Co-encapsulation of Pimpinella anisum and Coriandrum sativum essential oils based synergistic formulation through binary mixture: Physico-chemical characterization, appraisal of antifungal mechanism of action, and application as natural food preservative

https://doi.org/10.1016/j.pestbp.2022.105066Get rights and content

Highlights

  • Preparation of novel synergistic formulation of essential oils by binary mixture.

  • Encapsulation of formulation into chitosan nanobiopolymer and its characterization.

  • Encapsulation enhanced antifungal and AFB1 inhibitory efficacy of formulation.

  • Encapsulated binary formulation retarded lipid peroxidation in rice.

  • Encapsulated formulation displayed safety to mammalian system.

Abstract

The present study aimed to co-encapsulate binary synergistic formulation of Pimpinella anisum and Coriandrum sativum (PC) essential oils (0.75:0.25) into chitosan nanoemulsion (Nm-PC) with effective inhibition against fungal proliferation, aflatoxin B1 (AFB1) secretion, and lipid peroxidation in stored rice. Physico-chemical characterization of Nm-PC by SEM, FTIR, and XRD confirmed successful encompassment of PC inside the chitosan nanomatrix with efficient interaction by functional groups and reduction in crystallinity. Nm-PC showed superior antifungal, antiaflatoxigenic, and antioxidant activities over unencapsulated PC. Reduction in ergosterol biosynthesis and enhanced leakage of Ca2+, K+, Mg2+ ions and 260, 280 nm absorbing materials by Nm-PC fumigation confirmed irreversible damage of plasma membrane in toxigenic Aspergillus flavus cells. Significant diminution of methylglyoxal in A. flavus cells by Nm-PC fumigation illustrated biochemical mechanism for antiaflatoxigenic activity, suggesting future exploitation for development of aflatoxin resistant rice varieties through green transgenic technology. In silico findings indicated specific stereo-spatial interaction of anethole and linalool with Nor-1 protein, validating molecular mechanism for AFB1 inhibition. In addition, in situ investigation revealed effective protection of stored rice against fungal occurrence, AFB1 biosynthesis, and lipid peroxidation without affecting organoleptic attributes. Moreover, mammalian non-toxicity of chitosan entrapped PC synergistic nanoformulation could provide exciting potential for application as eco-smart safe nano-green food preservative.

Introduction

Fungi are opportunistic contaminant of food commodities, basically cereals, in the storage conditions leading to deterioration of nutritional qualities by production of different toxic secondary metabolites (mycotoxins), thereby, becoming a burning issue in 21st century (Shanakhat et al., 2018; Devi et al., 2021). Aspergillus flavus is one of the most common and competent fungal species, frequently contaminate rice (Oryza sativa L.) during postharvest storage by sporulation and production of aflatoxin B1 (AFB1), especially in tropical and subtropical regions of the world (Das et al., 2021a). Recent investigation of Food and agricultural organization (FAO) suggested approximate 15% loss of harvested rice due to improper storage resulting in fungal and AFB1 contamination (Al-Zoreky and Saleh, 2019). More importantly, International agency for research on cancer (IARC) has categorized AFB1 as class 1 human carcinogen on the basis of mutagenic, immunosuppressive, teratogenic and carcinogenic properties. Methylglyoxal, a reactive carbonyl component produced as a degradation product of glyceraldehyde 3-phosphate and dihydroxy acetone phosphate has shown major deleterious effects on foods during storage by inducing the AFB1 biosynthesis and generation of advanced glycation end products (Upadhyay et al., 2018).

Although, the deployment of chemical preservative easily inhibit the contamination of fungi and their associated aflatoxins in foods during storage conditions, however, overuse of these synthetic chemicals may result in increasing pathogen resistance, adverse health effects and environmental non-sustainability which made the food scientist to shift their idea towards the plant based products (Nikkhah and Hashemi, 2020). In this context, application of essential oils has been progressively emphasized for preparation of green fungicides based on their less residual toxicity, volatile nature, and mammalian non-toxicity. However, utilization of higher doses of essential oils during practical application in food commodities provides unfavorable organoleptic properties (due to absorption of essential oil components), therefore, optimization of dosage concentration are very important. Recently, use of essential oils in combination or formulation has been practiced in different food and agricultural industries due to their synergistic effects and significant antifungal activity at very low concentrations (Songsamoe et al., 2017). Binary mixture of essential oils has special benefit for their synergistic action and maximum participation of major components of different essential oils (Reyes-Jurado et al., 2019) with wide scale practical applicability in food industries for mitigation of fungal, aflatoxins and oxidative biodeterioration mediated postharvest problem of food commodities.

