BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Freeze-dried microalgae of Nannochloropsis oculata improvesoybean oil's oxidative stability

Ya-Lin Lee & Yao-Chun Chuang & Huei-Meei Su &

Fang-Sheng Wu

Received: 12 April 2013 /Revised: 5 August 2013 /Accepted: 7 August 2013 /Published online: 10 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Marine microalga Nannochloropsis oculata pos-sesses nutrients valuable for human health. In this study, weadded freeze-dried N. oculata powder to soybean oil andobserved a remarkable inhibition in oil oxidation. The amountof microalgae powder added was positively correlated to theincrease in oil stability. The addition of 5.0 % (w /w )microalgae powder increased the oil stability index (OSI)values of soybean oil more than twofold at the tested temper-atures 120 and 130 °C.N . oculata contains high levels of bothphenolic compounds and α-tocopherols that could be thecontributors to such an increase of the OSI. Two methodswere conducted to assay the active ingredients released frommicroalgae: one employed three solvent systems to extract themicroalgae and the other was the soybean oil added withmicroalgae. Analyses of free radical scavenging and reducingpower suggested that the phenolic compounds dominated theantioxidation activities in soybean oil when it was infusedwith the microalgae powder. Our results suggest that N.oculata could potentially be used as an additive in cookingoil to increase the shelf life and nutritional value of the oil and

to reduce the production of free radicals from lipid oxidationwhen the oil is used at high-temperature cooking processes.

Keywords Nannochloropsis oculata . Microalgae powder .

α-Tocopherol . Oil stability index . Soybean oil . Phenoliccompounds . Lipid oxidation

Introduction

Unicellular microalgae contain nutrients that serve as an im-portant source of live feed in aquaculture (Brown 2002). Theenormous biodiversity of microalgae has yielded various com-pounds that are widely used in nutritional supplements, phar-macological formulations, and many other biotechnologicalapplications for both humans and animals (Pulz and Gross2004; Spolaore et al. 2006). Various healthy and valuableingredients isolated from the dry mass of microalgae haveshown a high market demand that exceeded 5,000 tons andvalued US $1.2 billion annually (Pulz and Gross 2004;Spolaore et al. 2006).

The green marine microalga Nannochloropsis oculata ,previously known as “marine Chlorella” and belonging tothe family Eustigmatophyceae, has been shown to contain allessential vitamins for aquaculture (Brown et al. 1999). Inaddition, N. oculata possesses polyunsaturated fatty acids(PUFA) (Vazhappilly and Chen 1998) with as much as 5 %of its dry biomass consisting of eicosapentaenoic acid (20:5 n-3) (Roncarati et al. 2004; Khozin-Goldberg and Boussiba2011), as well as various phytopigments and carotenoids(Lee et al. 2006; Del Campo et al. 2007). Due to its high n-3PUFA levels and its ability to grow easily and rapidly, N.oculata is commonly utilized as feed to rear rotifers that arethen fed to marine finfish larvae for growth transformation(Chaturvedi et al. 2004).

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-5183-4) contains supplementary material,which is available to authorized users.

Y.<L. Lee (*) :Y.<C. ChuangBiotechnology Division, Taiwan Agricultural Research Institute,41362 Wufeng District, Taichung, Taiwan, Republic of Chinae-mail: ylleet@tari.gov.tw

H.<M. SuTungkang Biotechnology Research Center, Fisheries ResearchInstitute, Donggang, 92845 Pingtung, Taiwan, Republic of China

F.<S. Wu (*)Department of Biology, Virginia Commonwealth University,Richmond, VA 842012, USAe-mail: fwu@vcu.edu

Appl Microbiol Biotechnol (2013) 97:9675–9683DOI 10.1007/s00253-013-5183-4

Methanol extracts from N. oculata , as well as 13 othermicroalgal species from fresh, brackish, or marine waters inSouth Asia, were found to possess the ability to inhibit linoleicacid oxidation (Natrah et al. 2007). The application of antiox-idants isolated from natural sources is an emerging methodol-ogy for controlling lipid oxidation to extend the shelf life of oil(Samotyja and Malecka 2007; Sultana et al. 2007; DeLeonardis et al. 2007; Bouaziz et al. 2008). Various solventshave been tried to extract natural antioxidants. However, thewide arrays of natural antioxidants possess wide ranges ofphysicochemical characteristics and no single solvent canextract them all. In addition, it is costly to remove solventresidues which can be harmful to human health.

