Nanoencapsulated Levisticum officinale Koch. essential oil as a shelf life enhancer for functional foods based on their antifungal and antialfatoxigenic potential and antioxidant activity
The present study reports the nanoencapsulation of Levisticum officinale Koch. essential oil (LOEO) in chitosan polymer and its efficacy as an antifungal and anti-aflatoxigenic preservative to improve shelf life of stored functional foods. Nanoencapsulated LOEO was assessed against Aspergillus flavus in terms of MIC (Minimum Inhibitory Concentration) and MAIC (Minimum Aflatoxin Inhibitory Concentration), which were found to be 0.80µL/mL. The probable antifungal mode of action of LOEO based nanoemulsion was studied in terms of inhibition of the ergosterol content in the plasma membrane. Further, to elucidate the antiaflatoxigenic mode of action, reduction in the cellular methylglyoxal level has been assessed. Antioxidant activity of nanoencapsulated LOEO in terms of IC50 value was found to be 26.10µL/mL through DPPH assay. The in situ practical applicability was tested in stored functional food system and fungitoxicity spectrum was also performed to strengthen the potential use as a safe plant based shelf life enhancer for functional foods.
Keywords: Levisticum officinale, Aspergillus flavus, Essential oil, Antioxidant, Preservative
AFB1- Aflatoxin B1
FAO- Food and Agriculture Organization
WHO- World Health Organization
IARC- International Agricultural Research Centre
PDA- Potato Dextrose Agar
LOEO- Levisticum officinale Essential oil
MIC- Minimum Inhibitory Concentration
MAIC- Minimum Aflatoxin Inhibitory Concentration
BOD- Biochemical Oxygen Demand
ºC- Degree Celsius
Chia seeds, belonging to the family Lamiaceae and originated in Gultemala and Mexico, have been used in traditional food system since 5500 years because of its health benefits to human kind. Thus, the cultivation of this plant has also extended from tropical to subtropical regions (Ullah, et al., 2016; Cahill, 2003; Mohd Ali et al., 2012). The chemical composition of chia seed composes 35% dietetic fiber, 32% fat, 25% protein, 6% moisture and 2% carbohydrates and is rich in PUFAs (Poly Unsaturated Fatty Acids) (Rosas-Mendoza et al., 2017). The plant bears high ethnomedicinal importance as well. Containing substantial omega 3 fatty acids, it helps to prevent cardiovascular diseases and also controls diabetes, hypertension and kidney disorders (Muñoz et al., 2013). Besides, chia seeds and its oil harbors a rich array of natural antioxidants such as tocopherol, phytosterol and carotene, phenolic compounds as chlorogenic and caffeic acid which protects from many diseases, promoting human health. (Munoz et al., 2013). But, a broad spectrum of molds and their associated mycotoxins have been reported to contaminate agri-commodities during storage causing huge quantitative and qualitative loses worldwide. The FAO has estimated that 25 per cent of the world’s food crops are affected by the mycotoxins, produced by storage fungal biota such as Aspergillus sps, Penicillium sps and Fusarium sps (WHO,1999).
Statement of the Problems
In spite of all available improved technologies, food commodities are still facing major risk of bio-deterioration from different microorganisms especially fungal and mycotoxin contamination during storage. Aflatoxin, ochratoxin, zearalenone, patulin, alternic acids are the common mycotoxins contaminating stored food (Fung & Clark, 2004). Among all the diverse group of mycotoxins, aflatoxins produced by Aspergillus sp. mostly A.flavus, A.nomius and A.parasiticus has gained more attention. Aflatoxin B1, which is a polyketide derived secondary metabolite causes both acute and chronic toxicity to humans and animals upon exposure, due to the hepatocarcinogenic (liver cancer), teratogenic (foetal defect), mutagenic and immunosuppressive (weaken immune system) properties (Fung& Clark, 2004). According to the International Agency for Research on Cancer (IARC, 1993) and WHO, aflatoxins are categorized under human class I carcinogen. Hence, there is an urgent need to eliminate the fungal infestation and mycotoxin contamination from stored functional foods.
Objectives of the Research
1. Screening of Levisiticum officinale essential oil (LOEO) against A. flavus LHP-SH-1 strain, nanoencapsulation in chitosan matrix and characterization of prepared nanoparticles.
