P. gymnosprora in India contains only 5.704% protein and 0.02% fat 16 , and P. tetrastomatica contains 10.5% protein and 1.14% fat. 17 These differences could result from dissimilarity in harvest age and weather condition at the rearing period. 7 Furthermore, carbohydrate content in P. australis was higher than in Tidung island, Kupang Bay, only 8.78% 7 and in Souttheast Coast of India, 14.73% 18 , but lower than carbohydrate content in P. gymnospora from Sabah Malaysia, 84.54% 19 and from Tamilnadu, India, 118.14% 16 , and than that in P. minor found in Pahuwato waters, Gorontalo, 41.88 %. 5 The interspecific difference in carbohydrate content reflects that nutrients of macroalgae could be affected by species and habitat.
The water content of P. australis from Kelapa Lima coastal waters, Kupang Bay, was 11.21%, and it is different from that reported by Fitrya 20 , only 6.4%, and Maharany et al. 7 , 87.25%. This difference could be influenced by light intensity and temperature at the drying process. The drying method affect the proximate content of Sargassum polycystum (brown macroalgae), including water content. Ash content of P. australis was high (34.58%), if compared with P. australis collected in Tedung Island, Seribu islands, only 2.3%. 7 It is also different from other species, such as P. minor, 30.53% 5 , and P. tetrastomatica, 27%. 17 Higher ash content was found in P. gymnospora, 45.04%. 19 Ash content in a material could be related with number mineral components. 21 The mineral content of macroalga could be affected by the processing method as well. 22 Futhermore, the level of each mineral component is determined by species, physiological factor, geographic condition, wave frequency, and the method used in mineralization. 23 P. australis collected in Kupang Bay waters contained 15 amino acids with the highest content in aspartic acid, 1.16 %, and glutamic acid, 1.32%, and the lowest in histidine, 0.12, and methionine, 0.20%. These results are not different from those in P. gymnospora from Tamildanu, India, with the highest in aspartic acid, 12.7%, and glutamic acid, 13.9%, and the lowest in histidine, 2.7% and methionine, 1.5%, despite indifferent amount. However, Shanmuganathan and Devi 16 found that P. gymnospora, in India, had different content of amino acid with the highest in glycine (0.605) and tyrosine (0.504) and the lowest in arginine (0.103). Protein content variations in macroalgae could influence the amino acid content.
The highest mineral content of P. australis was calcium (10.22% w/w), followed by potassium (1.48% w/w) and the lowest was iron (0.125% w/w). A similar result is also reported by Manteu et al. 5 , which the highest mineral content of P. minor was calcium (32.91 mg/g), potassium (26.9 mg/g), and the lowest was iron (1.00 mg/g). Also, Shanmuganathan and Devi 16 found that P. gymnospora had the highest mineral content in calcium, 156.2 mg 100 g DM -1 , then potassium, 122.3 mg 100 g DM -1 , and the lowest in iron, 6.78 mg 100 g DM -1 . Although the highest mineral content is found in the same mineral for the three Padina species from different localities, the concentrations are different. It means that the mineral content of Padina sp. is affected by species and habitat.
Phytochemical analysis indicated that P. australis contained several compounds such as alkaloid, saponin, flavanoid, tannin, steroid, and terpenoid. The present findings are slightly different from the previous study 19 that found only steroid, terpenoid, polyphenol, and saponin. P. tetrastromatica was reported holding alkaloid, terpenoid, steroid, phenol, and flavonoid, but did not contain saponin. 24 P. australis contains flavanoid, tannin, and saponin 2 , but Maharany 7 found flavonoid, phenol hydroquinone, triterpenoid, tannin, and saponin in the same species. Even though the bioactive compounds have different content, all the active compounds of Padina sp. can be used for pharmaceutics properties such as inhibit the growth of the pathogenic microorganisms.
The antibacterial activity of P. australis has been proved to have the ability to inhibit the growth of A. hydropilla in either methanol extract with an inhibition zone of 10 mm or water extract with the inhibition zone of 10.5 mm ( Table 3). The ability of antibacterial activity of P. australis against Vibrio harveyii in fish was also reported by Gazali and Saputra 2 with an inhibition zone of 12.55 mm at the concentration of 60%. Salosso et al. 9 also showed that the antibacterial activity of P. australis against V. alginoliticus as well with inhibition zone of 22 mm in acetone extract.