Microbial conversion of vanillin for enhanced antimicrobial activity: A comprehensive study

Introduction

Vanillin, a phenolic compound derived from Vanilla planifolia, plays a vital role in natural flavors and is extensively employed in the food, cosmetics, pharmaceutical, and medical sectors. It serves as a flavor enhancer and food preservative owing to its antimicrobial and antioxidant characteristics. Although traditionally obtained from beans, synthetic production is prevalent due to cost efficiency. Synthetic vanillin is also used in the manufacturing of herbicides, antifoaming agents, household items, deodorants, air fresheners, and cleaning supplies. Nevertheless, its potent scent at high concentrations restricts its possible uses. Vanillin and its related aromatic compounds have been shown to have inhibitory effects on the growth of fungi and bacteria (Adeboye et al., 2014) [1] . While a small amount of vanillin is naturally sourced, the majority is produced through chemical synthesis (Priefert et al., 2001) [9] . Current research is focused on developing new methods for producing natural vanillin through biosynthesis using microorganisms. Efforts are being made to find efficient ways to convert various precursors into vanillin through microbial biotransformation. Microbial transformation involves the specific modification or chemical alteration of a compound using biological catalysts such as fungi, bacteria, dead microorganisms, cellular extracts, and isolated enzymes (Bains, 1993; Lilly, 1994) [3,5] . The resulting compounds may have enhanced properties compared to the original compound or may be identical to its metabolites. Therefore, microbial transformation can be used as a substitute for chemical synthesis to produce drug metabolites, resulting in the discovery of new products with distinct activities. In recent years, there has been a significant increase in consumer demand for natural ingredients. As a result, alternative natural sources of ingredients have been explored, and production methods involving microbial biotransformation of natural precursors have become more appealing. This approach is essential for the sustainable use of natural resources to develop commercially valuable products (Alvarado et al., 2003; Mishra et al., 2016) [2,6] . The breakdown of vanillin and its derivatives by microorganisms has been extensively researched, as vanillin can be toxic to many microorganisms even at low levels. Therefore, the ability to break down vanillin is crucial for microorganisms in natural settings or industrial processes where vanillin is present. Vanillin and ethyl vanillin have antibacterial properties due to their phenolic groups. Ongoing efforts are being made to enhance their antimicrobial effects, as bacteria are highly vulnerable. A combination of vanillin and cinnamon has been found to have a synergistic impact on antibacterial activity against E. coli and antifungal activity against L. monocytogenes (Roda et al., 2012) [11] . The oxidation of vanillin to produce its carboxylic acid has been carried out to enhance its antibacterial properties (Mourtzinos et al., 2009) [8] . A vanillin-derived piperidin-4-one oxime ester has been synthesized and shown to have superior antibacterial activity compared to the positive control (Harini et al., 2012) [4] . Acetyl vanillin derivatives have also been synthesized and displayed antibacterial properties (Yadav et al., 2018) [14] . This research investigates the transformation of vanillin using fungal species to improve its effectiveness and evaluate its antimicrobial properties against silkworm pathogens.

Materials and Methods

Chemicals and fungal strains

Culture media, solvents, and 4-hydroxy-3methoxybenzaldehyde (vanillin) were obtained from HiMedia Laboratories (Mumbai, India). Thin-layer chromatography precoated plates (Silica gel, Merck, PF254) were utilized. Fungal cultures Debaromyces species (MCC 1072) and Paecilomyces variotii (NCIM 1217) were acquired from the culture collection centers NCCS and NCL in Pune, India, respectively. These cultures were sub cultured on Potato Dextrose Agar and allowed to incubation for 5 days, with the marginal area culture being chosen for the conversion process.

Conversion and product extraction

The conversion experiments were carried out in 100 ml of Potato Dextrose Broth with fungal cultures. Vanillin was added to the established culture medium at a concentration of 20 mg/ml. The culture control consisted of only fungal culture, while the substrate control contained only the parent compound. The reaction was conducted at 120 rpm at 26±1ºC in a shaking incubator for seven days. Product formation was monitored starting from the second day. Products were extracted by adding solvents to the culture medium. The fungal mycelium and culture medium were separated by filtration using Whatman No. 3 filter paper. The filtered reaction mixture was extracted three times with an equal volume of ethyl acetate. The combined organic layer extract was dried with anhydrous Na 2 SO 4 and evaporated under reduced vacuum pressure in a rotary evaporator to obtain the crude extract.

Analytical methods

The converted products were detected through Thin Layer Chromatography (TLC) (Merck, Germany) and compared with the original compound. The extracts were applied to TLC silica plates and eluted with a mixture of ethyl acetate and hexane in an 8:2 ratio. The products were visualized using iodine vapor. The crude extract of the products was then further purified using column chromatography with silica gel (60-100 mesh size). The metabolites were confirmed through 1 H NMR analysis using Bruker Biospin (IISc, Bangalore, India). The NMR spectra were analyzed using MestreNova 12.0.0 software.

