Effects of Supplemental Light Spectra on the Composition, Production and Antimicrobial Activity of Ocimum basilicum L. Essential Oil

This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil (Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% (v/w, dry). The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4–90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150–200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p < 0.001) in basil biomass and oil production with the highest values found in the oils of basil under two conditions of (1) 71% Red: 20% Blue: 9.0% UVA in at 120 μmol·m−2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 μmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil.


Introduction
Basil (Ocimum basilicum L.) is an herb, probably native to the tropical and subtropical regions of India, well known for its aromatic and medicinal properties. This herb contains a distinctive essential oil and is used in both fresh and dried culinary dishes, providing a variety of positive health benefits when consumed. Some of the reports demonstrated that basil has pharmacological effects such as anti-inflammatory [1][2][3], antioxidant [4,5], and bronchodilatory properties [6]. In addition, basil has strong immunomodulatory, antibacterial, antimutagenic, and chemopreventive effects [7]. Basil was used in the treatment of obstructive lung diseases, e.g., chronic obstructive pulmonary disease (COPD), asthma, and other respiratory disorders such as bronchitis, aspergillosis, tuberculosis, and lung cancer [8]. Due to the unique properties of smell, taste, and biological activities, Basil is highly appreciated in the world market with a steady increase of 1.3% cumulative average growth reported by Kang et al. (2022) [24]. UV-A light (365-399 nm) at four dosages of 0, 35, 65 and 97 µmol·m −2 ·s −1 with the photoperiod of 16 h day/8 h night was added to the background RGB light for 14 days. It was reported that at the mild UV-A radiation (dosages of 35 and 65 µmol·m −2 ·s −1 ) supplemented with the main light, both yield and quality (the contents of acids, sugars, anthocyanins, and antioxidants) of sweet basil were increased.
The impact of mixed light based on R and B regions with UV-A, G and FR radiation for basil in terms of enhancing its composition, production, and antimicrobial activity of essential oil of basil planted in Vietnam has not been addressed yet. The rapid development of light-emitting diode (LED) technologies improves the economic output of plant production and quality. Therefore, the growth, composition, production, and antimicrobial activity of essential oils of basil planted in a net-house can be improved by supplemental light. In the present study, we focus on evaluating the effect of supplemental light sources in terms of spectral distribution and daily light dose (duration and illuminance) on the growth, composition, production, and antimicrobial activity of essential oils of basil planted in the net-house in Hanoi, Vietnam.

Results and Discussion
In this paper, we report on effects of different supplemental light spectra and doses on the essential oil productivity, composition and antimicrobial activity of basil cultivated in the net-house in Vietnam. The supplemental light conditions used for basil growing during four months were different in (i) ratio of light spectra:  Under different supplemental light conditions, the biomass and essential oil yield of basil showed differences ( Table 1). The highest plant height was found in the lighting formula F9 (118.55 cm/plant), followed by the one in the lighting formula F5 (104.50 cm/plant). These two values were significantly different (p = 0.001) with each other and with the ones in the other 8 formulas that ranged from 89.63 to 97.50 cm/plant. The formula F9 also gave the highest plant fresh weight (513.32 g/plant), followed by the formula F8 (390.65 g/plant) and F2 (399.50 g/plant). While the values varied from 290.40 to 334.62 g/plant from other formulas compared with 295.56 g/plant from the control formula F10.
The supplemental light conditions affected the water content of basil fresh biomass. The plant water content of two formulas F8 and F9 was similar to the control formula F10. However, significant reductions (p < 0.001) in the plant water content were observed in all other formulas that varied from 73.30 % (in F2) to 77.04 % (in F1). The essential oil yields of plants in the supplemental light formulas were significantly different (p < 0.001) ranging from 0.88 to 1.30% (v/w), calculated on a dry weight (DW) basis, compared with 1.21% (v/w) in the control formula F10. In line with data of plant height and biomass, the highest essential oil content was found in F9 (1.30%) that indicates the optimal supplemental light condition for basil growth and oil production. The increase in the essential oil content was also found in the formula F4 (1.26%) as well as formula F8 (1.25%).
The estimated production of basil in the supplemental light formulas F2 and F9 showed the significant higher values than the other formulas at 0.05 level (p < 0.001), namely respective productions of fresh biomass (15.58 ton/ha and 20.02 ton/ha), dry biomass (4.16 ton/ha and 3.55 ton/ha), and oil (44.03 L/ha and 46.11 L/ha).
Thus, supplemental light condition of formulas F2 (71% R: 20% B: 9% UVA at 120 µmol·m −2 ·s −1 in 6 h/day) and F9 (43.5% R:43.5% B:8.0% G:5.0% FR at 100 µmol·m −2 ·s −1 in 6 h/day) were the most suitable for the growth of basil that produced the highest biomass and essential oil productivity among all supplementary light conditions of the present study (Table 1). The ranges of basil fresh biomass, dry biomass and oil production in this research are slightly higher than the ones previously reported [25]. It was reported that the fresh and/or dry weight of basil under different lighting conditions can be varying depending on the time of year [26]. Other research indicated a positive relationship between basil biomass and the Daily Light Integrals (DLIs) observed [27,28] when growing basil under DLI conditions from 7.0-17.8 mol·m −2 ·d −1 that is not clear in the present study with the range of total DLIs (sum of average PPFD value and daily supplemental light) from 11.21-15.53 mol·m −2 ·d −1 .

