We had conducted study to discover whether or not there was any antioxidant compound in extracts of algal (Scenedesmus dimorphus and Spirulina fusiformis) and yeast (Phaffia rhodozyma) or fungus (Monascus purpureus). The purpose of this study was to determine activity of respective culture extracts as sources of natural antioxidant through their inhibition activities on bleaching of β-carotene against free radicals derived from oxidation of linoleic acid. Analysis of chemical constituents of the respective cultures was performed using GC-MS and the results showed that extract of S. dimorphus and S. fusiformis contained terpenoids (phytol), and benzofuranon that might be potential as antioxidant compound. The assay for antioxidant activity indicated that extracts of algal, yeasts and fungal cultures demonstrated considerably high activity to retain the bleaching of β-carotene, however, they were lower than that of exhibited by butylated hydroxy toluene (BHT) as positive control for a synthetic antioxidant
Keywords: Culture extract; Antioxidant activity; carotene; linoleic acid.
Nowadays, free radicals and antioxidants are important concerns in the medical world, since free radical is suspected can cause cell damage and underlie a wide variety of pathological conditions, such as coronary heart disease, diabetes mellitus, heart disease, and is also suspected to play a role in the aging process, whereas the benefits and usefulness of antioxidant is ward off the free radicals [31]. Free radicals are considered dangerous since it will be very reactive in attempt to get a © pair of electrons resulting in formation of new free radicals. As a result of these chain reactions, it may cause damage to various parts of cells and organs of body, and plays a major role in development of a wide range of degenerative diseases [11].
During this time, butylated hydroxy toluene (BHT) and butylated hydroxy anisole (BHA) are commonly used as synthetic antioxidants. However, their use are increasingly avoided by community, since some studies have shown a toxic and carcinogenic effects against human body. Therefore lately, more attention is drawn to natural antioxidants, such as bioactive compounds derived from plants and microbes, since they are better than synthetic antioxidants when viewed in terms of food safety, security, and health [21]. Potential microbial secondary metabolites that may be used as a source of natural antioxidants are necessarily to be studied furthermore. Search for new antioxidant compounds that are more effective and safer is consequentally encouraged. Microbial resources that may be empowered as a source of natural antioxidants can be obtained from yeasts, fungi and algae including P . rhodozyma, M . purpureus, S . dimorphus and S . fusiformis [27]. The purpose of study was to determine the bioactive compounds derived from algal culture of S. dimorphus and S. fusiformis, yeast culture of P. rhodozyma and fungal culture of M. purpureus as source of microbial antioxidants, which was assayed regarding with preventing on bleaching of β-carotene caused by oxidizing of free radicals derived from linoleic acid.
Proliferation of algal culture of S. fusiformis applied for this study had been carried out on Zarrouk media using 2500 o lux light intensity at 25 C, whereas culture of S. dimorphus was grown on PHM media with 2500 lux of light o intensity at 25 C [9].
The media consisted of 1g KNO and 0.2g 3 MgSO4.7H2O was dissolved into distilled water (1000 ml), and then added with 1mL trace metal solution and 1 mL soil extract and a 150 ml of each solution was poured onto three conical flask (300 ml) o and autoclaved at 121 C for 20 min. After cooling down, medium was added with 0,15 mL Fe and 0,15 mL K2HPO4 solution, and culture of S. dimorphus was then inoculated and incubated onto these three growth media and stirred continuously at 25°C and 2500 lux light intensity.
The media containing 3g KNO , 1g NaCl, 3 1g K2SO4, and 16.8g NaHCO3 was dissolved into distilled water (1000 ml) and poured onto two conical flask (500 ml), and added with 0.25mL H3PO4, and o sterilized by autoclaving at 121 C for 20 minutes. After cooling down, medium was then added with 0.005 g FeSO4.7H2O, 0.5 mL trace elements of Za and and Zb. The culture of S. fusiformis was then inoculated and incubated onto these two growth media and stirred continuously at 25°C and 2500 lux light intensity.
The media containing 0.9 g yeast extract, 0.9g malt extract, 3g glucose, 1.5g peptone was dissolved into acetate buffer (pH 5.5) in a 500 mL conical flask, and then o autoclaved at 121 C for 15 min. After cooling down, medium was inoculated with culture of P. rhodozyma and then o incubated at 22 C for 5 days by continuously shaking on a rotary shaker at 120 rpm.
Media containing 100 ml potato extract and 10 g glucose was dissolved into acetate buffer (pH 5.5) in a 500 mL conical o flask, and then autoclaved at 121 C for 15 min. After cooling down, medium was inoculated with culture of M. purpureus and then incubated at 30°C for 5 days by continuously shaking on the rotary shaker at 120 rpm.
