Evaluation of the volatile compound basil leaf ( Ocimum basilicum ) intervention on Spirulina platensis

Microalgae Spirulina platensis contains high protein which can be used as functional food, but it has several undesirable volatile compounds . Ocimum basilicum contains various essential oils from the monoterpene class that can be used to reduce volatile compounds causing an off - odour in Spirulina platensis . This study was conducted to evaluate the intervention of O. basilicum on nature and microencapsulated S. platensis powder after O. basilicum treatment against the volatile compounds. The research consisted of two intervention models. S. platensis powder soaked with basil extract (1:4, w/v) and then dried by freeze - drying method without microencapsulation (DSB). The S. platensis powder was soaked in basil extract (1:4, w/v) then dried by freeze - drying method and microencapsulated (MSB) after. Spirulina platensis powder was used as a control (SP). A solid - phase microextraction was used with the combination of GC - MS to identify volatile compounds. The results showed that the main volatile compounds in Spirulina are medium - chain alkanes. Other compounds that contributed to this off - odour are geosmin (0.02%), 2 - Methylisoborneol (0.28%), β - ionone (4.94%). The intervention of O. basilicum in S. platensis resulted in a decrease in these volatile compounds. Geosmin was not detected by intervention with O. basilicum either by microencapsulation (MSB) or without microencapsulation (DSB). Therefore, the intervention of O. basilicum can reduce off - odour in S. platensis and can be applied as a functional food.


Introduction
Spirulina platensis is a biomass of cyanobacteria as a single cell protein producer which is rich in protein, essential vitamin and minerals, making it very appropriate to be used as a food source (Jung et al., 2019;Kameshwari et al., 2020).The amino acids composition Spirulina protein is among the best, exceeding the amino acids in soybeans, useful as a protein supplement in the treatment of malnutrition in children (Saranjaj and Sivasakthi, 2014).Spirulina platensis contains bioactive compounds such as phenols, phycocyanin and polysaccharide which have bioactive as antioxidants, anti-inflammation and immunostimulants.The use of Spirulina as a functional food provides various human health benefits (Finamor et al., 2017).However, Spirulina added to food can reduce sensory characteristics in the presence of an off-odour (Agustini et al., 2019).
Ocimum basilicum is an aromatic plant that produces essential oils with a distinctive mint aroma.Essential oil is used in providing flavour and aroma to food to increase consumer acceptance of a product (Shaaban et al., 2012;Trivedi et al., 2014).Aromatic plants contain essential oils that play a specific role in aromas, such as chavicol, linalool and eugenol.The compounds are used in the food industry to reduce off-odours and act as an antioxidant (Kiferle et al., 2011;Barros et al., 2013;eISSN: 2550eISSN: -2166eISSN: © 2021 The Authors.Published by Rynnye Lyan Resources FULL PAPER Koroch et al., 2017).The instability of essential oil in O. basilicum characterised by its susceptibility to oxygen, temperature and light whereby it requires microencapsulation to maintain its aroma and bioactivity (Majeed et al., 2015).
Microencapsulation can be used to protect active compounds during the processing and storage stages.This technique has been widely used to mask taste and aroma to enhance the organoleptic properties of a product.Microencapsulation is also used in a significant reduction in aromatic odours considering it as a more user-friendly product when dealing with an unpleasant odour (Gondal et al., 2017).Limited information on the intervention of a substance against volatile compounds to increase Spirulina consumption is available.Several studies on Spirulina to date were used as nutraceuticals and pharmaceuticals (Sowjanya and Manjula, 2016).However, the aroma and taste of S. platensis have not been studied further.The objective of this study was to evaluate the intervention of O. basilicum extract on Spirulina powder and microencapsulation treatment against volatile compounds.