Antimicrobial activity assays

A bacterial pathogen (1x10 9 cells/ml) was applied to a Muller Hinton agar plate, while a fungal pathogen (1x10 7 conidia/ml) was applied to a potato dextrose agar plate. Discs with a diameter of 6 mm, soaked in varying concentrations of the converted product solution, were placed on the inoculated plates and then incubated at 32±1 °C for 24 hours. The fungal plates were incubated at 25±1 °C for 5 days. The presence of a zone around the disc was measured to assess antibacterial properties.

Systemic activity of the converted product

Various ratios of a mixture of derived product and mulberry leaf juice were combined and tested in vivo against pathogens. Third instar first-day silkworm larvae were orally administered one milliliter of silkworm pathogens at a concentration of 1x10 9 cells/ml (Bacillus, Streptococcus, Serratia, and Staphylococcus), and rearing continued until infection onset. In the control group, live pathogens were administered separately for comparison. Three replicates were maintained, with 200 larvae in each treatment group. Infected larvae were fed mulberry leaves sprayed with 0.5% and 1% concentration of the blend for three days, and observations were made on disease progression and mortality.

Statistical analysis

Statistical analysis of the data was carried out using standard procedures and reported as the mean±standard deviation. Post-hoc analysis was performed using Duncan's test, with a significance level of p<0.05.

Results

Screening of fungal strains and conversion of vanillin

A total of twenty-two fungal cultures were tested with the vanillin compound to investigate microbial conversion for novel products. Paecilomyces variotii and Debaromyces species were found to be capable of converting vanillin (Fig. 1) and producing two distinct new metabolites. Paecilomyces variotii transformed vanillin into metabolites 1 (R f 0.78) and metabolite 2 (R f 0.92), while Debaromyces sp. specifically converted metabolite 1 (R f 0.78) from the parent compound (Rf 0.82) (Fig. 2). Metabolite 1 was produced on the 3 rd day, and metabolite 2 on the 5 th day of the reaction. The parent compound was completely consumed on 7 th day, leaving only the newly formed products.

Structural elucidation of products

The parent compound and its metabolites were purified and analyzed using NMR spectroscopy.

Antimicrobial activity of converted metabolites

The antibacterial activity of the compounds was tested using in vitro well diffusion method against both Gram-positive and Gram-negative bacteria. The diameter of the inhibition zone around the disc in Metabolite No.1 demonstrated the highest antibacterial activity against Staphylococcus sp. with a 22.67 mm, while the control vanillin had an activity of 12.5 mm. The minimum inhibitory concentration of the metabolites effectively inhibited the growth of all pathogens at 1%, indicating the presence of bioactive compounds in the extract. Furthermore, metabolite No.1 exhibited highest antifungal activity against Alternaria alternata with a 20.0 mm inhibition zone compared to the control 15.0 mm (Table 1&2).

In vivo efficacy studies using Silkworm larvae

The efficacy of the metabolites was assessed through in vivo experiments on PMxCSR2 Silkworm breeds. A blend of modified compounds mixed in mulberry leaf juice was administered to fresh Mulberry leaves at varying concentrations (0.5 and 1%), which were readily consumed by silkworms in infected groups. Pupal survival rates increased in treated groups exposed to Bacillus by 15% and followed by Staphylococcus 13% in PMxCSR2 breeds at a 1% concentration compared to the pathogen inoculated control group. There was significant enhancement in survival rates observed against pathogens except Serratia species (Table 3).

Discussion

The study demonstrates the biotransformation of vanillin by Paecilomyces variotii and Debaromyces fungal strains, resulting in the formation of two major metabolites with enhanced antimicrobial activity. The identified metabolites, vanillic acid and vanillyl alcohol, represent the oxidation and reduction products of vanillin, respectively. These biotransformation reactions are consistent with the known metabolic pathways of aromatic aldehydes in microorganisms, where aldehydes can be either oxidized to carboxylic acids by aldehyde dehydrogenases or reduced to alcohols by aldehyde reductases (Ziadlou et al., 2020; Moon and Liu, 2015) [15,7] . The ability of P. variotii to efficiently transform vanillin may be attributed to their robust enzymatic systems for metabolizing aromatic compounds (Shashwati et al., 2006) [12] . These fungi are known to inhabit diverse ecological niches, including plant materials rich in phenolic compounds, and have evolved efficient detoxification mechanisms for such compounds (Mishra et al., 2016) [6] . The differential production of vanillic acid and vanillyl alcohol by various fungal strains likely reflects the relative activities of aldehyde dehydrogenase and aldehyde reductase enzymes in these organisms. The enhanced antimicrobial activity of the converted products compared to vanillin is a significant finding of this study. Vanillic acid exhibited stronger antibacterial activity, particularly against Gram-positive bacteria (Vale et al., 2019) [13] while vanillyl alcohol showed lesser antifungal activity. These differences in antimicrobial spectrum may be related to the structural features and physicochemical properties of these compounds, which influence their interactions with microbial cell components. Vanillic acid, with its carboxylic acid group, may disrupt bacterial cell membranes more effectively, possibly by interfering with membrane proteins or by chelating essential metal ions (Mourtzinos et al., 2009) [8] . The stronger activity against Gram-positive bacteria compared to Gram-negative bacteria is consistent with previous reports (Qian et al., 2020) [10] on phenolic acids and may be attributed to differences in cell wall structure and composition between these bacterial groups (Roda et al., 2012) [11] . Vanillyl alcohol, with its hydroxyl group, may have better penetration into fungal cell walls, possibly due to its higher lipophilicity compared to vanillic acid. The antifungal mechanism may involve disruption of membrane integrity, inhibition of ergosterol biosynthesis, or interference with cell wall synthesis (Harini et al., 2012) [4] . The synergistic antimicrobial effect observed with the combination of vanillic acid and vanillyl alcohol is particularly interesting. This synergism may result from the complementary modes of action of these compounds, targeting different cellular components or metabolic pathways in the microbial cells. Such synergistic effects have been reported for other phenolic compounds and are of considerable interest for developing more effective antimicrobial formulations (Yadav et al., 2018) [14] . The in vivo efficacy studies using silkworm larvae provide valuable insights into the potential therapeutic applications of these compounds. The enhanced survival rates observed in infected larvae treated with vanillic acid, vanillyl alcohol, or their combination, along with the reduced microbial loads confirm the in vivo antimicrobial efficacy of these compounds. This study demonstrates that fungal strains have the ability to convert vanillin into vanillic acid and vanillyl alcohol, both of which exhibit enhanced antimicrobial properties. These derivatives have the potential to serve as potent broad-spectrum antimicrobial agents effective against a range of bacteria and fungi.