In this study, we directly added freeze-dried N . oculatamicroalgae into soybean oil and analyzed the effects on oilstability. In order to further clarify the alterations and possiblecauses by the addition, the oils as well as three differentsolvents extracts from microalgae were assayed. Bioactivecomponents, phenols and vitamin E contents, andantioxidation activities were analyzed.

Materials and methods

Materials

Freeze-dried N . oculata microalgae powder was provided byTungkang Biotechnology Research Center (67 Fengyu St.,Donggang Township, Pingtung County 928, Taiwan). TheN . oculata strain (category no. CCAP 849/1) used in thisstudy is available from CultureCollection of Algae and Proto-zoa online (http://www.ccap.ac.uk/index.htm).

The microalgae were cultured outdoors in 130-L plasticbags with semicontinuous and batch modes that wereharvested every 3 and 4 or 7 and 8 days, respectively, untilthe cell concentration in each mode reached 3–6×107 cells/mL. The harvested microalgae were concentrated 20–30 timesinto a slurry in a continuous-flow Lavin Centrifuge 12–413 V(AML Industries, Inc., Hatboro, PA, USA). The residual waterwas then removed with another centrifuge (Kubota 5800,Kubota, Tokyo, Japan) before freeze-drying, and the driedmicroalgae powder was preserved at −20 °C until use.

Oil stability index and peroxide value of oils with N. oculatamicroalgae added

Oil stability index (OSI) is one of the most common methodsused to quantify oil oxidative stability. It employs vigorousaeration to agitate oil at a high temperature in order to accel-erate the rate of oxidation, which allows the study of oilstability within a short-time period without waiting for thelong “aging” time of real environments. The OSI value is thetime (hour, h) when a sample reaches the point of maximum

change in conductivity caused by the aerated sample's volatilecompounds that were produced during oxidation at 110 or130 °C (Lampert 1999). This method is useful in comparingthe stability of parallel oil samples and to predict their stableperiod at varied temperatures with a formula t =A ×e(B×T).This formula is derived based on a first-order chemical reac-tion, where t is time of OSI, T is heating temperature, and Aand B are deduced from experimental data.

The freeze-dried microalgae powder was added into 3.00 gof commercial soybean oil (Uni-President Co., Taiwan) atweights 0, 0.030, 0.075, and 0.150 g for final concentrationsof 0, 1, 2.5, and 5 % (w /w), respectively. A Rancimat appa-ratus (873 Biodiesel Rancimat, Metrohm AG, Switzerland)was used to assay oil samples for OSI and to predict oilstability with one set of temperatures 120, 130, 140, and150 °C. Briefly, four to twelve samples were used for eachset of the temperatures to achieve a high regression coefficientr> 0.9 (randomly replicated) under continuous aeration at 10-L/h of air flow (Fig. S1 and Table S1). The effluent of volatilegas was led into a test cup containing 60 mL of distilled water,and the electrical conductivity during the entire heating pro-cess was monitored.

For the oil peroxide value (POV) assay, the microalgaepowder was added into the soybean oil at 0, 1.0, 2.5, and5.0 % (w /w) in triplicate, placed on a shaker for constantshaking at 100 rpm for 15 h at room temperature (about23 °C), and then centrifuged at 10,000 rpm for 20 min toremove undissolved particles. The clear oils (supernatants)were preserved at 4 °C until use. Analyses of the POV wereperformed following the AOCS Official Method 965.33(Horwitz 2002) except that the weights of oil samples werereduced to one-half (2.50 g). The same oil samples were alsoused for the analyses of the total phenols and reducing powerdescribed below.