The Effect of Light Spectra on Essential Oil Composition of O. basilicum
In addition to the oil yield, the quality of basil is also evaluated by the chemical composition of its essential oil. Hydrodistillation of the shredded aboveground biomass of basil harvested from ten formulas produced pale yellow oils with the average relative densities of the oils d 20 ranged from 0.951 to 0.961 g/mL, the refractive indices n 20 ranged from 1.509 to 1.512 and the equal optical rotations [α]D 20 = [+] 0.26 • (Table 2). These values of oil densities are roughly equal to the ones previously reported but the values of refractive indices are slightly higher [29]. The identification of compounds present in the basil essential oils harvested from ten formulas was carried out using mass spectral (MS) and retention index (RI) data. Table 3 presents the identified compounds in order of their elution on the HP-5MS column used for the GC-MS analysis.   A total of 20 to 27 compounds representing from 99.1% to 99.9% (by mass intensity) of the compositions were identified in the essential oils of basil cultivated under 10 different supplemental light conditions. These were comprised of dominant benzenoid aromatics ranging from 88.4 to 91.6% of the oils. While monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes of the oils were at very low concentrations.
The difference between composition of the basil oils in 10 different supplemental lighting formulas is negligible with respect to the concentrations of methyl chavicol (=estragole); the main component ranged from 87.4 to 90.6%. The order of methyl chavicol content in basil oils is as follows: (Table 3) [30,31] suggested that the optimal lighting condition for basil is 16-18 h/day at PPFD 200-250 µmol·m −2 ·s −1 , which is comparable to the formulas F2 and F5 in the present study with supplemental lighting conditions of 6 h/day (total lighting duration of 20 h/day) at PPFD 120 µmol·m −2 ·s −1 . In the formulas F7 and F8, the highest methyl chavicol content in basil can be attributed, in addition to lighting duration and illuminance, which is 6 h/day (total lighting duration is 20 h/day) at PPFD 150-200 µmol·m −2 ·s −1 -equivalent to DLI 14.45-15.53 mol·m −2 ·d −1 , the composition of the light spectra has a higher ratio of B (43.5%). Its value is in agreement with the results reported by Hosseni (2018) [13] where the highest methyl chavicol was obtained under light B at DLI 14.4 mol·m −2 ·d −1 (16 h/day and PPFD 250 µmol·m −2 ·s −1 ).