Acknowledgement

AcknowledgmentsThe authors express their gratitude to the Central Silk Board for their financial support (Project ARP 3634) and appreciate the assistance of Smt. K. R. Rathnamma, Senior Technical Assistant and Harish H.N, Field Assistant of Silkworm Seed Technology Laboratory, in conducting experiments and rearing of silkworms.

References

1. Adeboye PT, Bettiga M, Olsson L. The chemical nature of phenolic compounds determines their toxicity and International Journal of Advanced Biochemistry Research https://www.biochemjournal.com induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates. AMB Express. 2014;4:1-10.
2. Alvarado E, Navarro D, Record E, Asther M, Asther M, Lesage ML. Fungal biotransformation of 4-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant. World Journal of Microbiology and Biotechnology. 2003;19:157-160.
3. Bains W. Biotransformation of aromatic compounds by halophilic bacteria. Journal of Chemical Technology and Biotechnology. 1993;57(1):75-84.
4. Harini ST, Kumar HV, Rangaswamy J, Naik N. Synthesis, antioxidant and antimicrobial activity of novel vanillin derived piperidin-4-one oxime esters: Preponderant role of the phenyl ester substituents on the piperidin-4. Bioorganic and Medicinal Chemistry Letters. 2012;22:7588-7592.
5. Lilly MD. Advances in biotransformation processes. Chemical Engineering Science. 1994;49:151-159.
6. Mishra S, Kullu M, Sachan A, Vidyarthi AS, Sachan SG. Bioconversion of ferulic acid to vanillic acid by Paenibacillus lactis SAMS-2001. Annals of Microbiology. 2016;66(2):875-882.
7. Moon J, Liu ZL. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae. Yeast. 2015;32(4):399-407.
8. Mourtzinos I, Konteles S, Kalogeropoulos N, Karathanos VT. Thermal oxidation of vanillin affects its antioxidant and antimicrobial properties. Food Chemistry. 2009;114:791-797.
9. Priefert H, Rabenhorst J, Steinbüchel A. Biotechnological production of vanillin. Applied Microbiology and Biotechnology. 2001;56:296-314.
10. Qian W, Yang M, Wang T, Sun Z, Liu M, Zhang J, et al. Antibacterial mechanism of vanillic acid on physiological, morphological, and biofilm properties of carbapenem-resistant Enterobacter hormaechei. Journal of Food Protection. 2020;83(4):576-583.
11. Roda RM, Taboada-Rodríguez A, Valverde-Franco MT, Marín-Iniesta F. Antimicrobial activity of vanillin and mixtures with cinnamon and clove essential oils in controlling Listeria monocytogenes and Escherichia coli O157:H7 in milk. Food and Bioprocess Technology. 2012;5:2120-2131.
12. Shashwati G, Ashish S, Adinpunya M. Formation of vanillic acid from ferulic acid by Paecilomyces variotii MTCC 6581. Current Science. 2006;90(6):825-829.
13. Vale J, Ribeiro M, Abreu AC, Soares-Silva I, Simões M. The use of selected phytochemicals with EDTA against Escherichia coli and Staphylococcus epidermidis single-and dual-species biofilms. Letters in Applied Microbiology. 2019;68(4):313-320.
14. Yadav R, Saini D, Yadav D. Synthesis and evaluation of vanillin derivatives as antimicrobial agents. Turkish Journal of Pharmaceutical Sciences. 2018;15:57-62.
15. Ziadlou R, Barbero A, Martin I, Wang X, Qin L, Alini M, et al. Anti-inflammatory and chondroprotective effects of vanillic acid and epimedin C in human osteoarthritic chondrocytes. Biomolecules. 2020;10(6):932.