403
The culture of S. dimorphus and S. fusiformis were extracted by using centrifuge and their supernatants were then analyzed with GC-MS to determine composition of organic compounds that contained in the respective extract of cultures. One mg of sample was dissolved into 1.0ml methanol, and filtered with a 0.22 μm filter membrane. A 1μl of samples was injected onto GC-MS using column of HP-5ms ( 0,25mm and film 0,25μl) eluted with 5% diphenyl and 95% methyl o polysiloxane as stationary phase at 300 C of injection and interface temperature. The oven was set at initial temperature of 100°C for 2 min at a rate of temperature o rise of 15 C/min and then stabilized at o 300 C for 20 min with helium as a mobile phase was set at a constant rate of 1 ml/min. Mass spectrometry was set at ion o source temperature of 250 C and o quadrupole temperatures at 50 C, energy 70ev and Mass Setting Range from 30 to 1050 amu.
Biomass cells was measured using gravimetric method by measuring 1.0mL of respective culture in eppendorf tubes where its dry weight of cells had been determined. The cells were centrifuged for 15 min at 4000 rpm. Supernatant was discarded and obtaining pellets were o dried using oven at 80 C for 36 h and weighed. The dry weight of cells was calculated by deducting the obtaining total weight with the weight of eppendorf tubes [30].
Media containing skim milk (2g) was dissolved into 150 ml Na-phosphate buffer pH 7 and heated for 60 min. Simultaneously, 0.5g peptone and 2.2g agar were dissolved into 150 ml Naphosphate buffer pH 7 and sterilized by autoclaving and then mixed and poured onto sterilized petridish and allow to solidify. The cultures were grown on the solidified media and incubated for observation regarding with a clear zone formation [8].
Media containing yeast extract (0.2g), 0.5g peptone, 0.3g KH2PO4, 0.05g MgSO4.7H2O, 0.01g CaCl2, and 2.0g soluble starch in conical flask (300 ml), diluted with 50 ml Na-phosphate buffer pH 7. Simultaneously, agar (2.2g) in 100 mL conical flask was dissolved into 50 ml Naphosphate buffer pH 7 and homogenized using microwave and then mix them all and sterilized by autoclaving. The cultures were grown on the solidified media and incubated for observation regarding with a clear zone formation [7].
Media containing 0.5g beef extract, 1g peptone, 0.5g NaCl, 0.3g yeast extract, 0.1g polyvinyl alcohol and 2.2g agar was dissolved into 100 mL Na-phosphate buffer pH 7 and then poured into a large test tube (15 ml) and sterilized by autoclaving. After sterilizing 0.3mL tributyrin was added into petridish then poured with sterilized media. The cultures were grown on the solidified media and incubated for observation regarding with clear zone formation [20].
Antioxidant activity of respective culture extracts were tested using β-carotene as their substrates and linoleic acid as oxidator by dissolving 2.0mg β-carotene into 10 mL chloroform. Of the solution was pipetted as amount of 1 mL into 100 mL conical flask and then evaporated upto dry at 40°C. Furthermore, it was successively added into the flask with 20 404 mL linoleic acid, 184mL Tween 80 as emulsifiers and 50 ml distilled water which had been aerated, and then stirred to form emulsion. As amount of 5.0 ml of emulsion was pipetted into each tubes which had contained antioxidants at various concentrations. Absorbances were observed at time-0 and at each 30 min o interval time after incubation at 50 C and λ 470 nm. Antioxidant activity was expressed as a protective factor (Fp) could be calculated based on ratio of absorbance of sample and absorbance of control at 30 min [1].
Figure 1 showed a visual observation dealing with enzymatic activities of yeast and fungal cultures by performance of clear zone surrounding their colonies. The result showed that culture of P. rhodozyma exhibited lipolytic activity on medium containing tributyrin, but it did not show amylolytic and proteolytic activities in media containing starch and skim milk. While the culture of M. purpureus showed lipolytic, amylolytic and proteolytic activities as it could perform some clear zones surrounding colonies on media containing tributyrin,
biochemical constituents of algal culture extracts using GC-MS. The results showed a presumption on existence of compounds that containing vitamins such as terpenoid (phytol), benzofuranon and fatty acids. High content of phycocyanin pigment may become main attraction in cultivation of algae, since it exhibited antioxidant characteristics [24, 25], and therefore it is considered to have a potential market in the field of healthcare related industry. The antioxidant activity of these compounds are mainly attributed to scavenging activity against superoxide and hydroxyl radicals, chelating ability, quenching singlet and triplet oxygen [3, 26]. It is important to develop, identify and utilize new source of safe and effective antioxidants of natural origin [17, 22].