Inspite of proven antifungal efficacy of essential oils and combinations, their practical application is non-economical and limited due to easy degradation of volatile components by environmental factors like heat, oxygen, light and pressure, aqueous phase insolubility and volatile nature (Gavahian et al., 2020). Nanoencapsulation is a novel cutting edge advantageous technology for entrapment of essential oils/formulations inside any biopolymer facilitating controlled as well as targeted delivery with enhanced bioefficacy in food system. Among different synthetic and natural polymers, chitosan (a cationic polysaccharide extracted by deacetylation of chitin) is effectively used as nanoencapsulant or wall material in food and pharmaceutical industries due to biocompatibility, biodegradability, non-toxic nature, easy binding ability, better electrostatic interaction with negatively charge molecules, film, gel, and emulsion forming properties and more importantly, consideration under GRAS category (Das et al., 2021b). Plenty of researches have been performed on nanoencapsulation of essential oils; however, the electrostatic interaction based ionic gelation coupled with high speed homogenization and sonication produce nano-range emulsionic particles, better aqueous phase dispersibility, physical stability and controlled delivery, a prerequisite for stored food preservation against fungal infestation and aflatoxin contamination.

Myristica fragrans is a commonly cultivated aromatic spice plant in Asiatic and European countries as flavoring agent and condiments. Essential oil isolated from seeds, seed coats and fruits (MFEO) showed antimicrobial, hepatoprotective, anti-inflammatory, carminative, spasmolytic and antioxidant properties (Piaru et al., 2012). Coriandrum sativum is an herbaceous plant usually grown in Middle East and Medditeranean regions of the world. Essential oil of C. sativum (CSEO) is effective against fungi, bacteria, dyspepsia, diarrhea and digestive disorders (Burdock and Carabin, 2009). Pimpinella anisum is an annual aromatic and medicinal herb cultivated in Medditeranean area of Asia as flavoring agent and essential oil (PAEO) is used as diuretic, neuroprotective, antiulcer, and anticonvulsant effects (Hashem et al., 2018). Anethum graveolens is an herbaceous medicinal plant and essential oil isolated from different plant parts (AGEO) is widely used as antifungal, antidiabetic, antihyperlipidemic, and antisecretory properties (Kaur et al., 2019). To date, few reports are available regarding antifungal activity of MFEO, CSEO, PAEO and AGEO, however, essential oil formulation based on binary mixture combination with potential fungitoxic efficacy against food contaminating fungi, aflatoxin secretion, and enhancement in overall biological efficacy after chitosan based nanoencapsulation, especially focusing on biochemical and molecular mechanisms are completely lacking.

Hence, the aim of the present investigation was to prepare binary formulation of effective essential oils by checkerboard assay and co-encapsulation of the most effective binary synergistic formulation into chitosan nanomatrix with resultant enhancement in antifungal and antiaflatoxigenic activities. More importantly, the study was focused on relevant biochemical and molecular mechanism associated with antifungal and AFB1 inhibitory efficacy. In addition, in situ fungitoxic, AFB1 inhibition, lipid peroxidation, and organoleptic property assessment of essential oil nanoformulation was also performed in rice as a model food system. Furthermore, safety profile of prepared nanoformulation was determined in animal model (mice) for its recommendation as safe, green, and smart preservative in food and agricultural industries.

Section snippets

Chemicals and solvents

Potato dextrose agar (PDA), SMKY (Sucrose, 200 g; MgSO4. 7 H2O, 0.5 g; KNO3, 0.3 g; yeast extract, 7 g), Methanol, acetonitrile, isoamylalcohol, perchloric acid (HClO4), DPPH, ABTS, toluene, acetone, KOH, K2S2O8, NaCl, thiobarbeturic acid (TBA), tricholoroacetic acid (TCA), HCl, chitosan, Tween-20, Tween-80, dichloromethane (DCM), diaminobenzene (DAB), n-heptane, and chloroform were procured from Sisco Research laboratory (SRL), Mumbai, India.

Fungal strains

AFB1 producing strain of Aspergillus flavus (AF LHP

Extraction of MFEO, AGEO, PAEO, CSEO and their chemical characterizations

Fresh plants of A. graveolens, P. anisum, C. sativum and seed coats of M. fragrans were used for extraction of AGEO, PAEO, CSEO, and MFEO, respectively. GC–MS of MFEO revealed elemicine, and myristicine as major components. A total of 16 components were found in GC–MS of AGEO; among them, dill apiol, and carvone were reported as major components. Chemical characterization of CSEO presented 12 components, among them, linalool and geranyl acetate were found as major ingredient. GC–MS of PAEO

Conclusion

Encompassment of PC synergistic formulation into chitosan nanobiopolymer improved the antifungal, antiaflatoxigenic and antioxidant activities as compared to unencapsulated PC. Controlled volatilization of PC from chitosan nanomatrix facilitated long term protection of rice against fungal infestation, AFB1 production, and ROS mediated biodeterioration. The investigation also explored the biochemical mechanisms for inhibition of fungal growth and diminution of methylglyoxal production, a

Declaration of Competing Interest

Authors reported no conflict of interest.

Acknowledgements

Somenath Das is thankful to Council of Scientific and Industrial Research (CSIR) [File No.: 09/013(0774)/2018-EMR-I], New Delhi, India, for the financial support. The authors wish to thank the head and coordinator CAS in Botany, DST-FIST, DST-PURSE, ISLS, and CIFC-IIT, Banaras Hindu University (BHU) and Principal, Burdwan Raj College, Purba Bardhaman, West Bengal, India for laboratory facilities.

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