2. Determination of MIC and MAIC of LOEO loaded chitosan nanoemulsion against most toxigenic A. flavus strain and fungitoxicity spectrum against 14 mold species.
3. Elucidation of the probable antifungal and antiaflatoxigenic mode of action of prepared LOEO nanoemulsion in terms of ergosterol inhibition and reduction in the cellular methylglyoxal level of treated A. flavus cells respectively.
4. Assessment of the antioxidant potential of LOEO in terms of DPPH free radical scavenging activity and a ‘short term’ in vivo and phytotoxicity test of nanoencapsulated LOEO as fumigant in stored functional food system to confirm the practical applicability
The literature is silent over the antifungal and antiaflatoxigenic activity of LOEO against A. flavus. Moreover, the nanoencapsulation to improve the preservative potential and in vivo trials in functional food system have been done for the first time in the present study. Hence, the study was designed to evaluate the bioactivity as well as the mode of action of the nanoencapsulated LOEO against infestations of A. flavus and toxin production in stored functional food. The encapsulation of LOEO for enhancement in the fungicidal potential, in vivo trials and phytotoxicity assessment has also been performed to boost the possible recommendation against post-harvest losses`
Nearly 70% of total production of food grains is affected by the fungal attack and their mycotoxins in tropical and subtropical countries. The contaminated stored food commodities are unfit for human consumption as fungal contamination retards their nutrition values by producing poisonous mycotoxins. To overcome these economic losses of stored food commodities, different synthetic pesticides have been recommended. But, due to carcinogenicity of some synthetic antioxidant such as BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole) have been banned for use (Dwivedy et al., 2016). Further, over usage of synthetic pesticides has not been accepted by the consumers, because of their residual toxicity, bioaccumulation, and lack of eco-friendliness (Kordali et al., 2008). Therefore, there is an urgent need to move towards the application of some green or botanical pesticides to meet out the demerits of synthetic preservatives. Recently, Essential oils (also called volatile or ethereal oils) are aromatic oily liquids obtained from plant material (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). A large number of plant EOs have been reported to have antimicrobial, antimycotoxin, antioxidant activity thereby preserve food item from microbes and their associated toxin as well as by toxic free radicals (Kordali et al., 2008). Levisticum officinale (Lovage), an aromatic plant, belongs to family Apiaceae is known for its anti-cancerous activity. It has also used from centuries to centuries due to its carminative, spasmolytic and diuretic in nature (Sertel et al., 2011).
Although EOs possesses potential activities against pest but also it has some demerits like instability and undergoes oxidation under various abiotic conditions (Singh et al., 2017). So, Nanoencapsulation of bioactive compounds represents the workable and effective approach due to their sub cellular size, physical stability, utilization of lower dosage, controlled and target release of the compound.
· Mohammad et al., (2015) investigates the nanoencapsulation of Zataria multiflora essential oil preparation and characterization with enhanced antifungal activity for controlling Botrytis cinerea, the causal agent of gray mould disease.
· Dwivedy et al., (2018) investigated the nanoencapsulated Illicium verum Hook.f. essential oil as an effective novel plant-based preservative against aflatoxin B1production and free radical generation.
· Herculano et al., (2015) investigated the physicochemical and antimicrobial properties of nanoencapsulated Eucalyptus staigeriana essential oil.
3.1 Chemicals and Equipments
The chemicals viz. methanol, Dichloromethane, Chitosan, Acetic acid, Tween-20, potato dextrose agar (PDA; potato, 200 g; dextrose, 20 g; agar, 18 g and distilled water 1,000 mL), SMKY(sucrose 200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g and yeast extract, 7 g; 1000 mL distilled water), were procured from Hi-Media Laboratories Pvt. Ltd, Mumbai, India. The equipments viz. Centrifuge (Bio Line, India), UV-transilluminator (Zenith Engineers, Agra, India) and spectrophotometer Systronics India Ltd, Mumbai, India) and Sonicator were used. Levisticum officinale (Lovage) essential oil was procured from MRK natural oils, Delhi, India.