There are several synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Propylgallate (PG) and butylated hydroxyquinone (TBHQ) are available commercially and currently used. However, the use of these synthetic antioxidants for food or medicine components has been stricted by the toxicity and safety that can lead to the problems of the potential health in human. Due to the reasons, many researchers have tried to find the more effective oxidation inhibitors that may be used as antioxidants for food or medicine compositions without the side effects for the past several years. They have paid attention to many kinds of natural antioxidants that can be used without toxicity in human.
Algae are known to contain reactive antioxidant molecules, such as ascorbate and glutathione (GSH) when fresh, as well as secondary metabolites, including carotenoids (α- and β-carotene, fucoxanthin, astaxanthin), mycosporinelike amino acids (mycosporine-glycine) and catechins (e.g., catechin, epigallocatechin), gallate, phlorotannins (e.g., phloroglucinol), tocopherols (α, χ, δ- tocopherols) [29].
Among algal natural antioxidants, terpenoids, phlorotannins, polyphenols, phenolic acids, anthocyanins, hydroxycinnamic acid derivatives, and flavonoids are important [4]. Recently, researchers have isolated various types of antioxidant compounds from different 405 algal species, including fucoxanthin in Hijikia fusiformis [28]; phycocyanin and phycocyanobilin in Spirulina platensis [5, 6, 13], phenolic acids, tocopherols, and - carotene in Spirulina maxima [18], fatty acids, polyphenols, and phlorotannin in Sargassum siliquastrum, Rhodomela confervoides, Symphjocladia latiuscula and Kappaphycus alvarezzi [10, 14], lutein in Botryococcus braunii [23], and carotenoids in Dunaliella salina [12, 15].
Assay for antioxidant activity of algal culture extracts had been carried out using reaction mixture containing β- carotene and linoleic acid. The bleaching time towards changing color of β-carotene indicated level of antioxidant activity exhibited by these culture extracts. The longer the time required for bleaching, the better the quality of the antioxidants possessed. [19]. Linoleic acid was utilized as a substrate that played a role on oxidizing free radicals which resulted in bleaching the color of β-carotene. Existence of antioxidant which has activity as free radical scavengers are potentially protecting β-carotene from the effect of free radicals thus its bleaching time will be longer. The antioxidant activity expressed as a protective factor (Fp) which is ratio of absorbance value of samples towards absorbance value of control in 30 min of incubation. Figure 4 showed the Fp value of respective samples of algal culture extracts at concentration of 100 ppm compared to BHT as the synthetic antioxidant. The result showed that the antioxidant activity of the algal culture extract of S . fusiformis showed higher antioxidant activity than the culture extract of S . dimorphus. The test results showed that the extract of S . fusiformis chemically protective factors was higher (3.85) compared to extracts of S . dimorphus (1.78) after 30 min incubation, however lower than that of
respective yeast and fungal culture extracts at concentration of 100 ppm compared to BHT as the synthetic antioxidant. The result showed that the antioxidant activity of the algal culture extract of P . rhodozyma showed higher antioxidant activity than the culture extract of M . purpureus. The results showed that the extract of P . rhodozyma chemically protective factors was higher (2.66) compared to extracts of M . purpureus (1.43) after 30 min incubation, however lower than that of BHT synthetic antioxidant (3.48).
Butylated hydroxytoluene (BHT) is a material that is often used as synthetic antioxidant that has the highest protective factor compared to the natural antioxidants contained in microbial culture extracts including algal culture of Scenedesmus, Spirulina those were cultivated on media of Zarrouk and PHM. It showed that the BHT is the most powerful antioxidative compound that has activity in capturing free radicals that cause damage in various products containing oils and fats. Although some synthetic antioxidant, including BHT is commonly used in processed foods, which have some adverse effects [2].
There is also concern that BHT may convert to other substances that may be harmful for human body since it is carcinogenic. It was reported that a conversion product of BHT had been shown to disrupt chemcial signals that are sent from cell to cell [16], and therefore the use of BHT as antioxidant dealing with food preservation has become obsolete. Hence the use of algal culture extracts of S. dimorphus and S. fusiformis, as well as yeast and fungal culture extracts of P . rhodozyma and M . purpureus are potentially to be applied as a source of 406
407
From the results of this study indicated that culture extracts of S. fusiform is, S.
dimorphus, P. rhodozyma and M. purpureus showed Fp value of antioxidant activity on inhibiting the bleaching of β-carotene against free radicals derived from oxidation of linoleic acid those were at 3.85, 1.78, 2.66 and 1.43, respectively, although they all showed lower than that of antioxidant activity showed by BHT (3.48 to 4.22) as the synthetic antioxidant. These algal, yeast and fungal culture may be considered to have an important role since they experimentally exhibited antioxidant activity, and therefore they are potentially useful for healthcare industrial market.
408
We thank Mrs Pritta Oktaviani and Mrs Iis Aprilia for their assistance in carrying out research during their study at Institute of Industrial Technology and Pharmaceuticals, Bogor, Indonesia.