Materials
Spirulina powder was obtained from Brackishwater Aquaculture Fisheries (BBPBAP) Jepara (Central Java, Indonesia) in 2019.O. basilicum was purchased from a traditional market in Semarang, Central Java.This study covers treatments of Spirulina platensis powder that was not soaked in O. basilicum as a control sample (SP), S.platensis powder soaked with basil extract (1:4, w/v) then dried by freeze-drying method without microencapsulation (DSB) and S. platensis powder soaked with basil extract (1:4, w/v) then dried by freezedrying method and then microencapsulated (MSB).Spirulina powder was soaked with O. basilicum leaf extract (1:4, w/v) and microencapsulation was conducted using maltodextrin (CV.Multi Kimia Raya, Semarang, Indonesia) combined with gelatin (Xian, Biof Bio-Technology, Cina) with the concentration of 9:1 (w/w).The drying process used was the freeze-drying method (Heto Powerdry LL 1500, Germany) with a temperature of -100 o C for 48 hours.Sample preparation for the intervention of O. basilicum in Spirulina was conducted as previously described by Handiani et al. (2019).

Solid-phase micro extraction analysis
Solid-phase micro extraction (SPME) analysis was carried out with 30 mins exposure to a 2 cm DVB/ CAR/ PDMS fibre followed by analyte desorption at 220 o C for 3 mins.Briefly, 3.5 g of S. platensis powder in 22 mL SPME bottles are heated to 80 o C. 10 ppm of methanol (Sigma-Aldrich, Saint Louis, MO, USA) was added as an internal standard.Sample preparation stages such as extraction, purification, and concentration are combined into one step and one device that was directly connected to the Gas Chromatography Mass Spectrophotometer (Hulburt et al., 2009).

GC-MS analysis
The samples were run on a GC Agilent 7890A and MS 5975C with a Triple Axis detector.A 30 m x 250 µm i.d.capillary column HP-5MS (J and W Scientific, folsom, USA) with 0.25 µm stationary phase was used.Helium was used as a carrier gas at a flow rate of 1.8 mL/min.The oven was initially held for 1 min at 50 o C, then gradually increased by 3 o C/min to 120 o C for 1 min.Then, at a temperature of 5 o C/min to 220 o C which was retained for 0 min.The ions of volatile compounds in S. platensis were observed over a period of 100 µs.The quantity of each volatile compound from Spirulina and intervention O. basilicum in powder and Spirulina microencapsulation were evaluated.

Identification and quantification of volatile compounds
Identification of all volatile compounds obtained SPME analysis on the comparison of experimentally obtained compounds mass spectra with mass spectra available in NIST14 database.The quantification analysis was performed using Advanced Chemistry Development through the integration of the peak area of the chromatograms.