Extraction with organic solvents from microalgae

Three organic solvent systems, including acetone, methanol,and chloroform/methanol in a ratio of 2:1 [v /v, abbreviated toCM (2:1)] were used to extract the antioxidant componentsfrom the microalgae. The sequence of log octanol–waterpartition coefficients for these three solvents is indicative oftheir hydrophobic tendencies: CM (2:1) > acetone >methanol.For each extraction, 0.10 g of microalgae powder was loadedinto a silica-gel membrane spin column (Viogene Co., Tai-wan), steeped for 5 min in 0.2-mL of the solvent, and thencentrifuged for 2 min at 2,000×g in a tabletop microcentrifugeto collect the filtrate. These extraction steps were repeateduntil the filtrate became colorless, and all filtrates were thencombined and evaporated to dryness in a centrifugal concen-trator (Taiyo VC-36, Tokyo, Japan). The resulting total drymass is defined as the dry extract of that solvent, and its weightdivided by the weight of microalgae powder originally used is

9676 Appl Microbiol Biotechnol (2013) 97:9675–9683

the extraction yield, by percentage basis. These dry extractswere then separately dissolved in methanol for analyses ofvitamin E and total phenolic contents, free radical scavengingactivity, and reducing power. When resuspended in methanol,the extract from CM (2:1) formed some white crystals, whichwere removed by filtering through a 0.22-μmmembrane priorto analyses.

Vitamin E content

The vitamin E contents were identified and quantified byhigh-performance liquid chromatography (HPLC) as de-scribed (Chen and Bergman 2005) with a C18 reverse-phaseanalytical column (RP250-4.6, 250×4.6 mm, 5 μm, Kanto,Tokyo, Japan), a gradient pump (PU-2089 plus, Jasco, Tokyo,Japan), and a fluorescence detector (FP-2020 plus, Jasco).Prior to HPLC, all extracts were filtered through a 0.22-μmmembrane filter (PVDF; Critical Process Filtration Inc. NH,USA), and 20 μL of each sample was subjected to columnseparation analysis at 30 °C in a column oven (Hipoint,Kaohsiung, Taiwan). The mobile phase was programmed asfollows: (1) from 0 to 6 min, maintained in solvent A (45 %acetonitrile, 45 % methanol, 5 % isopropanol, and 5 % aceticacid); (2) from 6 min to 16 min, performed as a linear gradientfrom 100 % solvent A to 100 % solvent B (25 % acetonitrile,70 % methanol, and 5 % isopropanol); and (3) maintained in100 % solvent B for 12 min. The flow rate was set at 0.8 mL/min. The fluorescence detector was set to an excitation wave-length of 298 nm and an emission wavelength of 328 nm.Four vitamin E homologues (α-, β-, γ-, and δ-tocopherol,Eisai Co., Ltd. Tokyo, Japan) of standard solutions wereprepared in methanol and used for peak identification andquantification.

Total phenols

Following the method of Singleton and Rossi (1965), 10 μLof each extract was added into 160 μL of distilled water,followed by 10 μL of Folin–Ciocalteau phenol reagent (Sig-ma Co., Saint Louis, MO, USA) and 20 μL of 35 % Na2CO3

(Sigma Co). After incubating at room temperature for 30 min,the absorbance was measured at 750 nm. The total phenoliccontent was determined by interpolating the data to the stan-dard curve of gallic acid (GA) (Sigma Co.) dissolved indistilled water at 0, 0.25, 0.50, 0.75, and 1.00 mg/mL. Toanalyze the oils added with microalgae, the samples were firstmixed with isopropanol at 1:1 (v /v ), and then centrifuged at13,000 rpm for 2 min. The undissolvable oil that floated to themeniscus was removed and the remaining solution wassubjected to the determination of phenolic compounds asdescribed above. All the analyses were triplicated.

Scavenging activity of free radical 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+)

The ABTSmethod (Awika et al. 2003) can produce stable freeradical ABTS·+ and determine antioxidants' capacity based onthe reduction rate of absorption at 750 nm. In this study, thefree-radical stimulating agent potassium persulfate was re-placed by ammonium persulfate (APS, Sigma Co). The radi-cal cations were generated by adding 8 mMABTS and 3 mMAPS, mixed well, and then incubated in the dark for 16 h atroom temperature. Before detecting the antioxidant activity,the radical cation reagent was diluted to reach an absorbanceof 750 nm between 1.0 and ~1.5. Three microliters of eachextract was added into 250 μL of the diluted cationsolution, and the absorbance at 750 nm was monitored everyhour for 3 h. Vitamin E analog trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Aldrich ChemicalCo., Steinheim, Germany) solutions at1, 2, 3, and 4 mM wereprepared in methanol to build a standard curve for the freeradical ABTS·+ scavenging activity.