3.2 Preparation of LOEO loaded chitosan nanoemulsion.
LOEO loaded chitosan nanoparticles were prepared following Yoksan et al. (2010) with slight modifications. The chitosan solution was prepared within aqueous acetic acid solution at temperature range of 27±2ºC and kept on magnetic stirrer overnight for proper mixing. The next day, tween 80 which acts as a surfactant was added to the chitosan solution and stirred at 45ºC for 2 hrs to obtain homogenized mixture. Further, different volume of LOEO (0.0, 0.06, 0.12, 0.18, 0.24 and 0.30 mL) was dissolved separately in 4mL CH2Cl2 and gradually dropped into the aqueous chitosan solution. Thereafter, a homogenization process was done at a speed of 13,000 rpm for 10 min under ice condition to get an oil water emulsion. Proper agitation of obtained oil water emulsion along with equal volume of TPP was done for 45 min. The homogenized nano emulsions were further centrifuged at 10,000 rpm for 10 min at 4ºC. The formed nano-pellet was washed with deionized water thrice and re-suspended in sterile double distilled water. Finally the ultrasonication was performed by sonicator on an ice for 8 min (4 min pulse on and 4 min pulse off) resulting to get the homogenous suspension.
3.3 Encapsulation efficiency and loading capacity of LOEO loaded chitosan particles
The loaded LOEO in the chitosan nanoparticle was determined by using UV- visible spectrophotometry. 300μL nanoemulsion was added in 3mL of methanol and mixed for 5 min properly. The amount of oil in methanol was compared with its absorbance to standard curve (R2=0.9964s) constructed already at 222.5 nm (λmax) for the LOEO. A blank sample was prepared for the chitosan nanoparticle without loaded LOEO. The encapsulation efficiency (EE) and the loading capacity (LC) of LOEO were calculated following equations (1) and (2) respectively,
LC (%) =Total amount of loaded LOEO/Weight of nanoparticles after sonication x 100
EE(%) = Total amount of loaded LOEO/Initial amount of LOEO x 100
3.4 Efficacy of LOEO loaded chitosan nanoemulsion on AF LHP –SH-1 strain and AFB1 secretion:
The calculated quantity of LOEO loaded chitosan nanoemulsion were mixed in 25 mL of SMKY media (Sucrose, 200g; MgSO4.7H2O, 0.5g; KNO3, 0.3g; yeast extract, 7g; in 1L distilled water; pH 5.6±0.2) to get the appropriate concentration which was been equivalent to 0.1-0.8µL/mL. The control sets were prepared by only adding SMKY media. Thereafter, all the sets were added with 25µl of spore suspension of toxigenic strain AF LHP-SH-1 and incubate in BOD at 27±2ºC for a period of 10 days. After the incubation period got to over the concentration of LOEO loaded chitosan nanoemulsion which inhibit the complete growth of the fungal mass was called as the minimum inhibitory concentration (MIC) against AF LHP –SH-1 strain.
To check the minimum inhibitory aflatoxin concentration (MAIC) of LOEO loaded chitosan nanoemulsion against AFB1 secretion, the control and all treated sets with media were initially filtered through the Whatman No 1 filter paper after 10 days of incubation period at the temperature of 27±2ºC, and the separation was made by using 20 mL of chloroform in a separating funnel. Thereafter, the filtrates were placed on the water bath at 85±2ºC for evaporation process. Finally, the obtained residues were again re-dissolved in 1mL of methanol. After that, 50µl of the methanolic extract was spotted on TLC plate and the development of toxin was made in TIM (toluene:isoamyl-alcohol:methanol (90:32:2v/v/v)) solvent system. The plates were kept for air- drying and spots were observed under UV-transilluminator (Zenith Engineers, Agra, India) at 360 nm. All the blue colored spots were scrapped out, dissolved in 5mL methanol and centrifugation was made at 5000 rpm for 5 minutes. The supernatants were collected and absorbance was taken by using UV- visible spectrophotometer (U-2900 Hitachi, Japan) at 360 nm.The quantity of AFB1 was calculated (Reddy et al., 1970) using the formula
AFB1 = DXMX1000X1000/EXL
Where, D= absorbance; M= Molecular weight of AFB1 (312); E= Molar extinction coefficient of AFB1 (21800); L=Path length (1 cm)
3.5 Fungitoxic spectrum of LOEO
Fungitoxic asseessment of LOEO was examined against 14 storage molds viz. Aspergillus flavus, Aspergillus luchuensis, Aspergillus versicolor, Aspergillus niger, Aspergillus sydowii, Aspergillus repens, Aspergillus fumigatus, Aspergillus chevalieri, Aspergillus terreus, Alternaria grisea, Alternaria humicola, , Fusarium oxysporum, Penicillium italicum, Penicillium spinulosum and Mycelia Sterilia isolated during mycoflora analysis was done by poisoned food assay. 0.5 mL 5% Tween-20 were homogenized with LOEO to obtain 1.4 μL/mL concentrations and gently mixed with 9.5mL PDA containing plate. The PDA containing plate without LOEO serve as control set. A 5 mm disc was cut from a seven day old culture of each isolates and placed at the centre of respective plates and then incubated in BOD at 27 ± 2 °C for 7 days. The percent inhibition of each mold growth was calculated by the following formula:
%mycelial inhibition =dc-dt/dc x 100
Where, dc denotes average diameter of fungal colony in control sets and dt denotes average diameter of fungal colony in treatment sets.
