2.1 Biofilm Formation
To develop a fungal-bacteria biofilm, A. niger and S. maltophilia were used to represent the fungus and the bacterium, respectively. The two microbes were selected based on the results of a previous study in which they produced the highest EPS-biochemical diversity compared to the other biofilms types tested (U.M.B. Premarathna, unpublished). From each, one loopful of microbes was inoculated to 250 mL CCM  broth to prepare monocultures. Hundred microliters of each monoculture was taken and inoculated in to 15 ml sterilized centrifuge tube containing 10 ml of CCM medium, and was incubated for seven days to develop the fungal-bacterial biofilm.
2.2 Extraction of the BFEx
The extraction of BFEx was performed by combining physical and chemical methods . A NaCl solution was prepared by dissolving 5 g of NaCl in 100mL of sterilized distilled water. Ten microliters of the solution was poured to 15 ml centrifuge tube that contained the developed biofilm. Then, it was subjected to ultra-sonication for 10 minutes, followed by centrifugation at 5000rpm for 10 minutes. Finally, the supernatant in the centrifuge tube was taken for further experiments.
2.3 Potential of BFEx in breaking dormancy of gut microbes: in vitro gut simulation study with different dietary patterns
Five commonly found gut microbes [15, 16] viz. Bacillus clausii, Lactobacillus sporogenes, Lactobacillus reuteri, Bacillus subtilis, and Aspergillus niger were taken as test microbes (many of them are soil-based probiotics) that were isolated from fecal matter. They were grown as monocultures and mixed cultures in a simulated gut environment with eight different dietary patterns, i.e. low-carbohydrate, high-carbohydrate, low-protein, high-protein, low-lipid, high-lipid, low-fiber and high-fiber. Here, culture medium described by Parmanand et al. (2019)  and Macfarlane et al. (1998)  was used after modifying. The culture medium contained; casein 3 g/l, yeast extract 2 g/l, NaCl 0.1 g/l, K2HPO4 0.04 g/l, KH2PO4 0.04 g/l, MgSO4.7H2O 0.01 g/l, CaCl2.6H2O 0.01 g/l, NaHCO3 2 g/l, Tween-80 2 ml, glucose 10 g/l, vitamin K1 10 µl, cysteine HCl 0.5 g/l, bile salts 0.5 g/l, Starch 10 g/l, Pectin 2 g/l. The pH was controlled and maintained between 6.2–6.6. Temperature was set to 37 oC. Moreover, the culture medium was modified to have different levels of carbohydrate, protein, lipid, and fiber as diets. For that, starch, casein, tween 80, and pectin were selected to represent carbohydrate, protein, lipid, and fiber, respectively. Likewise, eight different media were prepared to represent relatively low and high concentrations (i.e. 50% and 150% of the above concentrations of each dietary source as the low and high concentrations, respectively) of the diets in the gut environment. Each concentration with or without BFEx formed two treatments. Each treatment had three replicates in a completely randomized design. Live microbial cell concentrations were determined after 24 and 48 hours of inoculation using LIVE/DEAD™ BacLight™ bacterial viability kit (Invitrogen Molecular Probes, Eugene, OR, USA) [19, 20, 21].
2.4 Brine shrimp lethality assay for BFEx
Brine shrimp (Artemia salina L.) eggs were hatched in a half covered beaker (250 mL) containing artificial sea water and were allowed to stay for two days at room temperature (RT) . During the hatching period, constant and continuous aeration was given through an oxygen pump and illumination was given with a 20 W bulb. After 48 hours, 2nd larval stage (nauplii) of brine shrimps was observed towards the illuminated side of the beaker. Test samples were prepared in artificial sea water having 0, 100 and 300-fold dilutions of the BFEx, triplicated for each concentration. The test samples of 2 mL each were poured in to a 24 well plate. Each concentration was added with 10 brine shrimp nauplii and allowed to stay for another 48 hours under illuminated conditions at RT. After 24, and 48 hours, observations were taken and the percentage lethality was calculated.