Reducing power

The reducing power was determined as described by Oyaizu(1986). Twenty microliters of each extract was added into20 μL of 0.2-M sodium phosphate buffer (pH 6.6), followedby adding 20 μl of 1 % potassium ferricyanide solution andincubating at 50 °C for 20 min. Samples of the soybean oiladded with microalgae were directly reacted with 1 % potas-sium ferricyanide prepared in 0.2-M sodium phosphate buffer(rather than in water). The reaction mixtures were then rapidlycooled down to room temperature in cold water, and thereactions were stopped by adding 20 μL of 10 % trichloro-acetic acid (TCA) solution into the mixture. The mixtureswere then centrifuged to clarify the solution. Fifty microlitersof each supernatant was diluted with 50 μL of distilled water,added with 10 μL of 0.1 % ferric chloride solution, andincubated at room temperature for 10 min; then its absorbanceat 690 nm was measured with a plate reader (MultiskanAscent Thermo Labsystems, Waltham, MA, USA). Troloxsolutions at 1, 2, 3, and 4 mM were used to build a standardcurve. An additional centrifugation step was performed afterthe addition of 10% TCA to the soybean oil sample to removethe undissolvable oil. All the analyses were triplicated.

Statistical analysis

Duncan's multiple range tests were employed to determine thesignificance of differences among the treated samples byusing the Statistical Analysis System software (SAS InstituteInc., Cary, NC, USA). For each treatment, three determina-tions were performed, and the means were calculated. Meanswere considered significantly different at p <0.05.

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Results

Stability of soybean oil with added N . oculata microalgae

The OSIs of the oil without adding microalgae powder were2.21, 1.16, 0.95, and 0.56 h at 120, 130, 140, and 150 °C,respectively (Fig. 1a). With an increasing amount ofmicroalgae powder added at 1, 2.5, and 5 % (w /w), the OSIvalues increased accordingly. At the addition of 5.0 %microalgae powder, the OSI reached the highest values of5.28, 2.65, 1.49, and 0.92 h at 120, 130, 140, and 150 °C,respectively. Using linear regression analysis, the line equa-tions and linear correlation coefficients at the four tempera-tures were 120 °C, y =62.13 x +2.030, r =0.9987; 130 °C, y =27.28 x +1.347, r =0.9621; 140 °C, y =11.29 x +0.868, r =0.9448; and 150 °C, y =7.797 x +0.522, r =0.9775. The var-iable x represented the percentage of microalgae powderadded in relative to the oil weight. The y -intercepts signifiedthe deduced OSI values of the oil without the microalgae,having values of 2.03, 1.35, 0.87, and 0.52 h at 120, 130, 140,and 150 °C, respectively. All r values are greater than 0.9,indicating that the deduced OSIs from the true values werehighly consistent and reliable.

In addition to OSI, POV was also analyzed. Following theincreasing microalgae addition at 0, 1.0, 2.5, and 5.0 %, POVsincreased slightly and gradually with values of 59.4±0.2, 60.9±0.6, 62.0±1.6, and 63.0±0.8 mEq peroxide/kg oil, respectively.The POV increment (y) increased by the powder addition (x)following a logarithmic equation (Fig. 1b).

Oil stable periods at different temperatures predicted by OSI

The OSI formula, t =A ×e(B×T), was used to predict the stableperiods of oil at different temperatures (Table S1). Withoutmicroalgae addition, the predicted stable periods of soybeanoil are 15 days, 6 days, 3.3 h, and 21 min at temperatures of 4,25, 110, and 160 °C, respectively (Table 1). Our results alsoshowed that as the amounts of microalgae added into thesoybean oil increased, the stable periods also increased ac-cordingly. At the addition of 5 % microalgae powder, thestable periods were 195 days, 57 days, 9.2 h, and 29 min at4, 25, 110, and 160 °C, respectively.

