Quince

The SILVA database v.106 and ARB software v. 5.2 were used to identify and test specificity of the different primers in silico[30] (link), [31] (link), [51] (link). An internally developed AmpliconGenerator v.0.1 (http://sf.net/projects/amplicongenerator) was used to characterize nucleotide variability along the reference sequence. The software was also used to generate in silico amplicons and to assess the conservation of biodiversity as a function of sequence similarity. IDT SciTools OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, IA) was used to estimate melting temperatures of the primers as well as the presence of hairpins, self- and hetero-dimers in the candidate primers. Sequences from the environmental samples were processed through the bioinformatic pipeline AmpliconNoise according to Quince and co-workers [6] . As a first step in the pipeline, low quality reads were removed, defined as shorter than 200 nt or having inadequate signal intensity. Subsequently, noise from the flowgram and PCR-generated errors were removed using established probabilistic iterative algorithms [6] , [52] (link) and chimeras removed using the program Perseus [6] . The reads were then clustered together using the complete linkage-clustering algorithm implemented in FCluster, based on pairwise distances as calculated by NDist [6] , and aligned to the SILVA and NCBI databases using BLAST for the purpose of taxonomic annotation. The same software package was also applied to build operational taxonomic units (OTUs). Since the choice of the pipeline can influence the results of metagenetic studies and because there is no consensus on which pipeline to use, the results produced with AmpliconNoise were compared to results produced by an alternative commonly used pipeline, QIIME. In QIIME, original reads were filtered based on the quality score, but no further denoising procedure was applied. Further, in order to remove potential chimeras, OTUs representing singletons were discarded.

Free-radical polymerization technique was used to fabricate a quince/mucin co-poly (methacrylate) hydrogel. Polymers, monomers and a cross-linker were incorporated at different ratios. The compositions of different formulations (QHM1–QHM12) are presented in Table 1. Specified amounts of each QH were dissolved in deionized water (5 mL) on a magnetic stirrer (VELP Scientifica) until the QH fragments were properly distributed. Similarly, mucin, APS, MAA and MBA were dissolved separately in deionized water (5 mL) on a hot plate magnetic stirrer. The solutions of the polymers (quince and mucin) were mixed together with continuous stirring (100 rpm), followed by APS incorporation. MBA was added to the solution of MAA with continuous stirring. This solution was transferred to the activated polymeric solution with continuous stirring. The whole mixture was sonicated for 5 min, transferred into glass tubes, and sealed with aluminum foil. The test tubes were kept in a thermostatically controlled water bath (Memmert, Tokyo, Japan) at 55 °C for 2 h and then at 65 °C for 8 h. After a specified period of time, the test tubes were removed from the water bath and placed at room temperature for some time. The prepared hydrogel was removed with the help of a spatula and impregnated with ethanol. The cutting of the prepared hydrogels in the form of discs (5 mm thick) was carried out with the help of a sharp blade. Washing was executed with an ethanol and water mixture (70:30) for 30 min to remove unreacted contents. The drying of the discs was accomplished in a hot air oven at 45 °C until a constant weight was achieved [20 (link),21 (link)].

Molar solutions with different concentrations of sodium chloride and calcium chloride (0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M) were prepared to assess the equilibrium swelling of the quince/mucin co-poly (methacrylate) hydrogel discs. Pre-weighed discs were immersed in the electrolyte solutions for 24 h, after which they were removed from the respective immersion mediums and reweighed on an analytical balance to assess their equilibrium swelling capacities as g/g [22 (link),23 (link)].

Quince seeds (100 g) were immersed in distilled water (500 mL) for 8 h to isolate the quince hydrogel (QH). To maximize the yield, heating at 50 °C for at least 30 min was performed. The extracted QH was separated with a cotton cloth and washed with n-hexane. After washing, the QH was transferred to Petri plates and dried in an hot air oven at 50 °C for 48 h. The dried QH was powdered using a mortar and pestle, sieved through a 60-mesh sieve, and stored in a well-closed plastic jar for further use. The yield of the QH was estimated at 11 gm/100 gm of dried seeds [14 (link)].