Screening of wheat grains enriched with wall-bound phenolic compounds

Phenolic compounds are dominant antioxidant factors in whole grains and are essential quality traits in future breeding programs. We proposed a robust set of methods for extraction, screening, and quantitative analysis of soluble and wall-bound (WB) phenolic compounds from fine powder and fine powder products using a 96 Wells UV Flat Bottom and subsequent UHPLC-DAD validation of candidate samples. The plate-UHPLC strategy significantly simplifies the screening of phenolic-enriched grains, reduces the screening cost, saves harmful organic chemicals, and contributes to developing novel health-promoting varieties.

a b s t r a c t Phenolic compounds are dominant antioxidant factors in whole grains and are essential quality traits in future breeding programs. We proposed a robust set of methods for extraction, screening, and quantitative analysis of soluble and wall-bound (WB) phenolic compounds from fine powder and fine powder products using a 96 Wells UV Flat Bottom and subsequent UHPLC-DAD validation of candidate samples. The plate-UHPLC strategy significantly simplifies the screening of phenolicenriched grains, reduces the screening cost, saves harmful organic chemicals, and contributes to developing novel health-promoting varieties.

Description of protocol: Methods details Background
The antioxidant characteristic of wholegrain phenolics benefits human health by lowering the risks of cardiovascular disease (CVD), gastrointestinal (GI) cancer, and diabetes [3] . Cell wall-bound (WB) phenolic acids, mainly ferulates (FA), account for over 70% of the total phenolic acid concentration [4] . FA primarily exists in the cell walls of the seed coat and aleuronic layer than in endosperm [5] . Many phenolic compounds have been detected and quantified in plant samples by ultra-high-performance liquid chromatography with a diode array detector (UHPLC-DAD) [6] .
We screened wheat mutants enriched with ferulic acid by UHPLC-DAD to increase the antioxidant potential of foods. Screening mutant grains led to identifying mutants accumulating ferulates in the grains [2] . The biochemical and metabolite study revealed that an SNP in the tetraploid wheat increased the FA content and the antioxidant potential, which benefits consumers [1] . Nevertheless, UHPLC experiments take a long time and consume many harmful organic chemicals, which are unfriendly to the environment. To resolve the above limitations, we added a UV absorbance-based plate screening step before the UHPLC validation of candidates to save time and chemicals.

Step 1: Wheat sample preparation
We recommend the following method for the rapid preparation of large quantities of trace samples (less than six grains): Table 1 The regent gradient that was used for the separation and identification of WB phenolic compounds. Step 6: Quantitative phenolic acid analysis The analysis was performed using an Agilent 1290 Infinity system with a CNW Athena C18-WP HPLC Column (3um, 4.6 × 250mm) coupled with a DAD detector (Agilent, Santa Clara, CA, USA) [ 7 , 8 ]. 5 μL injection volume was used with a total flow rate of 1 mL/min over a full run time of 18 min. Mobile phase A consisted of HPLC-grade ddH 2 O with 0.1% acetic acid (v/v), while mobile phase C was acetonitrile with 0.1% acetic acid (v/v). The gradient details are in Table 1 .

Results and discussion
Our data demonstrated that the UV-spectrophotometer screening method effectively screened phenolic-enriched grains, although its precision and sensitivity were weaker than UHPLC. Given the cost and time reductions, the UV spectrophotometer/UHPLC strategy improves efficiency. Analyzing 96 samples take less than 15 min compared with 1920 min by the UHPLC method [1] . If the top ten samples were selected and analyzed in detail, it would save 90% of chemicals by the UV spectrophotometer/UHPLC strategy. Therefore, the above scenario represents a time-saving, cost-effective, and environmentally friendly method for future health-promoting grain breeding programs (Fig. 1) .

Funding
This work was supported by the National Natural Science Foundation of China ( 31972350 ).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.