ABTS·+ free radical scavenging activities of extractablematerials from N . oculata by organic solvents

Three organic solvents were employed to extract the antioxi-dant compounds from themicroalgae. Results indicated that theCM (2:1) solvent produced the highest yield of total extractablematerials from freeze-dried powder of N . oculata , followed bymethanol and acetone at 48.9, 43.3, and 2.3 % (w /w), respec-tively (Fig. 2a).

The standard curves for the ABTS scavenging activitywere built with trolox equivalent antioxidant capacity(TEAC). We found that the 3-h reaction time exhibited thebest correlation coefficient (r2=0.9950) and was thereforechosen as the reaction time for all samples. The TEAC valuesof the extracts from acetone, methanol, and CM (2:1) were3.3, 21.5, and 21.2 μmol/g microalgae, respectively (Fig. 2b),indicating that the scavenging activities of methanol and CM(2:1) extracts were similar and six times more than that of theacetone extract.

Total phenols

Using gallic acid (GA) as a quantitative standard, the phenoliccompounds extracted by acetone, methanol, and CM (2:1)were 0.62, 4.63, and 5.50 mg GA equivalents per grammicroalgae, respectively (Fig. 2c). This result indicates thatthe methanol extract did not significantly differ from the CM(2:1) in phenols yields, but both were remarkably higher thanthat of the acetone (Fig. 2c). Analyses of the soybean oilinfused with microalgae powder showed that the totalphenols in oils increased as the amounts of microalgaewere added logarithmically (Fig. 3a, b). The logarithmicregression analysis (Fig. 3b) suggests that the amount ofphenolic compounds released from the microalgae into oilmay have a maximal threshold level at about 5 % whenfurther additions of the powder may not offer additionalbenefits.

Reducing power

The reducing power of the microalgal extracts as determinedby the Oyaizu method (Oyaizu 1986) revealed that the TEACvalues of acetone, methanol, and CM (2:1) extracts were 4.5,34.1, and 35.0 μmol TE per gram microalgae, respectively(Fig. 4a). These values indicated that the extracts of eithermethanol or CM (2:1) possessed seven times more reducingpower than the extract of acetone. Additionally, the increasesin the reducing power of the oil were in proportion to theamounts of microalgae added (Fig. 4b) in a logarithmic scalefashion (Fig. 4c) at a maximal threshold level of about 5 %,which is similar to that of the phenols content increments(Fig. 3b).

Tocopherol contents

In the reverse-phase HPLC system, β- and γ-tocopherolsshared the same retention time, and their emission intensitieswere similar at 328 nm (data not shown). Since γ form oftocopherol was more abundant in plants than the β form(Shintani and DellaPenna 1998), the γ form was selected forquantification to represent both of these two forms asβ/γ. Theyields of α-tocopherol per gram microalgae powder in

9678 Appl Microbiol Biotechnol (2013) 97:9675–9683

extracts of CM (2:1), methanol, and acetone solvents were836.6, 576.1, and 230.8μg, respectively (Table 2 and Fig. S2),indicating that the CM (2:1) was the most efficient extractionsolvent. Among the extracts of three solvents, the tocopherolprofiles were similar: 95.1~96.1 % for α form and 1.5~2.8 %for both the β/γ and δ forms. On the other hand, the soybeanoils added with microalgae did not show a detectable amount

of tocopherols that were released from the microalgae (datanot shown).

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Fig. 1 Soybean oil with N . oculata microalgae powder at 0, 1.0, 2.5, or5.0 % (w /w) was analyzed with a oil stability index (OSI) and b peroxidevalue (POV). OSI was tested at 120, 130, 140, and 150 °C and simplelinear regression was applied for each temperature treatment. POV incre-mental curve was simulated with logarithmic function

Table 1 Predicted oil stable periods deduced from OSI formula atdifferent temperatures

Microalgae addition(g/100 g oil)

Stable periods at varied temperatures

4 °C 25 °C 110 °C 160 °C(day/month) (day/month) (h) (min)