The Effect of Light Spectra on Antimicrobial Activity of Essential Oil of O. basilicum
The essential oils of basil grown in 10 different formulas were evaluated for antimicrobial activity against 7 strains: Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The essential oils of basil in 6 formulas from F2 to F7 showed weak inhibitory effects on B. subtilis with respective IC 50 Table 4 presents basil oil samples with inhibition activity against tested microbial strains. The presence of high concentration of methyl chavicol and low concentration of linalool in the Basil oils in the present study may explain for the weak antimicrobial activity of the oils. A previous study showed that methyl chavicol is devoid of antimicrobial activity [40]. Likewise, basil oil with major constituents identified as methyl eugenol (39.3%) and methyl chavicol (38.3%) was found to be active against bacteria and fungi [36]. On the other hand, some other studies demonstrated that higher linalool-containing oils of basil exhibited higher antimicrobial activity [41,42].
No relationship was found between supplemental lighting conditions and the antimicrobial activity of basil essential oil in this study. However, previous studies showed that the antimicrobial activity of essential oils depends on their main compounds. On the other hand, supplemental lighting for basil with different light spectra can alter the content of its oil main compounds. Therefore, in the future, it is possible to find a certain light spectrum/mixture of light spectra that drastically changes the contents of the main compounds in basil essential oil, thereby increasing the oil's antimicrobial activity.

Plant Materials and Growth Conditions
The experiments were set up in a net-house in Hanoi, VN (N 21 • 04 08 , E 105 • 45 50 ) with 40% diffused light transmission being determined using relating photosynthetic photon flux density (PPFD) (400-700 nm) inside to outside the net-house. The basil seeds, purchased from Duc Thang Company in Hanoi, were sown in March 2021, then the seedlings were transplanted in April and a supplemental light experiment was held from May to September 2021. Daylight illuminance in a net-house was measured by the DigiSense Data Logging Light Meter (Model 20250-00), which was converted into Daily Light Integral (DLI) using conversion factors as described in the literature [43,44]. An average PPFD value of 11.21 mol·m −2 ·d −1 was determined for experimental time (in May 11.34, June: 11.44, July: 12.04, August: 10.92 and September: 10.31 mol·m −2 ·d −1 ). The supplemental LED lighting was applied after sunset and before sunrise with ten different light formulations as described in Table 5 with 15 plants in each formula and 3 replications. LED lights generated a wide continuous spectrum with UV (peak at 365 nm), B (peak at 440 nm), G (peak at 530 nm, R (peak at 660 nm), and FR (peak at 730 nm) measured by USB2000+ Fiber Optic Spectrometer, shown in Figure 1. The techniques for planting, caring, fertilizing, and harvesting basil plants were carried out according to the previous document [45]. at 530 nm, R (peak at 660 nm), and FR (peak at 730 nm) measured by US Spectrometer, shown in Figure 1. The techniques for planting, caring, f vesting basil plants were carried out according to the previous docum

Water Content Determination
Each basil sample was determined the water content using A& 4714A General purpose moisture determination balance.

Water Content Determination
Each basil sample was determined the water content using A&D Weighing AD-4714A General purpose moisture determination balance.

Essential Oil Isolation
Each basil sample consisting of aerial biomass of 15 plants (4.3-7.7 kg) was shredded and hydrodistilled for 4 h using a Clevenger type apparatus according to the previously published procedure [46] (Ministry of Health of Vietnam, 2017). The essential oil was then separated and stored at -5 • C for further analysis.

Determination of Essential Oil Physical Properties
Three physical properties of the essential oil including: relative density, refractive index and optical rotation were determined using the methods from the standards ISO 279:1998, ISO 280:1998 and ISO 592:1998, respectively.

GC-MS and GC-FID Analysis
The essential oils were analyzed by GC/MS-FID using an Agilent GC7890A system with Mass Selective Detector (Agilent 5975C). An HP-5MS fused silica capillary column (60 m × 0.25 mm i.d. × 0.25 µm film thickness) was used. Helium was the carrier gas with a flow rate of 1.0 mL/min. The inlet temperature was 250 • C and the oven temperature program was as follows: 60 • C to 240 • C at 4 • C/min. The split ratio was 1:100, the detector temperature was 270 • C, and the injection volume was 1 µL. The MS analysis was carried out at interface temperature 270 • C, MS mode, E.I. detector voltage 1258 V, and mass range 35-450 Da at 1.0 scan/s. FID analysis was carried out using the same chromatographic conditions. The FID temperature was 270 • C. Essential oil constituents were identified by their relative retention indices, determined by co-injection of a homologous series of n-alkanes (C 5 -C 30 ), as well as by comparison of their mass spectral fragmentation patterns with those stored on the MS library NIST08, Wiley09, HPCH1607 [47,48] (Adams, 2017;Linstrom & Mallard, 2021). Data processing software was MassFinder 4.0 [49]. Component relative concentrations were calculated based on the area peak of FID chromatography without standardization.