3.6 Mode of action of LOEO loaded chitosan nanoemulsion against AF LHP –SH-1 strain.
3.6.1 Checking the efficacy of LOEO loaded chitosan nanoemulsion on ergosterol content in plasma membrane of AF LHP –SH-1 cell
Requisite amount of LOEO loaded nanoemulsions (0.01-0.08 µL/mL) with 25µl of spore suspension was inoculated in SMKY medium (24.5mL) and placed in BOD for 4 days at 27±2ºC. The control sets were prepared by adding only spore suspension. Then the grown mycelia were filtered out, washed with distilled water twice and the net weight of biomass of control and treated was measured. Thereafter, in each pellet, 5mL of 25% alcoholic potassium hydroxide (KOH) was added and the vortexing was done for 2 minutes followed by incubation in a water bath at 85ºC for 4 hours. Further, in each sample, 5mL of n-heptane and 2 mL of distilled water was added followed by 2 minutes of vortex mixing to extract sterol and leave for 1 hour for incubation at room temperature. The ergosterol content were analyzed between 230nm and 300nm by UV-Visible scanning spectrophotometry (U-2900, Hitachi, Japan).The presence of ergosterol was observed by characteristic four peak and the late sterol intermediate 24(28) dehydroergosterol in the n-heptane layer. The percentage of ergosterol was calculated based on absorbance and mycelial weight (Tian et al.,2012).
% Ergosterol =A (% Ergosterol + % 24 (28) dehydroergosterol ) - B(% 24 (28)dehydrosterol)
A (% ergosterol + % 24 (28) dehydrergosterol) = (Abs282/290)/pellet weight
B (% 24 (28) dehydroergosterol) = (Abs230/518)/pellet weight
Where, 290 and 518 are the E values (in percentage per cm) determined for crystalline ergosterol and 24 (28) dehydroergosterol respectively, and pellet weight is the net weight (g).
3.6.2 Efficacy of LOEO loaded chitosan nanoemulsion on cellular Methylglyoxal (MG) of AF LHP –SH-1 cell
The estimation of MG was done by the following method given by Upadhayay et al., 2018. SMKY medium was used to grow the fungal cell of strain (AF LHP –SH-1) and after 7 days the fungal mat was harvested. Then 0.3 g of spore mass weighed and it was again put into the 10 mL of SMKY medium treated in various concentration (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.16µL/mL ) of LOEO loaded chitosan nanoemulsion. Control sets were devoid of LOEO loaded chitosan nanoemulsion which is mixed with the spore mass in SMKY medium. It was kept for overnight incubation at 27±2ºC. The next day, cells were grinded in 3mL of pre-chilled perchloric acid (0.5 M) and the macerated samples were centrifuged at 13000 rpm for 10 minutes once the 15 minutes of incubation got over in ice condition. After centrifugation, pH was adjusted at neutral. Then re-centrifuged at 13000 rpm for 10 minutes of each sample was done. The quantitative measurement was further done by addition of 250 µL of 7.2 mM 1, 2-diaminobenzene with 100 µL of 5 M perchloric acid into 650 µL of neutralized supernatant. The standard curve for MG was prepared by using the various concentrations of pure MG (35% aqueous solution, Hi-media ) (10, 20, 30, 40, 50, 60, 70, 80,90 and 100µM).