0 15/0.5 6/0.2 3.3 21

1.00 55/1.8 18/0.6 4.9 21

2.50 169/5.6 48/1.6 7.3 22

5.00 195/6.5 57/1.9 9.2 29

OSI formula, t =A×e(B×T) ; t is time of OSI, T is heating temperature, Aand B are deduced from experimental data

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Fig. 2 Yields of N. oculata microalgae (a) extracted by acetone, meth-anol, and CM (2:1), and their extracts were analyzed with ABTS·+ freeradical scavenging activity (b) and total phenolic contents (c). The totalphenols used gallic acid for standard curve construction (y =1.0989 x +0.0381, r2=0.9971). Trolox equivalent (TE) was used to construct theABTS scavenging standard curve (y=−0.1953 x+0.9692, r2=0.9950).All experiments were conducted in triplicates from three independentextractions. Different letters (a , b , and c) on top of the columns indicatethat the values are statistically different from each other (Duncan's mul-tiple range tests, p <0.05, n =3). The range of the vertical bar on the topof each column is the standard deviation of mean

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Discussion

The predicted stable periods of oil based on OSI presumablyshould be shorter than the actual stable periods, because thereis no aeration involved in the ordinary storage and cookingprocess in the actual periods. At 5 % addition of microalgae,the stable period was prolonged 13-fold at 4 °C from 15 to195 days, and 9.5-fold at 25 °C from 6 to 57 days. The 5 %microalgae addition also reduced the oxidation rates by a factorof 2.8 (from 3.3 to 9.2 h) at 110 °C, which is the approximatetemperature reached during some high-temperature cookingprocesses such as stir-frying and steaming. The relationshipbetween the increases in stable periods and the microalgaeaddition as shown in Fig. 5a clearly indicates that there is apositive correlation between the stable periods and the amountof microalgae added into the oil. The changes in the OSI fromlow (4 or 25 °C) to high (160 °C) temperatures imply that theeffectiveness of the antioxidant components in microalgae canbe altered at different temperatures. Because the stable periodspredicted at 110 °C followed a linear regression pattern withthe amount of microalgae added into the oil and because at160 °C only the 5 % addition showed significant stabilizing

effect, we believe that there may be room to further improvethe oil stability at high temperature by adding the microalgaebeyond 5 %.

The phenolic contents of microalgae extracts were corre-lated to the antioxidation activities in both ABTS scavengingactivity and reducing power (Fig. 5b). The slope of the reduc-ing power was comparatively higher than that of ABTS,indicating that the reducing power methodwas more sensitive.

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Fig. 3 Total phenols of the oils addedwith microalgae powder (a) and itsincremental relationship with the addition (b). All experiments wereconducted in triplicates with three independent oil samples

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Fig. 4 Reducing power analysis of microalgae extracts in (a) variousextraction solvents and (b) in various amounts of microalgae added insoybean oil. (c) Effect of microalgae addition on the reducing powerincrement in oil. All experiments were conducted in triplicates with threeindependent samples (trolox equivalent was used to construct standardcurve, y =0.0706 x+0.6637, r2=0.9969; statistical analysis was the sameas in Fig. 2)

9680 Appl Microbiol Biotechnol (2013) 97:9675–9683

This result supports the proposal by Buratti et al. (2007) thatthe reducing power could be considered as a direct test of totalantioxidant power. Our results showed that both phenoliccontents and reducing power increased logarithmically bythe microalgae addition into oil (Figs. 3b and 4c). The incre-ments of the increases in stable periods at lower temperaturesalso followed a logarithm function with the microalgae addi-tion (Fig. 5a). Based on these observations, we suggest thatthe phenols in microalgae contributed to the oil stabilitythrough antioxidant activity, especially at low temperatures.Our results are similar to a study with olive oil which showedthat phenolic contents increased the oil stability (MartínezNieto et al. 2010) and resistance to autoxidation (Tsimidouet al. 1992). In plants, phenolic compounds such as simplebenzene derivatives, including hydroxycinnamates, tocoph-erols, and flavonoids, were found to be the primary contribu-tors to their antioxidative capability (Singleton et al. 1999), andthese compounds are soluble in methanol. Themost frequentlyused artificial antioxidants are butylated hydroxyanisole andbutylated hydroxytoluene because they can prevent fat rancid-ity caused by oxidation and are hydrophilic monophenols.Among all phenolic compounds, the bromophenols exhibitmany beneficial biological activities and are commonly foundin marine macroalgae, but there is a lack of study on theirpresence in microalgae (Liu et al. 2011). Folin–Ciocalteauphenol reagent employed in this study has been used to detectthe total phenolic contents in a wide spectrum of organismsincluding those of marine algae (Li et al. 2007; Liu et al. 2011;