Results and discussion
The chromatographic profile was investigated to identify peaks of major and minor volatile compounds in each sample as shown in Figures 1 -3.The result obtained from the determination of volatile compounds from the intervention of O. basilicum in Spirulina powder and microencapsulation was shown as % chromatographic peaks of the total ion chromatogram (Table 1).The result showed that medium-chain alkanes were the main component of the volatile compounds in Spirulina.It has been proven that cyanobacteria have the capacity to produce natural hydrocarbon from fatty acids (Coates et al., 2014).The enzyme responsible for alkane production in cyanobacteria are acyl-acyl carrier protein (Acyl-ACP) reductase (AAR) and aldehydedeformulating oxygenase (ADO).These two enzymes work in series and in a complex manner which efficiently converts long-chain fatty acyl ACP or fatty acyl CoA into hydrocarbons (Gao et al., 2020).Pentadecane, hexadecane and heptadecane are the main hydrocarbon compounds in cyanobacteria (Milovanović  The observation of SPME in the extraction of volatile compounds plays an important role in the formation of the distinctive aroma of Spirulina intervention of O. bacilicum, it can further be used for flavour analysis.The most important cause of the unpleasant taste and odour in Spirulina are geosmin and 2-methylisoborneol which are detected by humans in small concentration levels.The odour threshold for geosmin is 15 ng/L and 2-methylisoborneol is 35 ng/L (Juttner and Wasten, 2007).However, many people detect geosmin and 2-methylisoborneol at lower concentration levels.Spirulina in this study naturally produces low concentration of geosmin, 2methylisoborneol, 2-pentylfuran, β-cyclocitral and βionone (Table 1).2-pentylfuran is a volatile compound found in cyanobacteria (Milovanović et al., 2015).This compound is formed through the auto-oxidation of linoleic acid, the high amount of 2-Pentylfuran is in line   et al., 1967;Xu et al., 2017).The conjugated diene radicals produced from the radical cleavage of 9-hydroxy linoleate can react with oxygen to produce vinyl hydroperoxide, which will then undergo cyclization by alkoxy radicals form 2-pentylfuran (Frankel, 1983).
Ocimum basilicum intervention increased βcyclocitral content in Spirulina, because O. basilicum contains citral compounds (da Costa et al., 2014).βcyclocitral is volatile compound that displays lytic activity on cyanobacteria.In particular, β-cyclocitral causes an attractive colour change from green to blue during the lysis process.β-cyclocitral provides distinctive properties with maximum absorption, thereby rapidly breaking down chlorophyll a and β-carotene (Harada et al., 2009).The threshold for β-cyclocitral volatile compounds is 750 nM.β-cyclocitral was not detected in live Mycrocystic cells, it was present only at very low concentration of 2.6 amol/cell.Conversely, the rapid cell rupture activates the carotene oxygenase reaction, resulting in a high β-cyclocitral number, around 77±5.5 amol/cell calculated according to the maximum intracellular concentration of 2.2 mM (Juttner et al., 2010).Similar to β-cyclocitral, β-ionone is one of the volatile compounds produced through the degradation of β-caroten cyanobacteria (Garcia-Plazaola et al., 2017).
Ocimum basilicum leaf extract can reduce geosmin compounds in powder and microencapsulation of Spirulina.This is because the essential oil content in O. basilicum can dissolve geosmin compounds.Essential oil from grape seeds efficiently decreases the concentration of geosmin (Lisanti et al., 2014).Whereas, 2-Methylisoborneol cannot be reduced by the essential oil in O. bacilicum.This is due to the isoborneol content found in its essential oils (Joshi, 2013), therefore the 2methylisoborneol content increases in S. platensis with O. basilicum intervention.Microencapsulation is carried out to maintain the pleasant aroma created by the intervention of O. basilicum in S. platensis, therefore increasing its consumption and application in functional food as well as for nutraceutical and pharmaceutical products.da Silva et al. (2014) explained that microencapsulation is a technology used in the food industry, one of which is to maintain the aroma of the product.Microencapsulation maintains the stability of aroma or taste during the processing and storage stage to minimize degradation or loss of aroma.In addition, it can improve the quality of the finished product and increase customer satisfaction (Gupta et al., 2016).The aroma encapsulation of essential oils also allows for controlled release (Maes et al., 2019).(2019) describe the extraction of petrichor and essential oils for the production of new fragrance because they produce a pleasant odour from the linalool compound (Elsharif et al., 2015).Stanojevic et al. (2018), explained that O. basilicum contains 65 active compounds and the highest content are 31.6%linalool and 23.8% methyl chavicol.Toncer et al. (2017) explained further that when plants experience flower growth the content of monoterpenes such as linalool increases and decreases along with the completion of the flowering period.The volatile compounds have their respective aroma descriptions.The combination of volatile compounds will cause a distinctive flavour.Essential oils are volatile liquid aroma components that are usually obtained from plants (Hamid et al., 2011).

Conclusion
To conclude, the essential oil in O. basilicum reduces volatile compounds such as geosmin, βcyclocitral, β-ionone which causes an off-odour in the powder and Spirulina microcapsule.Linalool is a monoterpene compound useful for its purpose in flavour.Therefore, the intervention of O. basilicum in S. platensis can be applied as a functional food as well as a nutraceutical and pharmaceutical product.

Figure 1 .
Figure 1.Chromatogram of the volatile compounds of Spirulina platensis (SP)