3.7 Antioxidant activity of LOEO loaded chitosan nanoemulsion through DPPH free radical assay
The antioxidant activity of LOEO loaded chitosan nanoemulsion was measured by visualizing the colour change of DPPH from purple colour to yellow colour following Tohidi et al., 2017. Various concentrations of LOEO loaded chitosan nanoemulsion (10, 20, 30, 40, 50, 60, 70, 80, 90, 100µL/mL) were separately mixed with 5 mL of 0.004% DPPH solution in methanol. Then the mixture was shaked and incubated for 30 minutes at room temperature (25±2ºC). By using the blank the absorbance was recorded at 517nm by spectrophotometer. The IC50 value (the concentration which caused 50% neutralization of DPPH radicals) was calculated by plotting a graph between percent inhibition and concentration and calculated by using formula
Where, Ablank is the absorbance of the control (without test material) and Asample is the absorbance of the test material.
3.8 In situ efficacy of LOEO as fumigant
In situ practical applicability of LOEO was designed to fumigate chia seed for 7 days followingwith slight modification. 5g chia seed with 10µL spore suspension were added into four sets viz. inoculated MIC, inoculated 2MIC, un-inoculated MIC and un-inoculated 2MIC along with inoculated and un-inoculated control sets were prepared and kept in BOD for 10 days at laboratory conditions at a temperature range from 27±2ºC. After that, the percent protection in the un-inoculated and inoculated treatments was compared with control sets by following formula
Percent protection = dc-dt/dc x 100
Where, Dc denotes percent occurrence of total fungi of control set and Dt denotes percent occurrence of total fungi in treatment set.
3.9 Statistical analysis
The experiments were done in triplicate and all data were analyzed for mean±SE. Then one way ANOVA was performed. Means were separated by Turkey’s multiple range testes when ANOVA was significant (p<0.05). The analysis of data was performed with the SPSS program version 16.0 for windows (SPSS inc., IBM Corporation).
RESULTS AND DISCUSSION
4.1 Standard curve preparation of LOEO
The calibration curve of LOEO was prepared in methanol and λmax was found to be 222.5 nm.
4.2 Encapsulation Efficiency (EE) and Loading Capacity (LC) of LOEO loaded chitosan nanoemulsion
The percentage of EE and LC of LOEO loaded chitosan emulsion was calculated by using UV –Visible spectrophotometer at 222.5 nm. The loading capacity of LOEO was found to be maximum (0.740%) at 1:1 ratio, while the percentage of EE of loaded chitosan nano emulsions for 1:1 (w/v) ranges from 19.66 to 23.34%.
4.3 Antifungal and antiaflatoxigenic efficacy of LOEO loaded chitosan nanoemulsion
The LOEO loaded chitosan nanoemulsion retarded the growth of toxic strain at 0.8µL/mL and AFB1 secretion at 0.8µL/mL (fig 2). The significant reduction in MIC and MAIC values of LOEO loaded chitosan nanoemulsion has been observed. Due to lower toxic potential of chitosan to mammalian system, it is widely used with EO’s to preserve the storage food commodities against the storage fungi (Rasooli, et al., 2008). The result showed that there is negative correlation between the oil application and mycelia as well as mycotoxins production. The dose dependent decrement in aflatoxin production revealed a pronounced activity of encapsulated LOEO chitosan nanoemulsion. These inhibitory effects are interestingly getting connected with the prevention of mycotoxins contamination in food.
4.4 Mode of action of LOEO loaded chitosan nanoemulsion against AF LHP –SH-1 cells
Ergosterol is a vital one in fungal cell membrane belong sterol groups. It has the role to regulate the membrane fluidity, plasma membrane biosynthesis and its function (Yang et al., 2015). It also involves in structural, signaling processes and component of fungal extra celluar vesicles which act as a vehicle for the trans cell transport in fungi (Rodrigues, 2018). Part of this got indulged in transcription control of genes which codes for ergosterol biosynthetic enzymes and also proteins which is important for sterol uptake and processing. The ergosterol biosynthesis is the major target to produce good antifungals (Odds et al., 2003) and it can be used as a chemical marker for the presence of fungal contamination (Perkowski et al., 2008). Another reason behind is, there is so much of correlations between the content of ergosterol and fungal biomass which revealed the specificity of ergosterol as an indicator of fungi to detect and also to find the fungal colonization and their proliferation ( Lafi et al., 2018). So it is necessary to check its performance on the fungal cell wall with LOEO loaded chitosan nanoemulsion. The present study reveals that at 0.40µL/mL, the ergosterol content of the fungal cells have been inhibited as increasing concentration of LOEO loaded chitosan nanoemulsion in dose dependent manner and the percent inhibition for the respective concentration have been depicted in the fig 3 . This analysis is being utilized for different aspects including checking the agriculture quality, food deterioration during storage and also the gowth of molds (Lafi et al., 2018).