Bhuvaneswari et al. 2013). However, this method cannot dis-tinguish between different categories of phenolic compoundssuch as bromophenols. The biodiversity in microalgae is muchgreater than in land plants, but studies on the variety of phe-nolic contents that exist in microalgae are limited and differentfrom many land plants, and these phenolic contents may notnecessarily be the major source of natural antioxidants (Li et al.2007). Many phenolic compounds are known to have a bittertaste, although the human perception of food taste is largelygenetic which can vary greatly (Lesschaeve and Noble 2005;Garcia-Bailo et al. 2009). There is a lack of report on the tasteand flavor of the freeze-dried N . oculata , but in our experi-ence, the taste/flavor of the freeze-dried N . oculata is withoutsignificant astringency and bitterness, similar to the driedseaweed wakame commonly used in Asian cuisines. Thus,vegetable oils that contain a small percentage (5 %; w /w) ofmicroalgae mass, rather than purified forms of phenolic com-pounds, presumably would not adversely affect the quality ofthe oil flavor.

The tocopherols (vitamin E) are a class of phenolic com-pounds and are well-acknowledged antioxidants in plants. All

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y = 3153.8 ln(x) + 1102.4r² = 0.9614

y = 864.54 ln(x) + 325.21r² = 0.9741

y = 2.1512 x - 0.4669r² = 0.9978

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4.0

5.0

6.0

7.0

0

500

1000

1500

2000

2500

3000

3500

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4500

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1.0% 2.5% 5.0%

Stab

le p

erio

d in

crem

ent

at 1

10

C(h

)

Stab

le p

erio

d in

crem

ent

at 4

and

25

C (

h)

Microalgae addition (w/w)

4°C

25°C

110°C

y = 3.929x + 1.2372r² = 0.9671

y = 6.5852x + 0.9248r² = 0.9801

0.0

5.0

10.0

15.0

20.0

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0.00 1.00 2.00 3.00 4.00 5.00 6.00

Ant

ioxi

dant

act

ivity

(T

E u

mol

/g m

icro

alga

e)

Phenols content (GA equivalents/mg microaglae)

ABTS

Reducing power

Fig. 5 The incremental effect of microalgae addition on predicted stableperiods (a). The correlations between total phenols content and theantioxidant activity assayed by ABTSmethod and by reducing power (b)

Table 2 Yields of tocopherols in extracts prepared from N . oculatamicroalgae by different solvents

Solvent α-Tocopherol β/γ-Tocopherol

δ-Tocopherol Sum

(μg/g microalgae powder)a

Acetone 230.8±33.2 c 5.6±1.2 c 3.7±0.8 c 240.2

(27.6 %,96.1 %)

(27.7 %,2.3 %)

(26.1 %,1.5 %)

(27.6 %,100 %)

Methanol 576.1±29.3 b 17.2±1.0 b 12.5±0.4 b 605.8

(68.9 %,95.1 %)

(85.1 %,2.8 %)

(88.0 %,2.1 %)

(69.6 %,100 %)

CM (2:1) 836.6±55.8 a 20.2±0.6 a 14.2±0.6 a 871.0

(100 %,96.1 %)

(100 %,2.3 %)

(100 %,1.6 %)

(100 %,100 %)

Yields are expressed as mean ± standard deviation. The experiment wasconducted with three independent extractions of each organic solvent.Letters a, b, and c represent means of different tocopherol forms that aresignificantly different (p <0.05, n =3). The first and second percentagevalues inside parentheses are the ratio to the corresponding ingredientextracted with CM (2:1) and the ratio to the sum, respectivelya The data were produced with interpolation method. The equations of thelinear standard curves of vitamin E homologues are α-tocopherol, y=85.533x−15.566 (r2 =0.9999); γ-tocopherol, y=257.26 x−11.542 (r2 =0.9998);δ-tocopherol, y=273.01 x−9.4881 (r2 =1.0000)