Microbial Strains
The MIC and IC 50 values of the oils were determined using 3 strains of Gram (+) bacteria including Staphylococcus aureus (ATCC 13709), Bacillus subtilis (ATCC 6633) and Lactobacillus fermentum (VTCC N4), 3 strains of Gram (−) bacteria including Salmonella enterica (VTCC), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442), 1 strain of yeast Candida albicans (ATCC 10231). The ATCC strains were obtained from American Type Culture Collection; the VTCC strains were obtained from Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi.

Screening of Antimicrobial Activity
MIC and IC 50 of the essential oils were measured by the microdilution broth susceptibility assay [50,51]. Stock solutions of the oil were prepared in dimethylsulfoxide (DMSO). Dilution series were prepared from 16,384 µg/mL to 2 µg/mL (2 14 , 2 13 , 2 12 , 2 11 , 2 10 , 2 9 , 2 7 , 2 5 , 2 3 , 2 1 µg/mL) in sterile distilled water in micro-test tubes, from where they were transferred to 96-well microtiter plates. Bacteria grown in double-strength Mueller-Hinton broth or double-strength tryptic soy broth, and fungi grown in double-strength Sabouraud dextrose broth were standardized to 5 × 10 5 and 1 × 10 3 CFU/mL, respectively. The last row, containing only the serial dilutions of sample without microorganisms, was used as a negative control. Sterile distilled water and medium served as a positive control. After incubation at 37 • C for 24 h, the MIC values were determined at the well with the lowest concentration of agents that completely inhibited the growth of microorganisms. The IC 50

Statistical Analysis
The measurements of physiological parameters and essential oil contents of basil were analyzed by a single factor completely randomized analysis of variance (ANOVA), which compared the lighting treatments. For the significant values, means were separated by the least significant difference (LSD) test at p ≤ 0.05 using IRRISTAT ver. 5.0 (International Rice Research Institute, Los Baños, Philippines).

Conclusions
This is the first study to provide the information on the various aspects of basil cultivated under ten different supplemental light spectra and dosages in Vietnam. The average yields of basil essential oils from 0.88 to 1.30% (v/w), calculated on a DW basis, were obtained. Methyl chavicol (=estragole) was the unique main component of the oils ranging from 87.4 to 90.6%. Additional lighting gave out effects on the growth and essential oil content of basil, as shown by the significant differences (p < 0.001) in biomass, oil yield and oil production between the lighting formulas. Supplemental light conditions of formulas F2 (71% R:20% B:9.0% UVA at 120 µmol·m −2 ·s −1 in 6 h/day) and F9 (43.5% R:43.5% B:8.0% G:5.0% FR at 100 µmol·m −2 ·s −1 in 6 h/day) were the most suitable for the growth of basil that produced the highest biomass (fresh: 15.58 ton/ha and 20.02 ton/ha; dry: 4.16 ton/ha and 3.55 ton/ha) and essential oil productivities (44.03 L/ha and 46.11 L/ha) among all supplementary light conditions of the present study. Additional lighting time and light intensity may have influenced the concentration of the major component of basil essential oil with higher contents of methyl chavicol under conditions of 6 h/day (formulas F2 and F5) and 150-200 µmol·m −2 ·s −1 (formulas F7 and F8) than under the other conditions. The essential oils from basil in formulas from F2 to F7 showed weak inhibitory effects on only Bacillus subtilis. However, no effect of supplemental lighting conditions on the antimicrobial activity of basil essential oil was recorded. The results of present study can be the basis for future research to promote the increase in biomass yield, production and quality of essential oils of plants.