Furthermore, there was a study on methylglyoxal (MG) has been assessed aginst LOEO loaded chitosan nanoemulsion. It is an extremely very toxic compound which has been formed as an intrinsic intermediate in glycolysis pathway where free forms of DHAP and GA-3-P led to the formation of methylglyoxal which has the ability to react and modify the various molecular targets (Aguilera & Antonio Prieto., 2004). Methylglyoxal also stimulate the uptake of Ca2+ions. If there is any changes occurring in the methylglyoxal pathyway it ultimately disrupt the Ca2+ ions uptake and finally cellular functions of fungi got collapsed which leads to the death of the cell (Maeta et al., 2005). So the assay of methylglyoxal plays an important role to check the fungal growth. The effect of various concentrations of LOEO loaded chitosan nanoemulsion on MG level of AF LHP –SH-1 cells revealed, there is significantly reduction in MG content (fig 4) as dose dependent manner. MG level in control set was found to be 354.98±0.448 while at the MIC 202±0.433 and 2MIC 192.83±0.507 of LOEO loaded chitosan nanoemulsion.
4.5 Antioxidant activity of LOEO free oil through DPPH free radical assay
The aromatic plant products and spices have shown the property of antioxidant activity owing to the presence of the hydroxyl groups in their chemical structure (Shan et al., 2005). During DPPH radical assay, DPPH solution is mixed with a substance that has the capacity to donate hydrogen atom and gives rise to reduced form 1,1 diphenyl-2-picrylhydrazine molecules (non radical). The extent of the antioxidant activity is usually observed in the loss of violet colour the loss, converting it to yellow colour and measured in terms of the decrease in absorbance at 517 nm. The graphical picture represents the antioxidant activity of LOEO loaded chitosan nanoemulsion in terms of IC50 value. It was found to be 26.10µL/mL which is lower than Cinnamomum camphora (31.85µL/mL) and Pelargonium odoratissimum (96.63µL/mL) (Prakash et al., 2016).
4.6 In situ efficacy of LOEO
The conducted In situ practical fitness of the LOEO nanoemulsion in stored food system is an important determinant to check the preservative potential which showed 100% remarkable protection efficacy of inoculated as well as uninoculated Salvia hispanica seeds at MIC and 2MIC concentration against A. flavus LHP-SH-1 contamination. The result depicted its application at large scale plant based preservative during prolong storage condition.
4.7 Fungitoxicity assessment
A broad spectrum fungitoxicity was furthermore checked at MIC level of LOEO against 14 storage molds associated with food commodities and get 100% inhibition in all recorded fungal species and, thus, outcome strengthens to recommend LOEO for complete protection of preserved Salvia hispanica and other functional food commodities from a range of biodeteriorating fungus and their potential mycotoxins contamination.
The present investigation opened up the antifungal, antiaflatoxigenic and antioxidant activity of LOEO loaded chitosan nanoemulsion which has the potentiality as a plant based preservative and also as the shelf life enhancer of stored food commodities. The further findings also help to reveal the mystery of bioactivity and stability of LOEO for its long time duration, nanoencapsulation is a novel approach. The present study also proved the LOEO loaded chitosan nanoemulsion has caused significant enhancement in antifungal and antiaflatoxigenic ability to inhibit the AFB1 secretion at a concentration of about 0.80µL/mL. It also has the capacity to interfere or has the negative effects on the cell membrane and the intracellular components of the AF LHP-SH-1 cells. Therefore, the nanoemulsion of LOEO may be recommended as a plant based preservative in food and also in agriculture industry.
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