Appl Microbiol Biotechnol (2013) 97:9675–9683 9681

tocopherols are lipophilic and located in the biological mem-branes, and they possess the reducing power to quenchfree radicals through the donation of phenolic hydrogensand electrons (Gregory 1996). It is therefore essential for us toinvestigate the amounts of tocopherols both in microalgaepowder and in the oils added with the powder. Althoughtocopherols were not detected in the microalgae-infused oils,their effects on oil stability may not be excluded because theOSI method was carried out at above 100 °C, the temperaturesmight energize the microalgae to release tocopherols.

The α form among the four tocopherols bears the highestbioactivity for humans, especially when compared to d ,l-α-tocopheryl acetate, a synthetic racemic mixture with no anti-oxidant activity that is widely used in food fortification(Gregory 1996). N . oculata can be a good source of naturalα-tocopherol, themain form found in themicroalgae (Table 2).In comparison with the fruits and vegetables revealed byHarris et al. (1950), the microalgae powder possessed a rela-tively higher content of vitamin E. By modifying nitrogensources under common cultural conditions of N. oculata ,Durmaz (2007) found an increase of α-tocopherol productionby a factor of 2 to 3 (2,326 μg/g microalgae). Tocopherolsfrom soybeans are available as a commercial source of vita-min E supplements, but the variation in α-tocopherol contentsof different soybean genotypes was remarkable, ranging be-tween 8.7 and 33.2 μg/g in 20 selected genotypes (Seguinet al. 2009), which are all well below those in microalgae.Therefore, N. oculata may also be an economically feasiblesupply source of α-tocopherol for human nutritionalsupplements.

If the extraction of tocopherols from the microalgae withCM (2:1) was considered as 100 %, the methanol would be70 % and the acetone 28 % (Table 2). It is intriguing thatalthough tocopherols are highly hydrophobic, they can beextracted by the highly hydrophilic methanol more efficientlythan the hydrophobic solvent acetone. The reason may be thatmethanol can effectively denature membrane proteins (Mitakuet al. 1988) to release the tocopherols. Clearly, different sol-vents used during the extraction can significantly affect theextractability of different nutrients.

Dai et al. (2008) found a synergistic effect of α-tocopherol,vitamin C, and polyphenols in green tea; the combination ofthese chemicals inhibited the oxidation of linoleic acid muchmore efficiently than when each chemical was used alone.Their analyses of reaction kinetics demonstrated that the poly-phenols promoted the regeneration of α-tocopherol in tea,which further facilitated the inhibition of lipid peroxidation(Dai et al. 2008). It is yet to be determined whether there areother ingredients in the whole cells ofN. oculata other than thepredominate phenolic contents observed here which may alsoplay a synergistic effect to prolong the oil oxidative stability.

Although a positive correlation between the increments ofoil's POVand the added amounts of microalgae was observed

(Fig. 1b), the prolonged oil stable periods indicated that themicroalgae overall exhibited a positive influence on the oxi-dative stability. POV is defined as the amount of peroxideoxygen in 1 kg of oil, and it is determined by the capacity of anoil sample to oxidize iodide ions to iodine molecules. In otherwords, the microalgae contained some ingredients as electroncapturers that caused the increase of POVs.

To our knowledge, this is the first study to demonstrate thatthe direct addition of freeze-dried microalgae into vegetableoil improved the oxidative stability of the oil. Our resultssuggest that the phenolic compounds released from themicroalgae are the primary contributors for the antioxidativeactivities that increased the oil stability. This is not onlybecause microalgae contains rich antioxidant compounds butalso because they possess a high surface area to volume ratio,allowing these compounds to be easily released into the oil.We believe that unicellular organisms such as N. oculata areparticularly suitable for use as additives to food such as theedible oils.

Acknowledgments This study was supported by the Council of Agri-culture (97AS-3.1.3-AI-A2), Taiwan, Republic of China. We are verygrateful to Dr. Pei-Lan Tsou of Grand Valley State University for hervaluable suggestions to the manuscript.

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