Combinatorial chemistry has been a major focus of pharmaceutical and biotechnological research in drug discovery and optimization [1] . It has also been proposed for applications in agrisciences [2] - [4] . The basic concept of combinatorial chemistry is the synthesis of a vast population (combinatorial library) of structural variants of a parent molecule. The library is then screened in a high-throughput scheme for the few variants carrying targeted new properties of desirable function/activity. Recently, we have applied the concept of combinatorial chemistry to enzyme-catalyzed hydrolytic conversion of plant fibers to bioactive microfibrils/oligosaccharides . Plant cell walls contain fibrous polymers that are particularly suitable and useful substrates in this scheme. For example, xylan has a β-1,4-linked xlosyl main chain decorated with several side groups, including phenolic (ferulic acid), acetyl, glucuronyl, and arabinofuranosyl groups [6] . Specific enzymes targeting each side group individually or in various combinations under controlled reaction conditions constitute a combinatorial scheme . The enzymes for specific cleavage of these side groups are available commercially or produced by custom cloning, including feruloyl esterase, acetylxylan esterase, β-glucuronidase, and α-L-arabinofuranosidase. The cleavage of the side groups, their positions on the main chain, and types of linkages would affect the cleavage pattern of the main chain and vice versa.

In our previous investigations, we enzymatically hydrolyzed wheat insoluble arabinoxylan (WIA) and screened for bioactive feruloyl oligosaccharides [7] [8] . The present work describes a preparative scale fractionation of combinatorial enzyme digest of hot water pretreated WIA to recover antimicrobial FOS species also showing antioxidant capacity based on the electron transfer method using Trolox as the standard.

The current study used Amberlite XAD preparative column chromatography to isolate FOS fractions from combinatorial enzyme digestion of hot water pretreated WIA as outlined recently [8] [9] [15] . The lyophilized FOS pool produced a puffy white color material which chemically comprised of 241.00 ± 4.12 nmoles ferulic acid, about one feruloyl side group per 25 xylose units. The average size of the oligosaccharides was ~4 xylose units.

The current study confirms the inhibitory effect of FOS on the growth of the test E. coli strain (ATCC 8379). The inhibitory effect increased with concentration, and a complete suppression of cell growth was achieved at a MIC (minimum inhibitory concentration) value of 0.9% w/v ( Figure 1 ).

The antimicrobial activity of FOS was due to the ferulic acid moiety carrying reactive (electrophilic) double bond structures that can participate in inactivation of cell biomolecular reactions. The inhibitory mechanism involves damaging effects on cell wall permeability, disrupting metabolism in cell wall synthesis, and interfering with intracellular enzyme reactions important to cell constituents [16] [17] . In our previous studies on pectic hydrolysate obtained by enzymatic digestion of citrus pectin with endo-polygalacturonase and pectate lyase, we identified active pectic oligo species with antimicrobial properties . The inhibitory action was attributed to the reactive double bonds (formed by the elimination reaction of pectate lyase) the acidic nature of carboxylic side groups, and the small size range of the oligo molecule. In similar studies by others, it has been reported that enzyme digestion of birchwood xylan produces acidic (glucuronic acid-containing) xylo-oligosaccharides, particularly aldopentauronic acids, that are effective inhibitors of certain gram-positive bacteria [19] .

The FOS species is expected to possess antioxidant activity, which depends on the hydroxyl and methoxy groups attached to the phenolic acid ring. The existence of the ester bonds in FOS contributes to the high antioxidant activity compared to the free acid [20] .

The antioxidant properties of ferulic acids and its possible applications in pharmaceutical and food industry have been reviewed [21] - [23] . In the present study, using the Trolox equivalent antioxidant capacity (TEAC), is an electron transfer (ET) based method [24] . In this type of reaction, it detects the ability of a potential antioxidant to transfer one electron (reduction of Cu²⁺ to Cu⁺). The cuprous ion forms a colored complex with a dye reagent disodium [2,2’-biquinolino]4,4-dicarbnoxylate (= BCA bicinchoninic acid sodium salt). The discoloration assay measures the color intensity at 570 nm proportional to the total antioxidant capacity (TAC) of the sample. Based on the Trolox standard curve, the TAC of FOS at 1% was calculated to be 636 ± 7 μM ( Figure 2 ).

The production and use of non-digestible oligosaccharides (NDO) has been a thriving industry producing prebiotics for food applications. The health cause-effect of these products is generally linked to the effects on beneficial bacteria in the gut microbiome, due to modification of the physiological environment of the intestinal digestive system [25] . This biological effect of the FOS will be analyzed using metagenomic cell culture studies.

Wheat insoluble arabinoxylan was treated by the approach of combinatorial enzyme digestion. Bioactive FOS species in the digest were recovered by preparative chromatographic fractionation. Antimicrobial and antioxidant activities were detected and analyzed. This study revealed the dual-function health benefits of FOS.

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